Vitamin B-i 2, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia13

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1 Vitamin B-i 2, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia13 Johan B Ubbink, WJ Hayward Vermaak, Annatjie van der Merwe, and Piet J Becker ABSTRACT We measured the vitamin B-6, vitamin B-l2, and folic acid nutritional status in a group ofapparently healthy men (n = 44) with moderate hyperhomocysteinemia (plasma homocysteine concentration > 16.3 zmol/l). Compared with control subjects (n = 274) with normal plasma homocysteine (f;; 16.3 mol/l) concentrations, significantly lower plasma concentrations of pyridoxal-5 -phosphate (P < 0.001), cobalamin (P < 0.001), and folic acid (P = 0.004) were demonstrated in hyperhomocysteinemic men. The prevalence of suboptimal vitamin B-6. B-l2, and folate status in men with hyperhomocysteinemia was 25.0%, 56.8%, and 59.1%, respectively. In a placebo-controlled follow-up study, a daily vitamin supplement (10 mg pyridoxal, 1.0 mg folic acid, 0.4 mg cyanocobalamin) normalized elevated plasma homocysteine concentrations within 6 wk. Because hyperhomocysteinemia is implicated as a risk factor for premature occlusive vascular disease, appropriate vitamin therapy may be both efficient and cost-effective to control elevated plasma homocysteine concentrations. Am J C/in Nutr 1993:57: KEY WORDS Homocysteine, occlusive vascular disease, hyperhomocysteinemia, pyridoxal-5 -phosphate, folate, cobalamin Introduction Because ofwidespread vascular disorders in patients with cystathionine-f3-synthase (EC ) deficiency (1-3), it has been suggested that hyperhomocysteinemia increases the risk for premature occlusive vascular diseases. Circulating homocysteine concentrations have now been shown to be elevated in coronary heart disease (CHD) (4-9) and in cerebral vascular (8, 10-13) as well as peripheral vascular diseases (8, 14-16). For CHD, it has been demonstrated that an elevated plasma homocysteine concentration is an independent risk factor (6), which may be even more common than hypercholesterolemia (9). Moreover, patients with elevated plasma homocysteine concentrations were more likely to demonstrate clinical progression of peripheral and coronary artery disease (1 5), indicating a possible prominent role for homocysteine in atherogenesis. Because the prevalence of hyperhomocysteinemia is between 20% and 40% in different populations with CHD (7-9), the therapeutic control of elevated homocysteine concentrations may become important in the prevention of premature vascular diseases. However, it is not always clear which factors cause hyperhomocysteinemia and which therapy should be used to lower circulating homocysteine concentrations. This is illustrated by the observation that although classical genetic hyperhomocysteinemia is the result of homozygous cystathionine-fl-synthase deficiency, heterozygotes for this enzyme deficiency often present with only a small or no elevation in basal circulating homocysteine concentrations (17, 1 8). Heterozygosity for cystathionine-fl-synthase deficiency can clearly not fully explain the prevalence of hyperhomocysteinemia in the population and other factors are also involved. Cofactors required for homocysteine metabolism may be important determinants of circulating homocysteine concentrations. Intracellular homocysteine is metabolized by either the transsulfuration pathway or by remethylation to methionine (19). The condensation ofserine and homocysteine, catalyzed by cysthathionine-fl-synthase in the first reaction ofthe transsulfuration pathway, is dependent on pyridoxal-5 -phosphate (PLP) as cofactor, whereas remethylation of homocysteine requires vitamin B- 12 and methyltetrahydrofolate