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Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I). In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had AI-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity. Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP IA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP IA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera. Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.

Patients with autoimmune polyendocrine syndrome type I (APS I) have autoantibodies against the enzyme aromatic L-amino acid decarboxylase (AADC) of pancreatic beta-cells. The aim of the present study was to investigate the presence of anti-AADC antibodies in a large cohort of patients with APS I, and in patients with isolated insulin-dependent diabetes mellitus (IDDM). We found autoantibodies against AADC in 35 of 69 patients (51%) with APS I but in none of 138 patients with isolated IDDM or 91 healthy controls. Among the patients with APS I, anti-AADC antibodies were more often found in those with hepatitis (11/12, 92%), than in those without hepatitis (24/57, 42%) (P = 0.003). Similarly, of 15 patients with vitiligo, 12 (80%) had anti-AADC antibodies, compared with 23/54 (43%) without vitiligo (P = 0.021). Of the 9 APS I patients with IDDM, 5 had antibodies against both AADC and glutamate decarboxylase, 2 against AADC only, and 2 against glutamate decarboxylase only. Interestingly, AADC is present in relatively large amounts in the liver, where its function is unknown. Thus, an autoimmune reactivity against AADC may be involved in the pathogenesis of autoimmune chronic active hepatitis and vitiligo in APS I patients, whereas the role of AADC in the development of IDDM in these patients remains to be determined.

The newborn infant must mobilize endogenous substrate stores to meet the requirements of glucose-dependent organs. High concentrations of free fatty acids and glycerol, and a rapid decrease in the respiratory quotient, indicate that lipids are an important fuel soon after birth. The purpose of the present study was to determine the onset of lipolysis and gluconeogenesis from glycerol in healthy, term, unfed infants. Eight infants were studied from a postnatal age of 3.5 +/- 0.5 h to 7.4 +/- 0.2 h using [6,6-2H2] glucose and [2-13C]glycerol analysed by gas chromatography/mass spectrometry. Plasma concentrations of glucose, glycerol and insulin averaged 2.9 +/- 0.4 mM, 369 +/- 89 microM and 9.4 +/- 9.4 +/- 3.7 microU.ml-1, respectively. The hepatic glucose production rate averaged 25.0 +/- 3.5 mumol.kg-1 min-1 (4.5 +/- 0.6 mg.kg-1.min-1) and the endogenous plasma appearance rate of glycerol 8.7 +/- 1.2 mumol.kg-1.min. On average, 57.9 +/- 8.4% of the glycerol was converted to glucose, representing 11.1 +/- 2.3% of hepatic glucose output. Thus, lipolysis and gluconeogenesis from glycerol are established within the first 8 h of life in term infants.

Extremely preterm infants have been shown capable of producing glucose at a rate comparable to that of term infants, but virtually no data are available on their capacity for lipolysis and gluconeogenesis. To address this issue, we studied the flux of glycerol and its gluconeogenic contribution to hepatic glucose output by determining the endogenous plasma appearance rate of glycerol (glycerol Ra) and its conversion to glucose in 10 newborn infants, 24-27 wk of gestational age. The study was performed during the 1st d of life by tracer dilution technique using [6,6-2H2]glucose and [2-13C]glycerol given as constant rate i.v. infusions. Plasma isotopic enrichments of the tracers were obtained by gas chromatography/mass spectrometry. Endogenous glycerol Ra ranged from 2.4 to 21.6 (median 5.0) mumol.kg-1.min-1, of which 31.5% (25.6-64.4%) was converted to glucose. The glucose production rate averaged 17.5 +/- 5.4 mumol.kg-1.min-1 (3.2 +/- 1.0 mg.kg-1.min-1), of which 5.0% (1.6-37.6%) was derived from glycerol. The results show that extremely preterm infants, despite limited fat stores, are capable of generating glycerol at a rate within the range reported for term and near term newborns. The infants were also capable of converting part of this glycerol to glucose, providing a contribution to hepatic glucose production comparable to that found in more mature newborns.

Growth retardation and gonadal insufficiency are well-known features of Down syndrome. In this longitudinal study, 44 home-reared children and adolescents with Down syndrome, aged 10-24 years, living in the county of Uppsala, were followed yearly. The male patients had a mean final height above that reported previously, and a close correlation between target and final heights was found. The mean final height in the female patients was below that reported earlier. Mean peak height velocities in males and females were 8.5 and 7.3 cm year-1, respectively, significantly lower than in healthy children. The mean ages at peak height velocity were 12.3 and 10.8 years, respectively, indicating early growth spurts. The serum follicle-stimulating hormone concentrations, the small testes and the negative correlation between luteinizing hormone and testicular volume in the males may indicate some primary gonadal insufficiency. For the girls, mean menarcheal age corresponded closely to that of their mothers.

An increased risk of developing precocious puberty has been reported in children with myelomeningocele. In order to evaluate this further and to study factors associated with early or precocious puberty the medical records of all girls with myelomeningocele, born from 1970 onwards (n = 64), who were admitted to our unit were reviewed. Early/precocious puberty (E/PP) was defined as breast development or pubic hair corresponding to Tanner stage 2 before the age of 9.2 years. In 32 out of 62 cases data were sufficient for evaluation of the timing of puberty. Twenty girls had E/PP and 12 girls normal timing of puberty. In the girls who had reached the age of 9.2 years the incidence of E/PP was at least 52%. Girls with E/PP had a higher incidence of hydrocephalus, were treated with intraventricular shunts more often, and had significantly higher frequency of increased intracranial pressure during the perinatal period (p < 0.05, p < 0.01, and p < 0.001, respectively). The group of girls developing E/PP was also more severely disabled with respect to motor and urological function and had more shunt revisions. In conclusion, E/PP in girls with myelomeningocele is strongly associated with increased intracranial pressure particularly during the perinatal period.

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high-performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivatization, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.

Uteroglobins, i.e. proteins with similar three-dimensional structure and ligand binding specificity to uteroglobin from rabbit uterus, have been found in rat, mouse and human lung. We have recently demonstrated the binding of calcium by human uteroglobin, and we have therefore tried to find potential binding sites for metals in the three-dimensional structure of uteroglobin by the use of two different computational procedures. A putative binding site for calcium in uteroglobin was identified by means of a hydrophobic contrast function. The spatial disposition of atoms that could ligand calcium in the putative calcium-binding site appears similar to that of the primary calcium-binding site of secretory phospholipase A2 enzymes, consisting of the carboxyl group of an aspartic acid residue and a loop providing three backbone carbonyl oxygens. From inspection of their primary sequences and three-dimensional structures, it became clear that this putative calcium-binding motif is conserved among uteroglobins from different species. The potential significance of the predicted site was investigated by site-directed point mutagenesis of human uteroglobin in which Asp46 was replaced by Asn or Lys. In both mutants, the ruthenium red and 45Ca2+ binding was significantly reduced. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis under non-reducing conditions indicated that the mutant proteins had the expected molecular masses and that their ability to dimerize was not disturbed by these mutations. Valence calculations also identified the putative calcium-binding site, but only after optimization of its conformation by the use of molecular dynamics with a restrained calcium ion. Our results support the notion that Asp46 of uteroglobins acts as a "cap" residue in a calcium-binding site structurally similar to the primary calcium binding sites of phospholipases A2.

The spontaneous growth process in Turner's syndrome is characterized by a progressive decline in height velocity during childhood and no pubertal growth spurt. Therefore, therapy aimed at improving height during childhood as well as increasing final height is desirable for most girls with Turner's syndrome. Forty-five girls with Turner's syndrome, 9-16 yr of age (mean age, 12.2 yr), were allocated to three study groups. Group 1 (n = 13) was initially treated with oxandrolone alone; after 1 yr of treatment, GH without (group 1a; n = 6) or with (group 1b; n = 7) ethinyl estradiol was added. Group 2 (n = 17) was treated with GH plus oxandrolone. Group 3 (n = 15) was treated with GH, oxandrolone, and ethinyl estradiol. The dosage were: GH, 0.1 IU/kg.day; oxandrolone, 0.05 mg/kg.day; and ethinyl estradiol, 100 ng/kg.day. A height of 150 cm or more was achieved in 61%, 75%, and 60% of the girls in groups 1, 2, and 3, respectively. The most impressive increase in height was seen in group 2. In this group the mean final height was 154.2 cm (SD = 6.6), which is equivalent to a mean net gain of 8.5 cm (SD = 4.6) over the projected final height. In group 3, in which ethinyl estradiol was included from the start of therapy, the initially good height velocity decelerated after 1-2 yr of treatment. Their mean final height was 151.1 (SD = 4.6) cm, equivalent to a mean net gain of 3.0 cm (SD = 3.8). A similar growth-decelerating effect of ethinyl estradiol was seen in group 1b. We conclude that in girls with Turner's syndrome who are older than 9 yr of age, treatment with GH in combination with oxandrolone results in significant growth acceleration, imitating that in normal puberty, leading to a more favorable height during childhood. This mode of treatment also results in a significantly increased final height, permitting a great number of the girls to attain a final height of more than 150 cm. However, early addition of estrogen decelerates the height velocity and reduces the gain in height.

Ten healthy males inhaled monodisperse Teflon particles (geometric diameter 3.6 microns, aerodynamic diameter 5.3 microns) labeled with 195Au (half-life 183 days). The leakage of 195Au from the particles in vitro in water was less than 0.2% per year. Retention over the thorax was followed for about 900 days using two separate detector systems. One system consisted of four Ge detectors placed close to the front of the chest over the upper and lower regions of the lungs. The other system consisted of three NaI crystals placed in a ring around the thorax at some distance from the chest wall. Activities of 195Au in feces (24- or 48-h samples) could be measured as long as activities in the thorax could be measured. For the period 7-250 days, the half-times were similar for the two detectors, on the average 740 days for the NaI detectors and 680 days for the Ge detectors. The average half-times estimated from measurements from about 250 days to about 900 days were 1750 days with the NaI detectors and 880 days with the Ge detectors. Clearance curves constructed from measurements from feces agreed very well with clearance measured with the NaI detectors. The excretion via feces was well described by a power function with days after exposure as base. This total clearance from the thoracic region was slower than in earlier studies. No activity could be measured in the urine. The measurements with the two detector systems show that a translocation within the thoracic region occurred. This might be explained by transportation of particles from the lung parenchyma to the regional lymph nodes. The accumulation of particles in the regional lymph nodes was tentatively calculated on the basis of that assumption.

