New Insight in Loss of Gut Barrier during Major Non-Abdominal Surgery.

BACKGROUND: Gut barrier loss has been implicated as a critical event in the occurrence of postoperative complications. We aimed to study the development of gut barrier loss in patients undergoing major non-abdominal surgery. METHODOLOGY/PRINCIPAL FINDINGS: Twenty consecutive children undergoing spinal fusion surgery were included. This kind of surgery is characterized by long operation time, significant blood loss, prolonged systemic hypotension, without directly leading to compromise of the intestines by intestinal manipulation or use of extracorporeal circulation. Blood was collected preoperatively, every two hours during surgery and 2, 4, 15 and 24 hours postoperatively. Gut mucosal barrier was assessed by plasma markers for enterocyte damage (I-FABP, I-BABP) and urinary presence of tight junction protein claudin-3. Intestinal mucosal perfusion was measured by gastric tonometry (P(r)CO2, P(r-a)CO2-gap). Plasma concentration of I-FABP, I-BABP and urinary expression of claudin-3 increased rapidly and significantly after the onset of surgery in most children. Postoperatively, all markers decreased promptly towards baseline values together with normalisation of MAP. Plasma levels of I-FABP, I-BABP were significantly negatively correlated with MAP at (1/2) hour before blood sampling (-0.726 (p<0.001), -0.483 (P<0.001), respectively). Furthermore, circulating I-FABP correlated with gastric mucosal P(r)CO2, P(r-a)CO2-gap measured at the same time points (0.553 (p = 0.040), 0.585 (p = 0.028), respectively). CONCLUSIONS/SIGNIFICANCE: This study shows the development of gut barrier loss in children undergoing major non-abdominal surgery, which is related to preceding hypotension and mesenterial hypoperfusion. These data shed new light on the potential role of peroperative circulatory perturbation and intestinal barrier loss.

In vivo evidence that human adrenal glands possess 11 beta-hydroxysteroid dehydrogenase activity.

Blood samples were collected intraoperatorily from inferior vena cava (VC) and adrenal vein (AV) of 8 male and 9 female consenting adult patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for kidney cancer, and steroid-hormone concentrations were assayed by quantitative HPLC. Hormonal concentrations were significantly higher in AV than in VC (systemic) blood and did not display significant differences between males and females. Higher levels not only of the main glucocorticoids cortisol and corticosterone, but also of their inactive oxidized forms corticosterone and 11-dehydrocorticosterone (DH-B), respectively, were detected in AV than in VC blood. Highly significant inverse correlations between cortisol and cortisone, and corticosterone and DH-B concentrations were observed in AV, but not in VC blood. Moreover, in AV blood the concentration of the main cortisol precursor 11-deoxycortisol correlated inversely with those of both cortisone and DH-B. Taken together, these findings are in keeping with previous in vitro evidence that human adrenal glands possess 11beta-hydroxysteroid dehydrogenase activity, which is engaged in the inactivation of newly formed glucocorticoids and is probably negatively regulated by the local concentrations of non-11beta-hydroxylated steroid-hormone precursors.

[Defects of adrenal steroidogenesis in patients with hirsutism]. / Defectos de la esteroidogénesis adrenal en pacientes con hirsutismo.

BACKGROUND: To determine the frequency and the type of adrenal steroidogenic abnormalities in hirsute women. SUBJECTS AND METHODS: ACTH test was performed during follicular phase in 127 hirsute and 40 normal (control) women. Before ACTH injection we measured in serum by RIA: 17-OH-pregnenolone (17-OH-P5), 17-OH-progesterone (17-OH-P4), androstenedione (AN), cortisol (CT), 11-deoxycortisol (DCT), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), total (TT) and free (FT) testosterone, oestradiol (E2), progesterone (PR), androstenediol glucuronide (AG), LH, FSH and prolactin. After 60 min of ACTH injection 17-OH-P5, 17-OH-P4, AN, DHEA, CT and DCT were measured. Net increment of stimulated steroids and the ratios 17-OH-P5/17-OH-P4, DHEA/AN, 17-OH-P4/CT, 17-OH-P5/CT and DCT/CT were calculated. Pelvic ultrasonographic exploration was done when irregular menses were reported. RESULTS: Up to 31% of the patients presented enzymatic defects in adrenal steroidogenesis. Diagnostic criteria for enzyme defects were established. Late-onset 21-hydroxylase deficiency was diagnosed in 6 (4.5%) patients, HLA typing of these patients demonstrated that 4 out of 6 had B14-DR1. Sixteen women (12.6%) displayed a 17-OH-P4 response and the net increment 2 SD above the normal mean concentration, which are diagnostic criteria for late-onset 21-hydroxylase deficiency carriers. We diagnosed a 3 beta-hydroxysteroid dehydrogenase defect when 17-OH-P5 and DHEA responses, their net increment and the 17-OH-P5/17-OH-P4 and 17-OH-P5/CT ratios were 2 SD above the normal mean after ACTH: 14 women were diagnosed. 11 beta-hydroxylase deficiency diagnosis was made when DCT response, its net increment and the DCT/CT ratio after ACTH were 2 SD above the normal mean: 7 women were detected. Associated biosynthetic defects were described. CONCLUSIONS: One third of our patients with hirsutism presented anomalous response to ACTH, consistent with enzymatic abnormalities in adrenal steroidogenesis.

