A possible explanation for the occurrence of increased beta-hexosaminidase activity in pregnancy serum

The enzymic and immunological properties of a novel gamma-glutamyl transpeptidase found in human renal carcinoma tissues were investigated further in comparison with those of the normal kidney enzyme. On isoelectric focusing, the novel gamma-glutamyl transpeptidase separated into two main forms, having pI values below 3.6, while the normal kidney enzyme separated into multi-molecular forms with pI values of 4.0-5.0. Neuraminidase treatment diminished the difference between these two enzymes, the products of the novel enzyme and the normal kidney having pI values of 5.4 and 5.6, respectively. The percentages of the total activity of the novel gamma-glutamyl transpeptidase binding with Con A before and after neuraminidase treatment were about 40% and 80%, respectively, while the corresponding percentages of the activity of the normal kidney enzyme were less that 10% and about 25%, respectively. The novel gamma-glutamyl transpeptidase was immunologically identical with the normal kidney enzyme in the activity inhibition test and the double diffusion test. The present data suggest that the novel gamma-glutamyl transpeptidase has the same antigen site as the normal kidney enzyme, but differs from the latter at least in its sialic acid content and in some other carbohydrate moieties.     

Serum alpha-amylase isozymes in mumps: estimation of salivary and pancreatic isozymes by isoelectric focusing

Serum alpha-amylase isozymes were separated into three major isozymes by thin-layer gel isoelectric focusing and detected by a starch-iodine zymogram procedure. Of the three groups of isozymes, one (S isozyme) corresponded to a salivary specific form, one (P isozyme) to a pancreatic specific form and the third (SP isozyme) to isozymes of similar isoelectric point common to both secretions. The levels of total alpha-amylase and of these three isozymes were estimated in the sera of 54 patients with mumps. Total alpha-amylase and salivary isozyme concentrations were greatly increased in the sera of all patients compared with controls. Pancreatic isozyme concentrations however, were only slightly increased and did not correlate with clinical pancreatitis. Indeed, in patients with mumps associated with pancreatitis, meningoencephalitis or orchitis, levels of total serum amylase, although higher than controls, were lower than levels in patients who presented solely with mumps sialadenitis.     

Effects of albumin and other proteins during assay of amylase activity.

Addition of albumin, gamma-globulin, alpha-casein or submaxillary mucin to the assay system for chromogenic measurement of human or rat amylase with blue starch increased the amylase activity, albumin having the most effect. These proteins seemed to increase the activity by protecting amylase from inactivation. Amylase activity was higher in urine samples showing proteinuria than in urine samples without detectable protein. It is concluded that amylase assay is more reliable when a final concentration of albumin of 1 mg per ml is added at the dilution step and at the incubation step.     

Use of magnesium silicate before gas-liquid chromatography for determination of "total estrogens" in urine during pregnancy.

The authors propose a simple, rapid, reproducible and reliable method for determination of "total estrogens" in urine during the last three months of pregnancy. The procedure consists of separation of free urinary estrogens, obtained after rapid hydrolysis, on a column of magnesium silicate. The estrogens are adsorbed on the column at acid pH and eluted by 1 M potassium hydroxide. Following extraction of the eluate by diethyl ether and formation of trimethylsilyl ether derivatives, the steroids are analysed by gas-liquid chromatography. This new procedure is used routinely in our laboratory, one assay being carried out in less than three hours. The results appear to be comparable to those obtained with classic methods. We wish to report the elimaination curves of "total estrogens" during normal pregnancies and their allowable limits.     

Enzyme immunoassay for total oestrogens in pregnancy plasma or serum.

We have developed an enzyme immunoassay for total oestrogens in pregnancy serum of plasma, using horseradish peroxidase (HRP) as the marker enzyme. A test combination consisting of an antiserum against oestriol-16/17-monosuccinyl-albumin and oestriol-16/17-monosuccinyl-HRP yielded a sensitive system, which reacted to approximately the same extent with oestrone, oestradiol, oestriol and their 16- and 17-conjugates. Samples had to be diluted 1 to 10 to avoid interference of plasma factors with the immune reaction. Bound/free separation was achieved with the double antibody solid phase (DASP) method. The HRP activity of the bound fraction was measured, after washing, to eliminate plasma factors disturbing the HRP reaction. The detection limit of the assay system was approx. 0.1 pmol/tube, while the index of precision lambda ranged from 0.02 to 0.06. To measure total oestrogens, including the 3-conjugated ones, we used an enzymatic hydrolysis with an extract of Helix pomatia. Hydrolysis was found to be optimal after 1 h at 50 degrees C and pH 5.0. The method was used on serum samples from normal pregnancies. The results showed a very good correlation (r=0.98) with those obtained by radioimmunoassay. Normal values for total oestrogens during pregnancy were determined in a multicentre clinical trial.     

Human serum amine oxidase. Enzyme activity in severely burnt patients and in patients with cancer

During a study of serum amine oxidase (SAO) levels in patients with fibrotic disease processes, 127 patients (100 adults and 27 children) with moderate to severe burns were investigated. Of these, 55 adults and 20 children, classified as severe, had enzyme levels of 2.7 ± 0.3 and 4.6 ± 0.9 McEwen Units (McE.U) (mean ± S.E.) respectively, which, compared with those for normal healthy adults (18.3 ± 0.6 McE.U), gave a difference of striking significance (t = 21.05 for 130 d.f., and t = 10.33 for 95 d.f., respectively).Subsequently, 125 patients with proven cancer were studied. 52% of the untreated and 41% of the treated showed remarkably low enzyme activity. The mean of the group of untreated patients was 9.6 ± 1.1 McE.U, that of the treated 11.6 ± 1.0 McE.U, and that of the entire group 10.7 ± 0.7 McE.U. The difference between these values and normal activity is highly significant (p < 10^-6).Sequential SAO estimations on severely burnt patients and those with cancer established the time course of enzyme activity in these patients.Our observations suggest that estimation of SAO activity may be useful not only for the classification, monitoring and prognosis of severe bums, but also in the diagnosis of cancer and the evaluation of its treatment.     

An improved procedure for isolation of thyroxine-binding globulin from human pregnancy serum.

A convenient method for isolation of TBG from human pregnancy serum is reported. The method is based on batch-wise treatment of serum with hydroxyapatite followed by batch-wise adsorption of TBG to an affinity gel consisting of Sepharose to which thyroxine was bound with a spacer group. After selective desorption the TBG-preparation was practically pure. The small amounts of contaminating proteins were easily removed by affinity chromatography on Concanavalin A-Sepharose. The resulting preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and immunological techniques.     

Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-sachs heterozygotes during pregnancy

Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEXA) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I dose not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays we examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassay of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power.     

A method for identification and follow-up of patients with a steroid-21-hydroxylase deficiency.

A method is described for the determination of 17 alpha-hydroxyprogesterone from blood samples obtained by heel prick and dried on filter paper. Discs (10 mm diameter) were cut from the filter paper and extracted in the assay tubes with 1 ml of a methanol/diethyl ether/ethyl acetate (50 :45 :5, v/v) solvent mixture. Antibody, tritium-labelled tracer and dextran-coated charcoal were added to assay tubes using a multichannel dispenser. The approach used permits one technician to analyze two series, each of 60 duplicate samples, within one working day. Thus the method is applicable for the centralized screening of suspected cases, while emergency samples may be analyzed at the same time within 4 h. Comparisons with a highly specific but more elaborate technique for the determination of blood 17 alpha-hydroxyprogesterone showed a correlation coefficient of 0.99, and the regression equation for the present method (y) against the established method (x) was y = 1.07x + 2.72. The calculated upper reference limit (mean +/- S.D.) for 17 alpha-hydroxyprogesterone in healthy infants from two days to eight years of age of 7.5 ng/ml of serum.     

Human leucocyte cerebroside sulphate sulphatase.

