ORIGINAL INVESTIGATION

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1 ORIGINAL INVESTIGATION Vitamin B 12 and Folate and the Risk of Anemia in Old Age The Leiden -Plus Study Wendy P. J. den Elzen, MSc; Rudi G. J. Westendorp, MD, PhD; Marijke Frölich, PhD; Wouter de Ruijter, MD; Willem J. J. Assendelft, MD, PhD; Jacobijn Gussekloo, MD, PhD Background: Screening for deficiencies in vitamin B 12 and folate is advocated to prevent anemia in very elderly individuals. However, the effects of vitamin B 12 and folate deficiency on the development of anemia in old age have not yet been established. Methods: The current study is embedded in the Leiden -Plus Study, a population-based prospective study of subjects aged years. Levels of vitamin B 12, folate, and homocysteine were determined at baseline. Hemoglobin levels and mean corpuscular volume (MCV) were determined annually during 5 years of follow-up. Results: We analyzed data from 423 subjects who did not use any form of cyanocobalamin, hydroxocobalamin, or folic acid supplementation, neither at baseline nor during follow-up. Folate deficiency ( 7 nmol/l; n=34) and elevated homocysteine levels ( 13.5 µmol/l; n=194) were associated with anemia at baseline (adjusted odds ratio [OR], 2.44; 95% confidence interval [CI], ; and adjusted OR, 1.82; 95% CI, , respectively), but vitamin B 12 deficiency ( 150 pmol/l; n=68) was not (adjusted OR, 1.51; 95% CI, ). Furthermore, vitamin B 12 deficiency was not associated with the development of anemia during follow-up (adjusted HR, 0.92; 95% CI, ) or with changes in MCV (adjusted linear mixed model; P=.77). Both folate deficiency and elevated homocysteine levels were associated with the development of anemia from age years onward (adjusted HR, 3.33; 95% CI, ; and adjusted HR, 1.70; 95% CI, , respectively), but not with an increase in MCV over time (P.30). Conclusion: In the general population of very elderly individuals, anemia in -year-old subjects is associated with folate deficiency and elevated homocysteine levels but not with vitamin B 12 deficiency. Arch Intern Med. 2008;168(20): Author Affiliations: Departments of Public Health and Primary Care (Ms den Elzen and Drs de Ruijter, Assendelft, and Gussekloo), Gerontology and Geriatrics (Dr Westendorp), and Clinical Chemistry (Dr Frölich), Leiden University Medical Center, Leiden, the Netherlands. THE PREVALENCE OF ANEMIA in elderly individuals aged 65 years or older is around 10% and increases with age to above 20% in subjects aged years or older. 1,2 Evidence of coincidental nutritional deficiencies (iron, vitamin B 12, and folate deficiency) can be found in clinical examinations of about one-third of older persons with anemia. 2 Because deficiencies in vitamin B 12 and folate are associated not only with macrocytic anemia but also with dementia, peripheral neuropathy, subacute combined degeneration, and cardiovascular disease (owing to elevated homocysteine levels), 3-6 low levels of these vitamins are usually supplemented. 4,5,7 And, because of the high prevalence of both deficiencies in elderly individuals ( 10% among people aged 75 years), 8-12 screening for deficiencies in vitamin B 12 and folate as part of a geriatric workup has also often been recommended. 13,14 Although the prevalence of vitamin B 12 and folate deficiency and the prevalence of anemia are highest in the oldest age groups ( years), to our knowledge, the association between vitamin B 12 and folate deficiency and anemia in old age has not been prospectively studied previously. Therefore, we investigated whether deficiencies in vitamin B 12 and folate are associated with the occurrence of anemia in the general population of very elderly persons ( years). Because serum levels of vitamin B 12 and folate alone may not accurately reflect vitamin status at tissue level, we also studied the effect of elevated serum homocysteine concentration on anemia, which is considered to be a more sensitive marker of these deficiencies at tissue level. 6,15,16 METHODS STUDY POPULATION AND PROCEDURES The present study is embedded in the Leiden - Plus Study, a population-based prospective follow-up study of -year-old inhabitants of 2238

