Implications of iron deficiency/anemia on the classification of diabetes using HbA1c

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1 OPEN Cittion: Nutrition & Dibetes (201), e166; doi: /nutd ORIGINAL ARTICLE Implictions of iron /nemi on the clssifiction of dibetes using SM Attrd 1, AH Herring 2,3, H Wng 4, A-G Howrd 2,3, AL Thompson 2,, LS Adir 1,2, EJ Myer-Dvis 1,6 nd P Gordon-Lrsen 1,2 BACKGROUND/OBJECTIVES: Nonglycemic fctors like iron (ID) or nemi my interfere with clssifiction of dibetes nd predibetes using hemoglobin A1c (). However, few popultion-bsed studies of dibetes in res with endemic ID/nemi hve been conducted. We imed to determine how mutully exclusive ctegories of ID lone, nemi lone nd iron- nemi (IDA) were ech ssocited with predibetes nd dibetes prevlence using fsting blood glucose () versus in popultion-bsed study of dults with endemic ID/nemi. SUBJECTS/METHODS: We used dt from the Chin Helth nd Nutrition Survey, longitudinl, popultion-bsed study cross 228 communities within nine provinces of Chin. This nlysis included 7308 dults seen in the 2009 survey ged 18 7 yers. We used descriptive nd covrite-djusted models to exmine reltive risk of predibetes nd dibetes using lone, lone, nd, or neither (normoglycemi) by nemi lone, ID lone, IDA or norml iron/hemoglobin. RESULTS: Approximtely 6% of individuls with dibetes in our smple were concordntly clssified with dibetes using both nd, while 3% hd discordnt dibetes clssifiction: they were clssified using either or, but not both. Fewer prticipnts with ID lone versus norml iron/hemoglobin were clssified with dibetes using only. From covrite-djusted, multinomil regression nlyses, the djusted prevlence of predibetes using only ws 22% for men with nemi lone, but 13% for men with norml iron/hemoglobin. In contrst, the predicted prevlence of predibetes using only ws 8% for women with ID lone, compred with 13% for women with norml iron/hemoglobin. CONCLUSIONS: These findings suggest potentil misclssifiction of dibetes using in res of endemic ID/nemi. Estimting dibetes prevlence using my result in under-dignosis in women with ID nd over-dignosis in men with nemi. Nutrition & Dibetes (201), e166; doi: /nutd ; published online 22 June 201 INTRODUCTION Fsting blood glucose () nd hemoglobin A1c () re importnt dignostic mesures to ssess glycemi; however, is incresingly recommended for use in popultion-bsed settings becuse it does not require fsting nd hs low intrindividul vribility. 1 In the generl popultion, nd do not clssify dibetes identiclly becuse they reflect glycemi sttus over different time periods. 2,3 It is well estblished tht levels cn be ffected by conditions unrelted to dibetes including nemi, blood loss nd iron (ID). 1 Anemi ffects n estimted five million women of reproductive ge in the US 4 nd 1.6 billion individuls worldwide, 4 substntil portion of whom hve concurrent ID. Thus, there is potentil for dibetes misclssifiction cross mny popultions worldwide. To understnd the clinicl implictions of ID nd/or nemi for dibetes prevlence estimtes when using, popultion-bsed reserch in res with high prevlence of ID nd/or nemi is needed. The mgnitude nd direction of dibetes misclssifiction due to ID nd nemi is not well understood. The mechnism through which ID nd nemi influences hs yet to be fully elucidted; 8 however, most epidemiologic studies suggest tht iron- nemi (IDA) cn result in spuriously high vlues, 6,7,9,10 though some suggest there is lower mong individuls with IDA 11 or nemi. 12 These differences my relte to the multiple etiologies for nemi, which include ID, sickle cell disese or other thlssemis, vitmin B12 or folte. ID my be cused by insufficient iron intke, inbility to bsorb iron, blood loss, menstrul blood loss or pregnncy. With these multiple etiologies, IDA my represent two seprte disese processes, nemi nd ID. Furthermore, susceptibility to, or severity of, ID versus nemi my differ by sex becuse of menstrution in women. Few popultion-bsed studies hve seprted ID nd nemi into mutully exclusive ctegories to exmine how ech my differentilly impct predibetes nd dibetes prevlence estimtes using. Thus, we exmined how nemi lone, ID lone nd IDA were ech ssocited with the prevlence of predibetes nd dibetes when using nd in popultion-bsed smple of 7308 Chinese dults ged 18 7 yers from the Chin Helth nd Nutrition Survey. We exmined the vrition in the prevlence of predibetes or dibetes using descriptive nd covrite-controlled nlyses, hypothesizing tht IDA would result in over-dignosis of dibetes using versus. 1 Deprtment of Nutrition, Gillings School of Globl Public Helth, UNC-Chpel Hill, Chpel Hill, NC, USA; 2 Crolin Popultion Center, UNC-Chpel Hill, Chpel Hill, NC, USA; 3 Deprtment of Biosttistics, Gillings School of Globl Public Helth, UNC-Chpel Hill, Chpel Hill, NC, USA; 4 Ntionl Institute of Nutrition nd Food Sfety, Chinese Center for Disese Control nd Prevention, Beijing, Chin; Deprtment of Anthropology, UNC-Chpel Hill, Chpel Hill, NC, USA nd 6 Deprtment of Medicine, UNC-Chpel Hill, Chpel Hill, NC, USA. Correspondence: Dr P Gordon-Lrsen, Crolin Popultion Center, UNC-Chpel Hill, 137 Est Frnklin Street, 6th Floor, Chpel Hill, NC , USA. E-mil: pglrsen@unc.edu Received 9 September 2014; revised 21 Jnury 201; ccepted 29 April 201

