Altered maternal hypothalamic-pituitary-adrenal axis activity in obese pregnancy is associated with macrosomia and prolonged pregnancy.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) axis is important for fetal growth and timing of parturition. Maternal obesity is associated with macrosomia (birthweight ⩾4000g) and prolonged pregnancy (⩾41weeks). We aimed to characterise HPA axis hormones in obese pregnancy and to test associations with these pregnancy outcomes. METHOD: Fasting cortisol was measured by radioimmunoassay in venous blood at 16, 28 and 36 weeks of gestation in 286 obese (BMI 44.05±3.98kg/m(2)) and 137 lean (BMI 22.71±1.66kg/m(2)) pregnant women. In subsets (n=20 obese, 20 lean) we measured corticosteroid binding globulin (CBG) and CRH by radioimmunoassay; progesterone, estradiol (E2), estriol (E3) and sex-hormone-binding-globulin (SHBG) by ELISA; and albumin by bromocresol green binding. Free cortisol levels were calculated using Coolen's equation. RESULTS: Cortisol, CBG, calculated free cortisol, CRH, E2, E3, progesterone and SHBG levels rose similarly during pregnancy in obese and lean, but were significantly lower in obese (p<0.05). In obese, lower free cortisol at 16 weeks was associated with higher birthweight (r=-0.46, p<0.05). Cortisol was not associated with labour onset. CRH was significantly lower at 36 weeks in women who delivered at ⩾41weeks and in women with macrosomic babies (p<0.05); and correlated negatively with gestation at delivery in obese (r=-0.557, p<0.05). CONCLUSION: Our findings suggest that decreased HPA axis activity in obese pregnancy may be a mechanism underlying macrosomia and prolonged pregnancy.

Characterization of the canine 3beta-hydroxysteroid dehydrogenase and its expression in the corpus luteum during diestrus.

3beta-Hydroxysteroid dehydrogenase (3betaHSD) is a key enzyme in the synthesis of bioactive steroid hormones. Objectives of the present study were to clone canine 3betaHSD and to investigate its expression in dog corpora lutea (CL) covering the periods of their formation, early and late regression (days 5, 15, 25, 35, 45, 65 after ovulation). Complete complementary DNA sequence was amplified by RACE PCR. Subsequent cloning revealed that the canine ovarian 3betaHSD transcript was composed of a 5'-untranslated region (5'-UTR) of 126 nucleotides, an open reading frame (ORF) of 1122 nucleotides and a 3'-UTR of 441 nucleotides. The putative ORF encoded a 374 amino acid protein which remains highly conserved (79-85% identity) between species. The transient expression of the cloned canine 3betaHSD in a mammalian heterologous cell expression system (HEK293T cells) identified the 3betaHSD activity as the only activity of this canine enzyme (absence of any detectable 17-hydroxysteroid dehydrogenase activity). Qualitative RT-PCR revealed expression of 3betaHSD on all days investigated and the signals were strongest on days 5 and 15, with day 25 intensity tending to decrease. However, variability between individual animals was high. The significant decrease in the expression of 3betaHSD towards the end of diestrus as indicated by Real Time PCR (p<0.01) and immunhistochemistry may indicate that the provision of progesterone is controlled by availability of the enzyme rather than the substrate.

Anti-inflammatory and proliferative responses in human and ovine ovarian surface epithelial cells.

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11betahydroxysteroid dehydrogenase (11betaHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1alpha. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1alpha stimulated a concomitant increase of 11betaHSD type 1 mRNA (31-fold; P <0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P <0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P <0.05) and >1.5-fold (P <0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.

Promiscuous 3beta-hydroxysteroid dehydrogenases: testosterone 17beta-hydroxysteroid dehydrogenase activities of mouse type I and VI 3beta-hydroxysteroid dehydrogenases.

3Beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is essential for the synthesis of all classes of steroid hormones, converting various delta5-3beta-hydroxysteroids into hormonally active delta4-3-ketosteroids in NAD+ -dependent reactions. Certain 3beta-HSD isoforms have been reported to exhibit additional dehydrogenase character (e.g., 17-hydroxysteroid dehydrogenase/reductase). We have investigated whether mouse type I (adrenal/gonadal) and type VI 3beta-HSDs (uterine/embryonic) display significant 17beta-HSD-like activity. Nonsteroidogenic HEK 293T cells were transiently transfected with pCMV-based expression vectors containing mouse type I and type VI 3beta-HSDs. Transfected cells expressing either mouse type I or type VI 3beta-HSD converted testosterone to androstenedione, albeit at rates one-tenth of those of pregnenolone to progesterone in similarly transfected 293T cells. Our findings demonstrate that the mouse 3beta-HSD I and VI isoforms can inactivate testosterone within an intact cell milieu. These findings are important not only in establishment of structure-function relationships, but also whenever murine systems are used for developmental/reproductive paradigms associated with human disorders.

Aromatase in skeletal muscle.

Aromatase gene expression and activity have been studied in human skeletal muscle. Using two separate rounds of RT-nested PCR, transcripts from the coding region of aromatase mRNA were detected in 32 of 34 samples. In terms of the non-coding exon I, PCR product for I.4 was detected in 13 cases (38%), I.3 in 10 cases (29%), P.II in 6 cases (18%) and I.1 was not detected in any case. No transcripts for any studied variants of exon I were detected in 18 samples (53%). Aromatase activity was assessed using two different methodologies: in 19 cases by definitive product isolation (DPI) and in 42 cases by tritium-release assay (TRA). Using both methods detectable activity was present in 52% of cases. Average values for the cases with detectable activity were 2.2 fmol/mg protein/h for DPI and 6.5 fmol/mg protein/h for TRA. In the cohort studied by TRA, a positive correlation of aromatase activity with age of the donor was observed (r=0.34, P=0.03). In six cases paired comparison of aromatase activity in muscle and associated fat tissue were performed by DPI. When expressed per milligram of protein the activity was always higher in fat. However, this difference disappeared when activity was based on the gram of wet tissue. Taking into account bulk in the body it is concluded that muscle can be an important source of estrogens in men and post-menopausal women and its contribution to the circulating pool of estrogens may be comparable to that of adipose tissue. The nature of mRNA transcripts in muscle suggests that estrogen formation may be controlled by glucocorticoid- as well as cAMP-dependent promoters of the aromatase gene.

Selenoprotein expression in endothelial cells from different human vasculature and species.

Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.

Effect of spinal anaesthesia on plasma concentrations of glutathione S-transferase.

BACKGROUND: Plasma glutathione S-transferase (GST) concentration measurement is a sensitive and specific index of hepatocellular injury. GST concentration increases after anaesthesia with most volatile anaesthetic agents, but not after propofol. Such increases are thought to result from reduced liver blood flow. The effect on GST concentration of spinal (subarachnoid) anaesthesia, which might also reduce liver blood flow, is not known. METHODS: We studied the effects of spinal anaesthesia on GST concentrations measured by specific radioimmunoassay in 33 patients undergoing intermediate orthopaedic, general or gynaecological surgery. GST concentrations were measured before anaesthesia and 3, 6 and 24 h after induction of anaesthesia. Hypotension (systolic blood pressure <70% of pre-induction value) was rapidly corrected with i.v. ephedrine. RESULTS: Mean duration of surgery was 41 min (range 11-80). No increase in GST concentration was observed at any time, but at 24 h GST concentration was significantly reduced (P<0.05). One patient in whom hypotension was not treated developed a greatly increased GST concentration at 3 h. CONCLUSION: We found no association between spinal anaesthesia and disturbance of hepatocellular integrity when hypotension does not occur or is rapidly corrected.

Thioredoxin reductase and cytoplasmic glutathione peroxidase activity in human foetal and neonatal liver.

Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.

Selenite protects human endothelial cells from oxidative damage and induces thioredoxin reductase.

The ability of selenium to protect cultured human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) from oxidative damage induced by 100 microM t-butyl hydroperoxide (t-BuOOH) was compared. Preincubation of human endothelial cells for 24 h with sodium selenite at concentrations as low as 5 nM provided significant protection against the harmful effects of 100 microM t-BuOOH, with complete protection being achieved with 40 nM selenite. The preincubation period was required for selenite to exert this protective effect on endothelial cells. When compared with selenium-deficient cells, the activities of cytoplasmic glutathione peroxidase (GPX-1), phospholipid hydroperoxide glutathione peroxidase (GPX-4) and thioredoxin reductase (TR) were each induced approx. 3--4-fold by 40 nM selenite. HCAEC and HUVEC showed great similarity in their relative abilities to resist oxidative damage in the presence and absence of selenite, and the activities of TR and the GPXs were also similar in these cell types. BAEC were more susceptible to damage by 100 microM t-BuOOH than were human endothelial cells, and could not be protected completely by incubation with selenite at concentrations up to 160 nM. The activity of TR in human endothelial cells was approx. 25-fold greater than that in BAEC of a similar selenium status, but GPX-1 and GPX-4 activities were not significantly different between the human and bovine cells. These studies, although performed with a small number of cultures, show for the first time that selenium at low doses can provide significant protection of the human coronary artery endothelium against damage by oxidative stress. TR may be an important antioxidant selenoprotein in this regard, in addition to the GPXs. The data also suggest that HUVEC, but not BAEC, represent a suitable model system in which to study the effects of selenium on the endothelium of human coronary arteries.

An improved ex vivo method of primary porcine hepatocyte isolation for use in bioartificial liver systems.

INTRODUCTION: Primary porcine hepatocytes are commonly, used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time-consuming and expensive, requiring specialist surgical facilities. AIM: To develop a simple, low-cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. METHODS: Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra-hepatic inferior vena cava was clamped and the supra-hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open-loop and re-circulating perfusion method (at 37 degrees C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum-free chemically-defined medium and function was assessed. RESULTS: The time interval from when the animals were killed to the final cell wash was 105+/-5 min (n = 20). Cell viability was 85+/-6% with a yield of (2.4+/-0.5) x 10(10) from 12+/-1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2+/-0.1 mg/10(6) cells (n = 5) and 1.2+/-0.3 mg/10(6) cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9x10(6) hepatocytes per dish. The percentage of total LDH released into the medium was 13+/-4% for day 0 and 8+/-4% at day 2; conversely, intracellular LDH activities were 87+/-4% and 92+/-4% of the total, respectively. The urea synthesis rate was 196+/-36 nmol/h/mg total protein at day 0 (n = 5) and 292+/-62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99+/-11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89+/-35 pmol/mg total protein in day 2 cultures. CONCLUSIONS: This ex vivo method provides a high viability, high yield, cost-effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques.

Detection of steroidogenic acute regulatory protein in equine ovaries.

A steroidogenic acute regulatory (StAR) protein has been identified in several species as a probable important rate-limiting step in steroidogenesis. This protein is believed to be responsible for transporting cholesterol from the outer to the inner mitochondrial membrane. It is known that equine chorionic gonadotrophin (eCG) stimulates steroidogenesis in the corpora lutea of early pregnant mares and that eCG also upregulates StAR mRNA in bovine ovaries. In the present study, ovarian tissue from cyclic and early pregnant mares was immunostained to detect the distribution of the StAR protein. Western blot analysis was performed, followed by phosphor imaging to establish whether the onset of eCG secretion in pregnancy was associated with increased expression of the StAR protein. Immunostaining for StAR was confined to the theca interna of growing and preovulatory follicles, but 24 h after treatment with hCG, some granulosa cells were positively stained. Positive staining was confined to the large luteal cells of the equine corpus luteum. There was no difference in the distribution of immunostaining before or after onset of eCG secretion in pregnant mares, but increased amounts of StAR were detected in corpora lutea from mares at day 40 or day 41 of pregnancy compared with non-pregnant mares and mares at days 20-30 of pregnancy.

