Concentrations of B vitamins and homocysteine in children with sickle cell anemia.

Background: Elevated concentration of serum homocysteine contributes to thrombosis, a frequent event in patients with sickle cell anemia. We aimed to test whether children with sickle cell anemia have elevated concentrations of serum homocysteine with diminished levels of folate or B vitamins from accelerated blood cell turnover.

Methods: We conducted a case-control study of children with homozygous sickle cell anemia (n = 17) and unaffected children (n = 11). We measured serum and red blood cell folate, vitamin [B.sub.6], vitamin [B.sub.12], and homocysteine concentrations, and assessed micronutrient intake.

Results: Children with sickle cell anemia had concentrations of homocysteine slightly higher than those of unaffected children. They had lower vitamin [B.sub.6] concentrations and comparable concentrations of folate and vitamin [B.sub.12]. Homocysteine concentration was inversely related to vitamin [B.sub.12] concentration and was not independently associated with levels of vitamin [B.sub.6] or folate.

Conclusion: Despite comparable intake, children with sickle cell anemia had lower concentrations of vitamin [B.sub.6] than unaffected children. Larger studies are needed to determine if chronically low serum vitamin [B.sub.6] concentration contributes to hyperhomocysteinemia in this population.

**********

Children with sickle cell anemia (SCA) are at an increased risk for thrombotic events, primarily stroke. Lifetime risk of overt stroke in patients with SCA ranges from 13 to 17%, (1) and may be as high as 7.8% by 14 years of age. (2) The risk of overt stroke in those with SCA who are younger than 20 years has been estimated to be 7.6 per 1,000 person-years. (3) A large, cooperative study of children in the 1980s found the age-adjusted incidence of stroke in children with homozygous hemoglobin S to be 6.1 per 1,000 person-years. (4) Thrombotic stroke, in this population, involves the large vessels. While the mechanisms contributing to stroke are incompletely understood in SCA, a chronic, low-grade elevation in thrombin activity has been demonstrated in these patients, suggesting an environment conducive to thrombosis. (5)

In patients who do not have SCA, elevated concentration of homocysteine has been linked prospectively with thrombotic events, including stroke, in middle-aged men (6) and in young people with systemic lupus erythematosus. (7) In addition, case-control and prospective studies have demonstrated strong positive associations between serum homocysteine concentration and atherosclerotic disease, including coronary artery disease, (8), (9) peripheral vascular disease, (10) and carotid artery stenosis. (11), (12) We hypothesized that hyperhomocysteinemia may also be a thrombotic risk factor in patients with SCA. As an initial step, we sought to determine if hyperhomocysteinemia occurs in children with SCA.

In studies of large populations, hyperhomocysteinemia has been linked to low serum concentrations of folate and vitamin [B.sub.12], important intermediaries in homocysteine metabolism. (13-15) The relationship between vitamin [B.sub.6] and homocysteine has been inconsistent in observational studies, although some reports suggest that low vitamin [B.sub.6] concentration contributes to hyperhomocysteinemia. (13), (15-18) Because patients with SCA have accelerated hemolysis and increased red blood cell production, they are at risk of depletion of these nutrients, particularly folate. We hypothesized that a plausible mechanism by which patients with SCA may have hyperhomocysteinemia is due to depletion of folate, vitamin [B.sub.12], or vitamin [B.sub.6].

We aimed to investigate whether children with SCA have elevated serum homocysteine concentration, compared with unaffected black children, and if homocysteine concentrations vary inversely with serum concentrations of folate, vitamin [B.sub.12], or vitamin [B.sub.6] in this population. A secondary goal was to assess whether children with SCA were meeting the recommended dietary needs for these micronutrients.

Patients and Methods

The Institutional Review Board of the Johns Hopkins Medical Institutions approved this study. Parents or guardians of the participants provided informed consent, and all children involved assented to participate.

Study Design

We conducted a case-control study of children with homozygous SCA and unaffected black children. Consecutive children with homozygous hemoglobin S and their parents or guardians were approached regarding participation in this study at their outpatient visits between December 1998 and November 1999. Healthy black children were recruited from among the unaffected siblings of the patients with SCA, as we anticipated that siblings' diets were fairly comparable. Additional healthy children were recruited from the general pediatrics clinic at the time of their well-child visits. Patients were scheduled for a fasting blood draw, dietary assessment, and medical history review.

