Unpredictable intra-individual variations in serum homocysteine levels on folic acid supplementation

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1 European Journal of Clinical Nutrition (1997) 51, 188±192 ß 1997 Stockton Press. All rights reserved 0954±3007/97 $12.00 in serum homocysteine levels on folic acid supplementation CR Santhosh-Kumar 1, JC Deutsch 1,2, JW Ryder 3 and JF Kolhouse 1 1 Department of Medicine, Divisions of Haematology and 2 Gastroenterology, and 3 Department of Pathology, University of Colorado Health Sciences Center and Denver Veterans' Administration Hospital, 4200 East Ninth Avenue, B-170, Denver, Colorado Objective: To determine if changes in serum homocysteine values during folic acid supplementation can identify objectively healthy subjects with subclinical folate de ciency. Design: Blood drawn and processed in a regimented fashion from fasting subjects. Serum homocysteine values determined by gas chromatography-mass spectrometry twice before and on days 5 and 8 of daily folic acid Setting: Outpatient University Hospital Clinical Research Center, Denver, Colorado. Subjects: Subjectively healthy adults with normal hematologic and biochemical screening tests. Intervention: Folic acid 1 mg daily for eight consecutive days. Results: Homocysteine values of the group fell signi cantly during folic acid Values presupplementation were and mmol/l while values on days 5 and 8 of supplementation were and mmol/l. However, the homocysteine values of any given individual varied up to 60% (rises up to 7 mmol/l and falls of 5 mmol/l) during folic acid supplementation despite the controlled circumstances of blood handling, and an assay coef cient of variation of 8%. Conclusions: Although group values of serum homocysteine fall during folic acid supplementation, intraindividual variation is so great that subjects with subclinical folate de ciency can not be identi ed using this study design. Furthermore, these data suggest than an individuals homocysteine values vary enough that single values must be interpreted with caution. Sponsorship: Supported in part by The Marcellus S. Merrill Memorial Research Fund, the Muscular Dystrophy Association and the Clinical Research Center at the University of Colorado Health Sciences Center. Descriptors: homocysteine; folic acid Introduction Epidemiological data have implicated hyperhomocystinemia as an independent risk factor in atherosclerotic vascular disease since the original observation by McCully, 1969 that homocysteine (Hcy) or one of its metabolites may be causal in atherosclerosis (Boushey et al, 1995; Clark et al, 1991; McCully, 1969; Selhub et al, 1995; Stampfer et al, 1992): Estimates suggest that 20±40% of the population may be folate de cient without any recognized clinical or laboratory manifestations (subclinical folate de ciency) (Subar et al, 1989; Tsui et al, 1990). It is believed that the majority of cases of hyperhomocystinemia are due to overt or subclinical folate de ciency. These cases could therefore be corrected by folic acid supplementation (Green and Jacobsen, 1995). It is important to note, however, that folate de ciency can either be due to dietary de ciency (global folate de ciency) or due to metabolic defects affecting availability of speci c coenzyme forms of folates (relative folate de ciency): Several investigators and governments agencies across the world have advocated supplementation of foodstuffs Correspondence: Dr JC Deutsch. Received 19 September 1996; revised 21 November 1996; accepted 22 November 1996 with folic acid with a view to prevent several illnesses including neural tube defects and atherosclerotic vascular disease (Anonymous, 1993; Oakley et al, 1995). The effect of such supplementation on Hcy levels in normal populations is not known. There are several potential concerns to folic acid forti cation of foods, including development of neurologic disease in B 12 de cient subjects and potential progression of malignancy (Chanarin, 1994; Farber et al, 1948; Zimmerman and Shane, 1993): It is well known that treatment of clinically obvious folate de ciency with folic acid causes a rapid fall in the abnormally elevated serum Hcy levels (Brattstrom et al, 1988). We hypothesized that some apparently normal subjects would have unsuspected subclinical folate de ciency and normal Hcy values and that those with folate de ciency would show a drop in Hcy levels on folic acid supplementation, providing a method to diagnose subclinical folate de ciency. Methods Approval for human studies was obtained from the Colorado Multi-Institutional Internal Review Board. Normal subjects of either gender between the ages of 18 and 65 y were recruited through advertisements. Subjects were screened by one of the investigators through a telephone

