Research ArticleThe Consumption of Bicarbonate-Rich Mineral WaterImproves Glycemic Control

Shinnosuke Murakami12 Yasuaki Goto3 Kyo Ito4 Shinya Hayasaka35 Shigeo Kurihara3

Tomoyoshi Soga12 Masaru Tomita12 and Shinji Fukuda12

1Systems Biology Program Graduate School of Media and Governance Keio University 5322 Endo FujisawaKanagawa 252-0882 Japan2Institute for Advanced Biosciences Keio University 246-2 Mizukami Kakuganji Tsuruoka Yamagata 997-0052 Japan3Onsen Medical Science Research Center Japan Health and Research Institute 1-29-4 Kakigaracho Nihonbashi Chuo-kuTokyo 103-0014 Japan4Ito Medical Office 7985-5 Nagayu Naoirimachi Taketa Oita 878-0402 Japan5Faculty of Human Life Sciences Tokyo City University 8-9-18 Todoroki Setagaya-ku Tokyo 158-8586 Japan

Correspondence should be addressed to Shinnosuke Murakami mushinsfckeioacjpand Shinji Fukuda sfukudasfckeioacjp

Received 29 September 2015 Revised 4 November 2015 Accepted 22 November 2015

Academic Editor Jenny M Wilkinson

Copyright copy 2015 Shinnosuke Murakami et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hot spring water and natural mineral water have been therapeutically used to prevent or improve various diseases Specificallyconsumption of bicarbonate-rich mineral water (BMW) has been reported to prevent or improve type 2 diabetes (T2D) in humansHowever the molecular mechanisms of the beneficial effects behind mineral water consumption remain unclear To elucidatethe molecular level effects of BMW consumption on glycemic control blood metabolome analysis and fecal microbiome analysiswere applied to the BMW consumption test During the study 19 healthy volunteers drank 500mL of commercially available tapwater (TW) or BMW daily TW consumption periods and BMW consumption periods lasted for a week each and this cycle wasrepeated twice Biochemical tests indicated that serum glycoalbumin levels one of the indexes of glycemic controls decreasedsignificantly after BMW consumption Metabolome analysis of blood samples revealed that 19 metabolites including glycolysis-related metabolites and 3 amino acids were significantly different between TW and BMW consumption periods Additionallymicrobiome analysis demonstrated that composition of lean-inducible bacteria was increased after BMWconsumption Our resultssuggested that consumption of BMW has the possible potential to prevent andor improve T2D through the alterations of hostmetabolism and gut microbiota composition

1 Introduction

Self-medication is an important approach to maintain andpromote human health There are many strategies of self-medication such as improving onersquos lifestyle andor dietaryhabits consumption of functional foodsbeverages and get-ting adequate exercise [1 2] Hot spring water and naturalmineral water are traditionally used in public baths andbalneotherapy in many countries Balneotherapy is the use ofthermal andor mineral water derived from natural springsor drilled wells for treatment of human health by employing

various methods such as bathing drinking mud therapyand inhalation [3] According to previous studies it has beenreported that balneotherapy has beneficial effects for variousdiseases such as type 2 diabetes (T2D) [4ndash6] rheumatism[7] low back pain [3] and cardiovascular disease [8 9]Particularly consumption of bicarbonate-rich mineral water(BMW) has been reported to prevent or improve T2D[4 6] However as the reported benefits of BMW weredetermined epidemiologically and clinically the molecularmechanisms of the beneficial effects behind BMW remainunclear

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 824395 10 pageshttpdxdoiorg1011552015824395

2 Evidence-Based Complementary and Alternative Medicine

Metabolomics is a method of comprehensive measure-ment of metabolites and it has been known to be a powerfultool to gain new insights into various research fields suchas metabolic syndrome [10] cancer [11 12] chronic kidneydisease [13] and biomarker screening [14] A recent studyhas reported that plasma metabolome profiles were alteredbetween healthy people and T2D patients [15] Moreoverit has been also reported that gut microbial compositionand function in T2D patients are different from that ofhealthy subjects [16ndash18] Therefore preventive andor ther-apeutic effects for T2D derived from BMW consumption areexpected via influencing blood metabolites concentrationsand gut microbial compositions

For this reason blood metabolome analysis and gutmicrobiome analysis were included in our study to eluci-date the molecular level effects of BMW consumption onglycemic control In this study BMW consumption test wasconducted amongst 19 healthy volunteers Here we show thatserum glycoalbumin levels were decreased and 19metabolitesincluding glycolysis-related metabolites and 3 amino acidswere changed after consumption of BMW Additionallymicrobiome analysis demonstrated that composition of lean-inducible bacteria family Christensenellaceae was increasedafter BMW consumption This current study is an importantstudy reporting the molecular level effects derived fromconsumption of BMW

2 Materials and Methods

21 Sample Preparation This study was approved by theEthical Committees of Japan Health and Research Instituteand Keio University Shonan Fujisawa Campus All subjectswere informed of the purpose of this study and writtenconsent was obtained from all subjects

In this study BMW consumption test was conductedamongst 19 healthy subjects (7 men and 12 women agesfrom 26 to 59 47 years old on the average) Initiallyindividual identification numbers were randomly assignedto 26 volunteers from N01 to N26 however N12 and N25dropped out due to personal reasons Additionally N01 N02N14 N18 and N21 were excluded from analysis because theyforgot to drink TW andor BMW at least once BMW wasobtained from Nagayu hot spring (Taketa Oita Japan) asit contains one of the highest bicarbonate concentrations inJapan TW was purchased from Nishikawa water treatmentplant (Yamagata Japan) Mineral contents and pH of the TWandBMWused in this studyweremeasured by theHot SpringResearch Center (Tokyo Japan) and are as shown in Table 1BMW were collected in 500mL plastic bottles and storedin refrigerator until consumption All BMW were consumedwithin 10 days from bottling TW bottles were also similarlystored in refrigerator until consumption During the testvolunteers opened 1 plastic bottle of TW or BMW every dayand drank the 500mL of TW or BMW divided into thricedaily (30ndash60 minutes before breakfast lunch and dinner)TW consumption periods and BMW consumption periodslasted for a week each and this cycle was repeated twice Vol-unteers were instructed to keep to their normal dietary habits

Table 1 Mineral contents and pH of TW and BMW used in thisstudy

Minerals Formula Conc (mgkg)TW BMW

Bicarbonate ion HCO3

minus 28 2485Chlorine ion Clminus 11 182Sulfate ion SO

4

2minus 69 355Carbonate ion CO

3

2minuslt01 12

Nitrate ion NO3

minus 07 12Fluoride ion Fminus lt01 03Hydrogen sulfide ion HSminus lt01 lt01Iodide ion Iminus lt01 lt01Bromide ion Brminus lt01 lt01Sodium ion Na+ 10 412Magnesium ion Mg2+ 19 291Calcium ion Ca2+ 61 177Potassium ion K+ lt01 80Aluminum ion Al3+ 02 06Manganese ion Mn2+ lt01 04Ferrous ion Fe2+ lt01 23Ferric ion Fe3+ lt01 lt01Metasilicic acid H

2SiO3

10 207Metaboric acid HBO

208 62

Carbon dioxide CO2

09 161Hydrogen sulfide H

2S lt01 lt01

Mercury Hg lt00005 lt00005Arsenic As lt0005 0005Copper Cu lt005 lt005Zinc Zn lt01 lt01Lead Pb lt005 lt005Cadmium Cd lt001 lt001pH 758 707

during the test but consumption of medicinal drugs wasprohibited

Blood and fecal samples were collected on the first dayof the test and last days of every week Blood samplingsthat were collected from the same subject were performed atapproximately the same time during the test Body weightbody mass index (BMI) height abdominal circumferenceand blood pressure were measured on the first day of thetest (see Table S1 of the Supplementary Material availableonline at httpdxdoiorg1011552015824395) A schematicrepresentation of the experimental design is as shown inFigure S1 of Supplementary Material

22 Clinical Blood Tests Clinical blood tests were performedat each sampling point including measurement of fastingplasma glucose serum glucose glycoalbumin insulin totalcholesterol HDL cholesterol LDL cholesterol triglycerideurate sodium chlorine calcium magnesium and cortisolThe measurement of the concentrations of these parameterswas outsourced to RINTEC Co Ltd (Fukuoka Japan)

Evidence-Based Complementary and Alternative Medicine 3

Plasma glucose measurement was performed usingmorning fasting blood samples obtained from only 8 volun-teers For these samples the homeostasis model assessmentratio (HOMA-R) was calculated from plasma glucose andinsulin levels

23 Metabolome Analysis Metabolome analysis was con-ducted as described previously with some modifications[13] In brief to extract metabolites from blood 400 120583L ofmethanol including the internal standards (20120583M each ofmethionine sulfone and D-camphor-10-sulfonic acid (CSA))was added to the 40 120583L of blood samples Next this mixturewas then mixed with 120120583L of ultrapure water and 400 120583Lof chloroform before centrifuging at 10000timesg for 3minat 4∘C Subsequently the aqueous layer was transferred toa centrifugal filter tube (UltrafreeMC-PLHCC 250pk forMetabolome Analysis Human Metabolome Technologies)to remove protein and lipid molecules The filtrate wascentrifugally concentrated and dissolved in 20120583L of ultrapurewater that contained reference compounds (200120583M each of3-aminopyrrolidine and trimesic acid) immediately beforecapillary electrophoresis with electrospray ionization time-of-flight mass spectrometry (CE-TOFMS) analysis

The measurement of extracted metabolites in both posi-tive and negative modes was performed by CE-TOFMS AllCE-TOFMS experiments were performed using the AgilentCE capillary electrophoresis system (Agilent Technologies)Annotation tables were produced from measurement ofstandard compounds and were aligned with the datasetsaccording to similar 119898119911 value and normalized migrationtime Then peak areas were normalized against those of theinternal standards methionine sulfone or CSA for cationicand anionic metabolites respectively Concentrations of eachmetabolite were calculated based on their relative peak areasand concentrations of standard compounds

After statistical analysis metabolite set enrichment anal-ysis (MSEA) [19] was performed using the metabolites thatwere significantly different between TW and BMW con-sumption periods

