Joyce L. Bell

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.

PHOSPHOGLUCONATE DEHYDROGENASE ESTIMATION IN VAGINAL FLUID IN THE DIAGNOSIS OF CERVICAL CANCER

THE early diagnosis of cervical cancer is a field in which there has been increasing interest since the work of Papanicolaou and Traut in the United States (1943). In 1962 Bonham and Gibbs, in a study of a series of selected patients, 46 of whom had cervical cancer, claimed that the estimation of 6-phosphogluconate dehydrogenase (6-PGD) in vaginal fluid was a simple and accurate aid to the diagnosis of this condition. Values of 0 to 30 units of 6-PGD were found in patients with no malignancy and of over 100 units in patients with cervical cancer. We have carried out a survey on the value of this test in 397 unselected patients from the gynaecological and obstetric clinics at the Royal Free Hospital. A few patients had more than one enzyme estimation. Clinical and cytological examinations were done in parallel with the enzyme test, and biopsies when indicated.

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

Comparative study of immunological tests for pregnancy diagnosis.

The reliability of five commercially produced immunological pregnancy diagnosis methods has been investigated. The tests used were Pregnosticon (a tube test) and four slide tests, Hyland, Pregslide, Gravindex, and Planotest. In the series described, Planotest gave 0·5% false positives, Gravindex had 2·1%, Pregnosticon 2·6%, and Pregslide 4·7%, Hyland A (sensitivity 4,500 iu/l.) gave 3·6% and Hyland B (sensitivity 2 to 3,000 iu/l.) had 8·7% false positives. Pregnosticon had 0·5% false negatives, Planotest 2·0%, Gravindex 3·5%, Hyland B 6·5%, Pregslide 9%, and Hyland A 19·5% false negatives. Planotest and Pregnosticon were found to be less influenced by protein and blood in the urine than the other pregnancy tests investigated.

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.

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.