Some of the properties of the cerebroside sulphate sulphatase of human leucocyte extracts have been studied. The enzyme has an apparent KM of 2.9 mmol/l and is inhibited by the products of the reaction, sulphate and galactocerebroside. A number of divalent metal ions including manganese and magnesium stimulated the reaction only slightly at 5 mmol/l but inhibited strongly at 20 mmol/l. Triton X-100 present in leucocyte extracts also inhibited, increasing both the apparent KM and Vmax. The enzyme activity was dependent on the presence of anionic detergents. At low substrate concentrations a crude taurocholate preparation was the most active of all bile acids examined. Pure taurocholate and sodium cholate were considerably less active. At highter substrate concentrations however, sodium cholate produced the greatest stimulation of enzyme activity. These data suggest that the bile acid/substrate ratio may be a critical factor in determining cerebroside sulphate sulphatase activity at different substrate concentrations.     

Modification of human pancreatic amylase isozymes by peptidoglutaminase I and II

The effects of peptidoglutaminase (PGln-ase) I and II on human pancreatic juice amylase purified as an isozyme were investigated. Several amylase isozymes were formed which corresponded to minor components of pancreatic amylase isozymes, indicating that appearance of amylase isozymes are due to enzymic deamidation. A similar result was observed when the purified amylase isozyme was incubated with the supernatant of human pancreatic juice whose amylase was previously removed by adsorption onto raw corn starch. These findings are discussed in connection with amylase isozymes in the sera of the patients suffering from pancreatic inflammation.     

Routine identification and determination of 11 barbiturates in biological samples.

A very easy, reliable and specific gas-chromatographic method for identification and dosage of 11 barbiturates in plasma is presented. Methylene unit values determined on two types of column (one polar, one non-polar) allows very accurate identification with a minimum risk of error due to fluctuations in operating conditions. Sensitivity of the method is 0.5 microgram/ml plasma with a flame ionization detector. Derivatization procedure is complete, without any degradation phenomenon as tested by mass spectrometry. Such a method can be easily used as routine procedure for toxicological or pharmacological applications.     

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.     

Kinetic immunoturbidimetry: the measurement of pregnancy specific beta 1 glycoprotein.

The extension of kinetic immunoturbidimetry to the measurement of low concentration proteins has been described using the protein pregnancy-specific beta 1 glycoprotein. The technique has a precision superior to that of all other methods currently available. The assay is rapid, cheap and compares well with other published methods. The assay further emphasises the usefulness of the technique of kinetic immunoturbidimetry.     

Isolation and identification of some urinary inhibitors of calcium phosphate formation.

Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.     

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.     

A comparison of reaction conditions for the automated determination of gamma-glutamyltransferase activity in serum.

The methods of Rosalki and Tarlow and the Scandinavian Committee on Enzymes for the determination of gamma-glutamyltransferase activity in serum have been compared, with an automatic reaction-rate analyzer in which the reaction is initiated by the addition of a concentrated solution of gamma-glutamyl-p-nitroanilide. Results are approximately 4% lower by the Scandinavian method, because of its lower substrate concentration. However, both methods are of comparable reproducibility. The greater stability of the more dilute substrate solution specified in the Scandinavian method has been confirmed, but attempts to stabilize solutions of gamma-glutamyl-p-nitroanilide at the higher concentrations recommended by Rosalki and Tarlow by the addition of organic solvents were accompanied by some enzyme inhibition. The comparative instability of the substrate solutionof Rosalki and Tarlow is unlikely to lead to erroneous results; however, the Scandinavian formulation offers the advantage of a more economical use of reagents, at the expense of a small and probably unimportant reduction in sensitivity.     

Isoelectric focusing of serum in cystic fibrosis: Failure to distinguish between homozygote and heterozygote sera

Thin-layer isoelectric focusing was performed on samples of sera from patients with Cystic Fibrosis, siblings and obligate heterozygotes (parents), and children without cystic fibrosis (controls). The protein band with an isoelectric point of pH 5.48, previously reported to be absent in homozygote cystic fibrosis sera, was found to have a pI of 5.25. It was present in approximately one-half of the homozygote and heterozygote sera tested, and absent from 18 percent of control sera. The presence of this band is not therefore a reliable marker for the normal gene, and cannot be used to identify the heterozygous carrier for cystic fibrosis.