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Proportion of individuals with low serum vitamin B-12 concentrations without macrocytosis is higher in the post–folic acid fortification period than in the pre–folic acid fortification period^1,2

¹ From Rush University Medical Center, Chicago, IL (KFW), and the Division of Nutrition, College of Health and Human Sciences, Georgia State University, Atlanta, GA (VG)

See corresponding editorial on page 897.

² Reprints not available. Address correspondence to V Ganji, Department of Nutrition, School of Health Professions, College of Health and Human Sciences, PO Box 3995, Georgia State University, Atlanta, GA 30302-3995. E-mail: vijaykganji{at}yahoo.com.

	ABSTRACT

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Background: Large intakes of folic acid may delay the diagnosis of vitamin B-12 deficiency, which could lead to irreversible neuropathy.

Objective: The objective of this study was to determine whether the proportion of individuals with low serum vitamin B-12 without macrocytosis (undiagnosed vitamin B-12 deficiency) has increased in the post–folic acid fortification period.

Design: Individuals aged 19 y with low serum vitamin B-12 (<258 pmol/L) and mean corpuscular volume (MCV) measured between 1995 and 2004 were identified from medical records. The proportion and odds ratios of individuals with low serum vitamin B-12 without macrocytosis by sex, race, and age according to prefortification (n = 86), perifortification (n = 138), and postfortification (n = 409) periods were determined.

Results: MCV was significantly lower in the postfortification period (88.6 fL) than in the prefortification (94.4 fL; P < 0.001) and perifortification (90.6 fL; P = 0.007) periods. The proportion of subjects with low serum vitamin B-12 without macrocytosis was significantly higher in the postfortification (87%) and perifortification (85%) periods than in the prefortification period (70%; P < 0.001). In a sex-, race-, and age-adjusted analysis, the odds ratio for having low serum vitamin B-12 without macrocytosis was 3.0 (95% CI: 1.7, 5.2) in the postfortification period relative to the prefortification period.

Conclusions: Subjects with low serum vitamin B-12 were likely to be without macrocytosis in the postfortification period. MCV should not be used as a marker for vitamin B-12 insufficiency. It is possible that folic acid fortification may have led to a correction of macrocytosis associated with vitamin B-12 insufficiency.

Key Words: Anemia • folic acid fortification • mean corpuscular volume • MCV • macrocytosis • masking of vitamin B-12 deficiency • megaloblastic anemia • serum vitamin B-12

	INTRODUCTION

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In the United States, fortification of cereals with folic acid began on 1 January 1998. Folic acid fortification was made mandatory by the Food and Drug Administration (FDA), at a level of 140 µg/100 g food, and was intended to increase the dietary intake of folic acid in individuals by 70–130 µg/d (1, 2). The intent of folic acid fortification was to reduce the risk of a woman having a child with a neural tube defect, a positive effect of folic acid found previously (3, 4). The prevalence of neural tube defects was reduced by 19% after fortification of cereals with folic acid in the United States (5, 6).

Numerous studies have shown the effect of folic acid fortification on improved serum and red blood cell (RBC) folate (7-10) and folate intakes in the United States (11). Secondary to improved folate status, folic acid fortification has lowered circulating total homocysteine (tHcy) (7, 8, 12), which has provided a possible role for folic acid in reducing the risk of cardiovascular disease (CVD). Higher levels of folic acid have been suggested to further reduce the prevalence of NTD (13). However, high folic acid intakes may lead to the correction of hematologic abnormalities associated with vitamin B-12 deficiency (14, 15). This may delay the diagnosis (patient's delay or patient not feeling tired or exhausted and hence not or less likely to see a physician) of vitamin B-12 deficiency, which could result in irreversible neurologic damage.

