Journal of the American College of Nutrition, Vol. 19, No. 5, 608-612 (2000)
Published by the American College of Nutrition
Homocysteine Elevation in Sickle Cell Disease
Elizabeth A. Lowenthal, DO,
Matthew S. Mayo, PhD,
Philip E. Cornwell, PhD and
Denyse Thornley-Brown, MD
Nutrition Sciences (P.E.C.), University of Alabama at Birmingham, Birmingham, Alabama
Division of Nephrology (D.T.-B.), University of Alabama at Birmingham, Birmingham, Alabama
Kansas Cancer Institute, Department of Preventive Medicine, University of Kansas Medical Center (M.S.M.), Kansas City, Kansas
Address reprint requests to: Denyse Thornley-Brown, M.D., Assistant Professor, Division of Nephrology, University of Alabama at Birmingham, 728 Richard Arrington, Jr. Blvd., Suite 238, Birmingham, Alabama 35233-6959. E-mail: dtb{at}nrtc.dom.usb.edu
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ABSTRACT
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Objective: Ischemic complications are common in patients with sickle cell disease. Hyperhomocysteinemia is a risk factor for arteriosclerosis and venous thrombosis, and given the propensity of patients with sickle cell disease to develop ischemic complications, we hypothesized that they might have elevated plasma homocysteine concentrations.
Methods: Plasma concentrations of homocysteine, vitamin B12 and folate were measured in 49 adults with sickle cell disease and 16 normotensive Black controls. All subjects with sickle cell disease had been prescribed folic acid 1 mg by mouth daily.
Results: The median plasma concentration of homocysteine of subjects with sickle cell disease was approximately 1.5-fold higher than that of controls (p=0.0008). This difference persisted, even when subjects with renal insufficiency were excluded. Plasma folate levels were 1.5-fold higher in subjects with sickle cell disease than in controls (p=0.0498). There was no significant difference in plasma vitamin B12 concentrations between the two groups. There was no difference in plasma homocysteine concentrations between transfused and non-transfused sickle cell subjects.
Conclusions: Patients with sickle cell disease have elevated plasma concentrations of homocysteine in spite of elevated plasma folate levels and vitamin B12 concentrations similar to those observed in controls. Based on these data, we hypothesize that the concentration of folate required to normalize plasma homocysteine levels in patients with sickle cell disease may be higher than that of normal controls and that patients with sickle cell disease have a higher nutritional requirement for folic acid than the general population.
Key words: homocysteine, sickle cell disease, folic acid, vitamin B12
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INTRODUCTION
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Ischemic complications are a major cause of morbidity and mortality in patients with sickle cell disease [1]. The pathogenesis of these complications is poorly understood. Ischemic events in these patients have been attributed to the effects of hemoglobin polymerization, resulting in abnormal erythrocyte shape and deformability and secondary microvascular occlusion. Several observations suggest that other factors may also play a pathogenic role. If ischemia resulted solely from the occlusion of vessels by sickle erythrocytes, then tissue injury should be a consequence of microvascular occlusion; however, strokes in these patients most often occur as the result of occlusion of large and medium-sized arteries [2,3]. Moreover, at least in some studies, there is poor correlation between the extent of HbS polymerization and the frequency of episodes of painful crises [4,5].
Elevation in the plasma concentration of homocysteine is an established risk factor for venous thrombosis and arteriosclerosis [6,7]. Nutritional deficiencies of vitamin B12 and folic acid can lead to an elevated plasma homocysteine concentration [6–8]. Since patients with sickle cell disease are prone to ischemic complications, we hypothesized that these may have elevated plasma homocysteine concentrations. Consistent with this hypothesis is the observation that patients with sickle cell disease have higher physiologic requirements for folic acid due to accelerated erythropoiesis [9]. In order to obtain preliminary data regarding this hypothesis, we performed a pilot study measuring plasma levels of homocysteine, vitamin B12 and folate in a group of 49 adults with sickle cell disease and 16 controls.