2 interview. Subjects who smoked, who consumed more than 20 g of alcohol per week or who were on any medications or vitamin supplements for at least one month prior to blood collection were excluded. All subjects had blood samples drawn after an overnight fast and the blood was analyzed for complete blood count, creatinine, liver-associated enzymes, bilirubin, cobalamin and Hcy. For inclusion in the folic acid supplementation part of the study, subjects were required to have normal values for all the laboratory parameters mentioned above. Sera from subjects with serum cobalamin levels below 200 pmol/l were assayed for serum methylmalonic acid (MMA) in the Laboratory of Robert H. Allen at the University of Colorado Health Sciences Center, Denver, CO; and only those subjects with normal MMA below 271 nmol/l were included (Allen et al, 1990; Marcell et al, 1985). All of the above mentioned analyses were conducted during a 5 week period prior to study day 1 at a mean of 17 7 d. On day 1, fasting samples were collected for serum and whole blood folates and a second pre therapy serum Hcy level. One tablet containing 1 mg folic acid each was then administered orally to each of the subjects for 7 d. A second blood sample was collected 3 h, after ingestion of the rst folic acid dose. Subjects were required to have a rise in serum folate of at least 5 nmol/l in order to eliminate any subjects with folate malabsorption. Further fasting samples were collected on days 5 and 8 for serum Hcy. Blood samples were kept in ice and sera were separated within 30 min of phlebotomy and stored at 780 C till assay. Hcy was measured using a modi cation of previously described methods (Stabler et al, 1987). Brie y, 100 ml of serum is diluted to a nal volume of 900 ml of 200 mmol/l mercaptoethanol containing 5.5 mmol/l of [ 2 H 8 3,3, ,4, ]homocystine. The samples are heated to 100 C for 20 min, cooled to 22 C, alkalinized with 750 ml of concentrated NH 4 OH, passed over 100 mg of Bio-Rad AGMP-1, and the resin washed with 15 ml of 0.05M NH 4 OH/MeOH, followed by 15 ml H 2 O. The homocysteine is eluted with 1.2 ml of 4 mol/l acetic acid/0.1 mol/l of HCl into glass tubes containing 3 ml of undiluted mercaptoethanol. The samples are dried in a Savant Drying Centrifuge, and derivatized with tertbutyldimethylsilyltri uoroacetamide at 90 C for 1 h. After derivatization, samples were injected onto a 10 meter Supelco dimethylsiloxane capillary column and analyzed by mass spectrometry as described (Santhosh-Kumar et al, 1995). Hcy measurement refers to total Hcy, namely sum of free, protein bound and mixed disul de forms. Serum folates and serum B 12 were measured using the Bio-Rad QuantaPhase II kit by a commercial laboratory. Red cell folates were determined by gas chromatography/mass spectrometry (Santhosh-Kumar et al, 1995). Subjects were instructed to keep a strict dietary record for two week days and two weekend days during the study period. Dietary surveys were analyzed using Food-processor Plus software. Statistical analysis Results are given as mean standard deviation. The group means were compared using two-tailed Student's t±tests. Pearsons correlation coef cients were calculated comparing folate levels, dietary intakes and Hcy levels at different time points. Chi square analyses were performed for comparison of proportions. Signi cance was de ned as P < Results One hundred and twentyone apparently normal people (85 females and 36 males) were screened for study eligibility. Test results for 13 variables derived from complete blood counts, cobalamin, creatinine, liver-associated enzymes, bilirubin, GGT, folate absorption and Hcy had to be within the normal reference range. If all laboratory values were independent we would expect P ˆ 1 7 (1 7 P) 13 percent of subject of subjects to be eligible (49%). However, since laboratory values are not totally independent, 65% of screened subjects (N ˆ 79) were eligible. There were 52 females and 27 males. Their ages ranged from 18± 65 y. There was no signi cant difference in the data between males and females except for the pre-treatment Hcy levels which were higher in males (8.26 vs 7.23 mmol/ L, P ˆ 0.03). The results of serum folates, Hcy, cobalamin and whole blood folate levels, the dietary folate and pyridoxine estimates are shown in Table 1. The Pearson correlation coef cients between dat one Hcy levels and the following variables: 0.47 for molar whole blood folate, for serum cobalamin, 0.12 for dietary folate and 0.19 for dietary pyridoxine. There was no difference in the paired t±test of Hcy values obtained in subjects on two occasions prior to folic acid supplementation (P ˆ 0.25). The mean values were and mmol/l. Furthermore, there was no difference in the paired t±test of Hcy values obtained in subjects between 5 and 8 