Vitamin B-12 is necessary for the function of methionine synthase. Methionine synthase is responsible for the remethylation of tHcy to methionine and for the conversion of N⁵-methyltetrahydrofolate to tetrahydrofolate (THF). In vitamin B-12 deficiency, folate is "trapped" as N⁵-methyl THF and is not able to convert to THF. Thus, N⁵, N¹⁰-methylene THF needed for DNA synthesis is not produced, which leads to macrocytosis characterized by elevated mean corpuscular volume (MCV). If the diet is supplemented with a large dose of folic acid, this "trap" is bypassed and the macrocytosis associated with vitamin B-12 deficiency is corrected. This occurs because a large dose of dietary folic acid is converted to dihydrofolate (DHF). DHF is converted to THF, which is further converted to N⁵, N¹⁰-methylene THF and allows enough N⁵, N¹⁰-methylene THF to be available for DNA synthesis and leads to the correction of macrocytosis of vitamin B-12 insufficiency. The exact amount of folic acid needed to correct the macrocytosis of vitamin B-12 deficiency has not been defined because, knowingly, patients are not given folic acid to treat vitamin B-12 deficiency. However, the Upper Intake Level (UL) for folic acid was set by the FDA at 1000 µg/d (16, 17).

From a public health perspective, it is important to study the relation between folic acid fortification and low vitamin B-12 without macrocytosis because the improvement in folic acid intake due to folic acid fortification is greater than anticipated. Very limited data are available on whether increased folic acid intake as a result of fortification has corrected macrocytosis associated with vitamin B-12 deficiency in the post–folic acid fortification period. The aim of this study was to determine whether the proportion of individuals with low serum vitamin B-12 without macrocytosis has increased after initiation of the folic acid fortification of cereals in 1998.

	SUBJECTS AND METHODS

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Subjects
Individuals aged 19 y with serum vitamin B-12 and MCV measured at Rush University Medical Center (RUMC) between 1 January 1995 and 31 December 2004 were retrospectively identified and collected from laboratory records. Approval was obtained from the Institutional Review Board at RUMC. Only subjects with low serum vitamin B-12 concentrations (<258 pmol/L) and MCV measurements that were completed within 14 d of the serum vitamin B-12 measurement were included in the study. A cutoff of <258 pmol/L was used to define low serum vitamin B-12 deficiency because this concentration is strongly associated with metabolic indicators of vitamin B-12 deficiency, such as serum methylmalonic acid and total homocysteine (tHcy) concentrations (18). Only the first low serum vitamin B-12 measurement was used; no repeat measures were used. MCV measurements 96.7 fL were used to indicate macrocytosis (19). Pregnant and lactating women and individuals who had previous surgery of the gastrointestinal tract were excluded from the study.

Methods
Serum vitamin B-12 concentrations were measured by using the VITROS competitive binding immunoassay method (Ortho-Clinical Diagnostics Inc, Rochester, NY). MCV was measured as part of the complete blood count. Data on low serum vitamin B-12 concentrations and MCV measurements in addition to demographic information, admit and discharge dates, pregnancy and lactation status, and history of gastrointestinal surgery were obtained from the patients’ medical records. Subjects whose race was not available and whose race was reported as other than white or black were listed in a separate category called "other/unknown."

On the basis of low serum vitamin B-12 concentrations (<258 pmol/L), a total of 758 subjects were eligible for the study. These subjects had both serum vitamin B-12 and MCV measured between January 1995 and December 2004. Of 758 subjects, 20 were excluded because they were pregnant or lactating and 35 were excluded for having had previous gastrointestinal surgery. Additionally, complete data for 70 subjects were unable to be located by the medical records department; therefore, these subjects were also excluded. Thus, the final study sample consisted of 633 subjects.

Subjects were placed into 1 of 3 folic acid fortification periods on the basis of the date of the serum vitamin B-12 measurement. Subjects were placed into the prefortification period if the serum vitamin B-12 measurement was made before 1 March 1996 (n = 86), the perifortification period if the measurement was made between 1 March 1996 and 31 December 1997 (n = 138), and the postfortification period if the measurement was made on or after 1 January 1998 (n = 409). Within each fortification period, subjects were categorized by sex, race (white, black, or other/unknown), and age (<65 y and 65 y) for the purpose of statistical analysis.