as coenzyme and cosubstrate, respectively (19). Subclinical deficiencies ofthe above-mentioned vitamins may result in hyperhomocysteinemia (1 7, 20-22). Stabler et al (20) reported that 77 of a total of 78 patients with vitamin B- 1 2 deficiency had serum homocysteine concentrations above the normal range, whereas Lindenbaum et al (2 1 ) found that patients with vitamin B-l2 deficiency, but without anemia or macrocytosis, had markedly elevated total homocysteine concentrations. Similar studies demonstrated that 84% of patients with subnormal serum folate concentrations also had hyperhomocysteinemia (20, 22). Little is known about the effect of vitamin B-6 deficiency on circulating homocysteine concentrations. Park and Linkswiler (23) reported that urinary homocystine excretion increased considerably when six male volunteers consumed a diet depleted ofvitamin B-6, whereas several studies on experimental animals suggested that a vitamin B-6 deficiency results in homocysteine accumulation (24-26). Because of these reports (1 7, 20-22) implicating specific vitamin deficiencies in causing hyperhomocysteinemia, we decided I From the Department ofchemical Pathology, Faculty of Medicine, University ofpretoria, and the Institute for Biostatistics, Medical Research Council, Pretoria, South Africa. 2 Supported by the Atherosclerosis Risk Factor Research Programme, Vesta Medicines Pty Ltd. and the HF Verwoerd Research Trust (JBU). 3 Address reprint requests to JB Ubbink, Department of Chemical Pathology, Faculty of Medicine, University of Pretoria, P0 Box Pretoria 0001, South Africa. Received January 21, Accepted for publication July 15, ilin J C/in Nuir 1993:57: Printed in USA American Society for Clinical Nutrition 47

2 48 UBBINK ET AL 60 Frequency < >41 Homocysteine (micromol/l) FIG I. Frequency distribution of plasma total homocysteine concentrations in adult Caucasian men (n = 349). to investigate the prevalence of suboptimal vitamin B-6. folate and vitamin B- 12 status in men with elevated plasma homocysteine concentrations, and to study the effect ofan oral vitamin supplement as a therapeutic agent to normalize elevated circulating homocysteine concentrations. Subjects and methods Subjects Fasting venous blood samples with EDTA as anticoagulant were obtained from 349 ambulatory Caucasian men aged between 19 and 7 1 y, who were employed by five different major employers in the vicinity of our laboratory. The blood samples were chilled on ice and transported to the laboratory, where plasma and cells were separated within 4 h after the samples had been obtained. We showed previously that the increase in plasma homocysteine concentrations, which occur before blood separation, is effectively inhibited for 4 h when the blood samples are kept in an ice bath (27). Regular consumers ofany medication or vitamin supplements were excluded from the study. Fortynine men had hyperhomocysteinemia (plasma homocysteine concentration > 16.3 imol/l): from this group 30 men gave informed consent to participate in a vitamin supplementation TABLE 1 Vitamin B-6, B-l2, and folate nutritional status ofparticipants with normal and elevated plasma total homocysteine concentrations* Group Age pyridoxal-5 -phosphate cobalamin folate.1 n,nol/l jnnol/l i,no//l Homocysteine > 16.3 zmol/l (n = 44) 40.6 ± ± ± ± 2.9 Homocysteine 16.3 mol/l (n = 274) 41.8 ± ± ± ± 3.6 P = 0.002t P < P = C SD. Reference ranges for plasma vitamin concentrations are as follows: Pyridoxal-5 -phosphate : cobalamin pmol/ L: folate, t P values (Student s I test) indicate that values for the group with normal ( 16.3 Mmol/L) homocysteine concentrations are significantly greater than the group with homocysteine concentrations > 16.3 mol/l.