2 2 MATERIALS AND METHODS Study design nd prticipnts The Chin Helth nd Nutrition Survey is longitudinl study cross 228 communities within nine provinces of Chin. Surveys begn in 1989, with subsequent surveys every 2 4 yers, for totl of nine rounds between 1989 nd The Chin Helth nd Nutrition Survey ws designed to provide representtion of rurl, urbn nd suburbn res vrying substntilly in geogrphy, economic development, public resources nd helth indictors, 13 nd it is the only lrge-scle, longitudinl study of its kind in Chin. The originl survey in 1989 used multistge, rndom cluster design in eight provinces (Lioning, Jingsu, Shndong, Henn, Hubei, Hunn, Gungxi nd Guizhou) to select strtified probbility smple; ninth province, Heilongjing, ws dded in 1997 using similr smpling strtegy. Essentilly, two cities (one lrge nd one smll city usully the provincil cpitl nd lower income city) nd four counties (strtified by income: one high, one low nd two middle income counties) were selected in ech province. Within cities, two urbn nd two suburbn communities were selected; within counties, one community in the cpitl city nd three rurl villges were chosen. Twenty households per community were then selected for prticiption. The study met the stndrds for the ethicl tretment of prticipnts nd ws pproved by the Institutionl Review Bords of the University of North Crolin t Chpel Hill nd the Institute of Nutrition nd Food Sfety, Chinese Center for Disese Control nd Prevention. More detiled survey procedures cn be found elsewhere. 13 Adults ged 18 7 yers with blood drw t the 2009 Chin Helth nd Nutrition Survey exm were eligible for inclusion (n = 8102). Of these 8102 dults, individuls were excluded due to: pregnncy t the time of survey (n = 62), hving nonfsting blood mesurement (n = 34) or missing dt on one or more mesures: (n = 6), (n = 19), hemoglobin (n = 32), ferritin (n = 11), trnsferrin receptor (n = 11), C-rective protein (n = 1), wist circumference (n = 222), household income (n = 101) or smoking sttus (men only; n = 30), with some individuls missing informtion for more thn one vrible (totl n = 378), leving 7308 individuls in our nlytic smple. Of the full eligible smple (n = 8102), greter proportion of those included (versus excluded) in the nlytic smple (n = 7308) were younger, hd lower nd were from lower urbnicity res. Mesures After n overnight fst, blood ws collected by venipuncture (12 ml). Whole blood ws immeditely centrifuged nd serum tested for glucose using Hitchi 7600 nlyzer using glucose oxidse phenol 4-minontipyrine peroxidse kit (GOD-PAP; Rndox, Crumlin, UK). ws mesured t centrl lb in ech province with high-performnce liquid chromtogrphy system (D10 HLC, Bio-Rd, Hercules, CA, USA; PDQ HPLC, Primus, Knss City, MO, USA; Model HLC-723 G7, Tosoh Corportion, Tokyo, Jpn). All the smples were clibrted to the D10 HLC from Bio-Rd. Self-report questionnires were used to scertin medicl history nd current mediction use. We compred prevlent dibetes using ( 6.% (48 mmol mol 1 )) versus ( 126 mg dl 1 (7.0 mmol l 1 )). 1 We considered individuls reporting dibetes dignosis (nswering yes to the question hs doctor ever told you tht you suffer from dibetes? ) nd tretment (reporting dibetes mediction use) s hving dibetes. For prevlent predibetes, we used ( =.7 6.% (39 48 mmol mol 1 )) versus ( = mg dl 1 (. 7.0 mmol l 1 )) ccording to the Americn Dibetes Assocition guidelines, 1 excluding individuls clssified with dibetes ccording to ny criteri (,, dibetes dignosis or dibetes mediction). In descriptive nlyses nd multinomil logistic regression models, we ctegorized individuls ccording to their dibetes sttus (nondibetic, dibetes (discordnt, by only), dibetes (discordnt, by only) or dibetes + (concordnt by nd )) nd predibetes sttus (normoglycemi, predibetes, predibetes or predibetes + ). Hemoglobin nd iron sttus mrkers were mesured t ntionl centrl lb in Beijing (medicl lbortory ccredittion certificte ISO 1 189:2007) with strict qulity control. From serum, soluble trnsferrin receptor ws mesured vi nephelometry on Siemens BNP (Seimns Chin, Beijing, Chin) nd ferritin ws mesured vi rdioimmunossy on Gmm counter XH-6020 (North Institute of Bio-Tech, Beijing, Chin). From whole blood, hemoglobin ws mesured vi VCS on Beckmn Coulter LH70 (Beckmn, Bre, CA, USA). Becuse cute infection rtificilly rises ferritin, we followed WHO recommendtions nd multiplied the mesured ferritin vlues in 802 individuls with C-rective protein mgl 1 by Anemi lone ws defined s hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men) ccording to WHO guidelines without ID (defined s inflmmtion-djusted ferritin o1 μgl 1 or trnsferrin receptor 1.76 mg l 1 without nemi). IDA ws defined s hving nemi nd ID ccording to WHO recommendtions. 