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1 The Joural of Nutritio Nutritioal Epidemiology Supplemetal Material ca be foud at: 9.DC1.html Estimatio of Treds i Serum ad RBC Folate i the U.S. Populatio from Pre- to Postfortificatio Usig Assay-Adjusted Data from the NHANES Christie M. Pfeiffer, 4 * Jeffery P. Hughes, 5 David A. Lacher, 5 Rega L. Bailey, 6 R. J. Berry, 7 Midy Zhag, 4 Elizabeth A. Yetley, 6 Jeae I. Rader, 8 Christopher T. Sempos, 6 ad Clifford L. Johso 5 4 Natioal Ceter for Evirometal Health, CDC, Atlata, GA; 5 Natioal Ceter for Health Statistics,CDC,Hyattsville,MD; 6 Office of Dietary Supplemets, NIH, Bethesda, MD; 7 Natioal Ceter for Birth Defects ad Developmetal Disabilities, CDC, Atlata, GA; ad 8 Food ad Drug Admiistratio, College Park, MD Abstract The NHANES has moitored status of the U.S. populatio from prefortificatio ( ) to postfortificatio ( ) by measurig serum ad RBC cocetratios. The Bio-Rad radioassay (BR) was used from 1988 to 2006, ad the microbiologic assay (MBA) was used from 2007 to The MBA produces higher cocetratios tha the BR ad is cosidered to be more accurate. Thus, to bridge assay differeces ad to examie treds over time, we adjusted the BR results to be comparable to the MBA results. Postfortificatio, assay-adjusted serum ad RBC cocetratios were 2.5 times ad 1.5 times prefortificatio cocetratios, respectively, ad showed a sigificat liear tred (P, 0.001) to slightly lower cocetratios durig The postfortificatio prevalece of low serum (,10 mol/l) or RBC (,340 mol/l) cocetratios was #1%, regardless of demographic subgroup, compared with 24% for serum ad 3.5% for RBC prefortificatio, with substatial variatio amog demographic subgroups. The cetral 95% referece itervals for serum ad RBC varied by demographic subgroup durig both pre- ad postfortificatio periods. Age ad dietary supplemet use had the greatest effects o prevalece estimates of low cocetratios durig the prefortificatio period. I summary, the MBA-equivalet blood cocetratios i the U.S. populatio showed first a sharp icrease from pre- to postfortificatio, the showed a slight decrease (17% for serum ad 12% for RBC ) durig the 12-y postfortificatio period. The MBA-equivalet pre- ad postfortificatio referece cocetratios will iform coutries that pla folic acid fortificatio or that eed to evaluate its impact. J. Nutr. 142: , Itroductio The status of the U.S. populatio has bee assessed for may years as part of the NHANES by measurig serum ad RBC cocetratios, first ( ) with the Bio-Rad radioassay (BR) 9, ad the ( ) with the microbiologic 1 Supported by fudig from the Office of Dietary Supplemets, NIH. The fidigs ad coclusios i this report are those of the authors ad do ot ecessarily represet the official views or positios of the CDC ad Prevetio/Agecy for Toxic Substaces ad Disease Registry, the NIH, or the Departmet of Health ad Huma Services. 2 Author disclosures: C. M. Pfeiffer, J. P. Hughes, D. A. Lacher, R. L. Bailey, R. J. Berry, M. Zhag, E. A. Yetley, J. I. Rader, C. T. Sempos, ad C. L. Johso, o coflicts of iterest. 3 Supplemetal Tables 1 6 are available from the Olie Supportig Material lik i the olie postig of the article ad from the same lik i the olie table of cotets at 9 Abbreviatios used: BR, Bio-Rad radioassay; DFE, dietary equivalets; MA, Mexica America; MBA, microbiologic assay; NCHS, Natioal Ceter for Health Statistics; NHB, o-hispaic black; NHW, o-hispaic white; PIR, poverty-to-icome ratio. * To whom correspodece should be addressed. cpfeiffer@cdc.gov. growth assay (MBA) usig Lactobacillus rhamosus (formerly kow as Lactobacillus casei) (1). Because the MBA produces much higher cocetratios tha does the BR (2 4), a 2010 roudtable of experts o NHANES ad vitami B-12 measuremet