3 VITAMINS AND HYPERHOMOCYSTEINEMIA 49 TABLE 2 vitamin concentration distribution in volunteers with normal or elevated plasma homocysteine concentrations Cobalamin Pyridoxal-5 -phosphate Folate Vitamin concentration cutoff points <200 pmol/l 200 pmol/l <30 30 <5 5 % Homocysteine > 16.3 pmol/l (n = 44) Homocysteine 16.3 amol/l (n = 274) P<0.00l* P= P=0.02l C p values (chi-square test) for dependence between plasma homocysteine concentration and cobalamin, pyridoxal-5 -phosphate, and folate status. study. The participants were randomly assigned to one of two groups. Group V (n = 15) received a vitamin supplement (Royl- Cynor, obtained from Vesta Medicines, Johannesburg, which contains 1.0 mg folic acid, 0.4 mg cyanocobalamin, 12.2 mg pynidoxal. HC1, and 6.0 mg /3-carotene per tablet) whereas group P (n = 1 5) received placebo tablets (containing 6.0 mg /3-carotene per tablet) similar in appearance. Participants were instructed to take one tablet daily after dinner. Fasting venous blood samples were again obtained 2, 4, 6, and 8 wk later. After the 6-wk blood samples had been obtained, participants were instructed to increase the tablet intake from one to two tablets a day; the 8-wk blood samples therefore reflect the effect ofa higher vitamin (or placebo) dose on circulating homocysteine concentrations. The study design was double blind; neither the participants nor the laboratory staffwere aware ofthe identity ofthe tablets used in the study. Four participants (two from each group) opted to withdraw during the study. The study was approved by the University Human Ethics Committee. Laboratori investigations homocysteine was derivatized with ammonium7-fluoro 2-oxa-l.3 diazole-4-sulfonate (SBD-F; supplied by Wako, Neuss, Germany) according to the method of Araki and Sako (28). The method entails complete reduction of homocystine, the mixed disulfide (cysteine-homocysteine) and release of protein-bound homocysteine. This method therefore measures total (free + protein-bound) plasma homocysteine concentrations. The SBD derivative of homocysteine was determined by HPLC (29). pyridoxal-5 -phosphate (PLP) was also determined by HPLC as previously described (30), whereas plasma vitamin B- 1 2 and folate concentrations were determined by a commercially available radioassay kit (Becton Dickinson and Co. Orangeburg, NY). From the total population sample (n = 349), adequate plasma was available from 3 18 subjects (including 44 with hyperhomocysteinemia) for complete vitamin profiles. Statistica/ analicis Hotelling s T2 test (3 1), Student s t test at the Bonferroni levels ofsignificance (32), Student s t test adjusted for unequal variance with Levene s test for equal variance (if required), Wilcoxon s matched-pairs signed-ranks test, and the Mann Whitney U test were used to analyze the data. Results The mean (±SD) plasma total homocysteine concentration of the group of Caucasian men (n = 349) screened for homocysteinemia was I 3.9 ± 9.0 mol/l with a range between 4.8 and 78.6 mol/l. The SD is considerably larger when compared with results obtained in a previous study (9); this can be explained by the inclusion of several men with very high plasma total homocysteine concentrations in the present study. The distribution of data points is skewed to the right, with several men (n = 49; 14% of the total population sample) presenting with total homocysteine concentrations greater than the upper normal cutoffpoint of 16.3 tmol/l (Fig 1). This upper cutoffpoint was calculated from epidemiological data obtained from a wellnourished community with a very low incidence ofchd (9). Table I compares the vitamin nutritional status of participants with elevated plasma total homocysteine concentrations (> 16.3 mol/l) with those with normal total homocysteine concentrations. The multivariate data were slightly skewed to the right and therefore logarithmic-transformed data were used to compare the two groups. Hotelling s T2 test (3 1) was used and the two groups were significantly different (P < 0.001) with respect to the vector (PLP, cobalamin, folate). In particular, the Student s t test, at the Bonferroni (32) adjusted level of significance (0.5/ 3 = 0.016), showed that the group with elevated plasma total homocysteine concentrations had significantly lower mean plasma concentrations of all