1 Age, sex, smoking sttus nd pregnncy sttus/history were selfreported t ech survey. Household income ws derived from individul nd household questionnires from time-use, sset nd economic ctivity t ech survey nd inflted to 2009 Yun currency in nlysis for comprbility over time, nd ctegorized into tertiles. Wist circumference ws mesured midwy between the lowest rib nd the ilic crest using nonelstic tpe. Urbnicity, which reflects the degree of moderniztion of ech of the Chin Helth nd Nutrition Survey communities, ws mesured using multicomponent scle incorporting infrstructure, economic nd socil service domins (rnge: 0 120). The scle hs high relibility nd vlidity, 16 with higher urbnicity score corresponding to hving more urbn chrcteristics cross multiple domins. We ctegorized urbnicity into tertiles. We controlled for region (North, Centrl, South) due to regionl differences in geogrphy nd diet cross Chin. Sttisticl nlyses Descriptive nlyses. All nlyses were conducted in Stt 13 (Stt Corp, College Sttion, TX, USA). First, we exmined the differences in smple chrcteristics by sex, using χ 2 or nlysis of vrince tests for ctegoricl or continuous vribles, respectively. Second, we exmined sex-specific differences in dibetes clssifiction (nondibetic, dibetes, dibetes or dibetes + ) or predibetes clssifiction (normoglycemi, predibetes, predibetes or predibetes + ) ccording to ID nd/or nemi sttus (norml iron/hemoglobin, nemi lone, ID or IDA), testing group-level differences in the clssifiction vi χ 2 tests. Third, mong individuls with dibetes or predibetes, we exmined the mismtch between the prevlence of dibetes nd predibetes (seprtely) on the bsis of versus (concordnt by nd ; discordnt by only; discordnt by only) using pirwise χ 2 tests compring ctegories of ID lone, nemi lone or IDA with norml iron/hemoglobin s referent. Sttisticl significnce ws set t the Po0.0 level for ll descriptive nlyses. Sttisticl modeling. We used sex-specific multinomil logistic regression models to exmine the estimted reltive risk rtios (RRR) of dibetes using concordnt nd discordnt clssifiction by nd (dibetes, dibetes, dibetes + nd nondibetic (referent)) cross ctegories of ID nd/or nemi sttus, controlling for confounding by demogrphic, socil nd environmentl fctors. An identicl set of models predicted reltive risk of concordnt nd discordnt predibetes clssifiction using nd (predibetes, predibetes, predibetes + nd normoglycemi (referent)), excluding n = 737 individuls with dibetes ccording to either,, doctor dignosis or dibetes mediction use. We strtified models by sex becuse cutpoints for nemi nd ID differ by sex nd becuse interction terms between ctegories of ID/nemi sttus nd sex were sttisticlly significnt (Po0.0). Finl models included ge (liner), urbnicity (low, medium, high), number of cigrettes smoked per dy (liner nd qudrtic; men only due to low smoking prevlence (3%) in women), pregnncy history (ever versus never; women only), household income (low, medium, high), region (North, Centrl, South) nd wist circumference (liner). Models were clustered t the community level using Huber White type vrince estimtor to correct stndrd errors. 17 In figures, we present reltive risk rtios (RRRs) from these models nd model-bsed predicted prevlence of dibetes or predibetes using only, only or both nd cross ID/nemi ctegories. Sensitivity nlysis. Becuse individuls with the genetic hemoglobin E (HbE) vrint could hve n rtificilly lower reltive to individuls without HbE, 18 we conducted n dditionl nlysis excluding prticipnts from the South region (where the HbE trit is most common) to determine whether reltionships mong ID, nemi, IDA nd dibetes clssifiction by versus were different in the presence of the HbE genetic vrint. Becuse provinces of Chin vry gretly in climte, ltitude, nd terrin, potentilly ffecting biochemicl prmeters relted to hemoglobin nd nemi, 19 we conducted two sensitivity nlyses: first, we repeted our nlysis strtified by region to see whether the mgnitude or direction of

3 ssocition differed by region. Second, we redefined our region vrible ccording to the four regions identified in pper by Mio, et l. 19 to exmine whether our results were sensitive to the clssifiction of region. Role of the funding source The sponsors of the study hd no role in study design, dt collection, dt nlysis, dt interprettion, writing of the report nd the decision to submit for publiction. The corresponding uthor hd full ccess to ll the dt in the study nd hd finl responsibility for the decision to submit for publiction. RESULTS Prevlence of predibetes, dibetes, ID nd nemi Approximtely 3% of men nd women hd or bove the predibetes thresholds. Although 11.1% of men nd 9.2% of women were clssified s hving dibetes by either,, physicin dignosis or mediction use, only 6.2% of men nd 4.8% of women hd dibetes using both nd, physicin dignosis or mediction use (Tble 1). The prevlence of ID nd/or nemi ws ~ 20% for men nd 40% for women. Differences in prevlence of dibetes by ID nd/or nemi sttus In