2 Leiden, the Netherlands. From September 1997 through September 1999, 705 inhabitants of Leiden reached the age of years and were eligible for participation in the study. No other selection criteria were applied. Fourteen subjects died prior to enrollment in the study, and 92 subjects refused to participate. Seven subjects died before blood sample collection, and 30 subjects refused blood sampling. Vitamin B 12 levels could not be measured for 3 subjects owing to analytical difficulties. As a result, data for 559 subjects were available at baseline. Subjects were visited annually (at ages - years) at their place of residence to participate in face-to-face interviews, to perform several (cognitive) tests (eg, Mini-Mental State Examination 17,18 ), and to obtain a venous blood sample. General practitioners and nursing home physicians were interviewed annually to obtain information on patients medical history. Mortality data were obtained from the municipal registry. All subjects gave their informed consent, and the medical ethics committee of Leiden University Medical Center approved the study. Pharmacy records were studied annually, and subjects were interviewed to evaluate the use of any form of (over-the-counter) supplements containing cyanocobalamin, hydroxocobalamin, or folicacid(includingmultivitamins)atbaselineandduringthefollowup period. A total of 136 subjects who used any form of cyanocobalamin, hydroxocobalamin, or folic acid supplements at baseline (n=92) or during follow-up (n=44) were excluded from the present study. Treating physicians were not informed about the individual levels of vitamin B 12, folate, and homocysteine during the study because the analysis of serum samples was performed 2 to 3 years after blood samples were drawn. Figure 1 shows the annual number of subjects from ages through years for whom hemoglobin (Hb) levels and mean corpuscular volume (MCV) were determined, starting with 423 subjects at age years (baseline). The annual mortality rate was approximately 10%, and the annual refusal rate was below 5%. MAIN LABORATORY MEASUREMENTS The Hb levels and MCV were determined annually (at ages - years) with an automated analysis system (Coulter Counter; Coulter Electronics, Hialeah, Florida). Anemia was defined according to World Health Organization criteria 19 (Hb 12 g/dl for women and Hb 13 g/dl for men). An MCV higher than µm 3 was considered to be elevated. (To convert Hb to millimoles per liter, multiply by 0.625; to convert Hb to grams per liter, multiply by 10.0; to convert MCV to femtoliters, multiply by 1.0.) Serum samples were stored at 80 C. Serum levels of vitamin B 12 and folate at age years were determined in 1 batch using the Dual Count Solid Phase No-Boil Assay (Diagnostic Products Corp, Los Angeles, California). Homocysteine levels for all subjects at age years were measured in 1 batch with a fluorescence polarization immunoassay on an IMx analyzer (Abbott, Abbott Park, Illinois) after reduction to the free form. Vitamin B 12 deficiency was defined as serum vitamin B 12 levels lower than 150 pmol/l. Folate deficiency was defined as serum folate levels lower than 7 nmol/l. Serum homocysteine levels higher than 13.5 µmol/l were considered to be elevated. (To convert serum vitamin B 12 to picograms per milliliter, divide by ; to convert folate to nanograms per milliliter, divide by ) POSSIBLE CONFOUNDERS Sex, level of education, income, institutionalization, the presence of (chronic) disease, and renal function were considered possible confounding variables in the present study. Information on the presence of (chronic) disease at age years (stroke, myocardial infarction, severe cognitive impairment, diabetes mellitus, Parkinson disease, hip fracture, arthritis, 423 Aged years 368 Aged years 316 Aged years 275 Aged years 226 Aged years 1 Aged years 24 Refused 31 Died 10 Refused 42 Died 8 Refused 33 Died 13 Refused 36 Died 13 Refused 23 Died Figure 1. Annual number of subjects for whom hemoglobin levels and mean corpuscular volume were determined from age through years. obstructive lung disease, and malignant lesion) was obtained from general practitioners, nursing home physicians, and pharmacy records. 20 The presence of severe cognitive impairment was based on diagnosis by the general practitioner or on a Mini-Mental State Examination score of less than 19. C-reactive protein (CRP) levels were measured as an additional marker of health status with a fully automated Hitachi 747 system (Hitachi, Tokyo, Japan). Creatinine clearance was measured as a proxy for renal function and was determined by the Cockcroft-Gault equation. 