Cloning and expression of a pig liver taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21): a novel member of the CYP4A subfamily.

A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6alpha-hydroxylase. This enzyme catalyzes a 6alpha-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6alpha-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6alpha-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.

Plasma metabolites after a lipid load in infants with congenital heart disease.

Growth retardation is common in infants with congenital heart defects. The aim of this study was to investigate whether growth retardation or type of heart defect in infants with congenital heart defects is related to disturbances in lipid metabolism. Sixteen infants with ventricular septal defects and six infants with transposition of the great arteries were given an intravenous load of lipid emulsion (Intralipid 20 mg/ml) corresponding to 0.5 g fat/kg body weight for 5 min after fasting for 8 h. Blood samples were drawn immediately before the infusion and 3, 20, 60, 120 and 240 min after the infusion was completed. Plasma concentrations of triglycerides (TG), free fatty acids (FFA), ketones, lactate, pyruvate, alanine, glycerol and glucose were determined. The fatty acid patterns in the TG and FFA fractions were measured using gas chromatography. Severe growth retardation in infants with defects of these kinds was correlated to higher fasting and maximum levels of linoleic acid in plasma FFA. The maximum levels of linoleic acid in the TG fraction were positively correlated to weight SD score, and maximum glycerol levels were higher in the most growth-retarded infants, indicating faster intravascular lipolysis. Linoleic acid in the TG fraction was still elevated at 120 and 240 min after the lipid load. Some differences between the cyanotic and VSD groups could be noted. These indicate decreased metabolic capacity to utilize released FFA in the cyanotic group. Infants with cyanotic heart defects also had higher lactate and alanine levels compared to infants with VSD. Our results support the hypothesis that lipid metabolism is disturbed in infants with congenital heart defects.

Glucose metabolism and insulin secretion in children with cyanotic congenital heart disease.

The aim of the study was to reveal differences in carbohydrate metabolism in children with cyanotic congenital heart diseases (CHD). Thirteen children with diseases of these kinds were investigated with regard to glucose tolerance and insulin secretion and comparisons were made with healthy controls of the same age. Investigations included an intravenous glucose tolerance test, insulin response to the glucose load in plasma and insulin secretion rate. The results reveal lower fasting glucose levels and signs of a higher insulin secretion rate in the relatively few patients in the CHD group where C-peptide measurements were performed, but no differences in glucose tolerance. The reasons for the differences are unclear, but the chronic increases in circulating catecholamines in combination with the impaired nutritional status of these children with CHD are probably the most important factors. We conclude that these divergences in carbohydrate metabolism should be emphasized in the care of children with CHD.

Skeletal muscle energy substrates, metabolic products and enzyme activity in infants with symptomatic ventricular septal defect.

Muscle biopsy studies were performed on 26 infants with symptomatic ventricular septal defect (VSD) (mean age 4.7 months) and 10 healthy infants (mean age 7.8 months). Analyses were made of muscle energy substrates, metabolic products, muscle enzyme activity, fibre types and fibre sizes. Relatively few differences were noted between the groups. The most important difference was a reduced ATP level in the VSD group. Glucose 6-phosphate concentrations were also lower in the VSD group. These differences could indicate a low metabolic activity in skeletal muscle in infants with heart failure. Most muscle enzyme activity was comparable with the exception of lactate dehydrogenase (LD), which was lower in the VSD group. Within the VSD group, no differences were revealed in muscle substrate concentrations for muscle enzyme activity in terms of the degree of heart failure. We conclude that low energy levels are probably explained by undernourishment and/or reduced blood flow to skeletal muscle and that the lack of other discrepancies in muscle metabolism indicates a desirable relatively normal motor activity in these infants with symptomatic VSD.

Glucose metabolism and insulin secretion in infants with symptomatic ventricular septal defect.

Nineteen infants with symptomatic ventricular septal defect (VSD) were examined on, altogether, 26 occasions, when each was given an intravenous glucose tolerance test (IVGTT); concentrations of insulin- and C-peptide in plasma were determined. Comparisons were made with 14 healthy infants of the same age. The VSD infants were growth retarded with lower weight/age and length/age ratios. Glucose tolerance as indicated by glucose fasting levels and response to intravenous glucose tolerance test, including glucose disappearance rate, did not differ between the two groups. In response to the glucose load, insulin in plasma was significantly less in VSD infants. In response to the IVGTT, insulin secretion rate calculated from C-peptide levels in plasma was significantly elevated in the VSD group. We conclude that compared to healthy infants, those with symptomatic VSD have normal glucose tolerance, increased secretion rate of insulin, but decreased levels of circulating insulin in response to an intravenous glucose load. We suggest this is so because binding of insulin to peripheral receptors and/or insulin extraction in the liver somehow increases.