Participants

Inclusion criteria were age between 4 and 18 years and, for the cases, a diagnosis of homozygous SCA. Exclusion criteria were use of folate supplementation therapy within the previous 4 months and pregnancy. Patients were also excluded if they were taking phenylbutyrate or hydroxyurea or using medications known to affect homocysteine and folate concentrations (such as methotrexate and select anticonvulsants) within the 30 days preceding phlebotomy. In addition, patients regularly undergoing erythrocytapheresis were excluded for logistical reasons, although patients receiving long-term transfusions were not. There were no exclusions based on clinical status or sex. Unaffected children were not excluded because of the presence of sickle cell trait.

Specimens

Blood was collected by venipuncture from fasting participants at least 4 weeks after the participant's most recent transfusion. One tube with EDTA was immediately centrifuged (1500 X g for 15 min), and plasma was aliquoted into an amber plastic vial and stored at 4[degrees]C, for measurement of vitamin [B.sub.6] concentration by radioimmunoassay (Quest Diagnostics, Teterboro, NJ). (19) Coefficient of variation (CV) was 9.5% at 10 ng/ml. A separate aliquot was stored at 4[degrees]C for measurement of plasma homocysteine concentration by high-pressure liquid chromatography (Quest Diagnostics, Teterboro, NJ) on the next day. (20) CV was 11% at 9 [micro]mol/L. A preservative-free specimen was allowed to clot for 30 minutes, centrifuged (1500 X g for 15 min), and used for measurement of serum vitamin [B.sub.12] and folate concentrations by enzyme-linked immunosorbent assay (ELISA), with CVs of 6.9 and 5.4%, respectively. (21) In addition, samples of EDTA blood were used for complete blood cell count and reticulocyte count (using a Coulter counter) and for measurement of erythrocyte folate concentration by ELISA (CV, 7.7%).

Dietary History 

A research dietitian administered a 24-hour dietary recall, a validated method of assessing micronutrient intake in adults and children. (22-25) To increase the accuracy of the recalled information, the participants were alerted to this before the visit. The initial recall was administered in person, with memory aids including food models, pictures, and standard household measures. Each participant was informed that there would be three additional dietary recalls administered by telephone over the subsequent year at approximately 3-month intervals to capture seasonal variation in diet. The participants were not told the dates in advance, to prevent significant alterations in the diet. The nutrient content of each participant's diet was analyzed using the Nutrition Data System 4.0 software (NDS, Minneapolis, MN). This software is updated biannually, although our version was not yet programmed to account for the addition of folate to grain that began with the national Food Folate Fortification Program in 1998. The micronutrients ingested in multivitamins were included in the total nutrient content of the diet. We determined the adequacy of nutrient intake by comparing the average daily intake to the U.S. Recommended Dietary Allowance for children. (26)

Medical History

One investigator (JBS) interviewed each patient or family member. Patients with SCA and the unaffected children were asked about use of multivitamins and their medical history, including frequency of hospitalizations, current medical therapy, and transfusion history.

Statistical Analysis

We examined the distribution of all measurements. Daily nutrient intakes, assessed up to four times per participant, were averaged to approximate the participant's average daily intake. Variables with skewed distributions were log transformed for normality, if appropriate, or reported as medians with interquartile ranges (IQRs).

Data were analyzed using Stata version 6.0 software (Stata Corp., College Station, TX). Concentrations of homocysteine, folate, vitamin [B.sub.12] and vitamin [B.sub.6], and reported nutrient intakes were compared between patients and unaffected participants by the Wilcoxon rank-sum test. Pairwise Pearson correlation coefficients (r) were calculated between average micronutrient intakes, vitamin concentrations, homocysteine concentration, and reticulocyte count. Folate, vitamin [B.sub.12], and vitamin [B.sub.6] concentrations were evaluated independently as predictors of homocysteine concentration using robust regression analyses. Sickle cell anemia status was entered as a predictor variable into the robust regression model.