Statistical analysis
The data analysis was completed by using SPSS software (version 13.0; SPSS Inc, Chicago, IL). Because serum vitamin B-12 data were not normally distributed, we reported median and interquartile difference for serum vitamin B-12 concentrations. A significant difference in serum vitamin B-12 concentrations between fortification periods was determined with the Kruskall-Wallis test. A pairwise comparison was performed by using the Mann-Whitney U test and then Bonferroni adjustment was applied for multiple comparisons (P < 0.017). MCV measurements across pre-, peri-, and postfortification periods were compared with a post hoc Scheffe test for multiple comparisons after the hypothesis was tested with analysis of variance. The difference in proportion of subjects with low serum vitamin B-12 concentrations without macrocytosis in the pre-, peri-, and postfortification periods was determined by using a chi-square test. The association between the proportions of individuals classified as having low serum vitamin B-12 concentrations without macrocytosis and folic acid fortification periods was determined with multivariate logistic regression. Odds ratios and 95% CIs were calculated after adjustments for sex, race, and age. Additionally, we determined interactions between folic acid fortification period and sex, age, and race for serum vitamin B-12, MCV, and MCV < 96.7 fl (absence of macrocytosis). A P value <0.05 was considered significant.

	RESULTS

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Characteristics of the study sample are presented in Table 1. Of 633 subjects, 41% were men and 59% were women. White, black, and other/unknown race-ethnicities were 53%, 23%, and 24%, respectively. When a cutoff of 65 y was used, 42% of subjects were aged <65 y and 58% were aged 65 y. The mean (±SD) age of the subjects was 65.3 ± 18.9 y. The mean age was not significantly different between all subjects and in any demographic group across the fortification periods (data are not shown).

Table 2

Table 3

Table 4

	DISCUSSION

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To our knowledge, only one study has examined the temporal association between folic acid fortification and possible masking of vitamin B-12 deficiency in the United States (19). Mills et al (19) reported that the proportion of subjects without anemia did not significantly change from the prefortification period (39%) to the optional (46%) or postfortification (38%) periods, which suggested no increase in the masking of vitamin B-12 deficiency as a result of folic acid fortification. In support of our findings, Ray et al (20), in 15 000 Canadian elderly women from 2 provinces, found that the prevalence of high serum folate and low serum vitamin B-12 increased from 0.09% in the prefortification period to 0.61% in the postfortification period (prevalence ratio: 7.0; 95% CI: 2.6, 19.2), which suggested that a few elderly persons might be at risk of masked vitamin B-12 deficiency. In this study, we found significantly more cases of low serum vitamin B-12 without macrocytosis in the postfortification period (87%) and in the perifortification period (85%) than in the prefortification period (70%) (P < 0.001). The proportion of cases with low serum vitamin B-12 without macrocytosis between the peri- and postfortification periods was not significant because it is likely that the majority of the foods were fortified with folic acid between 1 March 1996 and 31 December 1997 (8), although the fortification was not mandatory until 1 January 1998, which lead to increased folic acid intakes during the perifortification period itself.

The proportion of subjects with low serum vitamin B-12 without macrocytosis in the age group <65 y significantly increased from 45% in the prefortification period to 85% in the postfortification period. When adjusted for sex and race, the association between the proportion of cases with low serum vitamin B-12 and fortification period was significant in both the younger and older individuals. It was previously thought that correction of macrocytosis associated with vitamin B-12 deficiency would mostly be seen in older individuals (21), because of the increased prevalence of vitamin B-12 deficiency primarily due to a reduced absorption of vitamin B-12 through aging gut (22). Mills et al (19) found that subjects aged < 60 y were more likely to have a low vitamin B-12 concentration without anemia than were the older subjects (<60 y: 50.5%; 60–74 y: 38%; >74 y: 30.9%; P < 0.0001). Their finding indicates the correction of macrocytosis associated with vitamin B-12 deficiency in younger subjects rather than in older subjects; whereas our observation suggests a possible correction of macrocytosis associated with vitamin B-12 deficiency in both younger (<65 y) and older (65 y) subjects. Caution needs to be used when comparing the results from our study with those of Mills et al (19) because of the differences in subject characteristics. Consideration must be given for screening for vitamin B-12 deficiency when younger patients (<65 y) are presented with underlying neurologic symptoms without macrocytosis.

