Comparison of various β-glucosidase assays used to diagnose gaucher's disease

Gaucher's diselse is an inborn error of metabolism in which glucocerebroside accumulates in cells of the reticuloendothelial system due to a marked deficiency in the activity of lysosomal glucocerebrosidase. Five different published fluorometric assay procedures that utilize 4-methylumbelliferyl-β-D-gluco-pyranoside as the substrate to determine β-glucosidase activity in fibroblasts, leukocytes and liver from patients and heterozygote carriers of Gaucher's disease were compared with the results obtained using a glucocerebrosidase assay that employed authentic radiolabeled glucocerebroside as the substrate. When cultured skin fibroblasts were used as a source of enzyme, fluorometric β-glucosidase assays performed either at pH 4–4.05 in the absence of sodium taurocholate or at pH 5.4–5.5 in the presence of the bile salt, all proved effective in confirming the diagnosis of Gaucher's disease and in identifying heterozygous carriers of the disease. The fluorometric β-glucosidase assay procedures that were most effective in evaluating relative glucocerebrosidase activity in leukocytes from patients and carriers of Gaucher's disease were those that included sodium taurocholate in the incubation medium. When human liver biopsy served as the source of enzyme, only the glucocerebrosidase assay employing authentic glucocerebroside substrate proved effective in confirming the diagnosis of Gaucher's disease: fluorometric assays performed at pH 4 or pH 5.4–5.5 in the presence or absence of the bile salt sodium taurocholate do not always identify patients with Gaucher's disease.     

An improved procedure for the diagnosis of chronic granulomatous disease, using concanavalin A and cytochalasin E.

Urine samples from 18 individuals with various types of dicarboxylic acid-urias have been investigated by mass fragmentography for N-dicarboxyl-monoglycines (dicarboxylglyeines). One patient with methylmalonic acidemia excreted 14–20 μg methylmalonylglycine/mg creatinine, three patients with glutaric aciduria excreted 20–60 /gmg glutarylglycine/mg creatinine, and one patient with C₆-C₁₀-dicarboxylic aciduria excreted 120–365 μg succinyl-glycine/mg creatinine. Excretion of C₆-C₁₀-dicarboxylic acids in patients with ketosis and glycogenosis and in neonates were not accompanied by excretion of C₈-Cio-dicarboxylglycines in measurable amounts (>1μg/mg creatinine). Nor did patients with succinic aciduria excrete succinylglycine in amounts larger than 1 μg/mg creatinine. On the basis of these data it is argued that production of short- and medium-chain dicarboxylglycines is not a metabolic pathway of biological significance for the elimination of short- and medium-chain dicarboxylic acids from individuals with dicarboxylic acidurias.     

A rapid and sensitive gas-liquid chromatographic procedure for the micro determination of sodium valproate (sodium di-N-propylacetate) in plasma or serum.

A gas-liquid chromatographic procedure for the micro determination of sodium valproate is described. Valproic acid is extracted from acidified plasma or serum into n-heptane containing an internal standard (octanoic acid) and after phase separation an aliquot is analysed by gas-liquid chromatography. The procedure is rapid, reliable, sensitive and specific. It requires 25--50 microliter sample for a single estimation, has a detection threshold of less than 10 mumol/l, and is suitable for routine clinical use.     

A sensitive enzyme immunoassay for plasma cortisol

A sensitive enzyme immunoassay for plasma cortisol has been developed, using cortisol-beta-D-galactosidase conjugate as labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as fluorescence substrate. Sensitivity and precision were compared with those of radioimmunoassay with 3H-labelled cortisol and the same antiserum. The minimal detectable level for the enzyme immunoassay was 10 pg/tube or 1 microgram/dl of plasma, which makes it slightly more sensitive than the radioimmunoassay and 10 times more sensitive than previously reported enzyme immunoassays. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

A sensitive fluorometric assay for the simultaneous estimation of pepsin and pepsinogen in gastric mucosa

An assay for pepsin has been developed based on the fluorometric measurement of trichloroacetic acid-soluble peptides released from casein at pH 5.3. The increase in relative fluorescence was most sensitive in the range 10–50 μg pepsin/1 and casein hydrolysis was not affected by the addition of up to a 1000-fold molar excess of pepsinogen. This assay has been used to measure the free and total acid proteinase content of biopsies (< 5 mg) from different areas of the gastric mucosa of rat and man. Interference by the major lysosomal acid hydrolase. cathepsin D, could be eliminated by the differential stability of pepsin and cathepsin D at acid and neutral pH. The free acid proteinase activity of biopsies from the corpus were almost identical in these species whereas the total acid proteinase activity was approximately 5-fold greater in man.     

A simplified procedure for the estimation of arginine in plasma and urine using arginase

To ensure reliable arginine estimation, other compounds giving positive results with Chinard's ninhydrin reaction were removed by simple ion-exchange chromatography. Eluted arginine was converted to omithine by arginase and estimated using Chinard's ninhydrin reaction. Plasma arginine level in adults was in the range 56–98 μmol/1, and urine arginine in the range 0.58–1.77 mg/ day.     

A sensitive colorimetric assay for human urinary kallikrein

l-Prolyl-l-phenylalanyl-l-arginine-α-naphthylester (Pro-Phe-Arg-NE) was synthesized as a new substrate for use in the assay of kallikreins. An assay was developed based on the colorimetric determination of α-naphthol released by the enzyme reaction.With Pro-Phe-Arg-NE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001 KU. Thus use of Pro-Phe-Arg-NE provides a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25-μl sample of human urine.Zymograms of human urinary kallikrein were prepared using Pro-Phe-Arg-NE as substrate. Six bands were separated by polyacrylamide disc gel isoelectrophoresis.     