issues agreed that a adjustmet equatio based o a crossover study was ecessary for time-tred evaluatios (5). The MBA is cosidered more accurate because it recovers vitamers equally, which is ot always the case with cliical protei-bidig assays (3,4,6). The MBA is sometimes used i research studies ad populatio surveys other tha NHANES because it requires oly small sample volumes, ca be coducted i a high-throughput format, ad is a comparatively iexpesive assay (7). It is also the assay agaist which the accuracy of other assays is evaluated (5,8). For all of these reasos, it is of iterest to scietists ad public health officials worldwide to have iformatio o pre- ad postfortificatio status of the U.S. populatio based o MBA-equivalet serum ad RBC data. Previous reports have assessed the impact of folic acid fortificatio o serum ad RBC cocetratios i the geeral U.S. populatio ad i wome of childbearig age durig ã 2012 America Society for Nutritio. 886 Mauscript received December 21, Iitial review completed Jauary 30, Revisio accepted February 20, First published olie March 21, 2012; doi: /j

2 the first few years postfortificatio by usig data geerated with the BR (9 12). No reports are yet available for data geerated after 2006 or by expressig the data as MBA-equivalet cocetratios. I this article we preset the ewest serum ad RBC data geerated with the MBA for NHANES ad the ew MBA-equivalet blood data for 8 previous postfortificatio years ( ) ad 6 prefortificatio years ( ) by sociodemographic variables. We preset log-term treds i status of the U.S. populatio ad provide prevalece estimates of low blood cocetratios for pre- ad postfortificatio time periods. Last, we provide multistratified cetral 95% referece itervals by age, geder, ad race/ethicity for prefortificatio, early postfortificatio ( ), ad late postfortificatio ( ) time periods. A separate article describes the process by which the BR data were adjusted to MBA-equivalet data ad the impact of the adjustmet (13). Participats ad Methods Survey desig ad participats. The NHANES, which is coducted by the Natioal Ceter for Health Statistics (NCHS) at the CDC, collects cross-sectioal data o the health ad utritioal status of the civilia oistitutioalized U.S. populatio (14). Prior to 1999, the survey was coducted periodically; sice 1999, NHANES has become a cotiuous survey with data released i 2-y cycles. NHANES obtais a stratified, multistage, probability sample desiged to represet the U.S. populatio o the basis of age, geder, ad race/ethicity. The race/ethicity categories are based o self-reported data. NCHS persoel first iterview survey participats i their homes. Durig this household iterview, iterviewers collect iformatio o demographic characteristics, dietary supplemet use, ad some health-related issues. Participats udergo a physical examiatio ad blood collectio i a Mobile Examiatio Ceter ;1 2 wk after the household iterview. They also complete a 24-h dietary recall. All respodets provided iformed coset, ad the NHANES protocol was reviewed ad approved by the NCHS Research Ethics Review Board. Iterview ad examiatio respose rates for each survey period are publically available (15). Laboratory methods. Durig NHANES , whole-blood hemolysate ad serum samples from participats aged $1 y were aalyzed for by the CDC laboratory by use of the MBA method (16,17). A short descriptio of the assay ad of steps coducted to verify the comparability of results over time is provided elsewhere (13,18). Log-term quality-cotrol CV for serum were % for ad % for (19,20). For RBC, CV were % for ad % for (19,20). The Bio-Rad Quataphase I radioassay was used durig ad the Quataphase II was used durig (Bio-Rad Laboratories). Appropriate assay adjustmets were applied to