The association structures formed by cationic liposomes and DNA-plasmids have been successfully employed as gene carriers in transfection assays. In the present study such complexes was studied by cryo-TEM (cryo-transmission electron microscopy). Cationic liposomes made up by DOPE (dioleoylphosphatidylethanolamine) and various amounts of three different cationic surfactants were investigated. The cryo-TEM analysis suggests that an excess of lipid in terms of charge, leads to entrapment of the DNA molecules between the lamellas in clusters of aggregated multilamellar structures. With increasing amounts of DNA free or loosely bound plasmids were found in the vicinity of the complexes. The importance of the choice of surfactant, as reported from many transfection assays, was not reflected in changes of the type of DNA-vesicle association. A tendency towards polymorphism of the lipid mixtures is reported and its possible implications are discussed.

Autoimmune adrenal insufficiency is associated with premature ovarian failure at frequencies of 10-20%. In these patients a reactivity against an unknown steroid cell antigen in both the adrenal glands and gonads has been described. We have recently identified the cytochrome P450 enzyme 21-hydroxylase and the side-chain cleavage enzyme (SCC) as the major adrenal autoantigens in Addison's disease and the rare autoimmune polyendocrine syndrome type I (APS-I), respectively. In an attempt to identify the steroid cell antigen, sera from patients with Addison's disease (n = 13) and APS-I (n = 7) that reacted with Leydig cells were selected. Preparations of isolated human granulosa cells, isolated rat Leydig cells, and fractions of human placenta were used in Western blots. All sera were also tested against bacterially expressed 21-hydroxylase, SCC, and 17 alpha-hydroxylase. The SCC was recognized by sera from patients with Addison's disease and those with APS-I. In addition, a majority of the sera (n = 12) reacted with a novel 51-kilodalton autoantigen present in granulosa cells and placenta. The results show that the steroid cell autoantigen consists mainly of the SCC and a novel protein of unknown function.

The observation that thyroid disease is frequent in mothers of children with Down syndrome (DS) has suggested that maternal thyroid antibodies could be a factor predisposing to trisomy 21 in their offspring. In this study, the incidences of thyroglobulin (Tg) and thyroid peroxidase (TPO) antibodies were analysed with a sensitive solid-phase immunosorbent radioassay in sera from 29 mothers giving birth to children with trisomy 21 and 87 control mothers. The serum samples were collected at delivery. There was no statistical difference regarding the proportion of thyroid antibodies (against Tg and/or TPO) in the two groups. Thyroid antibodies were detected in 6/29 (20.7 per cent) of the DS mothers and in 23/87 (26.4 per cent) of the control mothers. Among the women with thyroid antibodies, 4/6 (66.7 per cent) of the DS mothers and 12/23 (52 per cent) of the control mothers had antibodies against both Tg and TPO. There was no increase in the relative risk of having a child with DS if the titre of either Tg or TPO antibodies or both were positive, i.e. > or = 1/5. The results indicate that the presence of thyroid antibodies in the serum of a pregnant women has no prognostic value for the birth of an infant with DS.

The prevalence of thyroid autoantibodies, i.e. thyroglobulin antibodies and antibodies to thyroid peroxidase, was analyzed in 89 girls, aged 3-16 years (mean age 10 years), with Turner's syndrome. The analyses were performed before the start of growth-promoting treatment and during a follow-up period of 1-5 years. The patients were divided into four groups according to karyotype as follows: group 1, 45, X (n = 63); group 2 with structural abnormalities of the X chromosome (n = 10); group 3 with mosaicism but no structural abnormalities of the X chromosome (n = 10); and group 4, with isochromosome X of the long arm (n = 12): 199 healthy girls aged 12 years, served as controls. Thyroid autoantibodies were demonstrated in 46 of 89 (52%) patients with Turner's syndrome compared with 34 of 199 (17%) age-matched control girls (p < 0.001), thus confirming the relationship between thyroid abnormalities and Turner's syndrome. There was also an increase in the prevalence of thyroid antibodies with age. Simultaneous presence of both autoantibodies was significantly more frequent in group 1 (45, X) and group 4 (isochromosome X of the long arm) than in group 3 (mosaicism) (p = 0.04 and p < 0.002, respectively) and significantly more frequent in group 4 than in group 1 (p < 0.05). During 12-60 months of growth-promoting treatment, no increase in the prevalence of thyroid antibodies was observed. The findings demonstrate the importance of continuous monitoring of thyroid function in girls with Turner's syndrome.

Small substrate stores and immature enzyme systems make preterm infants prone to develop hypoglycemia. Hyperglycemia may also occur, particularly when these infants are given i.v. glucose. To evaluate the capacity for regulation of glucose production in response to glucose infusion, 10 newborn infants, born after 25-30 wk of gestation, were studied. Their glucose production rates (GPR) were calculated and the concentrations of glucose, insulin, and glucagon in plasma were measured during infusion of glucose at a rate of first 1.7 +/- 0.2 and then 6.5 +/- 0.3 mg.kg-1.min-1 (9.4 +/- 1.1 and 36.1 +/- 1.7 mumol.kg-1.min-1) (mean +/- SD). GPR was determined by use of D-6,6-2H2-glucose. When the rate of infusion of glucose was increased, GPR decreased from 4.3 +/- 1.3 to 1.4 +/- 1.1 mg.kg-1.min-1 (23.9 +/- 7.2 to 7.8 +/- 6.1 mumol.kg-1.min-1) (mean +/- SD) (p = 0.00006). In addition, the plasma insulin concentration increased from 6 +/- 2 to 11 +/- 4 microU.mL-1 (p = 0.006) and the plasma glucose concentration from 3.6 +/- 1.1 to 6.1 +/- 1.3 mM (mean +/- SD) (p = 0.0002), whereas the glucagon concentration remained unchanged. Only the insulin concentration in plasma was significantly related to GPR. The results show that very immature newborn infants have an incomplete and varying capacity to respond to glucose infusion with suppression of glucose production. Insulin seems to be more important than plasma glucose in the regulation of glucose homeostasis in these infants.

