Transketolase Activity Research Articles

Sensitivity of transketolase to the thiamin status of juvenile marine shrimp ( Penaeus monodon)

Muscle, hemolymph and hepatopancreas transketolase activities and their thiamin pyrophosphate (TPP) effects were assessed for their potential to determine the thiamin status of juvenile Penaeus monodon after a 9-week feeding trial. Transketolase activity increased in response to increasing thiamin supplementation, while TPP effects decreased with increasing dietary thiamin levels. The TPP effect showed a significant increment when the dietary thiamin was reduced from 20 mg/kg diet to no supplement. Thiamin requirement assessed by TPP effect as the criterion was lower than that by transketolase activity; the thiamin requirement estimated by the TPP effect of the muscle (13.3 mg/kg) and hemolymph (18.3 mg/kg) was similar to that of the growth results (12.9 mg/km). These data suggest that, like vertebrates, measurement of the TPP effect in the tissues of the marine crustacean is a more sensitive indicator of thiamin status than measurement of transketolase activity. Among all criteria examined, the hemolymph TPP effect was the most sensitive and specific indicator of thiamin status.

An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth

We show that a tktA tktB double mutant, which is devoid of the two known transketolase isoenzymes of Escherichia coli K-12, requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth. This pyridoxine requirement can also be satisfied by 4-hydroxy-L-threonine or glycolaldehyde. These results provide direct evidence that D-erythrose-4-phosphate is a precursor of the pyridine ring of pyridoxine. In addition, they show that the two major E. coli transketolase isoenzymes are not required for the biosynthesis of D-1-deoxyxylulose, which is thought to be another precursor of pyridoxine.

Thiamin intake, erythrocyte transketolase (EC2.2.1.1) activity and total erythrocyte thiamin in adolescents

The relationships between thiamin intake, erythrocyte transketolase (EC 2.2.1.1) activity coefficient (ETK-AC) and total erythrocyte thiamin were investigated in a group of adolescents (13 to 14 years old; nineteen boys, thirty-five girls). Thiamin intakes were calculated from 7 d weighed records, using food composition tables, and compared with those obtained by direct analysis of duplicate diets. Average 7 d calculated thiamin intakes were significantly lower than analysed intakes for both sexes. On an individual basis, calculated intakes ranged from 30 to 143% of corresponding analysed values. Analysed and calculated intakes were significantly correlated when expressed as mg/d; however, when expressed in terms of energy intake, the correlation was significant for males only. Thiamin intake appeared largely adequate when compared with current UK dietary recommendations (Department of Health, 1991), but the limitations of such comparisons are considered. The major food groups contributing to thiamin intake were examined and showed breakfast cereals to contribute more than 25% of dietary thiamin. A proportion of the subjects had ETK-AC values in ranges usually associated with marginal or severe thiamin deficiency. There was, however, no statistically significant relationship between erythrocyte thiamin and basal or stimulated transketolase activity, or between thiamin intake and either of the methods used to assess status. The need to re-evaluate indices of thiamin status is discussed.

Vitamin status of infants receiving long-term peritoneal dialysis

The oral vitamin intakes and blood vitamin concentrations of seven infants receiving long-term peritoneal dialysis were measured. The serum concentrations of vitamin A, vitamin B12, vitamin C and folic acid were determined. Thiamine and riboflavin were assessed by the activation of erythrocyte transketolase and erythrocyte glutathione reductase, respectively. Vitamin B6 was measured as plasma pyridoxal phosphate. All patients received a daily vitamin supplement devoid of vitamin A. Dietary vitamin intake was derived from infant formula. In all cases, the patients' blood concentrations of the water-soluble vitamins were equal to or greater than normal infant values. Serum vitamin A levels were elevated despite the lack of supplementation. The combined dietary/supplemental water-soluble vitamin intake of the patients exceeded the recommended daily allowance in all but one patient. These preliminary data emphasize the need to further evaluate the vitamin requirements of infants receiving long-term peritoneal dialysis.

Pentose utilization by the ruminal bacterium Ruminococcus albus.

Ruminococcus albus is an important fibrolytic ruminal bacteria which degrades hemicellulose and ferments the resulting pentose sugars. However, little information is available on the utilization of pentoses by this organism or the effect of hexose sugars on pentose metabolism. Enzymatic studies indicated that R. albus metabolized pentoses via the pentose phosphate pathway and possessed constitutive transketolase activity. Cellobiose was preferred over xylose and arabinose, and it appeared that the disaccharide decreased pentose metabolism by repression of transport activity and catabolic enzymes (isomerases and kinases). Glucose and xylose were co-utilized, and transport studies suggested that there was a common transport system for both sugars. In contrast, glucose was preferred over arabinose and the hexose noncompetitively inhibited the transport of arabinose. Since R. albus lacks a glucose phosphotransferase system, the inhibition of arabinose uptake could not be explained by previously described models of inducer exclusion involving such a system. Because accumulation of radiolabeled xylose, arabinose, and glucose proceeded in the absence of a proton motive force and since transport was correlated with the intracellular ATP concentration, it appeared that monosaccharide uptake was driven by ATP hydrolysis.

Polioencephalomalacia induced with amprolium in buffalo calves--clinicopathologic findings.

Polioencephalomalacia was induced in eight buffalo calves, 6-12 months old, by drenching amprolium (300 mg/kg body weight per day) for 29-55 days. Four buffalo calves of the same age group were drenched with tap water only and served as control. Blood samples were collected at different intervals during amprolium administration until the onset of clinical signs. Cerebrospinal fluid was also collected prior to amprolium administration and at the onset of clinical signs. A significant progressive decrease in erythrocyte transketolase (TK) activity and an increase in the percent of thiamine pyrophosphate (TPP) effect were observed in amprolium-fed calves during amprolium administration until the onset of clinical signs. There was a significant increase in blood lactate and blood pyruvate concentrations and a significant decrease in lactate/pyruvate ratio at the onset of clinical signs. Serum electrolyte (Na, Ca, P, Mg) concentrations showed no significant changes. However, the serum potassium concentration had decreased significantly at the onset of signs. The cerebrospinal fluid analyses revealed a significant increase in lactate and pyruvate concentrations and lactate/pyruvate ratio in amprolium-fed calves. The electrolytes (Na, K, Ca, P and Mg) of cerebrospinal fluid did not show any change. It is concluded that oral administration of amprolium (300 mg/kg body weight daily) for 4-6 weeks produces biochemical changes characteristic of polioencephalomalacia in buffalo calves.

Thiamin status of the elderly: dietary intake and thiamin pyrophosphate response.

The present study was undertaken to investigate the relationship between thiamin intake and its biochemical status in 60 free-living (30 male, 30 female) elderly subjects (> or = 65 years). Dietary intake was estimated using a 3-nonconsecutive-day food record. Biochemical status was assessed by measuring the stimulating effect of thiamin pyrophosphate (TPP) on transketolase activity (TPP effect) in hemolyzed erythrocytes, which is a functional test indicative of metabolic availability of thiamin. Average daily thiamin intake was above the recommended requirement (> 0.4 mg/1000 Kcal) for each gender; however, almost half of the total study population had TPP effect > 14%, suggesting thiamin deficiency. There was no correlation between thiamin intake and TPP effect. These findings raise questions about the reliability of dietary intake in assessing metabolic availability of thiamin in the elderly.

Biochemical and pathological findings on sheep and calves dying of experimental cerebrocortical necrosis.

For observing biochemical and morphopathological changes in experimental cerebrocortical necrosis, 6 sheep and 6 calves were given amprolium (600 mg/kg/day). All of the amprolium-dosed animals showed neurological signs and died on days 35 to 57 after the onset of daily administration. They were pathologically confirmed as cerebrocortical necrosis. Total thiamine levels in blood and tissues were markedly reduced at death. Blood total thiamine level decreased significantly on day 7 of amprolium administration, and reached to the level as low as that seen at death about 2 weeks before the onset of abnormal electroencephalograms. Significant decrease in a thiamine-dependent enzyme, erythrocyte transketolase activity, and increase in thiamine pyrophosphate effect were detected about 2 weeks before the onset of abnormal electroencephalograms. Pathological changes and cerebral autofluorescence observed under ultraviolet light were also examined at death.

Pentose-phosphate pathway in Saccharomyces cerevisiae: analysis of deletion mutants for transketolase, transaldolase, and glucose 6-phosphate dehydrogenase

Deletion mutants for the yeast transketolase gene TKL1 were constructed by gene replacement. Transketolase activity was below the level of detection in mutant crude extracts. Transketolase protein could be detected as a single protein band of the expected size by Western-blot analysis in wild-type strains but not in the deletion mutant. Deletion of TKL1 led to a reduced but distinct growth in synthetic medium without an aromatic amino-acid supplement. We also isolated double and triple mutants for transketolase (tkl1), transaldolase (tal1), and glucose 6-phosphate dehydrogenase (zwf1) by crossing the different mutants. A tal1 tkl1 double mutant grew nearly like wild-type in rich medium. Only the tkl1 zwf1 double and the tal1 tkl1 zwf1 triple mutant grew more slowly than the wild-type in rich medium. This growth defect could be partly alleviated by the addition of xylulose but not ribose. The triple mutant still grew slowly on a synthetic mineral salts medium without a supplement of aromatic amino acids. This suggests the existence of an alternative but limited source of pentose phosphates and erythrose 4-phosphate in the tkl1 zwf1 double mutants. Hybridization with low stringency showed the existence of a sequence with homology to transketolase, possibly a second gene.

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

Thiamin status, diuretic medications and the management of congestive heart failure

Thiamin deficiency in malnourished patients is known to have adverse cardiovascular effects: peripheral vasodilation leading to high output cardiac failure, biventricular low output failure and retention of sodium and water resulting in edema. Results from previous investigations suggest increased risk of thiamin deficiency in patients with congestive heart failure (CHF) on long term loop diuretics due to excessive urinary excretion. The purpose of this study was to assess the prevalence of thiamin deficiency in patients with CHF who are receiving loop diuretic therapy. This was a prospective investigation of thiamin status including dietary assessment and biochemical measurements via transketolase activity (TKA) with and without thiamin pyrophosphate (TPP) cofactor in consecutive patients of a cardiology clinic. Thiamin status was measured biochemically by in vitro erythrocyte TKA before and after addition of exogenous TPP. TPP effect (TPPE) was expressed as percent stimulation of TKA with exogenous TPP. Thiamin status was defined by TPPE as: normal 0–15%, mild deficiency 15–25%, severe deficiency >25%. Dietary assessment was obtained by a quantitative food frequency questionnaire of thiamin-rich foods. Adequacy of dietary intake was classified as: normal >30 servings/week, mild inadequacy 20–30 servings/week, severe inadequacy <20 servings/week. Thirty-eight patients (mean and median of 56 years; range 20–79 years); 29 males, 9 females; and 30 white, 8 nonwhite were included in the study. Inclusion criteria: 18–80, New York Heart Association class II, III, IV, left ventricular ejection fraction <40%, Lasix 80mg/day or Bumex 2mg/day, treatment with ace inhibitor, nitrate digoxin, zaroxolyn was acceptable. Measurements were compared with values obtained from nine control subjects. Logistic regression was used to evaluate relationships between thiamin status and cardiac function, clinical laboratory data, patient characteristics and confounding variables. Biochemical evidence of thiamin deficiency was found in 21% (838) of patients. Overall prevalence of dietary thiamin inadequacy was 20%. Of the eight patients with biochemical evidence of thiamin deficiency, seven had levels of dietary thiamin which appeared to meet or exceed RDA levels. Biochemical evidence of thiamin deficiency was found to be more likely to occur in males (vs. females) and nonwhites (vs. nonwhites) (p=0.005). In conclusion, thiamin deficiency may occur in one-fifth of patients with CHF in a cardiology clinic. Of this group, male and nonwhite CHF patients appear to be at highest risk for thiamin deficiency. Dietary intake of thiamin-rich foods does not appear to prevent biochemical thiamin deficiency in this population. Status of other micronutrients may also be affected by loop diuretics and deserves further study.

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.

Thiamine pyrophosphate effect and erythrocyte transketolase activity during severe alcohol withdrawal syndrome.

The thiamine pyrophosphate (TPP) effect and erythrocyte transketolase activity (ETKA) in a group of 28 patients admitted to a psychiatric emergency ward because of severe alcohol withdrawal syndrome were compared with the TPP effect and ETKA in a control group of 20 healthy nonalcoholic volunteers. The patients were treated with 300 mg thiamine 3 times daily as intramuscular injections, and the TPP effect and ETKA were measured after 1 and 4 days of treatment. No difference was found between the patient group and the control group with regard to the TPP effect and ETKA and no decline in the TPP effect was found in the patient group after 4 days of intensive treatment with thiamine. ETKA increased with intensive thiamine treatment, which suggests that ETKA is a sensitive indicator of thiamine deficiency. Serum magnesium, which is a cofactor for thiamine pyrophosphate, decreased significantly with the disappearance of alcohol from the blood in patients with high initial blood alcohol levels, but this shift did not interfere with biological thiamine activity.

Water soluble vitamins in chronic hemodialysis patients and need for supplementation

Forty-three patients on chronic hemodialysis who before the present study had only received a low-dose supplement of folic and ascorbic acid were studied prospectively for one year. After baseline values were obtained in month one, increasing doses of postdialysis vitamin supplements were prescribed for the vitamins which were found to be insufficient in order to determine the minimal amount of oral postdialysis supplement necessary to normalize vitamin levels. According to our results no systematic supplement was indicated for biotin, riboflavin or vitamin B12. For folic acid and vitamin C, supplementation with lower doses than those prescribed in many dialysis units allowed optimal vitamin levels in the majority of patients; 2 to 3 mg/week (300 to 400 micrograms/day) of folic acid and of 1000 to 1500 mg/week (150 to 200 mg/day) of vitamin C was considered sufficient. A severe pyridoxine deficiency was present in most (> 80%) unsupplemented patients, either as judged by pyridoxal-5-phosphate determinations in plasma or determination of specific enzyme activation in erythrocytes (EGOTo and alpha-EGOT); a postdialysis supplement of at least 100 to 150 mg/week of pyridoxine hydrochloride (> 15 to 20 mg/day) corrects this deficiency. The activity of the thiamine-dependent enzyme transketolase in erythrocytes (ETKo) was insufficient in 35% and marginal in 21% of the patients, while whole blood thiamine determined simultaneously in 10 of the ETKo-deficient patients was within the normal range. These results suggest that in uremia insufficient transketolase activity may be related to inhibition of the enzymatic system rather than to true vitamin deficiency. On a long-term basis a supplement of 200 to 300 mg/week of thiamine hydrochloride (30 to 45 mg/day) restored ETKo to satisfactory levels in most patients; whether this supplement is to be recommended warrants further studies.

Étude des transcétolases et des gluthation réductases au cours de la maladie d'Alzheimer

Erythrocyte transketolase activity coefficient (AC) and affinity for coenzyme (Km) are significantly increased in 16 patients with senile dementia of Alzheimer's type, whereas gluthation reductase AC and Km are unchanged.

Lactic acidosis in thiamine deficiency

Two chronically ill patients with limited nutritional intake during several weeks developed prolonged lactic acidosis. As no other causes of hyperlactaemia could be identified, thiamine deficiency was suspected. Supplementation of 600 mg thiamine resulted in a rapid normalisation of serum lactate levels (in patient 1 from 10.9-2.4 mmol/l; in patient 2 from 11.8-2.0 mmol/l) and acid base status (patient 1: pH from 7.11-7.30, bicarbonate from 8.6–21.2 mmol/l; patient 2: pH from 7.24-7.46, bicarbonate from 16–28 mmol/l; before and after treatment, respectively). Thiamine deficiency was confirmed by the degree of stimulation of erythrocyte transketolase activation by adding thiamine pyrophosphate, evaluated before and after thiamine replacement therapy. Stimulation decreased in patient 1 from 170% to 17% and in patient 2 from 20% to 0%, respectively. In addition to the metabolic derangement right ventricular heart failure was confirmed by echocardiography in both patients and again this was rapidly reversible by thiamine supplementation. We conclude that in malnourished patients unexplained prolonged lactic acidosis may result from thiamine deficiency, which is rapidly reversible by thiamine replacement therapy.

An enzymatic and immunochemical analysis of transketolase in fibroblasts from Wernicke-Korsakoff syndrome

Transketolase in cultured skin fibroblasts from three patients with Wernicke-Korsakoff syndrome (GM7504, 7505 and 7506) and matched controls was analyzed enzymatically and immunochemically with specific antisera generated against transketolase purified from human liver or red blood cells. The transketolase activity decreased by 45% in fibroblasts from the three Wernicke-Korsakoff patients, when compared to the activity in control cells. On immunoblots after SDS-PAGE, fibroblasts from the Wernicke-Korsakoff patients exhibited a 69-kDa species, a size similar to that of normal transketolase. The level of immunoreactivity was similar in the patient and control cells. The immunoblots of isoelectric focusing gels showed a major species of p I 8.6 with additional minor bands. However, the isolectric focusing pattern of transketolase from the Wernicke-Korsakoff patients was also found in the majority of the control fibroblasts. Thus transketolase in fibroblasts from these Wernicke-Korsakoff patients is catalytically defective, but appears to be immunochemically normal.

Effect of cow's milk factors on the bioavailability of non-milk thiamine in rats

Summary - The influence of milk factors on the bioavailability of non-milk thiamine was assessed using thiamine-deficient young rats. Twenty-eight h dialysed raw cow's milk (3.5% fat) was used as the source of milk factors. Repletion test diets provided were (A) dialysed milk alone (59 119)bound thiamine, (B) dialysed milk (59 119) plus pure thiamine (256 119) or (C) pure thiamine (315 119) as thiamine source, for 21 d. Total hepatic thiamine content, erythrocyte transketolase (ETK) activity and TPP (thiamine pyrophosphate) effect on ETK activity were selected as response criteria. Group B receiving dialysed milk supplemented with pure thiamine had significantly (P 2-fold in the presence of milk factors.

Modification of liver and serum enzymes by paraquat treatment in rabbits.

Paraquat (PQ) is known to cause progressive interstitial fibrosis in the lungs. Previous investigations have indicated that PQ acts by lipid peroxidation of the membrane. However, there are few reports on the action of PQ on hepatic enzymes. This work was carried out to investigate the modulation of various hepatic enzymes by PQ in rabbits. Paraquat was administered at a dose of 3, 6 or 12 mg/kg b. wt/day intraperitoneally to male rabbits for different periods of time. Administration of paraquat resulted in a significant decrease in plasma activities of transaminase enzymes, alkaline phosphatase and liver transketolase. No significant change was found in the activities of plasma and hepatic lactate dehydrogenase and alkaline phosphatase. A marked increase in blood glucose was noticed 48 hours after paraquat administration.

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. In cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640 mU/mg), phosphoribose isomerase (1600 mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO2 and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly.