Development and evaluation of a simplified method to collect blood samples to determine hemoglobin and hematocrit using chromatographic paper discs.

A simplified method for the simultaneous determination of hemoglobin and hematocrit using dried capillary blood collected on chromatographic paper discs was developed and evaluated under laboratory and field conditions. The paper disc method (PDM) was compared with traditional laboratory methods (TM) in blood samples collected from human subjects with a wide range of hemoglobin and hematocrit values. Associations between PDM and TM were highly significant (p less than 0.001). Correlation coefficients were 0.90454 for hemoglobin and 0.9266 for hematocrit and there were no significant differences between mean values obtained by both procedures. Hemoglobin levels in discs remained unchanged during a storage for 1 mo. Hematocrit values, however, were constant throughout a 6-mo evaluation. On average, the percent coefficients of variation were 3.4 and 8.3% for determinations of hemoglobin and hematocrit, respectively. It is concluded that PDM represents a practical alternative approach for assessing hematological status in the field.

Effects of training and methandrostenolone (an anabolic steroid) on energy metabolism in the guinea pig: changes in enzyme activities in gastrocnemius muscle and myocardium.

Modifications of enzyme activities (creatine kinase and its B subunit; adenylate kinase; hexokinase; phosphofructokinase; lactate dehydrogenase; malate dehydrogenase, isocitrate dehydrogenase; citrate synthase; acetylcarnitine transferase; beta-hydroxyacetyl-CoA dehydrogenase; cytochrome c oxidase) in gastrocnemius muscle and myocardium were reported after two forms of training with or without administration of anabolic steroid. Endurance training was on a horizontal motor-driven treadmill, 2 km X hr-1, 5 days a week for 0.5 hr per day for 5 weeks. In the case of power endurance training there was a slope of 45 degrees. Enzyme activities in controls and treated guinea pigs, as well as treatment-induced enzyme activity changes are time dependent. Some of these activities correlate linearly with one another; such correlations characterize the effect of adaptation. Endurance training and power endurance training in this study induce similar modifications and seem to differ essentially in the daily work load. The anabolic steroid methandrostenolone (dianabol) induces modifications which training does not bring about but which training at least partially eliminates.

Electrochemical detection of catecholamines in urine and plasma after separation with HPLC.

The catecholamines in plasma and urine were determined by electrochemical detection after separation on a HPLC column RCM 100 C 18. Linear calibration plots of epinephrine, norepinephrine and dopamine have been obtained in the range expected to appear in urine or plasma. Coefficients of variation (0.5 to 10%), recovery rates (60 to 80%) and the standard deviation were satisfactory, whereas plasma dopamine showed a greater variation (10-15%). The values ascertained by this technique were compared with those determined by the radioenzymatic assay of Da Prada et al. They showed a good correlation for norepinephrine r = 0.92, p less than 0.001 and epinephrine r = 0.80, p less than 0.01, but less for dopamine r = 0.1. Experimental details of isolation from plasma with Al2O3 adsorption and desorption with HClO4, and from urine with ion exchange chromatography on Bio-Rex 70 and elution with boric acid are described. Technical modifications which improve the method are reported. The optimized assay could reliably be employed in investigations of more than 3000 urine and plasma samples obtained from patients and athletes before and after exercise.

ATP-ADP-dependent phosphorylations of glycolysis metabolites, creatine and glycerol: their compartition and thermodynamic relationship in gastrocnemius muscle cell of exercised guinea pigs.

The concentrations of following metabolites were determined in freeze-clamped gastrocnemius muscle samples: glucose 1-phosphate, glucose 6-phosphate, glucose, fructose 1,6-diphosphate, fructose 6-phosphate, D-glyceraldehyde 3-phosphate. dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, glycerol 3-phosphate, glycerol, creatine phosphate, creatine, glycerate 3-phosphate, glycerate 2-phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, inorganic phosphate. The results showed that within the limits of experimental error, concentration homeostasis for this metabolites is founded at least in some cases on equilibria between enzymic transformations. Discrepancies between constant mass ratios measured in this study and equilibrium constants allow the free energy variation delta G to keep creatine phosphate at high concentration to be calculated. For the phosphoglycerate mutase system, the equilibrium constant in controls and trained animals is unchanged and corresponds to that in vitro. Training hindered glycolysis and favoured phosphorylation of creatine by glycerol 3-phosphate. Metabolites of the pyruvate kinase and hexokinase system cannot be homogeneously distributed in one space. The creatine kinase system is also separated from the hexokinase und pyruvate kinase system. A compartition of glycolytic process in gastrocnemius muscle seems to be inferred from these results.

Ultrastructural and morphometric investigations on the effects of training and administration of anabolic steroids on the myocardium of guinea pigs.

The effects on the myocardium of guinea pigs of a training program, of the administration of anabolic steroids (Dianabol), and of both combined were investigated at the ultrastructural level. Quantitative electron microscopy showed an enlargement of the sarcoplasmic space in all experimental groups and a disbalance of the mitochondrial/myofibrillar ratio in favor of the mitochondria, which was most pronounced after administration of anabolic steroids. The augmentation of the mitochondrial volume was assumed to occur by fusion, hypertrophy, and development of new mitochondria. When the administration of anabolic steroids and training were combined, some myocardial cells were pathologically altered; destructed mitochondria and aberrant myofibrils were found. Focally dehiscent intercalated discs and necrotic cells were also observed. It is suggested from these findings that the administration of anabolic steroids in competitive sports may lead to pathological alterations in the myocardium.

Extraction of two different protein kinase activities from bovine rod outer segments.

An optimization of the rod outer segment (ROS) preparation technique is described. The protein responsible for ATP-gamma 32P binding to bovine ROS was separated from the protein active with protamine on a DEAE Sephadex column. Molecular weight evaluation on a G 100 Sephadex column gave a value of 75,000 for the protein active with ROS, and 42,000 for that active with protamine. 1.25 mM c-AMP or c-GMP reduced the activity to 0.7 or 0.8 of the control respectively. 10 mM c-GMP doubled the yield of the active protein extracted from ROS.

Bioluminescent and fluorometric techniques for determinations of 19 metabolites of ADP/ATP-dependent transformations in energy metabolism in 200 (or 400) mg muscle.

A procedure is described for the determination of the following 19 metabolites in 200 mg gastrocnemius muscle from guinea pig: glucose 1-phosphate and 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, 3-phospho- and 2-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, glycerol, glycerol 3-phosphate, AMP, ADP, ATP, inorganic phosphate, creatine, creatine phosphate. Bioluminometric and fluorometric techniques for mono- and dinucleotide determinations were used. In the case of fluorometric measurement for NADH, 400 mg tissue were necessary. The coefficient of variation for assays on the same sample was 0.04 for bioluminometric techniques and 0.10 for fluorometric techniques.

Bioluminescent assay for determination of picomole amounts of AMP, ADP, and ATP in adipose and muscle tissue biopsies.

A method is described for quantitative determinations of AMP, ADP, and ATP in tissue samples weighing a few milligrams by utilizing constant bioluminescent light intensity after reagent mixing. The smallest measurable quantity of adenosine nucleotide amounts to 0.2 pmol (coefficient of variation = 0.05) in the bioluminescent assay.

Determination of metabolite compartition in hepatic cells by varying the redox state in vivo.

In the present study, metabolite (lactate, pyruvate, glycerol 3-phosphate, dihydroxyacetone phosphate) concentrations were measured in various redox states. The mathematical relations between metabolite concentrations in various redox states were expressed algebraically and studied. Models which provided separate lactate/pyruvate (L/P) and glycerol 3-phosphate/dihydroxyacetone phosphate (G/D) spaces correspond to the experimental results in the case of "reductants" (e.g. ethanol, acetaldehyde, dihydroxyacetone and acetate) or of "oxidizing agents" (e.g. pyruvate) of the cytosolic NAD-NADH. Crotonate injection caused an oxidation of cytosolic redox couples, but no separation of the lactate/pyruvate space from the glycerol 3-phosphate/dihydroxyacetone phosphate space may necessarily be inferred. Furthermore, the following statements could be made in both first cases: (i.e. of "reductants" and "oxidizing agents"): (a) Redox couples in L/P space and in G/D space (together L/P-G/D system) are in equilibrium; (b) Redox-equivalent transport from the L/P space to the G/D space is not subject to any velocity-limiting mechanism; (c) Substrates which transports redox-equivalents into and from the L/P-G/D system reach concentrations to values, which are in a linear relation to each other in this system; (d) It is possible that these substrates are regenerated in another system which is also in equilibrium and subject to statement c.