The relationship between serum concentrations of glucose, insulin and glucagon during the first two days of life was studied in healthy newborns. The first capillary blood sample was obtained at 3-15 h of age (median 6 h; day 0) and a second sample approximately 24 h later (day 1). Serum glucose concentrations in the first sample averaged 2.1 +/- 0.07 mmol/l (mean +/- SEM; n = 60) and were positively correlated with postnatal age (p < 0.01). Serum glucagon concentrations in the first sample averaged 570 +/- 32 pg/ml and were inversely correlated with glucose concentrations (p < 0.0001). At the second sampling, serum glucose concentrations had increased to 2.9 +/- 0.07 mmol/l (p < 0.001; n = 57) and serum glucagon concentrations had decreased to 403 +/- 22 pg/ml (p < 0.001). Serum insulin concentrations were 11.7 +/- 0.3 microU/ml and 10.2 +/- 0.3 microU/ml at the two samplings and did not correlate with serum glucose concentrations. The relationship of serum glucose and hormone concentrations to maternal and infant characteristics was studied by stepwise regression analysis. Serum glucose concentration on day 0 was positively correlated with postnatal age (p < 0.01) and birth weight (p < 0.05) but inversely correlated with duration of labour (p < 0.05). Serum glucose concentration on day 1 was positively correlated with birth weight (p < 0.0001) and inversely correlated with maternal prep-pregnancy weight (p < 0.05). Similar analyses of serum hormone concentrations did not demonstrate any relationships with maternal or infant characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural tube defects (NTD) are known to occur at a higher rate in pregnancies of women with epilepsy. Antiepileptic drugs (AEDs), notably valproate (VPA) and carbamazepine (CBZ), have been identified as risk factors, but a familial aggregation of this condition also occurs in the absence of pharmacologic teratogens. Spina bifida occulta, defined as a nonsymptomatic nonfusion of vertebral arches, has been suggested to be genetically determined, with an increased prevalence in patients with primary generalized epilepsy, and that the presence of this trait in fetal development can be enhanced pharmacologically to produce NTD such as meningomyelocele. In this study, plain abdominal radiographs were obtained from 56 patients with juvenile myoclonic epilepsy (JME) and 56 age- and sex-matched controls. The radiographs were presented in a random order to an unbiased radiologist. No difference in prevalence of vertebral arch nonfusion (VAN) was noted between the two groups. Even if it has no increased frequency in patients with epilepsy, however, VAN is a common radiologic finding, and its relation to symptomatic neural tube defects should be clarified in future studies.

Beta-cell function and islet cell antibodies were studied in six patients with autoimmune polyendocrine syndrome type I. All suffered from mucocutaneous candidiasis, five had adrenocortical insufficiency and three hypoparathyroidism. All sera contained high titres of antibodies staining islets of Langerhans. Reactivity against glutamate decarboxylase, predominantly the 65 kDa isoform, was detected by immunoprecipitations and Western blots in five of the six sera, and all six sera immunoprecipitated a 51 kDa antigen from [35S]-methionine labelled rat islet cell lysates. No reactivity against this latter antigen was found in sera of patients with Type 1 (insulin-dependent) diabetes mellitus (n = 9), Graves' disease (n = 5), autoimmune gastritis (n = 4), idiopathic Addison's disease (n = 7), or stiff-man syndrome (n = 2). The 51 kDa antigen was also detected by Western blots using homogenates of rat islets and autoimmune polyendocrine syndrome type I patient sera, whereas no such reactivity was found with homogenates of testes, adrenals, small intestine, spleen, exocrine pancreas or brain. Moreover, the 51 kDa antigen was present in the rat insulinoma cell line RINm 5F but not in the SV-40 transformed, monkey kidney cell line COS, when examined by immunoprecipitations of [35S]-methionine labelled cell lysates and by Western blots. None of the patients with autoimmune polyendocrine syndrome type I had symptoms of diabetes and their insulin responses to glucose challenge were normal. The data illustrate that patients with autoimmune polyendocrine syndrome type I present an autoimmune response against islets of Langerhans, which is apparently different from that associated with classic Type 1 diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune polyendocrine syndrome type I (APS I) and idiopathic Addison's disease are both disorders with adrenal insufficiency but with differences in genetic background, clinical presentation, and extent of extraadrenal manifestations. In this study the major adrenal autoantigen identified with sera from patients with APS I was characterized by analyses using indirect immunofluorescence, Western blots of adrenal subcellular fractions and of recombinant proteins, immunoprecipitations of [35S]methionine-labeled lysates of a human steroid-producing cell line, and studies of enzymatic activity. Sera from patients with APS I, identifying cells in adrenal glands and testes involved in steroid synthesis, reacted in Western blots with a 53-kD antigen, which comigrated with the cytochrome P450 cholesterol side chain cleavage enzyme (SCC). The sera also immunoprecipitated this protein from lysates of radiolabeled adrenal cells. The enzymatic activity of SCC was inhibited by the APS I sera but not by control sera. Sera from patients with idiopathic Addison's disease did not react with the SCC. The results show that the autoimmune responses towards adrenal tissue in patients suffering from APS I and Addison's disease are remarkably selective and suggest that a determination of the antigen involved in a patient with autoimmune adrenal insufficiency will have diagnostic as well as prognostic implications.