undjusted nlyses, there ws sttisticlly significnt difference in dibetes (men: P = 0.04; women: P = 0.008) nd predibetes (men: Po0.001; women: Po0.001) clssifiction using versus cross ctegories of ID nd/or nemi sttus (Tble 2). Among individuls with dibetes, fewer individuls with ID lone (men: 12.9%; women: 19.1%) were estimted to hve dibetes using only (dibetes ) compred with individuls with norml iron/hemoglobin (men: 2.1%; women: 38.3%; Po0.0; Figure 1). However, mong individuls with predibetes, more men with nemi lone (66.1%) versus norml iron/hemoglobin (42.2%; Po0.0) were estimted to hve predibetes. Among individuls with predibetes, more 3 Tble 1. Survey Percent or men vlues for smple chrcteristics ccording to sex in Chinese dults ged 18 7 yers, 2009 Chin Helth nd Nutrition Men N = 340 Women N = 3903 P-vlue Men or % Stndrd error Men or % Stndrd error mg dl 1, men (s.e.) o0.001 %, men (s.e.) Dibetes sttus, b % (s.e.) o0.001 Nondibetic 88.9% 0.% 90.8% 0.% Dibetes 2.% 0.3% 3.0% 0.3% Dibetes 2.4% 0.3% 1.4% 0.2% Dibetes + 6.2% 0.4% 4.8% 0.3% Predibetes/dibetes sttus, c % (s.e.) 0.8 Normoglycemi 64.% 0.9% 6.% 0.8% Predibetes/dibetes 1.9% 0.7% 16.2% 0.6% Predibetes/dibetes 12.0% 0.6% 11.0% 0.% Predibetes/dibetes + 7.6% 0.% 7.3% 0.4% Age, men (s.e.) /nemi sttus, d % (s.e.) o0.001 iron nd hemoglobin 79.4% 0.7% 60.1% 0.8% Anemi lone 4.6% 0.4% 8.2% 0.4% lone 12.7% 0.6% 22.0% 0.7% - nemi 3.2% 0.3% 9.8% 0.% Wist circumference cm, men (s.e.) o0.001 Urbniztion, % (s.e.) e 0.43 Low 34.4% 0.8% 33.1% 0.8% Medium 33.7% 0.8% 33.9% 0.8% High 31.9% 0.8% 33.1% 0.8% Income, % (s.e.) 0.06 Low 31.3% 0.8% 33.8% 0.8% Medium 34.% 0.8% 33.6% 0.8% High 34.2% 0.8% 32.6% 0.8% Region, % (s.e.) 0.8 North 21.1% 0.7% 20.6% 0.6% Centrl 3.6% 0.8% 36.0% 0.8% South 43.4% 0.8% 43.4% 0.8% Abbrevitions:, fsting blood glucose;, hemoglobin A1c; s.e., stndrd error. P-vlue for χ2 (ctegoricl) or nlysis of vrince (continuous) test for difference in smple chrcteristics by sex. b Dibetes ws clssified s nondibetic ( o126 mg dl 1 nd o6.% (48 mmol mol 1 )), dibetes ( 6.% (48 mmol mol 1 ) nd o126 mg dl 1 ), dibetes ( 126 mg dl 1 nd o6.% (48 mmol mol 1 )) or dibetes + ( 126 mg dl 1 nd 6.% (48 mmol mol 1 ), doctor dignosis or reported dibetes mediction use). c Predibetes/dibetes clssified s norml ( o100 mg dl 1 nd o.7% (39 mmol mol 1 )), predibetes/dibetes (.7% (39 mmol mol 1 ) nd o100 mg dl 1 ), predibetes/ dibetes ( 100 mg dl 1 nd o.7% (39 mmol mol 1 )), or predibetes/dibetes + ( 100 mg dl 1 nd.7% (39 - mmol mol 1 ), doctor dignosis or reported dibetes mediction use). d The four ctegories of iron nd/or nemi sttus re defined s follows: norml iron nd hemoglobin (hemoglobin 12 g dl 1 (women) or 13 g dl 1 (men), ferritin 1 μgl 1, soluble trnsferrin receptor o1.76 mg l 1 ), nemi lone (hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men) without iron ), iron lone (ferritin o1 μgl 1 or trnsferrin receptor mg l 1 without nemi) nd iron- nemi (nemi: hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men) s well s iron-: ferritin o1 μgl 1 or trnsferrin receptor 1.76 mg l 1 ). e Column percents my not dd to 100% due to rounding.

4 4 Tble 2. Proportion of the smple clssified with dibetes nd predibetes by only, only, both nd, or neither ccording to ctegories of iron nd/or nemi sttus, Chinese dults ged 18 7 yers, 2009 Chin Helth nd Nutrition Survey Dibetes clssifiction Nondibetic Dibetes Dibetes Dibetes + P-vlue b Percent Stndrd error Percent Stndrd error Percent Stndrd error Percent Stndrd error Men N = 3027 N = 86 N = 81 N = iron/hemoglobin c 80.1% 0.7% 81.4% 4.2% 7.3% 4.8% 70.1% 3.2% Anemi lone c 4.% 0.4%.8% 2.% 4.9% 2.4%.7% 1.6% lone c 12.3% 0.6% 9.3% 3.2% 18.% 4.3% 18.% 2.7% - nemi c 3.1% 0.3% 3.% 2% 1.2% 1.2%.7% 1.6% Women N = 342 N = 117 N = 3 N = iron/hemoglobin c 9.7% 0.8% 7.2% 4% 4.7% 6.9% 9.8% 3.6% Anemi lone c 7.9% 0.% 6.8% 2.3% 11.3% 4.4% 12.7% 2.4% lone c 22.3% 0.7% 11.1% 2.9% 26.4% 6.1% 21.7% 3% - nemi c 10.1% 0.% 6.8% 2.3% 7.% 3.7%.8% 1.7% Predibetes clssifiction Normoglycemi Predibetes Predibetes Predibetes + P-vlue b Percent Stndrd error Percent Stndrd error Percent Stndrd error Percent Stndrd error Men N = 192 N = 480 N = 364 N = 231 o0.001 iron/hemoglobin c 78.8% 0.9% 78.8% 1.9% 82.7% 2% 90.% 1.9% Anemi lone c 4.1% 0.4% 7.7% 1.2% 2.7% 0.9% 3.9% 1.3% lone c 13.7% 0.8% 10.0% 1.4% 12.4% 1.7% 4.8% 1.4% - nemi c 3.4% 0.4% 3.% 0.8% 2.2% 0.8% 0.9% 0.6% Women N = 2320 N = 72 N = 391 N = 29 o0.001 iron/hemoglobin c 7.6% 1% 66.6% 2% 8.1% 2.% 6.3% 3% Anemi lone c 7.9% 0.6% 8.0% 1.1% 9.0% 1.4%.8% 1.% lone c 23.% 0.9% 1.2% 1.% 2.1% 2.2% 22.8% 2.6% - nemi c 10.9% 0.6% 10.1% 1.3% 7.9% 1.4% 6.2% 1.% Abbrevitions:, fsting blood glucose;, hemoglobin A1c. Dibetes ws clssified s nondibetic ( o126 mg dl 1 nd o6.% (48 mmol mol 1 )), dibetes ( 6.% (48 mmol mol 1 ) nd o126 mg dl 1 ), dibetes ( 126 mg dl 1 nd o6.% (48 - mmol mol 1 )) or dibetes + ( 126 mg dl 1 nd 6.