The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT): initial aims and impact of the family history of type 1 diabetes mellitus in Japanese children.

The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT) was established in July 1994 with the chief aim to improve the quality of therapy for type 1 diabetes in children, an entity far less common in Japan than in Europe. We proposed four initial research topics: (i) to determine the current status of medical care and glycemic control in Japanese children with type 1 diabetes mellitus; (ii) to standardize the measurement of hemoglobin A1c; (iii) to establish a registry of a large cohort of patients in order to enable prospective studies to improve the quality of therapy for children with type 1 diabetes in Japan; and (iv) to enable participants of the JSGIT to hold a workshop twice annually. We registered a total of 736 patients from 45 hospitals throughout Japan. Intervention via insulin treatment was instituted after 2 yr for those patients whose hemoglobin A1c level was more than 8.1%. The proportion of patients receiving multiple insulin injections increased after intervention; however, average hemoglobin A1c in females remained significantly higher than in males. We identified two forms of diabetes in Japanese children: a rapidly progressive form and a more slowly progressive form. There was a significantly higher prevalence of a family history of diabetes in first-degree relatives in the slowly progressive form. These preliminary findings are the result of the first collaborative study of childhood diabetes in Japan.

Effect of sports activity on carnitine metabolism. Measurement of free carnitine, gamma-butyrobetaine and acylcarnitines by tandem mass spectrometry.

The effects of sports activity on carnitine metabolism were studied using mass spectrometry. Serum levels of free carnitine, acylcarnitines (acetylcarnitine, propionylcarnitine, C4-, C5- and C8-acylcarnitine) and gamma-butyrobetaine, a carnitine precursor, were determined by tandem mass spectrometry in liquid secondary ion mass ionization mode. The coefficients of variation at three different concentrations were 2.8-7.9% for gamma-butyrobetaine, and 1.2 to approximately 6.7% for free carnitine. The recoveries added to serum were 109.1% for gamma-butyrobetaine, 89.3% for free carnitine. Sports activity caused increased serum levels of gamma-butyrobetaine, acetylcarnitine, C4- and C8-acylcarnitines and decreased serum levels of free carnitine. This method requires a small amount of sample volume (20 microl of serum) and short total instrumental time for the analysis (1 h for preparation, 2 min per sample for mass spectrometric analysis). Therefore, this method can be applied to study carnitine metabolism under various conditions that affect fatty acid oxidation.

Measurement of carnitine precursors, epsilon-trimethyllysine and gamma-butyrobetaine in human serum by tandem mass spectrometry.

Methods using tandem mass spectrometry for measurement of epsilon-trimethyllysine and gamma-butyrobetaine in human serum are described. Precursor ion scan analysis of a methylated sample was applied for gamma-butyrobetaine measurement. However, for epsilon-trimethyllysine measurement, homoarginine interfered with the methylated sample during precursor ion scan analysis. To overcome this interference, the sample was propylated and acetylated prior to precursor ion scan analysis. The obtained values resembled those obtained by enzymatic or HPLC measurement. Using tandem mass spectrometry, all members of the carnitine family, free carnitine, acylcarnitines, gamma-butyrobetaine, epsilon-trimethyllysine can be analyzed in 0.1 ml of serum. Thus, the proposed method appears to be suitable for clinical application, especially in the pediatric field.

Carnitine import to isolated hepatocytes and synthesis are accelerated in pivalate-treated rats.

To investigate the effect of pivalate on carnitine import and carnitine synthesis in the liver, we measured carnitine uptake in isolated rat hepatocytes with L-[(14)C] carnitine and concentrations of free carnitine, gamma-butyrobetaine and acylcarnitines using tandem mass spectrometry. Hepatocytes from rats treated with 20 mmol/L of pivalate for 4 wk had greater L-[(14)C] carnitine uptake than those of unsupplemented rats after 5, 10, 30 and 90 min. Addition of 1 mmol/L of pivalate or 1 mmol/L of pivaloylcarnitine to control cell suspensions did not affect L-[(14)C] carnitine uptake. The K(m) values for L-[(14)C] carnitine uptake for pivalate-treated rats were significantly lower than control (2.9 +/- 0.7 mmol/L for pivalate-treated rats, 6.2 +/- 1.1 mmol/L for controls). The concentration of free carnitine was not reduced in the liver of pivalate-treated rats, whereas the concentrations of acetylcarnitine and gamma-butyrobetaine were significantly lower than controls. In the heart and muscle the concentration of free carnitine was significantly lower and that of gamma-butyrobetaine was higher than controls. These results suggest that carnitine transport from plasma into the liver and synthesis in the liver are accelerated in rats with secondary carnitine deficiency induced by the administration of pivalate.

Serum levels of thyroid-stimulating hormone receptor antibodies during IFN-alpha2a treatment of chronic hepatitis C.

