A glucokinase (EC 18.104.22.168) was purified from Bacillus stearothermophilus, a moderate thermophile. The native glucokinase, Mr about 68, 000, consists of two identical subunits. This enzyme is relatively temperature- and pH-stable, the optimal pH range for the enzymatic activity being within the stable pH range. It selectively catalyzes the phosphorylation of glucose rather than other kinds of hexoses. The Km values were estimated to be 1×10-4M for glucose and 0.5×10-4M for ATP. It shows no ATPase activity. The high stability, high substrate specificity, low Km values for glucose and ATP, and the lack of ATPase activity make this enzyme advantageous for use in clinical chemical analysis.
We applied glucokinase (EC 22.214.171.124) from the thermophilic bacterium Bacillus stearothermophilus that we described in a previous paper, to the analysis of glucose in serum and urine, in combination withglucose-6-phosphate dehydrogenase (EC 126.96.36.199). We found the analytical precision, specificity, and sensitivity to be satisfactory. Results obtained with this method correlated well (r=0.998 for serum and r=0.997 for urine) with those obtained by a currently available method in which hexokinase (EC 188.8.131.52) is used (hexokinase method), and the working solution of the reagent consisting of the enzyme was far more stable than that of the reagent based on the hexokinase method.
An enzymic reagent, that has long-term stability even in the liquid state, was successfully employed for the measurement of serum creatine kinase (CK, EC 184.108.40.206) activity. The enzyme used was the thermostable glucokinase (GlcK, EC 220.127.116.11) obtained from the thermophile Bacillus stearothermophilus. The reagent was found to be stable in solution for about one month at 6°C and for about one week at 30°C. This substitution of glucokinase for the hexokinase of the most commonly used hexokinase-glucose-6-phosphate dehydrogenase (HK-G6PDH) method results in a remarkable improvement of the method. The CK activity measured by the GlcK-G6PDH method was linear up to about 2, 000U/liter at 37°C. The GlcK-G6PDH method was found to give a satisfactory precision and reproducibility (coefficient of variation less than 2.17%). Over a wide range of CK activity, an excellent agreement was obtained between the GlcK-G6PDH and the HK-G6PDH methods. Furthermore several coexistents and anticoagulants were found to have little effect on the measured value of CK activity by the GlcK-G6PDH method.
High density lipoprotein 3 (HDL3=1.12<d<1.21) was modified with dilinoleoylphosphatidylcholine, and the effects of these modified HDL3 on the removal of [3H]cholesterol from rat peritoneal macrophages were studied. On incubation of HDL3 with polyenephosphatidylcholine (PPC) vesicles, phospholipid-rich high density lipoproteins (1.18<d<1.24) were formed. The apolipoprotein AI (apo AI) and AII (apo AII) contents of these modified HDL3 were about 3 to 40% of those of native HDL3. Removal of [3H]cholesterol from macrophages was investigated using rat peritoneal macrophages prelabeled with [3H]cholesterol emulsion or with [3H]cholesterol oleate-labeled acetylated low density lipoproteins (1.019<d<1.063). The modified HDL3s facilitated the removal of [3H]cholesterol from macrophages. The extent of removal of [3H]cholesterol from macrophages was higher with modified HDL3 than with native HDL3, and among modified HDL3 subfractions, fractions IV and V, whose ratio of apo AI to phospholipid (AI/PL) were 0.44 and 0.58, respectively, were more efficient [3H]cholesterol acceptors than native HDL3 (AI/PL=1.46). These results indicate that the effectiveness of HDL3 in removing [3H]cholesterol from macrophages was enhanced by modification with PPC.
The relationship between the blood NAD and NADP levels and the niacin equivalent intake was investigated using women students. The daily niacin equivalent intake was 151±64μmol (18.5±7.5mg in terms of nicotinamide, mean±SD). The blood NAD and NADP levels were 34.7±7.0 and 19.3±2.3nmol/ml whole blood, respectively. The blood NAD level was proportional to the blood NADP level. Both blood levels were also proportional to the hemoglobin content, and the NAD and NADP levels per g hemoglobin were 233.7±44.7 and 156.3±25.5nmol. The blood NAD and NADP levels increased with an increase in the niacin equivalent intake. From these results, it is concluded that the blood total pyridine nucleotide level (NAD+NADP) reflects the niacin equivalent intake. Accordingly, the blood pyridine nucleotide level may be useful as an index for niacin nutrition in humans.
Dietary fat varies widely in both quality and quantity. In diets of poor income groups in India half of the total fat intake is obtained from edible oils. Also, there are regional preferences for these oils. The levels of essential fatty acids (EFA) vary widely in different edible oils. Hence a study was undertaken to investigate the effects of intake of different edible oils on EFA nutritional status in rats. Weanling rats were fed different edible oils in a casein-based diet for a period of five weeks. The EFA nutritional status as assessed from the plasma total fatty acid composition was normal in rats fed safflower (SO) or groundnut (GNO) oils. However, the fatty acid profile was suggestive of EFA deficiency in rats fed coconut (CO) or hydrogenated coconut (HCO) oils. In rats fed palmolein (PO), the EFA nutritive status was found to be marginally adequate. In the habitual cereal-pulse-based diets of Indians, a major part (80%) of dietary EFA is obtained from the invisible fat and only one fifth is derived from the visible fat. Therefore the marginally adequate levels of linoleic acid present in PO may be compensated by the linoleic acid of cereals and pulses present in the diet. It appears therefore that replacement of GNO or SO with PO in the Indian diets may not materially affect the EFA nutriture of the population. However, the effect of PO on a cereal-pulse-based diet in comparison to that of other edible oils needs to be evaluated.
Adult cats, adapted for 19 consecutive days to isocaloric semi-purified diets, were used to study the effects of three different levels of two types of dietary protein source (casein and soya) and the effects of a 2h immobilization period on various blood parameters. The ad libitum food intake and the daily dietary protein intake of cats fed casein diets were significantly higher than those of cats fed soya diets. Blood glucose content was not influenced by dietary protein source and daily protein intake, but pyridoxal-5′phosphate was decreased in cats fed the higher casein and soya levels (40.7% and 29.3%, respectively). Blood insulin and cortisol levels were significantly lower in soya than in casein groups, whereas pyridoxal-5′phosphate content increased progressively with the decreasing soya and casein levels in the diet. The serum dopamine-β-hydroxylase activity was found to be significantly higher in cats fed soya diets (22.1% and 25.7%, respectively) than in all cats fed casein diets. Following a 2h immobilization period, glucose and cortisol levels were significantly increased in casein and soya groups, and pyridoxal-5′phosphate content significantly decreased, whereas dopamine-β-hydroxylase activity and insulin levels remained unchanged. These results suggest that dietary protein sources influence blood parameters and that an acute stressful situation such as induced by a 2h immobilization, permits the dissociation on a biochemical basis of the physiological responses of blood pyridoxal-5′phosphate, glucose, and cortisol content from those of insulin level and dopamine-β-hydroxylase activity, whatever the dietary protein source and the protein level of the diet.
To evaluate the oxygen dependence of chick embryos at high and low oxygen concentrations, the gas exchange of embryos was measured with an oxygen consumption measuring apparatus. Oxygen consumption and the lipid peroxide levels in the blood, heart, and liver of chick embryos were measured after exposure of the embryos on the 13th day to high or low concentrations of oxygen (15-100%). Exposure to low concentrations (15-20%) resulted in a marked depression in oxygen consumption during the exposure time, which was followed by an increase in consumption to a level near the control. After exposure to high or low concentrations, a significant decrease in consumption was seen from day 14-21. A close relationship between oxygen consumption and the lipid peroxide level was observed in the blood of chicks on the 21st day.
Serum amino acid levels in young, adult, pregnant (day 12 and 21) and lactating (day 20) rats have been described and compared with plasma concentrations. Serum values systematically overestimate the amino acid plasma content. Changes reported for plasma amino acid levels in midpregnant and lactating rats are well reflected in the serum values. This is not the case for adult and late pregnant rats.
In order to explain decreased synthesis of myelin and reduced functional activity of brain as a result of early undernutrition, studies were undertaken to determine the activities of glycerol phosphate dehydrogenase and glutamine synthetase as markers of oligodendrocytes and astrocytes and that of lactic dehydrogenase as a multifunctional enzyme, at different stages of brain development in different brain regions, viz. cerebrum, cerebellum, and brain stem. Undernutrition was imposed in mother rats by feed restriction during the last week of pregnancy and through lactation. The undernourished group received 50% of the calories consumed by the control group which was reared on Lab Chow (22% protein) ad libitum. Results showed progressive increase in the activities of these enzymes in rat pups as a function of age. Undernutrition caused a significant decrease in the activities of glycerol phosphate dehydrogenase and glutamine synthetase in all the regions, especially during the 2nd and 3rd week of post-natal age. The magnitude of difference due to nutritional stress with respect to lactic dehydrogenase was relatively low. These results have possible implications regarding myelinogenesis and glutamic acid metabolism in undernourished rats.