Results

Twenty-eight children were enrolled in the study (17 children with SCA and 11 without the disease). The principal reasons for nonparticipation were use of folate supplementation, hydroxyurea therapy, or erythrocytapheresis. The participants ranged in age from 4 to 18 years, with a mean of 9.2 years; 61% were boys. Clinical characteristics of the participants are presented in Table 1. We obtained an average of 2.3 dietary recalls per child, with 19 children contributing two dietary histories and 9 children contributing three recalls.

Homocysteine and Vitamin Concentrations

Homocysteine concentration was higher in the children with SCA than in the unaffected children (5.4 [micro]mol/L [IQR, 4.6-5.9] versus 4.3 [micro]mol/L [IQR, 3.4-5.7]), although this was not a significant difference (P = 0.2). One 16-year-old boy with SCA had markedly elevated homocysteine concentration (15 ng/ml). In this sample with little age variance, homocysteine concentration was not significantly influenced by age (P = 0.35). Serum and red blood cell folate and vitamin [B.sub.12] levels were similar in the two groups (Table 2). Children with SCA, however, had lower plasma concentrations of vitamin [B.sub.6] than healthy children (6.4 ng/ml [IQR, 4.5-9] versus 11 ng/ml [IQR, 9.3-12]; P = 0.01). Among all of the measurements, only erythrocyte folate concentration showed age dependence. Erythrocyte folate content decreased significantly with age among the children with SCA (P = 0.008).

Dietary Intake

Daily intakes of folate and vitamins [B.sub.6] and [B.sub.12] were similar in both groups (Table 3). The percentage of children with SCA meeting national nutritional recommendations for age were 47% for folate consumption, 88% for vitamin [B.sub.6] intake, and 94% for vitamin [B.sub.12] intake. This was comparable to the rates among healthy children, in whom age-adjusted nutritional requirements were met by 64% for folate, 82% for vitamin [B.sub.6], and 73% for vitamin [B.sub.12]. Daily protein requirements were met by 94% of the children with SCA and 91% of the healthy children. All but one child met the Recommended Dietary Allowance for vitamin [B.sub.6] when expressed as milligrams per gram of protein (0.02 mg/g protein for children and adolescents).

Relationship between Intake and Serum Concentrations

There was a significant positive correlation between self-reported daily intake of folate and serum folate concentrations. Individuals with higher intake of folate had higher serum folate concentrations (r = 0.43, P = 0.03), but not higher erythrocyte folate concentrations (Table 4). This relationship was similar for those with and without disease, but was not seen in either group for concentrations of vitamins [B.sub.6] and [B.sub.12]. Children with SCA reported a daily consumption of vitamin [B.sub.6] equivalent to that of the unaffected children.

Relationship between Vitamins and Homocysteine

When patients and healthy children were analyzed together, vitamin [B.sub.12] concentration was a strong predictor of homocysteine concentration. For every decrease in vitamin [B.sub.12] level by 100 pg/ml, homocysteine concentration increased by 0.43 [micro]mol/L (P = 0.03) (Fig. 1). This relationship was similar in magnitude in the children with SCA and the unaffected children. We found a modest dependence of homocysteine concentration on the amount of daily vitamin [B.sub.6] intake. Patients with a higher average [B.sub.6] intake had higher homocysteine concentrations, even after controlling for serum vitamin [B.sub.12] concentrations. This may have been confounded by reticulocytosis, however, because when we controlled for reticulocytosis, this relationship disappeared. Homocysteine was not associated with folate concentrations in either serum or erythrocytes.

Discussion

We found that homocysteine concentrations were not significantly higher in children with SCA than in unaffected children and below the concentrations in healthy adults, in whom normal is considered up to 12 [mu]mol/L. Homocysteine concentrations among the unaffected children were similar to those reported in the Child and Adolescent Trial for Cardiovascular Health, (27) a large sample in which the mean age of subjects was 14 years and mean nonfasting homocysteine concentrations in black children was 5.5 [mu]mol/L.

Of the six published studies examining homocysteine concentration in patients with SCA, (28-33) three reported concentrations in children. (28), (29), (32) In one, the mean homocysteine concentration in 27 children with hemoglobin SS disease (mean age, 8 yr) was 12.5 [+ or -] 5.0 [mu]mol/L, which decreased after supplementation with vitamins [B.sub.6] and [B.sub.12] and folate. (28) In 120 children with SCA in Texas (mean age, 9 yr), the mean homocysteine concentration was only 5.2 [+ or -] 2.5 [mu]mol/L. (32) In another sample of 40 children, one-fourth of whom had a history of stroke, mean homocysteine concentration was 5.8 [mu]mol/L, which did not differ from the study children without SCA. (29)

Although we anticipated that children with SCA who were not given folate supplements might be folate-deficient due to accelerated red blood cell turnover, this was not observed. The mean serum folate concentration of children with SCA in our study was similar to that of the subjects in the other study of children not given supplementation therapy (9 and 8 ng/ml, respectively). (28) These levels were considerably higher than the concentrations seen in children in the Texas study, where the mean serum folate concentration was 5.5 [+ or -] 4.4 ng/ml. (32) The Texas study, however, occurred before the institution of national food fortification with folate (140 mg of folic acid per 100 g of grain).

[FIGURE 1 OMITTED]

Earlier studies of patients with SCA reported lower serum folate concentrations in these patients than in individuals with sickle trait and healthy adults, although the erythrocyte folate concentrations tended to be similar in all groups, possibly due to the high folate content of reticulocytes. (34), (35) A recent study concurred that young erythrocytes have high folate concentrations and that there is an inverse relationship between concentrations of hemoglobin erythrocyte folate in patients with hemoglobin SS disease (as we found). (36) Although it is challenging to define what should be considered adequate intake, we found dietary intake of folate to be below national daily recommendations in 53% of the patients with SCA. This is very similar to a recent study of 70 children with SCA, in which 57% were found to have dietary folate intakes below recommendations, despite supplementation therapy. (37)

In adults who do not have SCA, folate concentration is generally a strong predictor of homocysteine concentrations. (14), (18), (38) The association between folate and homocysteine concentrations in patients with SCA has been less consistent across studies. In the study involving supplementation, the authors found a strong correlation between folate and homocysteine concentrations, (28) while the Texas study found no association between homocysteine concentration and concentration of either serum or red blood cell folate. (32) Our study also found no association between folate and homocysteine levels, although it may not have had the statistical power to show this.

We did see an association between concentrations of vitamin [B.sub.12] and homocysteine in these patients, which differed from results in the supplementation trial, (28) but which was similar to the relationship between vitamin [B.sub.12] and homocysteine concentrations seen in most population-based studies. (13), (18) Other studies have found, as we did, that serum vitamin [B.sub.12] concentrations in patients with SCA tend to be normal. (37)

A surprising finding in our study was lower vitamin [B.sub.6] concentration in the children with SCA than in the healthy children, despite similar intake. This differs from previous studies that found similar vitamin [B.sub.6] concentrations in children with SCA and healthy black children, (28), (39) although one preliminary report suggested that plasma [B.sub.6] concentrations in children with SCA may be lower than in controls. (40) Adults with SCA were shown to have lower plasma [B.sub.6] concentrations than healthy adults, although they had higher concentrations of erythrocyte pyridoxal 5'-phosphate (PLP) than healthy controls. (41) No significant relationship was found in the single published study that examined the influence of vitamin [B.sub.6] on homocysteine concentration in patients with SCA, as we also found in our study. (28)

We hypothesize that utilization of vitamin [B.sub.6] was higher in the SCA patients than in the healthy children, as the intake of vitamin [B.sub.6] was similar but resulted in lower plasma concentrations. Alternatively, bioavailability of this nutrient may be lower among the children with SCA than among healthy children, although this would be unlikely. Vitamin [B.sub.6] is absorbed from the gut by passive diffusion and is thought to be 75% bioavailable from the diet. There are no known instances of vitamin [B.sub.6] malabsorption associated with a disease state, although there may be some decrement in absorption among the elderly. (42)

We observed that children with SCA who had a higher vitamin [B.sub.6] intake had a greater degree of reticulocytosis, even after controlling for hematocrit level, than those with lower concentrations of intake. The unexpected positive correlation between serum homocysteine concentration and vitamin [B.sub.6] intake no longer predicted homocysteine concentrations when we controlled for the reticulocyte count. It is difficult to know if the association between reticulocyte count and homocysteine concentration is due to higher concentrations of homocysteine in young erythrocytes or if it reflects a modest micro-nutrient deficit in patients with accelerated red blood cell turnover. Few other studies have investigated the relationship between reticulocytosis and homocysteine concentrations. (32)

Some limitations of this study need to be addressed. This study was statistically powered to detect a difference of 2.0 [mu]mol/L in homocysteine concentrations between patients with SCA and controls, and we anticipated that the participants would have higher homocysteine concentrations than we observed. Therefore, our study had only 40% power to detect a significant difference in homocysteine concentrations between the patients with SCA and their healthy siblings, and even less power to detect a significant difference in mean serum folate concentrations between the two groups. Regarding the generalizability of these results, a greater proportion of the children included in this study had a cerebrovascular accident than would be expected in a general population of children with SCA. We did not exclude these children with severe disease from the study, as we thought that they might be more likely to have hyperhomocysteinemia. Thus, although the results may not be generalizable to a general population of children with SCA, the study has internal validity and is applicable to the population of patients with more severe disease.

Despite the small study size, we demonstrated low serum concentrations of vitamin [B.sub.6] in the children with SCA, despite intake that can be considered to be nearly adequate by current standards. This suggests that larger studies to assess the adequacy of dietary intake of micronutrients may be indicated, particularly if these micronutrients are proved to contribute to hyperhomocysteinemia. Future studies of vitamin [B.sub.6] deficiency in this population should include measurement of cystathionine concentration, as it is more sensitive to vitamin [B.sub.6] than is homocysteine concentration.

Key Points

* Compared with black children without sickle cell anemia, children with this illness have lower plasma vitamin [B.sub.6] concentrations and comparable serum concentrations of folate and vitamin [B.sub.12].

* Children with sickle cell anemia have plasma homocysteine concentrations minimally higher than black children without sickle cell anemia.

* Homocysteine concentration is inversely related to plasma vitamin [B.sub.12] concentration in this population and does not vary with vitamin [B.sub.6] or folate concentrations.

Table 1. Clinical data for sickle cell patients and healthy black
children (a)

                                SCA patients      Healthy children
Variable                         (n  = 17)           (n = 11)

Age yr (mean [+ or -] SD)     9.8 [+ or -] 3.9    8.3 [+ or -] 3.7

Boys (%)                             53                73

Daily multivitamin use (%)           24                36

History of stroke (%)                35                0

Chronically transfused (%)           35                0

Days since last transfusion,   27 (18-2,099)           -
  median (IQR)

No. of crises in lifetime,        4 (0-40)             -
  median (IQR)

No. of blood transfusions in     8 (0-101)             -
  lifetime, median (IQR)

(a) SCA, Sickle cell anemia: IQR, interquartile range: -, no data.

Table 2. Laboratory values in sickle cell patients and healthy black
children (a)

                                    SCA patients (n = 17)

Variable                            Median        IQR

Homocysteine ([mu]mol/L)              5.4       4.6-5.9
Serum folate (ng/ml)                  9         5.8-15
Red blood cell folate (ng/ml)       389         257-533
Serum vitamin [B.sub.12] (pg/ml)    560         468-765
Plasma vitamin [B.sub.6] (ng/ml)      6.4       4.5-9
Hemoglobin (mg/dl)                    8.8       8.0-9.8
Reticulocyte count (X 1[0.sup.9]L)  243         145-310
Creatinine (mg/dl)                    0.4       0.3-0.4

                                    Healthy children (n = 11)

Variable                            Median          IQR       P value

Homocysteine ([mu]mol/L)              4.3         3.4-5.7       0.19
Serum folate (ng/ml)                 11           7.2-15        0.39
Red blood cell folate (ng/ml)       321           296-393       0.33
Serum vitamin [B.sub.12] (pg/ml)    569           429-758       0.96
Plasma vitamin [B.sub.6] (ng/ml)     11           9.3-12        0.012
Hemoglobin (mg/dl)                   13            12-13       <0.001
Reticulocyte count (X 1[0.sup.9]L)   59            49-66        0.002
Creatinine (mg/dl)                    0.5         0.4-0.5       0.17

(a) SCA, Sickle cell anemia; IQR, interquartile range.