the data before their public release to accout for method differeces betwee the Quataphase I ad II (21). The performace of the BR has bee discussed i previous reports (2,10). Log-term CV were % for serum ad % for RBC (10). Statistical aalyses. Statistical aalyses were performed by usig SAS (versio 9; SAS Istitute, Ic.) ad SUDAAN (versio 9; RTI Iteratioal) software. I each NHANES survey period we used sample weights to accout for differetial orespose or ocoverage ad to adjust for oversamplig of some groups. We used the followig age groups: 4 11 y (childre), y (adolescets), y (adults; i some aalyses y ad y), ad $60 y (older persos; i some aalyses y ad $80 y). Wome of childbearig age (15 44 y) were cosidered as a separate category for most aalyses. We used the 3 mai race/ethicity categories Mexica America (MA), o-hispaic black (NHB), ad o-hispaic white (NHW) that ca be compared over the time period covered i this aalysis, but we icluded other racial/ethic groups i overall estimates. We applied regressio equatios to serum ad RBC results aalyzed by the BR durig to derive MBA-equivalet data (13,16,17). This allowed the assessmet of log-term treds i status that were ucofouded by assay differeces over this log time period. The BR measured cocetratios that were 31% lower tha those from the MBA for whole-blood samples with the MTHFR (5,10-methyleetetrahydro reductase) T/T geotype, but 48% lower for samples with the C/C ad C/T geotypes (4). This is because the BR recovered the various forms differetly compared with the MBA assay (4). MTHFR geotype iformatio is ot available for NHANES participats. We could therefore ot use geotype-specific regressio equatios ad had to use a all geotype equatio (13). The oly participats excluded from our aalyses were those for whom data were missig: we excluded 2 participats i because their uadjusted serum cocetratios were,1 mol/l ad the adjustmet formula requires logarithmic trasformatio (which produces a egative umber) ad the calculatig the square root. Also, we excluded a small fractio (1 2%) of participats i (556 of 23,402), (101 of 7491), (116 of 8336), (82 of 7618), ad (173 of 7751) because the RBC adjustmet formula requires serum, RBC, ad hematocrit, ad for these participats oe of these tests was missig. All aalyses preseted i this article were performed by usig MBA-equivalet data. We determied pre- ad postfortificatio geometric mea blood cocetratios by sociodemographic variables [age, geder, race/ethicity, ad family poverty-to-icome ratio (PIR)] ad dietary supplemet use, which are variables kow to ifluece blood cocetratios. We used geometric meas because distributios of blood cocetratios were skewed. For the postfortificatio time period ( ), we also determied geometric meas by survey period. The prefortificatio time period ( ) was cosidered oe survey period. We tested which of the variables had a sigificat effect o blood cocetratios by usig simple liear regressio: we used PROC REGRESS i SUDAAN (RTI Iteratioal) with a subgroup statemet, ad we used EFFECT statemets to test each hypothesis. We limited the postfortificatio blood data to whe we evaluated the effect of supplemet use because dietary supplemet use iformatio is ot yet available for By usig multiple liear regressio, we evaluated whether adjustmet for the above variables had a effect o the relatioship betwee blood cocetratios ad each variable of iterest. Model 1 icluded the sociodemographic variables for prefortificatio data ad the sociodemographic variables ad survey period for postfortificatio data ( ). Model 2 icluded the sociodemographic variables ad supplemet use for prefortificatio data ad the sociodemographic variables, supplemet use, ad survey period for postfortificatio data ( ). All