Rat-liver cell compartition as revealed by correlations between redox-quotient changes following alloxan treatment, starvation, carbohydrate- and fat-diet.

As reported elsewhere (FERAUDI, 1976a & b), we have studied the mathematical relations between metabolite concentrations in the rat liver at various redox states and expressed them algebraically. In the present work we have measured the liver-cell concentrations of lactate, pyruvate, glycerol 3-phosphate, dihydroxyacetone phosphate, malate, oxaloacetate, beta-hydroxybutyrate, acetoacetate, 2-oxoglutarate, ribulose 5-phosphate as pentose phosphates, gluconate 6-phosphate, isocitrate, aspartate in untreated and treated rats (alloxan-diabetic, insulin-treated alloxan-diabetic or starved rats as well as rats fed on carbohydrate- or fat diet). Through analysis of the algebraic correlation between metabolite concentrations, we arrived at the following statements: 1. Under certain physiological conditions the concentration of some metabolites in one compartment determines their total quantity in the cell; 2. NADP and NADPH are comparted within the cytosol; 3. Reduced cosubstrate/oxidized cosubstrate ratios of some enzymic reactions are under certain physiological conditions in mutual equilibrium; 4. Such relationships are first verified after treatment and therefore characterize the metabolite status.

Steady-state study of horse-liver ADH: detection of two kinetically heterogeneous components.

New theoretical considerations and a new approximation strategy were applied to the kinetic analysis of the experimental relationship between the reaction velocity in the steady state and the concentrations of ethanol and NAD. It could be shown that horse-liver ADH consists of two kinetically heterogeneous components.

Role of rat-liver alcohol dehydrogenase in the glycerol pathway to L-lactate in homogenates.

14C-Distribution in the C3-chain of L-lactate after incubation of various 14-C-labelled prescursors ([1-14C] and [3-14C] glycerol; [3-14C] glyceraldehyde and glycerid acid; D- and L-[3-14C] serine; [1-14C] fructose and [6-14C] glucose) with homogenate showed that (1) most of the glycerol is metabolized to L-lactate via D-glyceraldehyde; the remainder may possibly form L-lactate via dihydroxyacetone; (2) a part of D-glyceraldehyde and D-glyceric acid may produce glycerol before L-lactate is formed; (3) D and L-serine do not form measurable amounts of L-lactate via D-glyceric acid; (4) rat-liver alcohol dehydrogenase (E.N.1.1.1.1) does not contribute to conversion to L-lactate of dihydroxyacetone phosphate from fructose nor of D-glyceraldehyde phosphate from glucose.

Mechanism of action of D-glyceraldehyde-3-phosphate dehydrogenase.

A computer approximation with polynomial quotients was used to evaluate from experimental data the dependence of the initial velocity of D-glyceraldehyde-3-phosphate dehydrogenase reaction on the concentration of substrates. The initial velocity values were determined at optimum conditions, over a wide range of substrate concentrations and by interpolating the time curve of enzyme reaction as t leads to 0. A further computer approximation with polynomial quotients, without any implied hypothese, gave the best fit to the experimental results. The analysis of this final equation shows that two types of catalytic sites may exist. Due to the complexity of the system, the results are compatible either with the ordered binding or with rapid equilibrium random binding of substrates to each separate, but interacting type of sites. Previous experimental data showing the formation of abortive and dead-end complexes can be interpreted as kinetic effects, inherent in the mechanism. Results at variance with earlier data can be explained by the different experimental conditions.