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MATERIALS AND METHODS
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Subjects
Human investigation approval was obtained from the University of Alabama at Birmingham Institutional Review Board. Adult subjects over the age of 17 years were recruited from The University of Alabama at Birmingham Comprehensive Sickle Cell Center and The University of Alabama at Birmingham Department of Internal Medicine. Subjects were informed of the purpose of the study and consent was obtained on the same day as the sample collection. All patients attending the UAB Adult Comprehensive Sickle Cell Clinic during a six-month period were invited to participate. Sixteen healthy Black controls and 49 subjects with sickle cell disease participated in the study. The characteristics of the study participants are summarized in Table 1. The hemoglobin mutations of the subjects with sickle cell disease were characterized using hemoglobin electrophoresis, quantification of HbA2 and determination of erythrocyte volume and hemoglobin content. None of the subjects had hemoglobin S ß° thalassemia or Sß+ thalassemia. All of the patients with sickle cell disease had been prescribed oral folic acid at a dose of 1 mg daily, and none of the subjects was experiencing a vaso-occlusive crisis at the time of sample collection. Control samples were derived from normotensive Black individuals without a known hemoglobinopathy. The medical records of all subjects with sickle cell disease were reviewed for evidence of recent transfusion. Subjects who had evidence of hemoglobin A on hemoglobin electrophoresis or who had a blood transfusion documented in the medical record within six weeks prior to sample collection were considered to have been recently transfused. Since many subjects received primary care outside of UAB, this information was not available for all subjects.
Study Design
Subjects were on an ad libitum diet. On the morning of the study, one 7-ml sample of blood was collected in a tube containing EDTA. All samples were collected in the morning, since Guttormsen et al. [10] have shown that food intake makes little difference in plasma homocysteine concentrations when samples are collected at that time of day. The blood was immediately centrifuged at 4°C for 10 minutes and the plasma frozen at -20°C for subsequent analysis.
Total plasma homocysteine concentration was determined by high performance liquid chromatography according to the method of Cornwell et al. [11]. In brief, plasma disulfides, mixed disulfides and protein bound homocysteine were reduced with tri-n-butylphosphine. The total reduced homocysteine was then derivatized with 7-fluorobenzo-2-oxa-1,3,-diazole-4-sulfonamide (ABDF). The resulting homocysteine-ABDF conjugate was then quantified fluorometrically, with an excitation wavelength of 385 nm and an emission wavelength of 515 nm. Separation of the thiols was accomplished by high performance liquid chromatography on a reverse phase column. Quantification was accomplished by comparison of sample peak heights with those of authentic samples. Vitamin B12 and folate levels were determined using a commercially available radioimmunoassay (MAGIC Vitamin B12/folate kit: Ciba Corning Diagnostic Corp., Medfield, MA).
All results (except gender) were expressed as median (range). Since subjects with both sickle cell genotypes (i.e. hemoglobin SS and hemoglobin SC) did not differ from each other with respect to age, gender, plasma homocysteine, vitamin B12 or folate concentrations, the data from patients with both sickle cell genotypes were combined. Differences in continuous variables between subjects with sickle cell disease and controls were evaluated using the Wilcoxon Rank Sum Test, and differences in gender were evaluated using the Fisher’s Exact Test. Multiple regression analysis was used to determine if adjusting for age had an effect on the comparisons between sickle cell patients and controls. Spearman’s correlation coefficient was used to assess the relationship between these variables.
All statistical analyses were generated on a Gateway P5-120 personal computer. The analyses were performed using the Statistical Analysis System Windows Version 4.0.950 Release 6.12.
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RESULTS
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Subjects with sickle cell disease had significantly elevated plasma homocysteine concentrations, with a median value that was approximately 1.5 fold higher than that of controls (p=0.008, Fig. 1A and Table 2). The median plasma folate level of subjects with sickle cell disease was 1.4-fold higher than that of controls (p=0.0498, Fig. 1B and Table 2). None of study participants had plasma folate concentrations below the reference range (lower limit of normal 3.0 µg/l). There was no statistically significant difference in plasma vitamin B12 levels between subjects with sickle cell disease and controls. The difference in plasma homocysteine concentration between subjects with sickle cell disease and controls persisted, even when subjects with a serum creatinine concentration greater than 1.2 mg/dL were excluded (median homocysteine concentration 13.5 µmol/L in the 37 subjects with serum creatinine 
1.2 mg/dL, p < 0.0009 compared to controls). Among the 40 subjects with sickle cell disease in whom transfusion data were available, nine had recently been transfused. There was no significant difference in plasma homocysteine concentrations between subjects with sickle cell disease who had been recently transfused (median value 13.7 µmol/L, range 7.5 – 19.6) and those who had not (median value 12.9 µmol/L, range 7.2–34.8; p=0.81). The between-group difference in plasma homocysteine concentration remained statistically significant when age and gender were controlled for in multiple regression analysis.
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Fig. 1. Scattergram of plasma homocysteine and folate concentrations in pateints with sickle cell disease (HbSS) and controls (HbAA). Horizontal bars indicate median values. 1A. Plasma homocysteine concentration. Figure 1B. Plasma folate concentration.
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Table 2. Plasma Homocysteine, Vitamin B12 and Folate Levels in 49 Patients with Sickle Cell Disease and 16 Controls
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Among subjects with sickle cell disease, there was an inverse correlation between plasma homocysteine and folate levels (r=-0.39, p=0.01). There was no statistically significant correlation between the plasma concentration of homocysteine and that of vitamin B12, cysteinylglycine or creatinine. There was also no correlation between plasma homocysteine concentration and age or percent fetal hemoglobin. Plasma folate concentration was directly correlated with the percentage of fetal hemoglobin (r=0.32, p=0.04) and with age (r=0.36, p=0.02).
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DISCUSSION
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We have shown that the plasma concentration of homocysteine in patients with sickle cell disease is significantly elevated compared with that of normal controls. An unexpected finding was that the homocysteine concentrations were elevated despite the fact that the patients with sickle cell disease had significantly elevated plasma folate concentrations compared with controls. This suggests that folic acid deficiency is an unlikely predisposing factor for the high homocysteine levels observed in this population or that patients with sickle cell disease have a nutritional requirement for folic acid that is higher than previously thought. Plasma vitamin B12 concentrations were not significantly different from those of controls, making vitamin B12 deficiency an unlikely causative factor in this population.
The association between renal insufficiency and elevated plasma homocysteine levels has been well described [8]; however, in our subjects, plasma homocysteine elevations persisted, even when subjects with an elevated plasma creatinine were excluded form the analysis. Malinow et al. have shown that erythrocytes are a source of homocysteine, raising the possibility that transfusion might increase plasma homocysteine concentrations in patients with sickle cell disease [12]. Although the number of recently transfused subjects was small, the observation that there was no difference in plasma homocysteine concentrations between transfused and non-transfused subjects with sickle cell disease suggests that the elevated plasma homocysteine concentrations were not due to a transfusion effect.
Only one other published paper has evaluated plasma homocysteine concentrations in adult patients with sickle cell disease. In a study designed to examine the relationship between stroke and plasma homocysteine concentration, patients with sickle cell disease and a history of stroke had significantly higher plasma concentrations of homocysteine than those without a stroke history [13]. In that study, there was an inverse relationship between plasma homocysteine and red cell folate concentrations [13], a finding similar to ours. The study population was very heterogeneous, however, and included children and adults as well as patients who were acutely ill. Moreover, the data presented were pooled; therefore, it is unknown whether there is a difference in plasma homocysteine concentrations in children and adults. No healthy subjects were studied as a control group, and plasma vitamin B12 concentrations were not measured.
Nutritional deficiencies of folic acid and vitamin B12 are well-described acquired causes of elevated plasma homocysteine concentrations [14–16]. Although folic acid deficiency is a well-recognized complication of sickle cell disease [9,17], many authors do not recommend giving routine folic acid supplements, since most patients have plasma and red cell folate concentrations within the usual reference range [18]. In our study population, plasma homocysteine levels were elevated in the subjects with sickle cell disease despite the use of oral folic acid supplementation and despite significantly elevated plasma folate concentrations (Table 2). The concentration of folate in red blood cells is a more sensitive marker of folic acid deficiency than is the plasma concentration of folate, and in the study by Houston et al. [13] red cell folate concentrations in subjects with sickle cell anemia and hyperhomocysteinemia were well within the normal range. Oral supplementation with folic acid has been shown to lower the plasma homocysteine concentration even in the absence of folic acid deficiency [19], and this has been recently demonstrated in children with sickle cell disease.
Children from Curaçao with sickle cell disease who were not receiving folic acid supplementation had a slight, but statistically significant elevation in plasma homocysteine cencentrations compared to controls (12.7 µmol/L in patients with sickle cell disease vs. 10.9 µmol/L in controls), despite similar plasma folate levels. Oral supplementation with 2 to 4 milligrams of folic acid daily led to a 53% reduction in plasma homocysteine concentrations in the children with sickle cell disease [20]. In contrast, in a study of Texan children, there were no statistically significant differences in plasma homocysteine or plasma or red blood cell folate concentrations in sickle cell patients compared to controls [21]. It is not known whether higher doses of oral folic acid would normalize plasma homocysteine in adults with sickle cell anemia.
While the results of the present study fail to demonstrate significant vitamin B12 deficiency in patients with sickle cell disease, subtle neurologic changes related to cobalamin deficiency may be difficult to detect in this group of patients, and measurement of methylmalonic acid levels in the urine or blood may be a more sensitive technique to evaluate vitamin B12 status [15,22]. Oral supplementation with vitamins B6 and B12 did not lower the plasma homocysteine concentration in children with sickle cell disease, however [20].
Congenital hyperhomocysteinemia, which has been associated with an increased risk of arteriosclerosis and venous thrombosis, has been associated with mutations in several of the enzymes involved in homocysteine catabolism. The prevalence of these mutations among peoples of African origin is unknown. Mild cases of congenital hyperhomocysteinemia have been demonstrated using methionine loading [23], a procedure that was not used in the current study. Further studies are indicated to determine whether mutations in the homocysteine catabolic pathway may be responsible for some of the elevated plasma homocysteine concentrations seen in patients with sickle cell disease.
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CONCLUSION
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Plasma homocysteine levels are elevated in the plasma of adult subjects with sickle cell disease who are free of vaso-occlusive crisis, despite seemingly adequate folate concentrations. Since hyperhomocysteinemia has been associated with vascular complications in other patient populations, it may contribute to the vascular complications of patients with sickle cell disease. We observed an inverse correlation between plasma folate and homocysteine concentrations. Other authors have been able to normalize plasma homocysteine levels in children, using folic acid supplementation at doses higher than those usually given to adults. These observations suggest that the dose of folic acid required to normalize plasma homocysteine levels in adult sickle cell patients may higher than the 1-mg daily oral dose that is usually prescribed, and that patients with sickle cell disease may have a higher nutritional requirement for folic acid than previously thought. Further studies are indicated to determine the optimal nutritional requirement of folic acid intake in adult patients with sickle cell disease.
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ACKNOWLEDGMENTS
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The authors wish to thank Dr. Michael Allon, Dr. Josef Prchal and Dr. Brian Adler for their for helpful suggestions and critical review of the manuscript and Ms. Barabara Lawson for assistance in manuscript preparation. Financial Support provided by NIH Cancer Research Training Grant #K12CA01719-05 and NIH DRR General Clinical Research Center Grant RR-32.
Received February 7, 2000.
Accepted July 27, 2000.
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ABSTRACT
INTRODUCTION