statistical comparisos were evaluated at a sigificace level of a = To visualize pre- ad postfortificatio treds, we plotted the media ad IQR of serum ad RBC cocetratios for by survey period. We described treds of the upper ed of the serum ad RBC distributios over time by plottig the 95th percetile cocetratios for by survey period ad by supplemet use. We used the WHO-recommeded cutoffs of,10 mol/l for serum ad,340 mol/l for RBC (22) to assess low status, ad we determied the proportio of low serum ad RBC cocetratios for pre- ad postfortificatio data by sociodemographic variables ad dietary supplemet use. We also determied proportios by survey period for the postfortificatio time period. Because blood cocetratios varied by age, geder, ad race/ ethicity, we calculated multistratified cetral 95% referece itervals ( th percetiles) for the prefortificatio ( ), early postfortificatio ( ), ad late postfortificatio ( ) periods. Six years of NHANES data provided a sufficiet sample size to allow 3 levels of stratificatio ad still exceed a cell size of 448 i the majority of subgroups. This cell size is eeded for robust estimates of the 2.5th ad 97.5th percetiles at a assumed mea desig effect of 1.4. We plotted frequecy distributio curves of MBA-equivalet serum ad RBC cocetratios for the pre-, early post-, ad late postfortificatio periods. Results Variables ifluecig blood cocetratios. Durig the prefortificatio period, age, geder, race/ethicity, PIR, ad supplemet use affected serum ad RBC cocetratios (P # 0.001) (Table 1). All effects were maitaied after we Pre- ad postfortificatio treds i serum ad RBC 887

3 TABLE 1 Prefortificatio ( ) ad postfortificatio ( ) blood cocetratios by sociodemographic variable, dietary supplemet use, ad survey period i participats aged $4 y (NHANES ) Serum Serum RBC RBC mol/l mol/l mol/l mol/l Total 23, , , , Age group 4 11 y y , , y , , y y $80 y P 2,0.001,0.001,0.001,0.001 P-model 1 2,3,0.001,0.001,0.001,0.001 P-model 2 2,4,0.001,0.001,0.001,0.001 Geder 11, , , , , , , , Age y P 5,0.001, ,0.001 P-model 1 3,5,0.001, ,0.001 P-model 2 4, , ,0.001 Race/ethicity MA , , NHW , , NHB , , P 6,0.001,0.001,0.001,0.001 P-model 1 3,6,0.001,0.001,0.001,0.001 P-model 2 4,6,0.001,0.001,0.001,0.001 PIR, , , , , , , $ P 7,0.001,0.001,0.001,0.001 P-model 1 3,7,0.001,0.001,0.001,0.001 P-model 2 4, , ,0.001 Supplemet use 8 Yes , , No 15, , , , P 9,0.001,0.001,0.001,0.001 P-model 1 3,9 NA NA NA NA P-model 2 4,9,0.001,0.001,0.001,0.001 Survey period NA NA NA NA NA NA NA NA NA NA NA NA P 10 NA,0.001 NA,0.001 P-model 1 3,10 NA,0.001 NA,0.001 P-model 2 4,10 NA,0.001 NA, Values are geometric meas 6 SE. Serum ad RBC cocetratios were measured from by the Bio-Rad radioassay ad assay-adjusted to be comparable to the microbiologic assay that was used from (13,16,17). MA, Mexica America; NHB, o-hispaic black; NHW, o-hispaic white; NA, ot applicable; PIR, poverty-to-icome ratio. 2 Test comparig group meas across age categories. 3 P-value for multiple liear regressio model 1 icludig age, geder, race/ethicity, ad PIR for prefortificatio data ad age, geder, race/ethicity, PIR, ad survey period for postfortificatio data ( ). 4 P value for multiple liear regressio model 2 icludig age, geder, race/ethicity, PIR, ad supplemet use for prefortificatio data ad age, geder, race/ethicity, PIR, supplemet use, ad survey period for postfortificatio data ( ). 5 Test comparig males ad females. 6 Test comparig group meas across race/ethicity categories. 7 Test of liear tred across PIR categories. 8 Geometric meas by dietary supplemet use were limited to data from for the postfortificatio period. 9 Test comparig group meas across supplemet use categories. 10 Test of liear tred across survey periods. 888 Pfeiffer et al.