three vitamins mentioned above. The plasma vitamin concentration distribution in men with normal and men with elevated total homocysteine concentrations are summarized in Table 2. We considered the nutritional status of vitamin B-l2 and folate to be suboptimal when the plasma concentrations were < 200 pmol/l (33) and 5 (34), respectively. A plasma PLP concentration < 30 was considered as indicative of a low vitamin B-6 nutritional status (35). The Yates corrected chi-square test (36) indicated that the distribution ofdata across the different defined cutpoints for vitamin B- 12 and folate status was significantly different (P < and P = 0.022, respectively) when the elevated homocysteine concentration group was compared with the group with normal homocysteine concentrations. For vitamin B-6 status, the data distribution in the two groups was only marginally not significant (P = 0.067). Table 3 lists the plasma concentrations of folate, cobalamin, and PLP for each individual with hyperhomocysteinemia. folate concentrations were negatively correlated (r ; P = 0.062) with plasma homocysteine concentrations whereas no significant correlation between plasma cobalamin or PLP and plasma homocysteine concentrations could be demonstrated. Five individuals with hyperhomocysteinemia were

4 50 UBBINK ET AL TABLE 3 Vitamin B-6, vitamin B- 12, and folate status of adult men with hyperhomocysteinemia Subect andage (y) I, 50 2, 33 3, 36* 4, 29 5, 28 6, * 8, 26 9, 46 10, 48 I 1, 51 12, t 14, 52 15, 49* 16, 49* 17, 30 18, 25* 19, 35 20, 58t 21, 45 22, 31 23, 44t 24, 54 25, , 32t 28, 43 29, 54t 30, 47 31, 48 32, 44 33, 47 34, 40 35, 37 36, 42 37, 35 38, 56 39, 47* 40, 21 41, , 47 44, 31* and homocysteine tmoi/l I I I I I I 8.0 folate nmo//l cobalamin pmo//l I I pyridoxal- 5 -phosphate nmo//l I C Individuals with normal plasma concentrations of folate, cobalamin, pyridoxal-5 -phosphate. t Individuals deficient in all three vitamins mentioned above. - individuals with normal plasma concentrations of the three vitamins; the plasma total homocysteine concentration was 18.6 ± 0.9 mol/l and the maximum homocysteine concentration was not > 20 tmol/l. Wilcoxon s matched-pairs signed-rank test indicated that plasma homocysteine concentrations were not significantly different (P > 0.05) when the subgroup with depressed concentrations of all three vitamins was compared with the subgroup with vitamin concentrations in the normal range. Table 4 summarizes the effect of vitamin supplementation on elevated plasma total homocysteine concentrations. Groups P and V were compared by using Student s t test, which, if necessary, was adjusted for unequal variances as established by Levene s test (37). Because groups P and V were relatively small, results obtained by Student s t test were also confirmed by the Mann-Whitney U test. concentrations of PLP, folate, cobalamin, and total homocysteine were not significantly different when group P was compared with group V before supplementation began (t = 0; Table 4). Two weeks later, plasma concentrations offolate, PLP and vitamin B-l2 were significantly higher in group V when compared with group P; plasma vitamin concentrations in group V remained significantly elevated for the remainder of the study period. After 4 and 6 wk of vitamin or placebo treatment, the mean plasma total homocysteine concentration was significantly lower in group V than in group P (P < ). After the 6-wk blood specimens had been obtained, the daily vitamin dose was doubled; this resulted in considerable higher circulating concentrations of PLP, folate, and vitamin B- 12, but had no additional lowering effect on total plasma homocysteine concentrations. Note also that after 2 wk of vitamin supplementation, mean plasma total homocysteine concentrations were 36.7% lower than basal concentrations (t = 0). This was however not significantly different when compared with placebo-treated control subjects (Group P). Discussion Numerous studies (4-16) have indicated that elevated plasma homocysteine concentrations are associated with increased risk for premature occlusive vascular disease. The reasons for hyperhomocysteinemia may be varied; it may be due to enzyme polymorphisms and variants, ie, cystathionine-f3-synthase deficiency ( 1 7, 1 8, 38) or possession of a thermolabile variant of