The role of homocysteine (Hcy) in promoting the development and progression of atherosclerotic vascular diseases is receiving growing attention in the medical literature. 1 Possible therapies to prevent or reduce hyperhomocystinemia include supplements of vitamin B6, B12, and folate. 2 However, few authors reported the presence of low serum folate (s-F) levels in patients on dialysis, 3,4 which shows that folate therapy should maintain folate levels within the normal interval. Other investigators, including some of the current authors, found normal s-F levels in the large majority of patients on dialysis. Nevertheless, according to some studies, the administration of folate alone or in combination with vitamin B6 or B12 effected a significant decrease or even normalization in plasma Hcy levels. 5–8 Only two studies included serial measurements of erythrocyte folate concentrations, which represent tissue folate storage and, therefore, the patient’s real folate status. Serum folate, in fact, is loosely bound to albumin, from which it is taken by high affinity folate receptors present in our cells. Erythrocyte folate concentrations represent a stable folate pool and reflect vitamin availability, (erythrocytes being nonproliferating and metabolically inactive cells), whereas s-F levels reflect recent dietary intake. 9 In patients on peritoneal dialysis, we 8 showed that plasma Hcy is correlated with serum and erythrocyte folate concentrations, although the majority of patients with folate concentrations within the reference interval had high or very high plasma Hcy levels.
In a previous study with patients on peritoneal dialysis with normal erythrocyte folate levels, we demonstrated that oral folate supplementation can reduce plasma Hcy levels even in the presence of normal serum or erythrocyte folate levels. 10 Therefore, on the basis of the available data, we cannot establish whether folate therapy would normalize serum or erythrocyte folate levels or increase concentrations to a normal level. Therapy with folate is not devoid of possible side effects, 11 and folate supplementation, even if cheap in itself, becomes quite expensive for long-term therapies. Therefore, in the absence of guidelines based on evidence, the clinician might be reluctant to give high folate doses to any patients on peritoneal dialysis, irrespective of the serum and erythrocyte levels of this vitamin, without a definite target.
In an attempt to clarify some of these points, we gave incremental doses of folate supplementation to 56 patients on peritoneal dialysis with normal erythrocyte folate levels. The aim of our study was to evaluate the effects of incremental doses of supplementation on serum and erythrocyte folate, serum vitamin B12, and plasma Hcy concentrations and the possible role of the thermolabile mutation of methylenetetrahydrofolate reductase (tMTHFR) genetic variant in modifying the response to this therapy.
Patients and Methods
The study was proposed to all nondiabetic patients on continuous ambulatory peritoneal dialysis (CAPD) who fulfilled the following inclusion criteria: plasma Hcy levels greater than 20 μmol/L in two consecutive samples, peritoneal dialysis for more than 6 months, no use of icodextrin or amino acid dialysis solutions, no active infections including peritonitis in the 3 months previous to the study, no malignant neoplasm for 3 years before the study, no acute illness, no malnutrition, and no treatment with vitamin or folate supplementation. Sixty-two patients, all on CAPD, had no exclusion criteria. Fifty-six of them accepted inclusion in the study.
All patients were treated with three to five daily exchanges of 2 to 2.5 L each. The exchanges were performed by different double bag systems. All the patients had undergone dialysis with solutions containing lactate buffer and glucose of different concentrations as an osmotic agent. At the time of the study or during the previous 2 months, none of the patients experienced exchanges of dialysis solutions containing amino acid, icodextrin, or bicarbonate buffer. After giving written informed consent, 56 patients (group A) were given 5 mg/day folic acid oral supplementation for 3 months. After this period, patients whose plasma Hcy levels were still greater than 20 μmol/L were asked to increase the dose of folate to 10 mg/day. Nineteen patients (group B) accepted. Three months later, patients with high serum Hcy were again asked to increase the dose of folate to 15 mg/day, and 10 (group C) accepted and completed the third part of the study. The patients whose plasma Hcy levels decreased to less than 15 μmol/L or by more than 50% were arbitrarily defined as “responders.”