Since mandatory folic acid fortification began on 1 January 1998, individuals who had vitamin B-12 and MCV concentrations measured after this date were treated as subjects exposed to folic acid fortification. Folic acid intakes have drastically increased during the postfortification period (23). Reported actual folic acid intakes increased by 215–240 µg/d (10), which was much more than originally predicted by the FDA. Also, many individuals may be receiving more than the UL of 1000 µg/d (10). Hence, the changes we have seen in MCV concentrations from the pre- to the postfortification period were more likely due to increased intakes of folic acid resulting from folic acid fortification. Overall, MCV has decreased by 6.5% from the pre- to the postfortification period. It is not surprising that the MCV has decreased in the postfortification period, relative to that in the prefortification period, given the role of folic acid in correcting macrocytosis associated with folate deficiency. Hence, the lower MCV in the postfortification period may be unrelated to the masking of vitamin B-12 deficiency and may also be likely due to the removal of folate deficiency from those with vitamin B-12 deficiency.

In contrast with our findings, Hirsch et al (24) determined that MCV measurements increased from 89.6 to 90.6 fL after folic acid fortification in Chile. Although Hirsch et al (24) did not evaluate the effect of folic acid fortification on the possible correction of macrocytosis of vitamin B-12 insufficiency, their results may indicate an ongoing vitamin B-12 deficiency. It is possible that, in the prefortification period, the Chilean population had a better folate nutritional status than did our study population.

It is plausible that some clinicians use MCV as a diagnostic tool for vitamin B-12 insufficiency. This study clearly showed that MCV is not a precise diagnostic measurement for vitamin B-12 deficiency. Overall, 84% (532 out of 633) subjects presented no macrocytosis despite their low serum vitamin B-12 concentrations. Use of MCV as a diagnostic tool not only delayed the diagnosis of vitamin B-12 deficiency but may also have accelerated the neurologic damage associated with vitamin B-12 deficiency (14). Furthermore, elevated MCV is often not related to vitamin B-12 insufficiency (25). In making a diagnosis of vitamin B-12 deficiency, it is prudent to consider serum vitamin B-12 measurements, serum methylmalonic acid measurements or both (18). Also, the circulating tHcy concentration is a good indicator of vitamin B-12 insufficiency in individuals with optimum folate status.

In this study, we used subjects from an academic medical center; therefore, results of this study should be used with caution when applied to the general population. It is possible that individual underlying medical conditions could have affected MCV (25). It is important to note that we investigated trends over time. Each time period contained different subjects with possible different medical conditions and dietary habits. The procedure for measuring serum vitamin B-12 changed in 2002. Whether or not this had any effect on the results is not known. We lack data on folate status and on subjects with normal vitamin B-12 and that such data would have helped in determining whether the change in MCV was due to improved folate status or masking. Because of the retrospective nature of the study, we were unable to obtain the subjects’ dietary or supplement intake to determine true folic acid intakes and the reasons for testing serum vitamin B-12; therefore, the results of this study should be interpreted with caution.

In conclusion, in the post–folic acid fortification period, subjects with low serum vitamin B-12 concentrations are 3 times as likely to be without macrocytosis (anemia) than are those in the prefolic acid fortification period. Additionally, MCV measurements should not be used as a diagnostic tool for vitamin B-12 deficiency. The absence of hematologic abnormalities, such as macrocytosis, is not an indicator of the absence of vitamin B-12 insufficiency. It is important to not only screen for serum and hematologic indicators, but also for clinical symptoms such as neuropathy and mood disorders before ruling out vitamin B-12 deficiency in the absence of macrocytosis. Although no controlled data are available on whether folic acid is harmful to patients with clinical vitamin B-12 deficiency, it is important to be aware of the possible correction of macrocytosis of vitamin B-12 deficiency in the era since folic acid fortification began, especially in those with high folic acid intakes. If the correction of macrocytosis associated with vitamin B-12 insufficiency (so called, patient's delay) is widespread in the post–folic acid fortification era, it is appropriate to add vitamin B-12 to foods currently fortified with folic acid.

	ACKNOWLEDGMENTS

 
The data presented in this article come from the masters thesis of KFW, who acknowledges her thesis committee.

The authors’ responsibilities were as follows— KFW and VG: responsible for the study design, data collection, data analysis, and draft of the manuscript. Neither author had a conflict of interest. At the time of the study, KFW was a graduate student and VG was a faculty member in the Departments of Clinical Nutrition and Food and Nutrition, Rush University Medical Center, Chicago, IL.

	REFERENCES

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