24 DNA Isolation Fecal DNA isolation was performed asdescribed previously with some modifications [20] Brieflyfecal samples were initially lyophilized by using VD-800Rlyophilizer (TAITEC) for at least 18 hours Freeze-driedfeces were disrupted with 30mm Zirconia Beads by vig-orous shaking (1500 rpm for 10min) using Shake Master(Biomedical Science) Fecal samples (10mg) were suspendedwith DNA extraction buffer containing 200120583L of 10 (wv)SDSTE (10mMTris-HCl 1mMEDTA and pH80) solution400 120583L of phenolchloroformisoamyl alcohol (25 24 1) and200120583L of 3M sodium acetate Feces in mixture bufferwere further disrupted with 01mm zirconiasilica beadsby vigorous shaking (1500 rpm for 5min) using ShakeMaster After centrifugation at 17800timesg for 5min at roomtemperature bacterial genomic DNA was purified by thestandard phenolchloroformisoamyl alcohol protocol RNAswere removed from the sample by RNase A treatment and

then DNA samples were purified once more by the standardphenolchloroformisoamyl alcohol protocol

25 16S rRNA Gene Sequencing 16S rRNA genes in the fecalDNA samples were analyzed using the MiSeq sequencer(Illumina) The V1-V2 region of the 16S rRNA genes wasamplified from the DNA isolated from feces using bacterialuniversal primer set 27Fmod (51015840-AGRGTTTGATYMTGG-CTCAG-31015840) and 338R (51015840-TGCTGCCTCCCGTAGGAGT-31015840) [21] PCR was performed with Tks Gflex DNA Poly-merase (Takara Bio Inc) and amplification proceeded withone denaturation step at 98∘C for 1min followed by 20cycles of 98∘C for 10 s 55∘C for 15 s and 68∘C for 30 swith a final extension step at 68∘C for 3min The ampli-fied products were purified using Agencourt AMPure XP(Beckman Coulter) and then further amplified using forwardprimer (51015840-AATGATACGGCGACCACCGAGATCTAC-AC-NNNNNNNN-TATGGTAATTGT-AGRGTTTGATY-MTGGCTCAG-31015840) containing the P5 sequence a unique8 bp barcode sequence for each sample (indicated in N) Rd1SP sequence and 27Fmod primer and reverse primer (51015840-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAGCC-TGCTGCCTCCCGAGGAGT-31015840) con-taining the P7 sequence a unique 8-bp barcode sequencefor each sample (indicated in N) Rd2 SP sequence and338R primer After purification using Agencourt AMPure XPmixed sample was prepared by pooling approximately equalamounts of PCR amplicons from each sample Finally MiSeqsequencing was performed according to the manufacturerrsquosinstructions In this study 2 times 300 bp paired-end sequencingwas employed

26 Analysis of 16S rRNA Gene Sequences Initially to assem-ble the paired-end reads fast length adjustment of shortreads (FLASH) (v1211) [22] was used Assembled reads withan average 119876-value lt 25 were filtered out using in-housescript 5000 filter-passed reads were randomly selected fromeach sample and used for further analysis Reads were thenprocessed using quantitative insights into microbial ecology(QIIME) (v180) pipeline [23] Sequences were clustered intooperational taxonomic units (OTUs) using 97 sequencesimilarity and OTUs were assigned to taxonomy using RDPclassifier

27 Statistical Analysis To analyze the intraindividualalterations statistical evaluation between two groups wasperformed by Wilcoxon signed-rank test (nonparametricpaired test) using the R package exactRankTests (availableat httpscranr-projectorgwebpackagesexactRankTestsindexhtml) or XLSTAT (v2014604) (Addinsoft) For mul-tiple comparisons the data were analyzed using Friedmanrsquostest and post hoc Nemenyi test using XLSTAT All statementsindicating significant differences show at least a 5 level ofprobability

28 Nucleotide Sequence Accession Number Themicrobiomeanalysis data have been deposited at the DDBJ SequenceRead Archive (httptraceddbjnigacjpdra) under acces-sion number DRA004008

4 Evidence-Based Complementary and Alternative Medicine

TW BMW

092

094

096

098

100

102

104

Relat

ive g

lyco

albu

min

leve

ls

lowastlowast

(a)

TW

1 2 3Week

40

TWBMW BMW

lowastlowast

lowastlowast

lowastlowast

092

094

096

098

100

102

Rela

tive g

lyco

albu

min

leve

ls

(b)

Figure 1 Comparisons of relative serum glycoalbumin levels between TW and BMW consumption periods Glycoalbumin levels wereexpressed as a relative value to week 0 (a) Individual data of mean relative glycoalbumin levels of weeks 1 and 3 (TW) and weeks 2 and4 (BMW) were shown in dot plots overlaid on box plots Plots corresponding to the same individuals were connected with red blue or graylines when the values were decreased increased or not changed in BMW consumption periods as compared with TW consumption periodsrespectively Plots were also colored in the same color as their lines (b) Records of weekly glycoalbumin levels Data were expressed asmean plusmnstandard deviation (SD) Significances between week 0 and each week were shown at top of the graph lowastlowast119875 lt 0005

3 Results

31 Serum Glycoalbumin Levels Were Decreased after BMWConsumption Firstly intraindividual alterations of clinicalparameters were analyzed Serum glycoalbumin levels one ofthe indexes of glycemic controls were significantly decreasedduring BMW consumption periods as compared with TWconsumption periods (Figure 1(a)) Serumglucose levelswerenot decreased significantly but tended to be lowered (119875 value= 0092) Other parameters related to glycemic controls likeplasma glucose levels insulin concentrations and HOMA-R were not different between TW and BMW consumptionperiods In this study other biochemical parameters involvedwith hypercholesterolemia hyperuricemia mineral con-sumption and stress were also measured but consumptionof BMW did not have any impact on the above-mentionedparameters apart from blood calcium levels (Table 2) Theseresults suggested that BMW consumption has the possiblepotential to prevent andor improve T2D without changinginsulin secretion and insulin resistance

Although the serum glycoalbumin levels were signifi-cantly decreased during BMW consumption periods as com-pared with TW consumption periods reduction of serumglycoalbumin levels was also observed after TW consump-tion periods as compared with before the test (week 0)

(Figure 1(b)) Relative serum glycoalbumin levels decreasedafter the first TW consumption period (week 1) and furtherdecreased after first BMW consumption period (week 2)Subsequently it slightly increased after second TW con-sumption period (week 3) and then decreased again aftersecond BMW consumption period (week 4) Taken togetherthese results suggest that the habit of water consumptionbefore every meal has the possible potential to decreaseserum glycoalbumin levels but BMW consumption is moreeffective

32 BMW Consumption-Related Changes of Physiologi-cal Metabolism To evaluate the molecular level effectsof BMW consumption blood metabolome analysis wasperformed using CE-TOFMS A total of 152 metaboliteswere detected from blood samples at least from 1 subjectand 1 time point and concentrations of these metaboliteswere compared within subject Over 85 of metaboliteswere not significantly changed after BMW consumptionperiods and it was expected that the concentrations ofmost metabolites remained consistent due to physiologi-cal homeostasis (Figure 2(a)) However the concentrationsof 19 metabolites were significantly different between TWand BMW consumption periods (Figures 2(a) and 2(b))These effects were expected to be derived from BMW

Evidence-Based Complementary and Alternative Medicine 5

Week 0 Week 1 Week 2

P v

alue

(TW

ver

sus B

MW

)

Low

High

Week 3 Week 4TW consumption periods BMW consumption periods

005Ala

Isocitrate5-Oxoproline

UreaS7P

BetaineDodecanoate

Proline betaineSAH

cis-AconitateCholine

ADP-riboseCitrate

CitrullineGMP

HisPhosphorylcholine

4-HydroxymandelatePRPP

Syringate4-Methyl-2-oxopentanoate

MalatePro

ThymineGABA

OphthalmatePhe

AdenineGly-Leu

Arg2-Hydroxybutyrate

Sero-AcetylcarnitineGuanidinoacetate

HypotaurinePEP

TaurineADMA

LysUrate

gamma-ButyrobetaineOrnithine

GluGlycerophosphate

Asn3-Aminoisobutyrate

5-MethylthioadenosineN-epsilon-acetyllysine

SDMAGDP-mannose

Indole-3-acetamideLactate

Allantoinbeta-Ala-Lys

Ethanolamine phosphateHistamine

NN-DimethylglycineNicotinamide

ThrNADPH

5-Hydroxylysine3-Hydroxybutyrate

Ile4-Aminosalicylate

DecanoateAlliin

UrocanateGuanidinosuccinate

GlycerophosphorylcholineHydroxyproline

1-MethylnicotinamideCreatinine

PelargonateFumarate

Glutathione(red)Gln

SarcosineQuinateCreatine

Cysteine-glutathione disulphide-divalentKynurenine

PhosphonoacetateTrp

7-MethylguanineUDP-glucose

alpha-AminoadipateVal

GDPMethionine sulfoxide

ThymidineS-Methylmethionine

N-AcetylglucosamineGlutathione(ox)

ArgininosuccinateAla-Ala

N-AcetylneuraminateUridine

HippurateAsp

CarnitineDiethanolamine

F16P3-Methylhistidine

CMP-N-acetylneuraminateSuccinate

Azelate23-DPG

Indole-3-acetaldehydeN6N6N6-Trimethyllysine

ThreonateTaurocyamine

R5P2AB

Gluconate

Trigonellinebeta-Ala

Leu

O-AcetylserineN-Acetylglutamate

CysDihydrouracil

dAMPHexylamine

Indole-3-acetate1-Methyladenosine

Phenyl phosphateRiboflavin

Sorbitol 6-phosphateTrimethylamine N-oxide

Pseudopelletierine

GlyATP

GlucosamineHypoxanthine

UMPRu5P

PyruvateMet

4-OxopentanoateUDP-N-acetylglucosamine

Pipecolate3PGTyr

IMP2-Oxoisopentanoate

ITPADPAMP

dGTP

N03

N26

Subjects

NADP+

NAD+

SAM+

00 18minus18Z-score

(a)

