Research Article Metabolomic Analysis of Differential Changes in Metabolites during ... · 2019. 7. 31. · and uronic acid pathway oscillate in phase with ATP oscillations, while

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 213972 8 pageshttpdxdoiorg1011552013213972

Research ArticleMetabolomic Analysis of Differential Changes in Metabolitesduring ATP Oscillations in Chondrogenesis

Hyuck Joon Kwon1 and Yoshihiro Ohmiya2

1 Department of Physical Therapy College of Health Science Eulji University Gyeonggi 461-713 Republic of Korea2National Institute of Advanced Industrial Science and Technology Biomedical Research Institute Tsukuba 305-8566 Japan

Correspondence should be addressed to Hyuck Joon Kwon kwonhjeuljiackr

Received 18 April 2013 Accepted 30 May 2013

Academic Editor Andrei Surguchov

Copyright copy 2013 H J Kwon and Y Ohmiya 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

Prechondrogenic condensation is a critical step for skeletal pattern formation Recent studies reported that ATP oscillations playan essential role in prechondrogenic condensation However the molecular mechanism to underlie ATP oscillations remainspoorly understood In the present study it was investigated how changes in metabolites are implicated in ATP oscillationsduring chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) CE-TOF-MS detected93 cationic and 109 anionic compounds derived from known metabolic pathways 15 cationic and 18 anionic compounds revealedsignificant change between peak and trough of ATP oscillations These results implicate that glycolysis mitochondrial respirationand uronic acid pathway oscillate in phase with ATP oscillations while PPRP and nucleotides synthesis pathways oscillatein antiphase with ATP oscillations This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate inantiphase with the ATP-consuming PPRPnucleotide synthesis pathway during chondrogenesis

1 Introduction

Skeletal development in vertebrate limb beginswith chondro-genesis in which prechondrogenic cells condense and thendifferentiate into chondrocytes to form a variety of preciselyshaped cartilage elements that are ultimately replaced bybone tissues through endochondral ossification [1] Thisindicates that the prechondrogenic condensation is a criticalstep for skeletal pattern formation in limb development Werecently found that ATP oscillations play a key role in pre-chondrogenic condensation during chondrogenesis [2ndash4] Itwas demonstrated that extracellular ATP and cAMPPKAsignaling mediate ATP oscillations during chondrogenesis[5] However how metabolic pathways are liked with ATPoscillations remains poorly understood

Metabolites are the main effectors of phenotype andfunctional entities in the cell [6] Thus metabolomics whichis defined as the measurement of the level of all intracellularmetabolites is a powerful tool for gaining insight intocellular functions Indeed metabolomic analyses are beingincreasingly performed to clarify biochemical mechanisms

to underlie various physiological processes [7ndash9] Thereforecomprehensive profiles of differential metabolite changesduring metabolic oscillations can reveal the connection ofbiochemical networks during metabolic oscillations and thusprovide a system-level understanding of metabolic oscilla-tions during chondrogenesis

Although most metabolic analyses have been performedwith gas chromatography mass spectrometry (GC-MS) [10]GC-MS is limited by the need for multiple deprivation proce-dures for each chemical moiety and the fact that nonvolatilethermolabile and highly polar compounds are difficult to bedetermined However capillary electrophoresis mass spec-trometry (CE-MS) in which metabolites are separated by CEbased on charge and size and then selectively detected usingMS has the major advantages in extremely high resolutionhigh throughput and ability to simultaneously quantify allcharged low-molecular weight compounds [11ndash13]Thus CE-MS has emerged as a useful tool for analyzing polar andcharged molecules [13ndash15]

In the present work capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) was applied to the

2 BioMed Research International

metabolome profiling of differential metabolite during ATPoscillations in chondrogenesis This CE-TOF-MS methodcovered a wide (50ndash1000)119898119911 rangeThis system determined93 cationic and 109 anionic compounds derived from knownmetabolic pathways in prechondrogenic cell lineATDC5 cellsand revealed significant change of 15 cationic and 18 anioniccompounds between peak and trough of ATP oscillationsThis study provides information about how metabolic path-ways are involved in ATP oscillations during chondrogenesis

2 Materials and Methods

21 Cell Line The ATDC5 cell line was obtained from theRIKEN cell bank (Tsukuba)The cells were cultured in main-tenance medium consisting of a 1 1 mixture of Dulbeccorsquosmodified Eaglersquos medium and Hamrsquos F-12 medium (DMEM-F12) (Invitrogen) supplemented with 5 fetal bovine serum10mgmL human transferrin (Roche Molecular Biochemi-cals) and 3 times 10minus8M sodium selenite (Sigma-Aldrich) inpolystyrene dishes at 37∘C under 5 CO

2

For chondrogenicinduction whenATDC5 cellsmaintained in themaintenancemedium reached confluency the medium was replaced withthe chondrogenic medium supplemented with 10 120583gmLinsulin (Sigma-Aldrich)

22 Bioluminescence Monitoring Experiments Biolumines-cence reporter constructed by fusing the human actin pro-moter to a Phrixothrix hirtus luciferase gene (Toyobo) wasinserted into retrovirus vector (Clontech) ATDC5 cells weretransfected using retrovirus infection and then were selectedby puromycin The cells transfected stably with PACTIN-PxRewere seeded in 35mm dishes After replacing the mediumby a recording medium (DMEMF12 with 5 FBS 01mMluciferin (Wako) and 50mMHEPES-NaOH pH = 70) (time= 0 h) bioluminescence intensity was continuouslymeasuredat 37∘C in air using a dish-type luminescence Kronos(ATTO) for 1min at 30min intervals For examining theeffect of chemical compounds on ATP oscillations the cellswere treated with 01mM iodoacetate (Sigma-Aldrich) or5mM cyanide (Sigma-Aldrich) about 24 hours after PACTIN-PxRe oscillations initiated