21 STATISTICAL ANALYSIS Cross-sectional Analyses Differencesinproportionsofsubjectswithanemiaandproportions of subjects with elevated MCV between subjects with vitamin B 12 deficiency vs those without, with folate deficiency vs those without, and with elevated homocysteine levels vs those without at age yearsweretestedwith 2 tests.differencesinhblevelsandmcv between groups were tested with Mann-Whitney tests. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for the relation between vitamin B 12 deficiency and anemia, folate deficiency and anemia, and elevated homocysteine levels and anemia at the age of years. Logistic regression models were used to adjust for possible confounders (sex, level of education, income, institutionalization, the presence of 1 [chronic] diseases, CRP levels, and creatinine clearance). Prospective Analyses For the prospective analyses, subjects with anemia at baseline (ie, at age years) were excluded. The effects of vitamin B 12 deficiency, folate deficiency, and elevated homocysteine level at baseline (age years) on the development of anemia during follow-up were investigated with Kaplan-Meier curves and Cox proportional hazard models. If a new case of anemia was identi- 2239

3 Table 1. Baseline Characteristics of 423 Subjects Aged Years a Characteristic Value Sociodemographic characteristics Men 144 (34.0) Low level of education (elementary school only) b 276 (65.7) Low income ( $1/mo) c 220 (52.9) Institutionalized (20.3) Hematological characteristics Anemia d 110 (26.0) Macrocytic anemia (MCV µm 3 ) 2 (1.8) Normocytic anemia (MCV 80- µm 3 ) 102 (92.7) Microcytic anemia (MCV 80 µm 3 ) 6 (5.5) Hb, g/dl 13.0 ( ) MCV, µm 3 91 (-94) MCV µm 3 10 (2.4) Vitamin characteristics Serum vitamin B 12, pmol/l ( ) Vitamin B 12 deficiency ( 150 pmol/l) 68 (16.1) Serum folate, nmol/l 11.9 ( ) Folate deficiency ( 7 nmol/l) 34 (8.0) Serum homocysteine, µmol/l 13.1 ( ) Elevated homocysteine ( 13.5 µmol/l) 194 (45.9) Other clinical characteristics (Chronic) diseases, No. e None 101 (24.0) (41.9) (34.0) CRP, mg/l 4 (1-8) Creatinine clearance, ml/min f 44.3 ( ) Abbreviations: CRP, C-reactive protein; Hb, hemoglobin; MCV, mean corpuscular volume. Conversion factors: To convert serum vitamin B 12 to picograms per milliliter, divide by ; to convert C-reactive protein to nanomoles per liter, multiply by 9.524; to convert folate to nanograms per milliliter, divide by 2.266; to convert Hb to millimoles per liter, multiply by 0.625; to convert Hb to grams per liter, multiply by 10.0; to convert MCV to femtoliters, multiply by 1.0. a Continuous data are presented as median (interquartile range); categorical data are presented as number (percentage). b There are 3 missing values. c There are 7 missing values. d Anemia defined according to World Health Organization criteria 19 : Hb level lower than 12 g/dl for women and lower than 13 g/dl for men. e Stroke, myocardial infarction, severe cognitive impairment, diabetes mellitus, Parkinson disease, hip fracture, arthritis, obstructive lung disease, and malignant lesion; 3 missing values. f The Cockcroft-Gault formula was used; there are 11 missing values. fied, it was assumed that the anemia had developed halfway through the annual follow-up period. The Cox proportional hazard models were also adjusted for possible confounders. Between-group differences in changes in Hb levels and MCV during follow-up were estimated with linear mixed models, with adjustment for possible confounding variables. Results were presented as predicted means and their standard errors. For the models examining changes in MCV, subjects at age years with MCV µm 3 were excluded. Data analyses were performed using SPSS statistical software (version for Windows; SPSS Inc, Chicago, Illinois). RESULTS STUDY POPULATION Table 1 describes the baseline characteristics of the study population. At age years, 34.0% of the population were male, and 20.3% were institutionalized into care homes for the elderly. The prevalence of anemia at baseline was 26.0% (n=110). Most subjects with anemia had normocytic anemia(mcv,80-µm 3 [92.7%]).Macrocyticanemia(MCV µm 3 ) was observed in only 2 subjects (1.8%). Six subjects (5.5%) had microcytic anemia (MCV 80 µm 3 ). The median serum vitamin B 12 level was pmol/l (interquartile range [IQR], pmol/l); 68 subjects (16.1%)hadvitaminB 12 deficiency( 150pmol/L).Themedian folate level was 11.9 nmol/l (IQR, nmol/l); 34 subjects (8.0%) had folate deficiency ( 7 nmol/l). Elevated homocysteine levels ( 13.5 µmol/l) were observed in 194 subjects (45.9% of the study population). CROSS-SECTIONAL DATA At age years, vitamin B 12 deficiency and folate deficiency were both associated with the presence of elevated homocysteine levels (ORs, 1. [95% CI, ] and 10.3 [95% CI, ], respectively). Vitamin B 12 deficiency was not associated with the presence of anemia (crude OR, 1.23 [95% CI, ]; adjusted OR, 1.51 [95% CI, ]; Table 2). There were no differences in Hb levels between subjects with vitamin B 12 deficiency and those with vitamin B 12 levels within reference range (P=.59). Subjects with vitamin B 12 deficiency had a higher median MCV than those with vitamin B 12 levels within reference range (93 µm 3 [IQR, -96 µm 3 ]vs91µm 3 [IQR, -94 µm 3 ]; P=.01). Folate deficiency was associated with anemia at age years (crude OR, 2.79 [95% CI, ]; adjusted OR, 2.44 [95% CI, ]). Subjects with folate deficiency had lower Hb levels (P.01) and higher MCV (P=.07) compared with subjects with folate levels within reference range. Subjects with elevated homocysteine levels had an increased risk of anemia (crude OR, 2.81 [95% CI, ]; adjusted OR, 1.82 [95% CI, ]) and lower Hb levels compared with subjects with homocysteine levels within reference range (P.01). No differences were observed in MCV between subjects with elevated homocysteine levels and subjects with homocysteine levels within reference range (P=.91). In an adjusted logistic regression model including vitamin B 12 deficiency, folate deficiency, and elevated homocysteine levels, no independent association with anemia was found (ORs, respectively, 1.40 [95% CI, ], 2.02 [95% CI, ], and 1.56 [95% CI, ]). PROSPECTIVE DATA Of the 313 subjects without anemia at baseline, 72 subjects developed anemia in 4 observed person-years (incidence rate: 7.2 per person-years at risk). Figure 2 shows the occurrence of anemia during followup, with respect to vitamin B 12, folate, and homocysteine status at baseline. Vitamin B 12 deficiency was not associated with the incidence of anemia during follow-up (crude HR, 0. [95% CI, ]). Furthermore, adjustment for sex, level of education, income, institutionalization, the presence of 1 or more (chronic) diseases, CRP levels, and creatinine clearance did not change the estimate 2240

4 Table 2. Hemoglobin (Hb) Levels and MCV Depending on the Presence of Vitamin B 12 Deficiency, Folate Deficiency, and Elevated Homocysteine Levels at Age Years a Vitamin B 12 Folate Homocysteine RR Deficiency P RR Deficiency P RR Elevated P Parameter (n=355) (n=68) b Value (n=3) (n=34) c Value (n=229) (n=194) d Value Median Hb level, 13.0 ( ) 13.0 ( ) ( ) 12.4 ( ) ( ) 12.8 ( ).01 g/dl (IQR) Anemia, % e Median MCV, µm 3 91 (-94) 93 (-96) (-94) 92 (-96) (-94) 91 (-95).91 (IQR) MCV µm 3, % Abbreviations: IQR, interquartile range; MCV, mean corpuscular volume; RR, reference range. Conversion factors: To convert folate to nanograms per milliliter, divide by 2.266; to convert Hb to millimoles per liter, multiply by 0.625; to convert serum vitamin B 12 to picograms per milliliter, divide by ; to convert Hb to grams per liter, multiply by 10.0; to convert MCV to femtoliters, multiply by 1.0. a Data are presented as median (IQR) or percentage; P values were obtained by Mann-Whitney (continuous variables) or 2 tests (dichotomous variables). b Vitamin B 12 level lower than 150 pmol/l. c Folate level lower than 7 nmol/l. d Homocysteine level greater than 13.5 µmol/l. e Anemia defined according to World Health Organization criteria 19 : Hb level lower than 12 g/dl for women and lower than 13 g/dl for men. A Normal vitamin B 12 levels (n = 265) Vitamin B 12 deficiency (n = 48) B Normal folate levels (n = 295) Folate deficiency (n = 18) C Normal homocysteine levels (n = 1) Elevated homocysteine levels (n = 123) Cumulative Incidence of Anemia, % Figure 2. The effect of vitamin B 12 deficiency, folate deficiency, and elevated homocysteine levels on anemia during follow-up in subjects without anemia at age years (n=313). A, The effect of vitamin B 12 deficiency ( 150 pmol/l); hazard ratio (HR), 0.; 95% confidence interval (CI), B, Folate deficiency ( 7 nmol/l); HR, 3.16; 95% CI, C, Elevated homocysteine levels ( 