Between 6 months and 3 years of age, growth velocity in children with Down's syndrome (DS) is markedly reduced in comparison to that of healthy children. However, after 3 years of age, it is almost normal. Thus, growth retardation becomes pronounced during the period when growth hormone (GH) starts to regulate growth. The present authors report the long-term effects of GH-therapy in 16 children with DS, who are being treated for 3 years from the age of 6-9 months. The treatment, Genotropin, 0.1 U kg-1 BW day-1, was started at a mean age of 7.4 (6-9) months. The results after 12 (n = 16), 24 (n = 12) and 30 (n = 8) months are presented. The mean height standard deviation score, SDS (range; Swedish standard), before therapy was -1.8 (-0.5 to -3.1) and the mean head circumference was -1.2 (-0.4 to -3.5). After 12, 24 and 30 months, the mean height SDS were -1.1 (-0.8 to -1.9), -0.9 (0 to -1.5) and -0.9 (0.1 to -1.5) and the mean head circumference SDS were -1.1 (0 to -2.5), -1.1 (0 to -2.2) and -1.2 (-0.5 to -2.0), respectively. During hGH-treatment, the children with DS thus gained height during the first year, and then followed the growth rate of healthy Swedish children. When compared to growth charts for children with DS the mean height of these children started at the fiftieth centile and reached the ninety-fifth centile after 24 months of treatment. Head circumference only slightly increased during the therapy, and not to the same extent as height. This indicates that small head circumference in DS is not only an effect of growth retardation, but also due to microcephaly.

Neonatal hypoglycemia is a frequent complication in immature infants. This may be due to small substrate stores, a high brain:body weight ratio, and immature enzyme systems. The purpose of the present study was to investigate the rate of glucose production in newborn infants with gestational ages of less than 28 wk. The subjects were 10 newborn infants delivered after 25 to 26 gestational wk. Their mean birth weight was 772 g (range 588-1000 g), and their mean postnatal age at the time of the study was 15 h (range 4-24 h). An isotopic compound (D-6,6-2H2-glucose) was given as a constant-rate i.v. infusion. In addition to dideuteroglucose, eight of the infants also received an i.v. infusion of unlabeled glucose at a rate of 1.4-2.6 mg.kg-1.min-1. Blood samples for determination of the concentration and isotopic enrichment of plasma glucose were obtained every 15 min in a 2-h period. Isotopic enrichment, measured by gas chromatography/mass spectrometry, was used for calculating the glucose production rate. The mean glucose production rate related to body weight (+/- SD) was 6.1 +/- 1.5 mg.kg-1.min-1. The results show that infants born at < 28 gestational wk have a capacity to produce glucose on their 1st d of life at rates close to or even exceeding those reported in term infants.

Haplotypes of the complement 4 (C4) and steroid 21-hydroxylase [21-OHase; steroid hydrogen-donor: oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] repeated gene complex were studied in nine families with at least one member affected with a mild form of 21-OHase deficiency. DNA probes from different parts of the repeated C4/21-OHase unit were used to follow the segregation of hybridization patterns in the families. Ten structurally distinct haplotypes of the C4/21-OHase gene region were identified, and the encoded phenotype was assigned to 34 of the 36 C4/21-OHase haplotypes. Four structurally different haplotypes with three C4/21-OHase repeat units were found. Eight of the nine haplotypes found with triplications of the C4/21-OHase repeat unit encoded the mild form of 21-OHase deficiency, whereas one particular triplicated haplotype encoded a severe form of the disease. In one case the mild form of 21-OHase deficiency was encoded by a haplotype with a single C4/21-OHase repeat unit. Mild 21-OHase deficiency was predicted in a patient by the presence of a triplicated haplotype. The finding of deranged 21-OHase genes on all triplicated C4/21-OHase haplotypes indicate that most of these common haplotypes carry mutated 21-OHase genes, and thus may cause functional polymorphism of general importance in the population.

A method for assaying mono(2-ethylhexyl)phthalate (MEHP) uridine diphosphate (UDP) glucuronyl transferase activity in microsomal preparations from guinea pig liver is described. The quantitation of the MEHP-glucuronide was performed by HPLC after direct injection of a sample of deproteinized incubation mixture. Solubilization of microsomal UDP-glucuronyltransferase activity was achieved by use of Lubrol, and optimal conditions for glucuronidation of MEHP were established. To investigate whether there is competition between MEHP and bilirubin for glucuronidation, inhibition experiments were performed with solubilized enzyme preparations. In these incubations addition of bilirubin decreased the formation of MEHP-glucuronide. No change in the maximal conversion rate (Vmax) was observed, indicating the occurrence of competitive inhibition. This observation may have implications in clinical situations where patients with hyperbilirubinemia are exposed to MEHP, e.g. in exchange transfusions in newborn infants.