Cerebrospinal fluid concentrations of neurotensin and corticotropin-releasing factor in pediatric patients.

Cerebrospinal fluid (CSF) concentrations of neurotensin (NT) and corticotropin-releasing hormone (CRF)-like immunoreactive materials (LIM) were measured in 22 infants and children 6 days to 15 years of age. For both neuropeptides there was a marked age-related exponential decline in CSF concentrations. The most prominent decrease in CSF neuropeptide concentrations was seen during the first 24 months of postnatal life. From 1 year and on there was no or only minimal age-associated alteration in CSF neuropeptide concentrations. In this group of children (1-15 years) mean CSF concentrations of NT-LIM and CRF-LIM were 36.8 +/- 4.32 and 65.9 +/- 4.63 pg/ml, respectively. As CSF neuropeptide concentrations are apparently independent of circulating serum concentrations, they may reflect functional activity of neuropeptide-containing neurons and therefore may be of value in the assessment of the role of peptides in the human central nervous system function and behavior.

Age-dependent effects of beta 2-adrenergic-stimulating drugs on fetal rabbit lung liquid and adrenal catecholamines.

The effects of the beta 2-adrenergic-stimulating drug, terbutaline, were studied on fetal rabbit lung liquid (FLL) at gestational ages between 25 and 30 days. At delivery terbutaline reduced FLL in rabbit fetuses with a gestational age of 26-30 days. The most pronounced reduction of FLL was seen at 28 days. Terbutaline administration reduced the wet lung weight/body weight (WLW/BW) ratio at delivery in the 28- and 30-day-old rabbit pups. In the 26-day-old animals, beta 2-adrenergic stimulation had no significant effect on the WLW/BW ratio and at 25 days of gestational age the ratio was increased. After parturition, however, the difference in WLW/BW between the terbutaline and control animals, seen at birth, was attenuated and, at 60 min of postnatal age, the difference was no longer observed. Terbutaline decreased the adrenal content of noradrenaline and adrenaline most marked at 28 days of gestational age, but did not alter the adrenaline/noradrenaline ratio. Furthermore, adrenal dopamine was decreased after terbutaline, suggesting a decreased catecholamine synthesis. Our data show that a positive effect of terbutaline on FLL in rabbit pups was most marked during late but not early gestational age. beta 2-Adrenergic treatment may, however, reduce adrenal catecholamines.

Changes in cerebrospinal fluid homovanillic acid in children with Ondine's curse.

The cerebrospinal fluid (CSF) concentrations of three acid monoamine metabolites, two purines, and a group of amino acids were determined in two children with chronic central alveolar hypoventilation (Ondine's curse). The levels of all assayed neuroactive substances, metabolites, and amino acids, with one exception, were normal compared to an age-matched group of neurologically healthy children. The levels of the dopamine metabolite homovanillic acid in the children with Ondine's curse were approximately 2.4 times higher than expected for age range. The present findings may indicate a link between central nervous system dopamine activity and chronic central alveolar hypoventilation. Among other possible explanations, the changes seen might represent a primary alteration in dopamine activity or may reflect a change in dopamine turnover resulting from the chronic hypoventilation.

Developmental variations in CSF monoamine metabolites during childhood.

Cerebrospinal (CSF) fluid content of the stable metabolites homovanillic acid, 3-methoxy-4-hydroxyphenyl glycol and 5-hydroxyindoleacetic acid were measured in 19 children without neurological disease known to alter CSF monoamine metabolites. The CSF levels of all three metabolites were found to be up 6 times higher in early infancy compared to the values during adolescence. The levels decreased in a logarithmic fashion, and adult values (approx. 25-50 ng/ml) were reached at 3-5 years of age. Two different interpretations of the findings are discussed: (1) a higher release or turnover of central serotonin and catecholamine metabolites during early postnatal age, or (2) lower clearance of the stable acid metabolites from CSF during infancy due to relatively immature active transport mechanisms.

Regulation of hydroxylations in biosynthesis of bile acids: modulation of reconstituted 12 alpha-hydroxylase activity by protein fractions from rabbit liver cytosol and microsomes.

The activity of purified 12 alpha-hydroxylase from rabbit liver microsomes was modulated by including protein fractions from rabbit liver microsomes and cytosol into the system. The microsomal protein fraction stimulated 12 alpha-hydroxylation two times. The cytosolic fraction inhibited the reaction markedly. The microsomal 12 alpha-hydroxylase stimulatory activity was labile and the cytosolic 12 alpha-hydroxylase inhibitory activity was stable to mild heat treatment. Addition of ATP and MgCl2 or NaF had no effect on the activities of the two protein fractions. The activity of the microsomal stimulatory fraction decreased upon storage but could be reactivated by addition of reduced glutathione to the system.