% (48 mmol mol 1 ), doctor dignosis or reported dibetes mediction use). Predibetes clssifiction excluded N = 737 individuls with dibetes ccording to,, doctor dignosis or reported dibetes mediction use. Predibetes ws clssified s normoglylcemi ( o100 mg dl 1 nd o.7% (39 mmol mol 1 )), predibetes ( =.7 6.% ( mmol mol 1 ) nd o100 mg dl 1 ), predibetes ( = mg dl 1 nd o.7% (39 mmol mol 1 )) or predibetes + ( = mg dl 1 nd =.7 6.% (39 48 mmol mol 1 )). Column percents my not dd up to 100% due to rounding. b P-vlue for χ2 test for differences dibetes or predibetes clssifiction ccording to ctegories of iron nd/or nemi. c /nemi sttus ws ctegorized s norml iron nd hemoglobin (hemoglobin 12 g dl 1 (women) or 13 g dl 1 (men), ferritin 1 μgl 1, soluble trnsferrin receptor o1.76 mg l 1 ), nemi lone (s hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men) without iron ), iron lone (ferritin o1 μgl 1 or trnsferrin receptor mg l 1 without nemi) nd iron- nemi (nemi: hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men), s well s iron- ferritin o1 μgl 1 or trnsferrin receptor 1.76 mg l 1 ). individuls with IDA (men: 63%; women:.2%) versus norml iron/hemoglobin (men: 42.6%; women: 49.0%) were estimted to hve predibetes. Using covrite-djusted models, we exmined differences in the clssifiction of dibetes nd predibetes (seprtely) using versus cross mutully exclusive ctegories of ID nd/or nemi (Figures 2 4, with RRRs presented in Supplementry Tbles 1 nd 2). In comprison with nondibetic individuls, women with ID lone versus norml iron/hemoglobin hd lower reltive risk of being clssified with dibetes (RRR = 0.2; 9% confidence intervl (CI): 0.29, 0.9; Figure 2 nd Supplementry Tble 1). Similrly, in comprison with dibetes, women with ID lone versus norml iron/hemoglobin hd lower reltive risk of being clssified with dibetes (RRR = 0.37; CI: 0.1, 0.88; Figure 2 nd Supplementry Tble 1). In comprison with nondibetic individuls, men with IDA reltive to norml iron/hemoglobin hd higher reltive risk of being concordntly clssified with dibetes + (RRR = 2.38; CI: 1.20, 4.72). A similr pttern ws seen for ID lone reltive to norml iron/hemoglobin (RRR = 1.80; CI: 1.21, 2.67; Figure 2 nd Supplementry Tble 1). From the predibetes models, reltive to normoglycemi, men with nemi lone versus norml iron/hemoglobin were more likely to be clssified with predibetes (RRR = 1.81; CI: 1.16, 2.82; Figure 3 nd Supplementry Tble 2). Similrly, in comprison with predibetes, men with nemi lone hd higher reltive risk of being clssified with predibetes (RRR = 3.00; CI: 1.43, 6.30; Figure 3 nd Supplementry Tble 2). Men with ID lone or IDA hd higher reltive risk of predibetes thn predibetes +. Women with ID lone versus norml iron/hemoglobin were more likely to be clssified with predibetes reltive to predibetes (RRR = 1.76; CI: 1.2, 2.49). There ws reltively little difference in predibetes by versus for women with IDA. Using these sme covrite-djusted models, we clculted the predicted probbility of dibetes nd predibetes clssifiction using only (discordnt), only (discordnt) or both nd (concordnt; Figure 4). Among women with ID lone, smller percentge were predicted to hve dibetes (0.%) compred with dibetes (1.%). For men, concordnt clssifiction of predibetes (predibetes + ) ws less common in men with IDA (1.0%) versus norml iron/hemoglobin (4.0%). A higher

5 Percent 100% 90% 80% 70% 60% 0% 40% 30% 20% 10% 0% / Hemoglobin N=279 Men with Dibetes Anemi N=21 N=62 nemi N=16 100% 90% 80% 70% 60% 0% 40% 30% 20% 10% 0% / Hemoglobin N=230 Women with Dibetes Anemi N=38 Anemi N=68 N=23 Percent 100% 90% 80% 70% 60% 0% 40% 30% 20% 10% 0% / Hemoglobin N=888 Men with Predibetes Anemi N= N=104 nemi N=27 100% 90% 80% 70% 60% 0% 40% 30% 20% 10% 0% Women with Predibetes / Hemoglobin N=777 Anemi N=96 N=244 Anemi N=10 Concordnt + Discordnt, Discordnt, only Figure 1. Discordnce nd concordnce in clssifiction of dibetes nd predibetes using only, only or with both nd by iron/nemi sttus in Chinese dults ged 18 7 yers from the 2009 Chin Helth nd Nutrition Survey with dibetes or predibetes. Dibetes clssified s concordnt + ( 126 mg dl 1 nd 6.% (48 mmol mol 1 ) or doctor dignosis), discordnt lone ( 6.% nd o126 mg dl 1 (48 mmol mol 1 ) with no doctor dignosis) or discordnt lone ( 126 mg dl 1 nd o6.% (48 mmol mol 1 ) with no doctor dignosis). Predibetes clssified s concordnt + ( = mg dl 1 nd =.7 6.% (39 48 mmol mol 1 )), discordnt lone ( =.7 6.