Pyruvate

AMP ADP IMP Tyr 4-Oxopentanoate Met Hypoxanthine Glucosamine Gly

dGTP 2-Oxoisopentanoate ITP PipecolateUDP-N- Ribulose-5-

UMP ATPacetylglucosamine

minus10

minus05

00

05

10

Z-s

core

minus10

minus05

00

05

10

Z-s

core

3-Phosphoglycerate

phosphate

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast

TWBMWTWTW BMWTW TW BMWBMW BMWTW TWBMWTWBMW TW BMWBMW

BMW TW BMW TWBMWTW BMW TW BMW TW BMW TW BMW TW BMWTW TW TW BMWBMW

(b)

Figure 2 Continued

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Metabolomics is a method of comprehensive measure-ment of metabolites and it has been known to be a powerfultool to gain new insights into various research fields suchas metabolic syndrome [10] cancer [11 12] chronic kidneydisease [13] and biomarker screening [14] A recent studyhas reported that plasma metabolome profiles were alteredbetween healthy people and T2D patients [15] Moreoverit has been also reported that gut microbial compositionand function in T2D patients are different from that ofhealthy subjects [16ndash18] Therefore preventive andor ther-apeutic effects for T2D derived from BMW consumption areexpected via influencing blood metabolites concentrationsand gut microbial compositions

For this reason blood metabolome analysis and gutmicrobiome analysis were included in our study to eluci-date the molecular level effects of BMW consumption onglycemic control In this study BMW consumption test wasconducted amongst 19 healthy volunteers Here we show thatserum glycoalbumin levels were decreased and 19metabolitesincluding glycolysis-related metabolites and 3 amino acidswere changed after consumption of BMW Additionallymicrobiome analysis demonstrated that composition of lean-inducible bacteria family Christensenellaceae was increasedafter BMW consumption This current study is an importantstudy reporting the molecular level effects derived fromconsumption of BMW

2 Materials and Methods

21 Sample Preparation This study was approved by theEthical Committees of Japan Health and Research Instituteand Keio University Shonan Fujisawa Campus All subjectswere informed of the purpose of this study and writtenconsent was obtained from all subjects

In this study BMW consumption test was conductedamongst 19 healthy subjects (7 men and 12 women agesfrom 26 to 59 47 years old on the average) Initiallyindividual identification numbers were randomly assignedto 26 volunteers from N01 to N26 however N12 and N25dropped out due to personal reasons Additionally N01 N02N14 N18 and N21 were excluded from analysis because theyforgot to drink TW andor BMW at least once BMW wasobtained from Nagayu hot spring (Taketa Oita Japan) asit contains one of the highest bicarbonate concentrations inJapan TW was purchased from Nishikawa water treatmentplant (Yamagata Japan) Mineral contents and pH of the TWandBMWused in this studyweremeasured by theHot SpringResearch Center (Tokyo Japan) and are as shown in Table 1BMW were collected in 500mL plastic bottles and storedin refrigerator until consumption All BMW were consumedwithin 10 days from bottling TW bottles were also similarlystored in refrigerator until consumption During the testvolunteers opened 1 plastic bottle of TW or BMW every dayand drank the 500mL of TW or BMW divided into thricedaily (30ndash60 minutes before breakfast lunch and dinner)TW consumption periods and BMW consumption periodslasted for a week each and this cycle was repeated twice Vol-unteers were instructed to keep to their normal dietary habits

Table 1 Mineral contents and pH of TW and BMW used in thisstudy

Minerals Formula Conc (mgkg)TW BMW

Bicarbonate ion HCO3

minus 28 2485Chlorine ion Clminus 11 182Sulfate ion SO

4

2minus 69 355Carbonate ion CO

3

2minuslt01 12

Nitrate ion NO3

minus 07 12Fluoride ion Fminus lt01 03Hydrogen sulfide ion HSminus lt01 lt01Iodide ion Iminus lt01 lt01Bromide ion Brminus lt01 lt01Sodium ion Na+ 10 412Magnesium ion Mg2+ 19 291Calcium ion Ca2+ 61 177Potassium ion K+ lt01 80Aluminum ion Al3+ 02 06Manganese ion Mn2+ lt01 04Ferrous ion Fe2+ lt01 23Ferric ion Fe3+ lt01 lt01Metasilicic acid H

2SiO3

10 207Metaboric acid HBO

208 62

Carbon dioxide CO2

09 161Hydrogen sulfide H

2S lt01 lt01

Mercury Hg lt00005 lt00005Arsenic As lt0005 0005Copper Cu lt005 lt005Zinc Zn lt01 lt01Lead Pb lt005 lt005Cadmium Cd lt001 lt001pH 758 707

during the test but consumption of medicinal drugs wasprohibited

Blood and fecal samples were collected on the first dayof the test and last days of every week Blood samplingsthat were collected from the same subject were performed atapproximately the same time during the test Body weightbody mass index (BMI) height abdominal circumferenceand blood pressure were measured on the first day of thetest (see Table S1 of the Supplementary Material availableonline at httpdxdoiorg1011552015824395) A schematicrepresentation of the experimental design is as shown inFigure S1 of Supplementary Material

22 Clinical Blood Tests Clinical blood tests were performedat each sampling point including measurement of fastingplasma glucose serum glucose glycoalbumin insulin totalcholesterol HDL cholesterol LDL cholesterol triglycerideurate sodium chlorine calcium magnesium and cortisolThe measurement of the concentrations of these parameterswas outsourced to RINTEC Co Ltd (Fukuoka Japan)

Evidence-Based Complementary and Alternative Medicine 3

Plasma glucose measurement was performed usingmorning fasting blood samples obtained from only 8 volun-teers For these samples the homeostasis model assessmentratio (HOMA-R) was calculated from plasma glucose andinsulin levels

23 Metabolome Analysis Metabolome analysis was con-ducted as described previously with some modifications[13] In brief to extract metabolites from blood 400 120583L ofmethanol including the internal standards (20120583M each ofmethionine sulfone and D-camphor-10-sulfonic acid (CSA))was added to the 40 120583L of blood samples Next this mixturewas then mixed with 120120583L of ultrapure water and 400 120583Lof chloroform before centrifuging at 10000timesg for 3minat 4∘C Subsequently the aqueous layer was transferred toa centrifugal filter tube (UltrafreeMC-PLHCC 250pk forMetabolome Analysis Human Metabolome Technologies)to remove protein and lipid molecules The filtrate wascentrifugally concentrated and dissolved in 20120583L of ultrapurewater that contained reference compounds (200120583M each of3-aminopyrrolidine and trimesic acid) immediately beforecapillary electrophoresis with electrospray ionization time-of-flight mass spectrometry (CE-TOFMS) analysis

The measurement of extracted metabolites in both posi-tive and negative modes was performed by CE-TOFMS AllCE-TOFMS experiments were performed using the AgilentCE capillary electrophoresis system (Agilent Technologies)Annotation tables were produced from measurement ofstandard compounds and were aligned with the datasetsaccording to similar 119898119911 value and normalized migrationtime Then peak areas were normalized against those of theinternal standards methionine sulfone or CSA for cationicand anionic metabolites respectively Concentrations of eachmetabolite were calculated based on their relative peak areasand concentrations of standard compounds

After statistical analysis metabolite set enrichment anal-ysis (MSEA) [19] was performed using the metabolites thatwere significantly different between TW and BMW con-sumption periods

24 DNA Isolation Fecal DNA isolation was performed asdescribed previously with some modifications [20] Brieflyfecal samples were initially lyophilized by using VD-800Rlyophilizer (TAITEC) for at least 18 hours Freeze-driedfeces were disrupted with 30mm Zirconia Beads by vig-orous shaking (1500 rpm for 10min) using Shake Master(Biomedical Science) Fecal samples (10mg) were suspendedwith DNA extraction buffer containing 200120583L of 10 (wv)SDSTE (10mMTris-HCl 1mMEDTA and pH80) solution400 120583L of phenolchloroformisoamyl alcohol (25 24 1) and200120583L of 3M sodium acetate Feces in mixture bufferwere further disrupted with 01mm zirconiasilica beadsby vigorous shaking (1500 rpm for 5min) using ShakeMaster After centrifugation at 17800timesg for 5min at roomtemperature bacterial genomic DNA was purified by thestandard phenolchloroformisoamyl alcohol protocol RNAswere removed from the sample by RNase A treatment and

then DNA samples were purified once more by the standardphenolchloroformisoamyl alcohol protocol

25 16S rRNA Gene Sequencing 16S rRNA genes in the fecalDNA samples were analyzed using the MiSeq sequencer(Illumina) The V1-V2 region of the 16S rRNA genes wasamplified from the DNA isolated from feces using bacterialuniversal primer set 27Fmod (51015840-AGRGTTTGATYMTGG-CTCAG-31015840) and 338R (51015840-TGCTGCCTCCCGTAGGAGT-31015840) [21] PCR was performed with Tks Gflex DNA Poly-merase (Takara Bio Inc) and amplification proceeded withone denaturation step at 98∘C for 1min followed by 20cycles of 98∘C for 10 s 55∘C for 15 s and 68∘C for 30 swith a final extension step at 68∘C for 3min The ampli-fied products were purified using Agencourt AMPure XP(Beckman Coulter) and then further amplified using forwardprimer (51015840-AATGATACGGCGACCACCGAGATCTAC-AC-NNNNNNNN-TATGGTAATTGT-AGRGTTTGATY-MTGGCTCAG-31015840) containing the P5 sequence a unique8 bp barcode sequence for each sample (indicated in N) Rd1SP sequence and 27Fmod primer and reverse primer (51015840-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAGCC-TGCTGCCTCCCGAGGAGT-31015840) con-taining the P7 sequence a unique 8-bp barcode sequencefor each sample (indicated in N) Rd2 SP sequence and338R primer After purification using Agencourt AMPure XPmixed sample was prepared by pooling approximately equalamounts of PCR amplicons from each sample Finally MiSeqsequencing was performed according to the manufacturerrsquosinstructions In this study 2 times 300 bp paired-end sequencingwas employed

