Decrease in tetrahydrobiopterin as a possible cause of nephropathy in type II diabetic rats.

A decrease in renal synthesis of nitric oxide (NO) in the progression of diabetic nephropathy has been documented. As (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor of NO synthase, we investigated whether BH4 deficiency is involved in the pathogenesis of nephropathy. Ten-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a type II diabetic model, and Long-Evans Tokushima Otsuka (LETO) rats as the healthy controls. OLETF rats were orally treated with BH4 (10 mg/kg daily) or with water from 10 to 61 weeks of age. In another experiment, OLETF rats were treated orally with a calcium channel blocker, benidipine (5 mg/kg daily), or with 0.3% carboxymethyl cellulose (nontreated) from 10 to 52 weeks of age. Proteinuria was observed periodically, and at the end of the study, BH4 level and GTP cyclohydrolase I (GTPCH) activity in the kidney were measured. Proteinuria was observed at 13 weeks of age in the OLETF rats, and deteriorated until 61 weeks of age. Supplemental BH4 reduced the proteinuria. At 52 weeks of age, GTPCH activity and the BH4 level were decreased in the plasma and kidneys of OLETF rats, whereas they were significantly higher in the benidipine group than in the nontreated group. Proteinuria was milder in the benidipine group than in the nontreated group, without a concomitant decrease in blood pressure. Histologically observed glomerulosclerosis was mild in the BH4 and benidipine groups. In type II diabetic rats, renal BH4 is considered to play a crucial role in the pathogenesis of diabetic nephropathy. Benidipine was found to preserve BH4 levels, suggesting therapeutic renoprotective effects.

Disorders of tetrahydrobiopterin metabolism and their treatment.

Tetrahydrobiopterin (BH4) deficiencies are disorders affecting phenylalanine metabolism in liver and neurotransmitters biosynthesis in brain. BH4 is the essential cofactor in the enzymatic hydroxylation of 3 aromatic amino acids (phenylalanine, tyrosine, and tryptophan). BH4 is synthesized from guanosine triphosphate (GTP) catalyzed by GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase (SPR), and in aromatic amino acids hydoxylating system is regenerated by pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). To date, 4 enzyme deficiencies (GTPCH, PTPS, DHPR, PCD) have been reported and they all follow an autosomal recessive mode of inheritance. The incidence of BH4 deficiency is at 1 in 1,000,000, except that in Taiwanese (much higher than in Japanese and Caucasians). BH4 deficiency has been diagnosed in patients with hyperphenylalaninemia (HPA) by neonatal mass-screening based on BH4 oral loading tests, analysis of urinary or serum pteridines, and measurement of dihydropterindine reductase (DHPR) activity in blood from a Guthrie card. BH4 deficiency without treatment causes combined symptoms of HPA and neurotransmitter (dopamine, norepinephrine, epinephrine, and serotonin) deficiency, such as red hair, psychomotor retardation, and progressive neurological deterioration. Treatment of BH4 deficiencies consists of BH4 supplementation (2-20 mg/kg per day) or diet to control blood phenylalanine concentration and replacement therapy with neurotransmitters precausers (L-dopa/CarbiDOPA and 5-hydroxytryptophan), and supplements of folinic acid in DHPR deficiency.

Brain magnetic resonance imaging in 23 patients with mucopolysaccharidoses and the effect of bone marrow transplantation.

A longitudinal study of cranial magnetic resonance imaging (MRI) was carried out in 23 patients with mucopolysaccharidoses (MPS); 1 each of types IH, VI, and VII; 2 of type IS; 10 of type II; and 4 each of types IIIB and IVA. Six types of distinct abnormalities were 1) cribriform changes or spotty changes in the corpus callosum, basal ganglia, and white matter; 2) high-intensity signal in the white matter on T2-weighted image; 3) ventriculomegaly; 4) diffuse cerebral cortical atrophy; 5) spinal cord compression; and 6) megacisterna magna. The cribriform changes that corresponded to dilated perivascular spaces were found in the patients with MPS IS, II, and VI. The patchy and diffuse intensity changes were found in the patient with MPS II and IIIB, respectively. MPS IH and the severe type of MPS II showed marked ventriculomegaly. Marked cerebral atrophy was observed in all MPS IIIB patients and in the severe type of MPS II patients. Spinal cord compression was a feature usually observed in MPS IH, IVA, VI, and VII. Megacisterna magna was frequent in the patients with MPS II (6/10). In two of five patients, the therapeutic effect of bone marrow transplantation (BMT) was remarkable. Both the cribriform changes and the intensity change of the white matter in a MPS VI patient disappeared eight years after the BMT. Slight improvement of cribriform change was noted in one patient with MPS II three years after the BMT. MRS was not sufficient to estimate the accumulation of glycosaminoglycans but was useful for evaluating neuronal damages.

