J. lnher. Metab. Dis. 15 (1992) 419-422 9 SSIEM and KluwerAcademicPublishers. Printed in the Netherlands

Short Communication

Lipid Peroxidation in Homocysteinaemia H. J. BLOM1, D. P. E. ENGELEN2, G. H. J. BOERS3, A. M. STADHOUDERS2, R. C. A. SENGERS1, R. DE ABREU1, M. T. W. B. TEPOELE-POTHOFF 1 and J. M. F. TRIJBELS1 IDepartment of Pediatrics, 2Department of Cell Biology and Histology, 3Division of Endocrinology, Department of Internal Medicine, University Hospital Nijmegen, P.O. Box 9101, 6500 liB Nijmegen, The Netherlands Homocysteinaemia due to cystathione synthase deficiency (CSD: McKusick 236200) is a rare autosomal recessive inborn error of methionine metabolism. The most lifethreatening complications caused by CSD are thromboembolism and vascular abnormalities. Boers et al (1985) provided evidence that even mild homocysteinaemia, as seen in heterozygote CSD patients, is predisposing for development of premature peripheral and cerebral occlusive arterial disease. Recently, Clarke et al (1991) demonstrated that homocysteinaemia due to intermediate CSD is an independent risk factor for vascular disease, including coronary disease. Furthermore, their data suggest that hyperhomocysteinaemia is a higher risk factor for development of vascular disease than hypercholesterolaemia, hypertension and cigarette smoking. Wide acceptance of hyperhomocysteinaemia as a risk factor for vascular disease is hampered by the poor knowledge of the underlying pathobiochemical mechanism. Starkebaum and Harlan (1986) provided evidence that the toxic effects of homocysteine on cultured venous endothelial cells result from the formation of hydrogen peroxide and, therefore, raised the possibility of free radical involvement and increased lipid peroxidation in homocysteinaemia. To test this hypothesis, in the present study lipid peroxidation was measured by two different methods in eight homozygote CSD patients. PATIENTS AND METHODS

Patients: Eight adult homozygote CSD patients were diagnosed by increased serum homocysteine and methionine concentrations and in seven of them by the low cystathionine synthase activity in fibroblasts ( < 10% of normal). Cystathione synthase activity in patient M.S. (see Table i) has not yet been measured. All patients suffered from one or more clinical manifestations typical for the classical homocysteinuria syndrome. Amino acid analysis: Methionine and homocysteine concentrations were measured as described previously (Blom et al 1988). The total amount of circulating non419

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protein-bound free homocysteine in serum was calculated as twice the concentration of homocysteine (homocysteine-homocysteine disulphide) plus the concentration of homocysteine cysteine mixed disulphide.

Determination of serum fluorescent lipid peroxidation products (FLPP)." Water-soluble protein-bound FLPP in serum were measured in triplicate according to Tsuchida et al (1985) with some minor modifications. In glass tubes 50#1 serum was added to 2 ml ethanol/ether mixture (3:1 v/v). After mixing and centrifugation (1000g, 5 min), the solvent was discarded and the precipitated proteins were washed. The precipitated proteins were dissolved in 1 ml phosphate buffer (67 mmol/L, pH 7.0). The fluorescence intensity of this solution was measured at 435 nm emission with an excitation of 340 nm (Perkin Elmer LS 5, Charles Goffin, The Netherlands). A solution of quinine sulphate (0.1/~g/ml in 50 mmol/L H2SO4) was used for calibration. The concentrations of serum FLPP are expressed in pmol quinine-equivalents per mg serum protein (pmol qeq/mg serum protein). Protein content of the samples was determined fluorimetrically. The serum precipitates were diluted 20 times with the phosphate buffer and the fluorescence intensity was measured at 330 nm after excitation at 278 nm. This fluorimetric protein measurement had been calibrated with the Lowry protein assay. Determination of serum lipid peroxides by the colorimetric thiobarbituric acid method: Lipid peroxidation products were also measured in triplicate according to the method of Wade et al (1987). The concentrations are expressed in /~mol malondialdehyde (MDA) per litre of serum. RESULTS The two homozygote homocysteinaemia patients without therapy exhibited very high free homocysteine concentrations in serum of 149 and 94#mol/L (Table 1). Serum homocysteine concentrations of the homozygote patients on therapy ranged from virtually normal values up to 68 #mol/L. The concentrations of the lipid peroxidation products FLPP and MDA in serum of the homocysteinaemia patients were all within the range of the mean + 2SD of the control values and, therefore, not significantly different from the controls. No correlation between the homocysteine concentrations and the lipid peroxidation products was observed. DISCUSSION Starkebaum and Harlan (1986) provided strong evidence that the toxic effects of homocysteine on cultured endothelial cells result from the formation of hydrogen peroxide. Oxidation of homocysteine to homocystine in a cell-free system yielded hydrogen peroxide in the presence of low concentrations copper or ceruloplasmin and even in the presence of normal human serum. Cultured endothelial cells were lysed by homocysteine (1 mmol/L) plus copper (4/~mol/L), and this toxic effect was prevented by the specific hydrogen peroxide scavenger catalase. These data indicate J. Inher. Metab. Dis. 15 (1992)

Lipid Peroxidation in Homocysteinaemia

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J. Inher. Metab. Dis. 15 (1992)

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that lipid peroxidation may be involved in the mechanism responsible for vascular disease in homocysteinaemia. In the present study lipid peroxidation was investigated by two different methods in homozygote CSD patients. The concentrations of the lipid peroxidation products F L P P and MDA were measured in serum of eight homozygote homocysteinaemia patients and compared with normal age-matched controls. The concentrations of F L P P and MDA were not increased in serum of homozygote patients. Even those patients with very high homocysteine levels showed normal concentrations of lipid peroxidation products. Also, therapy of hyperhomocysteinaemia with vitamin B 6 in patient C.J. hardly influenced the concentrations of F L P P and MDA. Obviously, high homocysteine concentrations are not associated with increased lipid peroxidation products in serum. Therefore, this study does not support the hypothesis that lipid peroxidation is involved in the pathogenesis of hyperhomocysteinaemia. ACKNOWLEDGEMENT

This study was supported in part by The Netherlands Heart Foundation. REFERENCES

Boers GHJ, Smals AGH, Trijbels JMF et al (1985) Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 313: 709-715. Blom HJ, Boers GHJ, van den Elzen PAM, van Roessel JJM, Trijbels JMF, Tangerman A (1988) Differences between premenopausal women and young men in the transamination pathway of methionine catabolism and the protection against vascular disease. Eur J Clin Invest 18: 633-639. Clarke R, Daly L, Robinson K et al (1991) Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324: 1149-1155. Starkebaum G, Harlan JM (1986) Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77: 1370-1376. Tsuchida M, Miura T, Mizutani K, Aibara K (1985) Fluorescent substances in mouse and human sera as a parameter of in vivo lipid peroxidation. Biochim Biophys Acta 834: 196-204. Wade CR, Jackson PG, Highton J, van Rij AM (1987) Lipid peroxidation and malondialdehyde in the synovial fluid and plasma of patients with rheumatoid arthritis. Clin Chim Acta 164: 245-250.

J. Inher. Metab. Dis. 15 (1992)

Lipid peroxidation in homocysteinaemia.

J. lnher. Metab. Dis. 15 (1992) 419-422 9 SSIEM and KluwerAcademicPublishers. Printed in the Netherlands Short Communication Lipid Peroxidation in H...
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