Properties of an adrenal medullary protein immunorelated to steroid acute regulatory (StAR) protein.

Immunohistochemistry using a StAR peptide antiserum had previously revealed strong staining in rat and bovine adrenal medulla, suggesting the presence of a protein immunogenically related to StAR. Western blots of bovine medulla tissue homogenates showed the principal adrenal medullary immuno-reactive species to have a higher molecular weight (50 kDa) compared to StAR protein (30 kDa). Subcellular fractionation localised the 50 kDa species principally to the medulla cytosol. StAR peptide antiserum binding to both the 30 kDa and 50 kDa species could be specifically competed by the peptide antigen. These data suggest that the adrenal medullary immuno-reactive species and StAR protein are distinct entities, which share some features in common.

Thioredoxin reductase is the major selenoprotein expressed in human umbilical-vein endothelial cells and is regulated by protein kinase C.

Damage to the endothelium by reactive oxygen species favours atherogenesis. Such damage can be prevented by selenium, which is thought to exert its actions through the expression of selenoproteins. The family of glutathione peroxidases (GPXs) may have antioxidant roles in the endothelium but other intracellular and extracellular selenoproteins with antioxidant actions may also be important. The selenoproteins expressed by cultured human umbilical-vein endothelial cells (HUVECs) were labelled with [(75)Se]selenite and separated using SDS/PAGE. HUVECs secreted no extracellular selenoproteins. There were distinct differences between the intracellular selenoprotein profile of (75)Se-labelled HUVECs and those of other tissues. A single selenoprotein with a molecular mass of 58 kDa accounted for approx. 43% of the intracellular (75)Se-labelled proteins in HUVECs. This protein was identified by Western blotting as the redox-active lipid-hydroperoxide-detoxifying selenoprotein, thioredoxin reductase (TR). TR expression in HUVECs was down-regulated by transiently exposing cells to the phorbol ester PMA for periods as short as 1 min. However, there was a delay of 48 h after PMA exposure before maximal down-regulation of TR was observed. The protein kinase C (PKC) inhibitor bisindolylmaleimide I hydrochloride had no effect on TR expression when added alone, but the agent prevented the down-regulation of TR expression seen with PMA. The calcium ionophore A23187 increased TR expression in HUVECs after a 12-h exposure, but the maximal effect was only observed after a 35-h exposure. These findings suggest that TR may be an important factor in the known ability of Se to protect HUVECs from peroxidative damage. Furthermore, the results also suggest that TR expression can be negatively regulated through PKC. It is possible that TR expression may be positively regulated by the calcium-signalling cascade, although TR induction by A23187 may be due to toxicity.

The use of human glutathione S-transferase A1 in the detection of cystic fibrosis liver disease.

OBJECTIVE: To determine the value of serum human glutathione S-transferase A1 (hGST A1) in the detection of cystic fibrosis liver disease (CFLD). METHODS: Sixty-three children (aged 0.5-16 years) with cystic fibrosis (CF) were screened prospectively for evidence of hepatobiliary abnormalities between February 1993 and February 1996. Comparison was made between clinical examination, abdominal ultrasonic scan, measurement of conventional liver enzymes (LFTs) and serum hGST A1 concentration in the detection of hepatobiliary abnormalities in children with CF. RESULTS: The 5-95% concentration of serum hGST A1 was 1.7-4.27 micrograms L-1 for the control group. The hGST A1 levels in the CF patients were significantly higher than in the non-CF group. Thirty-eight (60%) children had detectable hepatobiliary abnormalities. Ultrasound scanning detected the highest number of abnormalities (41%), followed by hGST A1 (30%). The presence of clinical liver disease was found in 19% of the children. The estimated sensitivities of detecting CFLD by clinical method, ultrasound scan, serum hGST A1, and LFTs would be 32%, 68%, 50% and 16%, respectively. CONCLUSIONS: Serum hGST A1 measurement increases the sensitivity of detecting hepatic abnormalities when included with clinical and ultrasound evaluation although, in some cases with advanced liver disease, serum hGST A1 may be normal. Conventional liver enzyme tests add little information in the detection of CF liver disease.