Infantile Zellweger syndrome belongs to the group of peroxisomal disorders that lack peroxisomes. Both trihydroxycoprostanic acid (THCA), the precursor to cholic acid, and dihydroxycoprostanic acid (DHCA), the precursor to chenodeoxycholic acid, accumulate in this disease. In previous studies, we have shown that liver peroxisomes are required for the conversion of THCA into cholic acid both in vitro and in vivo by measuring a defective conversion in infants with Zellweger syndrome. In our present study, the conversion of DHCA into chenodeoxycholic acid has been measured in an infant with Zellweger syndrome to evaluate the importance of liver peroxisomes for the formation of chenodeoxycholic acid. Coprostanic acidemia was present from the second day of life with high levels of THCA and only trace amounts of DHCA. The conversion of i.v. administered [3H]DHCA into chenodeoxycholic acid was only 7% compared with the 80% conversion in an analogous study in an adult. There was, however, a rapid incorporation of 3H into biliary THCA and, after a lag phase, the 3H was incorporated into biliary cholic acid. After 72 h, 15% of [3H]DHCA was converted to cholic acid. The pool size of DHCA was 1.2 mg/m2 and the pool size of both cholic acid and chenodeoxycholic acid was markedly reduced. The renal excretion of cholic acid was more efficient than that of the less polar chenodeoxycholic acid. More polar metabolites of DHCA and THCA are formed in alternative metabolic pathways facilitating renal excretion of these toxic intermediates. We conclude that liver peroxisomes are essential for a normal conversion of DHCA into chenodeoxycholic acid.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalyses the rate-limiting step in cholesterol synthesis. Glutathione (GSH) has been postulated to be an important activator of HMG-CoA reductase in vivo. HMG-CoA reductase activity was assayed in cultured fibroblasts from healthy children. Solubilized enzyme preparations were prepared by ultracentrifugation after freezing and thawing of fibroblasts. Such treatment increased the relative enzyme activity markedly. Enzymological assay conditions were established. Addition of GSH stimulated the reaction, whereas there was inhibition after addition of glutathione disulphide (GSSG). The inhibitory effect of GSSG could be reversed by the addition of excess GSH. Fibroblast preparations, deficient in GSH, were obtained from children with glutathione synthetase deficiency or from normal subjects after the growth of fibroblasts in the presence of buthionine sulphoximine. Solubilized enzyme preparations from GSH-deficient fibroblasts had HMG-CoA reductase activities lower than or comparable with those of control preparations. The results indicate only some reduction in the capacity for cholesterol synthesis in subjects with glutathione deficiency. The existence of additional activation mechanisms in vivo, alternative to GSH, for thiol-dependent modulation of HMG-CoA reductase activity seems likely.

The metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid was studied in the bile fistula rats and in preparations from rat liver homogenates. In the bile fistula rats, the main products were chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid. Only small amounts of cholic acid were formed. Incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and NADPH yielded as the main product 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholestanoic acid. The formation of small amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid was shown. The major product in incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and the 100,000 g supernatant fluid fortified with ATP was identified as 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid. This compound was converted into chenodeoxycholic acid and its metabolites in the bile fistula rat.

In a therapeutic trial, the effect of short-term low-dosage N-acetylcysteine supplementation on glutathione metabolism was investigated in two patients with hereditary glutathione deficiency (5-oxoprolinuria). Clinical and neurophysiological examinations of the patients indicated progressive neurological damage. The pretreatment concentrations of total and free glutathione in leukocytes were 15-20% of normal, whereas the corresponding gamma-glutamylcysteine levels were increased. In plasma, the glutathione concentrations were similarly decreased, but no gamma-glutamylcysteine was detected. Total glutathione in erythrocytes was markedly decreased. Low urinary excretion of cysteinylglycine, cyst(e)ine, taurine, N-acetylcysteine, mercaptolactate and mercaptoacetate and reduced leukocyte taurine levels constituted additional evidence of decreased intracellular availability of cysteine, i.e. glutathione. Oral supplementation with N-acetylcysteine (5 mg/kg x 3/day) had no effect on acid-base balance, erythrocyte glutathione levels or 5-oxoproline concentrations in plasma and urine. In leukocytes, the glutathione concentrations were increased by 20-30%, whereas the gamma-glutamylcysteine levels were essentially unaltered. In parallel, the urinary excretion of cysteinylglycine was increased and the leukocyte levels and urinary outputs of sulphur amino acids were restored. No side-effects of the treatment were noted. The results indicate that N-acetylcysteine may be of value in increasing the low intracellular glutathione concentrations and cysteine availability in patients with hereditary glutathione synthetase deficiency.

Male conventional rats have been treated for five days with benzylpenicillin, neomycin, kanamycin, erythromycin, bacitracintneomycin, succihylsulfathiazole or metronidazole. Total fecal bile acids were analyzed in samples collected during periods of three days during the pretreatment period and during the eight weeks following drug treatment. Metronidazole or succinylsulfathiazole had no or minor effects on the conventional bile acid pattern and the "bile acid index" (ratio beta-muricholic acid/deoxycholic acid) remained low. Benzylpenicillin, neomycin or kanamycin induced a germfree bile acid pattern, i.e. increased the relative amounts of alpha-and beta-muricholic acid in feces and eliminated deoxycholic acid and hyodeoxycholic acid from feces. The high bile acid index was normalized within three weeks after termination of drug treatment but the excretion of alpha- and beta-muricholic acid was not normalized until a normal flora had been established by giving an enema with intestinal contents from intact, oncentional rats. Treatment with eythromycin or bacitracintineomycin also produced a germfree bile acid pattern. In these cases, the bile acid index was not back to normal until after five to eight weeks and the excretion of the muricholic acids was not normalized until an enema with intestinal bacteria had been given. It is suggested that these long-lasting effects of antibiotics on the metabolism of bile acids in the intestinal tract should be considered after short-term antibiotic therapy in humans.