26 Analysis of 16S rRNA Gene Sequences Initially to assem-ble the paired-end reads fast length adjustment of shortreads (FLASH) (v1211) [22] was used Assembled reads withan average 119876-value lt 25 were filtered out using in-housescript 5000 filter-passed reads were randomly selected fromeach sample and used for further analysis Reads were thenprocessed using quantitative insights into microbial ecology(QIIME) (v180) pipeline [23] Sequences were clustered intooperational taxonomic units (OTUs) using 97 sequencesimilarity and OTUs were assigned to taxonomy using RDPclassifier

27 Statistical Analysis To analyze the intraindividualalterations statistical evaluation between two groups wasperformed by Wilcoxon signed-rank test (nonparametricpaired test) using the R package exactRankTests (availableat httpscranr-projectorgwebpackagesexactRankTestsindexhtml) or XLSTAT (v2014604) (Addinsoft) For mul-tiple comparisons the data were analyzed using Friedmanrsquostest and post hoc Nemenyi test using XLSTAT All statementsindicating significant differences show at least a 5 level ofprobability

28 Nucleotide Sequence Accession Number Themicrobiomeanalysis data have been deposited at the DDBJ SequenceRead Archive (httptraceddbjnigacjpdra) under acces-sion number DRA004008

4 Evidence-Based Complementary and Alternative Medicine

TW BMW

092

094

096

098

100

102

104

Relat

ive g

lyco

albu

min

leve

ls

lowastlowast

(a)

TW

1 2 3Week

40

TWBMW BMW

lowastlowast

lowastlowast

lowastlowast

092

094

096

098

100

102

Rela

tive g

lyco

albu

min

leve

ls

(b)

Figure 1 Comparisons of relative serum glycoalbumin levels between TW and BMW consumption periods Glycoalbumin levels wereexpressed as a relative value to week 0 (a) Individual data of mean relative glycoalbumin levels of weeks 1 and 3 (TW) and weeks 2 and4 (BMW) were shown in dot plots overlaid on box plots Plots corresponding to the same individuals were connected with red blue or graylines when the values were decreased increased or not changed in BMW consumption periods as compared with TW consumption periodsrespectively Plots were also colored in the same color as their lines (b) Records of weekly glycoalbumin levels Data were expressed asmean plusmnstandard deviation (SD) Significances between week 0 and each week were shown at top of the graph lowastlowast119875 lt 0005

3 Results

31 Serum Glycoalbumin Levels Were Decreased after BMWConsumption Firstly intraindividual alterations of clinicalparameters were analyzed Serum glycoalbumin levels one ofthe indexes of glycemic controls were significantly decreasedduring BMW consumption periods as compared with TWconsumption periods (Figure 1(a)) Serumglucose levelswerenot decreased significantly but tended to be lowered (119875 value= 0092) Other parameters related to glycemic controls likeplasma glucose levels insulin concentrations and HOMA-R were not different between TW and BMW consumptionperiods In this study other biochemical parameters involvedwith hypercholesterolemia hyperuricemia mineral con-sumption and stress were also measured but consumptionof BMW did not have any impact on the above-mentionedparameters apart from blood calcium levels (Table 2) Theseresults suggested that BMW consumption has the possiblepotential to prevent andor improve T2D without changinginsulin secretion and insulin resistance

Although the serum glycoalbumin levels were signifi-cantly decreased during BMW consumption periods as com-pared with TW consumption periods reduction of serumglycoalbumin levels was also observed after TW consump-tion periods as compared with before the test (week 0)

(Figure 1(b)) Relative serum glycoalbumin levels decreasedafter the first TW consumption period (week 1) and furtherdecreased after first BMW consumption period (week 2)Subsequently it slightly increased after second TW con-sumption period (week 3) and then decreased again aftersecond BMW consumption period (week 4) Taken togetherthese results suggest that the habit of water consumptionbefore every meal has the possible potential to decreaseserum glycoalbumin levels but BMW consumption is moreeffective

32 BMW Consumption-Related Changes of Physiologi-cal Metabolism To evaluate the molecular level effectsof BMW consumption blood metabolome analysis wasperformed using CE-TOFMS A total of 152 metaboliteswere detected from blood samples at least from 1 subjectand 1 time point and concentrations of these metaboliteswere compared within subject Over 85 of metaboliteswere not significantly changed after BMW consumptionperiods and it was expected that the concentrations ofmost metabolites remained consistent due to physiologi-cal homeostasis (Figure 2(a)) However the concentrationsof 19 metabolites were significantly different between TWand BMW consumption periods (Figures 2(a) and 2(b))These effects were expected to be derived from BMW

Evidence-Based Complementary and Alternative Medicine 5

Week 0 Week 1 Week 2

P v

alue

(TW

ver

sus B

MW

)

Low

High

Week 3 Week 4TW consumption periods BMW consumption periods

005Ala

Isocitrate5-Oxoproline

UreaS7P

BetaineDodecanoate

Proline betaineSAH

cis-AconitateCholine

ADP-riboseCitrate

CitrullineGMP

HisPhosphorylcholine

4-HydroxymandelatePRPP

Syringate4-Methyl-2-oxopentanoate

MalatePro

ThymineGABA

OphthalmatePhe

AdenineGly-Leu

Arg2-Hydroxybutyrate

Sero-AcetylcarnitineGuanidinoacetate

HypotaurinePEP

TaurineADMA

LysUrate

gamma-ButyrobetaineOrnithine

GluGlycerophosphate

Asn3-Aminoisobutyrate

5-MethylthioadenosineN-epsilon-acetyllysine

SDMAGDP-mannose

Indole-3-acetamideLactate

Allantoinbeta-Ala-Lys

Ethanolamine phosphateHistamine

NN-DimethylglycineNicotinamide

ThrNADPH

5-Hydroxylysine3-Hydroxybutyrate

Ile4-Aminosalicylate

DecanoateAlliin

UrocanateGuanidinosuccinate

GlycerophosphorylcholineHydroxyproline

1-MethylnicotinamideCreatinine

PelargonateFumarate

Glutathione(red)Gln

SarcosineQuinateCreatine

Cysteine-glutathione disulphide-divalentKynurenine

PhosphonoacetateTrp

7-MethylguanineUDP-glucose

alpha-AminoadipateVal

GDPMethionine sulfoxide

ThymidineS-Methylmethionine

N-AcetylglucosamineGlutathione(ox)

ArgininosuccinateAla-Ala

N-AcetylneuraminateUridine

HippurateAsp

CarnitineDiethanolamine

F16P3-Methylhistidine

CMP-N-acetylneuraminateSuccinate

Azelate23-DPG

Indole-3-acetaldehydeN6N6N6-Trimethyllysine

ThreonateTaurocyamine

R5P2AB

Gluconate

Trigonellinebeta-Ala

Leu

O-AcetylserineN-Acetylglutamate

CysDihydrouracil

dAMPHexylamine

Indole-3-acetate1-Methyladenosine

Phenyl phosphateRiboflavin

Sorbitol 6-phosphateTrimethylamine N-oxide

Pseudopelletierine

GlyATP

GlucosamineHypoxanthine

UMPRu5P

PyruvateMet

4-OxopentanoateUDP-N-acetylglucosamine

Pipecolate3PGTyr

IMP2-Oxoisopentanoate

ITPADPAMP

dGTP

N03

N26

Subjects

NADP+

NAD+

SAM+

00 18minus18Z-score

(a)

Pyruvate

AMP ADP IMP Tyr 4-Oxopentanoate Met Hypoxanthine Glucosamine Gly

dGTP 2-Oxoisopentanoate ITP PipecolateUDP-N- Ribulose-5-

UMP ATPacetylglucosamine

minus10

minus05

00

05

10

Z-s

core

minus10

minus05

00

05

10

Z-s

core

3-Phosphoglycerate

phosphate

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast

TWBMWTWTW BMWTW TW BMWBMW BMWTW TWBMWTWBMW TW BMWBMW

BMW TW BMW TWBMWTW BMW TW BMW TW BMW TW BMW TW BMWTW TW TW BMWBMW

(b)

Figure 2 Continued

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Plasma glucose measurement was performed usingmorning fasting blood samples obtained from only 8 volun-teers For these samples the homeostasis model assessmentratio (HOMA-R) was calculated from plasma glucose andinsulin levels

23 Metabolome Analysis Metabolome analysis was con-ducted as described previously with some modifications[13] In brief to extract metabolites from blood 400 120583L ofmethanol including the internal standards (20120583M each ofmethionine sulfone and D-camphor-10-sulfonic acid (CSA))was added to the 40 120583L of blood samples Next this mixturewas then mixed with 120120583L of ultrapure water and 400 120583Lof chloroform before centrifuging at 10000timesg for 3minat 4∘C Subsequently the aqueous layer was transferred toa centrifugal filter tube (UltrafreeMC-PLHCC 250pk forMetabolome Analysis Human Metabolome Technologies)to remove protein and lipid molecules The filtrate wascentrifugally concentrated and dissolved in 20120583L of ultrapurewater that contained reference compounds (200120583M each of3-aminopyrrolidine and trimesic acid) immediately beforecapillary electrophoresis with electrospray ionization time-of-flight mass spectrometry (CE-TOFMS) analysis

The measurement of extracted metabolites in both posi-tive and negative modes was performed by CE-TOFMS AllCE-TOFMS experiments were performed using the AgilentCE capillary electrophoresis system (Agilent Technologies)Annotation tables were produced from measurement ofstandard compounds and were aligned with the datasetsaccording to similar 119898119911 value and normalized migrationtime Then peak areas were normalized against those of theinternal standards methionine sulfone or CSA for cationicand anionic metabolites respectively Concentrations of eachmetabolite were calculated based on their relative peak areasand concentrations of standard compounds

After statistical analysis metabolite set enrichment anal-ysis (MSEA) [19] was performed using the metabolites thatwere significantly different between TW and BMW con-sumption periods