% (39 48 mmol mol 1 ) nd o100 mg dl 1 ) or discordnt lone ( = mg dl 1 nd o.7% (39 mmol mol 1 )). /nemi sttus ws ctegorized s norml iron nd hemoglobin (hemoglobin 12 g dl 1 (women) or 13 g dl 1 (men), ferritin 1 μgl 1, soluble trnsferrin receptor o1.76 mg l 1 ), nemi lone (s hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men) without iron ), iron lone (ferritin o1 μgl 1 or trnsferrin receptor 1.76 mg l 1 without nemi) nd iron- nemi (nemi: hemoglobin o12 g dl 1 (women) or o13 g dl 1 (men), s well s iron- ferritin o1 μgl 1 or trnsferrin receptor 1.76 mg l 1 ). Po0.0, denotes sttisticlly significnt difference reltive to norml iron/hemoglobin in the percent of individuls clssified s concordnt nd, discordnt only or discordnt only using chi-squred test. Percents my not dd up to 100% due to rounding. percentge of men with nemi lone were predicted to hve predibetes (22.0%) versus predibetes (6.8%). Sensitivity nlyses. In our nlysis to exmine differences in findings in the presence of the HbE genetic vrint, we excluded prticipnts from the South region (n = 3323) where the HbE vrint is most common. We did not see ny differences in the direction or mgnitude of ssocitions between ID, nemi or IDA (versus norml iron/hemoglobin) with dibetes or predibetes clssifiction vi or, lthough fewer results were sttisticlly significntduetothesmllersmplesizeinthissubsmple. In our nlysis with strtified models by region (North, Centrl nd South), we did not see differences in the direction or mgnitude of ssocitions, however, estimtes were imprecise due to smll smple size. In our lst sensitivity nlysis, we redefined our region vrible ccording to the four regions identified in pper by Mio, et l. 19 to exmine whether our results were sensitive to the clssifiction of region nd did not see differences in the direction or mgnitude of ssocitions. DISCUSSION ID-relted chnges in the hemoglobin molecule s well s nemiinduced erythrocyte turnover differences my result in inccurte mesurements for estimtes of dibetes prevlence. Though levels my not ccurtely reflect glycemi sttus if individuls hve certin nemis, blood loss or ID, the mgnitude nd direction of the ssocitions between ID nd/or nemi with dibetes prevlence using hs not been determined. Furthermore, we re wre of no popultion-bsed studies exmining differences in the prevlence of predibetes nd dibetes ccording to mutully exclusive ctegories of ID lone, nemi lone or IDA versus norml iron/hemoglobin. We

6 6 Anemi Deficiency Deficiency Anemi Reltive Risk Rtios predicting dibetes RRR (9% CI) Men Reltive Risk Rtios predicting dibetes RRR (9% CI) Women Nondibetic Nondibetic Nondibetic Figure 2. Multinomil logistic regression models predicting discordnce nd concordnce in clssifiction of dibetes using only, only, or both nd reltive to nondibetic ccording to iron nd/or nemi sttus, from the 2009 Chin Helth nd Nutrition Survey. Dibetes outcome ws clssified s nondibetic ( o126 mg dl 1 nd o6.% (48 mmol mol 1 )), only ( 6.% (48 mmol mol 1 ) nd o126 mg dl 1 ), only ( 126 mg dl 1 nd o6.% (48 mmol mol 1 )) or + ( 126 mg dl 1 nd 6.% (48 mmol mol 1 ), doctor dignosis of dibetes or reported dibetes mediction use). Min exposure vrible ws ctegories of iron /nemi sttus. Models were clustered t the community level nd djusted for ge, urbnicity level (low, medium, high), number of cigrettes smoked per dy nd number of cigrettes squred (men only), ever pregnnt (ever versus never; women only), household income (low, medium, high), region (North, Centrl, South) nd wist circumference. Po0.0, denotes sttisticlly significnt difference in the reltive risk rtios using chi-squred tests., fsting blood glucose; IDA, iron- nemi; RRR, reltive risk rtio. + Reltive Risk Rtios predicting predibetes RRR (9% CI) Men Anemi Deficiency Deficiency Anemi Reltive Risk Rtios predicting predibetes RRR (9% CI) Women Figure 3. Multinomil logistic regression models predicting discordnce nd concordnce in clssifiction of predibetes using only, only or both nd reltive to normoglycemi ccording to iron nd/or nemi sttus, from the 2009 Chin Helth nd Nutrition Survey. Predibetes model excluded N = 737 individuls with dibetes ccording to,, doctor dignosis or reported dibetes mediction use. Predibetes outcome ws clssified s norml ( o100 mg dl 1 nd o.7% (39 mmol mol 1 )), only ( =.7 6.% (39 48 mmol mol 1 ) nd o100 mg dl 1 ), only ( = mg dl 1 nd o.7% (39 - mmol mol 1 )) or + ( = mg dl 1 nd =.7 6.% (39 48 mmol mol 1 )). Min exposure vrible ws ctegories of iron /nemi sttus. Models were clustered t the community level nd djusted for ge, urbnicity level (low, medium, high), number of cigrettes smoked per dy nd number of cigrettes squred (men only), ever pregnnt (ever versus never; women only), household income (low, medium, high), region (North, Centrl, South) nd wist circumference. Po0.0, denotes sttisticlly significnt difference in the reltive risk rtios using chi-squred tests., fsting blood glucose; IDA, iron- nemi; RRR, reltive risk rtio. + + ddressed this gp, finding tht fewer women with ID lone versus norml iron/hemoglobin were predicted to hve dibetes reltive to dibetes, dibetes + or s nondibetic. Compred with men with norml iron/hemoglobin, however, more men with nemi lone were predicted to hve predibetes reltive to predibetes, predibetes + or normoglycemi. The direction of ssocition between IDA nd dibetes/predibetes using versus were mixed cross our sttisticl models. These findings hve importnt implictions for dibetes prevlence estimtes cross developing countries with high prevlence of both ID nd nemi, 4 s well s for developed countries, where is often considered preferble to for glycemi ssessment. 1 Multiple smll, clinic-bsed studies hve exmined IDA reltionship, however, inconclusive findings from these studies my be becuse ID nd nemi represent two different disese processes. ID, when mild, does not hve cliniclly relevnt effects on hemoglobin levels, wheres severe ID results in concurrent ID nd nemi. Though most nemi is due to ID, lrge proportion of nemi cses re due to sickle cell disese or other thlssemis, vitmin B12 or folte. Thus, combining ID nd nemi in reserch studies my msk

