Pediat. Res. 6: 187-196 (1972) Cystathionine homocystinuria synthase 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 de- ficiency. Pyridoxine responsiveness was dose-dependent but differed in the two pa- tients. 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, syn- thase 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 par- tial correction of his underlying synthase deficiency and that EY's response to pyri- doxine 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 stimula- tion of defective cystathionine synthase activity? Our results suggest a negative answer to this question and emphasize the need for further clinical and biochemical investi- gation of such patients. Introduction the fact that cystathionine synthase catalyzes one of the steps in the major degradative pathway by which me- Two years after the clinical description of homocystin- thionine is converted to cystine and ultimately to its uria in 1962 [5, 12], the enzymatic basis of the disease end product, inorganic sulfate [20] (Fig. 1). The rela- was demonstrated by Mudd et al. [24] who showed tion between these biochemical findings and the clini- that cystathionine synthase activity was absent in the cal hallmarks of homocystinuria which include ectopia livers of homocystinuric patients. This deficiency, also lentis, skeletal abnormalities, mental retardation, and demonstrated in cultured skin fibroblasts [34], leads to thrombotic vascular disease is unknown. increased concentrations of methionine and homocys- Two forms of therapy have been tried in homocys- tine in plasma and urine. The absence or markedly tinuria. Limitation of dietary methionine with admin- reduced concentration of cystine in these fluids reflects istration of supplemental cystine has resulted in bio- 187 188 SEASHORE, DURANT, AND ROSENBERG L-METHIONINE < ther the nature of pyridoxine-responsive homocystinu- ria. The effect of pyridoxine administration on plasma I and urinary amino acid concentrations and urinary S-ADENOSYL-L-METHIONINE inorganic sulfate excretion was studied during normal \ dietary intake and under conditions of methionine S-ADENOSYL-L-HOMOCYSTEINE loading. Cystathionine synthase activity in cell-free ex- L-HOMOCYSTINE \ tracts of cultured skin fibroblasts was determined, and 5: L-HOMOCYSTEINE the influence of varying pyridoxine concentrations in • serine cystathionine the growth medium and of pyridoxal phosphate con- synthase centration in the in vitro assay system was studied. (B6) Our results suggest two different mechanisms of pyri- L-CYSTATHIONINE doxine responsiveness in this disease. L-CYSTINE Patients Two young men, 16 and 18 years, with well docu- mented homocystinuria were studied. JK was an 18- year-old white male with lenticular dislocation, mild pectus excavatum, kyphoscoliosis, and minimal osteo- porosis. IQ as measured by the Wechsler Adult Intelli- Fig. 1. Pathway of methionine catabolism to sulfate. In homo- cystinuria, methionine and homocystine accumulate because o£ gence Scale was 71 verbal, 88 performance. EY was a an inherited deficiency of cystathionine synthase, the enzyme 16-year-old white male with lenticular dislocation, which catalyzes the condensation of serine and homocysteine to slight kyphoscoliosis, and mild intention tremor. IQ as form cystathionine. Note that the enzymatic conversion of homo- measured by the Wechsler Adult Intelligence Scale was cysteine to cystathionine appears to require vitamin Bo in its 70, full scale. Neither patient was anemic or demon- coenzyme form (pyridoxal phosphate). strated signs of vitamin B6 deficiency. Both patients had positive urinary nitroprusside tests and increased chemical improvement in several patients [3, 7, 13, 19, plasma and urine concentrations of methionine and 27], but it will require years to determine whether homocystine (Table I). Plasma cystine was undetecta- such improvement alters the clinical course of the dis- ble in both [36]. ease. The second therapeutic approach, oral adminis- During the study, the patients were hospitalized on tration of pyridoxine, is based on the increasing, al- a clinical research unit. Dietary intake of methionine though not unequivocal, evidence that cystathionine