Oral magnesium supplementation reduces insulin resistance in non-diabetic subjects - a double-blind, placebo-controlled, randomized trial.

The incidence of insulin resistance and metabolic syndrome correlates with the availability of magnesium (Mg). We studied the effect of oral Mg supplementation on insulin sensitivity and other characteristics of the metabolic syndrome in normomagnesemic, overweight, insulin resistant, non-diabetic subjects. Subjects were tested for eligibility using oral glucose tolerance test (OGTT) and subsequently randomized to receive either Mg-aspartate-hydrochloride (n = 27) or placebo (n = 25) for 6 months. As trial endpoints, several indices of insulin sensitivity, plasma glucose, serum insulin, blood pressure and lipid profile were determined. Mg supplementation resulted in a significant improvement of fasting plasma glucose and some insulin sensitivity indices (ISIs) compared to placebo. Blood pressure and lipid profile did not show significant changes. The results provide significant evidence that oral Mg supplementation improves insulin sensitivity even in normomagnesemic, overweight, non-diabetic subjects emphasizing the need for an early optimization of Mg status to prevent insulin resistance and subsequently type 2 diabetes.

Alterations of ionized Mg2+ in human blood after exercise.

Magnesium (Mg) is the second most abundant intracellular cation with modulating properties in a number of metabolic processes, e.g. in glycolysis, and intracellular signalling processes, e.g. regulation of ion channels and transporters. There are conflicting data available about the regulation of Mg in blood cells during exercise. Moreover, there are no data available about changes of the metabolic important fraction of ionized Mg(2+) both in blood and in blood cells during exercise. The present study investigated the changes of ionized Mg(2+) and total Mg concentration in different compartments after a stepwise treadmill ergometer test. Intracellular ionized Mg(2+) of thrombocytes and erythrocytes was determined by the magnesium sensitive fluorescent dyes mag-fura-2 and Mag-Green using fluorescence spectroscopy and flow cytometry, respectively. Ionized Mg(2+) in blood/serum was measured by an ion-sensitive microelectrode. Total cellular and serum Mg concentration were investigated using atomic absorbance spectroscopy and photometry, respectively. The present results shown that at the end of the ergometer test, ionized Mg(2+) in both blood and serum and total serum Mg decreased. In contrast, intracellular concentration of ionized Mg increased in both thrombocytes and erythrocytes. Total intracellular Mg was unchanged making a Mg(2+) shift between the intra- and extracellular compartment unlikely. The present study therefore demonstrated opposite changes of the ratio [ionized Mg(2+)]/[total Mg] in the intracellular and the extracellular compartment after anaerobic exercise. In in vitro experiments, similar changes of ionized Mg(2+) in both compartments could be mimicked by application of weak acids like propionic and lactic acid. It is concluded changes in the fraction of ionized Mg(2+) should be high enough to influence intracellular signalling and metabolic processes.

Effect of magnesium on granulocyte function and on the exercise induced inflammatory response.

Magnesium status is a well-known modulator of the immune system. In the present study we investigated the effect of magnesium on granulocyte signalling and function. Furthermore, we performed a double-blinded randomised study investigating the effect of a two-month magnesium supplementation period on the exercise-associated alterations in immune function. In vitro incubation of granulocytes in media of different magnesium composition resulted in significant changes in chemotactic peptide-induced calcium transients while basal calcium levels were not affected. Likewise, the stimulus-induced formation of free radicals was affected by extracellular magnesium while phagocytosis of granulocytes was not affected. In the second part of the study we investigated whether a two-month period of magnesium supplementation was able to diminish alterations in immune cell counts and functions after an exercise test until exhaustion. The magnesium status was similar in both human and placebo groups and did not change significantly after the supplementation period. Exhaustive exercise induced an activation of the immune system as indicated by an increase in granulocyte count and a post-exercise lymphopenia. In addition, chemotactic peptide-induced cellular calcium transients were enhanced post-exercise while oxidative burst and phagocytosis were decreased. These results suggest that magnesium is an important modulator of immune cell function under in vitro conditions. However, a magnesium supplementation seems to be unable to prevent any exercise-associated alterations in immune cell function in athletes with balanced magnesium status.

On the significance of magnesium in extreme physical stress.