The case history of a 4 year old girl with primary hypoparathyroidism is reported. Treatment with oral 1 alpha-hydroxyvitamin D3 did not result in normal calcium and phosphate levels, whereas treatment with oral 1 alpha,25-dihydroxyvitamin D3 did. During the treatment period, the patient developed signs of severe liver disease and died in a picture of increased intracranial pressure. Post-mortem examination revealed a giant cell hepatitis and severe cirrhosis. The clinical course is consistent with a liver vitamin D3 hydroxylation defect.

Conversion of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid into 3 alpha, 7 alpha, 12 alpha 24-tetrahydroxy-5 beta-cholestanoic and cholic acids was catalyzed either by the mitochondrial fraction fortified with coenzyme A, ATP, MgCl2 and NAD or by the combination of microsomal fraction and 100,000 x g supernatant fluid fortified with coenzyme A, ATP and nad. 24-hydroxylation and formation of cholic acid occurred at similar rates with the 25R- and the 25S-forms of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid. The 25R- and 25S-forms of 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acids were administered to bile fistula rats. Labeled cholic acid was isolated from the bile. The initial specific radioactivity of cholic acid was higher and the disappearance of radioactivity more rapid after administration of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid than of 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.

The present study was directed toward providing additional information in man on the nature of a potential alternative pathway to cholic acid not involving an initial 7 alpha-hydroxylation of cholesterol. Two bile fistula patients and one normal subject each received 25-hydroxy[G-3H]cholesterol; [14C]cholic and [14C]chenodeoxycholic acids were also simultaneously administered to one bile fistula patient and normal subject. The labeled 25-hydroxycholesterol was found to be poorly converted to primary bile acids by all three patients; the range of conversion was 9.7 to 18.9%. Cholic acid was favored over chenodeoxycholic acid by a margin of about 1.4/1. It is concluded that a pathway to primary bile acid via the 25-hydroxylation of cholesterol is of minor importance under conditions of normal or accelerated synthesis in man.

Several hydroxylase activities in bile acid biosynthesis were assayed in subcellular fractions of human fetal liver. The livers were obtained at legal abortions performed between gestational weeks 14 and 24. Microsomal 12 alpha-hydroxylase and mitochondrial 12 alpha- and 26-hydroxylase activities were detected from week 14. The microsomal fraction also had capacity for 25-hydroxylation, whereas 7 alpha- and 26-hydroxylase activities were hardly detectable. The variation of the hydroxylase activities between different experiments can be explained by inactivation during the abortion or workup procedure. The results are discussed with respect to earlier studies of bile acid biosynthesis during development and adult life.

The present study was designed to obtain more definitive information in man on the metabolic pathways to chenodeoxycholic acid and to cholic acid via a pathway not involving an initial 7 alpha-hydroxylation of cholesterol. Four bile fistula patients were administered consecutively two or more of the following 3H-labeled bile acid intermediates: 7 alpha-hydroxycholesterol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane 3 alpha,7 alpha,26-triol, 26-hydroxycholesterol,7 alpha,26-dihydroxy-4-cholesten-3-one, and 5-cholestene-3 beta,12 alpha-diol. Both 7 alpha-hydroxy[7 beta-3H]cholesterol and 7 alpha-hydroxy-4-[6 beta-3H]cholesten-3-one were efficiently converted to bile acids and preferred chenodeoxycholic acid over cholic acid. The specific activity time curves indicated that a portion of cholic acid synthesis did not pass through 7 alpha-hydroxycholesterol. [3H]26-Hydroxycholesterol and [3H]-5-cholestene 3 beta,12 alpha-diol, two potential intermediates of this bypass pathway to cholic acid, were poorly converted to primary bile acids (10 to 27%). The [3H]26-hydroxycholesterol preferred chenodeoxycholic over cholic acid by about 4 to 1. The [3H]5-cholestene 3 beta,12 alpha-diol formed cholic acid in low yield (10 to 20%). It is concluded that pathways to primary acids from cholesterol through 26-hydroxycholesterol and 5-cholestene 3 beta,12 alpha-diol are probably of minor quantitative significance. A selective pathway to chenodeoxycholic acid via 26-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one was also investigated. The 5 beta-cholestane 3 alpha,7 alpha,26-triol was converted in about equal amounts to cholic and chenodeoxycholic acids. The 7 alpha-hydroxy-4-cholesten-3-one was also efficiently converted to both bile acids but preferred chemodeoxycholic acid. The most efficient precursor of chenodeoxycholic acid was 7 alpha,26-dihydroxy-4-cholesten-3-one, which was efficiently converted to primary bile acids; chenodeoxycholic acid was preferred over cholic acid by approximately 7 to 1. These findings suggest the presence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one and intermediate formation of 7 alpha,26-dihydroxy-4-cholesten-3-one.