4 adjusted the prefortificatio serum ad RBC cocetratios for sociodemographic variables (P-model 1 # 0.001). However, whe we also adjusted for supplemet use, the differece betwee males ad females i prefortificatio RBC cocetratios disappeared (P-model 2 = 0.98), whereas all other effects remaied sigificat. Durig the postfortificatio period, age, geder, race/ethicity, PIR, ad supplemet use had a sigificat (P, 0.001) effect o serum ad RBC cocetratios (Table 1). All effects were maitaied after we adjusted the postfortificatio serum ad RBC cocetratios for sociodemographic variables ad survey period (P-model 1, 0.001) or for sociodemographic variables, survey period, ad supplemet use (P-model 2, 0.001; data limited to ). Postfortificatio geometric meas showed a sigificat (Ptred, 0.001) liear tred to lower cocetratios from 1999 to 2010, with serum ad RBC decliig up to 17% ad 12%, respectively, from to (Table 1). The liear tred remaied sigificat for serum ad RBC after adjustmet for sociodemographic variables (P-tred model 1, 0.001) or whe additioally adjusted for supplemet use (Ptred model 2, 0.001; data limited to ), idicatig that potetial chages i these variables over time did ot explai the decrease i blood cocetratios. Log-term treds i blood cocetratios. Geometric mea serum ad RBC cocetratios postfortificatio were 2.5 times ad 1.5 times prefortificatio cocetratios, respectively (Table 1). Media serum ad RBC cocetratios sharply icreased from pre- to postfortificatio, the showed small fluctuatios durig 12 y of postfortificatio (Fig. 1). The upper ed of the serum ad RBC distributios (95th percetiles) showed as expected a icrease from preto postfortificatio i both users ad ousers of dietary supplemets, ad the small fluctuatios from 1999 to 2008 (Fig. 2). Durig each survey period (pre- ad postfortificatio), serum cocetratios were ;30 mol/l higher i supplemet users tha i ousers, ad RBC cocetratios were ;750 mol/l higher. Pre- ad postfortificatio prevalece estimates of low blood cocetratios. The prefortificatio prevaleces of low serum (,10 mol/l) ad RBC (,340 mol/l) cocetratios were 24% ad 3.5%, respectively (Table 2). Prevalece varied by demographic subgroup, with the biggest differece observed i differet age groups: compared with all other age groups, childre (age 4 11 y) had the lowest prevalece (2.8% for serum ad 0.7% for RBC ), whereas youg adults (age y) had the highest prevalece (33% for serum ad 4.5% for RBC ). The differece i prevalece betwee supplemet users (13.5% for serum ad 1.5% for RBC ) compared with ousers (37.2% for serum ad 5.7% for RBC ) was also high. Postfortificatio, the prevalece of low serum ad RBC cocetratios was very low (geerally #1%), regardless of age, geder, race/ethicity, PIR, dietary supplemet use, or survey period (Table 2). Referece itervals ad distributios of blood cocetratios for pre-, early post-, ad late postfortificatio time periods. We combied 6 y each of NHANES serum (Table 3) ad RBC (Table 4) data to allow the geeratio of multistratified cetral 95% referece itervals by age, geder, ad race/ethicity. For childre (age 4 11 y) ad adolescets (age y), we observed little differece i serum FIGURE 1 Selected percetiles for pre- ad postfortificatio serum (A) ad RBC (B) cocetratios by survey period (NHANES ). Cocetratios measured from ad from by the Bio-Rad radioassay were adjusted to make them comparable to the cocetratios measured by microbiologic assay (13,16,17). Error bars represet 95% CIs. Sample sizes () for serum were as follows: 23,359 ( ), 7411 ( ), 8242 ( ), 7692 ( ), 7639 ( ), 7705 ( ), ad 8184 ( ). Sample sizes () for RBC were as follows: 22,846 ( ), 7393 ( ), 8220 ( ), 7618 ( ), 7578 ( ), 7728 ( ), ad 8222 ( ). pctl, percetile. referece itervals betwee males ad females (pre- ad postfortificatio), race/ethic groups (pre- ad postfortificatio), ad early versus late postfortificatio periods. For adults (age y) ad older persos (age $60 y), we observed little chage i serum referece itervals from early to late postfortificatio. However, wome geerally had higher serum cocetratios tha did me (pre- ad postfortificatio) ad NHW persos had higher cocetratios tha did NHB or MA persos (pre- ad postfortificatio). We oticed differet effects for RBC referece