7 3 Dibetes Men 3 Dibetes Women Percent Predibetes Men 3 Predibetes Women Percent iron/hemoglobin Anemi lone IDA Figure 4. Multinomil logistic regression model-predicted percent of individuls clssified with dibetes or predibetes using only, only or both nd ccording to iron nd/or nemi sttus, from the 2009 Chin Helth nd Nutrition Survey. Dibetes outcome ws clssified s nondibetic ( o126 mg dl 1 nd o6.% (48 mmol mol 1 )), only ( 6.% (48 - mmol mol 1 ) nd o126 mg dl 1 ), only ( 126 mg dl 1 nd o6.% (48 mmol mol 1 )) or + ( mg dl 1 nd 6.% (48 mmol mol 1 ), doctor dignosis of dibetes or reported dibetes mediction use). Predibetes model excluded N = 737 individuls with dibetes ccording to,, doctor dignosis or reported dibetes mediction use. Predibetes outcome ws clssified s normoglycemi ( o100 mg dl 1 nd o.7% (39 mmol mol 1 )), only ( =.7 6.% (39 48 mmol mol 1 ) nd o100 mg dl 1 ), only ( = mg dl 1 nd o.7% (39 mmol mol 1 )) or + ( = mg dl 1 nd =.7 6.% (39 48 mmol mol 1 )). Min exposure vrible ws ctegories of iron /nemi sttus. Models were clustered t the community level nd djusted for ge, urbnicity level (low, medium, high), number of cigrettes smoked per dy nd number of cigrettes squred (men only), ever pregnnt (ever versus never; women only), household income (low, medium, high), region (north, centrl, south) nd wist circumference. Ptterns of sttisticl significnce cn be found in Figures 2 nd 3. ID, iron ; IDA, iron- nemi. 0 heterogeneous disese processes underlying the reltionship between IDA nd. For exmple, ID my rtificilly increse by inducing chnges to the shpe of the hemoglobin molecule, promoting glyction of the terminl vline 8 or by lowering erythrocyte turnover, llowing more time for glyction of hemoglobin to occur. 6,7 Some nemis, on the other hnd, my rtificilly lower becuse of incresed erythrocyte turnover. 20 We know of no popultion-bsed studies to dte tht hve ddressed these potentilly different mechnisms by using two seprte mesures of glycemi sttus nd mutully exclusive ctegories of ID lone, nemi lone, IDA or norml iron/hemoglobin. We found tht in our smple, the prevlence of nemi ws 7.8% for men nd 18.0% for women, which is comprble to ntionl representtive dt from Chin. 21 This prevlence is similr to the prevlence of nemi in Mexico nd is much lower thn the prevlence for Indi (~0% for women of reproductive ge), but is much higher thn the prevlence of nemi in the United Sttes (~7% for women of reproductive ge). 4 Previous studies in Chin suggest tht ID in Chin my be endemic due to the fct tht the mjority of the iron consumed in the Chinese diet is from plnts, source of non-heme iron of lower biologic vilbility compred with niml-source foods. 22 The higher observed prevlence of ID in women versus men suggests tht menstrul blood loss nd pregnncy history my be lrge contributor to ID in this popultion. We lso found tht ~ 0% of individuls with nemi hd concurrent ID, similr to proportions observed in mny popultions. It is possible tht the other 0% of individuls with nemi could result from genetic hemoglobinopthies, thlssemis or other vitmin deficiencies. From our multivrible model results, nemi lone nd IDA were ssocited with greter reltive risk of predibetes versus normoglycemi in men but not women. A previous popultion-bsed study from the US found tht mong individuls without dibetes, those with nemi versus norml iron/hemoglobin hd higher men. 23 Another found nonsttisticlly significnt increse in men mong individuls with nemi. 9 However, studies using clinic-bsed smples from developing countries hve shown lower men in individuls with IDA 11 or nemi 12 versus norml iron/

8 8 hemoglobin. These study findings my differ becuse they were bsed in regionl, smll or clinic-bsed smples. Sex-bsed differences in our study my be due to the lower prevlence of nemi lone thn ID lone in women. In our smple, we observed tht women with ID lone hd lower reltive risk of being clssified with dibetes versus s nondibetic. Similr, lthough nonsignificnt, reltionships were seen in men, perhps owing to sex-bsed differences in the prevlence nd severity of ID nd nemi. Our finding confirms results from clinic-bsed smple of Indin women, which found lower in women with ID versus norml iron, 11 but contrdict findings from popultion-bsed study of US dults, which observed positive ssocition between ID nd. 9 With the exception of our model predicting predibetes in women, the reltionship of IDA (versus norml iron/hemoglobin) with dibetes or predibetes (versus normoglycemi), though not sttisticlly significnt, ws in the sme direction s the ssocitions we observed