Dual activity at an enzyme active site: 3 beta,20 alpha-hydroxysteroid oxidoreductase from fetal blood.

An enzyme exhibiting both 3 beta and 20 alpha steroid reductase activities from calf fetal red blood cells was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3 beta,20 alpha-Hydroxysteroid oxidoreductase (3 beta,20 alpha-HSD) was found to be a single-stranded polypeptide with a molecular weight of 55 000 +/- 1 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-100 chromatography. The amino acid composition of 3 beta,20 alpha-HSD was obtained. 17 beta-Hydroxy-5 alpha-androstan-3-one and progesterone were substrates for the enzyme's 3 beta and 20 alpha reductase activities, respectively, which required NADPH for both 3 beta [Km = 9.4 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] and 20 alpha [Km = 2.5 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] reductase activities. 17 beta-Hydroxy-5 alpha-androstan-3-one competitively inhibited (Ki = 35 microM) 20 alpha reduction of progesterone. Incubating 3 beta,20 alpha-HSD with 19-nortestosterone 17-bromoacetate at pH 7.0 and 25 degrees C caused simultaneous, time-dependent, and irreversible losses of 3 beta and 20 alpha activities by a first-order kinetic process. Similar incubations with either of the 3 beta or 20 alpha substrates present at concentrations equal to their respective Km values practically doubled the time required for loss of 3 beta and 20 alpha enzyme activities. These data lead us to conclude that the active site of 3 beta,20 alpha-HSD contains 3 beta and 20 alpha dual activity.

20 alpha-Hydroxysteroid oxidoreductase activity and 20 alpha-dihydroprogesterone concentration in human placenta before and after parturition.

The objective of this investigation was to determine if decreased biosynthesis or increased catabolism of progesterone (P) during labor was responsible for the decreased concentration of hormone observed in the human placenta after labor and vaginal delivery. No significant difference was found in P biosynthesis by placental tissues examined before and after labor as evidenced by a similar activity of 3 beta-hydroxysteroid dehydrogenase: delta 5-isomerase. In contrast, there was a marked increase in P catabolism during labor, as shown by a significant (p less than 0.05) change in placental 20 alpha-hydroxysteroid oxidoreductase activity which increased from 835.5 +/- 103 (mean +/- SEM) pmoles of 20 alpha-dihydroprogesterone (20 alpha-DHP) generated per milligram of protein per hour before labor to 1,160.4 +/- 101 pmoles/mg/hr after labor. A similar significant (p less than 0.02) increase in enzyme activity was noticed in parallel assays of the reverse (20 alpha-DHP leads to P) reaction. As a consequence of increased P catabolism, placentas after labor had a 20 alpha-DHP concentration of 63.7 +/- 9.5 (mean +/- SEM) ng/mg protein, a value which was significantly (p less than 0.05) larger than that found before labor (37.8 +/- 8.3 ng/mg protein). These changes resulted in a modification of the placental tissue P/20 alpha-DHP ratio which decreased from 2/1 before to 1/1 after labor. The results indicate that the catabolism of P to 20 alpha-DHP increases significantly during human parturition. This phenomenon may be of importance in the mechanism of initiation and continuation of labor.

Stabilization and assay of a 17beta-hydroxysteroid dehydrogenase in the serum of pregnant women.

Up to the present the activity of the 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the blood of pregnant women has not been accurately measured because of its great instability. The enzyme was stabilized by mixing fresh serum with an equal volume of potassium phosphate buffer (100 mM, PH 7.4), containing 40% (v/v) glycerol, 40 muM oestradiol-17beta (Oe2) and 0.4% (v/v) ethanol. Under these conditions the 17beta-HSD was stable for several days at -20, +4 and +20 degrees C and resistant to heat denaturation at 60 degrees C. The Km-value for Oe2 with NAD or NADP as cosubstrate was 5 X 10(-6) or 2 X 10(-6) M, respectively. In the presence of NAD, Oe2 was oxidized twice as rapidly as with NADP. The Km-value for NAD was 10(-5) M. Under the conditions of assay [3H]oestrone formation was linear with time (3 h) and with protein concentration. Serum enzyme activities in normal pregnancies (between 20 and 60 muU/ml serum at the end of gestation) were nearly twice as high as those reported by other investigators. In normal single and twin births highest activities were obtained at the expulsion of the placenta which in all cases was followed by a rapid decrease in activity.

Serum levels and biochemical characterization of oestradiol-17 beta dehydrogenase in umbilical cord blood.

The properties of the enzyme oestradiol-17 beta dehydrogenase from human umbilical cord serum have beem compared with those of oestradiol-17 beta dehydrogenase present in the maternal peripheral blood. On the basis of studies on stability, specificity, optimum pH, rate of enzymic reaction and Km determinations it can be concluded that oestradiol-17 beta dehydrogenase in the umbilical cord arteries has an activity between 5 and 15 times higher than in the umbilical cord vein and about 65% of the activity of the enzyme present in the maternal peripheral blood. The present results strongly suggest that in pregnancy the enzyme oestradiol-17 beta dehydrogenase is elaborated not only by the placenta but also by the foetus.