A radiochemical method for the determination of transketolase activity in erythrocyte hemolysates

A radiochemical method for the determination of transketolase activity is reported. It is based on incubation of erythrocyte hemolysates with radioactive ribose 5-phosphate followed by isolation of sedoheptulose 7-phosphate on anion-exchange columns. The experimental conditions are discussed and the presence of phosphatase activity in the incubation mixture is demonstrated. The proposed method is compared with a current colorimetric method. Reference values are given for the transketolase activity and the thiamine pyrophosphate effect.     

Estimation of hydroxylysine in urine and serum of patients with chronic uremia

Free hydroxylysine and hydroxylysine glycosides were separated from urine and serum extracts on cation exchange resin and assayed spectrophotometrically. The method in conjunction with gel filtration on BioGel P2 allowed to separate from urine also polypeptidic hydroxylysine and hydroxylysine bound in small molecules of neutral or acidic character. Glucosylgalactosylhydroxylysine and galactosylhydroxylysine were separated by partition and/or ion exchange chromatography.Patients with chronic renal insufficiency had elevated serum levels and urinary excretion of hydroxylysine glycosides with increased excretion of hydroxylysine bound in polypeptides and in small molecules of neutral or acidic character. The excretion of free hydroxylysine was often within normal limits.When compared to values found in normal growing subjects and in adult patients with increased bone turnover and normal renal function the urinary excretion of hydroxylysine glycosides in chronic uremia was more markedly increased than excretion of hydroxyproline polypeptides and total hydroxyproline.     

Erythrocyte transketolase,a new semi-automated method

This paper describes a method to determine transketolase activity in erythrocytes, with or without addition of thiamine-pyrophosphate, by quantitating the formation of glucose-6-phosphate formed during the incubation of hemolysed blood with ribose-5-phosphate.Glucose-6-phosphate is determined enzymatically, after deproteinisation of the sample, using a centrifugal fast analyser.The proposed method gave a correlation of r = 0.89 with an accepted colorimetric method. Reference values for blood are in the range 32–101 U/l.     

Blood vitamin B1, transketolase and thiamine pyrophosphate (TPP) effect in beriberi patients,with studies employing discriminant analysis

In recent years, many cases of beriberi have been reported throughout Japan. One may assume that a great many healthy subjects suffer from a subclinical thiamine deficiency. The present study was carried out in order to examine thiamine status in 21 beriberi patients and 674 apparently normal subjects. In the beriberi patients the total vitamin b₁ in whole blood and transketolase activity in the hemolysate were significantly lower, and the thiamine pyrophosphate effect was significantly higher, compared to normal subjects. However, these two groups could not be clearly separated by these biochemical parameters because of significant overlap. On analyzing the results of these biochemical parameters by discriminant analysis, beriberi could be diagnosed with an accuracy of 87,7%. Thus, vitamin b₁ levels in blood, transketolase activity and the thiamine pyrophosphate effect in the hemolysate are useful biochemical indices for the diagnosis of beriberi. Above all, the thiamine pyrophosphate effect proved to be the most effective parameter in distinguishing the beriberi group from normal subjects.     

Erythrocyte glutathione synthetase in 5-oxoprolinuria: kinetic studies of the mutant enzyme and detection of heterozygotes.

The primary metabolic defect in 5-oxoprolinuria is a generalized deficiency of glutathione synthetase. The activity of this enzyme was determined in cell-free extracts of erythrocytes from patients with 5-oxoprolinuria, their parents and a sibling as well as from normal control individuals. The following activities (pkat/mg of hemoglobin) for glutathione synthetase were obtained: homozygotes mean 0.10 (range 0.07-0.12), heterozygotes mean 3.1 (range 2.8-3.7) and control individuals mean 6.1 (range 5.4-6.7). These results indicate that 5-oxoprolinuria, i.e. the defective gluthione synthetase gene(s), is transmitted by autosomal recessive inheritance. Studies of the kinetics of the low remaining activity of erythrocyte glutathione synthetase in patients with 5-oxoprolinuria failed to reveal defective affinity for glycine, gamma-glutamyl-alpha-aminobutyrate, ATP and Mg2+ ions. Furthermore, the pH optimum, time curves and temperature dependence for the mutant enzyme activity did not significantly differ from the corresponding parameters observed with normal enzyme.     

A sensitive radioassay for biotinidase activity: deficient activity in tissues of serum biotinidase-deficient individuals

A new, sensitive radioassay for the determination of biotinidase activity was developed which measures the release of [14C-carboxyl]-p-aminobenzoate from N-biotinyl-[14C-carboxyl]-p-aminobenzoate. Biotinidase activity in serum from normal individuals is comparable to that determined by the colorimetric assay, but the radioassay is approximately 100 times more sensitive. Biotinidase deficiency was confirmed in the serum of patients who were previously shown to have reduced activities by the colorimetric assay. Although biotinidase activity was not detectable in extracts of normal peripheral blood leukocytes and fibroblasts using the colorimetric assay, activities could be measured by the radioassay. Using this method we demonstrated deficient biotinidase activity in extracts of leukocytes and fibroblasts from affected patients.     

Plasma catecholamines and dopamine-beta-hydroxylase activity in chronic renal failure

Plasma and urinary catecholamines (CA) and plasma dopamine β-hydroxylase (DBH) activity were studied serially in 17 hypertensive patients with chronic renal failure (CRF) and in 15 age-matched patients with benign essential hypertension (EH). Resting levels of plasma epinephrine (E) plus norepinephrine (NE) in CRF patients were significantly greater than those in EH patients (P < 0.05), whereas plasma DBH activities in CRF patients tended to be lower than those in EH patients (P < 0.1). However, DBH activities were found to be similar for the two groups, when they were expressed in units per litre of blood instead of per litre of plasma. Urinary free E + NE and dopamine were significantly less in CRF than in EH, whereas no significant difference was noted in urinary excretion of conjugated E + NE and vanillylmandelic acid between the two groups. The ratio of conjugated E + NE/free E + NE and of vanillylmandelic acid/free E + NE were significantly greater in CRF than in EH patients (P < 0.05). Glomerular filtration rate correlated significantly with free E + NE, free and conjugated dopamine, and inversely with the ratio of conjugated E + NE/ free E + NE in the whole subjects. These findings suggest that raised plasma CA concentration associated with the relative enhancement of extraneuronal inactivations may be relevant to the retarded clearance of circulating CA rather than increased CA release in CRF patients. It is likely that many factors unrelated to sympathetic nerve discharge have a considerable influence on both plasma CA concentration and plasma DBH activity in CRF patients, making them unreliable for studying the role of sympathetic nerve activity in these patients.     

N-Acetyl-beta-D-glucosaminidase activity in normal and chronic lymphocytic leukaemic lymphocytes.

N-Acetyl-beta-D-glucosaminidase (EC 3.2.1.30) activity was measured fluorimetrically in: (a) lymphocytes from 20 normal donors, (b) enriched B and T lymphocyte populations prepared by E rosette sedimentation from 8 normal subjects, (c) lymphocytes from 15 untreated B cell chronic lymphocytic leukaemic patients. The pH profiles and optima (4.7) were similar in all preparations. Normal B lymphocytes had higher activity than normal T lymphocytes and both these preparations and normal unfractionated lymphocytes had significantly higher activity than chronic lymphocytic leukaemic lymphocytes. The apparent Michaelis constants were similar in normal unfractionated, B enriched and T enriched lymphocytes, whereas a reduced affinity for the enzyme was observed in the leukaemic lymphocytes. The difference in enzyme content between normal and chronic lymphocytic leukaemic lymphocytes cannot therefore be explained on the basis of a high B cell percentage in patients with chronic lymphocytic leukaemia.     

Serum glycylproline dipeptidyl aminopeptidase activity in human hepatic cancer.

A method for the assay of glycylproline dipeptidyl aminopeptidase activity in the serum is described. The enzyme activity determination in sera from patients with hepatic cancer opens a possibility in the differential diagnosis of hepatobiliary diseases, because of the significantly higher values found in some cases of hepatic cancer. No correlation with alpha-fetoprotein was found.     

Glycogen synthase activity in parodontal disease (author's transl)]

A biochemical study of an enzyme participating in the synthesis of glycogen is presented, with particular regard to the fluctuations in the amounts of this polysaccharide in human gingival epithelium, during inflammation. The increase in the activity of UDPglucose : glycogen glucosyltransferase can be related to the accumulation of glycogen. Some kinetic parameters of this enzyme are described.     

Serum glycylproline p-nitroanilidase activity in blood cancers

Glycylproline p-nitroanilidase activity in serum of patients with acute lymphatic leukemia, lymphosarcoma and Hodgkin's disease, and of normal neonates (umbilical blood) was significantly lower than that of normal adult controls. In contrast, the enzyme activity of patients with myelocytic leukemias did not differ significantly from that of normal controls.     

