Dehydroepiandrosterone sulfate in plasma: hydrolysis, extraction and radioimmunoassay.

At pH 4.5, the hydrolysis of 3beta-hydroxy-5-androsten-17-one 3-sulfate (DHA-SO4) to DHA was complete within 75 min at 120 degrees or 4h at 100 degrees. In the same conditions, the 3alpha-SO4 of androsterone was stable, and only 8.5% of the 3beta-SO4 of epiandrosterone hydrolysed to epiandrosterone. Of the sulfates of 5-androstene-3beta, 17beta-diol, 100% of the 3beta-mono-SO4, 2% OF THE 17BETA-MONO-SO4 and none of the 3beta, 17beta-di-SO4 was converted to 5-androstenediol. Denatured plasma proteins adsorbed DHA. The recovery of DHA from plasma diluted 1:100, 58.7 +/- 6.2% (mean +/- S.D.). In similar conditions the recovery of cholesterol from plasma diluted 1:20, was 0.12 - 1.76% (mean, 0.44%). A radioimmunoassay for DHA in extracts of hydrolysed plasma is described. Results for normal subjects in the age range 17-45y were 192 +/- 73mug/dl (22 men) and 158 +/- 57mug/dl (40 women).     

Enzymes and inhibitors in leu-enkephalin in metabolism in human plasma

The enzymes degrading leucine enkephalin in human plasma and the inhibitors active on these enzymes were studied by kinetic and chromatographic techniques. Data obtained evidence the existence of complex kinetics of leu-enkephalin hydrolysis and of formation of its hydrolysis byproducts. These appear to originate from the combined effect of further hydrolysis of the enkephalin's fragments after their release and of competition between the different enzymes present in plasma. Chromatographic separation of plasma proteolysis inhibitors indicates the existence of several pools of substances acting on all three enzyme groups that degrade leu-enkephalin. The partial specificity of these substances induces competition effects: consequently, the actual protection over leu-enkephalin is considerably lower that the total inhibitory activity. That notwithstanding, plasma inhibitors control enkephalin hydrolysis to a relevant extent, while they modify only slightly the ratio of hydrolysis between the different enzymes. This latter parameter—and specifically the large prevalence of aminopeptidases over dipeptidylaminopeptidases and dipeptidylcarboxypeptidases—appears controlled mainly by kinetic factors.     

Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism

Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.     

Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging.

Dehydroepiandrosterone (DHEA) is produced in prodigious quantities by the human adrenal, principally as the 3-sulfoconjugate DHEA sulfate (DS) during intrauterine life. The fetal zone and neocortex cells of the fetal adrenal express large amounts of DHEA sulfotransferase and minimal amounts, at least until very near the end of gestation, of 3beta-hydroxysteroid dehydrogenase. This pattern of enzyme expression favors substantial secretion of DHEA/DS with minimal cortisol produced; the DHEA/DS serves as the major precursor for placental estrogen formation in human pregnancy. Aside from adrenocorticotropin, other physiologic regulators of growth and steroidogenesis in the fetal adrenal have been postulated to exist, but have yet to be identified. Whereas intrauterine stressors may activate adrenal cortisol secretion, the fetal adrenal responds to many pregnancy conditions by suppressing DHEA/DS formation. After birth, the human adrenal undergoes reorganization whereby the large, inner fetal zone regresses, and DHEA/DS production is diminished. Just prior to gonadal maturation, the human adrenal undergoes morphologic and functional changes (adrenarche) that give rise to a prominent zona reticularis that is characterized by the presence of DHEA sulfotransferase, the absence of 3beta-hydroxysteroid dehydrogenase, and an enhancement of DHEA/DS production. The adrenal of the adult responds to stress in many instances like that of the fetus: increased cortisol secretion and diminished DHEA/DS secretion. The mechanisms for this divergence in the adrenocortical pathway is unknown. With aging, there is suppression of DHEA/DS secretion, possibly as the consequence of an involution of the zona reticularis, but corticosteroid production continues unabated.     

Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.     

Dehydroepiandrosterone and dehydroepiandrosterone sulfate dynamics in obesity.

Dehydroepiandrosterone (D) and dehydroepiandrosterone sulfate (DS) dynamics were studied in three obese female subjects following a single injection of [4-14-CA1D and [7 alpha-3-H]-DS tracers. Dynamic parameters were calculated simultaneously by both the urinary and blood method of compartmentalization; Estimates for the urinary secretion and production rates of D were found to be high, and those of DS varied within normal range. Calculation of the conversion factors, rho DDS and rho DSD, by the urinary method revealed a noraml extraglandular DS yields D conversion, while that for D yields DS appeared deficient in obese female subjects. Estimates of inner and outer pool distribution volumes were extremely increased for free D; in contrast to this, moderately increased inner and decreased outer pool volumes of DS were observed. The metabolic clearance rates of D were normal or decreased and those for DS were greater than normal. The blood production rates of both B and DS were higher in obese female subjects than those estimated for normal women in our previous study; These observations suggest a considerable uptake of unconjugated D by adipose tissue, an overall poor D yields DS conversion and an accelerated DS metabolism in obese female subjects.     

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were prepared by centrifugation from the plasma of apo E-3/3 or 4/3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50-150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 micrograms protein/10 micrograms IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B,E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with Kd values of 2.50, 0.93 micrograms protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2-3 molecules/particle) and enrichment of IDL with apo E-3 (to 4-5 molecules/particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.     

Prostaglandin E1 metabolism by human plasma

An enzymic prostaglandin E₁ metabolising system in human plasma is described. Various properties of the system have been investigated. Metabolism of prostaglandin E₁ added to whole human blood or plasma, particularly in low concentrations such as those found physiologically, can be extremely rapid and extensive. The importance of these findings in relation to the extraction of prostaglandins from human blood or plasma is discussed.     

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.     

Role of enzymes and inhibitors in leu-enkephalin metabolism in rabbit and human plasma

The hydrolysis of leucine enkephalin by the proteolytic enzymes present in human and rabbit plasma has been studied by kinetic and chromatographic techniques. Data obtained indicate the existence of noticeable intraspecific differences in the kinetics of leu-enkephalin degradation, and of formation of its hydrolysis by-products. The separation of the enzymes active on the substrate and of the inhibitors active on these enzymes evidences the existence of a species specific distribution of both groups of substances. Yet, the dissimilar kinetics of the substrate hydrolysis and of formation of its hydrolysis by-products appear to arise more from diversities in the competition between the enzymes present in plasma and in the role of inhibitors than from the differences in the enkephalin-degrading enzymes. It is suggested that differences observed may be related to the existence of species specific populations of the information-carrying plasma peptides.     

Dehydroepiandrosterone protects human keratinocytes against apoptosis through membrane binding sites

Although the epidermis is importantly affected by steroid hormones, little is known about the effects of dehydroepiandrosterone (DHEA) on human keratinocytes, in spite of its abundance in human serum. Here, we demonstrate for the first time a protective role of DHEA against apoptosis in keratinocytes, using non-cancerous immortalized human HaCaT cells. We show that DHEA transmits its signal via specific G protein-coupled, membrane binding sites and inhibits apoptosis, through prevention of mitochondrial disruption and altered balance of Bcl-2 proteins. DHEA conjugated to the membrane impermeable molecule BSA, as well as DHEA-S, the most abundant form of DHEA in human serum exhibit similar anti-apoptotic effect. Our data provide new insights in the treatment of the epidermis with steroid hormones in apoptosis-related conditions.     

Progesterone metabolism in human saliva in vitro.

Human salivary glands are known to be able to metabolize progesterone as well as other steroid hormones. The rate of progesterone metabolism in the salivary glands is so low that it is not thought to affect salivary progesterone concentrations. On the other hand it is usually recommended that saliva should be frozen quickly after the collection to prevent any kind of metabolism in saliva. When saliva is collected at home e.g. delayed freezing or partial thawing during to transport to laboratory may create circumstances where progesterone metabolism may occur. However, it is not known to which extent progesterone metabolism continues in saliva and whether this continued metabolism of progesterone affects salivary hormone levels. Paraffin-stimulated salivary samples were collected from female (N = 6) and male (N = 6) dental students and perimenopausal women (N = 8). The salivary samples were incubated with 14C-progesterone for 2 h at 37 degrees C in a shaking water bath. Metabolites were analyzed using thin-layer chromatography and autoradiography and quantified by liquid scintillation counting. Human saliva was found to be able to metabolize progesterone, but its metabolic activity was very low, 9.3 and 6.8 pmol/ml/h in young adults and perimenopausal women, respectively. Metabolic activity was higher in whole saliva than in the corresponding activities of the supernatant or sonicated fraction of the same saliva. The supernatant fraction, which was thought to be mainly representative of glandular saliva, was metabolically least active. The polar metabolites of progesterone predominated in all incubations. The metabolic activity of saliva is probably mainly due to its cellular content and the contribution of this activity to salivary progesterone concentrations is not significant.     

Oxidative metabolism of human platelets.

The reducing capacity toward cytochrome c present in human resting platelets increases upon platelet stimulation, and is partially inhibited by superoxide dismutase. This activity therefore represents the generation of superoxide anion. In order to evaluate hydrogen peroxide formation a quantitative assay by mean of dichlorofluorescin (DCFH) has been set up. The DCFH, trapped inside the cell, is oxidized by hydrogen peroxide to the fluorescent compound DCF. Basal DCF increases during activation of platelets by agonists. Arachidonic acid, calcium ionophore A23187 and to a lesser extent PMA and thrombin are the most effective. N-ethylmaleimide induces a dose-dependent DCFH oxidation and potentiates the effect of agonists. NAD(P)H--cytochrome c reductase enzyme, which catalyzes superoxide anion production, is present in platelets at high specific activity, as well as those enzymes who protect the cells from oxygen reactive species.