23 Sample Preparation for CE-TOF-MS Analysis ATDC5cells cultured to 1times106 cells needed pretreatmentThe culturemedium was removed from the plates and 10mL of 5mannitol solution was added to wash the cells After 2mL of5 mannitol solution was added to wash the cells 1300120583Lof methanol containing the internal standards was addedCells were scraped from the plate and then cell lysate (sim1000120583L) was used in the analysis Then 400120583L of Milli-Qwater and 1000120583L of chloroform were added to the samplesthoroughly mixed and then centrifuged for 5min at 2300 gand 4∘CThe 750120583L of upper aqueous layer was centrifugallyfiltered through a Millipore 5 kDa cutoff filter to removeproteins The filtrate was lyophilized and suspended in 25120583Lof Milli-Q water We prepared three samples at peak andtrough of PACTIN-PxRe oscillations respectively and usedeach sample for independent CE-TOF-MS analysis

24 Instrumentation for CE-TOF-MS Analysis Themeasure-ment of extracted metabolites was performed by using a cap-illary electrophoresis- (CE-) connected ESI-TOF-MS systemwith electrophoresis buffer (Solution ID H3302-1021 HumanMetabolome Technologies Inc Tsuruoka Japan) CE-TOF-MS was carried out using an Agilent CE Capillary Elec-trophoresis System equipped with an Agilent 6210 Time ofFlight mass spectrometer Agilent 1100 isocratic HPLC pumpAgilent G1603A CE-MS adapter kit and Agilent G1607ACE-ESI-MS sprayer kit (Agilent Technologies WaldbronnGermany) The system was controlled by Agilent G2201AAChemStation software version B0301 for CE (Agilent Tech-nologies Waldbronn Germany)

25 Experimental Conditions for CE-TOF-MS Analysis Forcationic metabolites capillary electrophoreses were per-formed using a fused silica capillary The electrolyte was 1Mformic acid Methanol-water (50 vv) containing 05mMreserpine (the lock mass for exact mass measurements)was delivered as the sheath liquid For anionic metabolitesseparations were performed using a fused silica capillaryThe electrolyte was 50mM ammonium acetate (pH 85)The sheath liquid for anionic metabolites was used as theelectrolyteThe capillary was pretreatedwith preconditioningbuffer including 25mM ammonium acetate and 75mMsodium phosphate at pH 85 Pressure of 5mbar was appliedto inlet capillary during run to reduce the analysis time Forall analytical modes inner diameter and total length of capil-lary are 50mm and 80 cm respectively The applied voltagewas set at +30 kV and minus30 kV for cation and anion moderespectively Electrospray ionization TOF-MS was operatedin the positive ion mode (4 kV) the negative ion mode(35 kV) for cationic metabolites anionic metabolites andnucleotides respectively A flow rate of heated dry nitrogengas (heater temperature 300∘C) was maintained at 5 psigExact mass data were acquired over a 50ndash1000119898119911 rangeSignal peaks corresponding to isotopomers adduct ions andother product ions of known metabolites were excluded allsignal peaks potentially corresponding to authentic com-pounds were extracted and then their migration time (MT)was normalized using those of the internal standards There-after the alignment of peaks was performed according tothe 119898119911 values and normalized MT values Finally peakareas were normalized against those of the internal standardsMetSul and CSA for cations and anions respectively Theresultant relative area values were further normalized bysample amount Annotation tables were produced from CE-ESI-TOF-MSmeasurement of standard compounds andwerealigned with the datasets according to similar119898119911 values andnormalized MT values

3 Results and Discussion

31 Relationship of Glycolysis and Mitochondrial Respirationwith ATP Oscillations In this study insulin which stimulateschondrogenesis of ATDC5 cells [16] was used in inductionof ATP oscillations during chondrogenesis IntracellularATP level of ATDC5 cells was monitored in real time by

BioMed Research International 3

Iodoacetate

Insulin

0 24 48 72 96Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Cyanide

Insulin

0 24 48 72 96 120 144Time (hours)

0

50000

100000

150000

200000

250000

300000

RLU

min

(b)

Figure 1 ATP oscillations are linked with both glycolysis andmitochondrial respiration Either a glycolysis inhibitor iodoacetateor an inhibitor of mitochondrial respiration cyanide eliminatesinsulin-induced PACTIN-PxRe oscillations in ATDC5 cells Biolu-minescence monitoring of PACTIN-PxRe activity in ATDC5 cellswas performed after chondrogenic induction with the chondrogenicmedium (time = 0 h) About 24 hours after PACTIN-PxRe oscillationswere initiated ATDC5 cells were treated with iodoacetate orcyanide

transfection of ATP-dependent Phrixothrix hirtus luciferase(PxRe) reporter gene fused to a constitutiveACTINpromoter(PACTIN-PxRe) into the cells Consistent with the previousstudies [2ndash4] we found that ATP oscillations were generated2ndash4 d after chondrogenic induction (Figure 1) Since ATP isproducedmainly by glycolysis andmitochondrial respirationwe examined whether ATP oscillations depend on glycolysis

or mitochondrial respiration It was shown that both a gly-colysis inhibitor iodoacetate and a mitochondrial respirationinhibitor cyanide suppressed insulin-induced ATP oscilla-tions (Figure 1) This result indicates that ATP oscillations inchondrogenesis dependonboth glycolysis andmitochondrialrespiration

32 Heatmap for Differential Changes of Metabolites betweenPeak and Trough of ATP Oscillations To elucidate themetabolic pathways to generate ATP oscillations duringchondrogenesis the differential changes in the metabolitesbetween peak and trough of ATP oscillations were analyzedby using CE-TOF-MS analysis system When PACTIN-PxReoscillations revealed peak or trough as shown in Figure 2the metabolites were extracted from the ATDC5 cells CE-TOF-MS was used for interrogating the relative levels ofmetabolites between peaks and troughs in PACTIN-PxRe oscil-lations At molecular weights range from 500 to 100011989811991193 cationic and 109 anionic compounds were detected andanalyzed in this analysis To delineate the metabolomicalterations between peak and trough in ATP oscillations theratio of metabolite concentrations between peak and troughof PACTIN-PxRe oscillations was represented in heatmap(Figure 3) Among total compounds 15 cationic and 18anionic compounds revealed statistically significant changebetween peak and trough of PACTIN-PxRe oscillations

33 Metabolites to Significantly Increase at Peak of ATPOscillations in Comparison with That at Trough of ATPOscillations ATP showed higher concentration at the peakof PACTIN-PxRe oscillations than at the trough (Table 1)which confirmed that PACTIN-PxRe oscillations reflect ATPoscillations Besides ATP 3 cationic compounds such asO-acetylcarnitine phosphorylcholine and tyramine and11 anionic compounds such as fructose 16-bisphos-phatemalate fumaric acid 2-oxoglutaratecis-aconitic acid 13-bisphosphoglycerate 2-phosphoglycerate 3-phosphoglycer-ate NADHUDP-glucuronic acid and glucuronic acid weresignificantly increased at the peak of PACTIN-PxRe oscilla-tions (Table 1) This result suggests that intracellular levels ofthese compounds oscillate in phase with ATP oscillations

These compounds include glycolytic intermediates suchas fructose 16-bisphosphate 3-phosphoglyceric acid and 2-phosphoglyceric acid and intermediates of the tricarboxylicacid cycle (TCA cycle) such as cis-aconitic acid 2-oxoglutaricacid fumaric acid and malate This result implicates thatglycolysis and TCA cycle oscillate during ATP oscillationsPrevious studies reported the similar results that glycolyticoscillations are coupled to oscillations in mitochondrialmembrane potentials in S cerevisiae and pancreatic b-cells [17ndash19] and suggested that glycolytic oscillations drivemitochondrial oscillations [20] However the present resultthat either a glycolysis inhibitor or a mitochondrial inhibitorprevented ATP oscillations implies that glycolytic and mito-chondrial oscillations are interdependent on each other Inaddition NADH was significantly increased in the peak ofATP oscillations indicating that NADH oscillates in phasewith ATP oscillations Although it is uncertain how NADH


Trough

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Peak

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(b)

Figure 2 Blue dots represent trough and peak of PACTIN-PxRe oscillations in which metabolites were extracted from ATDC5 cellsBioluminescence monitoring of PACTIN-PxRe activities was performed after chondrogenic induction (time = 0 h)

Table 1The fold changes in concentrations ofmetabolites increasedsignificantly in peak of ATP oscillations

Metabolite Ratio(peaktrough)

119875 value(Welchrsquos t-test)

Fructose 16-bisphosphate 177 0045Malate 165 0042Fumaric acid 158 00302-Oxoglutarate 157 004713-Bisphosphoglycerate 152 0041cis-Aconitic acid 151 00372-Phosphoglycerate 144 00423-Phosphoglycerate 144 0047NADH 143 0048UDP-glucuronic acid 134 0026O-Acetylcarnitine 132 0011ATP 131 0042Phosphorylcholine 130 0029Glucuronic acid 116 0023Tyramine 111 0049

level stems from glycolysis or mitochondrial respirationNADH oscillations support that glycolysis or mitochondrialrespiration oscillates during ATP oscillations in chondrogen-esis

O-acetylcarnitine level was also higher in the peak ofATP oscillations which indicates that O-acetylcarnitine leveloscillates in phase with ATP oscillations L-carnitine andacetyl-CoA are converted to O-acetylcarnitine and CoAinside mitochondria by carnitine O-acetyltransferase andthe O-acetylcarnitine is transported outside the mitochon-dria where it converts back to O-carnitine and acetyl-CoA[21] It was known that cycling of acetyl-CoA through

O-acetylcarnitine plays a key role in matching instanta-neous acetyl-CoA supply with metabolic demand [22] Sinceacetyl-CoA is required for TCA cycle in mitochondria O-acetylcarnitine oscillations may contribute to mitochondrialoscillations by inducing oscillations of acetyl-CoA cyclingO-acetylcarnitine oscillations may be driven by oscillatoryactivity of carnitine O-acetyltransferase There is evidencethat suggests that carnitine O-acetyltransferase activity isnecessary for the cell cycle to proceed from the G1 phase tothe S phase [23] Therefore oscillatory activity of carnitineO-acetyltransferase may mediate prechondrogenic conden-sation by modulating cellular proliferation

In addition both uridine diphosphate (UDP)-glucuronicacid and glucuronic acid were significantly increased in thepeak of ATP oscillations UDP-glucuronic acid is the activeform of glucuronic acid for the incorporation of glucuronicacid into chondroitin sulphate [24] UDP-glucuronic acidis produced from glucose via uronic acid pathway glucose6-phosphate is isomerized to glucose 1-phosphate whichthen reacts with uridine triphosphate (UTP) to form UDPglucose in a reaction catalyzed by UDP-glucose pyrophos-phorylase and subsequently UDP glucose is oxidized byNAD-dependent UDP-glucose dehydrogenase to yield UDP-glucuronic acid [25] Therefore the uronic acid pathway ofglucose conversion to glucuronic acid may oscillate duringATP oscillations Furthermore since NADH is producedin the uronic acid pathway the oscillatory uronic acidpathway may contribute to generating NADH oscillationsduring chondrogenesis In addition glucuronic acid andUDP-glucuronic acid are used for synthesis of glycosamino-glycans [26] and the oscillatory uronic acid pathway candrive oscillatory synthesis of proteoglycans which are majorextracellular matrix (ECM) in cartilage Our previous reportdemonstrated that ATP oscillations drive oscillatory secre-tion and proposed that ATP oscillations lead to prechon-drogenic condensation by inducing oscillatory secretion of


Phenylpyruvic acidSpermine

SpermidineFructose 16-diphosphate

Malic acidStreptomycin sulfate

Fumaric acid2-Oxoglutaric acid

23-Diphosphoglyceric acidCis-Aconitic acid

Citric acid3-Phosphoglyceric acid2-Phosphoglyceric acid

NADHIsocitric acid

PutrescineATP

UDP-glucuronic acid2-Hydroxypentanoic acid

O-AcetylcarnitineAdenine

PhosphorylcholineDihydroxyacetone phosphate

Uric acidUMP

5-OxoprolineHypoxanthine

2-Oxoisovaleric acid2-Hydroxybutyric acid

Phosphoenolpyruvic acidLauric acid

Dicrotalic acidMyo-Inositol 1 or 3-phosphate

Glucuronic acid

Succinic acidLactic acid

Diphenylcarbazide

Tyramine5-Methoxyindoleacetic acid

Decanoic acid6-Phosphogluconic acid

Ethanolamine phosphatePyruvic acid

NicotinamideUDP-glucose

CreatinineLeucinic acid

Glycerophosphocholine

KetoprofenN-Acetylglutamic acid

Undecanoic acidNN-Dimethylglycine

3-Methyl-2-oxovaleric acidUTP

Glyceric acidTromethamine

ThiaminePhosphocreatine

Homovanillic acidCyclohexanecarboxylic acid

N-AcetylphenylalaninePyridoxine

IMP3-Aminoisobutyric acid

O-Succinylhomoserine1-Methylnicotinamide

2-Deoxyglucose 6-phosphateHomoserinelactone

CMP-N-acetylneuraminate3-Hydroxybutyric acid

4-Oxovaleric acid5-Hydroxylysine

o-Hydroxybenzoic acid

Myo-Inositol 2-phosphatedTTP

Carnosine1-Phenylethylamine

UDP-N-acetylglucosamineIsethionic acid

2-Aminoadipic acidPelargonic acid

Gly-AspCTP

Taurine

CystineGly-Gly

3-Phenylpropionic acidADP-glucose

Ribulose 5-phosphateCystathionine

Ophthalmic acidGluconic acid

5-Aminovaleric acidGTP

Pantothenic acidOctanoic acid

PyridoxalN-Acetylleucine

GuanosineS-AdenosylmethionineArgininosuccinic acid

GDPTriethanolamine

Carnitine2-Carboxybenzaldehyde

UreaN-Acetylneuraminic acid

3-Methylhistidine

Glutaric acidDiethanolamine

GDP-galactose4-Guanidinobutyric acid

AMPCreatine

PRPPGlu

ADP3-Methylbutanoic acid

BetaineCys

Thiaproline3-Aminobutyric acid

ValSDMA

Glucose 1-phosphate

CitrullineN-Acetylaspartic acid

TrpGly

Ribose 5-phosphateIsobutylamine

GuaninePhe

dCTPMet

Cyclohexylamine

Glycerol-3-phosphateCitramalic acid

IleCDP

ADMAGln

Hexanoic acidImidazole-4-acetic acid

Choline1H-Imidazole-4-propionic acid

HisAsp

UDPHeptanoic acid

LeuTyr

HomoserineGlutathione (GSH)

Lys4-Acetylbutyric acid

2-Aminoisobutyric acidCysteine-glutathione disulphide

SerAzelaic acid

ArgBenzoic acid

Tiglic acidOrnithine

Fructose 6-phosphateGlucose 6-phosphate

Phthalic acidMethionine sulfoxide

AlaGMP

ProThr

3-Methylbenzoic acidSedoheptulose 7-phosphate

AsnButyric acid

HydroxyprolineCDP-choline

Adipic acidcGMP

1-Methyl-4-imidazoleacetic acid1-Pyrroline 5-carboxylic acid

Terephthalic acidUrocanic acid

Melamine

NADPH divalent

N 8-Acetylspermidine5998400-Methylthioadenosine

N-Acetyl-120573-alanine

FAD divalent

120574-Butyrobetaine

Glutathione (GSSG) divalent

NAD+

NADP+

120573-Imidazolelactic acid

120574-Aminobutyric acid

Acetyl CoA divalent

120573-Ala

N5-Ethylglutamine

CoA divalent

Exp no 1 Exp no 2 Exp no 3Ratio (peaktrough)


minus2

minus15

minus1

minus05

0

05

1

15

2Fo

ld ch

ange

(log

2)

Propionic acid

Phenaceturic acid

Figure 3 The result of the metabolite analysis is illustrated in a heatmap which represents the fold change of metabolite concentrations inpeaks of PACTIN-PxRe oscillations compared with those in troughs of PACTIN-PxRe oscillations Data are from three independent experiments(Exp nos 1ndash3) Gray means the case in which the metabolite was undetected in peak or trough of PACTIN-PxRe oscillations


Table 2 The fold changes in concentrations of metabolitesdecreased significantly in peak of ATP oscillations



Diethanolamine 088 0020PRPP 086 0005ADP 085 0036Glycine 081 0042Guanine 080 0016Cyclohexylamine 079 0025dCTP 079 0044Glutamine 078 0014Aspartic acid 077 0032UDP 076 00452-Aminoisobutyric acid 074 0034Methionine sulfoxide 069 0009GMP 069 0043Proline 067 0011Threonine 067 0022Asparagine 064 0032Hydroxyproline 063 0042cGMP 059 0037

adhesion molecules and ECM involved in cell-cell adhesion[2] Therefore it is suggested that glucuronic acid oscilla-tions can lead to oscillations of ECM secretion by inducingoscillatory synthesis of proteoglycan in cooperation withoscillatory secretion activity and consequently contribute toprechondrogenic condensation during chondrogenesis

34 Metabolites to Significantly Increase at Trough of ATPOscillations in Comparison with That at Peak of ATP Oscil-lations Our analysis showed that 12 cationic compoundssuch as diethanolamine glycine guanine cyclohexylamineglutamine aspartic acid 2-aminoisobutyric acid methioninesulfoxide proline threonine asparagine and hydroxypro-line and 6 anionic compounds such as phosphoribosylpy-rophosphate (PPRP) ADP dCTP UDP GMP and cGMPwere significantly increased in the trough of PACTIN-PxReoscillations (Table 2) This result suggests that intracellularlevels of these compounds oscillate in antiphase with ATPoscillations which is supported by the result that ADP wasincluded in these compounds because ADP used for ATPsynthesis should be in antiphase with ATP oscillations

PPRP which is produced from glucose via either theoxidative or nonoxidative pentose phosphate pathway is akey regulator of de novo nucleotide synthesis [27]Thereforenucleotide synthesis may oscillate in antiphase with ATPoscillations because the result that PRPPwas decreased in thepeak of ATP oscillations implies that PRPP level oscillates inantiphase with ATP oscillationsThis suggestion is supportedby the result that for glutamine glycine and aspartic acid toparticipate in nucleotide synthesis purines such as guanosinemonophosphate (GMP) cyclic GMP (cGMP) and guanine

and pyrimidines such as UDP and cCTP showed significantlyhigher level in the trough of ATP oscillations The fact thatATP is used for synthesis of PPRP and pruinepyrimidine[28] can explain the reason why PPRP and nucleotides oscil-late in antiphase toATP cGMPalso influencesCa2+ dynamicsin a number of different cell types [29] Since Ca2+ influx wasshown to mediate ATP oscillations in chondrogenesis [2 5]cGMP oscillations may be associated with ATP oscillationsvia Ca2+ influx Indeed cGMP was reported to regulate Ca2+influx in pancreatic acinar cells in growth factor-stimulatedNIH-3T3 cells [30 31] On the other hand it should be notedthat the essential amino acid threonine was also decreased inthe peak of ATP oscillations It was reported that the essentialamino acid-starved condition leads to decrease in PPRP leveland subsequent purine synthesis mainly by decreasing theactivity of nonoxidative pentose phosphate pathway [32]Thissuggests that the decreased threonine level in the peak of ATPoscillationsmay induce the decrease in PPRP and subsequentpurine level via nonoxidative pentose phosphate pathway

Proline hydroxyproline and asparagine were signifi-cantly increased in the trough of ATP oscillations It isknown that glutamine metabolism and proline metabolismare interconnected via glutamate and pyrroline-5-carboxylate[33] and asparagine are biosynthetically derived from glu-tamine and aspartic acid [34] Hydroxyproline is produced byhydroxylation of prolineTherefore the increase in glutamineand aspartic acid in the trough may lead to the increasein proline hydroxyproline and asparagine It was reportedthat hydroxyproline is a major component of collagen whichoccupies a large amount of extracellular matrix in cartilage[35] and hydroxyproline and proline play key roles for thecollagen stability [36 37]These facts indicate that oscillationsin hydroxyproline and proline play a crucial role in cartilagedevelopment by modulating expression level or structure ofcollagen during chondrogenesis

Taken together our results implicate that glycolysismitochondrial respiration and uronic acid pathway oscillatein phase with ATP oscillations while PPRP and nucleotidessynthesis pathways oscillate in antiphase with ATP oscilla-tions during chondrogenesis This suggests that the ATP-producing pathways such as glycolysis and mitochondrialrespiration oscillate in antiphase with the ATP-consumingPPRPnucleotide synthesis pathway Therefore we proposethat the antiphase synchronization of the ATP-producingand the ATP-consuming pathways can contribute to ATPoscillations during chondrogenesis (Figure 4) However ourresults cannot exclude the possibility that many other factorscan be involved in ATP oscillations

4 Conclusion

Although a number of previous studies on chondrogenesishave focused on regulation at gene expression level werecently found that metabolic oscillations play a key rolein prechondrogenic condensation during chondrogenesisThe present study analyzed the quantitative change of theintermediates and products of metabolism between peak andtrough of ATP oscillation in chondrogenesis and suggested


TCA cycle

Glycolysis

Glucose PPRP synthesis Nucleotide synthesis

ATP

Uronic acidpathway

Figure 4 Schematic representation shows that the synchronizedantiphase oscillations of the oscillatory ATP-producing metabolicpathways (glycolysis and mitochondrial respiration) and ATP-consuming pathways (PPRP and nucleotide synthesis) play a keyrole in generation or maintenance of ATP oscillations during chon-drogenesis White and black boxes represent oscillatory metabolicpathways in phase with ATP and in antiphase with ATP respectivelyQuestionmarks indicate the unknown factors which are involved inATP oscillations

that the catabolic processes such as glycolysis and mito-chondrial respiration and the biosynthetic processes suchas PPRPnucleotide synthesis pathway oscillate in antiphasewith each other and thus might mediate ATP oscillationsduring chondrogenesis Further study is necessary to eluci-date what underlies oscillations of the biochemical reactionsand their integration and how the integrated biochemicalreactions contribute to cartilage development in cooperationwith ATP oscillations

Acknowledgment

This paper was supported by Eulji University in 2013

References

[1] F V Mariani and G R Martin ldquoDeciphering skeletal pattern-ing clues from the limbrdquoNature vol 423 no 6937 pp 319ndash3252003

[2] H J Kwon Y Ohmiya K-I Honma et al ldquoSynchronized ATPoscillations have a critical role in prechondrogenic condensa-tion during chondrogenesisrdquo Cell Death and Disease vol 3 no3 article e278 2012

[3] H J Kwon ldquoTGF-120573 but not BMP signaling induces pre-chondrogenic condensation through ATP oscillations duringchondrogenesisrdquoAndBiophysical ResearchCommunication vol424 no 4 pp 793ndash800 2012

[4] H J Kwon ldquoATP oscillations mediate inductive action of FGFand Shh signalling on prechondrogenic condensationrdquo CellBiochemistry and Function vol 31 no 1 pp 75ndash81 2013

[5] H J Kwon ldquoExtracellular ATP signaling via P2X4 receptor andcAMPPKA signaling mediate ATP oscillations essential forprechondrogenic condensationrdquo Journal of Endocrinology vol214 no 3 pp 337ndash348 2012

[6] O Fiehn ldquoMetabolomicsmdashthe link between genotypes andphenotypesrdquo Plant Molecular Biology vol 48 no 1-2 pp 155ndash171 2002

[7] W R Wikoff G Pendyala G Siuzdak and H S FoxldquoMetabolomic analysis of the cerebrospinal fluid revealschanges in phospholipase expression in theCNSof SIV-infectedmacaquesrdquo The Journal of Clinical Investigation vol 118 no 7pp 2661ndash2669 2008

[8] H J Atherton M K Gulston N J Bailey et al ldquoMetabolomicsof the interaction between PPAR-120572 and age in the PPAR-120572-nullmouserdquoMolecular Systems Biology vol 5 article 259 2009

[9] M G Barderas C M Laborde M Posada et al ldquoMetabolomicprofiling for identification of novel potential biomarkers in car-diovascular diseasesrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 790132 9 pages 2011

[10] O Fiehn J Kopka P Dormann T Altmann R N Tretheweyand L Willmitzer ldquoMetabolite profiling for plant functionalgenomicsrdquo Nature Biotechnology vol 18 no 11 pp 1157ndash11612000

[11] T Soga and D N Neiger ldquoAmino acid analysis by capillaryelectrophoresis electrospray ionization mass spectrometryrdquoAnalytical Chemistry vol 72 no 6 pp 1236ndash1241 2000

[12] T Soga YUenoHNaraoka YOhashiM Tomita andTNish-ioka ldquoSimultaneous determination of anionic intermediates forBacillus subtilismetabolic pathways by capillary electrophoresiselectrospray ionization mass spectrometryrdquo Analytical Chem-istry vol 74 no 10 pp 2233ndash2239 2002

[13] T Soga Y Ueno H Naraoka K Matsuda M Tomita and TNishioka ldquoPressure-assisted capillary electrophoresis electro-spray ionization mass spectrometry for analysis of multivalentanionsrdquo Analytical Chemistry vol 74 no 24 pp 6224ndash62292002

[14] P Cao and M Moini ldquoAnalysis of peptides proteins proteindigests and whole human blood by capillary electrophore-siselectrospray ionization-mass spectrometry using an in-capillary electrode sheathless interfacerdquo Journal of the AmericanSociety for Mass Spectrometry vol 9 no 10 pp 1081ndash1088 1998

[15] S K Johnson L L Houk D C Johnson and R S HoukldquoDetermination of small carboxylic acids by capillary elec-trophoresis with electrospray-mass spectrometryrdquo AnalyticaChimica Acta vol 389 no 1ndash3 pp 1ndash8 1999

[16] C Shukunami C Shigeno T Atsumi K Ishizeki F Suzukiand Y Hiraki ldquoChondrogenic differentiation of clonal mouseembryonic cell line ATDC5 in vitro differentiation-dependentgene expression of parathyroid hormone (PTH)PTH-relatedpeptide receptorrdquo Journal of Cell Biology vol 133 no 2 pp 457ndash468 1996

[17] E Heart G C Yaney R F Corkey et al ldquoCa2+ NAD(P)H andmembrane potential changes in pancreatic 120573-cells by methylsuccinate comparison with glucoserdquo Biochemical Journal vol403 no 1 pp 197ndash205 2007

[18] A ZAndersen A K Poulsen J C Brasen and L FOlsen ldquoOn-line measurements of oscillating mitochondrial membranepotential in glucose-fermenting Saccharomyces cerevisiaerdquoYeast vol 24 no 9 pp 731ndash739 2007

[19] A K Poulsen A Z Andersen J C Brasen A M Scharff-Poulsen and L F Olsen ldquoProbing glycolytic and membranepotential oscillations in Saccharomyces cerevisiaerdquo Biochem-istry vol 47 no 28 pp 7477ndash7484 2008

[20] L F Olsen A Z Andersen A Lunding J C Brasen and A KPoulsen ldquoRegulation of glycolytic oscillations bymitochondrialand plasma membrane H+-ATPasesrdquo Biophysical Journal vol96 no 9 pp 3850ndash3861 2009


[21] C J Rebouche and H Seim ldquoCarnitine metabolism and itsregulation in microorganisms and mammalsrdquo Annual Reviewof Nutrition vol 18 pp 39ndash61 1998

[22] M A Schroeder H J Atherton M S Dodd et al ldquoThe cyclingof acetyl-coenzyme A through acetylcarnitine buffers cardiacsubstrate supply a hyperpolarized 13C magnetic resonancestudyrdquo Circulation Cardiovascular Imaging vol 5 no 2 pp201ndash209 2012

[23] S Brunner K Kramar D T Denhardt and R HofbauerldquoCloning and characterization of murine carnitine acetyltrans-ferase evidence for a requirement during cell cycle progres-sionrdquo Biochemical Journal vol 322 no 2 pp 403ndash410 1997

[24] J E Silbert and A C Reppucci Jr ldquoBiosynthesis of chon-droitin sulfate Independent addition of glucuronic acid and Nacetylgalactosamine to oligosaccharidesrdquo Journal of BiologicalChemistry vol 251 no 13 pp 3942ndash3947 1976

[25] F Eisenberg Jr P G Dayton and J J Burns ldquoStudies on theglucuronic acid pathway of glucose metabolismrdquoThe Journal ofbiological chemistry vol 234 no 2 pp 250ndash253 1959

[26] D Vigetti M Ori M Viola et al ldquoMolecular cloning andcharacterization of UDP-glucose dehydrogenase from theamphibian Xenopus laevis and its involvement in hyaluronansynthesisrdquo Journal of Biological Chemistry vol 281 no 12 pp8254ndash8263 2006

[27] M Sochor S Kunjara A L Greenbaum and P McLeanldquoChanges in pathways of pentose phosphate formation inrelation to phosphoribosyl pyrophosphate synthesis in thedeveloping rat kidney Effects of glucose concentration andelectron acceptorsrdquo Journal of Developmental Physiology vol 12no 3 pp 135ndash143 1989

[28] H G Khorana J F Fernandes and A Kornberg ldquoPyrophos-phorylation of ribose 5-phosphate in the enzymatic synthesis of5-phosphorylribose 1-pyrophosphaterdquoThe Journal of BiologicalChemistry vol 230 no 2 pp 941ndash948 1958

[29] E A Milbourne and F L Bygrave ldquoDo nitric oxide and cGMPplay a role in calcium cyclingrdquo Cell Calcium vol 18 no 3 pp207ndash213 1995

[30] T D Bahnson S J Pandol and V E Dionne ldquoCyclic GMPmodulates depletion-activated Ca2+ entry in pancreatic acinarcellsrdquo Journal of Biological Chemistry vol 268 no 15 pp 10808ndash10812 1993

[31] E Clementi C Sciorati andGNistico ldquoGrowth factor-inducedCa2+ responses are differentially modulated by nitric oxidevia activation of a cyclic GMP-dependent pathwayrdquo MolecularPharmacology vol 48 no 6 pp 1068ndash1077 1995

[32] G R Boss and R B Pilz ldquoPhosphoribosylpyrophosphatesynthesis from glucose decreases during amino acid starvationof human lymphoblastsrdquo Journal of Biological Chemistry vol260 no 10 pp 6054ndash6059 1985

[33] D EMatthewsMAMarano andRGCampbell ldquoSplanchnicbed utilization of glutamine and glutamic acid in humansrdquoAmerican Journal of Physiology vol 264 no 6 pp E848ndashE8541993

[34] M K Patterson Jr and G R Orr ldquoAsparagine biosynthesis bythe Novikoff Hepatoma isolation purification property andmechanism studies of the enzyme systemrdquo Journal of BiologicalChemistry vol 243 no 2 pp 376ndash380 1968

[35] R E Neuman and M A Logan ldquoThe determination ofhydroxyprolinerdquoThe Journal of biological chemistry vol 184 no1 pp 299ndash306 1950

[36] L Vitagliano R Berisio A Mastrangelo L Mazzarella and AZagari ldquoPreferred proline puckerings in cis and trans peptide

groups implications for collagen stabilityrdquo Protein Science vol10 no 12 pp 2627ndash2632 2001

[37] F W Kotch I A Guzei and R T Raines ldquoStabilization ofthe collagen triple helix by O-methylation of hydroxyprolineresiduesrdquo Journal of the American Chemical Society vol 130 no10 pp 2952ndash2953 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


metabolome profiling of differential metabolite during ATPoscillations in chondrogenesis This CE-TOF-MS methodcovered a wide (50ndash1000)119898119911 rangeThis system determined93 cationic and 109 anionic compounds derived from knownmetabolic pathways in prechondrogenic cell lineATDC5 cellsand revealed significant change of 15 cationic and 18 anioniccompounds between peak and trough of ATP oscillationsThis study provides information about how metabolic path-ways are involved in ATP oscillations during chondrogenesis

2 Materials and Methods

21 Cell Line The ATDC5 cell line was obtained from theRIKEN cell bank (Tsukuba)The cells were cultured in main-tenance medium consisting of a 1 1 mixture of Dulbeccorsquosmodified Eaglersquos medium and Hamrsquos F-12 medium (DMEM-F12) (Invitrogen) supplemented with 5 fetal bovine serum10mgmL human transferrin (Roche Molecular Biochemi-cals) and 3 times 10minus8M sodium selenite (Sigma-Aldrich) inpolystyrene dishes at 37∘C under 5 CO

2

For chondrogenicinduction whenATDC5 cellsmaintained in themaintenancemedium reached confluency the medium was replaced withthe chondrogenic medium supplemented with 10 120583gmLinsulin (Sigma-Aldrich)

22 Bioluminescence Monitoring Experiments Biolumines-cence reporter constructed by fusing the human actin pro-moter to a Phrixothrix hirtus luciferase gene (Toyobo) wasinserted into retrovirus vector (Clontech) ATDC5 cells weretransfected using retrovirus infection and then were selectedby puromycin The cells transfected stably with PACTIN-PxRewere seeded in 35mm dishes After replacing the mediumby a recording medium (DMEMF12 with 5 FBS 01mMluciferin (Wako) and 50mMHEPES-NaOH pH = 70) (time= 0 h) bioluminescence intensity was continuouslymeasuredat 37∘C in air using a dish-type luminescence Kronos(ATTO) for 1min at 30min intervals For examining theeffect of chemical compounds on ATP oscillations the cellswere treated with 01mM iodoacetate (Sigma-Aldrich) or5mM cyanide (Sigma-Aldrich) about 24 hours after PACTIN-PxRe oscillations initiated

23 Sample Preparation for CE-TOF-MS Analysis ATDC5cells cultured to 1times106 cells needed pretreatmentThe culturemedium was removed from the plates and 10mL of 5mannitol solution was added to wash the cells After 2mL of5 mannitol solution was added to wash the cells 1300120583Lof methanol containing the internal standards was addedCells were scraped from the plate and then cell lysate (sim1000120583L) was used in the analysis Then 400120583L of Milli-Qwater and 1000120583L of chloroform were added to the samplesthoroughly mixed and then centrifuged for 5min at 2300 gand 4∘CThe 750120583L of upper aqueous layer was centrifugallyfiltered through a Millipore 5 kDa cutoff filter to removeproteins The filtrate was lyophilized and suspended in 25120583Lof Milli-Q water We prepared three samples at peak andtrough of PACTIN-PxRe oscillations respectively and usedeach sample for independent CE-TOF-MS analysis

24 Instrumentation for CE-TOF-MS Analysis Themeasure-ment of extracted metabolites was performed by using a cap-illary electrophoresis- (CE-) connected ESI-TOF-MS systemwith electrophoresis buffer (Solution ID H3302-1021 HumanMetabolome Technologies Inc Tsuruoka Japan) CE-TOF-MS was carried out using an Agilent CE Capillary Elec-trophoresis System equipped with an Agilent 6210 Time ofFlight mass spectrometer Agilent 1100 isocratic HPLC pumpAgilent G1603A CE-MS adapter kit and Agilent G1607ACE-ESI-MS sprayer kit (Agilent Technologies WaldbronnGermany) The system was controlled by Agilent G2201AAChemStation software version B0301 for CE (Agilent Tech-nologies Waldbronn Germany)

25 Experimental Conditions for CE-TOF-MS Analysis Forcationic metabolites capillary electrophoreses were per-formed using a fused silica capillary The electrolyte was 1Mformic acid Methanol-water (50 vv) containing 05mMreserpine (the lock mass for exact mass measurements)was delivered as the sheath liquid For anionic metabolitesseparations were performed using a fused silica capillaryThe electrolyte was 50mM ammonium acetate (pH 85)The sheath liquid for anionic metabolites was used as theelectrolyteThe capillary was pretreatedwith preconditioningbuffer including 25mM ammonium acetate and 75mMsodium phosphate at pH 85 Pressure of 5mbar was appliedto inlet capillary during run to reduce the analysis time Forall analytical modes inner diameter and total length of capil-lary are 50mm and 80 cm respectively The applied voltagewas set at +30 kV and minus30 kV for cation and anion moderespectively Electrospray ionization TOF-MS was operatedin the positive ion mode (4 kV) the negative ion mode(35 kV) for cationic metabolites anionic metabolites andnucleotides respectively A flow rate of heated dry nitrogengas (heater temperature 300∘C) was maintained at 5 psigExact mass data were acquired over a 50ndash1000119898119911 rangeSignal peaks corresponding to isotopomers adduct ions andother product ions of known metabolites were excluded allsignal peaks potentially corresponding to authentic com-pounds were extracted and then their migration time (MT)was normalized using those of the internal standards There-after the alignment of peaks was performed according tothe 119898119911 values and normalized MT values Finally peakareas were normalized against those of the internal standardsMetSul and CSA for cations and anions respectively Theresultant relative area values were further normalized bysample amount Annotation tables were produced from CE-ESI-TOF-MSmeasurement of standard compounds andwerealigned with the datasets according to similar119898119911 values andnormalized MT values

3 Results and Discussion

31 Relationship of Glycolysis and Mitochondrial Respirationwith ATP Oscillations In this study insulin which stimulateschondrogenesis of ATDC5 cells [16] was used in inductionof ATP oscillations during chondrogenesis IntracellularATP level of ATDC5 cells was monitored in real time by


Iodoacetate

Insulin

0 24 48 72 96Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Cyanide

Insulin

0 24 48 72 96 120 144Time (hours)

0

50000

100000

150000

200000

250000

300000

RLU

min

(b)








Trough

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Peak

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(b)

















NADHIsocitric acid

PutrescineATP




Uric acidUMP





Glucuronic acid


Diphenylcarbazide
























Gly-AspCTP

Taurine

CystineGly-Gly








GDPTriethanolamine



3-Methylhistidine



AMPCreatine

PRPPGlu


BetaineCys


ValSDMA

Glucose 1-phosphate


TrpGly


GuaninePhe

dCTPMet

Cyclohexylamine


IleCDP

ADMAGln



HisAsp

UDPHeptanoic acid

LeuTyr




SerAzelaic acid

ArgBenzoic acid




AlaGMP

ProThr


AsnButyric acid


Adipic acidcGMP



Melamine

NADPH divalent



FAD divalent



NAD+

NADP+



Acetyl CoA divalent

120573-Ala

N5-Ethylglutamine

CoA divalent



minus2

minus15

minus1

minus05

0

05

1

15

2Fo

ld ch

ange

(log

2)

Propionic acid

Phenaceturic acid













4 Conclusion



TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Iodoacetate

Insulin

0 24 48 72 96Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Cyanide

Insulin

0 24 48 72 96 120 144Time (hours)

0

50000

100000

150000

200000

250000

300000

RLU

min

(b)








Trough

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Peak

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(b)

















NADHIsocitric acid

PutrescineATP




Uric acidUMP





Glucuronic acid


Diphenylcarbazide
























Gly-AspCTP

Taurine

CystineGly-Gly








GDPTriethanolamine



3-Methylhistidine



AMPCreatine

PRPPGlu


BetaineCys


ValSDMA

Glucose 1-phosphate


TrpGly


GuaninePhe

dCTPMet

Cyclohexylamine


IleCDP

ADMAGln



HisAsp

UDPHeptanoic acid

LeuTyr




SerAzelaic acid

ArgBenzoic acid




AlaGMP

ProThr


AsnButyric acid


Adipic acidcGMP



Melamine

NADPH divalent



FAD divalent



NAD+

NADP+



Acetyl CoA divalent

120573-Ala

N5-Ethylglutamine

CoA divalent



minus2

minus15

minus1

minus05

0

05

1

15

2Fo

ld ch

ange

(log

2)

Propionic acid

Phenaceturic acid













4 Conclusion



TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Trough

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(a)

Peak

0 20 40 60 80 100Time (hours)

0

50000

100000

150000

200000

250000

RLU

min

(b)

















NADHIsocitric acid

PutrescineATP




Uric acidUMP





Glucuronic acid


Diphenylcarbazide
























Gly-AspCTP

Taurine

CystineGly-Gly








GDPTriethanolamine



3-Methylhistidine



AMPCreatine

PRPPGlu


BetaineCys


ValSDMA

Glucose 1-phosphate


TrpGly


GuaninePhe

dCTPMet

Cyclohexylamine


IleCDP

ADMAGln



HisAsp

UDPHeptanoic acid

LeuTyr




SerAzelaic acid

ArgBenzoic acid




AlaGMP

ProThr


AsnButyric acid


Adipic acidcGMP



Melamine

NADPH divalent



FAD divalent



NAD+

NADP+



Acetyl CoA divalent

120573-Ala

N5-Ethylglutamine

CoA divalent



minus2

minus15

minus1

minus05

0

05

1

15

2Fo

ld ch

ange

(log

2)

Propionic acid

Phenaceturic acid













4 Conclusion



TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








NADHIsocitric acid

PutrescineATP




Uric acidUMP





Glucuronic acid


Diphenylcarbazide
























Gly-AspCTP

Taurine

CystineGly-Gly








GDPTriethanolamine



3-Methylhistidine



AMPCreatine

PRPPGlu


BetaineCys


ValSDMA

Glucose 1-phosphate


TrpGly


GuaninePhe

dCTPMet

Cyclohexylamine


IleCDP

ADMAGln



HisAsp

UDPHeptanoic acid

LeuTyr




SerAzelaic acid

ArgBenzoic acid




AlaGMP

ProThr


AsnButyric acid


Adipic acidcGMP



Melamine

NADPH divalent



FAD divalent



NAD+

NADP+



Acetyl CoA divalent

120573-Ala

N5-Ethylglutamine

CoA divalent



minus2

minus15

minus1

minus05

0

05

1

15

2Fo

ld ch

ange

(log

2)

Propionic acid

Phenaceturic acid













4 Conclusion



TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












4 Conclusion



TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TCA cycle

Glycolysis


ATP

Uronic acidpathway



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Metabolomic Analysis of Differential Changes in Metabolites during ... · 2019. 7. 31. · and uronic acid pathway oscillate in phase with ATP oscillations, while