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Research ArticleRelationship of Baseline Hemoglobin Level with SerumFerritin Postphlebotomy Hemoglobin Changes andPhlebotomy Requirements among HFE C282Y Homozygotes

Seyed Ali Mousavi Faiza Mahmood Astrid AandahlTeresa Risopatron Knutsen and Abid Hussain Llohn

Department of Immunology and Transfusion Medicine Akershus University Hospital University of Oslo Norway

Correspondence should be addressed to Abid Hussain Llohn abidlohnahusno

Received 18 June 2015 Accepted 17 August 2015

Academic Editor Kim Bridle

Copyright copy 2015 Seyed Ali Mousavi 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

Objectives We aimed to examine whether baseline hemoglobin levels in C282Y-homozygous patients are related to the degree ofserum ferritin (SF) elevation and whether patients with different baseline hemoglobin have different phlebotomy requirementsMethods A total of 196 patients (124 males and 72 females) who had undergone therapeutic phlebotomy and had SF and bothpre- and posttreatment hemoglobin values were included in the study Results Bivariate correlation analysis suggested that baselineSF explains approximately 6 to 7 of the variation in baseline hemoglobin The results also showed that males who had higher(ge150 gL) baseline hemoglobin levels had a significantly greater reduction in their posttreatment hemoglobin despite requiringfewer phlebotomies to achieve iron depletion than those who had lower (lt150 gL) baseline hemoglobin regardless of whetherbaseline SF was below or above 1000 120583gLThere were no significant differences between hemoglobin subgroups regarding baselineand treatment characteristics except for transferrin saturation betweenmale subgroupswith SF above 1000 120583gL Similar differenceswere observed when females with higher (ge138 gL) baseline hemoglobin were compared with those with lower (lt138 gL) baselinehemoglobin Conclusion Dividing C282Y-homozygous patients into just two subgroups according to the degree of baseline SFelevation may obscure important subgroup variations

1 Introduction

Hemochromatosis due to mutations in the hemochromatosisFe (HFE) gene is the most common autosomal recessiveform of inherited iron-overload disorder among peopleoriginating from northwestern Europe This form of hered-itary hemochromatosis (HH) is due to excessive intestinaliron absorption As there is no regulated pathway for ironexcretion in humans this leads to accumulation of iron inparenchymal cells of different tissues particularly the liverpancreas and heart [1 2] HFE-associated HH is character-ized biochemically by elevated transferrin saturation (TS)a measure of iron availability for erythropoiesis and highlevels of serum ferritin (SF) an iron storage protein reflectingbody iron stores [3] Two missense mutations C282Y (sub-stitution of tyrosine for cysteine at position 282) and H63D

(substitution of aspartic acid for histidine at position 63) arethe most frequent (sim8 and sim15 resp) among individualsof northern European ancestry [4] However the majorityof HFE-associated HH patients (gt90) are homozygous forthe C282Y variant about 6 are C282YH63D compoundheterozygotes and less than 1 of reported cases are H63Dhomozygotes [1 5 6]

Therapeutic phlebotomy when this is feasible has beenthe preferred treatment for clinical management of thispatient population Depletion of the excess iron by phle-botomy consists of removing one unit of blood (approxi-mately 450mL + 30mL for laboratory measurements) untilSF levels are lt50120583gL and when initiated early will preventsevere iron overload and iron-related disease such as livercirrhosis and cardiovascular problems [7]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 241784 9 pageshttpdxdoiorg1011552015241784

2 BioMed Research International

The present study was prompted by our observationthat some C282Y-homozygous patients with higher baselinehemoglobin had lower posttreatment hemoglobin levels inspite of requiring fewer phlebotomies than patients withlower baseline hemoglobin However this informal observa-tionwas not directly investigated at our institutionMoreoverto our knowledge whether baseline hemoglobin level variesdepending on the degree of SF elevation has not been prop-erly addressed in the literature about C282Y-homozygouspatients In this study we first examined whether there is arelationship between baseline hemoglobin and the degree ofSF elevation among C282Y-homozygous patients We thenevaluated the effect of therapeutic phlebotomy on changes inposttreatment hemoglobin levels among subgroups of C282Yhomozygotes

2 Methods

21 Study Subjects This was a retrospective study con-ducted at Akershus University Hospital blood bank in Nor-way In addition to collecting blood for transfusion theblood bank provides services for individuals that requiretherapeutic phlebotomy After obtaining approval from theInstitutional Review Board patient databases were used tocollect relevant data of HH patients who had undergonetherapeutic phlebotomyduring 1996 to 2013 For this analysispatients were included if they (1) had known gender (2)had available data on age at baseline (3) had undergonetesting for HFE mutations and were confirmed to be eitherhomozygote (C282YC282Y) or compound heterozygote(C282YH63D) (4) had elevated baseline SF (gt200120583gL infemales and gt300120583gL in males) and (5) had recordedvenous hemoglobin values at baseline and at the last phle-botomy when their SF was below 50 120583gL (referred to asposttreatment hemoglobin)

Genotype data were available on 254 patients Of these32 (126) had incomplete baseline data and were thereforeexcluded Five C282Y-homozygous males were excludedfrom analysis as a result of (1) alcohol abuse (119899 = 2) (2) cir-rhosis (119899 = 1) and (3) blood donation (119899 = 2 prior blooddonors) because these conditions are capable of affecting SFlevels Three H63D-homozygous males were also excludeddue to uncertainty aboutwhether theywere trueHH subjectsleaving 214 patients for this analysis Patients with low SF(females between 200 and 1000 120583gL males between 300and 1000 120583gL) and high (above 1000 120583gL) SF levels wereincluded in order to increase the statistical power for demon-strating the presence (or lack) of an association Transferrin-iron saturation could not be calculated for all of these patientsbecause of missing transferrin or serum iron data for 14(30214) of themNo patients were excluded from the analysisdue to missing TS values because this would yield smallsample sizes Transferrin saturation was calculated as 4 times(serum iron [in 120583molL])(serum transferrin [in gL]) or bydividing the serum iron by the total iron-binding capacity andmultiplying by 100

22 Statistical Analysis Unpaired 119905-test was used to com-pare means between continuous variables and Pearsonrsquos

chi-square or Fisherrsquos exact test to compare differencesamong group proportions Since serum ferritin values andthe number of phlebotomies were not normally distributedmean values were compared after logarithmic transforma-tion Paired 119905-test and Wilcoxon signed-rank test were usedcomparing baseline and posttreatment hemoglobin levelsPearsonrsquos correlation analysis was performed to assess therelationship between baseline levels of hemoglobin and otherbaseline characteristics and those significantly associatedwith baseline hemoglobin were included in multiple linearstepwise regression analysis Missing TS data were imputedby the mean of the two straddling values In all cases a 119901value oflt005was considered statistically significant Becauseof gender-specific differences in baseline characteristics sep-arate analyses were performed for male and female C282Yhomozygotes Statistical analyses were performed using SPSSsoftware version 21 (Chicago IL USA)

