A new Japanese case of succinyl-CoA: 3-ketoacid CoA-transferase deficiency

J. lnher. Metab. Dis. 18 (1995) 323-325 © SSIEM and Kluwer AcademicPublishers. Printed in the Netherlands

A new Japanese case of succinyl-CoA: 3-ketoacid CoA-transferase deficiency H. SAKAZAKI1., K. HIRAYAMA1, S. MURAKAMI1, S. YONEZAWAt, H. SHINTAKU2, Y. SAWADA3, T. FUKAO4, H. WATANABE4, T. ORIP and G. ISSHIKI2

1Department of Pediatrics, Izumi Municipal Hospital, Osaka; 2Department of Pediatrics, Osaka City Medical School, Osaka; 3Department of Pediatrics, Children's Medical Center, Osaka City General Hospital, Osaka; 4Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan *Correspondence: Department of Pediatrics, Izumi Municipal Hospital, 4-10-10 Fuchu-cho, Izumi City, Osaka 594, Japan MS received 3.11.94 Accepted12.1.94 Succinyl-CoA:3-ketoacid CoA-transferase (SCOT; EC 2.8.3:5) deficiency (McKusick 245050) is a rare inherited metabolic disease. This enzyme is a key enzyme for utilization in peripheral tissues of ketone bodies that are produced by the liver. The absence of SCOT activity blocks peripheral ketone body utilization and causes recurrent attacks of severe ketoacidosis beginning in the neonatal or infantile period. Five cases of SCOT deficiency have been reported (Tildon and Cornblath 1972; Spence et al 1973; Middleton et al 1987; Saudubray et al 1987; Perez-Cerda et al 1992). The prognosis of these patients seems to parallel the severity of the SCOT deficiency. Here we report a new case of SCOT deficiency in Japan. We also report SCOT activity in the lymphocytes of the patient and his family. CASE REPORT The patient, T.H., a boy, was born on 16 September 1993 of unrelated parents at 38 weeks gestation. His birth weight was 3280g. At 5 months of age he was admitted because of vomiting and upper respiratory tract infection. At 6 months of age he presented with vomiting, hypotonia, tachypnoea, and peripheral cyanosis. Severe metabolic acidosis (pH 7.08, BE -22.2, I-ICO 3- 5.1mmol/L) was detected, and urinary ketones were strongly positive. Serum glucose, lactate, pyruvate, creatine kinase, ammonia, and amino acids were normal. Blood concentrations of acetoacetate and 3-hydroxybutyrate were 4.5 and 7.7mmol/L, respectively. His karyotype was 46 XY. The patient was treated with intravenous bicarbonate and glucose infusion, and the acidosis resolved after 4 days. The ketonaemia (acetoacetate 1.2 retool/L, 3-hydroxybutyrate 1.7 mmol/L) persisted, however. Gas chromatography-mass spectrometry of the urine revealed high concentrations of acetoacetate and 3-hydroxybutyrate without any other significant organic acids. These findings indicated a defect in ketone body metabolism. At that point, an enzyme assay for SCOT was performed and the diagnosis was made. 323

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S a k a z a k i et al.

Table 1 Enzyme activities (nmoi/min per mg protein) in cultured skin fibroblasts of T.H. Acetoacetyl-CoA thiolase

Succinyl-CoA:3 ketoacid

Subject

+K +

-K ÷

+K+/-K÷

CoA-transferase

T.H. Normal controls* (n=20) (range)

11.9

5.7

2.1

3.3

16.4+2.3 (12.6-22.0)

8.1 +1.4 (5.8-11.0)

2.1 (1.8-2.5)

9.5+1.1 (8.1 - 11.8)

*Data are shown as mean+SD The patient, now aged 1 year, shows good psychomotor and physical development except for some episodes of ketosis. MATERIALS AND METHODS

Sodium succinate, acetoacetyl-CoA, and coenzyme A were purchased from Sigma Chemical Co. All other chemicals were reagent grade.

Materials:

Skin fibroblasts were cultured in MEM supplemented with 10% fetal calf serum. Cells were harvested after trypsinization and washed twice with 0.9% sodium chloride. Lymphocytes were obtained with the use of Ficoll-Paque and washed twice with 0.9% sodium chloride. They were then stored at - 8 0 ° C until assayed.