In a double-blind randomized study, 23 competitive triathletes competing in an event consisting of a 500-meter swim, a 20-km bicycle race, and a 5-km run were studied after 4-week supplementation with placebo or 17 mmol/d Mg orotate. The tests were carried out without a break. Blood was collected before and after the test, and between the different events for assaying energy stress and membrane metabolism. Swimming, cycling, and running times decreased in the Mg-orotate group compared with the controls. Serum glucose concentration increased 87% during the test in the control group and 118% in the Mg-orotate group, while serum insulin increased 39% in the controls and decreased 65% in the Mg-orotate group. Venous O2 partial pressure increased 126% during the test in the controls and increased 208% in the Mg-orotate group. Venous CO2 partial pressure after the bicycle race decreased 66% (significantly) in the Mg-orotate group compared with 74% in the controls. Blood proton concentration decreased to 90% in the Mg-orotate group (significantly) compared with 98% in the controls. Blood leukocyte count increased from 5.92/nL to 11.0/nL in the controls and from 5.81/nL to 9.10/nL in the Mg-orotate group, a significant difference. Serum cortisol was lower in the Mg-orotate group before and after the test compared with the controls. CK catalytic concentration after the test was elevated 140% in the controls compared with 122% Mg-orotate group. The stress-induced modifications of energy and hormone metabolism described in this study indicate altered glucose utilization after Mg-Orotate supplementation and a reduced stress response without affecting competitive potential.

Enzyme catalytic concentrations in human plasma after a marathon.

Blood was obtained from 11 males participating in the Berlin marathon 1986, directly before and after the marathon, and on the three following days. Several observations were made: a) catalytic concentrations (activity) of creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP) increased directly after the marathon or on the three following days; b) Cholinesterase (CHE), amylase (AML) and gamma glutamyltransferase (GGT) decreased directly after the marathon; c) the time course of AP and LDH isoenzyme activity after the race indicated an elimination from plasma to lower values than those originally observed before the run.

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver.

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.

Thin-layer chromatography--the forgotten alternative for the quantitative determination of steroids.

We have developed a high performance thin layer chromatography (HPTLC) system for quantitative determination of androgens, corticosteroids, mineralocorticoids and gestagens on silicagel KG-60 HPTLC-plates with different solvent systems. A complete separation of androgens, gestagens and metabolites was achieved with dichlormethane/cyclohexane/acetone (70:25:5). Corticosteroids, mineralocorticoids and their derivatives were completely separated with diethylether/isooctane/isopropanol (70:25:5). The quantitative in situ fluorescence determination was carried out after post-chromatographic derivatization with cinnamic aldehyde, 4-dimethylaminobenzaldehyde and sulfuric acid. The sensitivity of detection was found between 500 pg and 1 ng per spot. The steroid metabolism as catalysed by rat liver microsomal oxidoreductases was measured by these procedures, and was compared with determination of steroids by gas chromatography (GC). According to HPTLC, steroids were reduced by NADPH-5 alpha-reductase (EC 1.3.1.4) in the order progesterone greater than testosterone greater than aldosterone greater than cortisol greater than corticosterone. The enzyme activities as measured by HPTLC agree well with those obtained by GC (r = 0.94). When turnover of enzyme assays, speed of determination, detection limit, application to labile steroids and costs of steroid determination are considered, all points speak in favour of HPTLC.

Properties and biochemical characterization of NADH 5 alpha-reductase from rat liver microsomes.

NADH 5 alpha-reductase is present in microsomes of various rat organs: heart and skeletal muscle, liver, adrenal glands, kidney, testes and prostate. The enzyme from rat liver microsomes utilizes B-hydrogen from the coenzyme NADH for steroid reduction. After solubilization of the enzyme with the nonionic detergent lubrol, phosphatidylcholine is necessary to restore the activity. This reactivation of the enzyme activity is paralleled by a corresponding increase of Vmax for testosterone (17 beta-hydroxy-4-androsten-3-one). Km and Vmax for testosterone change, Km and Vmax for the coenzyme NADH remain constant with an alteration of phosphate concentration in the incubation medium. The NADH 5 alpha-reductase is inhibited by numerous substances: amytal, phenobarbital, mepacrin, thenoyltrifluoracetone, gallic acid propyl ester, dicoumarol, pentachlorophenol, NADP and antibodies against rat liver NADPH ferrihemoprotein reductase. Antibodies against rat liver cytochrome-b5 reductase cause an activation of NADH 5 alpha-reductase.

Effect of medroxyprogesterone acetate on steroid reductases in rat liver.

Male and female rats were treated with medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-pregn-4-ene-3,20-dione, MPA) 600 mg/kg body weight i.p. daily for seven days. The steroid metabolizing enzymes in liver microsomes and liver homogenate were measured following MPA treatment and in control rats. The specific activity of NADPH- and NADH-5 alpha-reductase in female rats and of NADPH-5 alpha-reductase in male rats decreased by the treatment. NADP+- and NAD+-3-hydroxysteroid dehydrogenase activities were lower in female MPA rats if compared to untreated animals. In male rats only NADP+- and NAD+-3 beta-hydroxysteroid dehydrogenase activities (substrate 5 beta-dihydrotestosterone) were diminished by MPA administration. There was no effect on the cytoplasmatic 5 beta-reductases. Plasma concentrations of luteinising hormone (LH), testosterone and androstenedione were lowered by MPA treatment.