3 Results

31 Baseline Characteristics of Patients The final study pop-ulation consisted of 214 patients of these 142 (663) weremales and 72 (337) were females Baseline characteristicsof patients in each genotype group according to gender aresummarized in Table 1 The majority of patients (119899 = 196916) were homozygous for C282Y as expected Of these124 (633) were males and 72 (367) were females AmongC282Y-homozygous patients males on average were 73 yearsyounger than females (119901 lt 00001) but they had significantlyhigher mean SF (119901 lt 00001) and slightly higher mean TSlevels (119901 = 017) than females Compound-heterozygous(C282YH63D) males had significantly lower SF (119901 = 0012)and TS (119901 lt 00001) levels than C282Y-homozygous males

32 Differences in Mean Baseline Hemoglobin Levels acrossGenotype Groups Most patients had baseline hemoglobinlevels that fell within the reference range for hemoglobin(Table 1) Among C282Y-homozygous patients males hadsignificantly higher mean hemoglobin values than females(119901 lt 0001) as might be expected because of a physiologi-cally higher hemoglobin concentration in men Compound-heterozygous males had a mean hemoglobin value that wasslightly higher than that of C282Y-homozygousmales but thedifference was not statistically significant

33 Relationship between Baseline Hemoglobin and SF LevelsFurther analysis of the data which was limited to C282Y-homozygous patients revealed that patients with lower SFhad higher baseline hemoglobin These patients were there-fore categorized as having low (lt1000120583gL mean 658 plusmn170 120583gL range 316ndash972 120583gL) or high (gt1000120583gL mean1680 plusmn 856 120583gL range 1030ndash4000 120583gL) SF in order to com-pare their baseline hemoglobin levels Males with SF levelslt1000 120583gL (685 85124) had higher mean hemoglobinconcentration than those with SF levels gt1000120583gL (1519 plusmn84 versus 1471 plusmn 126 120583gL 119901 = 0014) Similarlyfemales with SF lt1000 120583gL (847 6172) had highermean hemoglobin concentration than those with SF levels

BioMed Research International 3

Table 1 Baseline characteristics of the patients (119899 = 214) according to HFE genotype

Genotype groups Agelowast SF (120583gL) TS () Hb (gL)Males (119899 = 142)

C282YC282Y (119899 = 124) 473 plusmn 130 979 plusmn 688 782 plusmn 155 1504 plusmn 101(20ndash84) (316ndash4000) (39ndash100) (124ndash172)

C282YH63D (119899 = 18) 510 plusmn 95 632 plusmn 179dagger 592 plusmn 154dagger 1532 plusmn 67(29ndash69) (326ndash967) (31ndash88) (143ndash169)

Reference ranges [15ndash300] [le50] [130ndash165]Females (119899 = 72)

C282YC282Y 546 plusmn 103Dagger 646 plusmn 496Dagger 745 plusmn 168 1382 plusmn 81Dagger

(31ndash71) (210ndash3100) (29ndash100) (116ndash156)Reference ranges [15ndash200] [le45] [115ndash155]

Results are mean plusmn SD values in parentheses are range minimumndashmaximumlowastAge at the start of treatmentdagger

119901 lt 0001 comparison between male C282YH63D and male C282YC282YDagger

119901 lt 0001 comparison between female C282YC282Y and male C282YC282YHb hemoglobin SF serum ferritin TS transferrin saturation

Table 2 Association between baseline hemoglobin and other baseline characteristics in C282Y-homozygous males (119899 = 124) and females(119899 = 72)

Variables Males FemalesAge Log-SF TS () Log-PH Age Log-SF TS () Log-PH

Base-Hb minus029dagger minus025dagger minus023dagger minus037Dagger 008 minus027dagger 002 minus027dagger

Age 029dagger 001 046Dagger 007 021 000Log-SF 045dagger 071Dagger 034dagger 072Dagger

TS () minus036Dagger 029dagger

Base-Hb baseline hemoglobin SF serum ferritin Log-PH log number of phlebotomiesdagger

119901 lt 005Dagger

119901 lt 001

gt1000 120583gL (1390 plusmn 81 versus 1340 plusmn 66 120583gL) althoughthe difference was of borderline significance (119901 = 0059)(see Table 3) The unadjusted odds ratio (OR) of having SFgt1000 120583gL for males versus females was 254 (95 confi-dence interval [CI] 12ndash54 119901 = 0012)

Correlation analysis (Table 2) showed that baselinehemoglobin in C282Y-homozygous males was negativelycorrelated with age (119903 = minus029 119901 = 0001) log-SF (119903 = minus025119901 = 0006) and TS (119903 = minus023 119901 = 001) indicating thatincreased values of these variables are associated with lowerlevels of baseline hemoglobinWhen significant determinantsof baseline hemoglobin (as dependent variable) were exam-ined simultaneously in amultiple linear regressionmodel age(120573 minus029 119901 = 0001) and TS (120573 minus023 119901 = 0007) remainedsignificant but log-SF did not retain statistical significanceThe overall 1198772 for the model was 0123 (119901 = 0001) Howeverin a regression model that also included the age by TSinteraction age and TS did not remain independently sig-nificant (120573 039 119901 lt 00001 for the interaction term) Theoverall 1198772 for the model was 0144 (119901 lt 00001) indicatingthat approximately 144 of the variability in baselinehemoglobin values was accounted for by the effect of com-bined variation of age and TS In females only log-SF wascorrelated with baseline hemoglobin (119903 = minus027 119901 lt 001)

indicating that baseline SFmay explain approximately 73 ofthe variability in baseline hemoglobin values

34 Effect of Therapeutic Phlebotomy on Posttreatment Hemo-globin Levels Table 3 shows mean (95 CI) decreases andpercentage changes in posttreatment hemoglobin from base-line among male and female patients Among C282Y-homo-zygous males the mean hemoglobin decreased by 60 gL(119901 lt 00001 paired 119905-test) Compared with baseline valuesposttreatment hemoglobin levels decreased in 93 patients(75) increased in 30 patients (234) and remained thesame in one patient Similar results were also observedamong C282Y-homozygous females the mean hemoglobindecreased by 51 gL (119901 lt 00001 paired 119905-test) posttreat-ment hemoglobin levels decreased in 52 patients (722)increased in 17 patients (236) and remained unchanged in3 patients Among compound-heterozygous males the meanhemoglobin decreased by 99 gL (119901 lt 00001 paired 119905-test)one patient (48) had a rise in posttreatment hemoglobinand 2 patients had no change in their hemoglobin levels

