D. N. Baron

A colorimetric method for determination of isocitric dehydrogenase

Abstract A method is described for the colorimetric estimation of isocitric dehydrogenase, to replace the spectrophotometric method for routine purposes. Serum (0.2 ml) is incubated at 37° for 60 min with isocitrate substrate in the presence of Mn++ and coenzyme II (TPN) ; the α-oxoglutarate formed is determined by a colour reaction with 2,4-dinitrophenylhydrazine and sodium hydroxide, after addition of EDTA to complex the manganese which would otherwise precipitate on adding alkali. A separate blank for each serum containing all reagents except coenzyme II, and a coenzyme II blank, is set up. The colours are read at 390 mμ, and compared with colours produced from standard oxoglutaric acid solution. The effects of the following variations of the method are described: different quantities of serum; varying the metal ion co-factor; different quantities of substrate and coenzyme; varying incubation temperature and time. Comparison between results obtained with this method and with the standard spectrophotometric method were good. The normal range in adult serum was 3.0–8.5 mμmole α-oxoglutarate/ml serum/min at 37°.

A simple specific titration method for serum calcium

Abstract A rapid, accurate, and convenient method is described for the analysis of calcium in 0.1–1.0 ml of serum by titration in alkaline solution with sodium edetate using calcein-thymolphthalein indicator, and without protein precipitation.

COMPLEXIMETRIC DETERMINATION OF CALCIUM IN PATHOLOGICAL AND PHYSIOLOGICAL SPECIMENS

Titrimetric estimation of calcium with disodium ethylene diamine tetra-acetate (E.D.T.A., sodium edetate), as performed on serum (Baron and Bell, 1957), cannot be applied directly to most other biological specimens because of their high concentration of phosphate. A high ratio of phosphate to calcium in the material titrated causes a drawnout and inaccurate endpoint. A rapid procedure for calcium determination has been devised which gives accurate results after separation of phosphate. METHOD

BIOCHEMICAL STUDIES ON HEPATIC INVOLVEMENT IN INFECTIOUS MONONUCLEOSIS.

Eighty cases of infectious mononucleosis have been investigated by serum enzyme studies and other liver function tests. Maximum abnormalities occurred between the second and fourth weeks of illness and all tests were usually normal by the sixth week. Serum isocitric dehydrogenase activity was increased in 93% of cases and serum glutamic-oxaloacetic transaminase in 74%. Conventional liver function tests were less sensitive. Serum bilirubin was above normal in 40% of cases; in 17% of cases the increase was sufficient to show as clinical jaundice. No patient has developed chronic hepatitis.

Serum enzyme changes in patients receiving antituberculosis therapy with rifampicin or p-aminosalicylic acid, plus isoniazid and streptomycin

Abstract The hepatotoxicity of rifampicin in comparison with p -aminosalicylic acid was investigated by serial estimations of serum aspartate transaminase, isocitrate dehydrogenase, and alkaline phosphatase. Both drugs were given in combination with streptomycin and isoniazid for the treatment of pulmonary tuberculosis in Britain: in all, 54 patients were studied. A high proportion of patients receiving either regimen have a transient disturbance of liver function shown by increase in the aspartate transaminase and isocitrate dehydrogenase values, but only 2 patients, both receiving p -aminosalicylic acid, developed clinical jaundice. An increase in alkaline phosphatase, showing cholestasis, was rare.

An AutoAnalyzer method for estimating serum glyceride glycerol using a glycerokinase procedure.

The glyceride glycerol analysis depends, after saponification of triglycerides, on a linked enzymatic procedure using glycerokinase, pyruvate kinase, and lactate dehydrogenase: the final conversion of NADH to NAD+ is followed fluorimetrically. Twenty analyses can be performed per hour on the AutoAnalyzer; recoveries of added triglycerides ranged between 90 and 104%. In a mixed male and female group the normal range for glyceride glycerol was 2·5 to 15·5 mg/100 ml (0·2-1·4 mmol/l) fasting, and 2·5 to 18·0 mg/100 ml (0·2-1·6 mmol/l) postprandially using fresh serum. There was a significant rise postprandially in older men.

Quantitative Biuret Determination of Urine Protein

Collie and Fleck (1967) report that the Lowry et al. (1951) method is an accurate method for determination of urine protein. This method depends on a reaction with the tryptophane and tyrosine (Daughaday et al., 1952) and cysteine (Chou and Goldstein, 1960) content of the protein molecule. The proportions of these amino acids vary in different proteins, thus albumin and globulins give different colour equivalents (Greenberg and Mirobubova, 1936), and the type of protein present in the urine may vary in different disease states. The Lowry et al. (1951) method is thus an accurate method for estimation of albumin using an albumin standard, but is not an accurate method for measurement of mixed urine protein. The biuret reaction should theoretically be a more accurate method for determination of urine protein, being much less dependent on the nature of the protein present. It is not advisable to perform a biuret reaction directly on urine because of the presence of interfering substances, and for this reason, preliminary precipitation of protein with trichloracetic acid has been suggested (Kilbrick, 1958). Using a preliminary trichloracetic acid precipitation, recoveries of urine protein greater than 100% can still be obtained (Table I), This appears to be due to protein-bound urinary pigments and these can be eliminated by the use of appropriate blanks as in the method below. Another difficulty in determination of urine protein is in deciding on the amount of urine to be used for analysis. This difficulty may be overcome by the use of Albustix as a preliminary screen.

SODIUM CONTENT OF INJECTABLE β-LACTAM ANTIBIOTICS

Abstract Many users of antibiotics are unaware of their ionic content. The ionic content is a particularly important factor for injectable β-lactam compounds in patients on a restricted sodium intake. A table of the sodium content of commonly prescribed β-lactam compounds is provided.

Isoenzymes of Isocitrate Dehydrogenase

Estimation of 1.1.1.41 Ls-isocitrate: NADP enzyme by alkali. At pH 7·4 the enzyme was oxidoreductase (isocitrate dehydrogenase, not inactivated, but separation was poor. About (lCD» is of particular value in the investigation o· 05 ml. of supernatant, containing 20-50 milliof hepatocellular damage (Sterkel, Spencer, units (Report of the Commission on Enzymes, Wolfson and Williams-Ashman, 1958; Bell, 1961) were applied to the slot. Shaldon and Baron, 1962). ICD is present in cardiac muscle as well as in liver, but the serum After running, the gel could be cut into 3 mm. ICD only rises slightly or not at all after myoblocks, the starch dissolved with amylase. and cardial infarction. Other enzymes which are the lCD estimated (Bell and Baron, 1960). present in approximately equal concentration in Reagent-grade ex-amylase has been found to liver and in cardiac muscle are lactate dehydrocontain proteolytic activity, causing some inactigenase and aspartate transaminase (glutamicvation of the lCD. Alternatively the gel was oxaloacetic transaminase). The former is sliced and the strips stained either for protein sensitive primarily to heart muscle damage, (haemoglobin could be seen without staining), whereas the latter increases after both myoor for lCD. To visualise the enzyme a staining cardial injury and hepotocellular damage. The solution was used as follows : existence of isoenzymes, namely proteins with the same enzymatic activity but with other differing biochemical properties, occuring in different tissues, is an explanation for these findings.

A salicylsulphonic acid tablet test for protein in urine.

Conclusions Many questions connected with the problems tackled remain unsolved; nevertheless, while further investigations are necessary it seems that the following conclusions are justified: 1. The intravenous injection of a 50 % solution of sodium salicylate results in thrombosis appearing 6-12 hours later. The first evidence of organization is seen after seven days. 2. The administration of heparin, in amounts of 200 mg. daily, 6-24 hours after the production of thrombosis prevents the development of the thrombotic process. 3. Heparin treatment given three or more days after the production of thrombosis does not prevent its development. Microscopically, symptoms of the fibrinolytic action of heparin are absent. 4. Veins of dogs treated with heparin present in most cases papillomatous proliferations of the endothelium, whereas in dogs treated with procaine block the picture of hyaline degeneration of the intima is prevalent. 5. The application of procaine block less than six hours from the production of the thrombosis prevents the full development of the thrombus, provided the treatment lasts for at least four days. 6. Procaine block applied 24 or more hours after the time of the injection of sodium salicylate solution does not disperse the existing thrombus; nevertheless it accelerates organization of the thrombosis as compared with non-treated animals. 7. The degree of organization of the thrombosis in dogs treated with heparin or with procaine block is approximately the same. The proliferation of endothelial cells is more marked in thrombosis after heparin treatment. 8. The prothrombin time after 10 days of heparin or procaine block shows no significant changes from the normal. 9. The histopathological examinations are not sufficient for the evaluation of dynamic changes of blood supply of the extremity and should be supplemented by the analysis of the phlebographic appearances.