A sensitive enzymatic method for the determination of free and esterified tissue cholesterol

An enzymatic assay currently in use in clinical laboratories for quantitating total serum cholesterol has been modified for the determination of microgram quantities of tissue cholesterol. Two types of assays were developed. In the first, more generally useful method, lipids extracted with chloroform/methanol were solubilized by the addition of detergent and assayed for free and total cholesterol using laboratory prepared mixtures of reagents. Although different detergents were effective, it appears that Triton X-100 may be most universally applicable. In the second type of assay, commercially available reagent mixtures were employed for the determination of total cholesterol only. Results of both types of assays compare favorably with determinations using the colorimetric assay on 3β-hydroxysterols recovered from digitonides.     

A sensitive fluorescence assay for the simultaneous and separate determination of arylsulphatases A and B.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl sulphate has been developed for the simultaneous determination of arylsulphatases A and B from leucocytes, based on the differential effect of silver ions on the two enzymes. The procedure allows discrimination between normal cases and those with metachromatic leucodystrophy.     

Dihydropteridine reductase deficiency: diagnosis by leukocyte enzyme assay

An assay procedure for dihydropteridine reductase in peripheral leukocytes is described. The assay utilizes the tetrahydropterin-dependent reduction of ferri-cytochrome C in the presence of NADH and requires a smaller number of cells than assays described for cultured skin fibroblasts.Dihydropteridine reductase activity was not detectable in the peripheral leukocytes nor in the cultured skin fibroblasts from two adolescent patients with malignant hyperphenylalaninemia. The parents of the patients showed approximately 50% of normal dihydropteridine reductase activity in their peripheral leukocytes.Immunochemical experiments using antibodies against bovine liver dihydropteridine reductase suggest that normal leukocytes and skin fibroblasts contain dihydropteridine reductase which is immunologically similar to that of human liver.The present studies indicate that the determination of dihydropteridine reductase activity in peripheral leukocytes can be used to diagnose hyper-phenylalaninemia due to a defect in dihydropteridine reductase.     

A sensitive enzyme immunoassay specific for human chorionic gonadotrophin

A sensitive enzyme immunoassay procedure for the β subunit of HCG is described. The procedure should be suitable for the early diagnosis of pregnancy, the management of threatened abortion, and as a tumour marker in patients with trophoblastic neoplasms.     

A new form of the solubility test for hemoglobin s; results from a survey of 3000 cases

A new form of the solubility test for the detection of Hemoglobin S is described. Glass ampoules containing one ml of the test solutioff ready for use are employed; they can be stored for more than one year at 4°C. 3000 blood samples were analyzed by the solubility test and electrophoresis. The comparison between the two methods showed that the solubility test is highly satisfactory.     

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3, 17-dihydroxy-5β-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3, 11β-dihydroxy-5-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P₃) and 11-keto-pregnanetriol (11-keto-P₃). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3ξ, 16ξ, 17ξ, 20ξ-pregnanetetrol (16-OH-P₃) and surprisingly also a 21-hydroxylated compound, namely 3β, 20, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency.

The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of .the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.     

A screening procedure for dyslipoproteinemia in the newborn. Apoprotein quantitadon on dried blood spots

A new screening procedure for the detection of dyslipoproteinemia in the newborn is proposed, based on apoprotein quantitation. Blood is collected by heel-prick in infants 5–7 days after birth-when apoprotein and lipids have reached stable values-and adsorbed on filter paper. Blood spots are eluted with a detergent and apoprotein A-I and B are assayed by inununonephelometry. The apo A-I/B ratio is used as a screening parameter, given its high discriminative power between normals and individuals with cardiovascular disease. The quantitation of the apo A-I/B ratio in blood spots is independent of the volume of the blood spot and of the newborn hematocrit. In a follow-up study, plasma lipid and apoprotein concentrations were assayed in the infants with an apo A-I/B ratio lower than 1.0 at 5 days, and a family study was carried out. On screening 1500, 30 infants gave positive results at birth, and were controlled between 2 and 8 months. In eight families studied, six children had abnormally high cholesterol and apo B values and five children had abnormally low HDL cholesterol and apo A-I concentrations. This screening procedure is a new approach to the detection of familial dyslipoproteinemia in the newborn, as it is based on the quantitation of the apo A-I/B protein ratio, instead of cholesterol, LDL cholesterol or β-lipoprotein quantitation. It enables a differentiation to be made between various forms of dyslipoproteinemia, leads to a better characterization of various diseases, and decreases the percentage of false positive cases.     

The effect of pH on vesicles composed of phosphatidylcholines and N-acylamino acids: A calcein release fluorescence study

Release of the fluorescent anionic dye calcein from small unilamellar vesicles composed of mixtures of phosphatidylcholines (PC) and N-acylamino acids as “trigger” molecules was examined by fluorescence spectroscopy as a function of pH in phosphate buffered saline (PBS) containing 25% serum. These vesicle systems were maximally destabilized in response to pH when both the PC and the N-acylamino acid had similar chain lengths (c₁₆ and C₁₇). The ability of the amino acid headgroup to form a thiolactone ring was not required for dye release. Following exposure to pH 6, vesicles of ³H-labeled trigger and ¹⁴C-labeled PC co-eluted from Sepharose and Sephadex column chromatography. Thus, thiolactone formation and/or loss of trigger from the vesicle bilayers are not likely explanations for the mechanism of pH-dependent dye release.