Table 3. Daily intake and satisfaction of requirements (a)
                    Daily Intake

                  SCA patients (n = 17)  Healthy children (n = 11)

Nutrient            Median        IQR       Median         IQR

Protein (g)          59          49-73        62          47-76

Folate ([micro]g)   195        151-293       222         106-339

Vitamin [B.sub.6]     1.3       1.1-2.3        1.5         1-2.1
(mg)

Vitamin [B.sub.6]     0.061   0.037-0.080      0.038   0.025-0.064
(mg/g protein)

Vitamin [B.sub.12]    3.1       2.3-4.6        2.4       1.2-4.1

Nutrient            P value

Protein (g)          0.80

Folate ([micro]g)    0.88

Vitamin [B.sub.6]    0.64
(mg)

Vitamin [B.sub.6]    0.13
(mg/g protein)

Vitamin [B.sub.12]   0.36

                    Percentage meeting daily age-adjusted
                          requirement for nutrients

Nutrient            SCA patients (%)  Healthy children (%)  P value

Protein                    94                  91            0.63

Folate                     47                  64            0.39

Vitamin [B.sub.6]          88                  82            0.11

Vitamin [B.sub.6]         100                  91            0.20
(mg/g protein)

Vitamin [B.sub.12]         94                  73            0.75

(a) SCA, Sickle cell anemia; IQR, interquartile range.

Table 4. Pearson correlation coefficients, between measurements for all
participants (a)

                    Intake [B.sub.12]    Intake [B.sub.6]

Intake folate              0.64                0.82
                    P < 0.001 [dagger]  P < 0.001 [dagger]

Intake [B.sub.12]          1                   0.67
                                        P < 0.001 [dagger]

Intake [B.sub.6]                               1
Serum folate *

RBC folate

Serum [B.sub.12] *

Plasma [B.sub.6] *

Plasma Hcy *

Retic count

                      Serum folate *        RBC folate

Intake folate             0.43                0.19
                    P = 0.025 [dagger]

Intake [B.sub.12]         0.0029             -0.21

Intake [B.sub.6]          0.060              -0.092

Serum folate *            1                   0.48
                                        P = 0.014 [dagger]

RBC folate                                    1

Serum [B.sub.12] *

Plasma [B.sub.6] *

Plasma Hcy *

Retic count

                       Serum        Plasma        Plasma Hcy *
                   [B.sub.12] *   [B.sub.6] *

Intake folate          0.18         -0.23            0.15

Intake [B.sub.12]      0.12         -0.37            0

Intake [B.sub.6]       0            -0.24            0.37

Serum folate *         0.036        -0.05           -0.20

RBC folate             0.13         -0.22           -0.088

Serum [B.sub.12] *     1             0.21           -0.55
                                               P = 0.003 [dagger]


Plasma [B.sub.6] *                   1               0.02

Plasma Hcy *                                         1

Retic count

                      Retic           Hgb
                      count

Intake folate         0.22           0.047

Intake [B.sub.12]     0.11          -0.040

Intake [B.sub.6]      0.40           0.0072
                    P = 0.036

Serum folate *       -0.040         -0.001

RBC folate            0.26          -0.47
                               P = 0.019 [dagger]

Serum [B.sub.12] *    0.081          0.00

Plasma [B.sub.6] *   -0.29          -0.24

Plasma Hcy *          0.081         -0.086

Retic count           1              0.40
                               P = 0.036 [dagger]

* Serum and plasma measurements are log-transformed.
[dagger] P values for correlations are presented only when P < 0.05.
(a) RBC, red blood cell; Hcy, homocysteine; Retic, reticulocyte; Hgb,
hemoglobin.

Acknowledgments

We thank Celide Koemer, MS, RD, for assistance with the study design; Susan Dixon, BA, RN, for help with patient recruitment; the staff of the Pediatric Clinical Research Unit; and the Johns Hopkins Hospital pediatric hematology patients and physicians for their participation.

Accepted July 8, 2002.

Copyright [c] 2004 by The Southern Medical Association

0038-4348/04/9702-0149

References

(1.) Wood DH. Cerebrovascular complications of sickle cell anemia. Stroke 1978;9:73-75.

(2.) Balkaran B, Char G, Morris JS, et al. Stroke in a cohort of patients with homozygous sickle cell disease. J Pediatr 1992;120:360-366.

(3.) Powars D, Wilson B, Imbus C, et al. The natural history of stroke in sickle cell disease. Am J Med 1978;65:461-471.

(4.) Ohene-Frempong K, Weiner SJ, Sleeper LA, et al. Cerebrovascular accidents in sickle cell diseases: Rates and risk factors. Blood 1998;91:288-294.

(5.) Francis RB Jr. Elevated fibrin D-dimer fragment in sickle cell anemia: Evidence for activation of coagulation during the steady state as well as in painful crisis. Haemostasis 1989;19:105-111.

(6.) Perry IJ, Refsum H, Morris RW, et al. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 1995;346:1395-1398.

(7.) Petri M, Roubenoff R, Dallal GE, et al. Plasma homocysteine as a risk factor for atherothrombotic events in systemic lupus erythematosus. Lancet 1996;348:1120-1124.

(8.) Selhub J, Jacques PF, Bostom AG, et al. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med 1995;332:286-291.

(9.) van den Berg M, Stehouwer CD, Bierdrager E, et al. Plasma homocysteine and severity of atherosclerosis in young patients with lower-limb atherosclerotic disease. Arterioscler Thromb Vasc Biol 1996;16:165-171.

(10.) Nygard O, Nordrehaug JE, Refsum H, et al. Plasma homocysteine concentrations and mortality in patients with coronary artery disease. N Engl J Med 1997;337:230-236.

(11.) Malinow MR, Nieto FJ, Szklo M, et al. Carotid artery intimal-medial wall thickening and plasma homocysteine in asymptomatic adults: The Atherosclerosis Risk in Communities Study. Circulation 1993;87:1107-1113.

(12.) Boushey CJ, Beresford SA, Omenn GS, et al. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274:1049-1057.

(13.) Robinson K, Mayer EL, Miller DP, et al. Hyperhomocysteinemia and low pyridoxal phosphate. Common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825-2830.

(14.) Kang SS, Wong PW, Norusis M. Homocysteinemia due to folate deficiency. Metabolism 1987;36:458-462.

(15.) Mansoor MA, Kristensen O, Hervig T, et al. Plasma total homocysteine response to oral doses of folic acid and pyridoxine hydrochloride (vitamin [B.sub.6]) in healthy individuals: Oral doses of vitamin [B.sub.6] reduce concentrations of serum folate. Scand J Clin Lab Invest 1999;59:139-146.

(16.) Vermeulen EG, Stehouwer CD, Twisk JW, et al. Effect of homocysteine-lowering treatment with folic acid plus vitamin [B.sub.6] on progression of subclinical atherosclerosis: A randomized, placebo-controlled trial. Lancet 2000;355:517-522.

(17.) McKinley MC. Nutritional aspects and possible pathological mechanisms of hyperhomocysteinaemia: An independent risk factor for vascular disease. Proc Nutr Soc 2000;59:221-237.

(18.) Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693-2698.

(19.) Schrijver J, Speek AJ, Schreurs WH. Semi-automated fluorometric determination of pyridoxal-5'-phosphate (vitamin [B.sub.6]) in whole blood by high-performance liquid chromatography (HPLC). Int J Vitam Nutr Res 1981;51:216-222.

(20.) Ueland PM, Refsum H, Stabler SP, et al. Total homocysteine in plasma or serum: Methods and clinical applications. Clin Chem 1993;39:1764-1779.

(21.) Dunn RT, Foster LB. Radioassay of serum folate. Clin Chem 1973;19:1101-1105.

(22.) Bingham SA, Gill C, Welch A, et al. Comparison of dietary assessment methods in nutritional epidemiology: Weighed records vs. 24-hr recalls, food-frequency questionnaires and estimated-diet records. Br J Nutr 1994;72:619-643.

(23.) Buzzard IM, Faucett CL, Jeffery RW, et al. Monitoring dietary change in a low-fat diet intervention study: Advantages of using 24-hour dietary recalls vs food records. J Am Diet Assoc 1996;96:574-579.

(24.) Kuehneman T, Stanek K, Eskridge K, et al. Comparability of four methods for estimating portion sizes during a food frequency interview with caregivers of young children. J Am Diet Assoc 1994;94:548-551.

(25.) Haraldsdottir J, Hermansen B. Repeated 24-hr recalls with young school-children: A feasible alternative to dietary history from parents? Eur J Clin Nutr 1995;49:729-739.

(26.) Recommended Dietary Allowances. Washington, DC, National Academy Press, 1989, ed 10, pp 284-285.

(27.) Osganian SK, Stampfer MJ, Spiegelman D, et al. Distribution of and factors associated with serum homocysteine concentrations in children: Child and Adolescent Trial for Cardiovascular Health. JAMA 1999;281:1189-1196.

(28.) van der Dijs FP, Schnog JJ, Brouwer DA, et al. Elevated homocysteine concentrations indicate suboptimal folate status in pediatric sickle cell patients. Am J Hematol 1998;59:192-198.

(29.) Balasa VV, Gruppo RA, Gartside PS, et al. Correlation of the C677T MTHFR genotype with homocysteine levels in children with sickle cell disease. J Pediatr Hematol Oncol 1999;21:397-400.

(30.) Balasa VV, Gruppo RA, Glueck CJ, et al. The relationship of mutations in the MTHFR, prothrombin, and PAI-1 genes to plasma concentrations of homocysteine, prothrombin, and PAI-1 in children and adults. Thromb Haemost 1999;81:739-744.

(31.) Houston PE, Rana S, Sekhsaria S, et al. Homocysteine in sickle cell disease: Relationship to stroke. Am J Med 1997;103:192-196.

(32.) Rodriguez-Cortes HM, Griener JC, Hyland K, et al. Plasma homocysteine concentrations and folate status in children with sickle cell anemia. J Pediatr Hematol Oncol 1999;21:219-223.

(33.) Lowenthal EA, Mayo MS, Cornwell PE, et al. Homocysteine elevation in sickle cell disease. J Am Coll Nutr 2000;19:608-612.

(34.) Liu YK. Folate deficiency in children with sickle cell anemia. Am J Dis Child 1974;127:389-393.

(35.) Liu YK. Folic acid deficiency in sickle cell anemia. Scand J Haematol 1975;14:71-79.

(36.) Muskiet FA, van der Dijs FP, Fokkema MR, et al. Erythrocyte folate does not accurately reflect folate status in sickle cell disease. Clin Chem 2000;46:2015-2016.

(37.) Kennedy TS, Fung EB, Kawchak DA, et al. Red blood cell folate and serum vitamin [B.sub.12] status in children with sickle cell disease. J Pediatr Hematol Oncol 2001;23:165-169.

(38.) Appel LJ, Miller ER III, Jee SH, et al. Effect of dietary patterns on serum homocysteine: Results of a randomized, controlled feeding study. Circulation 2000;102:852-857.

(39.) Flores L, Pais R, Buchanan I, et al. Pyridoxal 5'-phosphate concentrations in children with sickle cell disease. Am J Pediatr Hematol Oncol 1988;10:236-240.

(40.) Balasa VV, Kalinyak KA, Gruppo RA. Homocysteine and vitamin co-factor levels in patients with sickle cell disease. Thromb Haemost 2001;86(1 Suppl):Abstract OC2515.

(41.) Natta CL, Reynolds RD. Apparent vitamin [B.sub.6] deficiency in sickle cell anemia. Am J Clin Nutr 1984;40:235-239.

(42.) Gregory JF III. Bioavailability of vitamin [B.sub.6]. Eur J Clin Nutr 1997;51(Suppl 1):S43-S48.

Jodi B. Segal, MD, MPH, Edgar R. Miller III, MD, PHD, Nga Hong Brereton, MS, RD, and Linda M.S. Resar, MD

From the Departments of Medicine and Pediatrics, the Welch Center for Prevention, Epidemiology, and Clinical Research, and the Pediatric Clinical Research Unit, The Johns Hopkins University School of Medicine and Medical Institutions, Baltimore, MD.

This study was supported by a grant from the General Clinical Research Center of The Johns Hopkins Medical Institutions.

Reprint requests to Jodi B. Segal, MD, MPH, Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross #1025, Baltimore, MD 21205. Email: [e-mail]