Blood samples were drawn in the morning, after overnight fast. Two blood specimens from each patient were collected in light protected plastic tubes, either with no additive (for B12 and s-F) or containing ethylenediaminetetraacetic acid (EDTA) to prevent coagulation, for erythrocyte folate and Hcy concentrations assays. EDTA specimens were immediately put on ice and centrifuged within 30 minutes after the collection of aliquots of whole blood for erythrocyte folate determination. 12 Serum and plasma samples were frozen and stored at −20° until analyzed.
Plasma total Hcy concentrations were measured by the semiautomatic immunoenzymetric fluorescence polarization immunoassay IMx Hcy method (Abbott Laboratories, Diagnostic Division, Abbott Park, IL). Samples with Hcy concentrations higher than the upper limit of calibration (50 μmol/L) were again tested after proper dilution. Erythrocyte and s-F concentrations were measured by the semiautomatic immunoassay MEIA IMx folate method (Abbott). Serum vitamin B12 was measured by the semiautomatic immunoassay MEIA IMx vitamin B12 method (Abbott), as described elsewhere. 13 Molecular analysis of the tMTHFR genetic variant had been performed in 38 patients by polymerase chain reaction (PCR) amplification and HinFI enzymatic digestion. 14 Determinations were performed on frozen stored samples when available. Molecular analysis of the tMTHFR genetic variant was limited to the first patients enrolled in the study. This choice was not randomized but was casual so as not to result in an unbalanced or biased selection.
The reference intervals of the variables considered were obtained from samples of healthy subjects and are currently used in our laboratories. The reference intervals were as follows: Hcy, cut-off of 10.5 μmol/L; erythrocyte folate, 420 to 1460 nmol/L; s-F, 7 to 28 nmol/L; and B12, 165 to 835 pmol/L. Hematologic values were determined by an STKS Beckman-Coulter counter (Fullerton, CA).
The comparison between mean values was determined with the Student’s t-test, and comparison between proportions with the chi-square test. Spearman coefficients were calculated where appropriate.
The characteristics of the patients enrolled in the study are reported in Table 1. Mean levels of plasma Hcy, serum vitamins B12, s-F, and erythrocyte folate measured in the three phases of the study are reported in Table 2. Serum and erythrocyte folate concentrations were all within the relevant reference interval after 5 mg/day folate administration and, after higher folate doses, these data were beyond the upper limit of method detection in all the patients, thus making any statistical evaluation unreliable.
During the first period (5 mg/day folate supplementation, group A), one patient began hemodialysis and did not comply with the prescribed therapy. Mean s-F and erythrocyte folate levels showed a statistically significant increase, whereas vitamin B12 remained unchanged (Table 2). After folate administration, s-F levels rose above the upper limit of detection in 23 patients and erythrocyte levels in 27 patients. Plasma Hcy levels decreased to less than 15 μmol/L in 6 (10%) patients and in another 12 (21%) patients by more than 50% (responders). The patients who responded to folate supplementation had higher mean baseline Hcy levels (62.4 ± 35.1 vs. 33 ± 15.9 μmol/L, p < 0.01), lower mean s-F levels (7.9 ± 4.8 vs. 12.1 ± 8.8, p < 0.05), and a higher Hcy/s-F ratio (9.8 ± 6.1 vs. 4.3 ± 4.2, p < 0.01) than the patients who did not respond. Molecular analysis of the tMTHFR genetic variant had been performed in 14 of 18 responders and 24 of 39 nonresponders. Among the responders, 8 of 14 (57%) were homozygotes, 5 of 14 (36%) heterozygotes, and 1 of 14 (7%) were wild type. In the 24 nonresponders, molecular analysis showed that 7 of 24 (29%) genotypes were homozygotes, 8 of 24 (30%) heterozygotes, and 9 of 24 (37%) were wild type. The distribution of genotypes showed no statistically significant difference between responders and nonresponders (chi-square 4.86, p > 0.9).
After 5 mg/day folate supplementation, mean Hcy concentrations decreased from 89 ± 47 to 27 ± 14 μmol/L (p < 0.01) in the eight homozygous responders and from 65 ± 35 to 18 ± 8 μmol/L (p < 0.01) in the six heterozygous and wild type responders. In nonresponders, mean Hcy plasma levels decreased from 44 ± 18 to 25 ± 9 μmol/L (p < 0.03) in the 7 homozygotes and from 28 ± 6 to 22 ± 5 μmol/L (p < 0.02) in the 17 nonhomozygotes.
Nineteen (49%) of the remaining 39 patients accepted an increase in the oral folate dose to 10 mg/day (group B). The data of patients continuing the study found in Table 1 (Hcy 32 ± 10 μmol/L, vitamin B12 372 ± 143 pmol/L, s-F 41 ± 15 nmol/L) did not differ from the data of those who stopped folate therapy (age 63 ± 15 years, body weight 64 ± 8 kg, dialysis duration 30 ± 29 months, Hcy 35 ± 22 μmol/L, vitamin B12 420 ± 93 pmol/L, s-F 36 ± 26 nmol/L). At the end of the second part of the study, both serum and erythrocyte folate concentrations were beyond the upper limit of method detection in all the samples, and therefore statistical evaluation was not reliable and no comparison was made. Plasma Hcy levels decreased to less than 15 μmol/L in one (6%) patient but increased in one and did not decrease by 50% in any patient.
Ten of the remaining 18 patients accepted an increase in the dose of folate to 15 mg/day (group C). The data of patients continuing the study shown in Table 1 (Hcy 29 ± 7 μmol/L, vitamin B12 353 ± 151 pmol/L) did not differ from the data of those who gave stopped folate therapy (age 63 ± 15 years, body weight 64 ± 8 kg, dialysis duration 30 ± 29 months, Hcy 38 ± 42 μmol/L, vitamin B12 347 ± 185 pmol/L). At the end of this (third) part of the study, serum and erythrocyte levels were above the upper detection limit in all the patients, and once again vitamin B12 levels did not change. No decrease in Hcy levels to less than 15 μmol/L was observed in any of the other patients.
Only a few patients said that they did not want to increase the dose because of the great number of therapies they were undergoing or because of the greater amount of water needed to take the additional pill, but a large majority of them gave no reason for refusing to continue the study. In a written interview, none of the patients complained of or had clinically evident symptoms that could be related to folate overload. 11
In patients on dialysis, cardiovascular diseases still remains the main cause of mortality and one of the most important causes of morbidity. Hyperhomocystinemia is an independent risk factor for cardiovascular diseases, and every effort should be made to lower plasma levels of this aminothiol. The comparative lack of any effect of vitamin B6 and B12 supplementation on these patients 15,16 and the response to folic acid supplementation 5–8 suggest some shortage of intracellular folate in patients on dialysis. However, this hypothesis was not confirmed by recent studies. 8,12
In small groups of patients on dialysis with high plasma Hcy concentrations, some authors report a definite decrease, others a mild decrease or normalization, in plasma Hcy. 5–8 In patients on peritoneal dialysis, folate deficiency is less frequent than in patients on hemodialysis, and mean levels of s-F are generally higher. 17 In our patients on CAPD, low s-F or plasma vitamin B12 levels are rare, and erythrocyte folate concentrations are normal in a large majority of cases. 8 Apparently, this result excludes the need for folate supplementation in reducing Hcy levels in these patients. However, in another study, daily supplementation with 5 mg oral folate produced a definite Hcy decrease in patients. 10 It is noteworthy that the plasma Hcy/folate ratio was significantly higher in patients who responded to folate supplementation than in nonresponders.
Because s-F and erythrocyte folate levels are unreliable in establishing the target dose of folate supplementation, the appropriate dose of folate is still undefined. With patients on hemodialysis, two studies (only 1 prospective study and neither of them controlled) reported similar effects of different doses of folate supplementation (1 vs. 5 and 2.5 vs. 5 mg daily) on plasma Hcy levels. 5,18 However, this result cannot be applied to patients on peritoneal dialysis because of the different mean folate levels. 17 In another study, we observed a definite Hcy decrease in 45% of patients on CAPD given 5 mg folate supplementation. However, to date, we do not know whether the folate dose was adequate or not.
In the current study, we attempted to increase folate dose up to 15 mg/day to evaluate possible effects of incremental doses, side effects, and levels of s-F and erythrocyte folate concentrations that were associated with lower Hcy levels. After oral folate administration (5 mg/day), we observed a large increase in s-F and erythrocyte folate concentrations in all the patients treated, which confirms their compliance with folate supplementation. Unfortunately, as a consequence of taking the highest doses of folate supplementation, nearly all the patients treated had s-F and erythrocyte folate levels above the upper detection limit of the laboratory method used, which biases the search for a statistical correlation between final folate and Hcy concentrations. This result does not allow us to determine whether folate concentration is adequate or not for Hcy metabolism to maintain the transmethylation pathway of Hcy metabolism. After 5 mg oral folate supplementation, less than one fourth of the patients showed a major (>50%) decrease in Hcy levels, and this effect was not accompanied by higher s-F or erythrocyte folate levels. These data are less promising than those previously reported by us 10 but confirm our previous results that suggest that neither s-F nor erythrocyte folate measurements can help in identifying dialysis patients requiring folate supplementation to keep Hcy levels within an acceptable interval. The dose increase to 10 mg/day was followed by a mild decrease in Hcy levels to less than 15 μmol/L in one patient, but, in every other patient, the mean Hcy variation was negligible or less than 20%. No additional effect was obtained by a further increase of 15 mg/dl in folate dose. About half the patients did not want to continue the study after the first step. Obviously, this high rate of patients giving up the study could bias the following steps of the study. However, the characteristics of patients stopping the study did not differ from those patients who continued, and we suspect that a larger acceptance rate could be associated with a lower compliance.
Only a small number of patients enrolled in this study received genetic mapping for tMTHFR, and the lack of statistical significance does not allow any definite conclusion or speculation on this point. Both homozygous and nonhomozygous responders were characterized by higher baseline Hcy levels than nonresponders. It is interesting to note that mean s-F levels were lower in responders than in nonresponders.
Unfortunately, because of the relatively small number of eligible patients, the study could not be controlled and randomized. In addition, the number of patients who accepted an increase in the dose was even smaller. Each patient was treated with different folate doses, but, in the absence of a control group, small spontaneous variations of Hcy levels could not be ruled out. However, in a previous controlled study, 10 we observed no change in Hcy plasma concentrations in untreated control patients, thus indicating that spontaneous Hcy variations levels are rather infrequent. In addition, to assess the efficacy of the therapy, only significant changes (>50%) or normalization of Hcy levels were considered.
Urticarial rash was the most commonly reported side effect of folate supplementation, 19 but Hunter et al.11 reported a high rate of disturbing side effects after administration of a high oral folate dose to healthy volunteers. In fact, most of these symptoms are common in patients on dialysis even in the absence of folate supplementation. Nevertheless, our patients did not complain of intensifying symptoms after increasing folate supplementation, which would seem to indicate that vitamin side effects were clinically negligible.
In conclusion, in the current study, the effect of 5 mg oral folate supplementation was small, and, in fact, only 31% of patients showed a significant lowering of Hcy concentration. The effect of increasing doses of folate was negligible in the majority of cases. However, in that folate supplementation is relatively inexpensive and free of side effects, a careful attempt to increase the dose up to 10 mg/day might be suggested in patients on peritoneal dialysis not responding to lower doses. If no effect is achieved, the vitamin supplementation should be either withdrawn or reduced to maintain serum and erythrocyte levels within the reference intervals.