Identification of a 57-kilodalton selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway.

Human thyrocytes incubated with the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-5)-10(-8) mol/L) and the calcium ionophore A23187 (10(-5)-10(-8) mol/L) showed a marked increase in the expression of a 57-kDa selenoprotein identified as thioredoxin reductase (TR). After the addition of A23187 with PMA, a significant induction in TR expression was observed after 6 h, with maximal induction occurring by 24 h. The addition of 8-bromo-cAMP (10(-4) mol/L) or TSH (10 U/L) alone had no effect on TR expression, nor did these agents influence the induction of TR brought about by the addition of A23187 and PMA. These data show that the calcium-phosphoinositol second messenger cascade that controls hydrogen peroxide generation in the human thyrocyte is also an important stimulator of TR expression. The role of TR in the thyrocyte is unclear, but the selenoenzyme has a high capacity to detoxify compounds, such as hydrogen peroxide and lipid hydroperoxides, that are produced in high concentration during thyroid hormone synthesis.

Antitumour activity and schedule dependency of 8-chloroadenosine-3',5'-monophosphate (8-ClcAMP) against human tumour xenografts.

8-Chloroadenosine-3',5'-monophosphate (8-ClcAMP) is a novel antitumour agent currently undergoing phase I clinical trials in several European centres. In this study, its antitumour activity against human tumour xenografts and its dependence on schedule were investigated. When administered by continuous infusion at doses of 100 or 50 mg/kg/day to nude mice bearing human tumour xenografts, 8-ClcAMP inhibited the growth of the HT 29 colorectal, ZR-75-1 breast, HOX 60 and PE04 ovarian and PANC-1 pancreatic carcinoma xenografts. However, these infusion schedules produced hypercalcaemia and severe weight loss. In an attempt to optimise antitumour activity and minimise toxicity, several other schedules were studied. In comparison with continuous administration of 8-ClcAMP at 50 mg/kg/day for 14 days which, although producing complete growth inhibition in the HOX 60 model, was associated with a marked body weight loss, schedules in which the infusion was interrupted (infusion on either days 0-4; 7-11 or days 0-2; 6-8) produced minimal weight loss but also reduced antitumour activity. However, co-administration of salmon calcitonin with continuous infusion of 8-ClcAMP prevented both hypercalcaemia and body weight loss in 3/6 animals while still producing marked inhibition of tumour growth. These data indicate that 8-ClcAMP has broad-spectrum antitumour activity and the major side-effect of hypercalcaemia may at least in part be ameliorated by the use of salmon calcitonin.

Differential expression of selenoproteins by human skin cells and protection by selenium from UVB-radiation-induced cell death.

The generation of reactive oxygen species has been implicated as part of the mechanism responsible for UVB-radiation-induced skin damage. In mice, evidence suggests that increased dietary selenium intake may protect skin from many of the harmful effects of UVB radiation. We sought to determine the selenoprotein profile of cultured human skin cells and whether selenium supplementation could protect keratinocytes and melanocytes from the lethal effects of UVB radiation. Labelling experiments using [75Se]selenite showed qualitative and quantitative differences in selenoprotein expression by human fibroblasts, keratinocytes and melanocytes. This was most noticeable for thioredoxin reductase (60 kDa) and phospholipid glutathione peroxidase (21 kDa); these proteins were identified by Western blotting. Despite these differences, we found that a 24 h preincubation with sodium selenite or selenomethionine protected both cultured human keratinocytes and melanocytes from UVB-induced cell death. With primary keratinocytes, the greatest reduction in cell death was found with 10 nM sodium selenite (79% cell death reduced to 21.7%; P<0.01) and with 50 nM selenomethionine (79% cell death reduced to 13.2%; P<0.01). Protection could be obtained with concentrations as low as 1 nM with sodium selenite and 10 nM with selenomethionine. When selenium was added after UVB radiation, little protection could be achieved, with cell death only being reduced from 88.5% to about 50% with both compounds. In all of the experiments sodium selenite was more potent than selenomethionine at providing protection from UVB radiation.