days of folic acid supplementation (P ˆ 0.56). The mean value at day 5 was and day 8 was mmol/l. However, there was a signi cant fall of mean Hcy values of approximately 1.4 mmol/l comparing subjects before folic acid supplementation to both 5 and 8 days of folic acid supplementation (P ˆ 0.02, P ˆ 0.01, respectively): The within-person variance prior to folic acid supplementation was 3.06 based on a mean change of mmol/l. The between-person variance was Likewise, the within-person variance after folic acid supplementation (comparing levels on day 5 to day 8) was 3.20 based on a mean change of , while the between-person variance was The individual Hcy levels appeared to randomly fall and rise between the pre-treatment values (Figure 1): Levels on the second pre-treatment blood sample ranged as low as 46% to as high as 165% of the values on the rst pretreatment blood sample. Likewise, blood levels ranged from 36±165% between days 5 and 8 of folic acid treatment (Figure 2): The change in Hcy levels on day 5 ranged from 48±150% of day 1 values and from 29±241% on day 8. Since the coef cient of variation (CV) for the assay of serum Hcy for two serum samples (each with a value of 5 and 10 mmol/l), assayed multiple times was less than 8% (maximum of 0.8 mmol/l), we de ned a change of twice Table 1 Homocysteine, folate and cobalamin values and dietary folate and pyridoxine estimates in 79 normal subjects Mean (s.d.) Range Serum homocysteine (mmol/l) a 7.62 (2.0) 3.4±13.3 Whole blood folate (nmol/g Hb) 3.44 (1.4) 1.51±8.79 Serum B 12 (pmol/l) 418 (156) 167±979 Serum folate (nmol/) 20.6 (8.6) 7.4± > 40 Dietary folate (mg/d) 309 (131) 102±700 Dietary pyridoxine (mg/d) 1.92 (0.4) 0.5±9.5 a Mean of two pre therapy values. 189

3 190 folate-de cient subjects on folic acid repletion (Allen et al, 1990), the changes in Hcy values observed between days 5 to 8 are not likely to be due to folic acid replacement. Furthermore, the proportion of subjects with a signi cant fall in Hcy values between days 5 and 8 (13, 17%) was above the same as the numbers of subjects with rises in Hcy in either the days 1 to 5 group (7, 9%), or the days 5 to 8 group (9, 11%) (w ˆ 2.2 with 2 degrees of freedom, P ˆ 0.33). The absolute change in Hcy values not due to folate repletion was as much as 7 mmol/l. On the other hand, there was an excess number of subjects (26, 33%) with a fall in Hcy between days 1 and 5. Although the variation in Hcy levels between the two pre-treatment samples is not greater than that found between days 5 and 8, more subjects had a change of Hcy levels of 1.6 mmol/l or more prior to folic acid supplementation than between 5 and 8 d of folic acid Comparing the two pre-treatment values, an increase was observed in 16 (33%) subjects, a decrease in 27 (34%) subjects and no changes were seen in 36 (46%) subjects. Chi-squared analysis showed that there is a signi cantly lower proportion of subjects who did not show a change pre-treatment (w 2 ˆ 10.5 with a 1 degree of freedom, P ˆ 0.001) when compared to changes between days 5 and 8. Figure 1 Paired serum homocysteine in 79 normal individuals measured on two separate occasions (17 7 d between tests) before folic acid the CV (16% or 1.6 mmol/l) between days 1 and 5 as signi cant and possibly due to folic acid replacement. The results of analysis of serum Hcy of three groups of subjects divided according to change in serum Hcy from days 1 to 5 and days 5 to 8 are shown in Figure 2. There were 7 subjects (9%) who showed rise (A) in serum Hcy by more than 1.6 mmol/l between days 1 and 5, with some values rising above the upper limit of normal. 26 (33%) subjects showed a fall (C) in Hcy greater than 1.6 mmol/l between days 1 and 5, while 46 (58%) showed no signi cant change (B). Average values for these data on day 1 are shown in Table 2. Analysis of variance and individual t±testing between groups showed there was no signi cant difference between the three groups in terms of serum or whole food folate levels, cobalamin levels, day 1 Hcy levels or dietary folate or pyridoxine estimates. Between day 5 and day 8, 9 subjects (11%) showed an increase, 13 subjects (17%) showed a decrease and 57 (72%) showed no signi cant change. Roughly proportionate changes were observed in the two sets of Hcy levels before folic acid supplementation (increase in 16 {20%}, decrease in 27 {34%} and no change in 36 {46%}). Because mean Hcy levels were no different (P ˆ 0.56) on days 5 and 8, and because previous literature suggests that homocysteine levels return to normal within 1±3 d in Figure 2 Changes in serum homocysteine on oral folic acid 1 mg daily in 79 individuals divided according to (A) rise, (B) fall or (C) no change on day 5. Change in de ned as > 1.6 mmol/l (twice the coef cient of variation of assay (either way).