24 DNA Isolation Fecal DNA isolation was performed asdescribed previously with some modifications [20] Brieflyfecal samples were initially lyophilized by using VD-800Rlyophilizer (TAITEC) for at least 18 hours Freeze-driedfeces were disrupted with 30mm Zirconia Beads by vig-orous shaking (1500 rpm for 10min) using Shake Master(Biomedical Science) Fecal samples (10mg) were suspendedwith DNA extraction buffer containing 200120583L of 10 (wv)SDSTE (10mMTris-HCl 1mMEDTA and pH80) solution400 120583L of phenolchloroformisoamyl alcohol (25 24 1) and200120583L of 3M sodium acetate Feces in mixture bufferwere further disrupted with 01mm zirconiasilica beadsby vigorous shaking (1500 rpm for 5min) using ShakeMaster After centrifugation at 17800timesg for 5min at roomtemperature bacterial genomic DNA was purified by thestandard phenolchloroformisoamyl alcohol protocol RNAswere removed from the sample by RNase A treatment and

then DNA samples were purified once more by the standardphenolchloroformisoamyl alcohol protocol

25 16S rRNA Gene Sequencing 16S rRNA genes in the fecalDNA samples were analyzed using the MiSeq sequencer(Illumina) The V1-V2 region of the 16S rRNA genes wasamplified from the DNA isolated from feces using bacterialuniversal primer set 27Fmod (51015840-AGRGTTTGATYMTGG-CTCAG-31015840) and 338R (51015840-TGCTGCCTCCCGTAGGAGT-31015840) [21] PCR was performed with Tks Gflex DNA Poly-merase (Takara Bio Inc) and amplification proceeded withone denaturation step at 98∘C for 1min followed by 20cycles of 98∘C for 10 s 55∘C for 15 s and 68∘C for 30 swith a final extension step at 68∘C for 3min The ampli-fied products were purified using Agencourt AMPure XP(Beckman Coulter) and then further amplified using forwardprimer (51015840-AATGATACGGCGACCACCGAGATCTAC-AC-NNNNNNNN-TATGGTAATTGT-AGRGTTTGATY-MTGGCTCAG-31015840) containing the P5 sequence a unique8 bp barcode sequence for each sample (indicated in N) Rd1SP sequence and 27Fmod primer and reverse primer (51015840-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAGCC-TGCTGCCTCCCGAGGAGT-31015840) con-taining the P7 sequence a unique 8-bp barcode sequencefor each sample (indicated in N) Rd2 SP sequence and338R primer After purification using Agencourt AMPure XPmixed sample was prepared by pooling approximately equalamounts of PCR amplicons from each sample Finally MiSeqsequencing was performed according to the manufacturerrsquosinstructions In this study 2 times 300 bp paired-end sequencingwas employed

26 Analysis of 16S rRNA Gene Sequences Initially to assem-ble the paired-end reads fast length adjustment of shortreads (FLASH) (v1211) [22] was used Assembled reads withan average 119876-value lt 25 were filtered out using in-housescript 5000 filter-passed reads were randomly selected fromeach sample and used for further analysis Reads were thenprocessed using quantitative insights into microbial ecology(QIIME) (v180) pipeline [23] Sequences were clustered intooperational taxonomic units (OTUs) using 97 sequencesimilarity and OTUs were assigned to taxonomy using RDPclassifier

27 Statistical Analysis To analyze the intraindividualalterations statistical evaluation between two groups wasperformed by Wilcoxon signed-rank test (nonparametricpaired test) using the R package exactRankTests (availableat httpscranr-projectorgwebpackagesexactRankTestsindexhtml) or XLSTAT (v2014604) (Addinsoft) For mul-tiple comparisons the data were analyzed using Friedmanrsquostest and post hoc Nemenyi test using XLSTAT All statementsindicating significant differences show at least a 5 level ofprobability

28 Nucleotide Sequence Accession Number Themicrobiomeanalysis data have been deposited at the DDBJ SequenceRead Archive (httptraceddbjnigacjpdra) under acces-sion number DRA004008

4 Evidence-Based Complementary and Alternative Medicine

TW BMW

092

094

096

098

100

102

104

Relat

ive g

lyco

albu

min

leve

ls

lowastlowast

(a)

TW

1 2 3Week

40

TWBMW BMW

lowastlowast

lowastlowast

lowastlowast

092

094

096

098

100

102

Rela

tive g

lyco

albu

min

leve

ls

(b)

Figure 1 Comparisons of relative serum glycoalbumin levels between TW and BMW consumption periods Glycoalbumin levels wereexpressed as a relative value to week 0 (a) Individual data of mean relative glycoalbumin levels of weeks 1 and 3 (TW) and weeks 2 and4 (BMW) were shown in dot plots overlaid on box plots Plots corresponding to the same individuals were connected with red blue or graylines when the values were decreased increased or not changed in BMW consumption periods as compared with TW consumption periodsrespectively Plots were also colored in the same color as their lines (b) Records of weekly glycoalbumin levels Data were expressed asmean plusmnstandard deviation (SD) Significances between week 0 and each week were shown at top of the graph lowastlowast119875 lt 0005

3 Results

31 Serum Glycoalbumin Levels Were Decreased after BMWConsumption Firstly intraindividual alterations of clinicalparameters were analyzed Serum glycoalbumin levels one ofthe indexes of glycemic controls were significantly decreasedduring BMW consumption periods as compared with TWconsumption periods (Figure 1(a)) Serumglucose levelswerenot decreased significantly but tended to be lowered (119875 value= 0092) Other parameters related to glycemic controls likeplasma glucose levels insulin concentrations and HOMA-R were not different between TW and BMW consumptionperiods In this study other biochemical parameters involvedwith hypercholesterolemia hyperuricemia mineral con-sumption and stress were also measured but consumptionof BMW did not have any impact on the above-mentionedparameters apart from blood calcium levels (Table 2) Theseresults suggested that BMW consumption has the possiblepotential to prevent andor improve T2D without changinginsulin secretion and insulin resistance

Although the serum glycoalbumin levels were signifi-cantly decreased during BMW consumption periods as com-pared with TW consumption periods reduction of serumglycoalbumin levels was also observed after TW consump-tion periods as compared with before the test (week 0)

(Figure 1(b)) Relative serum glycoalbumin levels decreasedafter the first TW consumption period (week 1) and furtherdecreased after first BMW consumption period (week 2)Subsequently it slightly increased after second TW con-sumption period (week 3) and then decreased again aftersecond BMW consumption period (week 4) Taken togetherthese results suggest that the habit of water consumptionbefore every meal has the possible potential to decreaseserum glycoalbumin levels but BMW consumption is moreeffective

32 BMW Consumption-Related Changes of Physiologi-cal Metabolism To evaluate the molecular level effectsof BMW consumption blood metabolome analysis wasperformed using CE-TOFMS A total of 152 metaboliteswere detected from blood samples at least from 1 subjectand 1 time point and concentrations of these metaboliteswere compared within subject Over 85 of metaboliteswere not significantly changed after BMW consumptionperiods and it was expected that the concentrations ofmost metabolites remained consistent due to physiologi-cal homeostasis (Figure 2(a)) However the concentrationsof 19 metabolites were significantly different between TWand BMW consumption periods (Figures 2(a) and 2(b))These effects were expected to be derived from BMW

Evidence-Based Complementary and Alternative Medicine 5

Week 0 Week 1 Week 2

P v

alue

(TW

ver

sus B

MW

)

Low

High

Week 3 Week 4TW consumption periods BMW consumption periods

005Ala

Isocitrate5-Oxoproline

UreaS7P

BetaineDodecanoate

Proline betaineSAH

cis-AconitateCholine

ADP-riboseCitrate

CitrullineGMP

HisPhosphorylcholine

4-HydroxymandelatePRPP

Syringate4-Methyl-2-oxopentanoate

MalatePro

ThymineGABA

OphthalmatePhe

AdenineGly-Leu

Arg2-Hydroxybutyrate

Sero-AcetylcarnitineGuanidinoacetate

HypotaurinePEP

TaurineADMA

LysUrate

gamma-ButyrobetaineOrnithine

GluGlycerophosphate

Asn3-Aminoisobutyrate

5-MethylthioadenosineN-epsilon-acetyllysine

SDMAGDP-mannose

Indole-3-acetamideLactate

Allantoinbeta-Ala-Lys

Ethanolamine phosphateHistamine

NN-DimethylglycineNicotinamide

ThrNADPH

5-Hydroxylysine3-Hydroxybutyrate

Ile4-Aminosalicylate

DecanoateAlliin

UrocanateGuanidinosuccinate

GlycerophosphorylcholineHydroxyproline

1-MethylnicotinamideCreatinine

PelargonateFumarate

Glutathione(red)Gln

SarcosineQuinateCreatine

Cysteine-glutathione disulphide-divalentKynurenine

PhosphonoacetateTrp

7-MethylguanineUDP-glucose

alpha-AminoadipateVal

GDPMethionine sulfoxide

ThymidineS-Methylmethionine

N-AcetylglucosamineGlutathione(ox)

ArgininosuccinateAla-Ala

N-AcetylneuraminateUridine

HippurateAsp

CarnitineDiethanolamine

F16P3-Methylhistidine

CMP-N-acetylneuraminateSuccinate

Azelate23-DPG

Indole-3-acetaldehydeN6N6N6-Trimethyllysine

ThreonateTaurocyamine

R5P2AB

Gluconate

Trigonellinebeta-Ala

Leu

O-AcetylserineN-Acetylglutamate

CysDihydrouracil

dAMPHexylamine

Indole-3-acetate1-Methyladenosine

Phenyl phosphateRiboflavin

Sorbitol 6-phosphateTrimethylamine N-oxide

Pseudopelletierine

GlyATP

GlucosamineHypoxanthine

UMPRu5P

PyruvateMet

4-OxopentanoateUDP-N-acetylglucosamine

Pipecolate3PGTyr

IMP2-Oxoisopentanoate

ITPADPAMP

dGTP

N03

N26

Subjects

NADP+

NAD+

SAM+

00 18minus18Z-score

(a)

Pyruvate

AMP ADP IMP Tyr 4-Oxopentanoate Met Hypoxanthine Glucosamine Gly

dGTP 2-Oxoisopentanoate ITP PipecolateUDP-N- Ribulose-5-

UMP ATPacetylglucosamine

minus10

minus05

00

05

10

Z-s

core

minus10

minus05

00

05

10

Z-s

core

3-Phosphoglycerate

phosphate

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast

TWBMWTWTW BMWTW TW BMWBMW BMWTW TWBMWTWBMW TW BMWBMW

BMW TW BMW TWBMWTW BMW TW BMW TW BMW TW BMW TW BMWTW TW TW BMWBMW

(b)