Serum glutathione S-transferase B1 activity as an index of liver function in cystic fibrosis.

AIMS: To evaluate serum glutathione S-transferase B1 (GST B1), a highly sensitive test of hepatocellular function, as a means of identifying liver disease in patients with cystic fibrosis (CF). METHODS: The presence of liver disease was sought over a three year period in 60 children with CF, using a combination of clinical assessment, ultrasound examination, conventional biochemical tests of liver function (LFTs), and measurement of GST B1. RESULTS: Reference ranges for serum GST B1 were established in a paediatric control population. The 95% value (4.55 micrograms/l) was similar to the upper limit of normal previously derived in adults. Mean (SE) serum GST B1 activities were higher in the CF population (9.0 (1.14) micrograms/l) than in age matched controls (2.4 (0.15) micrograms/l). Ten patients with CF showed clinical signs of liver dysfunction. All but one had a serum GST B1 > 4.55 micrograms/l. Twelve other patients had elevated LFTs without clinically evident liver dysfunction, six had abnormal ultrasound scans and two showed both of these anomalies. Thirty patients with CF had neither biochemical, ultrasonographic nor clinical signs of liver disease. On review three years later, clinically important liver disease was reaffirmed in eight of the 10 index cases and had become apparent in a further eight, all of whom had elevated GST B1 activities. Five (36%) of the patients with elevated LFTs and two (33%) with isolated ultrasound changes continued to show these abnormalities. CONCLUSIONS: The limitations of conventional LFTs and ultrasound scans were evident from this study. The results suggest that elevated GST B1 activities may be a better predictor of hepatic dysfunction in CF than conventional LFTs.

Thyroidal extracellular glutathione peroxidase: a potential regulator of thyroid-hormone synthesis.

Human thyrocytes were found to synthesize and secrete the selenoenzyme extracellular glutathione peroxidase (E-GPX), a process which was controlled by the Ca2+/phosphoinositol second-messenger cascade. The potential involvement of thyroidal E-GPX in the regulation of thyroid-hormone synthesis and in the protection of the thyrocyte from peroxidative damage is discussed.

Biological variation and the effect of fasting and halothane anesthesia on plasma glutathione S-transferase concentrations.

Using a specific RIA, we have investigated in patients and volunteers whether fasting, diminished hepatic clearance, hemoconcentration, or within-day biological variation might be responsible for the transient increases in plasma glutathione S-transferase (GST) concentration observed after anesthesia. GST concentration was measured in 44 healthy volunteers after an overnight fast and at 3, 6, and 24 h after the fasting sample. The concentration was significantly lower at 3 and 6 h after than in the fasting sample (P = 0.0019 and P = 0.015, respectively). The change in GST concentration caused by fasting was examined in 30 subjects by comparing pre- and postfasting values. Fasting had no significant effect on GST concentration overall (P = 0.4721), but two individuals showed a marked increase in GST concentration after fasting overnight. In a separate study of 10 patients, plasma amylase activity and plasma concentrations of GST and albumin were measured immediately before and 3 h after induction of halothane anesthesia. Although GST concentration was increased at 3 h in each of the 10 patients, plasma amylase activity and plasma albumin concentration were significantly decreased in all patients (P = 0.002). Apparently, increases in GST concentration after anesthesia do not result from incidental factors.