Search

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.

Changes in the concentrations of vitamin D and its metabolites in plasma of healthy subjects orally given physiological doses of vitamin D2 by multivitamin or vitamin D liquid preparations were determined and the bioavailability of vitamin D was studied. Separative assay on the D2 and D3 compounds of vitamin D, 25-hydroxyvitamin D (25-OH-D), 24R,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] was performed in plasma of eight healthy male volunteers. When the concentrations of vitamin D and its metabolites in plasma of volunteers were assayed after daily oral administration of 400 IU of vitamin D2 in a form of multivitamin tablet for 1 week, the variations of vitamin D3 and its metabolites in plasma levels were very small. In contrast, the concentrations of 25-OH-D2 and 1,25(OH)2D2 slightly increased after the administration, while neither vitamin D2 nor 24,25(OH)2D2 was detected. A single dose of 4,000 IU of vitamin D2 was orally given to the volunteers in a form of a vitamin D liquid preparation and the hourly variations were observed during 24 h. These concentrations of vitamin D2, 25-OH-D2, and 1,25(OH)2D2 were slightly higher than those of the repeated doses. The result suggests that even the high dose of 4,000 IU has little effect on the plasma levels of vitamin D2 and its metabolites by a single dose, indicating a low risk for hypervitaminosis D.

Xanthine oxidase activity in the rat brain was measured by means of high-performance liquid chromatography with electrochemical detection of uric acid. Cerebral ischemia was produced by a four-vessel occlusion method. In the control rat, the enzyme activity was 0.87 +/- 0.13 nmol/gm wet weight/min at 25 degrees C (mean +/- standard deviation), of which 92.4% was associated with the nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase form and only 7.6% with the oxygen-dependent superoxide-producing oxidase form. However, the ratio of the latter form increased to 43.7% after 30 minutes of global ischemia, despite the total xanthine oxidase activity remaining the same. Thus, it was revealed that uric acid can be synthesized in the rat brain and that cerebral ischemia induced the conversion of xanthine oxidase from an NAD-dependent dehydrogenase to an oxygen-dependent superoxide-producing oxidase. Although the xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in various ischemic organs other than brain, the results of the present study suggest the involvement of the oxygen free radicals generated from this pathway in the pathogenesis of the ischemic injury of the rat brain.

In order to assess the role of thiamin on ethanol metabolism, changes in the activity of hepatic microsomal ethanol oxidizing system (MEOS) were measured in rats fed thiamin deficient diet for 4-6 weeks. In thiamin deficient rats, the activity of hepatic MEOS was significantly decreased as compared with control rats. In vitro addition of thiamin or thiamin pyrophosphate (TPP) caused the restoration of the decreased MEOS activity, and this effect was dependent on the concentration of thiamin in rat liver microsomal fraction. Thus, thiamin partly involves in the oxidation of ethanol, and chronic thiamin deficiency predisposes to impair the ethanol oxidation, and consequently to increase the toxicity due to ethanol.

1. The binding kinetics for [35S]thiamine pyrophosphate to transketolase and the dependency of transketolase on divalent cations for activity were investigated. 2. With Scatchard analysis, dissociation constant (Kd) and n value were calculated to be 0.2 x 10(-6) M and 0.66 respectively. 3. The activity of the reconstituted enzyme increased in the order of Co2+ less than Mn2+ less than Ca2+ less than Mg2+. The native transketolase contained Mg2+ in its molecular structure.

To clarify the effect of ethanol on thiamine metabolism and activity of transketolase, a thiamine dependent enzyme, in vivo and in vitro experiments were carried out. For the in vivo experiment, rats were separated into four groups: Group 1, thiamine sufficient diet with no ethanol administered; group 2, thiamine sufficient diet and ethanol administered by stomach tube; group 3, thiamine deficient diet with no ethanol administered; group 4, thiamine deficient diet and ethanol administered. After three weeks, the rats were killed, then thiamine levels and the activity of transketolase were assayed. Thiamine concentration in brains and livers of ethanol fed rats were lower than that in control rats. Urinary thiamine was higher in ethanol administered rats than in controls. The increase in the liver transketolase activity by the in vitro addition of cocarboxylase (TPP effect) was higher in group 3 than in group 4 although original transketolase activity was lower in group 4. For the in vitro experiment, various amounts of ethanol or acetaldehyde were added to the supernatant of brain and liver of intact rats, and transketolase activity was determined. Inhibition by acetaldehyde to transketolas was about 10 times higher than that by ethanol.

We studied the utility of a trial multivitamin preparation 'NK-041' for total parenteral nutrition. This preparation was used in 260 surgical patients during total parenteral nutrition, and changes in their blood vitamin levels were analyzed. Blood levels of all vitamins administered varied within the respective normal ranges, with few exceptional patients. During the administration of NK-041, avitaminosis, hypervitaminosis and side-effects were not observed. Laboratory examinations demonstrated that no abnormalities attributable to this preparation had arisen in these patients. Thus, these results seem to warrant the conclusion that NK-041 is useful as a multivitamin preparation for total parenteral nutrition, with no adverse effects.

This experiment was carried out to investigate the effects of intravenous injection of an essential trace-element preparation (TE-5) on iron, zinc, copper and manganese concentrations, and blood biochemical and hematological parameters in rats. The rats were treated by intravenous injection of TE-5 for 7 days and the following results were obtained: 1) Neither a 0.04 nor a 0.4 ml/kg/day injection of TE-5 affected the iron, zinc, copper and manganese concentrations in tissues. 2) At a higher dose (1.2 ml/kg/day), iron concentrations in liver and spleen, zinc concentrations in liver, kidney, tibia and plasma, copper concentrations in heart, kidney and whole blood, and manganese concentrations in brain, heart, spleen, kidney, femoral muscle, tibia and whole blood increased. 3) At the highest does (4 ml/kg/day), all rats died and iron, zinc, copper and manganese concentrations in tissues increased remarkably. 4) With injections of TE-5 (0.04-1.2 ml/kg/day), hemoglobin, hematocrit, alkaline phosphatase activity and blood urea nitrogen decreased slightly. These results suggest that iron, zinc, copper and manganese concentrations, and blood biochemical and hematological parameters are maintained at doses up to 0.4 ml/kg/day of TE-5, but that doses higher than that destroy homeostasis.

In an attempt to elucidate the etiology and pathogenesis of 'Itai-Itai' disease, the relationship between bone and kidney lesions in cadmium-poisoned rats was determined using the standard renal clearance technique. Calcium and cadmium contents in the femur and kidney were also estimated. The severity of renal lesions was histologically evident both in the tubule and glomerulus, and paralleled the decrease in GFR and the increase in FF. There was no significant change in fractional excretion of phosphate, but fractional excretion of calcium increased in the cadmium-poisoned rats. The secondary hyperparathyroidism followed by uremic renal osteodystrophy did not result in an osteomalacia. Thus, it is apparent that disturbance in calcium reabsorption in the tubules plays a role in the loss of calcium, and cadmium associated with low levels of calcium has a direct effect on the bone-causing osteomalacia.

We investigated the differences in manganese concentration and distribution in the brains of rats administered MnCl2 perorally, intravenously and intraperitoneally for one week. Forty two male Wistar rats were divided into eight groups, designated A to H. Groups A (n = 5) and B (n = 7) were maintained with synthetic diets, with 50 and 1000 mg manganese per kg diets, respectively. Groups C (n = 5), D (n = 5) and E (n = 5) were administered manganese intravenously with dosages of 0.088, 0.88 and 2.2 mg/kg.b.w./day, respectively. Groups F (n = 5), G (n = 5) and H (n = 5) were administered manganese intraperitoneally with dosages of 0.088, 0.88 and 2.2 mg/kg.b.w/day, respectively. Four brain regions (cerebrum, cerebellum, basal ganglia and the remainder) were analyzed by atomic absorption spectrophotometry for manganese. The manganese concentrations in basal ganglia were increased proportionally to the administration dosages in intravenous administration groups (C, D and E). At the highest dosage of manganese administration, manganese concentrations in cerebrum and basal ganglia were higher with intravenous administration of manganese than with intraperitoneal administration. In addition, manganese concentrations were hardly increased in peroral administration groups. Therefore, we suggested that basal ganglia are vulnerable to manganese exposure and that liver and intestine might play the important roles in the reduction of manganese accumulation in the central nervous system.

In convulsive pigeons on a diet of polished rice, a significant decrease of thiamine and calcium in the central nervous system rather than in peripheral nervous system was observed. In subcellular fractions of telencephalon in these same pigeons, thiamine and calcium levels decreased in the myelin-membrane and synaptosomal fractions. When radioactive thiamine was injected into the pieon during a convulsion, radio-activity was most prominent in the telencephalon and in subcellular fractions, in the myelin-membrane and synaptosomal fractions of telencephalon immediately after recovery from the convulsion. When calsium was added to the polished rice, the pigeons never had such convulsions. Thiamine and calsium concentrations in myelin-membrane and synaptosomal fractions of telencephalon of calcium-rice fed pigeons were significantly higher than in convulsive pigeons. It is suggested that calsium plays a role in binding the protein and thiamine which is related to the sodium transport system of excitable membranes.

Primary hypomagnesemia with secondary hypocalcemia (PHSH) is a rare type of hypocalcemic disorder which occurs in early infancy and is clinically characterized by recurrent tetany and/or convulsion. In this paper, a male infant with PHSH who had frequent seizures at the age of 9 days is described. Besides PHSH, several illnesses in infancy are manifested by hypomagnesemia and hypocalcemia, i.e. transient neonatal hypomagnesemic hypocalcemia, congenital renal or hepatic insufficiencies, magnesium-losing nephropathy, combined impairments of intestinal absorption and renal reabsorption of magnesium. PHSH is to be differentiated from these illnesses by the demonstration of a combination of the following findings; hypocalcemia refractory to calcium but responsive to magnesium, continuous requirement for magnesium supplementation to maintain normocalcemia, lack of hypermagnesiuria and/or impaired intestinal absorption of magnesium. Twenty cases from the literature were found to exhibit these characteristics. The clinical, biochemical, and endocrine features of PHSH are summarized on the basis of a review of the data of these and the present case. No associated illness was known in the afflicted infants or mothers. Both male and female infants were afflicted at a male to female ratio of 15:6. Some siblings were afflicted but none of the parents or relatives. The onset of tetany and/or convulsion was between the 9th day and 4th month, which is later than that of other neonatal hypocalcemic illnesses. Hypocalcemia was more pronounced than other infantile hypocalcemic illnesses. The role of the parathyroid hormone in the pathogenesis of hypocalcemia has been studied in several studies but no unifying concepts have yet been established.

Thirty-one male SD rats, six weeks old, were fed a trace element-deficient diet for two weeks and then divided into three groups and maintained for 1 week as follows: group A with total parenteral nutrition (TPN) without supplementation of trace elements, group B with TPN supplemented with the following 5 trace elements ... iron, zinc, copper, manganese and iodine, and group C with a diet free of the above five trace elements. Another group of eight rats was fed a diet supplemented with the above five trace elements for three weeks as a control (group D). Feeding or TPN without supplementation of trace elements evoked microcytic hypochromic anemia and significant decreases in iron concentrations in plasma and tissues (groups A and C). Supplementation of trace elements in the TPN solution showed a tendency to cure anemia and a significant increase in the iron concentration in tibia (group B). Decreases in the zinc or copper concentrations in plasma and tissues during TPN without trace elements were prevented by supplementation of trace elements in the TPN solution (group B). The plasma zinc and copper concentrations correlated well with their levels in liver, kidney and tibia. Manganese deficiency was not recognized in this investigation (groups A and C), though supplementation of trace elements in the TPN solution increased tissue manganese concentration (group B). Feeding or TPN without supplementation of trace elements induced decreases in plasma triiodothyronine and thyroxine (groups A and C). Supplementation of trace elements in the TPN solution showed a tendency to increase plasma thyroxine (group B).(ABSTRACT TRUNCATED AT 250 WORDS)

Two children are described who suffered from episodes of metabolic acidosis and progressive mental and motor deterioration. The patients showed periodic elevation of blood lactate, pyruvate and alanine, which was accompanied by vomiting, hypotonia or convulsions. The concentrations of lactate and pyruvate in cerebrospinal fluid were found to be increased. Liver biopsies revealed a decrease in pyruvate carboxylase activity and normal pyruvate decarboxylase activity. No inhibitor of TPP-ATP phosphoryl transferase was detected in urine from the patients. These findings suggest that congenital lactic acidosis due to pyruvate carboxylase deficiency is probably a different disease entity from Leigh's encephalomyelopathy. A possible mechanism of brain damage caused by a defect in pyruvate carboxylase is postulated.

To clarify the effect of polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane (DDT) and other organochloride compounds on thiamine metabolism, rats were separated into six groups and placed on different admixed diets. Group 1 was fed a normal diet; group 2, a PCB-supplemented diet; group 3, a DDT-supplemented diet; group 4, a benzenehexachloride-supplemented (BHC) diet. After 50 days of these dietary regimens all animals were sacrificed and various studies were done on the excised tissues. In PCB-treated rats, thiamine levels in blood, liver and sciatic nerve decreased, transketolase activity in erythrocytes and liver decreased and TPP effect in erythrocytes and liver increased. In DDT-treated rats, thiamine levels in blood, brain and liver decreased, transketolase activity in brain and liver decreased and TPP effect in brain and liver increased. In other groups, no significant changes were observed regarding thiamine metabolism. Our findings provide direct evidence that changes occur in thiamine metabolism in PCB- and DDT-poisoned rats.

To clarify the mechanism of lipid peroxide formation in polychlorinated biphenyls (PCB)-poisoned rats, the following two experiments were carried out. Experiment No. 1: Rats were separated into three groups. Group 1 was fed a normal diet, group 2 was fed a PCB-supplemented diet, and group 3 was fed a dichlorodiphenyltrichloroethane (DDT)-supplemented diet. After 5 months, the rats were killed. The thiobarbituric acid (TBA) values in livers of the PCB- and DDT-exposed rats had increased. The activity of catalase was increased in the PCB-fed rats but decreased after the administered of DDT. The glutathione peroxidase activity was decreased only in the PCB-administered rats. These results indicate that PCB and DDT have some effects to enhance lipid oxidation. It is probable that the decrease in glutathione peroxidase is the major reason for the increase of lipid oxidation in PCB-poisoned rats. The mechanism of lipid peroxidate production in DDT-poisoned rats could be different from the case of PCB poisoning. Experiment No. 2: Rats were separated into two groups. To one group, normal diet was given and to the other group PCB-supplemented diet was given. After 1 month, the rats were killed. In PCB-exposed rats, activities of glutathione reductase and glutathione S-transferase were increased. The increase in glutathione reductase and glutathione S-transferase were increased. The increase in glutathione reductase could be a compensation for a decrease in glutathione peroxidase. It is probable that PCB is metabolized to make glutathione conjugates by the action of glutathione S-transferase.

Extracts of tissue fluids from a patient with subacute necrotizing encephalomyelopathy inhibit thiamine pyrophosphate-adenosine triphosphate phosphotransferase of rat brain. Brain tissue from the patient, in contrast to normal brain tissue, contained essentially no thiamine triphosphate, although thiamine and its other phosphate esters were present in normal concentrations. These findings suggest a relation between this disease and thiamine triphosphate.