To further examine the relationship between baselinehemoglobin serum ferritin and changes in posttreatmenthemoglobin levels C282Y-homozygous patients in eachof the two SF categories were divided into those with


Table 3 Comparisons of mean plusmn SD decreases (95 confidence interval) percentage changes in posttreatment hemoglobin from baselineand the mean plusmn SD number of phlebotomies among hemoglobin subgroups of C282Y homozygotes and male genotype groups

Base-Hb Post-Hb MD (95 CI) change Post lt Base Nr-PHgLdagger gLdagger gL gL 119873 () Mean plusmn SD

MalesC282YC282Y 1504 plusmn 101 1444 plusmn 110 minus60 (minus77 minus44) minus40 93124 (75) 217 plusmn 148

SF lt1000120583gL 1519 plusmn 84 1453 plusmn 107 minus66 (minus87 minus45) minus42 6585 (76) 160 plusmn 68SF gt1000120583gL 1471 plusmn 126 1422 plusmn 114 minus48 (minus77 minus19) minus33 2839 (72) 340 plusmn 194119901 value 0014 031 lt0001

Low SFhigh Hb 1574 plusmn 47 1490 plusmn 109 minus84 (minus114 minus55) minus53 4352 (83) 143 plusmn 51Low SFlow Hb 1433 plusmn 49 1396 plusmn 75 minus37 (minus62 minus12) minus26 2233 (67) 188 plusmn 81119901 value lt0001 0038 0038

High SFhigh Hb 1603 plusmn 56 1512 plusmn 94 minus91 (minus14 minus43) minus57 1316 (81) 267 plusmn 113High SFlow Hb 1378 plusmn 62 1360 plusmn 81 minus18 (minus51 minus15) minus13 1523 (65) 391 plusmn 222119901 value lt0001 0014 006

C282YH63D 1532 plusmn 67 1434 plusmn 96 minus99 (minus15 minus49) minus65 1518 (83) 112 plusmn 49Females

C282YC282Y 1382 plusmn 81 1331 plusmn 91 minus51 (minus73 minus30) minus37 5272 (72) 154 plusmn 109

SF lt1000120583gL 1390 plusmn 81 1332 plusmn 91 minus58 (minus80 minus36) minus42 4761 (77) 123 plusmn 63SF gt1000120583gL 1340 plusmn 66 1325 plusmn 98 minus15 (minus85 56) minus11 511 (45) 327 plusmn 147119901 value 0059 016 lt0001

Low SFhigh Hb 1443 plusmn 51 1363 plusmn 77 minus80 (minus105 minus55) minus55 3135 (89) 125 plusmn 75Low SFlow Hb 1312 plusmn 53 1289 plusmn 92 minus28 (minus67 minus10) minus21 1626 (62) 120 plusmn 41119901 value 0020 064

High SFhigh Hb 1420 plusmn 20 1367 plusmn 32 minus53 (minus91 minus15) minus37 33 (100) 207 plusmn 103High SFlow Hb 1310 plusmn 49 1310 plusmn 112 00 00 28 (25) 373 plusmn 139119901 value 0005 049 0036

daggerResults are mean plusmn SDBase-Hb baseline hemoglobin Post-Hb posttreatment hemoglobin MD mean difference between posttreatment hemoglobin and baseline hemoglobin Postlt Base number () of patients with decreased posttreatment hemoglobin compared with baseline values Nr-PH number of phlebotomiesThepercentage change is expressed as difference between posttreatment hemoglobin level and baseline hemoglobin level divided by baseline hemoglobin leveltimes100 and was used as a continuous variable for calculations of the statistical differences between subgroups

hemoglobin lt150 and ge150 gL or lt138 and ge138 gL basedon mean baseline hemoglobin values for male and femalepatients respectively The results of this subanalysis areshown in Table 3

Among the subgroups with higher (ge150 gL) hemoglo-bin the percentage change in hemoglobin from baseline formales with low (lt1000 120583gL) SF was minus53 and for those withhigh (gt1000120583gL) SF it was minus57 compared respectivelywith minus26 and minus13 in the two subgroups with lower(lt150 gL) hemoglobin (119901 = 0038 and 119901 = 0014 resp)Among the subgroups with high (gt1000 120583gL) SF males withhigher (ge150 gL) hemoglobin had lower TS than those withlower (lt150 gL) hemoglobin (809 plusmn 12 versus 919 plusmn 66

119901 = 0002) but no other differences between SF subgroupswere significant (Table 4)

A similar pattern of results was observed among femalesthe percentage change for females with higher (ge138)hemoglobin and low (lt1000120583gL) SFwasminus55 significantlydifferent from minus21 among those with lower (lt138 gL)hemoglobin and low (lt1000120583gL) SF (119901 = 0020) The per-centage change for females with higher (ge138 gL) hemo-globin and high (gt1000120583gL) SF was minus37 but there wasbasically no difference between baseline and posttreatmenthemoglobin for females with lower (lt138 gL) hemoglobinand high (gt1000 120583gL) SF except for the wider standarddeviation in their posttreatment hemoglobin values (Table 3)


Table 4 Baseline and treatment characteristics of hemoglobin subgroups of C282Y homozygotes by gender

Subgroups Age SF (120583gL) TS () Intervallowast Post-SFlowastlowast

MalesLow SFhigh Hb 434 plusmn 133 640 plusmn 169 741 plusmn 152 157 plusmn 612 421 plusmn 523Low SFlow Hb 472 plusmn 106 685 plusmn 167 725 plusmn 145 161 plusmn 410 423 plusmn 433119901 value 018 025 068 071 083

High SFhigh Hb 505 plusmn 137 1728 plusmn 931 809 plusmn 129 136 plusmn 408 420 plusmn 376High SFlow Hb 539 plusmn 122 1647 plusmn 819 919 plusmn 66 144 plusmn 341 417 plusmn 432119901 value 043 080 0002 052 073

FemalesLow SFhigh Hb 549 plusmn 102 466 plusmn 164 757 plusmn 164 158 plusmn 519 419 plusmn 596Low SFlow Hb 535 plusmn 114 480 plusmn 161 686 plusmn 150 170 plusmn 503 429 plusmn 451119901 value 072 062 031 039 048


Results are mean plusmn SDlowastInterval the time interval between the penultimate phlebotomy and the last phlebotomylowastlowastPost-SF posttreatment SF

There were no statistically significant differences in baselinecharacteristics between the two subgroups within each SFcategory (Table 4)

There was a higher proportion of males with baselinehemoglobin ge150 gL among patients with SF lt1000120583gLthan among patients with SF gt1000 120583gL (61 versus 41OR 22 95 CI 099ndash47 119901 = 0049) the correspondingproportions for females with baseline hemoglobin ge138 gLwithin the low and high SF categories were 57 and 27respectively (OR 36 95 CI 09ndash148 119901 = 010 by Fisherrsquosexact test)

The results from this subanalysis indicate that C282Y-homozygous patients with higher baseline hemoglobin hada larger decrease in their posttreatment hemoglobin levelsthan those with lower baseline hemoglobin levels regardlessof whether baseline SF was above or below 1000120583gL Thesignificant higher decrease in posttreatment hemoglobin lev-els among patients with higher baseline hemoglobin relativeto those with lower hemoglobin levels was not becausethey had higher phlebotomy requirements Among maleswithin both SF categories the mean number of phlebotomiesrequired to achieve iron depletion among the subgroups withhigher (ge150 gL) baseline hemoglobin was lower (143 plusmn52 [range 6ndash35 phlebotomies] and 267 plusmn 113 [range 7ndash52phlebotomies] resp for low and high SF categories) than thesubgroups with lower (lt150 gL) baseline hemoglobin (188plusmn81 [range 8ndash35 phlebotomies] and 391 plusmn 222 [range 14ndash94phlebotomies] 119901 = 0038 and 119901 = 006 resp for low andhigh SF categories) Among females within the low SF cate-gory the mean number of phlebotomies required to achieveiron depletion was similar between the two hemoglobinsubgroups (119901 = 064) whereas among those within the highSF category the mean number of phlebotomies requiredto achieve iron depletion among the subgroup with higher

(ge138 gL) baseline hemoglobin was significantly lower thanthemean among the subgroup with lower (lt138 gL) baselinehemoglobin (207 plusmn 103 [range 12ndash32 phlebotomies] versus373 plusmn 139 [range 23ndash68 phlebotomies] 119901 = 0036)

To ensure that subgroup differences in posttreatmenthemoglobin changes were not due to differences in treatmentintensity at the end of therapeutic phlebotomy and to controlfor the degree of phlebotomy-induced iron depletion wecompared the average time interval between the penultimatephlebotomy and the last phlebotomy and themean posttreat-ment SF value for each subgroup

The mean time intervals between penultimate phle-botomy and the last phlebotomy are shown in Table 4Among males the mean time intervals for subgroups withhigher (ge150 gL) baseline haemoglobin were somewhatthough not significantly shorter (157plusmn612 [range 7ndash34 days]and 136 plusmn 408 [range 7ndash21 days] resp for low and highSF categories) than those of subgroups with lower (lt150 gL)baseline haemoglobin (161 plusmn 410 [range 7ndash28 days] and144 plusmn 341 [range 7ndash21 days] resp for low and high SFcategories) The mean time interval for the female subgroupwith SF lt1000120583gL and with higher (ge138 gL) baselinehaemoglobin was slightly shorter than that of the femalesubgroup with SF lt1000120583gL and with lower (lt138 gL)baseline haemoglobin (158 plusmn 519 [range 7ndash28 days] versus170 plusmn 503 [range 7ndash28 days]) The female subgroup with SFgt1000 120583gL and with higher (ge138 gL) baseline haemoglobinhad a slightly longer mean interval than that of the femalesubgroup with SF gt1000120583gL and with lower (lt138 gL)baseline haemoglobin (187 plusmn 404 [range 14ndash21 days] versus161 plusmn 426 [range 10ndash21 days]) These differences were notsignificant (Table 4)

There were no statistically significant differences in thelevels of posttreatment SF between the two hemoglobin


subgroups within each SF category (Table 4) No patients ineither hemoglobin subgroup within each SF category haddeveloped iron deficiency (SF lt20120583gL) at the last phle-botomy Among males with SF lt1000 120583gL posttreatment SFlevels ranged from 27 to 50 120583gL in the subgroup with higher(ge150 gL) baseline haemoglobin and from 32 to 49 120583gL inthe subgroup with lower (lt150 gL) baseline haemoglobinwhereas they ranged from 35 to 48 120583gL in both subgroupswith SF gt1000 120583gL Among females with SF lt1000120583gLposttreatment SF levels ranged from 29 to 52 120583gL in the sub-group with higher (ge138 gL) baseline haemoglobin and from31 to 49 120583gL in the subgroup with lower (lt138 gL) baselinehaemoglobin whereas among those with SF gt1000120583gLthey ranged from 41 to 45 120583gL in the subgroup with higher(ge138 gL) baseline haemoglobin and from 38 to 48 in thesubgroup with lower (lt138 gL) baseline haemoglobin

The time interval between the penultimate phlebotomyand the last phlebotomy for compound-heterozygous malescombined was an average of 162plusmn76 days (range 7ndash28 days)and their posttreatment SF levels averaged 424 plusmn 58 120583gL(range 25ndash48120583gL) A comparison between these males andC282Y-homozygous males with comparable SF levels (seeTables 1 and 4) indicates that compound-heterozygous maleshad larger decrease in posttreatment hemoglobin levels inspite of requiring on average fewer phlebotomies thanC282Y homozygotes with similar baseline SF levels (Table 3)

35 Determinants of Phlebotomy Requirements Bivariatecorrelation of log number of phlebotomies with baselinevariables in C282Y-homozygous patients is shown in Table 2To examine whether baseline hemoglobin independentlypredicts the number of phlebotomies required to induce irondepletion multiple linear regression analysis was performedadjusting for age (only in males) log-SF and TS In malesthe multiple regression analysis showed that Log-SF (120573 062119901 lt 00001) baseline hemoglobin (120573minus025119901 lt 00001) andage (120573 017 119901 = 0004) were significantly associated with thelog number of phlebotomies BaselineTSwas not a significantpredictor however The overall 1198772 for the model was 0636(119901 lt 00001) The results indicated that an increase in SF of42120583gL was associated with one additional unit of blood tobe removed to achieve iron depletion whereas a decreasein baseline hemoglobin of 045 gL was associated with adecrease of phlebotomy requirements of one unit of bloodThe regressionmodel predicting the number of phlebotomiesin females showed that log-SF was significantly and positively(120573 062 119901 lt 00001) associated with the log number of phle-botomies but baseline hemoglobin and TS did not remainThe overall 1198772 for the model was 0528 (119901 lt 00001) Infemales an increase in SF of 33 120583gL was associated withone additional unit of blood to be removed to achieve irondepletion

4 Discussion

In this retrospective study among a cohort of C282Y-homo-zygous patients we examined the relation between baselineSF and hemoglobin In addition we evaluated the effect of

therapeutic phlebotomy on changes in posttreatment hemo-globin levels among subgroups with higher and lower base-line hemoglobin Our results showed that in C282Y-homo-zygous patients lower baseline serum ferritin was associatedwith higher baseline hemoglobin levels In bivariate analysisof C282Y-homozygous males there was weak but significantinverse relationship between baseline hemoglobin and log-SFage and TS However the association between hemoglobinand log-SF did not remain in a multiple regression modeladjusting for age and TS The adjusted model described thatmore than 85 of the variability in baseline hemoglobin wasaccounted for by variables not included in the model InC282Y-homozygous females baseline hemoglobin correlatednegatively only with log-SF explaining about 73 of the vari-ability in baseline hemoglobin Taken together these resultssuggest that differences in serum ironmarkersrsquo baseline levelsare inadequate to explain much of the variability in baselinehemoglobin Or in other words we do not understand a greatdeal about the determinants of baseline hemoglobin in thispatient population perhaps because the level of hemoglobinproduction in these patients is strongly influenced by otherfactors such as genetic modifiers (see below)

Further subanalysis showed that C282Y-homozygousmales who had higher baseline hemoglobin (ge150 gL) hadsignificantly higher decrease in posttreatment hemoglobinlevels compared to those with lower baseline hemoglobin(lt150 gL) regardless of baseline SF category Similar dif-ferences were observed when comparing high (ge138 gL)and low (lt138 gL) hemoglobin subgroups among C282Y-homozygous females Comparisons of baseline variables andtreatment characteristics between hemoglobin subgroupswithin each SF category did not indicate any significant groupdifferences except for the mean values of baseline TS withinthe group with high SF (gt1000120583gL)

Several studies in subjects with HFE gene mutationshave found significant increases in red blood cell parametersincluding hemoglobin andmean corpuscular volume as com-pared with normal controls [8ndash11] The elevated hemoglobinobserved in C282Y-homozygous subjects has been ascribedto increased uptake of transferrin-bound iron by erythroidprecursors possibly through upregulation of transferrinreceptor 1 (TfR1) [8] Assuming mean hemoglobin concen-trations in normal men and women are 150 gL and 140 gLrespectively that is 20 gL higher than the cutoff level foranemia [12] our results show that compound-heterozygousmales as a whole had a mean hemoglobin level that wassim32 gL above the normal mean value C282Y-homozygousmales as a whole had a mean hemoglobin level that was onlyslightly higher than the average whereas C282Y-homozygousfemales as a whole had a mean hemoglobin level that waslower than the average However about 54 (67124) ofC282Y-homozygous males in our patient population hadhemoglobin levels that were 75 to 103 gL above the normalmean value for men compared with about 43 (3072)females who had hemoglobin levels that were 2 to 43 gLabove the normal mean value for women

The retrospective nature of the study does not allow usto explain the cause(s) of differences in baseline hemoglobinlevels at a given SF among C282Y-homozygous patients


or to definitively determine why those with higher base-line hemoglobin have greater decrease in posttreatmenthemoglobin despite requiring lower phlebotomy require-ments to achieve iron depletion However we can speculateon some potential explanations for these observations Ironoverload associated with HFE mutations has been shown toresult from inadequate production (relative to the degree ofiron overload) of the hepatic peptide hormone hepcidin [1314] Circulating hepcidin is the central regulator of systemiciron metabolism by directly binding and downregulatingthe cell surface iron transporter ferroportin-1 (FPN1) andreducinginhibiting iron efflux from duodenal enterocytesreticuloendothelial macrophages and hepatocytes [15] Inhepatocytes the HFE protein appears to be part of an iron-sensing complex which has a key role in regulating hepcidinexpression This complex also involves TfR2 bone mor-phogenetic protein 6 (BMP-6) the BMP receptor complexcomposed of activin-like kinase receptor 2 (ALK2) and ALK3and its intracellular signaling pathway (Smad proteins) andhemojuvelin which is a coreceptor for BMP-6 signaling [16ndash21]

The normal HFE gene encodes a transmembrane glyco-protein that has interactionwith 1205732-microglobulin and intra-cellular TfR1 which is necessary for its stabilitytraffickingthrough the endoplasmic reticulum-Golgi pathway and cellsurface localization It is also a binding partner of TfR1 onthe cell surface lowering the apparent affinity of the receptorfor diferric transferrin (Fe

2-Tf) and thereby reducing the

amount of iron entering cells The presence of the C282Ymutation has been shown to render the HFE protein unableto interact with 1205732-microglobulinTfR1 thereby preventingits cell surface expression whereas the H63D variant inter-acts normally with TfR1 but reduces the affinity of TfR1for diferric transferrin to a lesser extent [22ndash24] Little isknown regarding the role of HFE protein in the regulationof hepcidin Some authors have proposed that increasedtransferrin saturation leads to dissociation of HFE proteinfrom TfR1 which subsequently forms a complex with TfR2which in turn upregulates hepatic hepcidin expression [25]More recent evidence suggests however that HFE proteinmay not bind to TfR2 [26 27] A recent study [28] suggestedthat the mechanism by which normal HFE protein regulateshepcidin expression is by binding intracellular ALK3 andmediating its cell surface localization Presumably C282Yinteracts normally with ALK3 but prevents it from reachingthe cell surface whereas interaction of H63D with ALK3leads to increased ALK3 ubiquitination with the subsequentproteasomal degradation

One hypothesis consistent with our findings is that dif-ferences in baseline levels of hemoglobin reflect differentialexpression of hepcidin It is possible for example that aproportion of untreated C282Y homozygotes have higherbaseline hemoglobin because they produce more hepcidinthan those with lower baseline hemoglobin Higher circu-lating hepcidin is expected to lead to a greater decrease inthe amount of FPN1 on erythroid precursors and thereby toincreased iron retention in cells which in turn will lead toan increase in the availability of iron to enhance hemoglobinsynthesis Subjects with lower circulating hepcidin levels

are expected to have higher transferrin saturation due toincreased iron efflux from erythroid precursors therebyreducing their ability to use transferrin-bound iron as effi-ciently as those with higher circulating hepcidin levelspotentially explaining the observation that the patient groupswith high SF (gt1000120583gL) and with lower mean hemoglobinvalues had higher mean values of TS (see Table 4) Thesefindings agree with a prior study by Brandao et al [29] of45 C282Y-homozygous patients in which mean hemoglobinconcentrations were significantly lower in patients with highTS (gt70) than in patients with TS between 50 and 70In support of this interpretation iron export from erythroidprecursors and enterocytes is shown to be mediated byFPN1B a product of a splice variant of the transcript forFPN1 that lacks an iron responsive element (IRE) in its 51015840untranslated region and thus is not subject to regulationby intracellular iron making hepcidin the main negativeregulator of FPN1 expression on erythroid precursors [30]