Systemic short-term fibrinolysis with high-dose streptokinase in acute myocardial infarction: time course of biochemical parameters.

The course of plasma catalytic activities of total creatine kinase, creatine kinase isoenzyme MB, total, cytoplasmatic and mitochondrial aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, glutamate dehydrogenase and concentrations of myoglobin, urea, acidic alpha 1-glycoprotein and creatinine were followed in 33 patients suffering from acute myocardial infarction. All patients were randomized in a double-blind, prospective study. One group (18 patients) was infused with streptokinase 1.5 X 10(6) units/90 minutes; the control group received routine continuous i.v. heparin treatment (1000 units/h). Ten hours after completion of the study protocol, treatment of both groups of patients was continued with heparin, 1000 units/h and Aspisol, 1 g/day2). Streptokinase treatment induced earlier wash-out and therefore earlier peak levels of several enzymes: total creatine kinase (11 hours), creatine kinase isoenzyme MB (6 hours), total and cytoplasmatic aspartate aminotransferase (6 hours) and lactate dehydrogenase (9 hours). Total creatine kinase peak catalytic activity and myoglobin peak concentration were higher in the group receiving thrombolytic therapy. A significantly different course of catalytic activity between both treatment groups was found for total creatine kinase and creatine kinase isoenzyme MB, total and cytosolic aspartate aminotransferase, lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase. The course of mitochondrial aspartate aminotransferase catalytic activity was different only 12 hours after the beginning of treatment. The shift of several catalytic activities to an earlier peak level in plasma may indicate reperfusion of ischaemic myocardium due to thrombolytic therapy.

Rat liver 3-oxo-5 alpha-steroid delta 4-dehydrogenase. Modulation of enzyme activity by changes in phosphorylation state.

3-Oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase ("NADH-5 alpha-reductase", EC 1.3.1.?) is rapidly inactivated in the presence of 17 beta-hydroxy-4-androsten-3-one (testosterone). This activation is prevented by increasing the phosphate concentration. When the enzyme assay is carried out in Tris-HCl, only a small activity (1.7 nmol X min-1 X mg-1) is observed which may be further decreased by addition of phosphatases. Addition of the phosphatase inhibitor dextran sulphate or ATP, Mg++ and c-AMP results in a significant increase of activity (228% and 273%, respectively) compared with the Tris-HCl control. Glycerol 2-phosphate and glycerol 3-phosphate have a stabilizing effect on 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase by decreasing the Km towards the substrate testosterone from 1.2 X 10(-5) mol/l to 3.3 X 10(-6) mol/l. V remains unchanged. Half maximal velocity (testosterone reduction) is achieved with 20 mumol/l glycerol 2-phosphate and glycerol 3-phosphate. Addition of c-AMP dependent protein kinase (EC 2.7.1.37) to a microsomal preparation pretreated with alkaline phosphatase (EC 3.1.3.1) results in a significant increase of 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase activity compared with the control.

Plasma aldosterone, cortisol and electrolyte concentrations in physical exercise after magnesium supplementation.

Plasma aldosterone, cortisol, sodium (Na), potassium (K), calcium (Ca), magnesium (Mg) as well as urine and sweat Na, K, Ca and Mg concentrations were measured in nine male healthy persons during an one hour ergometer exercise before and after a fourteen day magnesium aspartate (Mg) supplementation. The usual aldosterone and cortisol increase during exercise was not observed and cortisol concentration was significantly lower after Mg supplementation. Na and K in plasma increased during the exercise; these changes were not affected by Mg. The Mg concentration was elevated in plasma and erythrocytes after Mg supplementation. During the ergometer course plasma Mg was unchanged but decreased significantly in the red blood cells. Mg and K concentration in sweat decreased during the exercise. No influence of Mg on urinary electrolyte excretion was observed.

Creatine kinase isoenzyme MB determination on the ACA: dependence on serum matrix and other effectors.

Creatine kinase isoenzyme MB catalytic activities in human serum, determined by ACA ion exchange chromatography and immunoinhibition, differ significantly, the correlation coefficient being 0.88. The reasons for this variation are interference of antibodies with the creatine kinase B subunit in the immunoinhibition assay, nonreproducible elution of creatine kinase isoenzyme MB from the ion exchange resin in the ACA pack, due to varying protein concentrations in the serum samples and increasing elution of creatine kinase isoenzyme MM from the ion exchange column caused by a preceding partial inactivation of creatine kinase isoenzyme MM. Pretreatment of serum samples with a solution containing magnesium sulphate, maleate and 2-oxoglutarate (solution A) prior to determination of creatine kinase isoenzyme MB catalytic activities on the ACA significantly improves the sensitivity and specificity of the method; the correlation coefficient for the values from the ACA and immunoinhibition then becomes 0.92. Dilution of serum samples with bovine serum albumin solution is now practicable.

Circadian rhythm of acid phosphatase activity in rat liver microsomes and dependence of NADH 5 alpha-reductase on phosphatase activity.

The specific activity of acid phosphatase in male and female rats follows a circadian rhythm. Preincubation of liver microsomes with testosterone led to an increase of phosphatase activity and a loss of circadian rhythm. NADH 5 alpha-reductase was inactivated by several animal and bacterial acid and alkaline phosphatases while the acid phosphatase from potatoes was ineffective. The extent of inhibition depends on the course of circadian rhythm of NADH 5 alpha-reductase activity. Preincubation of microsomes in the presence of testosterone inhibited the NADH 5 alpha-reduction of testosterone. No such inhibition was observed after preincubation of microsomes with progesterone.

Effect of allylisopropylacetamide and Sedormid on enzymes of steroid metabolism in rat liver.

Female rats, treated with allylisopropylacetamide (AIA) showed a marked decrease of hepatic NADH-5 alpha-reductase, NADPH-5 alpha-reductase, NAD+- and NADP+-3 alpha-hydroxysteroid dehydrogenase activities and an increase of the activity of NADH- and NADPH-5 beta-reductase and NAD+ and NADP+-3 beta-hydroxysteroid dehydrogenase. Administration of Sedormid decreased the activities of 5 alpha-reductases and 3 alpha-hydroxysteroid dehydrogenases (substrate, 5 alpha-dihydrotestosterone) and increased the activity of NADH-5 beta-reductase, whereas no effect was seen on NADPH-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase.

Effect of 5 alpha-dihydrocorticoids on enzymes of gluconeogenesis in rat liver.

5 alpha-Dihydrocortisol (11 beta, 17, 21-trihydroxy-5 alpha-pregnane-3,20-dione), 5 alpha-dihydrocorticosterone (11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione) as well as cortisol (11 beta, 17, 21-trihydroxy-4-pregnene-3,20-dione) and corticosterone (11 beta, 21-dihydroxy-4-pregnene-3,20-dione) were administered for seven days to male rats. Blood glucose increased in cortisol- and corticosterone-treated rats and blood insulin decreased after 5 alpha-dihydrocorticosteroid treatment. In the liver, total protein was elevated after cortisol, corticosterone and 5 alpha-dihydrocorticosterone application. Phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in liver were significantly lowered after treatment with 5 alpha-dihydrocortisol and 5 alpha-dihydrocorticosterone.

Further evidence for the participation of 5 beta-steroids in the development of a porphyria induced by hexachlorobenzene.

Adult female rats were fed with a diet containing hexachlorobenzene to induce a porphyria. 5 alpha/5 beta-ratios of androstane steroids in blood were 0.61 +/- 0.13 in porphyric rats and 1.05 +/- 0.35 in normal rats. Etiocholanolone treatment of rats was ineffective in generation of a porphyria. The activity of microsomal glucuronyltransferase increased from 0.20 +/- 0.04 mU/mg to 0.77 +/- 0.23 mU/mg by this treatment. Administration of Flutamide (4'-nitro-3'-trifluoromethylisobutyranilide) to porphyric rats resulted in a decline of porphyrin excretion by 55%. The hepatic NADH-5 beta-reductase was strongly inhibited by this drug, whereas NADPH-5 beta-reductase displayed a slightly increased activity. These findings are further evidence for the involvement of 5 beta-steroids in the formation of porphyria.

Affinity labeling of rat liver microsomal NADH-5 alpha-reductase with a nucleoside analogue.

A time dependent irreversible loss of rat liver microsomal NADH-5 alpha-reductase activity is caused by incubation of microsomes with the nucleoside 5'-p-fluorosulfonylbenzoyladenosine (FSA). The decrease of activity is dependent on FSA concentration and shows first order kinetics. Presence of NADH partially stabilizes the NADH-5 alpha-reductase. Thioglycerol present before incubation prevents loss of activity, and stops decrease of activity when added during incubation. NADPH-5 alpha-reductase (E.C. 1.3.1.4) and NADPH-cytochrome c reductase (E.C. 1.6.2.4) are not influenced while NADH-cytochrome c reductase (E.C. 1.6.99.3) is inhibited by FSA. Evidently FSA causes inactivation of the enzymes by binding to the NADH-binding site. Affinity labeling by FSA thus clearly distinguishes between NADH- and NADPH-dependent 5 alpha-reductases from rat liver microsomes.

The involvement of porphyrogenic steroids in the development of experimental porphyria.

Hexachlorobenzene alters the hepatic steroid metabolism, and it was suggested that a porphyria was induced by overproduction of 5 beta-H-steroids. Structurally similar chlorinated hydrocarbons (pentachlorobenzene, pentachlorophenol, 2,4,5-trichlorophenol) without porphyrogenic activity did not affect the steroid metabolism in rat liver.