1. Rosenthal AF, Ginsburg MJ, Crawford JF: Homocysteine and heart disease in dialysis patients. Dial Transplant 27: 627–629, 1998.
2. Welch G, Loscalzo J: Homocysteine and atherothrombosis. N Engl J Med 338: 1042–1050, 1998.
3. Bostom AG, Shemin D, Lapane KL, et al: Folate status is the major determinant of fasting total plasma homocysteine levels in maintenance dialysis patients. Atherosclerosis 123: 193–202, 1996.
4. Vychytil A, Fodinger M, Wolfl G, et al: Major determinants of hyperhomocystinemia in peritoneal dialysis patients. Kidney Int 53: 1775–1782, 1998.
5. Dierkes J, Domrose U, Ambrosch A, et al: Response of hyperhomocystinemia to folic acid supplementation in patients with end-stage renal disease. Clin Nephrol 51: 108–115, 1999.
6. Westhuyzen J: Folate supplementation in the dialysis patients fragmentary evidence and tentative recommendations. Nephrol Dial Transplant 13: 2748–2750, 1998.
7. Jannsen MJ, van Guldener C, De Jong GM, et al: Folic acid treatment of hyperhomocystinemia in dialysis patients. Miner Electrolyte Metab 22: 110–114, 1996.
8. De Vecchi AF, Bamonti Catena F, Finazzi S, et al: Homocysteine, vitamin B12, serum and erythrocyte folate in peritoneal dialysis and hemodialysis patients. Perit Dial Int 20: 169–173, 2000.
9. Jacobsen DW: Serum and erythrocyte folates: a matter of life and premature death. Clin Chem 42: 1579–1580, 1996.
10. De Vecchi AF, Patrosso C, Novembrino C, et al: Folate supplementation in peritoneal dialysis patients with normal erythrocyte folate. Effect on plasma homocysteine. Nephron
89: 297–302, 2001.
11. Hunter R, Barnes J, Oakeley BF, Matthews DM: Toxicity of folic acid given in pharmacological doses to healthy volunteers. Lancet 1: 61–63, 1970.
12. Bamonti Catena F, Buccianti G, Porcella A, et al: Folate measurements in patients on regular hemodialysis treatment. Am J Kidney Dis
33: 492–497, 1999.
13. Bamonti-Catena F, Porcella A, Pomati M, et al: Whole blood folate concentrations: comparison between Stratus Folate (DADE) and radioassay (DPC) methods. Eur J Clin Chem Clin Biochem 35: 781–785, 1997.
14. Frosst P, Blom HJ, Milos R, et al: A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 10: 111–113, 1995.
15. Dierkes J, Domrose U, Ambrosch A, et al: Supplementation with vitamin B12 decreases homocysteine and methylmalonic acid but also serum folate in patients with end stage renal disease. Metabolism 48: 631–635, 1999.
16. Bostom AG, Shemin D, Lapane KL, et al: High dose B vitamin treatment of homocystinemia in dialysis patients. Kidney Int 49: 147–152, 1996.
17. Moustapha A, Gupta A, Robinson K, et al: Major determinants of hyperhomocystinemia in hemodialysis and peritoneal dialysis patients. Kidney Int 55: 1470–1475, 1999.
18. Spence JD, Cordy P, Kortas C, Freeman D: Effect of usual doses of folate supplementation on elevated plasma homocysteine in hemodialysis patients. No difference between 1 and 5 mg daily. Am J Nephrol
19: 405–410, 1999.
19. Butterworth CE, Tamura T: Folic acid safety and toxicity: a brief review. Am J Clin Nutr 50: 353–358, 1989.