4 Table 2 Day 1 Homocysteine, folate and cobalamin values in three groups of normal subjects who were segregated based on change in homocysteine after 5 days of folate supplementation 191 Mean change n Hcy Serum B 12 Serum folate Whole blood folate (mmol/l) (pmol/l) (nmol/l) (nmol/l/g Hb) Increase in Hcy > 1.6 mmol/l 2.24 (0.55) (3.0) 425 (115) 15.6 (8.8) 2.9 (0.9) No change a 0.29 (0.83) (1.9) 439 (184) 20.2 (10.4) 3.8 (1.6) Decrease in Hcy > 1.6 mmol/l (0.86) (1.6) 380 (100) 21.6 (9.2) 3.0 (1.1) Values are mean and numbers in parentheses are standard deviations. a Change less than 1.6 mmol/l in either direction. Discussion Our data show that even in highly selected normal individuals, there is a group of subjects (approximately 33%) who will show a Hcy lowering response to folic acid However, our data also show that there is a marked day to day variation in Hcy values, with both rises and falls which are not due to either technical problems of the assay, or due to the effects of folic acid Our data strongly suggest that any effect due to folic acid supplementation occurred only between days 1 and 5 and that changes that occurred between days 5 and 8 are due to biologic variation not associated with folic acid The observed variation of up to 7 mmol/l in Hcy levels (either rises in Hcy or falls in Hcy on days 5 to 8) actually exceeded any observed decreases in Hcy (5 mmol/l) which occurred between days 1 and 5, where a true folate effect occurs. Based on our data, we estimated that approximately 10± 15% of a group of normal subjects would have a signi cant rise or fall in Hcy between two assay times unrelated to folate Subtracting out this estimated nonfolate variation of 10±15%, leaves 10±20 subjects normal (15±25%) who had a Hcy response to folic acid Since, as discussed above, the maximal fall in that group (5 mmol/l) was less than the variation seen in the groups where folate repletion was not a factor, it does not appear to be possible to determine which subjects had a fall in Hcy due to folate replacement, and which subjects had a fall due to variations not related to folate. Obviously, the accuracy of our stable-isotope dilution assay in serum is a critical part of assessing our data. A recent report by Rasmussen et al, 1996 suggests that our assay is closely comparable to total plasma Hcy values reported from other laboratories. Our maximal analytical variation was 8% (0.8 mmol/l) on subjects with serum Hcy values of 10 mmol/l, where as Rasmussen et al, 1996 had analytical variation of 5±6% in plasma values over a similar range. We studied a highly selected group of individuals who would be expected to be similar to the `poor responders' to folic acid supplementation reported by Rasmussen et al, Our mean value of Hcy prior to folic acid supplementation was 7.77 mmol/l and folic acid caused a mean fall of 1.24 mmol/l. Rasmussen's `poor responder' group had a pretreatment value of 7.76 mmol/l and an average fall of 1.26 mmol/l with folic acid (Rasmussen et al, 1996). Furthermore, to minimize the possibility that analytical variation was responsible for the observed variation, we only considered a change to occur if the magnitude was greater than twice our maximally observed intra-assay variation. Furthermore, our patient preparation and blood collection were performed in a more stringent manner that what is done in routine medical practice. The blood samples for Hcy were uniformly collected after overnight fasts and sera were separated within 30 min of phlebotomy to minimize any possibility of random spurious elevations in Hcy (Andersson et al, 1992): Methionine intake during the previous meal could have played a role in the variability of Hcy levels since an oral methionine load (75±100 mg/kg) is well known to increase serum Hcy levels to more than twice the baseline level in 4±6 h, which does not return to normal until as late as 48 h (Andersson et al, 1992; Frontiera et al, 1994). Dietary methionine intake may vary from a few milligrams (vegetables) to more than 1.5 g (cottage cheese or sh) per