methylenetetrahydrofolate reductase (EC ), an enzyme required in the remethylation of homocysteine to methionine (39). It is also possible that nutritional deficiencies could contribute to hyperhomocysteinemia; effective metabolism of homocysteine requires an adequate supply ofvitamin B-6, vitamin B-l 2, and folic acid. It is known that losses of both vitamin B- 6 and folic acid during food refinement and processing may be considerable. Food folates are heat sensitive and 98% may be destroyed by cooking of food (40, 4 1). For vitamin B-6, losses between 10% and 50% have been reported during processing and/or storage of a wide variety of foods (35). Cobalamin deficiency may occur in persons consuming largely or exclusively found to have suboptimal plasma concentrations of all three of vegetarian diets, whereas several clinical conditions and drug the vitamins involved in homocysteine metabolism (Table 3). treatment may also result in cobalamin deficiency (42). The mean (±SD) plasma total homocysteine concentration in The data presented in Table 1 indicate that apparently healthy this subgroup was ± 22.2 tmol/l, with two individuals individuals with elevated plasma homocysteine concentrations (subjects 23 and 29, Table 3) presenting with plasma concen- had significantly lower plasma concentrations of PLP. cobalatrations > 70 zmol/l. On the other hand, Table 3 also lists seven mm, and folate when compared with persons with normal plasma

5 VITAMINS AND HYPERHOMOCYSTEINEMIA 51 TABLE 4 Effect of vitamin supplementation on plasma concentrations of homocysteine. cobalamin, pyridoxal-5 -phosphate. and folate* Treatment period index and group 0 wk 2 wk 4 wk 6 wk 8 wk Pyridoxal-5 -phosphate () P 44.5 ± ± ± ± ± 26.8 V 39.9± ±62.lt l57.0±72.4t ±69.Ot 297.3± 1l4.3t Cobalamin (pmol/l) P 215.7± ± ± ± ± 77.5 V ± ± 99.Of ± 1 lo.of ± I 1 l.7t ± l33.ot Folate (nmolll) P 6.2 ± ± ± ± ± 3.1 V 5.6 ± ± l5.6t 27.4 ± l3.8t 23.6 ± 14.6t 39.3 ± 2l.9t Homocysteine (tmol/l) P 24.0 ± ± ± ± ± 9.6 V 28.6 ± ± ± 4.74 I 1.5 ± 3.2t 10.9 ± 3.Ot C SD. Group P received placebo. whereas group V received a vitamin supplement. Vitamin and placebo doses were doubled after 6 wk. t Significantly different from group P: tp < 0.001, jp < homocysteine concentrations. Using concentration cutoff points to define suboptimal vitamin status, we found that 56.8%, 25.0%, and 59. 1% ofparticipants with hyperhomocysteinemia had suboptimal plasma concentrations of vitamin B-l 2, PLP, and folic acid, respectively (Table 2). The distribution of data across the different cutoffpoints for vitamin status was significantly different when compared with the distribution in the group with normal homocysteine concentrations. Cutoffpoints to define suboptimal vitamin status in our laboratory are plasma concentrations of < 200 pmol/l. < 30, and < 5 for cobalamin, PLP, and folate respectively. These cutoffpoints are very similar to those reported in the literature for vitamin B-6 (35), folate (33, 34), and vitamin B-l2 (33). Despite the depressed vitamin status in men with hyperhomocysteinemia, no significant correlation (cobalamin and PLP) or only a weak negative correlation (folate) between plasma concentrations of the individual vitamins and homocysteine could be demonstrated in hyperhomocysteinemic men. Although no direct relationship could be demonstrated between any single vitamin and the degree ofhyperhomocysteinemia, our data suggest that all three vitamins may be interdependent determinants of circulating homocysteine concentrations. Table 3 indicates that four offive individuals with a combined vitamin deficiency (subnormal concentrations of folate, PLP, and cobalamin) had very high plasma homocysteine concentrations, suggesting that a combined vitamin deficiency may result in severe hyperhomocysteinemia. In contrast, individuals with an adequate vitamin status (ii = 7: Table 3) had only mild hyperhomocysteinemia (< 20 omol/l). Other factors, which may also determine the degree ofhyperhomocysteinemia. could include dietary protein intake as well as the duration of the preceding period of low vitamin intake. On the basis ofthe results presented in Tables 1, 2. and 3, we investigated the effect of a daily vitamin supplement on circulating homocysteine concentrations. Daily supplementation of hyperhomocysteinemic men with 10 mg pyridoxal, 0.4 mg cyanocobalamin, and 1.0 mg folic acid normalized circulating homocysteine concentrations within 6 wk in all 1 3 participants from group V (Table 4). Our observations agree with those of previous studies, indicating that circulating homocysteine concentrations in patients with diagnosed cobalamin or folate deficiency were significantly reduced after supplementation with relatively large doses of the relevant vitamin ( ). Similarly, folic acid was effective in reducing circulating homocysteine concentrations in patients with renal insufficiency (46, 47) or occlusive vascular disease (48). However, in the studies mentioned above, high doses offolate (5-10 mg). cobalamin (1 mg), and pyridoxine ( mg) were administered. The vitamin supplement used in our study had only 20% of the folate. 25% of the vitamin B-6, and 40% of the cobalamin content when compared with the pharmaceutical preparations used in previous studies, yet considerable reductions in plasma homocysteine concentrations were observed by using this smaller vitamin supplement. In fact, an excess vitamin intake had no further beneficiary effect on plasma homocysteine concentrations. When we doubled the vitamin dose during the last 2 wk of the study. no further decline in plasma homocysteine concentrations were observed despite considerably higher circulating concentrations ofthe vitamins used as supplements (Table 4). Our results therefore indicate that hyperhomocysteinemia can often be treated with a relatively low dose ofa daily vitamin supplement. The high prevalence of suboptimal folate. vitamin B- I 2. and vitamin B-6 status in hyperhomocysteinemic men, as well as the efficacy of vitamin supplementation to normalize circulating homocysteine concentrations. indicates that hyperhomocysteinemia in the population sample studied could have been nutritionally induced by the lack ofadequate vitamins in the diet. It is important to realize that although nutritionally induced hyperhomocysteinemia will respond to a small. balanced vitamin supplement, the situation is completely different in other clinical conditions, ie, classical genetic hyperhomocysteinemia or pernicious anemia. For example, in classical genetic hyperhomocysteinemia the molecular defect resulting in cystathionine-f3- synthase deficiency is a lowered affinity of the enzyme for its cofactor, PLP (49). Hyperhomocysteinemia due to cystathionine- 13-synthase deficiency can often be treated by high-dose vitamin

6 52 UBBINK ET AL B-6 supplementation to elevate the intracellular PLP concentration as high as possible to facilitate association between the enzyme and PLP. In pernicious anemia there is no secretion of the mucosal intrinsic factor, which is required for intestinal vitamin B-l2 absorption (42). These patients suffer from a severe vitamin B-l2 deficiency and hyperhomocysteinemia (2 1, 42), which cannot be corrected by oral, low-dose cobalamin supplementation. Patients with pernicious anemia require either large, oral doses ( 1 mg) or intramuscular injections of cobalamin (33). It is unlikely that any patient with pernicious anemia was included in our study. Neither of our homocysteinemic men had plasma cobalamin concentrations < 70 pmol/l; these very low concentrations are usually encountered in pernicious anemia (42). However, it is important to realize that when elevated plasma homocysteine concentrations still exist after a reasonable period oflow-dose vitamin supplementation, appropriate followup tests should be initiated to establish the cause ofthe persistent hyperhomocysteinemia. In conclusion, we demonstrated a high prevalence of subclinical folate, vitamin B-l2, and vitamin B-6 deficiencies in men with hyperhomocysteinemia; however, this condition is easily reversed by a relatively low dose of daily vitamin supplementation. Increased risk for premature atherosclerotic vascular disease due to hyperhomocysteinemia should thus be easy to prevent by ensuring an adequate dietary vitamin intake. B A Schnell and L Goddard rendered excellent technical assistance. References I. Gibson JB, Carson NAJ. Neill DW. Pathological findings in homocystinuria. J Clin Pathol I964:17: McCully KS. Vascular pathology ofhomocysteinemia: implications for the pathogenesis of arteriosclerosis. 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