Figure 2 Continued

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

TW BMW

092

094

096

098

100

102

104

Relat

ive g

lyco

albu

min

leve

ls

lowastlowast

(a)

TW

1 2 3Week

40

TWBMW BMW

lowastlowast

lowastlowast

lowastlowast

092

094

096

098

100

102

Rela

tive g

lyco

albu

min

leve

ls

(b)

Figure 1 Comparisons of relative serum glycoalbumin levels between TW and BMW consumption periods Glycoalbumin levels wereexpressed as a relative value to week 0 (a) Individual data of mean relative glycoalbumin levels of weeks 1 and 3 (TW) and weeks 2 and4 (BMW) were shown in dot plots overlaid on box plots Plots corresponding to the same individuals were connected with red blue or graylines when the values were decreased increased or not changed in BMW consumption periods as compared with TW consumption periodsrespectively Plots were also colored in the same color as their lines (b) Records of weekly glycoalbumin levels Data were expressed asmean plusmnstandard deviation (SD) Significances between week 0 and each week were shown at top of the graph lowastlowast119875 lt 0005

3 Results

31 Serum Glycoalbumin Levels Were Decreased after BMWConsumption Firstly intraindividual alterations of clinicalparameters were analyzed Serum glycoalbumin levels one ofthe indexes of glycemic controls were significantly decreasedduring BMW consumption periods as compared with TWconsumption periods (Figure 1(a)) Serumglucose levelswerenot decreased significantly but tended to be lowered (119875 value= 0092) Other parameters related to glycemic controls likeplasma glucose levels insulin concentrations and HOMA-R were not different between TW and BMW consumptionperiods In this study other biochemical parameters involvedwith hypercholesterolemia hyperuricemia mineral con-sumption and stress were also measured but consumptionof BMW did not have any impact on the above-mentionedparameters apart from blood calcium levels (Table 2) Theseresults suggested that BMW consumption has the possiblepotential to prevent andor improve T2D without changinginsulin secretion and insulin resistance

Although the serum glycoalbumin levels were signifi-cantly decreased during BMW consumption periods as com-pared with TW consumption periods reduction of serumglycoalbumin levels was also observed after TW consump-tion periods as compared with before the test (week 0)

(Figure 1(b)) Relative serum glycoalbumin levels decreasedafter the first TW consumption period (week 1) and furtherdecreased after first BMW consumption period (week 2)Subsequently it slightly increased after second TW con-sumption period (week 3) and then decreased again aftersecond BMW consumption period (week 4) Taken togetherthese results suggest that the habit of water consumptionbefore every meal has the possible potential to decreaseserum glycoalbumin levels but BMW consumption is moreeffective

32 BMW Consumption-Related Changes of Physiologi-cal Metabolism To evaluate the molecular level effectsof BMW consumption blood metabolome analysis wasperformed using CE-TOFMS A total of 152 metaboliteswere detected from blood samples at least from 1 subjectand 1 time point and concentrations of these metaboliteswere compared within subject Over 85 of metaboliteswere not significantly changed after BMW consumptionperiods and it was expected that the concentrations ofmost metabolites remained consistent due to physiologi-cal homeostasis (Figure 2(a)) However the concentrationsof 19 metabolites were significantly different between TWand BMW consumption periods (Figures 2(a) and 2(b))These effects were expected to be derived from BMW

Evidence-Based Complementary and Alternative Medicine 5

Week 0 Week 1 Week 2

P v

alue

(TW

ver

sus B

MW

)

Low

High

Week 3 Week 4TW consumption periods BMW consumption periods

005Ala

Isocitrate5-Oxoproline

UreaS7P

BetaineDodecanoate

Proline betaineSAH

cis-AconitateCholine

ADP-riboseCitrate

CitrullineGMP

HisPhosphorylcholine

4-HydroxymandelatePRPP

Syringate4-Methyl-2-oxopentanoate

MalatePro

ThymineGABA

OphthalmatePhe

AdenineGly-Leu

Arg2-Hydroxybutyrate

Sero-AcetylcarnitineGuanidinoacetate

HypotaurinePEP

TaurineADMA

LysUrate

gamma-ButyrobetaineOrnithine

GluGlycerophosphate

Asn3-Aminoisobutyrate

5-MethylthioadenosineN-epsilon-acetyllysine

SDMAGDP-mannose

Indole-3-acetamideLactate

Allantoinbeta-Ala-Lys

Ethanolamine phosphateHistamine

NN-DimethylglycineNicotinamide

ThrNADPH

5-Hydroxylysine3-Hydroxybutyrate

Ile4-Aminosalicylate

DecanoateAlliin

UrocanateGuanidinosuccinate

GlycerophosphorylcholineHydroxyproline

1-MethylnicotinamideCreatinine

PelargonateFumarate

Glutathione(red)Gln

SarcosineQuinateCreatine

Cysteine-glutathione disulphide-divalentKynurenine

PhosphonoacetateTrp

7-MethylguanineUDP-glucose

alpha-AminoadipateVal

GDPMethionine sulfoxide

ThymidineS-Methylmethionine

N-AcetylglucosamineGlutathione(ox)

ArgininosuccinateAla-Ala

N-AcetylneuraminateUridine

HippurateAsp

CarnitineDiethanolamine

F16P3-Methylhistidine

CMP-N-acetylneuraminateSuccinate

Azelate23-DPG

Indole-3-acetaldehydeN6N6N6-Trimethyllysine

ThreonateTaurocyamine

R5P2AB

Gluconate

Trigonellinebeta-Ala

Leu

O-AcetylserineN-Acetylglutamate

CysDihydrouracil

dAMPHexylamine

Indole-3-acetate1-Methyladenosine

Phenyl phosphateRiboflavin

Sorbitol 6-phosphateTrimethylamine N-oxide

Pseudopelletierine

GlyATP

GlucosamineHypoxanthine

UMPRu5P

PyruvateMet

4-OxopentanoateUDP-N-acetylglucosamine

Pipecolate3PGTyr

IMP2-Oxoisopentanoate

ITPADPAMP

dGTP

N03

N26

Subjects

NADP+

NAD+

SAM+

00 18minus18Z-score

(a)

Pyruvate

AMP ADP IMP Tyr 4-Oxopentanoate Met Hypoxanthine Glucosamine Gly

dGTP 2-Oxoisopentanoate ITP PipecolateUDP-N- Ribulose-5-

UMP ATPacetylglucosamine

minus10

minus05

00

05

10

Z-s

core

minus10

minus05

00

05

10

Z-s

core

3-Phosphoglycerate

phosphate

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast

TWBMWTWTW BMWTW TW BMWBMW BMWTW TWBMWTWBMW TW BMWBMW

BMW TW BMW TWBMWTW BMW TW BMW TW BMW TW BMW TW BMWTW TW TW BMWBMW

(b)

Figure 2 Continued

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Week 0 Week 1 Week 2

P v

alue

(TW

ver

sus B

MW

)

Low

High

Week 3 Week 4TW consumption periods BMW consumption periods

005Ala

Isocitrate5-Oxoproline

UreaS7P

BetaineDodecanoate

Proline betaineSAH

cis-AconitateCholine

ADP-riboseCitrate

CitrullineGMP

HisPhosphorylcholine

4-HydroxymandelatePRPP

Syringate4-Methyl-2-oxopentanoate

MalatePro

ThymineGABA

OphthalmatePhe

AdenineGly-Leu

Arg2-Hydroxybutyrate

Sero-AcetylcarnitineGuanidinoacetate

HypotaurinePEP

TaurineADMA

LysUrate

gamma-ButyrobetaineOrnithine

GluGlycerophosphate

Asn3-Aminoisobutyrate

5-MethylthioadenosineN-epsilon-acetyllysine

SDMAGDP-mannose

Indole-3-acetamideLactate

Allantoinbeta-Ala-Lys

Ethanolamine phosphateHistamine

NN-DimethylglycineNicotinamide

ThrNADPH

5-Hydroxylysine3-Hydroxybutyrate

Ile4-Aminosalicylate

DecanoateAlliin

UrocanateGuanidinosuccinate

GlycerophosphorylcholineHydroxyproline

1-MethylnicotinamideCreatinine

PelargonateFumarate

Glutathione(red)Gln

SarcosineQuinateCreatine

Cysteine-glutathione disulphide-divalentKynurenine

PhosphonoacetateTrp

7-MethylguanineUDP-glucose

alpha-AminoadipateVal

GDPMethionine sulfoxide

ThymidineS-Methylmethionine

N-AcetylglucosamineGlutathione(ox)

ArgininosuccinateAla-Ala

N-AcetylneuraminateUridine

HippurateAsp

CarnitineDiethanolamine

F16P3-Methylhistidine

CMP-N-acetylneuraminateSuccinate

Azelate23-DPG

Indole-3-acetaldehydeN6N6N6-Trimethyllysine

ThreonateTaurocyamine

R5P2AB

Gluconate

Trigonellinebeta-Ala

Leu

O-AcetylserineN-Acetylglutamate

CysDihydrouracil

dAMPHexylamine

Indole-3-acetate1-Methyladenosine

Phenyl phosphateRiboflavin

Sorbitol 6-phosphateTrimethylamine N-oxide

Pseudopelletierine

GlyATP

GlucosamineHypoxanthine

UMPRu5P

PyruvateMet

4-OxopentanoateUDP-N-acetylglucosamine

Pipecolate3PGTyr

IMP2-Oxoisopentanoate

ITPADPAMP

dGTP

N03

N26

Subjects

NADP+

NAD+

SAM+

00 18minus18Z-score

(a)

Pyruvate

AMP ADP IMP Tyr 4-Oxopentanoate Met Hypoxanthine Glucosamine Gly

dGTP 2-Oxoisopentanoate ITP PipecolateUDP-N- Ribulose-5-

UMP ATPacetylglucosamine

minus10

minus05

00

05

10

Z-s

core

minus10

minus05

00

05

10

Z-s

core

3-Phosphoglycerate

phosphate

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast

TWBMWTWTW BMWTW TW BMWBMW BMWTW TWBMWTWBMW TW BMWBMW

BMW TW BMW TWBMWTW BMW TW BMW TW BMW TW BMW TW BMWTW TW TW BMWBMW

(b)