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.

The present investigation has aimed at defining the factor responsible for the differences in microsomal metabolism of steroids between germ-free and conventional rats. Cholesterol, cholic acid, taurocholic acid, and chenodeoxycholic acid were fed to conventional and germ-free male rats and the effects on liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]-cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, and [24-14C]lithocholic acid were studied. The most consistent effects were found with dietary cholesterol that stimulated the activities of several of the hydroxylases active on 4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol and that decreased the 5alpha reduction of 4-androstene-3,17-dione, increased the 7alpha hydroxylation of cholesterol, decreased the 12alpha hydroxylation of 7alpha-hydroxy-4-cholesten-3-one, and increased by 6beta hydroxylation of lithocholic acid. These effects of cholesterol feeding on the microsomal metabolism of steroids in conventional rats made the pattern of microsomal enzyme activities resemble that characteristic of germ-free rats. Cholesterol feeding led to a pronounced increase in the intestinal concentration of beta-muricholic acid in conventional rats. Furthermore, cholesterol feeding to conventional animals led to an intestinal ratio of chenodeoxycholic acid (including its metabolites alpha- and beta-muricholic acid and hyodeoxycholic acid) to cholic acid (including deoxycholic acid) that was almost identical to that in germ-free rats. Conventionalization of germ-free rats for a period of up to 56 days led only to a partial normalization of the liver microsomal metabolism of 5alpha-[4-14C]androstane-3alpha, 17beta-diol and 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one and of the liver microsomal concentration of cytochrome P-450. The concentration of cholesterol was higher in both total liver homogenate and liver microsomal fraction of germ-free rats than in corresponding preparations from conventional rats. In conclusion, it is suggested that cholesterol is one of the factors responsible for the different microsomal metabolism of steroids in germ-free and conventional rats. It is also suggested that cholesterol may play a role as regulator of microsomal enzyme activities.

Metabolic pathways involved in the conversion of cholesterol to cholic and chenodeoxycholic acids have been investigated in bile fistula patients treated with a number of labeled potential bile acid intermediates. The findings of the present report indicate that the human liver cell has the capacity to synthesize both primary bile acids via multiple routes from cholesterol and 7 alpha-hydroxycholesterol. Evidence has been obtained for the existence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one. Cholic acid is synthesized preferentially via pathways from 5 beta-cholestane 3 alpha, 7 alpha-diol and a pathway from cholesterol not involving an initial 7 alpha-hydroxylation.

Hydroxylation of 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol was studied in mitochondrial preparations from human fetal livers. The livers were obtained at legal abortions between weeks 14 and 24. In addition to hydroxylation in the 26-position, 5 beta-cholestane-3 alpha, 7 alpha-diol was hydroxylated in the 12 alpha-position. In one experiment, mitochondrial protein was solubilized and partially purified. Material with such chromatographic properties as those of cytochrome P450 showed 12 alpha-hydroxylase activity when combined with adrenodoxin and adrenodoxin reductase from bovine adrenal mitochondria. Because adrenodoxin and adrenodoxin reductase are components specific for mitochondrial hydroxylase systems, the results exclude microsomal contamination as the origin of this 12 alpha-hydroxylase activity. Further, there was no hydroxylase activity when NADPH-cytochrome P450 reductase from rat liver microsomes was added instead of adrenodoxin and adrenodoxin reductase. The microsomal fraction of fetal liver was also shown to possess 12 alpha-hydroxylase activity. Microsomal and mitochondrial hydroxylase activities per milligram of protein towards 5 beta-cholestane-3 alpha, 7 alpha-diol were of the same order of magnitude. The occurrence of an efficient sterol nucleus hydroxylase activity in liver mitochondria appears to be unique for fetal liver.

The present report has been directed toward providing additional information on the major defects in the bile acid pathways present in patients with cirrhosis and its relevance to the problem of how bile acid synthesis is regulated in man. An unusual patient with severe liver disease and a completely broken enterohepatic circuit was studied. The synthesis of cholic and chenodeoxycholic acids was examined over a 5-d period. The secretion rats and the incorporation of [3H]7 alpha-hydroxycholesterol and [3H]26-hydroxycholesterol into both primary bile acids in the cirrhotic bile fistula patient was cross compared to earlier data obtained on patients with and without liver disease and an intact enterohepatic circuit and patients with no liver disease and a bile fistula. The daily synthesis rate of cholic acid increased 7-fold and chenodeoxycholic acid 2-fold in the cirrhotic bile fistula patient. The incorporation of [3H]7 alpha- hydroxycholesterol into bile acids in the cirrhotic bile fistula patient was efficient (75%) and equal to bile fistula patients with no cirrhosis (76%); chenodeoxycholic acid synthesis was favored over cholic acid particularly in the cirrhotic patient. [3H]26-hydroxycholesterol was poorly incorporated in patients with no cirrhosis (25%) and the cirrhotic patient (20%); chenodeoxycholic acid was favored by a wide margin. It is concluded from this and previous reports that the profound reduction in bile acid synthesis present in patients with cirrhosis is not caused singly by a failure in the metabolic pathways from 7 alpha-hydroxycholesterol to cholic and chenodeoxycholic acid (i.e., 12 alpha-hydroxylation step), but rather due to a defect in the feedback control regulating bile acid synthesis.