E n z y m e source:

assays: Cells were thawed on ice and sonicated in 50mmol/L sodium phosphate, pH 8.0, 0.1% Triton X-100. Protein concentrations were determined by a modification (Markwell et al 1978) of the method of Lowry et al (1951). SCOT activity was measured in the direction of succinyl-CoA formation, as described by Williamson et al (1971), with some modifications. Briefly, the cuvettes contained, in a final volume of 1 ml: 50 mmol/L Tris-HC1 buffer, pH 8.5, 10mmol/L MgC12, 4mmol/L iodoacetamide, and 30gmol/L acetoacetyl-CoA, and enzyme sample (up to 50/11). After 3 min recording of AE303, sodium succinate (50 #mol) was added, and sodium succinate-dependent AE~o3 was measured. The millimolar extinction coefficient of acetoacetyl-CoA used was 21.0 (303 nm). Acetoacetyl-CoA thiolase (AAT; EC 2.3.1.9) activity was measured as described elsewhere (Yamaguchi et al 1988). Enzyme

RESULTS AND DISCUSSION

Fibroblast SCOT and AAT activities are shown in Table 1. The ratio of AAT activity in the presence of potassium ion to its activity in the absence of potassium ion was 2.1, indicating that mitochondrial AAT activity was normal. However, the SCOT activity of the patient (T.H.) was reduced to 35% of the control value, suggesting defective SCOT activity in his fibroblasts. We also performed lymphocyte enzyme assays, which have not been reported previously. As shown in Table 2, the patient's lymphocytes had only 10% of the SCOT activity of the control. Furthermore, the SCOT activity of the lymphocytes of his parents was about 50% of control. Based on these data, we diagnosed the patient as SCOT J. Inher. Metab. Dis. 18 (1995)

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Succinyl-CoA:3-ketoacid CoA transferase deficiency

Table 2

Enzyme activities (nmol/min per mg protein) in lymphoeytes of T.H.'s family

Subject

Acetoacetyl-CoA thiolase +K ÷

Succinyl-CoA:3-ketoacid CoA-transferase

T.H. Sister Mother Father

22.7 23.0 19.5 23.7

2.7 20.4 9.4 11.0

Normal controls

20.9

20.0

(n=3)

deficiency. It is noteworthy that the SCOT activity in lymphocytes was greater than in fibroblasts. The results of the present study suggest that lymphocytes are a better enzyme assay specimen for SCOT than fibroblasts, especially for heterozygote detection. A correlation between clinical severity and residual SCOT activity was suggested by Perez-Cerda et al (1992). Two of the 5 reported patients (Tildon and Cornblath 1972; Spence et al 1973), with less 5% residual activity, died while the other 3 patients (Middleton et al 1987; Saudubray et al 1987; Perez-Cerda et al 1992), who had more than 20% residual activity in their fibroblasts, are alive. Our patient has 10% residual activity in his lymphocytes and 35% in his fibroblasts. He is now well-developed but requires careful dietary management. Urinary ketone excretion is monitored daily by his mother to detect signs of metabolic decompensation. In this way, intravenous bicarbonate and glucose therapy can be initiated before severe metabolic acidosis develops. REFERENCES Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275. Markwell MAK, Hass SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206-210. Middleton B, Day R, Lombes A, Saudubray JM (1987) Infantile ketoacidosis associated with decreased activity of succinyi-CoA:3-ketoacid CoA-transferase. J Inher Metab Dis 10 (supplement 2): 273-275. Perez-Cerda C, Merinero B, Sanz R et al (1992) A new case of succinyl-CoA:acetoacetate transferase deficiency. J Inher Metab Dis 15:371-373. Saudubray JM, Specola N, Middleton Bet al (1987) Hyperketotic states due to inherited defects of ketolysis. Enzyme 38: 80-90. Spence MW, Murphy MG, Cook HW, Ripley BA, Embil JA (1973) Succinyl CoA:3-ketoacid CoAtransferase deficiency: a new phenotype. Pediatr Res 7: 394. Tildon JT; Cornblath M (1972) Succinyl-CoA:3-ketoacid CoA-transferase deficiency: a cause for ketoacidosis in infancy. J Clin Invest 51: 493-498. Williamson DH, Bates MW, Ann Page M, Krebs HA (1971) Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues. Biochem J 121: 47. Yamaguchi S, Orii T, Sakura N, Miyazawa S, Hashimoto T (1988) Defect in biosynthesis of mitochondrial acetoacetyl-CoA thiolase in cultured fibroblasts from a boy with 3-ketothiolase deficiency. J Clin Invest 81: 813-817.

J. Inher. Metab. Dis. 18 (1995)