Figure 2 Continued

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

NS3-Hydroxybutyrate

BMWTW

minus10

minus05

00

05

10

Z-s

core

(c)

Figure 2 Comparisons of blood metabolites between TW and BMW consumption periods (a) Relative concentrations of each metabolitein blood were transformed to 119885-score by subjects and shown as heat maps using blue-black-yellow scheme Gray color indicates that themetabolites were not detected in the sample A total of 152 metabolites were arranged in increasing order of 119875 value that was calculated byWilcoxon signed-rank test between TW and BMW consumption periods (b) Mean relative concentrations of each metabolite (119885-score) ofweeks 1 and 3 (TW) and weeks 2 and 4 (BMW) were shown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only19 metabolites that their concentrations were significantly increased (upper 9 metabolites) or decreased (lower 10 metabolites) after BMWconsumption (shown in upper block of panel (a)) were demonstrated (c) Relative concentrations of 3-hydroxybutyrate in blood were shownin dot plots overlaid on box plots in the same manner as in Figure 1(a) NS not significant lowast119875 lt 005 lowastlowast119875 lt 0005

Table 2 Results of clinical blood test

Tests TW BMW119875 value

Average SD Average SDFasting blood glucose (plasma) (mgdL) 912 43 886 47 0156Blood glucose (serum) (mgdL) 935 237 914 290 0092Glycoalbumin ( of total albumin) 144 17 142 17 0002lowastlowast

Insulin (120583UmL) 193 280 188 310 0294HOMA-R 12 05 12 04 1000Total cholesterol (mgdL) 2006 260 2024 303 0914HDL cholesterol (mgdL) 609 149 601 141 0685LDL cholesterol (mgdL) 1120 225 1123 241 1000Triglycerides (mgdL) 1512 1421 1807 1590 0123Urate (mgdL) 52 12 52 12 0396Na (mEqL) 1398 13 1403 12 0172Cl (mEqL) 1026 17 1029 19 0457Ca (mgdL) 93 02 94 02 0021lowast

Mg (mgdL) 23 01 23 01 0518Cortisol (120583gdL) 101 40 100 36 0623lowast119875 value is under 005 lowastlowast119875 value is under 0005

consumption As compared with TW consumption periods9 metabolites were significantly increased and 10 metaboliteswere significantly decreased in BMW consumption periods(Figure 2(b)) Metabolites that may be related to T2D such

as glycolysis-related metabolites (pyruvate ATP and ADP)amino acids (tyrosine methionine and glycine) and UDP-N-acetylglucosamine were included in significantly changedmetabolites In addition 3 amino acids were significantly

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Results of MSEA list of metabolite setspathways that are significantly different between TW and BMW consumption periods

Pathway Totallowast1 Hitslowast2 Expectlowast3 Fold change (hitsexpect) 119875 value FDRlowast4

Purine metabolism 45 7 098 71 lt0001 0001Ammonia recycling 18 4 039 102 lt0001 0014Urea cycle 20 4 044 92 lt0001 0014RNA transcription 9 3 020 153 lt0001 0014Intracellular signaling through prostacyclin receptor and prostacyclin 6 2 013 153 0006 0103Glycolysis 21 3 046 66 0009 0121Citric acid cycle 23 3 050 60 0012 0133Methionine metabolism 24 3 052 57 0013 0133Gluconeogenesis 27 3 059 51 0018 0163Amino sugar metabolism 15 2 033 61 0040 0290Mitochondrial electron transport chain 15 2 033 61 0040 0290lowast1Total numbers of metabolites that corresponded in each pathwaylowast2Observed numbers of metabolites that derived from given dataset in each pathwaylowast3Expected observed numbers of metabolites that are calculated by given dataset in each pathwaylowast4False discovery rate (FDR) according to the Benjamini and Hochberg method that was provided by MSEA software

decreased but almost all amino acids were also lowered afterBMW consumption periods (see Figure S2 of SupplementaryMaterial)

Additionally MSEA was performed using the 19 sig-nificantly changed metabolites (Table 3) These metaboliteswere involved with central metabolic pathways (glycoly-sis gluconeogenesis and citric acid cycle) and nitrogenmetabolism pathways such as ammonia recycling urea cycleand methionine metabolism These results suggested thatphysiological metabolism was modified by consumption ofBMW

On the other hand it was observed that 3-hydroxybu-tyrate one of the indexes of diabetic ketoacidosis was notaltered by BMW consumption (Figure 2(c))

33 BMWConsumption-Related Changes of Microbiota Com-positions Microbiome analysis was conducted using fecalsamples that were collectedweekly during the study A total of7075OTUs were constructed from 16S rRNA gene sequencesderived from 19 subjects These OTUs corresponded to 62families and whole structures of fecal microbiota are shown(Figure 3(a)) To investigate the intraindividual changes ofgut microbiota during the test relative abundances of eachmicrobial taxon were compared between TW and BMWconsumption periods within subjects From the results it wasobserved that over 85 of families were not altered similarlyto blood metabolites but relative proportions of 8 familiesespecially Christensenellaceae were significantly differentbetween TW and BMW consumption periods (Figure 3(b))These results suggested that consumption of BMW has apotential to change some gut microbial compositions

4 Discussion

In the present study it was shown that serum glycoalbuminlevels were significantly decreased after BMW consumptionas compared with TW consumption Although glycoalbuminlevels have been known to reflect blood glucose levels during

the last 14 days of the experiment [24] the mean bloodglucose levels were not decreased significantly but tended tobe lowered by BMW consumption as observed in our dataThis might be attributed to the fact that blood glucose levelsare influenced by external factors such as food intake andexercise [25] Although insulin is one of the major factorsinvolved in blood glucose control the reduction of glycoalbu-min level that was observed in this study was not expected tobe related to the changing of insulin secretion andor insulinresistance because insulin concentrations andHOMA-Rwerenot significantly changed after BMW consumption Previousstudies have also reported the reduction of blood glucoselevels by consumption of BMW or sulfate containing water[4 5] but this study is the first evidence of reduction of serumglycoalbumin levels by consumption of BMW Additionallyrelative glycoalbumin levels were partly decreased even afterTW consumption period Although it has been previouslyreported that body weights of the volunteers were decreasedby consumption of TW before every meal during 12 weeks[26] the reduction in serum glycoalbumin levels is a novelresult

Clinical tests also indicated significant increments inblood calcium levels This phenomenon was expected tobe attributed to BMW consumption because BMW used inthis study contains calcium (177mgkg) According to theprevious report calcium deficiency may apparently lead toinsulin resistance [27] Therefore calcium supplementationby drinking mineral water that includes calcium may beimportant as well as BMW consumption to better manageglycemic control

According to the metabolome analysis of blood sam-ples ATP and pyruvate were significantly increased whereasADP was decreased This result suggested that glycolysiswas upregulated after consumption of BMW Since bloodglucose levels were not decreased significantly but tendedto be lowered as observed in our results the effects ofglycolysis enhancement were expected On the other handconcentrations of 3 amino acids (tyrosine methionine and

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Other Coriobacteriaceae Erysipelotrichaceae Bifidobacteriaceae Veillonellaceae

Prevotellaceae Bacteroidaceae Lachnospiraceae Ruminococcaceae

100

80

60

40

20

0

N03

N04

N05

N06

N07

N08

N09

N10

N11

N13

N15

N16

N17

N19

N20

N22

N23

N24

N26

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Subjects

Rela

tive a

bund

ance

(of

tota

l seq

uenc

es)

Clostridialesmdashunclassified

(a)

Christensenellaceae PorphyromonadaceaeBacteroidaceaeDehalobacteriaceae Rikenellaceae Erysipelotrichaceae Oxalobacteraceae Bifidobacteriaceae

BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW BMWTW

lowastlowast

minus10

minus05

00

05

10

Z-s

core

lowast lowast lowast lowast lowast lowast lowast

(b)

Figure 3 Comparisons of fecal microbiota compositions between TW and BMW consumption periods (a) Family level compositions offecal microbiota during the test (b) Mean relative abundances of each taxa (119885-score) of weeks 1 and 3 (TW) and weeks 2 and 4 (BMW) wereshown in dot plots overlaid on box plots in the same manner as in Figure 1(a) Only 8 families that their compositions were significantlydifferent between TW and BMW consumption periods were demonstrated lowast119875 lt 005 lowastlowast119875 lt 0005

glycine) were significantly lowered after consumption ofBMW A previous study reported that high concentrationsof various amino acids especially tyrosine in blood are oneof the risk factors of T2D [28] Additionally it has beenalso reported that plasma concentrations of several aminoacids including tyrosine and methionine were significantlyhigh in hyperinsulinemia (it is often observed in early stageof T2D) patients as compared to healthy subjects [29] Ourresults demonstrated that concentrations of 3 amino acidsincluding tyrosine and methionine in blood were signifi-cantly decreased and that those of other standard amino acids

were tended to be lowered after consumption of BMW Assuch it can be suggested that the BMW consumption has apossible potential to prevent andor improve T2D throughthe alterations of metabolism in the body

After insulin resistance was increased it can be expectedthat proteolysis and ketogenesis would be enhanced [30]As a result the increment of blood concentrations of aminoacids or ketone bodies is expected Sincemetabolome analysisindicated that the concentrations of 3-hydroxybutyrate atype of ketone body between TW and BMW consumptionperiods were not changed consumption of BMW may not

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

affect generation of energy from free fatty acids As our resultsdemonstrated that concentrations of almost of all amino acidsespecially tyrosine methionine and glycine were decreasedbut that of 3-hydroxybutyrate was not changed BMWconsumption may influence energy metabolism throughproteolysis but not ketogenesis

UDP-N-acetylglucosamine is the substrate of O-linkedN-acetylglucosamine transferase and the relationshipbetween O-linked N-acetylglucosamine transferase andinsulin resistance has been previously reported [31]However it was also reported that concentration of UDP-N-acetylglucosamine in muscle tissue was increased afterreaching euglycemia by insulin treatment in obese subjects[32]These reports suggested that UDP-N-acetylglucosamineis related to glucose control andor insulin resistance butthe details are still unclear In the current study metabolomeanalysis indicated that blood concentrations of UDP-N-acetylglucosamine were significantly increased after BMWconsumption but future studies are required to understandthe meaning of this phenomenon