between nemi lone nd higher reltive risk of dibetes/predibetes. It is possible tht positive reltionship between IDA (which is severe ID with nemi) nd is ttenuted becuse ID is negtively ssocited with, while nemi is positively ssocited with. Another possibility is tht the low prevlence of dibetes nd IDA, prticulrly in men, could hve resulted in insufficient sttisticl power, or tht hemoglobinopthies, thlssemis or other confounding fctors could hve reduced the precision of our estimtes. However, our findings suggest tht different direction of ssocition between ID nd (negtive) versus tht between nemi nd (positive) my underlie inconsistent findings in the epidemiologic literture nd support the need to exmine both iron nd nemi sttus when ssessing dibetes using. Our study contributes to the literture on influences of nonglycemic fctors on, but there re some limittions of note. In our popultion-bsed smple, we do not hve n orl glucose tolernce test, the gold stndrd of glycemic mesurement 1 yet we do hve nd FGB, wheres most studies only collect one or the other. As nonclinicl study, we only hve one nd mesurement from prticipnts, which is stndrd for reserch studies in the field, 24 wheres clinicl dignostic test would include replicte mesures. We do not hve informtion on genetic hemoglobinopthies such s HbE, which ffects ~ 30% of Southest Asins nd my rtificilly lower. 18 However, in sensitivity nlysis, we excluded prticipnts from the South region of Chin where the HbE vrint is most prevlent, nd observed similr findings in the full smple, lthough estimtes were less precise due to the smller smple size. Our nlysis does not ddress pregnncy, time when women re t high risk of developing ID nd gesttionl dibetes. However, our study hs the gret strengths of reporting popultion-level ssocitions between ID lone, nemi lone nd IDA nd dibetes/predibetes clssifiction in popultionbsed smple, using stndrdized nd clibrted technique for mesurement, llowing us to compre cross lrge nd geogrphiclly diverse smple; discordnt smples nd techniques would mke this impossible. Using these unique dt, we report the impct of nemi lone, ID or IDA on dibetes nd predibetes prevlence estimtes in sex-strtified, popultion-bsed smple of Chinese dults with substntil vribility in iron, hemoglobin nd dibetes mesures. Our findings suggest tht ID my result in under-dignosis of dibetes nd nemi my result in over-dignosis of dibetes when using. Furthermore, differences in dibetes/predibetes clssifiction for individuls with IDA my be driven by the positive ssocition between nemi nd, but ttenuted by the opposing, negtive ssocition between ID nd. Our findings suggest tht inconsistent findings in the literture could be due to the complex disese etiology of IDA, nd confirm wrnings tht should only be used for glycemic ssessment in the bsence of ID nd/or nemi. These findings suggest tht concurrent mesurement of iron, hemoglobin nd is criticl to correctly interpret glycemi sttus in popultions with prevlent ID nd/or nemi. CONFLICT OF INTEREST The uthors declre no conflict of interest. ACKNOWLEDGEMENTS This work ws supported by NIH: the NIH Hert, Lung nd Blood Institute (R01- HL108427). This reserch uses dt from the Chin Helth nd Nutrition Survey, funded by NIH: The Eunice Kennedy Shriver Ntionl Institute of Child Helth nd Humn Development (NICHD; R01-HD30880), though no direct support ws received from grnt for this nlysis. SMA is supported by the Americn Hert Assocition (14PRE ). ALT is supported by NIH: NICHD (K01 HD ). We re grteful to the Crolin Popultion Center, University of North Crolin t Chpel Hill, for generl support (grnt R24 HD00924 from the NICHD). NIH hd no role in the design nd conduct of the study; collection, mngement, nlysis nd interprettion of the dt; nd preprtion, review or pprovl of the mnuscript. SMA nd PG-L re the gurntors of this work nd, s such, hd full ccess to ll the dt in the study nd tke responsibility for the integrity of the dt nd the ccurcy of the dt nlysis. AUTHOR CONTRIBUTIONS HW contributed to the collection of the dt. SMA, PG-L, EJM-D, LSA nd ALT contributed to the initil design of the nlysis. SMA, AHH, A-GH nd PG-L contributed to the nlysis. All uthors contributed to the interprettion of dt nlysis. SMA nd PG-L wrote the first drft of the mnuscript. All the uthors revised/edited the mnuscript. REFERENCES 1 Americn Dibetes Assocition. Dignosis nd clssifiction of dibetes mellitus. Dibetes Cre 2013; 36: S67 S74. 2 Bonor E, Tuomilehto J. The pros nd cons of dignosing dibetes with A1C. Dibetes Cre 2011; 34 (Suppl 2): S184 S Cowie CC, Rust KF, Byrd-Holt DD, Gregg EW, Ford ES, Geiss LS et l. Prevlence of dibetes nd high risk for dibetes using A1C criteri in the U.S. popultion in Dibetes Cre 2010; 33: World Helth Orgniztion. Worldwide Prevlence of Anemi : WHO Globl Dtbse on Anemi World Helth Orgniztion. Deficiency Anemi: Assessment, Prevention, nd Control. A guide for Progrmme Mngers Ahmd J, Rft D. nd iron : review. 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