serving (Pennington, 1989): Our ndings have implications for the interpretation of results of epidemiologic studies on serum Hcy and on nutritional policy on folic acid supplementation as applied to individuals. Several of the large epidemiological studies linking mild hyperhomocystinemia to accelerated vascular disease used single non fasting blood samples (Boushey et al, 1995; Clark et al, 1991; Selhub et al, 1995; Stampfer et al, 1992) and found mean differences in Hcy levels between cases and controls to be in the order of only 1± 2 mmol/l. Our data show that a change of up to 7 mmol/l can be due to biologic variation so that an increase of 1± 2 mmol/l in any individual could be due to factors not related to folate intake. It is possible that the cases consumed diets which caused temporary increases in Hcy compared to controls. Alternatively, if one assumes that the biologic variation is distributed equally between cases and controls, the epidemiologic studies may have underestimated the true magnitude of differences. A recent meta analysis of studies linking hyperhomocystinemia and vascular disease has suggested that a rise of 5 mmol/l of Hcy is as signi cant a risk factor as a serum cholesterol elevation of 20 mg/dl (Boushey et al, 1995). The authors of the meta analysis went on to suggest that folic acid supplementation in the dose of 400 mg per day will lower Hcy levels by 6 mmol/l in the general population. While this may be true to overt folate de ciency, this is unlikely to be the case in the majority of the population who are normal. In our subjects the mean decrease of serum Hcy was less than 1.5 mmol/l when a two and a half times higher dose of folic acid (1 mg) was used. This in turn suggests that folic acid supplementation to the effect of 400 mg per day may have little impact on the normal population Hcy levels. It has been shown the supplementation of elderly individuals (Naurath et al, 1995) and South African men (Ubbink et al, 1995) with doses similar to those used in this study (1.0±1.1 mg), resulted in a signi cant decrease in mean Hcy values to a similar degree as in our study of healthy normal individuals. At present there is no consensus about the upper limits

5 192 of normal for serum or plasma Hcy because of differences in assay techniques, diets, technical factors and the frequency of enzyme polymorphisms which may lead to mild hyperhomocystinemia (Andersson et al, 1992). Again, this suggests that single measurements of serum or plasma Hcy levels should be interpreted with caution especially if they are only mildly abnormal. It is also dif cult to compare studies where different assay techniques, reference ranges and different populations are use. Folic acid forti cation of foods to prevent hyperhomocystinemia should be based on randomized clinical trials of vitamin supplementation which make adequate provision for the large variation in Hcy levels observed in the current study as well as for other confounding variables. Prospective studies should also examine the effect of dietary methionine intake on Hcy levels. However, the intra-individual variations observed in this study in no way discredits epidemiologic studies that that show that mean elevation of blood levels of Hcy is a cardiovascular risk factor. Our data suggest that the extrapolation of these epidemiologic studies comparing blood levels of Hcy to cardiovascular risk to an individual may require repeated Hcy analyses. AcknowledgementsÐWe are grateful to NIH Research Resources for assistance in performance of part of the study. Dr P Archer and the University of Colorado Cancer Center Biostatistics Core for help with data analysis and Dr KL Hassell for helpful discussion. The authors also wish to thank Ms V Allen for secretarial assistance in preparing the manuscript. References Allen RH, Stabler SP, Savage DG & Lindenbaum J (1990): Diagnosis of Cobalamin De ciency I: Usefulness of serum methylmalonic acid and total homocysteine concentrations. Am. J. Hematol. 34, 90±98. 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