Studies of the Mechanism of Pyridoxine-Responsive Homocystinuria

Pediat. Res. 6: 187-196 (1972) Cystathionine synthase homocystinuria pyridoxine Studies of the Mechanism of Pyridoxine-Responsive Homocystinuria MARGRETTA R. SEASHORE, JOSEPH L. DURANT, AND LEON E. ROSENBERG140' Division of Medical Genetics, Departments of Pediatrics and Medicine, Yale University School of Medicine, New Haven, Connecticut, USA Extract Pharmacologic doses of pyridoxine corrected plasma amino acid abnormalities in two boys (JK and EY) with homocystinuria caused by cystathionine synthase deficiency. Pyridoxine responsiveness was dose-dependent but differed in the two patients. JK required 25 mg pyridoxine per day for correction of plasma methionine, homocystine, and cystine concentrations; EY required more than 50 mg pyridoxine per day. Gystathionine synthase assays on extracts of cultured skin fibroblasts were carried out to explore this apparent clinical difference. Under basal conditions, synthase activity in extracts from both patients was less than 5% of normal. Addition of saturating concentrations of pyridoxal phosphate to the assay mixture stimulated synthase activity fourfold in extracts from JK's cells. No detectable increase in enzyme activity was noted in extracts of EY's cells under identical conditions. These in vivo and in vitro differences suggest that JK's pyridoxine responsiveness is mediated by partial correction of his underlying synthase deficiency and that EY's response to pyridoxine may be produced by another mechanism, perhaps stimulation of alternate pathways of sulfur-amino acid metabolism. Speculation Can pyridoxine responsiveness in homocystinuria always be equated with stimulation of defective cystathionine synthase activity? Our results suggest a negative answer to this question and emphasize the need for further clinical and biochemical investigation of such patients. the fact that cystathionine synthase catalyzes one of the steps in the major degradative pathway by which methionine is converted to cystine and ultimately to its end product, inorganic sulfate [20] (Fig. 1). The relation between these biochemical findings and the clinical hallmarks of homocystinuria which include ectopia lentis, skeletal abnormalities, mental retardation, and thrombotic vascular disease is unknown. Two forms of therapy have been tried in homocystinuria. Limitation of dietary methionine with administration of supplemental cystine has resulted in bio- Introduction Two years after the clinical description of homocystinuria in 1962 [5, 12], the enzymatic basis of the disease was demonstrated by Mudd et al. [24] who showed that cystathionine synthase activity was absent in the livers of homocystinuric patients. This deficiency, also demonstrated in cultured skin fibroblasts [34], leads to increased concentrations of methionine and homocystine in plasma and urine. The absence or markedly reduced concentration of cystine in these fluids reflects 187 188 SEASHORE, D U R A N T , AND ROSENBERG L-METHIONINE < I S-ADENOSYL-L-METHIONINE \ S-ADENOSYL-L-HOMOCYSTEINE \ 5: L-HOMOCYSTEINE L-HOMOCYSTINE cystathionine synthase (B6) • serine L-CYSTATHIONINE ther the nature of pyridoxine-responsive homocystinuria. The effect of pyridoxine administration on plasma and urinary amino acid concentrations and urinary inorganic sulfate excretion was studied during normal dietary intake and under conditions of methionine loading. Cystathionine synthase activity in cell-free extracts of cultured skin fibroblasts was determined, and the influence of varying pyridoxine concentrations in the growth medium and of pyridoxal phosphate concentration in the in vitro assay system was studied. Our results suggest two different mechanisms of pyridoxine responsiveness in this disease. Patients L-CYSTINE Fig. 1. Pathway of methionine catabolism to sulfate. In homocystinuria, methionine and homocystine accumulate because o£ an inherited deficiency of cystathionine synthase, the enzyme which catalyzes the condensation of serine and homocysteine to form cystathionine. Note that the enzymatic conversion of homocysteine to cystathionine appears to require vitamin Bo in its coenzyme form (pyridoxal phosphate). chemical improvement in several patients [3, 7, 13, 19, 27], but it will require years to determine whether such improvement alters the clinical course of the disease. The second therapeutic approach, oral administration of pyridoxine, is based on the increasing, although not unequivocal, evidence that cystathionine synthase requires pyridoxal phosphate as a coenzyme [4, 18, 26]. Barber and Spaeth [1, 2] reported that plasma methionine and homocystine concentrations returned to normal in three patients given pharmacologic doses of pyridoxine (200-500 mg daily). This response, confirmed by other investigators [10, 15, 16, 33] in some but not all homocystinuria patients [16, 29], has also been associated with the appearance of cystine in plasma [11]. The biochemical basis for these findings is unclear. Yoshida et al. [35] reported that pyridoxal phosphate in vitro stimulated hepatic cystathionine synthase activity in one patient. Mudd and co-workers [23] observed that hepatic synthase activity in two pyridoxine-responsive patients was considerably greater when they were receiving pyridoxine than when they were not. Conversely, Hollowell [15], Gaull [11] and their co-workers found no such stimulation in other pyridoxine-responsive patients. The present study was carried out to examine fur- Two young men, 16 and 18 years, with well documented homocystinuria were studied. JK was an 18year-old white male with lenticular dislocation, mild pectus excavatum, kyphoscoliosis, and minimal osteoporosis. IQ as measured by the Wechsler Adult Intelligence Scale was 71 verbal, 88 performance. EY was a 16-year-old white male with lenticular dislocation, slight kyphoscoliosis, and mild intention tremor. IQ as measured by the Wechsler Adult Intelligence Scale was 70, full scale. Neither patient was anemic or demonstrated signs of vitamin B6 deficiency. Both patients had positive urinary nitroprusside tests and increased plasma and urine concentrations of methionine and homocystine (Table I). Plasma cystine was undetectable in both [36]. During the study, the patients were hospitalized on a clinical research unit. Dietary intake of methionine and cystine, estimated by a dietitian, was kept constant, and was similar to that ingested at home (Table I). Informed consent from both patients was obtained in accordance with the provisions set forth in the Declaration of Helsinki. Table I. Parameters of sulfur amino acid metabolism in two homocystinuric patients prior to pyridoxine administration Methionine Homocystine Patient Die- Plas- Urinary tary inexcretion3 take1 Die- Plas- Uritary nary inexcretake 1 tion3 Dietary mtaket Plas- Urinary excretion3 JK EY 35.8 9.7 28.3 7.8 3.5 2.9 21.6 19.2 0 0 0 0 1 65 71 0 0 111 216 Cystine Milligrams per kilogram per day; calculated (...truncated)