Connatal tuberculosis in an extremely low birth weight infant: case report and management of exposure to tuberculosis in a neonatal intensive care unit.

UNLABELLED: A case of connatal tuberculosis in an extremely low birth weight infant is reported. The patient was a female with a birth weight of 973 g born in the 27th week of pregnancy. She developed respiratory distress and signs of infection immediately after birth, which did not respond to mechanical ventilation, antibiotics, and corticosteroid therapy. Connatal tuberculosis was confirmed at 48 days of age by isolation of Mycobacterium tuberculosis from the infant's tracheal aspirate and the mother's menstrual discharge. The infant died of respiratory failure at 90 days of age. Mantoux tuberculin skin tests (TST) were performed on 99 infants, 144 medical staff members, and two family members. TST conversion occurred in three medical staff members, and preventive therapy with isoniazid was initiated. Eight exposed infants had normal chest X-rays and negative gastric aspirates for acid-fast bacilli and all received preventive isoniazid therapy. No case of tuberculosis developed during the 2-year follow-up period. CONCLUSION: Connatal tuberculosis should be considered in neonatal respiratory infection resistant to antibiotics. Prevention of transmission of tuberculosis on the neonatal intensive care unit by chemoprophylaxis is important.

Measurement of bone mineral density of lumbar spine and whole body in low-birth-weight infants: comparison of two methods.

In the present study, we compared lumbar spinal and whole-body bone mineral density (BMD) measurements to determine which is more suitable for evaluating the bone mineral status of low-birth-weight (LBW) infants. Lumbar spinal and whole-body BMD were assessed simultaneously in a prospective series including 152 Japanese LBW infants (birth weight 453-2400 g, gestational age 24-38 weeks) from the age of 40 weeks post-conception to 2 years of age. Lumbar spinal BMD at 40 weeks post-conception was significantly correlated with birth weight (r = 0.74; P < 0.0001), but wholebody BMD was not correlated with birth weight. No correlation was found between lumbar spinal and whole-body BMD at 40 weeks post-conception. However, after 40 weeks post-conception, a significant correlation was found between lumbar spinal and whole-body BMD (r = 0.65; P < 0.0001). For infants with a body weight of 4 kg or less at the time of measurement, no correlation was found between lumbar spinal and whole-body BMD. However, for infants with a body weight above 4 kg, a significant correlation was found between lumbar spinal and whole-body BMD (r = 0.65; P < 0.0001). Thus, lumbar spinal BMD is more suitable than whole-body BMD for evaluation of the bone mineral status of LBW in early infancy. Therefore, lumbar spinal BMD should be used for serial evaluation of changes in the bone mineral status of LBW infants.

Diagnosis and treatment of 6-pyruvoyl-tetrahydropterin synthase deficiency.

We detected a case of 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency during a neonatal mass screening and considered the differentiation and treatment of the peripheral form of PTPS deficiency. Although single treatment of BH4 had been started, because of the lowered biopterin (B) value, elevated neopterin (N) value, and N/B ratio in the cerebrospinal fluid (CSF), the peripheral form was judged negative and combined treatment with L-dopa and 5-hydroxy tryptophan (5-HTP) was started. Follow-up study will be necessary to confirm the diagnosis of PTPS deficiency.

Bone mineral density of the lumbar spine in very-low-birth-weight infants: a longitudinal study.

UNLABELLED: To examine osteopenia in very low birth weight (VLBW) infants we used repeated dual-energy X-ray absorptiometry in a prospective study of lumbar spinal bone mineral density (BMD) in Japanese VLBW infants (birthweight 426-1498 g; n = 61, group 1) aged 40 weeks postconception to 3 years of age. Control subjects were Japanese infants with birthweight 1500-1999 g (group 2), 2000-2499 g (group 3), or more than 2500 g (group 4). BMD in group 1 during the early period after birth was very low, increased rapidly for 1 year, and then gradually increased until 3 years of age (r = 0.931, P < 0. 0001). BMD at the age of 40 weeks postconception was 0.085 +/- 0.026, 0.132 +/- 0.039, 0.178 +/- 0.042, and 0.196 +/- 0.046 g/cm(2) in groups 1, 2, 3, and 4, respectively (P < 0.0001). However, at 1 and 2 years of age no differences were observed among the groups in BMD. CONCLUSION: This study shows that lumbar spinal BMD in VLBW infants can normalize by the age of 2 years.