and cystine, estimated by a dietitian, was kept con- synthase requires pyridoxal phosphate as a coenzyme stant, and was similar to that ingested at home (Table [4, 18, 26]. Barber and Spaeth [1, 2] reported that I). Informed consent from both patients was obtained plasma methionine and homocystine concentrations re- in accordance with the provisions set forth in the Dec- turned to normal in three patients given pharmaco- laration of Helsinki. logic doses of pyridoxine (200-500 mg daily). This re- sponse, confirmed by other investigators [10, 15, 16, 33] Table I. Parameters of sulfur amino acid metabolism in two in some but not all homocystinuria patients [16, 29], homocystinuric patients prior to pyridoxine administration has also been associated with the appearance of cystine in plasma [11]. The biochemical basis for these find- Methionine Homocystine Cystine et al. Pa- Die- Plas- Uri- Die- Plas- Uri- Die- Plas- Uri- ings is unclear. Yoshida [35] reported that pyri- tient tary nary tary nary tary nary in- excre3- in- 1 excre3- m- excre3- doxal phosphate in vitro stimulated hepatic cysta- take1 tion take tion taket tion thionine synthase activity in one patient. Mudd and co-workers [23] observed that hepatic synthase activity JK 35.8 9.7 65 0 3.5 111 21.6 0 0 in two pyridoxine-responsive patients was considerably EY 28.3 7.8 71 0 2.9 216 19.2 0 0 greater when they were receiving pyridoxine than 1 Milligrams per kilogram per day; calculated by a dietitian and when they were not. Conversely, Hollowell [15], Gaull kept constant during the study. [11] and their co-workers found no such stimulation in 2 Milligrams per 100 ml; value presented is representative of other pyridoxine-responsive patients. several fasting determinations. 3 Milligrams per 24 hr; value is representative of several 24-hr The present study was carried out to examine fur- collections. Pyridoxine-responsive homocystinuria 189 Methods cystathionine synthase activity. Protein concentration of this cell-free supernatant fluid was determined by Effect of Pyridoxine on Sulfur Amino Acid Metabo- the method of Lowry et al. [21]. Two different assay lism systems were employed. The first was the radioisotopic method of Mudd et al. [25, 34], in which the incorpo- Pyridoxine hydrochloride was given daily, by 14 14 mouth, in four divided doses. During administration ration of serine-3- C into cystathionine- C was meas- of the 500-mg daily dose, the patients were hospitalized ured. This technique employed a serine concentration for 2 weeks, and were then observed as outpatients for of 2.5 m.M and required the addition of 0.07 ^moles four consecutive months. The vitamin was then dis- cystathionine to the reaction mixture. The labeled cys- continued for 1 month. Subsequently, lower doses of tathionine formed during a 135-min incubation was pyridoxine were given for 2- to 3-week intervals and identified by paper chromatography [25] and quanti- increased progressively. Fasting plasma amino acid tated in a liquid scintillation spectrometer. Hepatic concentrations were determined at each pyridoxine cystathionine synthase activity has been shown to be dose after 2-3 weeks. Oral loads of L-methionine were linear with time and enzyme concentration when as- administered to both patients on two occasions: while sayed by this method [25]. Similarly, in our experi- on 500 mg pyridoxine daily for four consecutive ments, doubling the amount of control fibroblast cell months, and after receiving no supplementary pyridox- extract led to a doubling of cystathionine formation. ine for 60 days. L-Methionine was given as the pure, Boiled blanks were run daily for each cell line studied. crystalline amino acid in gelatin capsules, each con- In the second method, cystathionine formation was taining 0.3 g. The total daily methionine load of 0.75 quantitated by column chromatography on a Beckman mmole/kg was given in four divided doses for 4 days, 120C amino acid analyzer using the method of Scriver and was well tolerated. et al. [30]. The reaction mixture was identical with Urine was collected, without preservative, for 24-hr that employed for the isotopic method except that the periods, and kept refrigerated