The decreases in posttreatment hemoglobin in subgroupswith higher baseline hemoglobin were significantly greaterthan in subgroups with lower baseline hemoglobin Thesedifferences could not be explained by differences in thenumber of phlebotomies needed to achieve iron depletionbecause the subgroups with higher baseline hemoglobin onaverage needed fewer phlebotomies to achieve iron depletionthan the subgroups with lower baseline hemoglobin Norcould the differences be attributed to other baseline ortreatment characteristics such as treatment intensity at theend of therapeutic phlebotomy or the degree of posttreatmentSF reduction (Table 4) Several studies in HFE-related HHpatients have shown that phlebotomy therapy leads to afurther decrease in the circulating hepcidin levels or to upreg-ulation of divalent metal transport 1 (DMT1) leading toincreased intestinal iron absorption and increased ironrelease from stores [31 32] Thus it is conceivable that theextent to which phlebotomy-induced iron depletion influ-ences hepcidin expression andor the amount of iron abndashsorbed may contribute to the observed differences in post-treatment hemoglobin levels in treated patients

The finding that C282Y-homozygous patients with highermean baseline hemoglobin had lower phlebotomy require-ments to achieve iron depletion than those with lower meanbaseline hemoglobin probably reflects the relative amountof iron stored as ferritin and hemosiderin rather than aneffect of baseline hemoglobin per se The release of iron fromhemosiderin has been shown to be slower than that of ferritin[33] probably due to the time required for degradation ofhemosiderin The distribution of iron among parenchymalcells in different tissuesmay also affect ironmobilization fromstorage sites

In the present study we compared the importance ofbaseline hemoglobin level as a predictor of phlebotomyrequirement for reduction of iron overloadwith serummark-ers of iron Multiple linear regression analysis performed formale patients identified baseline SF as a strong predictorof phlebotomy requirement whereas baseline hemoglobinwas found as a poor albeit independent predictor Baselinehemoglobin was however not an independent predictor


of phlebotomy requirement in the smaller group of femalepatients

However the ability of baseline SF to predict phlebotomyrequirements to induce iron depletion inC282Y-homozygoussubjects with iron overload is less clear For example theamount of iron mobilized as estimated by phlebotomyassuming that each unit of blood (480mL) represents 240mgof iron in male patients averaged 52 g (645 g124) which isconsiderably less than 75 g (931 g124) calculated from base-line SF concentrations assuming that 1 120583g of SF is equivalentto 8mg of storage iron The corresponding values for femalepatients were 37 g (267 g72) and 52 g (372 g72) respec-tively In comparison the average amount of storage iron asestimated by phlebotomy in male compound heterozygoteswas 27 g (49 g18) compared with 51 g (91 g18) calculatedfrom baseline SF concentrations These observations whichare in agreement with previous studies [34 35] suggest thatbaseline SF levels as an index of iron stores are not ableto accurately predict the actual iron status and thus thephlebotomy requirement in HFE-related HH patients

Despite the strong association between the C282YC282Ygenotype and iron overload there is so much variabilitywithin this group that genotypic information alone is notsufficient to predict the severity of iron overload in anyindividual subject suggesting that common variants of othergenes involved in the iron metabolism andor environmentalfactors modify the penetrance of the C282YC282Y genotypein these subjects [36] Indeed a polymorphism (pD519G)in the gene encoding glyceronephosphate O-acyltransferase(GNPAT) was recently identified as one such modifier[37] The authors studied this polymorphism with regardto its effect on the severity of iron overload and foundthat 68 (1522) of C282Y-homozygous males with high-iron phenotypes carried one allele and one male carriedtwo alleles of GNPAT pD519G Importantly the authorsprovide data indicating that GNPAT pD519G modulates theseverity of iron overload through downregulation of hepcidinexpression

There are a number of limitations of this study Firstour study was retrospective and therefore does not infercausality between variables examined here Moreover theretrospective nature of the study limited the interpretationof our findings Second data for transferrin saturation werenot available for all patients Third our study contained fewfemale patients with SF above 1000 120583gL and was thereforenot powered to detect true differences Fourth we also didnot measure hepcidin as no frozen serum samples for assayswere available Finally although the data presented here canbe viewed as hypothesis-generating theymust be followed byconfirmation in an independent prospective study Howeverto our knowledge this is the first study inC282Y-homozygouspatients evaluating baseline hemoglobin to examine whetherthis red blood cell parameter is associated with body ironstores (as reflected in SF concentrations) and reportingdifferences in posttreatment changes in hemoglobin andphlebotomy requirements based on subgrouping patientsaccording to SF and baseline hemoglobin levels

In conclusion the results of this study show that treatingC282Y-homozygous patients as a homogeneous group or

dividing them into just two subgroups according to baselineSF levels may obscure important distinctions which onlybecome evident when patients are further divided intosubgroups based onother baseline characteristicsOur resultsmay also suggest that baseline TS levels and hemoglobinlevels in C282Y homozygotes may not necessarily run in thesame direction

Conflict of Interests

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

References

[1] P C Adams ldquoHemochromatosisrdquo Clinics in Liver Disease vol8 no 4 pp 735ndash753 2004

[2] C Fowler ldquoHereditary hemochromatosis pathophysiologydiagnosis and managementrdquo Critical Care Nursing Clinics ofNorth America vol 20 no 2 pp 191ndash201 2008

[3] P C Adams D M Reboussin J C Barton et al ldquoHemochro-matosis and iron-overload screening in a racially diverse pop-ulationrdquo The New England Journal of Medicine vol 352 no 17pp 1769ndash1778 2005

[4] J N Feder A Gnirke W Thomas et al ldquoA novel MHC classI-like gene is mutated in patients with hereditary haemochro-matosisrdquo Nature Genetics vol 13 no 4 pp 399ndash408 1996

[5] A Asberg K Hveem K Thorstensen et al ldquoScreening forhemochromatosis high prevalence and low morbidity in anunselected population of 65238 personsrdquo Scandinavian Journalof Gastroenterology vol 36 no 10 pp 1108ndash1115 2001

[6] W Burke E Thomson M J Khoury et al ldquoHereditaryhemochromatosis gene discovery and its implications forpopulation-based screeningrdquoThe Journal of the American Med-ical Association vol 280 no 2 pp 172ndash178 1998

[7] P C Adams and J C Barton ldquoHow I treat hemochromatosisrdquoBlood vol 116 no 3 pp 317ndash325 2010

[8] J C Barton L F Bertoli and B E Rothenberg ldquoPeripheralblood erythrocyte parameters in hemochromatosis evidencefor increased erythrocyte hemoglobin contentrdquo The Journal ofLaboratory and Clinical Medicine vol 135 no 1 pp 96ndash1042000