Twenty chronic hepatitis C patients with baseline levels of thyroid hormones, antithyroglobulin, and antimicrosomal antibodies within the normal range were monitored by thyroid testing during a 26-week treatment with interferon-alpha2a (IFN-alpha2a). The present study was aimed at retrospectively analyzing thyroid-stimulating hormone (TSH) receptor antibodies, employing stored serum samples obtained from these patients at baseline, at 12 weeks, and at the end of IFN-alpha2a treatment. Ten patients (group A) received IFN-alpha2a at a total dose of 474 million units (MU), and 10 patients (group B) at a total dose of 774 MU. None of the patients produced antithyroglobulin and antimicrosomal antibodies. Two patients in group A exhibited elevated TSH levels without free thyroid hormone alterations while on the treatment. At baseline, 1 patient in group A had a positive TSH binding inhibitory immunoglobulin, and 1 patient in group B was positive for thyroid-stimulating antibody at the end of treatment. Both patients remained euthyroid throughout the treatment. Treatment with IFN-alpha2a may infrequently induce the production of TSH receptor antibodies in chronic hepatitis C patients provided preexisting autoimmune thyroid disease is precluded.

Molecular basis of intermittent maple syrup urine disease: novel mutations in the E2 gene of the branched-chain alpha-keto acid dehydrogenase complex.

The E2 gene of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was studied at the molecular level in three patients with intermittent maple syrup urine disease (MSUD). All three patients had higher BCKDH activity than did those with the classical phenotype. In the first patient, a single base substitution from A to G in intron 8 created a new 5' splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3' splice site in the same intron. The predicted mRNA encoded a truncated protein with 282 amino acids including 4 novel ones at the carboxyl terminus, compared with the normal protein with 421 amino acids. In vitro, the region from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutation probably supports an exon definition model in which the spliceosome recognizes a 3' splice site and then scans downstream for an acceptable 5' splice site, thereby defining an exon. The second patient was homozygous for a G to T transversion at nucleotide 1463 in exon 11, which predicted a substitution of the termination codon by a leucine residue and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parents were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transition at nucleotide 1165 in exon 9, causing an Ile-to-Met substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, respectively. Taken together, these results indicate that the molecular basis of intermittent phenotype MSUD in some patients can be due to mutations in the E2 gene, giving rise to a low but significant residual activity of the BCKDH complex.

The effect of carnitine on ketogenesis in perfused livers from juvenile visceral steatosis mice with systemic carnitine deficiency.

Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

Does the presence of serum autoantibodies influence the responsiveness to interferon-alpha 2a treatment in chronic hepatitis C?

The serum autoantibodies, antinuclear antibody, anti-DNA antibody, anti-smooth muscle antibody, antithyroglobulin antibody, antimicrosomal antibody, antimitochondrial antibody, rheumatoid factor and antibody to deoxyribonucleoprotein were measured at the baseline and on completion of interferon-alpha 2a (IFN-alpha 2a) treatment in chronic hepatitis C (CHC) patients who did not present with any autoimmune disease prior to treatment. Of the 57 patients examined, 27 spontaneously manifested at least one autoantibody. Only the prevalence of rheumatoid factor (26%) was significantly higher in the CHC patients than in the control subjects. There were no differences in the prevalence of the 8 autoantibodies examined between hepatitis C virus (HCV) genotypes 1b and 2a/2b. Twenty-six patients responded to IFN-alpha 2a. Subclinical hypothyroidism developed in two patients with elevated antithyroid antibody titres during treatment. No relationship was observed between changes in the status of autoantibodies and either response to IFN-alpha 2a or HCV genotype. Irrespective of the HCV genotype, autoantibodies might be present in CHC patients before and during the IFN-alpha 2a treatment. The presence of such antibodies does not represent a contraindication to the use of IFN-alpha 2a in CHC patients not complicated by autoimmune diseases. Careful observations are necessary for CHC patients positive for antithyroid antibodies during the IFN-alpha 2a treatment. Preexisting or newly developed autoantibodies do not necessarily predict a poor response to IFN-alpha 2a.

Pivalate affects carnitine status but causes no severe metabolic changes in rat liver.

To investigate the carnitine deficiency induced by pivalate, rats had free access to drinking water with or without pivalate. Consumption of 20 mmol/L pivalate for 1 wk decreased the levels of both free and total carnitine in plasma to approximately 20% of levels before treatment. After 4 wk, the concentrations of free carnitine in the liver, heart and muscle of pivalate-treated rats were approximately 60-80% of the control, and in the kidney, 26% of the control. Fractional excretion of free carnitine (FEFC) in pivalate-treated rats was measured; however, the treatment for 3 or 8 d did not affect the values relative to those obtained before treatment. Treatment with pivalate for 4 wk did not affect plasma concentrations of glucose, ammonia and free fatty acids (FFA) in the rats; however, the concentration of 3-hydroxybutyrate (3-OHB) was higher, and the FFA/3-OHB ratio was lower than those of controls. In a liver perfusion study, ketogenesis from oleate and gluconeogenesis from lactate and pyruvate in rats treated with pivalate for 4 wk were not different from controls. These results suggest that administration of pivalate did not induce the excessive excretion of free carnitine in urine, and secondary carnitine deficiency induced by intake of 20 mmol/L pivalate for 4 wk did not cause severe metabolic changes in rat liver.

