Takahiro Inokuchi

Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic-mass spectrometric (GC-MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC-MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16,246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and alpha-aminoadipic aciduria/alpha-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16,246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.

Neonatal Dubin‐Johnson Syndrome with Severe Cholestasis: Effective Phenobarbital Therapy

ABSTRACT. We described Dubin‐Johnson syndrome (DJS) with severe cholestasis in a 20‐day‐old Japanese boy. Although neonatal DJS has been sporadically reported, DJS with severe cholestasis has not to our knowledge been described in the English literature. The ratio of urinary coproporphyrin isomer I to urinary total coproporphyrin in our patient was high (93%). Liver histology showed cytoplasmic pigment granules in the liver cells. Administration of phenobarbital (PB) significantly decreased the levels of bilirubin and bile acids in the serum. There was a significant elevation of 1β‐hydroxylated bile acids in the urine. It is predicted that severe cholestasis in neonatal DJS may cause metabolic abnormalities in both bilirubin and bile acids transport.

Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency

AbstractIsolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency appears to be the most frequent organic aciduria detected in tandem mass spectrometry (MS/MS) screening programs in the United States, Australia, and Europe. A pilot study of newborn screening using MS/MS has recently been commenced in Japan. Our group detected two asymptomatic MCC deficiency patients by the pilot screening and collected data on another three MCC deficiency patients to study the molecular bases of the MCC deficiency in Japan. Molecular analyses revealed novel mutations in one of the causative genes, MCCA or MCCB, in all five of the patients: nonsense and frameshift mutations in MCCA (c.1750C > T/c.901_902delAA) in patient 1, nonsense and frameshift mutations in MCCB (c.1054_1055delGG/c.592C > T) in patient 2, frameshift and missense mutations in MCCB (c.1625_1626insGG/c.653_654CA > TT) in patient 3, a homozygous missense mutation in MCCA (c.1380T > G/ 1380T > G) in patient 4, and compound heterozygous missense mutations in MCCB (c.569A > G/ c.838G > T) in patient 5. No obvious clinical symptoms were observed in patients 1, 2, and 3. Patient 4 had severe neurological impairment and patient 5 developed Reye-like syndrome. The increasing use of MS/MS newborn screening in Japan will further clarify the clinical and genetic heterogeneity among patients with MCC deficiency in the Japanese population.

Perinatal bile acid metabolism: bile acid analysis of meconium of preterm and full-term infants.

BackgroundOur purpose was to evaluate the metabolism of bile acids in the fetus by analyzing the bile acid composition of meconium of preterm (less than 30 weeks’ gestational age) and full-term infants and comparing the results with the bile acid composition of feces of preterm and full-term infants 6 days after delivery.MethodsThe concentrations of individual bile acids were determined by gas chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates.ResultsIn meconium, the main bile acids were chenodeoxycholic and hyocholic acids. The main bile acid of feces from preterm infants at 6 days of age was the same as that of meconium. We also detected large amounts of secondary bile acids, especially deoxycholic acid and ursodeoxycholic acid. The ratio of cholic acid relative to chenodeoxycholic acid in meconium of preterm and full-term infants and in feces of preterm infants was less than 1, 0.36, 0.55, and 0.55, respectively. The percentage of chenodeoxycholic acid relative to total bile acids in meconium of preterm (P < 0.05) and full-term (P < 0.01) infants was significantly higher than that in feces of 6-day-old full-term infants.ConclusionsMore than half of the main pathway, at least, for bile acid synthesis in preterm infants may be the acidic pathway until the infants reach about 7 days of age.

Effects of diethyl maleate (DEM), a glutathione depletor, on prostaglandin synthesis in the isolated perfused spleen of rabbits.

To investigate the role of glutathione (GSH) on prostaglandin (PG) synthesis, isolated rabbit spleens were perfused with Tyrodes solution with or without the addition of diethyl maleate (DEM) in concentrations up to 1 mM. In the absence of DEM, PG synthesis was stimulated by the Ca2+ ionophore A23187 (20 nmole) or arachidonate (0.4 μmole). Prostaglandin (PG) E2 was a major product, accounting for 60–70% of the total cyclooxygenase products. Small amounts of PGF2α, 6-keto-PGF1α, PGD2 and thromboxane (Tx) B2 were also produced. When DEM was added to the perfusion medium, GSH content decreased dose-dependently with increasing DEM concentration. Lactate dehydrogenase activity was not detected in the venous effluent, indicating that DEM depleted intrasplenic GSH without causing any lysis of cellular membranes. A23187-induced production of PGs and of Tx was decreased with increasing concentrations of DEM up to 0.5 mM, whereas at 1.0 mM DEM, these products showed a tendency to increase as compared with levels at 0.5 mM DEM. However, this increase was only significant for TxB2, which returned to levels obtained in the absence of DEM. DEM 1 mM did not cause cell lysis, but it appears to perturb the cell membrane to a degree similar to that which occurs with stimulation of phospholipase A2. The small but significant increase of TxB2 with 1.0 mM DEM could be a result of decreased PGE2 isomerase activity. Perfusion with arachidonate gave virtually identical results: 1.0 mM DEM attenuated the production of all prostanoids except for TxB2 as compared with untreated controls. These results suggest that GSH contributes to the regulation and/or maintenance of PGs synthesis.

Profile of urinary bile acids in familial intrahepatic cholestasis with Coombs’negative haemolytic anaemia

We present two male siblings with intrahepatic cholestasis and prolonged indirect hyperbilirubinaemia. Their familial intrahepatic cholestasis syndrome was characterized by Coombs’negative haemolytic anaemia, without giant cell transformation of hepatocytes and high concentrations of serum γ‐glutamyl transpeptidase and cholesterol. By gas chromatography‐mass spectrometry, we detected large amounts of 1β‐hydroxylated bile acids, especially lβ,3α,7α,12α‐tetrahydroxy‐5β‐cholan‐24‐oic acid (25.5‐67.9% of total urine bile acids) in the urine during phenobarbital therapy. However, the amount of urinary 1β‐hydroxylated bile acids gradually decreased as the disease progressed. At the end‐stage, we detected large amounts of 7α,12α‐dihydroxy‐3‐oxochol‐4‐en‐24‐oic acid (19.6% of total urine bile acids). The ratio of 7α,12α‐dihydroxy‐3‐oxochol‐4‐en‐24‐oic acid to cholic acid in the urine was 0.8. We conclude that in infants with end‐stage liver failure, the microsomal hydroxylation of bile acids is impaired and the excretion of Δ4‐3‐oxo bile acids is increased.Familial intrahepatic cholestasis, Coombs’negative haemolytic anaemia, 1β‐hydroxylated bile acids, unsaturated ketonic bile acids

Stimulation of prostaglandin synthesis by cholecystokinin in primary culture cells of bovine gallbladder muscle

Primary culture cells derived from bovine gallbladder muscles synthesize PGE2 as a major cyclooxygenase product with a trace amount of 6-keto-PGF1 alpha-like material. The synthesis of PGE2 and total cyclooxygenase products was enhanced in response to cholecystokinin (CCK). In the presence of indomethacin the synthesis of PG was inhibited and the release of arachidonic acid (AA) in response to CCK was enhanced. These data suggest that CCK may stimulate the release of AA, probably by activating phospholipase A2/C, from membrane phospholipids in the gallbladder muscle.

Developmental pattern of urinary bile acid profile in preterm infants

Background:  Bile acid metabolism in preterm infants is yet to be fully characterized. We compared the developmental pattern of urinary bile acid profiles in ten infants born at gestational ages from 25 to 33 weeks with previous data from full‐term infants from birth to about 7 months of age.

Generation of leukotrienes and hydroxyeicosatetraenoic acids in peritoneal macrophages of tumor-bearing mice.

To examine the potential role of lipoxygenase products in the pathophysiology observed after experimental tumor implantation, we examined the generation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in peritoneal macrophages. C57BL/6 mice were given subcutaneous inoculations of B16 melanoma cells, and peritoneal macrophages were isolated various days after the inoculation. Macrophages were incubated for 1 h at 37 degrees C in serum-free RPM11640 containing 10 microM calcium ionophore A23187, 10 microM exogenous arachidonic acid (AA), 5 mM cysteine hydrochloride and 1 mM reduced glutathione. LTs and HETEs were separately extracted, passed through Sep-Pak cartridges, then identified and quantitated with a HPLC system using UV absorbance. The B16 melanoma-cell-treated/untreated macrophages were found to produce substantial amounts of 15-HETE, 12-HETE and 5-HETE and LTC4 by enzymatic mechanisms. Thus, when determined under various conditions, the production of HETEs was dependent on substrate-concentration, incubation-time and cell-number. The production of LTC4 was dependent on incubation-time and cell number but not substrate-concentration, indicating utilization of endogenous AA stores. Of these products, 12-HETE and LTC4 showed a significant increase on the fourth day after the tumor cell inoculation and returned to the control level by the 11th day after the same treatment. These results suggest that in vivo tumor cell implantation may induce a transient increase of 12-HETE and LTC4 production in macrophages.

Prevention of 4-pentenoic acid-induced liver injury in rats by 16,16-dimethyl PGE2

16,16-Dimethyl PGE2 (dmPGE2) is known to protect against cellular damage in various tissues. Histological and biochemical approaches were used to examine the effect of this prostaglandin on hepatocellular damage in an experimental Reyes syndrome model produced in rats by 4-pentenoic acid. Chronic intraperitoneal administration of 4-pentenoic acid induced an accumulation of fatty droplets throughout the hepatic lobules along with mitochondrial abnormalities including swelling, disappearance of christae, and heterogeneity of matrix. These abnormalities were more intense in the marginal zone and successively decreased nearer to the central vein. Such hepatic abnormalities were markedly reduced by the combined administration of dmPGE2 with 4-pentenoic acid. Biochemical examination confirmed that dmPGE2 was able to inhibit the accumulation of hepatic triglyceride seen after the treatment with 4-pentenoic acid alone. These results indicated that dmPGE2 can prevent characteristic hepatocellular damage in this experimental Reyes syndrome model, suggesting that the involvement of prostaglandins should be taken into account in discussing the etiology and management of this syndrome.