[9] E Beutler V Felitti T Gelbart and J Waalen ldquoHaematolog-ical effects of the C282Y HFE mutation in homozygous andheterozygous states among subjects of northern and southernEuropean ancestryrdquo British Journal of Haematology vol 120 no5 pp 887ndash893 2003

[10] C Datz T Haas H Rinner F Sandhofer W Patsch andB Paulweber ldquoHeterozygosity for the C282Y mutation in thehemochromatosis gene is associated with increased serum irontransferrin saturation and hemoglobin in young women aprotective role against iron deficiencyrdquo Clinical Chemistry vol44 no 12 pp 2429ndash2432 1998

[11] C E McLaren J C Barton V R Gordeuk et al ldquoDeter-minants and characteristics of mean corpuscular volume andhemoglobin concentration in white HFE C282Y homozygotesin the Hemochromatosis and Iron Overload Screening StudyrdquoAmerican Journal of Hematology vol 82 no 10 pp 898ndash9052007

[12] J D Cook B S Skikne S R Lynch and M E ReusserldquoEstimates of iron sufficiency in the US populationrdquo Blood vol68 no 3 pp 726ndash731 1986


[13] K R Bridle D M Frazer S J Wilkins et al ldquoDisruptedhepcidin regulation in HFE-associated haemochromatosis andthe liver as a regulator of body iron homoeostasisrdquoThe Lancetvol 361 no 9358 pp 669ndash673 2003

[14] B A C van Dijk C M M Laarakkers S M Klaver etal ldquoSerum hepcidin levels are innately low in HFE-relatedhaemochromatosis but differ between C282Y-homozygoteswith elevated and normal ferritin levelsrdquo British Journal ofHaematology vol 142 no 6 pp 979ndash985 2008

[15] E Nemeth M S Tuttle J Powelson et al ldquoHepcidin regulatescellular iron efflux by binding to ferroportin and inducing itsinternalizationrdquo Science vol 306 no 5704 pp 2090ndash20932004

[16] B Andriopoulos Jr E Corradini Y Xia et al ldquoBMP6 is akey endogenous regulator of hepcidin expression and ironmetabolismrdquo Nature Genetics vol 41 no 4 pp 482ndash487 2009

[17] T Ganz and E Nemeth ldquoHepcidin and iron homeostasisrdquoBiochimica et Biophysica Acta vol 1823 no 9 pp 1434ndash14432012

[18] H Li and Y Z Ginzburg ldquoCrosstalk between iron metabolismand erythropoiesisrdquo Advances in Hematology vol 2010 ArticleID 605435 12 pages 2010

[19] D Meynard L Kautz V Darnaud F Canonne-Hergaux HCoppin and M-P Roth ldquoLack of the bone morphogeneticprotein BMP6 induces massive iron overloadrdquo Nature Geneticsvol 41 no 4 pp 478ndash481 2009

[20] A U Steinbicker T B Bartnikas L K Lohmeyer et alldquoPerturbation of hepcidin expression by BMP type I receptordeletion induces iron overload in micerdquo Blood vol 118 no 15pp 4224ndash4230 2011

[21] N Zhao A-S Zhang and C A Enns ldquoIron regulation byhepcidinrdquo The Journal of Clinical Investigation vol 123 no 6pp 2337ndash2343 2013

[22] J N Feder DM Penny A Irrinki et al ldquoThe hemochromatosisgene product complexes with the transferrin receptor andlowers its affinity for ligand bindingrdquoProceedings of the NationalAcademy of Sciences of the United States of America vol 95 no4 pp 1472ndash1477 1998

[23] C N Gross A Irrinki J N Feder and C A Enns ldquoCo-trafficking of HFE a nonclassical major histocompatibilitycomplex class I protein with the transferrin receptor implies arole in intracellular iron regulationrdquo The Journal of BiologicalChemistry vol 273 no 34 pp 22068ndash22074 1998

[24] AWaheed S Parkkila X Y Zhou et al ldquoHereditary hemochro-matosis effects of C282Y and H63D mutations on associationwith120573

2-microglobulin intracellular processing and cell surface

expression of theHFE protein in COS-7 cellsrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol94 no 23 pp 12384ndash12389 1997

[25] T Goswami and N C Andrews ldquoHereditary hemochromatosisprotein HFE interaction with transferrin receptor 2 suggestsa molecular mechanism for mammalian iron sensingrdquo TheJournal of Biological Chemistry vol 281 no 39 pp 28494ndash28498 2006

[26] G Rishi E M Crampton D F Wallace and V N Sub-ramaniam ldquoIn situ proximity ligation assays indicate thathemochromatosis proteinsHfe and transferrin receptor 2 (Tfr2)do not interactrdquo PLoS ONE vol 8 no 10 Article ID e772672013

[27] P J Schmidt and M D Fleming ldquoTransgenic HFE-dependentinduction of hepcidin in mice does not require transferrinreceptor-2rdquo American Journal of Hematology vol 87 no 6 pp588ndash595 2012

[28] X-G Wu Y Wang Q Wu et al ldquoHFE interacts with the BMPtype I receptor ALK3 to regulate hepcidin expressionrdquo Bloodvol 124 no 8 pp 1335ndash1343 2014

[29] M Brandao J C Oliveira F Bravo J Reis I Garrido andG Porto ldquoThe soluble transferrin receptor as a marker of ironhomeostasis in normal subjects and in HFE-related hemochro-matosisrdquo Haematologica vol 90 no 1 pp 31ndash37 2005

[30] D-L Zhang R M Hughes H Ollivierre-Wilson M C Ghoshand T A Rouault ldquoA ferroportin transcript that lacks an iron-responsive element enables duodenal and erythroid precursorcells to evade translational repressionrdquo Cell Metabolism vol 9no 5 pp 461ndash473 2009

[31] D Girelli P Trombini F Busti et al ldquoA time course of hepcidinresponse to iron challenge in patients with HFE and TFR2hemochromatosisrdquo Haematologica vol 96 no 4 pp 500ndash5062011

[32] K A Stuart G J Anderson D M Frazer et al ldquoDuo-denal expression of iron transport molecules in untreatedhaemochromatosis subjectsrdquo Gut vol 52 no 7 pp 953ndash9592003

[33] C Finch ldquoRegulators of iron balance in humansrdquo Blood vol 84no 6 pp 1697ndash1702 1994

[34] E Beutler V Felitti N J Ho and T Gelbart ldquoRelationshipof body iron stores to levels of serum ferritin serum ironunsaturated iron binding capacity and transferrin saturation inpatients with iron storage diseaserdquoActa Haematologica vol 107no 3 pp 145ndash149 2002

[35] J K Olynyk B A Luxon R S Britton and B R Bacon ldquoHep-atic iron concentration in hereditary hemochromatosis doesnot saturate or accurately predict phlebotomy requirementsrdquoAmerican Journal of Gastroenterology vol 93 no 3 pp 346ndash350 1998

[36] M JWood LW Powell andG A Ramm ldquoEnvironmental andgenetic modifiers of the progression to fibrosis and cirrhosis inhemochromatosisrdquo Blood vol 111 no 9 pp 4456ndash4462 2008

[37] C E McLaren M J Emond V N Subramaniam et al ldquoExomesequencing in HFE C282Y homozygous men with extremephenotypes identifies a GNPAT variant associated with severeiron overloadrdquo Hepatology 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The present study was prompted by our observationthat some C282Y-homozygous patients with higher baselinehemoglobin had lower posttreatment hemoglobin levels inspite of requiring fewer phlebotomies than patients withlower baseline hemoglobin However this informal observa-tionwas not directly investigated at our institutionMoreoverto our knowledge whether baseline hemoglobin level variesdepending on the degree of SF elevation has not been prop-erly addressed in the literature about C282Y-homozygouspatients In this study we first examined whether there is arelationship between baseline hemoglobin and the degree ofSF elevation among C282Y-homozygous patients We thenevaluated the effect of therapeutic phlebotomy on changes inposttreatment hemoglobin levels among subgroups of C282Yhomozygotes

2 Methods

21 Study Subjects This was a retrospective study con-ducted at Akershus University Hospital blood bank in Nor-way In addition to collecting blood for transfusion theblood bank provides services for individuals that requiretherapeutic phlebotomy After obtaining approval from theInstitutional Review Board patient databases were used tocollect relevant data of HH patients who had undergonetherapeutic phlebotomyduring 1996 to 2013 For this analysispatients were included if they (1) had known gender (2)had available data on age at baseline (3) had undergonetesting for HFE mutations and were confirmed to be eitherhomozygote (C282YC282Y) or compound heterozygote(C282YH63D) (4) had elevated baseline SF (gt200120583gL infemales and gt300120583gL in males) and (5) had recordedvenous hemoglobin values at baseline and at the last phle-botomy when their SF was below 50 120583gL (referred to asposttreatment hemoglobin)

Genotype data were available on 254 patients Of these32 (126) had incomplete baseline data and were thereforeexcluded Five C282Y-homozygous males were excludedfrom analysis as a result of (1) alcohol abuse (119899 = 2) (2) cir-rhosis (119899 = 1) and (3) blood donation (119899 = 2 prior blooddonors) because these conditions are capable of affecting SFlevels Three H63D-homozygous males were also excludeddue to uncertainty aboutwhether theywere trueHH subjectsleaving 214 patients for this analysis Patients with low SF(females between 200 and 1000 120583gL males between 300and 1000 120583gL) and high (above 1000 120583gL) SF levels wereincluded in order to increase the statistical power for demon-strating the presence (or lack) of an association Transferrin-iron saturation could not be calculated for all of these patientsbecause of missing transferrin or serum iron data for 14(30214) of themNo patients were excluded from the analysisdue to missing TS values because this would yield smallsample sizes Transferrin saturation was calculated as 4 times(serum iron [in 120583molL])(serum transferrin [in gL]) or bydividing the serum iron by the total iron-binding capacity andmultiplying by 100

22 Statistical Analysis Unpaired 119905-test was used to com-pare means between continuous variables and Pearsonrsquos

chi-square or Fisherrsquos exact test to compare differencesamong group proportions Since serum ferritin values andthe number of phlebotomies were not normally distributedmean values were compared after logarithmic transforma-tion Paired 119905-test and Wilcoxon signed-rank test were usedcomparing baseline and posttreatment hemoglobin levelsPearsonrsquos correlation analysis was performed to assess therelationship between baseline levels of hemoglobin and otherbaseline characteristics and those significantly associatedwith baseline hemoglobin were included in multiple linearstepwise regression analysis Missing TS data were imputedby the mean of the two straddling values In all cases a 119901value oflt005was considered statistically significant Becauseof gender-specific differences in baseline characteristics sep-arate analyses were performed for male and female C282Yhomozygotes Statistical analyses were performed using SPSSsoftware version 21 (Chicago IL USA)

3 Results

31 Baseline Characteristics of Patients The final study pop-ulation consisted of 214 patients of these 142 (663) weremales and 72 (337) were females Baseline characteristicsof patients in each genotype group according to gender aresummarized in Table 1 The majority of patients (119899 = 196916) were homozygous for C282Y as expected Of these124 (633) were males and 72 (367) were females AmongC282Y-homozygous patients males on average were 73 yearsyounger than females (119901 lt 00001) but they had significantlyhigher mean SF (119901 lt 00001) and slightly higher mean TSlevels (119901 = 017) than females Compound-heterozygous(C282YH63D) males had significantly lower SF (119901 = 0012)and TS (119901 lt 00001) levels than C282Y-homozygous males

32 Differences in Mean Baseline Hemoglobin Levels acrossGenotype Groups Most patients had baseline hemoglobinlevels that fell within the reference range for hemoglobin(Table 1) Among C282Y-homozygous patients males hadsignificantly higher mean hemoglobin values than females(119901 lt 0001) as might be expected because of a physiologi-cally higher hemoglobin concentration in men Compound-heterozygous males had a mean hemoglobin value that wasslightly higher than that of C282Y-homozygousmales but thedifference was not statistically significant

33 Relationship between Baseline Hemoglobin and SF LevelsFurther analysis of the data which was limited to C282Y-homozygous patients revealed that patients with lower SFhad higher baseline hemoglobin These patients were there-fore categorized as having low (lt1000120583gL mean 658 plusmn170 120583gL range 316ndash972 120583gL) or high (gt1000120583gL mean1680 plusmn 856 120583gL range 1030ndash4000 120583gL) SF in order to com-pare their baseline hemoglobin levels Males with SF levelslt1000 120583gL (685 85124) had higher mean hemoglobinconcentration than those with SF levels gt1000120583gL (1519 plusmn84 versus 1471 plusmn 126 120583gL 119901 = 0014) Similarlyfemales with SF lt1000 120583gL (847 6172) had highermean hemoglobin concentration than those with SF levels


















119901 lt 005Dagger

119901 lt 001










































4 Discussion

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































119901 lt 005Dagger

119901 lt 001










































4 Discussion

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































4 Discussion

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































4 Discussion

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4 Discussion

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Relationship of Baseline Hemoglobin Level ...downloads.hindawi.com/journals/bmri/2015/241784.pdf · Research Article Relationship of Baseline Hemoglobin Level with