itervals. Regardless of age, we observed race/ethicity differeces (pre- ad postfortificatio), with NHW persos havig higher RBC cocetratios tha NHB ad MA persos. Also, regardless of age for most subgroups, we observed a slight decrease i RBC referece itervals from early to late postfortificatio. The frequecy distributio curves of serum ad RBC cocetratios showed a distict shift of the etire distributio to higher cocetratios from the pre- to the early postfortificatio period, the a mior shift to slightly lower cocetratios from the early to the late postfortificatio period for serum, but hardly ay chage i RBC (Fig. 3). To allow a detailed review of multistratified (by age, geder, ad race/ Pre- ad postfortificatio treds i serum ad RBC 889

5 The data also allow officials i other coutries where the MBA is used to compare their coutry-specific populatio data to those of the U.S. populatio. However, other coutries that evaluate the impact of fortificatio must also cosider the extet of their fortificatio (e.g., food vehicles ad levels of fortificatio) ad the percetage of their populatio that fortificatio is reachig whe comparig biomarker data. We cofirmed fidigs from previous aalyses (9 12,25) that showed differeces i blood cocetratios by sociodemographic subgroup. The ew fidig i this aalysis is that the differece i prefortificatio RBC cocetratios betwee males ad females disappeared after adjustmet for supplemet use. It has bee show that a higher percetage of females use supplemets compared with males (26), ad this FIGURE 2 Pre- ad postfortificatio 95th percetile serum (A) ad RBC (B) cocetratios for users (solid lie) ad ousers (dashed lie) of dietary supplemets by survey period (NHANES ). Cocetratios measured from 1988 to 1994 ad from 1999 to 2006 by the Bio-Rad radioassay were adjusted to make them comparable to the cocetratios measured by microbiologic assay (13,16,17). Error bars represet 95% CIs. Sample sizes () were the same as show i Figure 1. pctl, percetile. ethicity) distributio curves for serum (Supplemetal Tables 1 3) ad RBC (Supplemetal Tables 4 6) for pre-, early post-, ad late postfortificatio time periods, we provided selected percetiles from the 2.5th to the 97.5th percetile. Discussio To our kowledge, this article presets the first aalysis of more tha a decade of post folic acid fortificatio treds i serum ad RBC i a represetative sample of the U.S. populatio aged $4 y usig MBA-equivalet data. The evaluatio of data from 12 y postfortificatio ( ) ad comparig it with prefortificatio ( ) data was oly possible because of the availability of regressio equatios that allowed us to express all data o a MBA-equivalet basis, eve though some of the data ( ) were geerated with the BR (13). Two past (21,23) ad oe recet (5) expert pael who assessed chages i assays as well as a recet expert pael o 25- hydroxyvitami D assays (24) have all come to the coclusio that whe laboratory methods chage, data eed to be adjusted to allow for meaigful iterpretatio ad tred aalysis. The ew MBA-equivalet data allow U.S. public health officials for the first time to directly assess the prevalece of iadequate blood cocetratios pre- ad postfortificatio by usig updated cutoffs from a 2005 WHO Techical Cosultatio (22). 890 Pfeiffer et al. TABLE 2 Prefortificatio ( ) ad postfortificatio ( ) proportios of low blood cocetratios by sociodemographic variable, dietary supplemet use, ad survey period i participats aged $4 y (NHANES ) 1 Serum,10 mol/l RBC,340 mol/l % Total Age group 4 11 y y y y $60 y Geder Age y Race/ethicity MA NHB NHW PIR, $ Supplemet use 4 Yes No Survey period NA NA NA NA NA NA NA NA NA NA NA NA Values are percetages 6 SE. Serum ad RBC cocetratios were measured by the Bio-Rad radioassay from 1988 to 2006 ad assay-adjusted to be comparable to the microbiologic assay, which was used from 2007 to 2010 (13,16,17). values are the same as i Table 1. MA, Mexica America; NHB, o-hispaic black; NHW, o- Hispaic white; NA, ot applicable; PIR, poverty-to-icome ratio. 2 Estimate suppressed because the relative SE was $40%. 3 Relative SE was.30% but,40%. 4 Because iformatio o dietary supplemet use is ot yet available for , postfortificatio prevalece estimates were limited to data from 1999 to 2008.

6 TABLE 3 Demographic variable Cetral 95% referece itervals for serum cocetratios by demographic variable i participats age $4 y (NHANES ) 1 Prefortificatio ( ) Serum Early postfortificatio ( ) Serum Late postfortificatio ( ) may explai the higher blood cocetratios we observed i females. However, durig postfortificatio, whe RBC cocetratios were ;50% higher i both males ad females compared with the prefortificatio time period, adjustmet for supplemet use did ot remove the geder differece. The relative icreases i serum (2.5 times) ad RBC (1.5 times) cocetratios from pre- to postfortificatio usig MBA-equivalet data were very similar to earlier reports usig the origial BR data (10,12). Those reports, limited to 6 y ( ) ad 8 y ( ) of postfortificatio data, foud a sigificat liear tred showig slightly lower cocetratios durig the postfortificatio period ad discussed potetial reasos for the decrease, such as chages i cosumer Serum mol/l mol/l mol/l MA 4 11 y y y $60 y y y y $60 y NHB 4 11 y y y $60 y y y y $60 y NHW 4 11 y y y $60 y y y y $60 y Values are referece itervals ( th percetile). Serum cocetratios were measured by the Bio-Rad radioassay from 1988 to 1994 ad from 1999 to 2006 ad assay-adjusted to be comparable to the microbiologic assay, which was used from 2007 to 2010 (13,16,17). MA, Mexica America; NHB, o-hispaic black; NHW, o-hispaic white. 2 Cell size is smaller tha required ( = 448) to estimate the 2.5th ad 97.5th percetiles with sufficiet precisio at a assumed mea desig effect of 1.4. behavior ad reduced cotet of fortified breads. The yearly USDA per capita disappearace data also show a slight declie i daily per capita dietary equivalets (DFE) betwee 2000 (927 DFE) ad 2006 (874 DFE), supportig the idea that the decliig postfortificatio blood cocetratios could be explaied by decliig itakes (27). Our aalysis showed that adjustmet for potetial chages i sociodemographic variables ad dietary supplemet use over the course of 12 y postfortificatio did ot explai the decreases i serum ad RBC cocetratios of #17% ad 12%, respectively. Despite this sigificat decrease, however, the prevalece of low blood cocetratios remaied #1% throughout the etire 12-y time period ad TABLE 4 Demographic variable Cetral 95% referece itervals for RBC cocetratios by demographic variable i participats aged $4 y (NHANES ) 1 Prefortificatio ( ) RBC Early post-fortificatio ( ) RBC Late post-fortificatio ( ) RBC mol/l mol/l mol/l MA 4 11 y y y $60 y y y y $60 y NHB 4 11 y y y $60 y y y y $60 y NHW 4 11 y y y $60 y y y y $60 y Values are referece itervals ( th percetile). RBC cocetratios were measured by the Bio-Rad radioassay from 1988 to 1994 ad from 1999 to 2006 ad assay-adjusted to be comparable to the microbiologic assay, which was used from 2007 to 2010 (13,16,17). MA, Mexica America; NHB, o-hispaic black; NHW, o-hispaic white. 2 Cell size is smaller tha required ( = 448) to estimate the 2.5th ad 97.5th percetiles with sufficiet precisio at a assumed mea desig effect of 1.4. Pre- ad postfortificatio treds i serum ad RBC 891

7 FIGURE 3 Frequecy distributio curves for serum (A) ad RBC (B) cocetratios for prefortificatio ( ), early postfortificatio ( ), ad late postfortificatio ( ) time periods (NHANES ). Cocetratios measured from by the Bio-Rad radioassay were adjusted by regressig them to be comparable to the cocetratios measured by microbiologic assay (13,16,17). The vertical dashed lies represet the 10-mol/L low serum (A) ad 340-mol/L low RBC (B) cutoffs. Sample sizes () for serum were as follows: 23,359 ( ), 23,345 ( ), ad 23,528 ( ). Sample sizes () for RBC were as follows: 22,846 ( ), 23,231 ( ), ad 23,528 ( ). did ot exceed 1% for ay demographic or socioecoomic subgroup. Thus, these declies are ulikely to have biological sigificace with respect to status ad are ot of public health cocer. We have show that the cetral 95% referece itervals varied over time as well as with demographic subgroup. This uderscores the difficulty of usig referece itervals to derive cutoffs for assessig prevalece of low status. The curretly \recommeded WHO cutoffs for low serum (,10 mol/l) ad RBC (,340 mol/l) cocetratios have both bee derived from iflectio poit aalyses of cross-sectioal data i which the oe-carbo metabolite homocysteie, a fuctioal idicator of iadequate status, starts to icrease (28). These cutoffs are higher tha the traditioal cutoffs of,7 mol/l for serum ad,305 mol/l for RBC as defied by the 1998 Istitute of Medicie Committee that reviewed the DRI for (29). The Istitute of Medicie committee used small, covetioal metabolic ad depletio/repletio studies as the basis for their cutoffs. Eve whe usig the higher, more coservative WHO cutoffs, we foud #1% of the U.S. populatio with iadequate status durig 12 y postfortificatio. For the prefortificatio time period, it is surprisig to see how differet the prevalece estimates of low serum (24%) ad RBC 892 Pfeiffer et al. (3.5%) cocetratios are, cosiderig that the WHO cutoff for serum was derived from the same study populatio as the cutoff for RBC by lookig at the value at which a optimum total homocysteie cocetratio is achieved. Possibly, the differet withi-perso to betweeperso ratios for serum (0.192) compared with RBC (0.043) may affect prevalece estimates to some degree because of a wider spread i the tails of the serum distributio compared with the RBC distributio (1). The prevalece of low blood cocetratios (,10 mol/l for serum ad,340 mol/l for RBC ) durig prefortificatio varied by sociodemographic subgroup ad was lower i supplemet users (14% for serum ad 1.5% for RBC ) compared with ousers (37% for serum ad 5.7% for RBC ). Data from the Framigham cohort study, which used a slightly higher cutoff for low RBC (,363 mol/l), showed similar prefortificatio estimates of 1.6% (supplemet users) ad 4.9% (ousers) as well as similar postfortificatio estimates of 0% (supplemet users) ad 1.9% (ousers) (30). A secod Framigham cohort aalysis (31), which used a lower cutoff for serum (,6.8 mol/l), showed lower prefortificatio estimates of 3.9% (supplemet users) ad 22% (ousers) but similar postfortificatio estimates of 0% (supplemet users) ad 1.7% (ousers). A MBA was used to measure blood cocetratios i the Framigham cohort study, which may explai the similarity of prevalece estimates compared to the NHANES MBA-equivalet data. The major limitatio with this aalysis relates to the use of statistically adjusted data ad was discussed as part of a article that preseted equatios o how to adjust U.S. pre- ad postfortificatio blood cocetratios geerated by the BR to make them comparable to the MBA (13). Aother limitatio is that whe we geerated the assay-adjusted MBAequivalet RBC data, we were ot able to use geotypespecific regressio equatios to accout for the differet relatioship betwee the BR ad MBA assays for samples with the MTHFR T/T geotype compared with the C/C or C/T geotype (13). The differet geotype frequecies by race/ ethicity make it difficult to provide a accurate descriptio of race/ethicity differeces. The stregths of this article iclude the followig: its large pre- ad postfortificatio data sets that allow multiple levels of stratificatio; its desig that crosses over multiple postfortificatio survey periods, allowig a more accurate estimatio of time treds; its use of cutoffs for low blood cocetratios that have bee updated by WHO; ad its results expressed as MBA-equivalets, which makes them directly relevat to the assay method geerally accepted as accurate for assessig status. I summary, we have preseted the ewest serum ad RBC data geerated with the MBA for NHANES as well as MBA-equivalet blood data for 8 previous postfortificatio years ( ) ad 6 prefortificatio years ( ). After the itroductio of folic acid fortificatio, there was a sharp icrease i blood cocetratios, more tha doublig serum cocetratios ad icreasig RBC cocetratios by ;50%. Over the ext 12 y postfortificatio, serum cocetratios decreased by ;17% ad RBC cocetratios by ;12%. These decreases did ot affect the very low postfortificatio prevalece (#1%) of iadequate blood cocetratios. The curret data provide a ivaluable corerstoe for U.S. ad foreig public health officials i guidig ad evaluatig folic acid fortificatio policy ad i assessig populatio status.

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