Serum glutathione S-transferase activity in liver diseases

Assay conditions of human liver glutathione S-transferase and its activity in human serum from liver disease patients were investigated. One mmol/l reduced glutathione, and 1 mmol/l-1-chloro-2,4-dinitrobenzene, pH 6.5, were used for the measurement, because of the very low non-enzymatic conjugation. Glutathione S-transferase activity was inhibited by bilirubin, but this inhibition was counteracted by the presence of a low concentration of albumin. The normal human serum glutathione S-transferase activity was 5.2 +/- 2.4 I.U./l (mean +/- S.D.), and was not influenced by any differences of age, sex or leukocyte count. A significant increase in serum enzyme activity was noted in cases of acute hepatitis with GPT exceeding 200 I.U./l, primary hepatoma and metastatic liver cancer. Some of the cases with fulminant hepatitis showed extremely high values. The degree of correlation between serum glutathione S-transferase and GOT or GPT was high in acute hepatitis, with GOT or GPT exceeding 200 I.U./l, in fulminant hepatitis, primary hepatoma and gall stones, while in chronic hepatitis and liver cirrhosis it was low. In cases of acute hepatitis and fulminant hepatitis, the disappearance of serum glutathione S-transferase from the blood was much faster than that of GOT and GPT. Serum glutathione S-transferase measurements will provide new and unique information for the diagnosis of acute liver diseases.     

Platelet monoamine oxidase: specific activity and turnover number in headache

Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [³H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction.     

Evaluation of metabolic pathway activity in cultured skin fibroblasts and blood leukocytes

Screening for a variety of blocked catabolic mutants can be achieved in cultured skin fibroblasts and in peripheral blood leukocytes with a simple radioisotope method that is reliable and convenient. The method measures the incorporation into trichloroacetic acid (TCA) insoluble macromolecules of a ¹⁴C-labelled intermediate in the pathway under question; incorporation of a ³H-labelled metabolite, not in the same pathway, is used as an internal control of metabolism in the cell population. The ¹⁴C/³H incorporation ratio is decreased in blocked pathways; use of the ratio method eliminates the delay required by radioautography (used in earlier adaptations of this method), the problems involved in CO₂, collection, and the need to standardize cultures for cell number. This method has been used to identify cells with biochemical lesions in the oxidation of propionate, galactose, hypoxanthine and pyruvate; it has allowed us to identify a new variant of methylmalonicaciduria; we believe it can be extended to include other metabolites and pathways.     

New enzymatic assay of cholinesterase activity.

A new enzymatic method for the determination of serum pseudo-cholinesterase activity is described. Choline, which is liberated from benzoylcholine as substrate by cholinesterase, is oxidized by choline oxidase to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximal absorbance at 500 nm. The calibration curve is linear up to 1500 units per liter of serum. The method is reproducible, and the results correlate well with those obtained by the method using butyrylthiocholine as substrate and 5,5'-dithiobis-(2-nitrobenzoic acid) as color reagent.     

Plasma somatomedin activity in vitamin A deficient children.

A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.     

Determination of enteropeptidase activity in human duodenal aspirates.

A sensitive procedure is described for the determination in duodenal aspirates of enteropeptidase activity based on the activation of trypsinogen and the estimation of trypsin formed with benzoyl-arginine-p-nitroanilide. Using the recovery approach where a known amount of purified human enteropeptidase is diluted in duodenal fluid and the recoverable activity determined, this method was shown to give a sensitive and reliable estimate of the enteropeptidase activity in duodenal fluid although it was shown that the enzyme was subject to a 10% activation by components in the duodenal fluid. The reported 5-fold stimulation of enteropeptidase activity by bile salts could not be demonstrated.     

The activity of gamma-glutamyltransferase after bile duct ligation in guinea pig.

The activity of gamma-glutamyltransferase was studied in guinea pig after bile duct ligation. In serum, an abrupt increase in activity up to 10--20 times the normal value was found 3 h after obstruction and the mean activity over the first 3 days following the operation was some 8 times the normal value. In liver, however, a small decline in activity could be demonstrated. The administration of cycloheximide did not influence the acute increase in serum activity. Bile duct ligation caused marked increases in serum bile acid levels which initially paralleled the serum gamma-glutamyltransferase activity. It is suggested that the increased serum activity may arise from the solubilization by bile acids of liver membrane-bound enzyme.     

An automated continuous-monitoring procedure for the determination of acid phosphatase activity in serum.

The method of Hillmann, in which hydrolysis of alpha-naphthyl phosphate by acid phosphatase is coupled to the formation of an alpha-naphthol-Fast Red TR azo-compound, has been adapted for use with the LKB Produkter AB 8600 reaction rate analyzer. Factors which affect the reproducibility of the method are described and its performance is shown to be superior to that of a manual phenyl-phosphate procedure.