13.5 µmol/l); HR, 1.96; 95% CI, To convert serum vitamin B 12 to picograms per milliliter, divide by ; to convert folate to nanograms per milliliter, divide by (adjusted HR, 0.92 [95% CI, ]). Subjects with folate deficiency had an increased risk of developing anemia over time compared with those with folate levels within reference range (crude HR, 3.16 [95% CI, ]; adjusted HR, 3.33 [95% CI, ]). Subjects with elevated homocysteine levels were also at an increased risk of developing anemia(crude HR, 1.96[95% CI, ]; adjusted HR, 1.70 [95% CI, ]). In an adjusted Cox regression model including vitamin B 12 deficiency, folate deficiency, and elevated homocysteine levels, only folate deficiency was independently associated with the incidence of anemia (HRs, respectively, 0. [95% CI, ], 2.77 [95% CI, ], and 1.48 [95% CI, ]). Figure 3 shows Hb levels and MCV during the 5-year follow-up period with respect to the presence or absence of vitamin B 12 deficiency, folate deficiency, and elevated homocysteine levels at baseline. The analyses were adjusted for sex, level of education, income, institutionalization, the presence of (chronic) diseases, CRP levels, and creatinine clearance. In subjects with vitamin B 12 deficiency, changes in Hb levels were similar to those with vitamin B 12 levels within reference range (P =.38). Subjects with folate deficiency had an accelerated decline in Hb levels compared with subjects with folate levels within reference range (additional annual decline, 0.19 g/dl [95% CI, 0.30 to 0.07]; P=.05). Subjects with elevated homocysteine levels also showed an accelerated decline in Hb levels when compared with subjects with homocysteine levels within reference range (additional annual decline, 0.10 g/dl [95% CI, 0.15 to 0.04]; P=.02). After excluding subjects with folate deficiency from the analyses, elevated homocysteine levels were no longer associated with the decline in Hb level (P=.13). 2241

5 A B 14.0 Predicted Mean (SE) Hb Level, g/dl Normal vitamin B 12 levels (n = 265) Vitamin B 12 deficiency (n = 48) P =.38 Predicted Mean (SE) MCV, µm Normal vitamin B 12 levels (n = 346) Vitamin B 12 deficiency (n = 67) P = n = 313 n = 281 n = 253 n = 222 n = 1 n = n = 413 n = 360 n = 309 n = 268 n = 220 n = 1 C D 14.0 Predicted Mean (SE) Hb Level, g/dl Normal folate levels (n = 295) Folate deficiency (n = 48) P =.05 Predicted Mean (SE) MCV, µm Normal folate levels (n = 381) Folate deficiency (n = 32) P = n = 313 n = 281 n = 253 n = 222 n = 1 n = n = 413 n = 360 n = 309 n = 268 n = 220 n = 1 E F 14.0 Predicted Mean (SE) Hb Level, g/dl Normal homocysteine levels (n = 1) Elevated homocysteine levels (n = 123) P =.02 Predicted Mean (SE) MCV, µm Normal homocysteine levels (n = 224) Elevated homocysteine levels (n = 1) P = n = 313 n = 281 n = 253 n = 222 n = 1 n = n = 413 n = 360 n = 309 n = 268 n = 220 n = 1 Figure 3. Changes in hemoglobin (Hb) levels and mean corpuscular volume (MCV) during follow-up. Left panels: Changes in hemoglobin levels during follow-up depending on the presence of vitamin B 12 deficiency ( 150 pmol/l) (A), folate deficiency ( 7 nmol/l) (C), and elevated homocysteine levels ( 13.5 µmol/l) (E) at age years. Subjects with anemia at age years (n=110) were excluded from the analyses. Right panels: Changes in MCV during follow-up depending on the presence of vitamin B 12 deficiency ( 150 pmol/l) (B), folate deficiency ( 7 nmol/l) (D), and elevated homocysteine levels ( 13.5 µmol/l) (F) at age years. Subjects with MCV greater than fl at age years (n=10) were excluded from the statistical analyses. All changes were adjusted for sex, levelof education, income, institutionalization, the presence of 1 or more (chronic) diseases (ie, stroke, myocardial infarction, severe cognitive impairment, diabetes mellitus, Parkinson disease, hip fracture, arthritis, obstructive lung disease, and malignant lesion), C-reactive protein levels, and creatinine clearance at baseline. To convert serum vitamin B 12 to picograms per milliliter, divide by ; to convert folate to nanograms per milliliter, divide by There were no differences in changes in MCV between subjects with vitamin B 12 deficiency vs those without (P=.77), or between subjects with folate deficiency vs those without (P=.39), or between subjects with elevated homocysteine levels vs those without (P=.73). COMMENT The present study shows that among very elderly individuals in the general population, both folate deficiency and elevated homocysteine levels were associated with 2242

