Diagnosis of Tay-Sachs disease by estimation of beta-N-acetylhexosaminidase activity using a radiolabeled hyaluronic acid-derived trisaccharide substrate

We have prepared a new radiolabeled substrate (N-[3H]acetylglucosamine-glucuronic acid-N-[3H]acetylglucosamine), from hyaluronic acid, for an assay of beta-N-acetylhexosaminidase activity. Using this substrate, we found a striking deficiency of beta-N-acetylhexosaminidase activity in cultured skin fibroblasts and in liver homogenates from patients with Tay-Sachs disease. DEAE-cellulose chromatography at pH 6.0 revealed that both isoenzymes A and B of beta-N-acetylhexosaminidase from normal liver participated in the catabolism of hyaluronic acid. There were, however, major differences in substrate specificities between isoenzymes A and B. Our results indicate that this substrate should be useful for enzymatic diagnosis of Tay-Sachs disease.     

Factor XII assay with the chromogenic substrate chromozym PK

A chromogenic assay for the determination of factor XII using the chromogenic substrate Chromozym PK was evaluated. The assay was linear in the range 10 U/l to more than 200 U/l. Using the assay, the normal range of factor XII (50 healthy volunteers at random) was 136 U/l +/- 27 U/l. Kallikrein-inhibiting concentrations of aprotinin did not influence factor XII. Comparison of the chromogenic with the clotting assay resulted in a correlation coefficient of r = 0.831 (p-value less than 0.001). In patients with deep vein thrombosis, factor XII level was found to be reduced to about 60% of normal activity.     

Thermodynamic characterisation of the mutated isoenzyme A of beta-N-acetylhexosaminidase in GM2-gangliosidosis B1 variant.

Here we report the determination of the activation energies of the plasma isoenzymes of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), isolated by chromatography in DEAE-cellulose, using the neutral chromogenic substrate 3,3'dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide. The activation energy of mutated Hex A isoenzyme (Ea approximately 71.5 kJ/mol) from a patient with GM2-gangliosidosis B1 variant, homozygote for the G533-->A (Arg178His) mutation, was significantly higher than that of normal Hex A (Ea approximately 41.8 kJ/mol) and analogous to that of Hex B isoenzyme (Ea approximately 75.1 kJ/mol). The determination of this thermodynamic variable of Hex in different biological specimens could allow for a straightforward biochemical characterisation of the GM2-gangliosidosis B1 variant.     

Plasma beta-N-acetylhexosaminidase isoenzyme composition and temperature conversion factors

Using the chromogenic substrate 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide for the activity determination of plasma beta-N-acetylhexosaminidase (Hex), the temperature conversion factors (TCF) offer a highly significant positive correlation with the relative proportion of Hex B isoenzyme (P< 0.001). The calculation of TCF 37 degrees/25 degrees C allows the isoenzyme composition of Hex to be determined quickly and cheaply. The results may be superimposed over those obtained in a previously described method based on the calculation of the enzyme's activation energy using four temperatures. However, the use of TCF 37 degrees/30 degrees C does not appear to comply with the required demands.     

Determination of alpha 2-macroglobulin-trypsin complex with a new synthetic substrate

We have developed a colorimetric assay for quantifying alpha 2-macroglobulin-trypsin complex (alpha 2M-TRY) in human serum, based on use of a new chromogenic substrate D-gamma-tert-butyloxy-Glu-Gly-Arg-3-carboxy-4-hydroxyanilide dihydrochloride (PS-3001). Within-run CVs by this assay were 4.76%, 1.57%, and 0.83% for trypsin complex concentrations of 3.1, 12.2, and 48.1 U/L, respectively (n = 10 each). Between-day CVs were 5.38%, 3.12%, and 2.20% at each concentration, respectively (n = 7). Mean analytical recoveries of alpha 2M-TRY added to serum were 100%, 105%, and 101% for 9.2, 15.1, and 46.3 U/L, respectively (n = 2). The standard curve obtained was linear up to 330 U/L. We applied this method to the study of alpha 2M-TRY activity in sera from 97 healthy subjects (group 1), from 27 patients with acute pancreatitis (group 2), and from 25 patients with other chylopoietic diseases (group 3); results ranged from 0 to 1.2 U/L (mean = 0.5, SD = 0.3), from 1.2 to 77.4 U/L (mean = 14.6, SD = 19.0), and from 0 to 1.3 U/L (mean = 0.4, SD = 0.3), respectively. Concentrations of enzymatically active alpha 2M-TRY were significantly greater in sera from group 2 than in groups 1 and 3. The determination of serum alpha 2M-TRY activity by this simple, rapid, colorimetric method may be useful for the diagnosis and evaluation of pancreatic disease.     

The measurement of sulphated and non-sulphated bile acids in serum using gas-liquid chromatography.

A method is described to assay sulphated and non-sulphated bile acids in serum using gas-liquid chromatography. Previously described techniques have been substantially modified to allow analysis of free and conjugated salts of the four major bile acids with particular care to ensure quantitative recoveries of lithocholic acid, its conjugates and sulphate esters. Losses of lithocholic acid inherent in some methods have been reduced by avoidance of column chromatography with alumina and extraction of lipid contaminants into heptane. Assay of the proportion of serum bile acids present as sulphate esters is achieved by the routine use of column chromatography to separate sulphated bile acids from non-sulphated bile acids followed by solvolysis of the sulphated bile acids before deconjugation. Careful selection of the conditions of strong alkaline hydrolysis ensures deconjugation of all bile salt conjugates including lithocholic conjugates which are not completely hydrolysed in weaker alkaline solutions. The trifluoroacetate derivatives of the methyl esters of the bile acids are chromatographed using 5-beta-cholanic acid as an internal standard with clear separation of the four major bile acids from the internal standard. In 10 fasting control subjects the mean serum total bile acid concentration was 5.3 muM (RANGE 1.1-16.4) including 0.7 mum sulphated bile acid (range 0-1.8). In 10 patients with acute viral hepatitis the total bile acid concentration was elevated in some but normal in others (mean 44.9 muM, range 2.7-80.3). The percentage of the total bile acid sulphated was not significantly different in the hepatitis patients compared to controls (controls 13%, range 0-35; hepatitis 23%, range 0-52). Lithocholic acid made up 13% of the total bile acid in controls (0-32%) and 18% in hepatitis patients (0-53%). Most of this lithocholic acid was sulphated (controls 81%, range 30-100; hepatitis 67%, range 37-100). Unconjugated bile acids were demonstrated in the serum of a few patients with acute viral hepatitis but in no control subjects.     

A simple isotopic assay method for human serum phospholipase A2 activity

An assay using 2-(1-14C)palmitoyl-labelled dipalmitoyl phosphatidylcholine substrate for the determination of serum phospholipase A2 (PLA2) activity is described and validated. The rapid determination of the enzyme activity is enabled by a simple liquid-liquid partition system to replace the laborious thin-layer chromatography used in earlier studies. The PLA2 activity of human pancreatic juice was used for the optimization of the assay. Interference by serum phospholipids can be avoided by using 10 microliter aliquots of serum in the assay, whereas larger amounts caused a progressive inhibition of the enzyme activity. Virtually no enzyme activity is determined in serum from normal healthy subjects (range from 1.2 to 3.0 IU/l). In acute haemorrhagic pancreatitis the PLA2 activity is markedly elevated (range from 10.7 to 42.0 IU/l). Due to the simple extraction of the reaction products the results can be obtained the same day. Therefore, the assay can conveniently be used for the rapid clinical identification of subjects with acute haemorrhagic pancreatitis.     

Marked variation in blood beta-hexosaminidase in Gaucher disease.

Gaucher disease is due to a primary deficiency of acid beta-glucosidase activity and is associated with secondary elevations of several plasma/serum lysosomal enzyme activities, including beta-hexosaminidase. We analyzed plasma and serum beta-hexosaminidase A & B activities in 55 patients with enzyme-documented Gaucher disease. The mean beta-hexosaminidase activity was increased and the percent of the A isozyme decreased, consistent with earlier studies. Gaucher disease patients had 2,067 +/- 1,491 nmol ml-1 h-1 units of beta-hexosaminidase activity with 51.9 +/- 15.5% beta-hexosaminidase A compared to 1,086 +/- 260 nmol ml-1 h-1 and 67.8 +/- 4.0% beta-hexosaminidase A in normal controls and 965 +/- 261 nmol ml-1 h-1 and 43.6 +/- 5.5% beta-hexosaminidase A in Tay-Sachs disease heterozygotes. Contrary to previous reports, marked heterogeneity of both total plasma/serum enzyme activity and isozyme pattern was noted, as some patients had normal enzyme levels and others had severe reductions in the percent of hexosaminidase A. These data argue against the suggestions of recent studies that routine serum beta-hexosaminidase testing done in Tay-Sachs disease heterozygote detection programs can be effectively used to screen for patients with Gaucher disease.     

Gamma-glutamyl transpeptidase activity in human urine

The gamma-glutamyl transpeptidase (EC 2.3.2.2) activity of the urine was determined in 369 cases, with L-gamma-glutamyl-p-nitranilide as substrate. Thermally stable, low molecular mass inhibitors were removed by dialysis. The enzyme activity was found to have a mean of 10.57 U/l for the normal neonate population, and a mean of 16.92 U/l for children. A significant increase in activity was observed in pyelonephritis, Alport's syndrome, Wilms' tumour and glomerulopathies. The highest activities were found in kidney diseases associated with renal insufficiency. This non-invasive test can be used for the evaluation of tubular disorders in childhood.     

A fluorimetric assay for angiotensin-I converting enzyme in human serum.

A fluorimetric method is described for a simple, sensitive and reproducible assay for angiotensin-I converting enzyme in human and guinea pig sera. The very weak fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline is enzymatically hydrolyzed, producing the highly fluorescent o-aminobenzoylglycine that is quantitatively determined by spectrofluorimetry. Dependence of activity on substrate concentration, amount of serum, time of incubation and pH were investigated. The KM value for the substrate is 0.1 and 0.032 mM for the human and guinea pig serum enzyme, respectively. The mean value of serum angiotensin-I converting enzyme for 16 normal adult persons was 2.56 +/- 0.10 (S.E.) with a standard deviation of 0.81 nmol/min/ml serum.     

Serum creatine kinase and CK-MB isoenzyme responses to acute and prolonged swimming in trained athletes

Six highly-trained male swimmers completed a maximum work capacity tethered swim and a 1-h continuous tethered swim at approximately 70% VO2max in order to evaluate total serum creatine kinase and CK-MB isoenzyme changes. Venous blood obtained before, 5 min post-, 6 h post-, and 24 h post-exercise was analyzed for total serum CK (kinetic UV method, normal = less than 100 U/l) and CK-MB isoenzyme (quantitative electrophoretic technique, normal = less than 5 U/l). VO2max averaged 4.59 +/- 0.28 l/min, with a mean total work time of 24.5 min to achieve maximum capacity. Mean resting total CK was 100.5 +/- 15.8 U/l. Compared to rest, neither swim bout produced a significant (p greater than 0.05) elevation in mean total creatine kinase. No CK-MB isoenzyme was observed in any post-exercise blood sample. Swimming, performed by highly-trained swimmers at high levels of intensity or for prolonged durations, may not impose sufficient degrees of trauma producing muscular stress. Therefore, the structural integrity of the cell membrane is maintained and the loss of intracellular creatine kinase to the bloodstream prevented.     

Clinical evaluation of high-performance affinity chromatography for the separation of bone and liver alkaline phosphatase isoenzymes

The newly described high-performance (HPLC) affinity chromatography method for the separation of human bone and liver alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes was clinically evaluated. The improved resolution of bone from liver isoenzyme and lower detection limit was achieved by conjugation of wheat-germ lectin (WGL) to a diol-bonded silica gel column, stepwise elution with N-acetylglucosamine (NAG) and post column derivatization using para-nitrophenyl phosphate substrate. To establish a reference interval, we measured bone ALP in 86 healthy women, ages 33 to 95 years. The normal reference interval is described by a piecewise linear regression on age (R2 = 0.20, P less than 0.01). For women less than or equal to 45 years, bone ALP, U/l = 8.495. For normal women between ages of 45 to 55 years, bone ALP, U/l = -12.765 + 0.472* age. If age greater than or equal to 55 years, then bone ALP, U/l 13.219. In all 10 patients with primary biliary cirrhosis, serum bone ALP levels were elevated. In addition, sera from 43 patients with diverse metabolic bone diseases were evaluated. As expected, the sera from all 6 patients with Paget's disease and 2 with osteolytic metastasis had bone ALP activity which was greater than 3 standard deviations (SD) from the mean. In all 10 patients with hypoparathyroidism, bone ALP levels were depressed. Only 1 of the 9 patients with glucocorticoid excess and 2 of the 7 patients with primary hyperparathyroidism had elevated bone ALP when compared to the 95% confidence interval for the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

An alternative approach to the prevention of succinyldicholine-induced apnoea

Succinyldithiocholine was utilized as a substrate analogue of succinyldicholine to study normal and atypical serum pseudocholinesterase (EC 3.1.1.8). In the method, the enzyme acts on succinyldithiocholine to release thiocholine, which reacts with 5,5'-dithio-bis-(2-nitrobenzoic acid) to produce a coloured compound with maximal absorbance at 410 nm. The procedure appears to be precise (between-day analysis gives a coefficient of variation between 1.1 and 3.7%) and amenable to automation, permitting routine use in any laboratory. The reference interval for 300 healthy adults with "usual" cholinesterase genotype was estimated to be 34-77 U/l, with a significant difference between males and females (40-78 U/l for men and 33-76 U/l for women, p less than 0.01). The median activity in 105 individuals with "heterozygous" cholinesterase genotype was 22 U/l (range 5-35 U/l), and for 14 "atypical" homozygotes 1.5 U/l (range 1-4 U/l). The assay with succinyldithiocholine may offer a direct procedure for preoperative screening of individuals with an abnormal response to the muscle relaxant succinyldicholine, thus avoiding the determination of genotype by measurement of inhibitor numbers.     

Separation and comparison of isoenzymes of N-acetyl-beta-D-hexosaminidase of pregnancy serum by polyacrylamide gel electrofocusing

Sera from normal and pregnant subjects as well as those from subjects with Tay-Sachs disease, hyperlipidemia and cancer were subjected to electrofocusing in the pH range of 3–10 in polyacrylamide gel (7.5%). The separated isoenzyme bands were stained for visualization on the gel for activity with $\text{α-}\text{naphthol-AS-BI}\text{-N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminide}$ as substrate.Elevation of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ (hexosaminidase) activity during pregnancy was found to be due to a progressive increase in number of isoenzymes: 1–2 bands in the early stage of pregnancy (5 weeks) to 6–8 bands in the last trimester. By comparison with the known isoenzymes of human aortic hexosaminidase, at least one of these bands corresponds to the A form, 4–5 bands to the P form and 1–2 bands to intermediate between the A and P forms. In the B form region only a single band with weak enzyme activity was observed, and its intensity of staining remained almost unchanged during pregnancy.Heat treatment (50° for 3 h at pH 4.4) resulted in inactivation of isoenzymes in the area on the gel of the A form and of the intermediates between the A and P forms but had no effect on the P and B forms. However, some bands found in the A area, between the A and P areas and in the P regions, transformed into forms having more alkaline isoelectric points (pIs) by this treatment. The new forms were found predominantly in the B area. The proportion of the thermostable (P and B) and the thermolabile (A) forms of the enzyme by such treatment was 32% and 68%, respectively, for normal serum, and 70% and 30%, respectively, for pregnancy serum. The value for pregnancy serum remained fairly constant throughout pregnancy (5 weeks to the last trimester).Tay-Sachs serum (one case) possessed three very minor bands corresponding to the P region and two major bands in the B region. When heat-treated, a partial transformation of minor bands and a complete transformation of one of the major bands in the B region occurred. One B band was transformed into the other B band having the most alkaline pI. Total hexosaminidase activity, meanwhile, did not show any change.Increase in number of isoenzymes of hexosaminidase in the P region was also found in sera of hyperlipidemic and cancer patients, suggesting that the P form may not be specific to pregnancy.     

The metabolism of Tay-Sachs ganglioside: catabolic studies with lysosomal enzymes from normal and Tay-Sachs brain tissue

The catabolism of Tay-Sachs ganglioside, N-acetylgalactosaminyl- (N-acetylneuraminosyl) -galactosylglucosylceramide, has been studied in lysosomal preparations from normal human brain and brain obtained at biopsy from Tay-Sachs patients. Utilizing Tay-Sachs ganglioside labeled with (14)C in the N-acetylgalactosaminyl portion or (3)H in the N-acetylneuraminosyl portion, the catabolism of Tay-Sachs ganglioside may be initiated by either the removal of the molecule of N-acetylgalactosamine or N-acetylneuraminic acid. The activity of the N-acetylgalactosamine-cleaving enzyme (hexosaminidase) is drastically diminished in such preparations from Tay-Sachs brain whereas the activity of the N-acetylneuraminic acid-cleaving enzyme (neuraminidase) is at a normal level. Total hexosaminidase activity as measured with an artificial fluorogenic substrate is increased in tissues obtained from patients with the B variant form of Tay-Sachs disease and it is virtually absent in the O-variant patients. The addition of purified neuraminidase and various purified hexosaminidases exerted only a minimal synergistic effect on the hydrolysis of Tay-Sachs ganglioside in the lysosomal preparations from the control or patient with the O variant of Tay-Sachs disease.     

Serum CK-MB isoenzyme after aortic and mitral valve replacements

A release of the MB fraction of creatine kinase (CK) enzyme into the serum due to myocardial manipulation and trauma occurs in patients undergoing cardiac surgery. Thus, the appearance of CK-MB activity as such is not sufficient to indicate of perioperative myocardial infarction. The mean (+/- SD) serum CK-MB isoenzyme level was 95 +/- 103 U/l 18 hours after aortic or mitral valve replacement in 76 patients. Thirteen patients undergoing closure of an atrial septal defect served as controls. They had a significantly lower (p less than 0.01) isoenzyme level postoperatively: 45 +/- 39 U/l. Two patients had the ECG changes of definite myocardial infarction after valve replacement and they also showed high CK-MB values, while the other patients with high enzyme level had no ECG signs suggesting acute infarction. CK-MB values correlated with the aortic cross-clamping time (r = 0.39, p less than 0.001) and weakly with the precordial ECG voltage of SV1 + RV5 (r = 0.25, p less than 0.01). While these findings may reflect the sensitivity of a thick myocardial wall to ischaemia during surgery, the postoperative recovery was not related to the serum CK-MB level.     

The stability of prostatic acid phosphatase, as measured by a capture immunoenzyme assay

A capture immunoenzyme assay (CIEA) for prostatic acid phosphatase (PAP) was developed and used to study the stability of this isoenzyme. Immunospecifically purified goat antibodies to PAP were covalently bound to special discs and used to capture the enzyme in serum samples in a weakly acidic medium during the first incubation (2 h) at 37 degrees C. The capture enzyme was then measured by its catalytic activity with p-nitrophenyl phosphate as substrate during the second incubation (1 h) at 37 degrees C. As much as 98% of the PAP in test specimens was captured and measured by this CIEA. The test results were expressed as enzymatic activity (U/l), extrapolated from a standard curve which was linear between 0.026 and 70 U/l. In test sera stored at 4 degrees C, the PAP was variably stable for 7 to 70 days, but the enzyme was quite stable in serum when stored at -20 degrees C for at least 156 days. At room temperature, when the sera were appropriately acidified, there was no loss of enzymatic activity for periods of 15 days, and in some cases, a large proportion of activity was still intact after 70 days. At 4 degrees C, as well as -20 degrees C, acidified serum and the partially purified PAP standard showed complete stability for at least 7 months. The CIEA reactivity of positive test specimens was inhibited by L(+)-tartaric acid, but not by cupric sulfate. The acid phosphatases of blood cell extracts were non-reactive in the CIEA procedure. The CIEA results of 224 serum samples from patients with and without prostate cancer correlated very well with those obtained by two direct enzymatic and two commercial RIA procedures, with correlation coefficients between 0.960 and 0.993, and diagnostic agreement between 86% and 100%.     

Synthesis of 4-methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate and its use in classification of GM2 gangliosidosis genotypes

Measurement of hexosaminidase A (Hex A) is an important clinical chemical procedure in the classification of GM2 gangliosidosis genotypes. We have synthesized a new substrate which may be useful in both the biochemical diagnosis of GM2 gangliosidosis and the detection of heterozygotes for the Tay-Sachs disease (TSD) allele. 4-Methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate (4MUGS) was synthesized by sulfation of 4MU-beta-D-N-acetylglucosamine (4MUG) with chlorosulfonic acid and purified through gel filtration and ion-exchange chromatography. The structure of 4MUGS was verified by elemental analysis and NMR. Hex A is approximately 100 times more active toward 4MUGS than Hex B. The advantage of this increased specificity is that Hex A can be determined in a one-step procedure which allows separation of normal control serum values from those of obligate heterozygotes. Alternatively, assay values obtained using both substrates can be transformed by application of an empirical equation that allows the calculation of both Hex A and Hex B without the requirement of thermal fractionation. Lower values for % Hex A in serum have been obtained for Tay-Sachs homozygotes using the 4MUGS assay procedure. The results of Hex A assays on fibroblast cell strains obtained from Tay-Sachs homozygotes, variant forms of GM2 gangliosidosis and normal controls are also discussed.     

A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide

Granulocyte elastase (GE, EC 3.4.21.37) is a key enzyme in tissue injury. To elucidate the role of GE in tissue injury, a new method of measuring GE activity in various inflammatory tissue fluids was developed using diazotization and the chromogenic synthetic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (S-2482). GE activity demonstrated first order kinetics in the range from 1.9 to 30 U/l. Other proteases, such as pancreatic elastase, trypsin, and chymotrypsin did not hydrolyze S-2484. This assay permits the determination of GE activity with a coefficient of variance less than 7.8% and 95.6 to 105.4% recovery. With this method, hydrolytic GE activity was found to be increased in bronchoalveolar lavage fluid from patients with ARDS or pneumonia, synovial fluid from patients with rheumatoid arthritis, and blister fluid from burn patients.     

Characterization of the isoenzyme profile of beta-N-acetylhexosaminidase in the urine of newborns.

The urinary isoenzymes of beta-N-acetylhexosaminidase (Hex) in newborn infants were characterised by chromatography, electrophoresis, thermodynamic analysis and through substrate specificity. No qualitative difference was found for the major Hex A and Hex B isoenzymes between full-term or premature newborns and adults, although in the latter group the relative proportion of Hex B is much lower (18.5 +/- 2.7% vs. 36.3 +/- 1.0%). An additional minor enzyme form was found in some premature newborns, which eluted from the DEAE-cellulose column at a higher concentration of NaCl than Hex A and, like this isoenzyme, is able to hydrolyse 4-methylumbellipheryl-2-acetamido-2-deoxy-beta-D-glucopyranoside-6 -sulphate, which would suggest that it has alpha subunits in its molecule. These results do not confirm the hypothesis of other authors about the existence of a unique fetal Hex isoenzyme in neonatal urine which eluted before the application of the NaCl gradient, similarly to the Hex B.