Arne N. Wick

The oxidation of mannitol.

Summary Radioactive mannitol has been administered to rats with the following results: 1. When given orally, about 50% of the mannitol carbon is recovered in the expired CO2 in 12 hours. 2. With intraperitoneal injection, the mannitol is largely recovered in urine with only 2-3% of the mannitol carbon recovered in the expired CO2. 3. By injection directly into the portal system by way of the spleen, oxidation comparable to or greater than that obtained by oral administration is obtained. It is concluded from these results that mannitol can be oxidized by the body if it is administered by a route that makes it first available to the liver, as in oral feedings.

NAD-dependent l-fucose dehydrogenase from sheep liver☆

Abstract l -Fucose dehydrogenase has been purified about 45-fold from the soluble fraction of sheep liver. The enzyme accepts either l -fucose or d -arabinose as substrates but is specific for NAD as coenzyme. Disc-gel clectrophoresis failed to separate the activities obtained with l -fucose and d -arabinose substrates. The pH maximum is 10.4; at this pH, the Km values are 1.5, 7.2, and 0.19 m m for l -fucose, d -arabinose, and NAD, respectively. The product obtained with the l -fucose substrate under these conditions was l -fuconic acid; with a d -arabinose substrate, d -arabonic acid was formed, d -Glucose, d -galactose, d -xylose, and l -arabinose were not substrates for this enzyme.

Stimulation of glucose oxidation by liver homogenates with hexose 6-phosphates.

Abstract Liver homogenates incubated with labeled glucose yield increasing values of 14CO2 as the concentration either of D -glucose 6-phosphate, 2-deoxy- D -glucose 6-phosphate, or D -glucosamine 6-phosphate is increased. The maximal stimulation of 14CO2 production with increasing hexose phosphate concentration comes from systems with added NADP+; added NAD+ does not significantly change values of expired 14CO2 relative to the controls, while added ATP caused dilution in the CO2 labeling. 2-Deoxy-glucose 6-phosphate was the most effective of the hexose phosphates stimulating 14CO2 production, followed by glucosamine 6-phosphate. The former was found to stimulate the production of an unidentified lactone, which, however, was not 6-phosphogluconolactone. 6-Phosphogluconate caused some 14CO2 increase, but this was less than that due to glucose 6-phosphate. None of the non-phosphorylated compounds tested, such as 2-deoxy- D -glucose, causes the stimulation of 14CO2 production, nor does inorganic phosphate or pyrophosphate. D -Gluconate addition results in a slight dilution of the CO2 labeling. Time studies on the rate of 14CO2 production indicate that the phosphate esters themselves are the cause of the increased 14 CO2 values. [I-14C]glucose, when used as substrate, gives about twice the value of expired 14CO2 relative to control as [6-14C]glucose in the presence of increasing concentrations of of glucose 6-phosphate, glucosamine 6-phosphate, or 2-deoxyglucose 6-phosphate. The stimulation of 14CO2 production by 2-deoxyglucose 6-phosphate in the presence of NADP+ was also found using kidney homogenates. Stimulation was not observed using homogenates prepared from heart, spleen or brain. The enzyme, glucose dehydrogenase (β- D -glucose:NAD(P) oxidoreductase, EC i.i.i. 47), has been found in kidney and, previously, in hepatic microsomes. No glucose dehydrogenase activity could be demonstrated in preparations from brain, spleen and heart. The results obtained are the opposite to those predicted on the basis of isotopic dilution by a metabolic intermediate, in addition to those predicted by enzyme inhibition caused by the phosphate esters employed in these experiments. This suggests the involvement of a pathway of glucose oxidation which does not involve glucose 6-phosphate as an intermediate. This pathway, under the conditions employed, is stimulated by NADP+ and certain hexose phosphates and inhibited by ATP.

The effect of fasting, alloxan diabetes, and streptozotocin diabetes on rat liver D-arabinose (L-fucose) dehydrogenase activity.

Abstract The specific activity of d -arabinose ( l -fucose) dehydrogenase is elevated 25, 16, and 20% in the livers of fasted, alloxan diabetic and streptozotocin diabetic rats, respectively, as compared to normal control values. The specific activity of hepatic glucose-6-phosphate dehydrogenase in both groups of diabetic animals was greatly decreased. The same results are obtained if the data are calculated on the basis of activity per gram of wet weight of liver or on the basis of activity per 100 grams body weight. The increase in specific activity of d -arabinose ( l -fucose) dehydrogenase under these conditions may indicate a preferential sparing of the enzyme reflecting a greater utilization of a pathway of l -fucose catabolism.

Dehydrogenation of glucose and xylose catalyzed by rat and sheep liver microsomal and soluble fractions

Abstract The soluble fraction (homogenates from which nuclei, mitochondria, and the microsomal fraction have been removed) of rat and sheep liver catalyze the formation of NADH and NADPH in the presence of a glucose substrate. The activity is obtained reproducibly from sheep liver preparations, but in rat liver soluble fractions it is extremely variable (0–3.8 μmoles NAD(P)H formed per hour per gram liver). The activity of the rat liver soluble fraction is stimulated by the presence of the bicarbonate anion. A comparison of the substrate specificity of purified microsomal glucose dehydrogenase and that of the rat liver soluble fraction, together with a study of the effect of glucose-6-P and the bicarbonate anion on the activity of the two preparations, shows that the glucose-dependent NAD(P)H formation catalyzed by the rat liver soluble fraction is not due to the presence of glucose dehydrogenase. Sheep liver soluble fractions may contain some glucose dehydrogenase activity, but 10-fold more activity is found in the sheep hepatic microsomal fraction. Glucose-6-P dehydrogenase, partially purified from the rat liver soluble fraction, catalyzes a slow oxidation of D-glucose. This activity is stimulated by the bicarbonate anion. However, the observed glucose oxidation catalyzed by glucose-6-P dehydrogenase is probably too low to account for the occurrence of glucose oxidative activity in the soluble fraction. A D-xylose dehydrogenase activity has been found in the soluble fraction of rate liver. This activity was formerly believed to be associated with microsomal glucose dehydrogenase alone. Data are reported which suggest that microsomal glucose dehydrogenase may play a metabolic role under conditions where glucokinase activity is low or absent. A role for the activity of the soluble fraction catalysis of glucose oxidation, at least in rat liver, is less likely.

Inhibition of Glucose Oxidation by 6-Deoxy-D-Glucose.

Summary 6-Deoxy-D-glucose has been shown to be an inhibitor of glucose oxidation in rat kidney slices, mouse adipose tissue, and rat diaphragm. The raio of 6-DOG to glucose which gave a 50% inhibition of glucose oxidation corresponded to 12:1 in rat kidney slices and rat diaphragm and 4:1 in mouse adipose tissue. The inhibition of glucose oxidation in mouse adipose tissue was shown to be reversible. Experiments using rat diaphragm tissue showed that 6-DOG inhibited uptake of glucose in proportion to decrease in glucose oxidation. Incubation of mouse adipose tissue with 6-DOG uniformly labeled with C14 did not result in formation of measurable amounts of C14O2. It is concluded that this compound is incapable of complete oxidation in this tissue. It is believed that the site of competitive inhibition is either at the cell entry mechanism or at the hexokinase reaction. Thus, the metabolic block produced by 6-DOG should be less complex than that produced by the 2-position modified sugars. The use of 6-DOG may be very useful for studying cell permeation mechanisms.

Effect of 6-deoxy-6-fluoroglucose on glucose permeation of the cell.

The processes involved in the permeation of glucose into muscle cells have received considerable attention in recent years. The possible ways of transfer may be roughly classified in two ways: (1) passage or diffusion in a chemically unmodified form, and (2) passage by the participation of a membrane carrier system possibly associated with coupling and uncoupling enzymes. It is questionable whether there is sufficient evidence a t present to support one hypothesis to the exclusion of the other. The use of glucose analogues has shown that the uptake of hexoses by muscle cells can be limited by factors of steric specificity.’ The use of modified sugars is not always a rewarding one, because one cannot always predict in advance whether or not a particular compound will be useful. Most of the compounds examined thus far have been of no value because of their inertness for one reason or another. However, 6-deoxy-6-fluoroglucose (hereafter referred to as 6-FG) is a compound with interesting physiological properties. In this report we present a summary of the effect of 6-FG on the intracellular transfer of glucose.

The occurrence of l-fucose (d-arabinose) dehydrogenase in selected vertebrate species

Abstract 1. 1. l -Fucose ( d -arabinose) dehydrogenase activity is found in the cytosol of the livers from eighteen species of vertebrates ranging from class Cyclostomata to class Mammalia. Some properties of selected enzymes are listed. 2. 2. The enzyme is found predominantly in liver, kidney and lung. No activity was found in brain, heart, pancreas, skeletal muscle, small intestines or testis. 3. 3. Most of the enzymes studied preferentially utilized NAD as coenzyme. Enzymes from rodents preferred NADP as coenzyme. It is suggested that the difference in coenzyme specificity may account for the differences in l -fucose metabolism observed between humans and rats.

Effect of Oxamate on Oxidation of Specifically Labeled Glucose by Ehrlich Ascites Carcinoma Cells.

Summary The oxidation of specifically labeled glucose (0.01 M) by whole Ehrlich ascites carcinoma cells in the presence of sodium oxamate (0.01 M, 0.03 M, 0.06 M) was investigated. With increase in concentration of oxamate, the expired C14O2 is strikingly increased with glucose-6-C14 substrate and essentially unchanged with glucose-1-C14 substrate. Simultaneously, the amounts of residual glucose increase and of accumulated lactate decrease.