Since recent studies reported the relationships betweengut microbiota and T2D andor obesity [33ndash35] we hypoth-esized that beneficial effect for glycemic control derivedfrom BMW consumption might involve gut microbiota Asexpected alterations of gut microbiota compositions derivedfromBMWconsumption were observed In this study familyChristensenellaceae was the most significantly increasedtaxon Previous study reported that Christensenellaceae wasenriched in lean group (BMI lt 25) as compared with obesegroup (BMI gt 30) [36] Additionally it was also reportedthat transplantation of Christensenella minuta to germ-freemice reduced weight gain Moreover abundance of thefamilyDehalobacteriaceae thatwas also increased after BMWconsumption has been reported to be positively correlatedwith Christensenellaceae Therefore our results suggestedthat consumption of BMWhas a possible potential to preventgetting obese via increments of the abundance of lean-inducible bacteria Christensenellaceae and Dehalobacteri-aceae

5 Conclusions

In this study we have shown that serum glycoalbumin levelswere significantly decreased after BMW consumption ascompared with TW consumption It was also observed that19 blood metabolites were significantly changed and lean-inducible bacteria were significantly increased after BMWconsumption (see Figure S3 of Supplementary Material) Thecurrent study is expected to become important evidencedetailing the molecular level effects of BMW consumptionFinally we believe that the molecular level elucidation ofthe beneficial effects derived from balneotherapy is vital forfurther progress and understanding of balneology and ourstudy is the first step towards this approach

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Ms Chiharu Ishii Ms YukaOhara and Mr Yuki Yoshida for their experimental andoranalytical support They also wish to thank Dr Wanping Awfor critical reading and editing of the paper As performingblood sampling they really thank Ms Mitsuyo Matsuo andmembers of Ito Medical Office They would like to offertheir gratitude to officials Ms Ryoko Washitsukasa MsKyoko Nakashita Ms Nozomi Mai Mr Toru Miyazaki MrYasuyuki Morita Mr Takahiro Kudo Mr Teiji Shimizu MrToshinori Hayashi Mr Hironori Shin and Mayor KatsujiShuto fromTaketa for their governmental supportThis studywas supported in part by research grants from the Ministryof Education Culture Sports Science and Technology Japan(26117725 and 15H01522 to Shinji Fukuda) Japan Society forthe Promotion of Science (254930 to Shinnosuke Murakamiand 24380072 to Shinji Fukuda) the Yamagata PrefecturalGovernment and the city of Tsuruoka and the Japan Healthand Research Institute (Shinji Fukuda)

References

[1] K Z Walker K OrsquoDea M Gomez S Girgis and R ColagiurildquoDiet and exercise in the prevention of diabetesrdquo Journal ofHumanNutrition and Dietetics vol 23 no 4 pp 344ndash352 2010

[2] J Salas-Salvado M Martinez-Gonzalez M Bullo and E RosldquoThe role of diet in the prevention of type 2 diabetesrdquoNutritionMetabolism and Cardiovascular Diseases vol 21 supplement 2pp B32ndashB48 2011

[3] M Karagulle andM Z Karagulle ldquoEffectiveness of balneother-apy and spa therapy for the treatment of chronic low back paina review on latest evidencerdquo Clinical Rheumatology vol 34 no2 pp 207ndash214 2015

[4] C Gutenbrunner ldquoKontrollierte Studie uber die Wirkungeiner Haustrinkkur mit einem Natrium-Hydrogencarbonat-Sauerling auf die Blutzuckerregulation bei gesunden Ver-suchspersonenrdquo Physikalische Medizin RehabilitationsmedizinKurortmedizin vol 3 no 4 pp 108ndash110 1993

[5] Y Ohtsuka J Nakaya K Nishikawa and N Takahashi ldquoEffectof hot spring water drinking on glucose metabolismrdquo TheJournal of the Japanese Society of Balneology Climatology andPhysical Medicine vol 66 no 4 pp 227ndash230 2003

[6] S Schoppen F J Sanchez-Muniz A M Perez-Granados etal ldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[7] F Annegret and F Thomas ldquoLong-term benefits of radon spatherapy in rheumatic diseases results of the randomised multi-centre IMuRa trialrdquo Rheumatology International vol 33 no 11pp 2839ndash2850 2013

[8] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[9] E D Pagourelias P G Zorou M Tsaligopoulos V G AthyrosA Karagiannis and G K Efthimiadis ldquoCarbon dioxide bal-neotherapy and cardiovascular diseaserdquo International Journal ofBiometeorology vol 55 no 5 pp 657ndash663 2011

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

[10] S Samino M Vinaixa M Dıaz et al ldquoMetabolomics revealsimpaired maturation of HDL particles in adolescents withhyperinsulinaemic androgen excessrdquo Scientific Reports vol 5article 11496 2015

[11] A Sreekumar L M Poisson T M Rajendiran et alldquoMetabolomic profiles delineate potential role for sarcosine inprostate cancer progressionrdquoNature vol 457 no 7231 pp 910ndash914 2009

[12] M Uetaki S Tabata F Nakasuka T Soga and M TomitaldquoMetabolomic alterations in human cancer cells by vitamin C-induced oxidative stressrdquo Scientific Reports vol 5 Article ID13896 2015

[13] E Mishima S Fukuda H Shima et al ldquoAlteration of theintestinal environment by lubiprostone is associated with ame-lioration of adenine-induced CKDrdquo Journal of the AmericanSociety of Nephrology vol 26 no 8 pp 1787ndash1794 2015

[14] M Sugimoto D T Wong A Hirayama T Soga and MTomita ldquoCapillary electrophoresis mass spectrometry-basedsaliva metabolomics identified oral breast and pancreaticcancer-specific profilesrdquo Metabolomics vol 6 no 1 pp 78ndash952010

[15] P Kaur N Rizk S Ibrahim et al ldquoQuantitative metabolomicand lipidomic profiling reveals aberrant amino acidmetabolismin type 2 diabetesrdquoMolecular BioSystems vol 9 no 2 pp 307ndash317 2013

[16] N Larsen F K Vogensen F W J van den Berg et al ldquoGutmicrobiota in human adults with type 2 diabetes differs fromnon-diabetic adultsrdquo PLoS ONE vol 5 no 2 Article ID e90852010

[17] J Qin Y Li Z Cai and et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[18] F H Karlsson V Tremaroli I Nookaew et al ldquoGutmetagenome in European women with normal impairedand diabetic glucose controlrdquo Nature vol 498 no 7452 pp99ndash103 2013

[19] J Xia and D S Wishart ldquoMSEA a web-based tool to identifybiologically meaningful patterns in quantitative metabolomicdatardquoNucleic Acids Research vol 38 no 2 pp W71ndashW77 2010

[20] Y Furusawa Y Obata S Fukuda et al ldquoCommensal microbe-derived butyrate induces the differentiation of colonic regula-tory T cellsrdquo Nature vol 504 no 7480 pp 446ndash450 2013

[21] S-W KimW Suda S Kim et al ldquoRobustness of gut microbiotaof healthy adults in response to probiotic intervention revealedby high-throughput pyrosequencingrdquo DNA Research vol 20no 3 pp 241ndash253 2013

[22] T Magoc and S L Salzberg ldquoFLASH fast length adjustment ofshort reads to improve genome assembliesrdquo Bioinformatics vol27 no 21 pp 2957ndash2963 2011

[23] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[24] M Koga and S Kasayama ldquoClinical impact of glycated albuminas another glycemic control markerrdquo Endocrine Journal vol 57no 9 pp 751ndash762 2010

[25] S Moebus L Gores C Losch and K-H Jockel ldquoImpact oftime since last caloric intake on blood glucose levelsrdquo EuropeanJournal of Epidemiology vol 26 no 9 pp 719ndash728 2011

[26] E A Dennis A L Dengo D L Comber et al ldquoWaterconsumption increases weight loss during a hypocaloric dietintervention in middle-aged and older adultsrdquo Obesity vol 18no 2 pp 300ndash307 2010

[27] T Fujita ldquoCalcium paradox consequences of calcium defi-ciency manifested by a wide variety of diseasesrdquo Journal of Boneand Mineral Metabolism vol 18 no 4 pp 234ndash236 2000

[28] T J Wang M G Larson R S Vasan et al ldquoMetabolite profilesand the risk of developing diabetesrdquoNatureMedicine vol 17 no4 pp 448ndash453 2011

[29] H Nakamura H Jinzu K Nagao et al ldquoPlasma amino acidprofiles are associated with insulin C-peptide and adiponectinlevels in type 2 diabetic patientsrdquoNutrition ampDiabetes vol 4 pe133 2014

[30] P Sonksen and J Sonksen ldquoInsulin understanding its action inhealth and diseaserdquo British Journal of Anaesthesia vol 85 no 1pp 69ndash79 2000

[31] X Yang P P Ongusaha P D Miles et al ldquoPhosphoinositidesignalling links O-GlcNAc transferase to insulin resistancerdquoNature vol 451 no 7181 pp 964ndash969 2008

[32] M-J J Pouwels P N Span C J Tack et al ldquoMuscle uridinediphosphate-hexosamines do not decrease despite correction ofhyperglycemia-induced insulin resistance in type 2 diabetesrdquoThe Journal of Clinical Endocrinology amp Metabolism vol 87 no11 pp 5179ndash5184 2002

[33] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[34] GMusso RGambino andMCassader ldquoObesity diabetes andgut microbiota the hygiene hypothesis expandedrdquo DiabetesCare vol 33 no 10 pp 2277ndash2284 2010

[35] V K Ridaura J J Faith F E Rey et al ldquoGut microbiota fromtwins discordant for obesity modulate metabolism in micerdquoScience vol 341 no 6150 Article ID 1241214 2013

[36] J K Goodrich J L Waters A C Poole et al ldquoHuman geneticsshape the gut microbiomerdquo Cell vol 159 no 4 pp 789ndash7992014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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BioMed Research International

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Oxidative Medicine and Cellular Longevity

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

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Stem CellsInternational

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MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom