Soo-Sang Kang

Homocysteinemia due to folate deficiency

We examined the relationship between serum folate and total homocyst(e)ine levels by determining protein-bound homocyst(e)ine in stored serum from 19 subjects with subnormal serum folate (less than 2 ng/mL), 137 subjects with low normal serum folate (between 2.0 and 3.9 ng/mL), 44 subjects with normal serum folate (between 4.0 and 17.9 ng/mL), and 38 subjects with high serum folate (above 18 ng/mL). Eighty-four percent of the subjects with subnormal serum folate and 56% of the subjects with low normal serum folate had more than 7.05 nmol/mL serum total homocyst(e)ine (ie, more than two standard deviations above the normal mean). Thirty-two percent of these subjects had more than a three-fold increase in serum total homocyst(e)ine. These observations support the hypothesis that depletion of tissue folate causes homocysteinemia in nonhomocystinuric subjects. Subnormal as well as low normal concentrations of serum folate appear to produce an accumulation of homocyst(e)ine. In addition, relatively normal levels of serum total homocyst(e)ine were observed in four pregnant women with low serum folate, supporting previous suggestions of an influence of female sex hormone(s) in homocysteine metabolism.

Relation of Plasma Homocyst(e)ine to Cerebral Infarction and Cerebral Atherosclerosis

BACKGROUND AND PURPOSE A number of investigations support the theory that the elevated plasma homocyst(e)ine is associated with occlusive vascular disease. The aim of this study is to examine whether moderate hyperhomocyst(e)inemia is an independent risk factor for cerebral infarction. In addition, we examined the association between plasma homocyst(e)ine and the severity of cerebral atherosclerosis. METHODS We conducted a hospital-based case-control study with 140 male controls and 78 male patients with nonfatal cerebral infarction, aged between 39 and 82 years. Plasma homocyst(e)ine levels were analyzed in 218 subjects. Fifty-five patients were evaluated for cerebral vascular stenosis by MR angiography. RESULTS The mean plasma level of homocyst(e)ine was higher in cases than in controls (11.8+/-5.6 versus 9.6+/-4.1 micromol/L; P=0.002). The proportion of subjects with moderate hyperhomocyst(e)inemia was significantly higher in cases than in controls (16.7% versus 5.0%; P=0.004). Based on the logistic regression model, the odds ratio of the highest 5% of homocyst(e)ine levels in control group was 4.17 (95% confidence interval, 3.71 to 4. 71)(P=0.0001). After additional adjustment for total cholesterol, hypertension, smoking, diabetes, and age, the odds ratio was 1.70 (95% confidence interval, 1.48 to 1.95) (P=0.0001). The plasma homocyst(e)ine levels of patients having vessels with 3 or 2 stenosed sites were significantly higher than those of patients having vessels with 1 stenosed site or normal vessels (14.6+/-1.4, 11.0+/-1.4 versus 7.8+/-1.5, 8.9+/-1.4 micromol/L respectively; P<0. 02). Multiple logistic regression analysis revealed that moderate hyperhomocyst(e)ienemia was significantly associated with the number of stenosed vessels (P=0.001). CONCLUSIONS These findings suggest that moderate hyperhomocyst(e)inemia is an independent risk factor for cerebral infarction and may predict the severity of cerebral atherosclerosis in patients with cerebral infarction.

Genetic and nongenetic factors for moderate hyperhomocyst(e)inemia

To assess the risk for homocyst(e)ine-associated vascular disease, overt hyperhomocyst(e)inemia should be demonstrated. In nonhomocystinuric subjects, clinical vascular disease must have developed after 40 or more years of persistent hyperhomocyst(e)inemia which may not be present without a genetic defect(s). Nongenetic factors, however, may amplify or mask phenotypic expression of a genetic defect, causing difficulties for the evaluation of hyperhomocyst(e)inemia based on plasma homocyst(e)ine concentration alone. Therefore, the search for genetic defects seems as important as the determination of plasma homocyst(e)ine concentration in evaluating the relationship between hyperhomocyst(e)inemia and the development of vascular disease. If genetic defect, such as heterozygous cystathionine synthase deficiency or thermolabile methylenetetrahydrofolate reductase is not detected, post-methionine homocyst(e)ine determination is a suitable means to identify genetic susceptibility to hyperhomocyst(e)inemia when the environmental factors are similar in the control and study groups.

Protein-Bound Homocyst(e)ine in Normal Subjects and in Patients with Homocystinuria

Summary: A method was developed to quantitate protein-bound homocyst(e)ine using 2-mercaptoethanol. Protein-bound homocyst(e)ine was discovered in the plasma from normal individuals, ranging from 0.5–2.2 nmole/ml. In two obligatory heterozygotes for classical homocystinuria, plasma protein-bound homocyt(e)ine was 3.5 and 4.8 nmole/ml, respectively. Untreated homozygotes showed approximately a 40-fold increase of plasma protein-bound homocyst(e)ine. Furthermore, using conventional methods, no free homocystine was detectable in the supernatant of plasma precipitate from two classical homocystinuric patients treated with pyridoxine, but plasma protein-bound homocyst(e)ine showed a 10-fold increase. Protein-bound homocyst(e)ine was also demonstrated in the liver, kidney, and brain tissues from a patient with methylenetetrahydrofolate reductase deficiency.Speculation: The results in this study suggest that determination of proteinbound homocyst(e)ine using 2-mercaptoethanol may provide a more reliable assessment of treatment in patients with homocystinuria and a potentially useful tool for the definition of the carrier state.Demonstration of protein-bound homocyst(e)ine in various tissues of homocystinuric patients suggests the possibility that this compound may be directly associated with the development of some of the pathologic changes in the tissues

Total homocyst(e)ine in plasma and amniotic fluid of pregnant women

Total homocyst(e)ine was determined by the quantitation of protein-bound homocyst(e)ine in the stored plasma and amniotic fluid from 25 pregnant women and in the stored plasma from 17 nonpregnant women. The mean +/- SE of plasma total homocyst(e)ine was 29.8 +/- 2.4 nmol/g protein in pregnant women and 52.4 +/- 3.8 nmol/g protein in nonpregnant women. In contrast, the mean +/- SE of total homocyst(e)ine in amniotic fluid obtained at 16 weeks of gestation was 36.3 +/- 2.9 nmol/g protein. There was a statistically significant difference in the plasma total homocyst(e)ine concentrations from pregnant and nonpregnant women (P less than 0.01). Similarly, there was also a statistically significant difference between plasma total homocyst(e)ine from nonpregnant women and amniotic fluid total homocyst(e)ine (P less than 0.01). These observations suggested that the metabolism of homocysteine to cysteine was more efficient in pregnant women. In addition, the concentrations of total homocyst(e)ine in amniotic fluids were within narrow limits in normal pregnancies. Hence, total homocyst(e)ine concentration might be very valuable as a rapid assessment of fetuses for congenital defects of homocysteine metabolism.

Pathogenicity of Thermolabile Methylenetetrahydrofolate Reductase for Vascular Dementia

Although the major biochemical abnormality due to methylenetetrahydrofolate reductase (MTHFR) deficiency is hyperhomocyst(e)inemia, its pathogenicity appears to involve more than homocysteine toxicity. In patients with severe MTHFR deficiency, a metabolite(s) other than hyperhomocyst(e)inemia also appears to be associated with its clinical manifestation in cerebrovascular disease. To elucidate the specific role of the TT genotype of MTHFR in the development of cerebral infarction with and without cognitive impairment, we determined the prevalence of hyperhomocyst(e)inemia and the C677T genotypes of MTHFR in 143 patients with vascular dementia, 122 patients with cerebral infarction, and 217 healthy subjects matched for age and sex. Prevalence of hyperhomocyst(e)inemia [homocyst(e)ine >/=15 micromol/L] was higher in cerebrovascular patients with or without dementia than in normal control subjects (42.6%, 20.5%, and 10.1%, respectively; P=0.001). In contrast, a higher frequency of MTHFR TT genotype was found only in demented patients compared with nondemented patients and healthy controls (25.2%, 9.8%, and 12.0%, respectively; P=0.01). When the study subjects were divided into normohomocyst(e)inemic and hyperhomocyst(e)inemic groups, the TT genotype was significantly associated with the risk for vascular dementia in the hyperhomocyst(e)inemic group (odds ratio 4.13, 95% CI 2.18 to 7.85; P=0.03) but not in the normohomocyst(e)inemic group. Demented patients with multiple infarcts had a higher frequency of TT genotype (odds ratio 3.13, 95% CI 2.23 to 4.39; P=0.0007), whereas those with a single infarct did not (odds ratio 2.03, P=0.15). In contrast, there was no significant association of the TT genotype with multiple infarcts in hyperhomocyst(e)inemic stroke patients. Taken together, these findings indicate a possible role of MTHFR TT genotype combined with hyperhomocyst(e)inemia in the pathogenesis of vascular dementia. Similar to the relationship between homocystinuria due to severe MTHFR deficiency and severe cystathionine beta-synthase deficiency, the TT genotype of MTHFR in hyperhomocyst(e)inemic subjects is differentiated from the cases of the TT genotype without hyperhomocyst(e)inemia or hyperhomocyst(e)inemia without the TT genotype in the development of cerebrovascular disease.

Cortisol and estradiol : Nongenetic factors for hyperhomocyst(e)inemia

A low plasma homocyst(e)ine concentration in premenopausal and pregnant women compared with postmenopausal women and men suggests that steroid hormones are nongenetic factors affecting homocysteine metabolism. This hypothesis was tested by determining plasma homocyst(e)ine levels in adult male rats treated with cortisol, estradiol, or a combination of both. Mean plasma homocyst(e)ine concentrations were 3.71 +/- 0.71, 5.26 +/- 1.76, and 4.28 +/- 0.84 nmol/mL in cortisol-treated, estradiol-treated, and cortisol plus estradiol-treated groups, respectively. These values were substantially low compared with the level of 7.32 +/- 0.89 nmol/mL plasma homocyst(e)ine in the control group, indicating a significant effect of steroid hormones on homocysteine metabolism.

The Effect of D-Penicillamine on Protein-Bound Homocyst(e)ine in Homocystinurics

Summary: There is considerable evidence that homocystine has a direct damaging effect on vascular endothelium and other tissues. The demonstration of the existance of protein-bound homocyst(e)ine has strengthened this hypothesis. In an attempt to remove bound homocyst(e)ine, D-penicillamine was given to three patients with pyridoxine-nonresponsive homocystinuria. Before the clinical trial, it had been demonstrated that 0.1 μmole per ml concentration of D-penicillamine or cysteamine released approximately 50% of the homocyst(e)ine bound to plasma proteins in vitro. Oral D-penicillamine effectively reduced both free and plasma protein-bound homocyst(e)ine in homocystinurics from the second day of treatment. The homocystine excreted in the urine was mainly in the form of homocysteine-penicillamine disulfide. No mixed disulfide was detectable in the plasma, indicating an extremely high renal clearance. These observations suggested that oral D-penicillamine removed a considerable quantity of the bound homocyst(e)ine accumulated in the tissue proteins.Speculation: D-Penicillamine treatment may be used on an experimental basis in pyridoxine-nonresponsive homocystinuric patients when dietary treatment is not practical. It may also be used in pyridoxine-responsive patients when control with pyridoxine is unsatisfactory. In addition, determination of protein-bound homocyst(e)ine should be used for the assessment of the effectiveness of therapy.

Thermolabile Methylenetetrahydrofolate Reductase

Thermolabile methylenetetrahydrofolate reductase (MTHFR) is a homozygous genetic defect that is defined by its in vitro heat sensitivity. Similar to heterozygotes for severe MTHFR deficiency, specific enzyme activity of thermolabile MTHFR is 50% of the normal mean. Population analysis of the inheritance pattern follows an autosomal recessive trait, and thermolabile MTHFR is the most common homozygous genetic defect. Molecular analysis shows a homozygous C to T transition at the 677th base of cDNA in the majority of thermolabile MTHFR. Genetic compounds of the 677 mutation and severe mutations are also identified as thermolabile MTHFR. There is a positive correlation between thermolabile MTHFR and the development of vascular disease. Thermolabile MTHFR causes hyper-homocysteinemia alone or in combination with other genetic or nongenetic factors. Compared with a single thermolabile mutation, the combination of two defects produces more striking hyperhomocysteinemia. A higher prevalence of thermolabile MTHFR in hyperhomocysteinemic patients with vascular disease than in nonclassified patients suggests a potential association of hyperhomocysteinemia with the development of vascular disease. Interrelationships between thermolabile MTHFR, hyperhomocysteinemia, and the development of vascular disease have yet to be thoroughly elucidated. The investigation of nonallelic heterozygotes with other defects, such as cystathionine β-synthase (CBS), methionine synthase (MHMT), and betaine homocysteine methyltransferase (BHMT), which are enzymes for the synthesis of cofactors, may be important in establishing this relationship.

Protein‐Bound Homocyst(e)ine in Patients With Rheumatoid Arthritis Undergoing D‐Penicillamine Treatment

Protein‐bound homocyst(e)ine was measured in the plasma of 38 nonhomocystinuric patients with rheumatoid arthritis. Nineteen of them were treated orally with d‐penicillamine 100‐1,500 mg/d for a period of one month to 15 years. For these patients, the mean ± standard deviation level of plasma protein‐bound homocyst(e)ine was 1.95 ± 1.07 nmol/mL. In contrast, the mean plasma level of protein‐bound homocyst(e)ine was 4.72 ± 1.11 nmol/mL in the 19 patients who had not been treated with oral d‐penicillamine. There was a statistically significant difference (P < .0001) in the plasma protein‐bound homocyst(e)ine concentrations between patients with and without oral d‐penicillamine therapy. Thus, it may be speculated that oral d‐penicillamine may be beneficial in protecting patients from the development of thromboembolism and arteriosclerosis.