Neopterin and biopterin concentrations in cerebrospinal fluid in controls less than 1 year old.

Neopterin and biopterin concentrations were measured in cerebrospinal fluid (CSF) and urine samples from controls less than 1 year old. This is the first time for CSF reference data for controls less than 1 year old to be reported. The ratio of neopterin to biopterin in CSF 0-30 days (n = 48) of age in control samples was 0.65 +/- 0.31 (SD), which was far lower than that in urine over the same time period, 4.0 +/- 1.9 (SD), (n = 51). This finding is very important when diagnosing 6-pyruvoyltetrahydropterin synthase (PTPS) deficiency and peripheral form of PTPS deficiency in the neonatal period. Our CSF reference data for controls should be useful in the diagnosis of PTPS deficiency.

Molecular characterization of 6-pyruvoyl-tetrahydropterin synthase deficiency in Japanese patients.

We identified three mutations in four Japanese patients with central type 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency. One missense mutation was a C-to-T transition, resulting in the substitution of Pro by Ser at codon 87 (P87S) in exon 5. Another missense mutation was a G-to-A transition, resulting in the substitution of Asp by Asn at codon 96 (D96N) in exon 5. A splicing mutation was found by skipping of exon 4 on PTPS mRNA analysis, and a G-to-A transition at the third base of codon 81 (E81E) and at the terminal base in exon 4 were detected on genomic PTPS DNA analysis. The E81E mutation affected the splice donor site of exon 4 and caused the splicing error. In COS cell expression analysis, the P87S and D96N mutant constructs revealed, respectively, 52% and 10% of wild-type activity. Patients with P87S/P87S (52%/52% in-vitro PTPS activity) exhibited 0.11 and 0 microU/g hemoglobin [Hb] in erythrocyte PTPS activity (wild-type control: 11-29 microU/gHb) erythrocyte PTPS activity, and the patient with P87S/D96N mutations (52%/10%) had 0.97 microU/gHb in PTPS erythrocyte activity. The PTPS erythrocyte activity did not coincide with the in-vitro PTPS activity based on patient genotype.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

Molecular analysis of glycogen storage disease type Ib: identification of a prevalent mutation among Japanese patients and assignment of a putative glucose-6-phosphate translocase gene to chromosome 11.

Glycogen storage disease type Ib (GSD-Ib) is an inborn error of metabolism with autosomal recessive inheritance, caused by defects in microsomal transport of glucose-6-phosphate. Recently, Gerin et al isolated a human cDNA encoding a putative transporter homologous to bacterial transporters of hexose-6-phosphate, and identified two mutations in its gene in two patients with GSD-Ib (9). Independently, a linkage analysis mapped the GSD-Ib gene on chromosome 11q23 (10). It remains to be elucidated whether the two genes are identical or GSD-Ib is genetically heterogeneous. We first mapped the transporter gene on chromosome 11 by using a DNA panel of human/hamster hybridoma cells. The result suggested that the GSD-Ib genes identified by the two distinct approaches may be identical and GSD-Ib was allelic. We then studied four unrelated Japanese families with GSD-Ib, and found three novel mutations: a four-base deletion/two-base insertion, a point mutation within a consensus splicing donor site, and a missense mutation (W118R). The W118R mutation was found in 4 out of 8 mutant alleles, suggesting that it is prevalent among Japanese patients.

Diagnosis of infections in newborns using a new particle-mediated immunoassay for serum C-reactive protein.

C-reactive protein (CRP) levels were measured using a new particle-mediated immunoassay. Tests for precision and linearity of this method gave satisfactory results. The minimum sensitivity of the assay was 1 ng/ml. Interference by bilirubin (<220mg/l) and haemoglobin (<20g/l) was not observed. Using this method, CRP was assayed as a means of monitoring for infection in newborns up to 72 h after delivery. The pattern of time course elevation curves was similar for both groups (10 healthy subjects and 26 patients), but the serum CRP (ng/ml) of infected newborns rose significantly higher than in healthy subjects at 24 h after birth. The rate of increase of CRP (CRP; ng/ml/h) may be a more useful parameter to detect infection, since a significant change in CRP was apparent only 12 h after birth. The reported method was reliable and the parameters obtained were considered clinically useful for early detection of infection.

Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT): development of an antibody to human SCOT and diagnostic use in hereditary SCOT deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization. Hereditary SCOT deficiency in humans (McKusick catalogue number 245050) is characterized by intermittent ketoacidotic attacks and permanent hyperketonemia. Since previously-available antibody to rat SCOT did not crossreact with human SCOT, we developed an antibody against recombinant human SCOT expressed in a bacterial system. The recombinant SCOT was insoluble except under denaturing conditions. Antibody raised to this polypeptide recognized denatured SCOT and proved useful for immunoblot analysis. On immunoblots, SCOT was easily detectable in control fibroblasts and lymphocytes but was detected neither in fibroblast extracts from four SCOT-deficient patients, nor in lymphocytes from two SCOT-deficient patients. These data indicate that immunoblot analysis is useful for diagnosis of SCOT deficiency in combination with enzyme assay.

Regulation of obese gene expression in KK mice and congenic lethal yellow obese KKAy mice.

To elucidate the regulation of obese (ob) gene expression in obesity and diabetes, we examined ob gene expression in KK mice and congenic lethal yellow obese KKAy mice. Northern blot analysis revealed that the ob mRNA levels are roughly equivalent in each of the epididymal, mesenteric, and subcutaneous white adipose tissue (WAT) from KK and KKAy mice at 4 wk of age, when the obese phenotype of KKAy mice was not apparent. Expression of the ob gene was augmented in the mesenteric and subcutaneous WAT but was unchanged in the epididymal WAT in KKAy mice at 12 wk of age, when KKAy mice developed marked obesity with hyperglycemia, hyperlipidemia, and hyperinsulinemia. The ob gene expression was also examined during fasting in 12-wk-old KK and KKAy mice. After 24 or 72 h of fasting in both mouse strains, ob gene expression was downregulated in the epididymal and mesenteric WAT but was unchanged in the subcutaneous WAT. The present study demonstrates that adipose tissue expression of the ob gene is regulated depending on the nutritional status in KK and KKAy mice.

Prenatal diagnosis of succinyl-coenzyme A:3-ketoacid coenzyme A transferase deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare disorder of ketone body catabolism. In the present study, we prenatally diagnosed SCOT deficiency in a fetus in a family of which the proband was the first patient with SCOT deficiency identified in Japan, by analysis of enzyme activity levels in samples of chorionic villi and cultured amniocytes. In the fetus of the family, SCOT activity was not detected in either chorionic villi or cultured amniocytes. Since the levels of SCOT activity in control chorionic villi were close to our minimal detectable level and were much lower than those in control cultured amniocytes, enzyme assay in cultured amniocytes was more feasible than that in chorionic villi for prenatal diagnosis of SCOT deficiency. No elevated accumulation of 3-hydroxybutyrate or acetoacetate was detected in the amniotic fluid of the fetus. To our knowledge, this report is the first of prenatal diagnosis of SCOT deficiency.

[A sensitivity of CRP determination of cord blood and neonatal serum for neonatal infection].

CRP was determined for 110 cord bloods and peripheral blood of 36 newborns collected within 72 hours after delivery for the early diagnosis of newborn infection. The determination of CRP was done by a counting immunoassay method using PAMIA-30(Sysmex, Kobe, Japan). Sample volume needed was small and the time for determination was short. Within-run and between-run precisions were satisfactory, with CV values being approximately 6%. The CRP of healthy newborns was lower than that of cord blood, and the mean value was 33.4 +/- 4.2 ng/ml and the value was not significantly different from that obtained from the newborn babies with turbid amnionic fluid or early rupture of a sac. The CRP gradually increased after delivery had a peak at 24 to 48 hours after delivery. This tendency was observed both in healthy and infected newborns. The data were divided into 6 groups depending on the time collected after delivery (6, 12, 24, 48, and 72 hours). The CRP of blood from infected newborns tended to have higher CRP than that of healthy newborns in each group. Increased amount of CRP (ng/ml/hrs) was calculated as ((CRP of peripheral blood at time x)--(CRP of cord blood))/x, and this value was significantly higher (p < 0.05) in infected newborns than in healthy newborns 12hrs and more after delivery. Thus, CRP might be useful for monitoring the newborn infection.