Occurrence of Graves' disease during retreatment with interferon-alpha 2a for chronic hepatitis C.

The present report describes a patient who developed Graves' disease 3 months after inception of retreatment with higher doses of interferon-alpha 2a for chronic hepatitis C, although the initial 6-month treatment caused no serious adverse reactions. Severe hyperthyroidism continued despite discontinuation of interferon-alpha 2a, and the patient was subsequently treated with 131I. This case suggests careful evaluation of the safety of retreatment to prevent manifestation of such a complication in the retreatment of chronic hepatitis C with interferon.

Changes of erythrocyte ouabain maximum binding after birth in neonates--in relation to erythrocyte sodium and potassium concentrations.

Erythrocyte ouabain maximum binding (Vmax level) represents the number of molecules of Na-K ATPase on the erythrocyte membrane. Erythrocyte Vmax levels and sodium potassium concentrations were studied in 38 mature and 57 premature infants, including 40 very low birth weight infants, 41 children and 16 adults, in order to clarify the development of erythrocyte Na-K ATPase related to mineral balance and thermogenesis. Vmax levels were the highest (22.9 +/- 8.9 nmol/l cells) for premature infants; correlated negatively with gestational age (r = -0.77, P < 0.01). Vmax levels correlated positively with the ratio of erythrocyte potassium to sodium concentration (IC-K/Na) at < 20 nmol/l cells of Vmax levels (r = 0.72, P < 0.01), but not at > or = 20 nmol/l cells (r = 0.23). Vmax levels in very low birth weight infants, especially < 28 weeks postconception, decreased gradually with postnatal days. Ln(Vmax) levels correlated negatively with postconceptional age (r = -0.75, P < 0.01). Under 28 weeks postconception, Vmax levels were high, but not correlated with IC-K/Na. It is probable that individual Na-K ATPase molecules may be less active, and the large numbers of Na-K ATPase molecules may compensate for the individual lower activity.

Erythrocyte sodium-potassium transport in hyperkalaemic and normokalaemic infants.

UNLABELLED: One of the causes of early onset hyperkalaemia in very low birth weight infants is presumed to be the dysfunction of K+ transport across the cell membrane. Sodium-potassium adenosine triphosphatase(Na(+)-K+ ATPase) is known to play a major role in K+ transport. We compared the concentrations of erythrocyte Na(+)-K+ ATPase (Vmax levels) for hyperkalaemic and normokalaemic infants of matched gestational age. In hyperkalaemic infants, the highest levels of Vmax were reached at 24-48 h after birth, but in normokalaemic infants, there were no significant changes in Vmax levels during the 1st week after birth. At 12-72 h after birth, erythrocyte K+ concentrations for hyperkalaemic infants were higher than those of normokalaemic infants. For both groups of infants, the highest levels of plasma K+ during the 1st week after birth showed a positive correlation with those of Vmax. CONCLUSION: Na(+)-K+ ATPase on the cell membrane is activated to compensate for hyperkalaemia; however, when this compensation is incomplete, hyperkalaemia occurs.

Simultaneous occurrence of adenomas in stomach, duodenal bulb, and colon: disappearance of gastric and duodenal adenomas during oral administration of 5'-deoxy-5-fluorouridine.

The present report concerns a patient who was first diagnosed as having adenoma in the duodenal bulb and subsequently was discovered to have adenomas in the stomach and colon. Surgical procedures including endoscopic resection could not be carried out because the patient had advanced liver cirrhosis. Follow-up colonoscopy no longer detected any polyp in the colon. Ultrasonography demonstrated a slowly growing hypoechoic mass in the liver, which was indicative of hepatocellular carcinoma. Then, single chemotherapy with oral administration of 5'-deoxy-5-fluorouridine was initiated. Surprisingly, repeated esophagogastroduodenoscopy revealed gradual regression of the gastric and duodenal tumors. Findings from the endoscopy and biopsy materials 10 months after the start of therapy showed disappearance of the gastric and duodenal adenomas. Despite enlargement of the hepatic mass, no recurrence of gastric and duodenal tumors has been noted during the past 1 1/2 yr.

Patient with unusual Hutchinson-Gilford syndrome (progeria).

A patient with unusual Hutchinson-Gilford syndrome (progeria) is reported. This 7-year-old boy had all the characteristics of progeria, except for coxa valga and the "horse-riding" stance. A previous cerebral infarction was detected in the right putamen on cranial magnetic resonance imaging. During treadmill exercise test electrocardiography, ST depression suggested the existence of arteriosclerotic lesions. Skin fibroblast culture exhibited 76% DNA-repair capacity compared to normal. He has not manifested endocrinologic abnormalities. From these findings it is concluded that this patient has an incomplete case of Hutchinson-Gilford syndrome and that a correlation may exist between the clinical features and the degree of DNA-repair capacity.