6 the presence of anemia at baseline and the development of anemia during follow-up. No such relationship was present between vitamin B 12 deficiency and anemia. Although (macrocytic) anemia is one of the most generally known consequences of vitamin B 12 deficiency, 3-6 we did not find an association between vitamin B 12 deficiency and the presence or development of anemia within the general population of very elderly persons. The present data are, however, in line with clinical trials in mainly elderly subjects with low or subnormally low vitamin B 12 levels (detected by screening), without any other clinical symptoms, which showed that cyanocobalamin administration had no effect on Hb levels In a previous study, 25 we also showed that low vitamin B 12 levels at age years do not predict accelerated deterioration in cognitive function. These observational findings were in line with other observational studies and with several randomized controlled trials that showed no effect of cyanocobalamin administration on cognitive function as well This accumulating evidence suggests that clinicians should reconsider starting cyanocobalamin or hydroxocobalamin supplementation in very elderly patients ( years) with low vitamin B 12 levels. Because the metabolic pathways of vitamin B 12, folate, and homocysteine are tightly entwined, it is difficult to unravel which deficiency is the direct cause of anemia. When combining vitamin B 12 deficiency, folate deficiency, and elevated homocysteine levels in 1 Cox proportional hazard model, only folate deficiency remained independently associated with the development of anemia in our study population. One possible explanation is that folate is more critical for DNA synthesis than vitamin B 12. Within the cell, folate is converted into 5-methyl-tetrahydrofolate; vitamin B 12 is converted into methylcobalamin, which is a coenzyme for methionine synthase. Methionine synthase converts homocysteine into methionine by transferring a methyl group from 5-methyl-tetrahydrofolate to homocysteine, thereby lowering homocysteine levels. This reaction converts 5-methyl-tetrahydrofolate into tetrahydrofolate (THF); THF is essential for the formation of thymidylate and purine synthesis, both of which are required for DNA replication. 5,29 When THF production is decreased, DNA synthesis is delayed, and anemia develops owing to ineffective erythropoiesis and destruction of abnormal circulating red cells. 30,31 Thus, folate is a direct precursor of the components of DNA, whereas vitamin B 12 is only indirectly involved in DNA synthesis by assisting in the conversion of 5-methyl-tetrahydrofolate into THF. Because vitamin B 12 is a precursor of a coenzyme, it can assist many subsequent enzymatic reactions, and even severely low levels of vitamin B 12 may be sufficient to produce adequate levels of THF. In this light, folic acid supplementation could increase the level of THF and directly restore erythropoiesis, but the effects of cyanocobalamin or hydroxocobalamin supplementation on erythropoiesis may be smaller. Because the strong association between elevated homocysteine levels and anemia disappears after adjustment for the presence of folate deficiency, elevated homocysteine levels will identify older subjects at increased risk of developing anemia, but homocysteine does not seem to be causally related to anemia in old age. Strong points of the present study are its prospective design, an almost complete follow-up, and the populationbased setting, allowing us to unravel the temporal relationship between vitamin B 12 deficiency, folate deficiency, and subsequent changes in Hb levels and MCV in the general population of older persons. In addition, we measured serum homocysteine levels because it is well known that serum levels of vitamin B 12 and folate may not accurately reflect the vitamins true status within tissues. 6,15,16 It may be considered a limitation of our study that we evaluated only subjects aged years or older. However, it is important to study anemia in very elderly subjects, not only because they are the fastest growing segment of the general population in industrialized societies but also because the prevalence of anemia and deficiencies in vitamin B 12 and folate rapidly increase with age. 1,2,8-11 More important, results from studies in elderly subjects who are younger than years cannot be extrapolated to those years or older because the risk of common determinants of disease and mortality in middle age, such as hypothyroidism, hypertension, and hypercholesterolemia, have already been shown to reverse or disappear in those years or older For the same reasons, the results of the present study should not be extrapolated to the population of those younger than years. The small number of subjects with folate deficiency, especially in the prospective analyses, may also be considered a potential limitation of our study. Yet, the fact that even with this low number of subjects we still find a statistically significant association between folate deficiency and the development of anemia (adjusted HR, 3.33; 95% CI, ) and changes in Hb levels over time (P =.05) suggests that folate deficiency is a strong risk factor for developing anemia in old age. Another limitation of our study could be that vitamin B 12, folate, and homocysteine levels were not measured annually. Nevertheless, we believe that correction of low vitamin B 12 and folate levels is not underlying the lack of association between low vitamin B 12 levels and the development of anemia during follow-up. First, subjects known to have used cyanocobalamin, hydroxocobalamin, or folic acid supplements at baseline or during follow-up were excluded from the analyses. Second, treating physicians were not informed about the results of the vitamin B 12, folate, and homocysteine measurements during the study because the laboratory analyses were performed 2 to 3 years after blood samples were drawn. In conclusion, anemia is an important health issue in elderly individuals because it is highly prevalent in this sector of the population and is associated with poor functional status and increased mortality. 1-3,37-43 Based on our findings, however, screening and subsequent treatment of low vitamin B 12 levels may not have any beneficial effect on the occurrence of anemia in old age, but early detection of folate deficiency by screening may identify elderly individuals at risk of developing anemia. Although the biochemical pathways suggest that folic acid supplementation is beneficial, it remains to be seen if folic acid fortification of grain and cereal products (a practice in the United States 44,45 ) has a positive effect on the incidence of anemia in the very elderly population. 2243

7 Accepted for Publication: April 20, Correspondence: Wendy P. J. den Elzen, MSc, Department of Public Health and Primary Care, Leiden University Medical Center, Post Zone V-0-P, PO Box 9600, 2300 RC Leiden, the Netherlands Author Contributions: Dr Gussekloo had full access to all of the data in the study and takes responsibility for the integrityofthedataandtheaccuracyofthedataanalysis.study concept and design: Westendorp and Gussekloo. Acquisition of data: Westendorp, Frölich, and Gussekloo. Analysis and interpretation of data: den Elzen, Westendorp, Frölich, de Ruijter, Assendelft, andgussekloo. Draftingofthemanuscript: den Elzen and Gussekloo. Critical revision of the manuscript for important intellectual content: den Elzen, Westendorp, Frölich, de Ruijter, Assendelft, and Gussekloo. Statistical analysis: den Elzen, Westendorp, and Gussekloo. Obtained funding: Westendorp and Gussekloo. Administrative, technical, and material support: den Elzen and Frölich. Study supervision: Westendorp, Assendelft, and Gussekloo. Financial Disclosure: None reported. Funding/Support: The Leiden -Plus Study was funded in part by the Dutch Ministry of Health, Welfare, and Sports. Homocysteine measurements were funded in part by an unrestricted grant from Abbott Laboratories. REFERENCES 1. Beghé C, Wilson A, Ershler WB. Prevalence and outcomes of anemia in geriatrics. Am J Med. 2004;116(suppl 7A):3S-10S. 2. Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004;104(8): Andrès E, Loukili NH, Noel E, et al. Vitamin B12 (cobalamin) deficiency in elderly patients. CMAJ. 2004;171(3): Wolters M, Strohle A, Hahn A. Cobalamin: a critical vitamin in the elderly. Prev Med. 2004;39(6): Babior BM, Bunn HF. Megaloblastic anemias. In: Kasper DL, Braunwald E, Fauci AS, et al, eds. Harrison s Principles of Internal Medicine. 16th ed. 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