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Objectives: The relative proportion in percentage of the isoenzyme A of beta-hexosaminidase (Hex) is the single discriminatory function most frequently used for the biochemical screening of heterozygote Tay-Sachs disease carriers. It has been suggested that the assay of the Hex isoenzymes in homogeneous cell preparations is preferable to that in mixed total leukocytes which present greater interindividual variation. The major aim of our study was the evaluation of this hypothesis., Design and Methods: Total Hex and its Hex A and Hex B isoenzymes were determined in different samples of serum and plasma (n = 81) as well as in lysates of platelets (n = 75), and mononuclear (n = 81), polymorphonuclear (n = 81) and mixed total leukocytes (n = 33)., Results: The interindividual variations found for % Hex A in the different biological samples were: plasma (CV = 23.4%), platelets (CV = 10.2%), mononuclear (CV = 5.7%), polymorphonuclear (CV = 5.3%) and total leukocytes (CV = 7.1%). Although the relative proportion of Hex A was significantly greater in polymorphonuclear than in mononuclear leukocytes (P < 0.001), a statistical significance was not attained for the correlation between the relative proportions of blood polymorphonuclear cells and Hex A in mixed total leukocytes (r = 0.220)., Conclusions: The use of total leukocyte lysates does not appear to introduce a significant increase for the interindividual variation of the Hex A isoenzyme relative proportion in relation to the use of homogeneous cell preparations.

In the GM2 gangliosidosis B1 variant, the mutated isoenzyme A of beta-hexosaminidase (Hex) is incapable of hydrolyzing ganglioside GM2 and negatively charged substrates. Biochemical characterization of this lysosomal disease is carried out using synthetic alpha-subunit-specific sulfated substrates, as heat-inactivation assays are not applicable. The apparent enzyme activation energy of Hex using the chromogenic substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide is related directly to the relative proportions of Hex A and Hex B isoenzymes. This thermodynamic variable was used for the study of Hex enzyme heterogeneity in 3 patients with the GM2 gangliosidosis B1 variant and 6 heterozygote carriers. Hex activity was determined at 25 degrees C, 30 degrees C, 35 degrees C, and 37 degrees C in a Cobas Bio analyzer (Roche Diagnostics, Basel, Switzerland), and Arrhenius plot slopes and apparent activation energies were calculated in plasma samples and mononuclear and polymorphonuclear leukocyte lysates. The determination of the Hex isoenzymes in plasma presented a high discrimination power for B1 variant patients but not for heterozygote carriers, in whom false-negative results may be obtained. However, thermodynamic evaluation of the isoenzyme composition of Hex in leukocyte lysates permits the biochemical identification of patients with the GM2 gangliosidosis B1 variant and of heterozygote carriers.

Using 3,3'-diclorophenolsulfoftaleinil N-acetyl-beta-D-glucosaminide as a substrate, the apparent activation energy of beta-N-acetylhexosaminidase (Hex) was determined in samples of plasma and urine, as well as in leukocyte and platelet lysates. Incubation with papain produced an increase in this thermodynamic variable for plasma Hex (precursor forms with high molecular mass) that would be caused by the proteolytic action of papain on the Hex A isoenzyme. However, digestion with papain did not significantly modify the activation energy of Hex in leukocyte and platelet lysates (mature enzymatic forms). In 11 healthy subjects and 28 patients with different renal diseases, no statistically significant differences were found with regard to the values obtained in cellular lysates for variations in the activation energy of urinary Hex, regardless of whether they presented normoalbuminuria, microalbuminuria or macroalbuminuria. These results support the hypothesis that even in patients with proteinuria, no significant amounts of plasma Hex precursor forms are found in urine samples, and the source of the enzyme activity is the kidney itself.

Ammonia is known to increase the secretion of beta-N-acetylhexosaminidase (Hex) to the extracellular medium in cultured human fibroblasts and Hep G2 cells. We examined 35 patients with liver cirrhosis and the results revealed a significant increase for the plasma activities of total Hex and its isoenzymes Hex A and Hex B (p < 0.001). The partial correlations, with other biochemical markers of liver injury constant, between plasma ammonia concentration and the activity (r = 0.658) and the proportion in percentage of the Hex B isoenzyme (r = 0.692) were statistically significant (p < 0.001). The increased concentration of ammonia could explain, at least partially, an increased secretion of Hex B isoforms to the plasma in patients with liver disease.

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.

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.

The determination of the activation energy of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), using 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide as substrate, allows its isoenzyme composition to be evaluated in different biological specimens. However, in the analysis of urine samples, it is necessary first to remove the endogenous low-molecular-mass components, as these provoke an over-estimation of the activation energy of the Hex and, consequently, of the relative proportion of Hex B isoenzyme. The study of this interference has allowed urea to be characterised as the only urinary metabolite that is responsible, and to establish a mathematical expression for the correction, in relation to the endogenous urea concentration, of the activation energy of the Hex obtained experimentally in samples of native urine. The results thus obtained for the isoenzyme composition of urinary Hex are similar to those found using an electrophoretic separation procedure.

The activation energy (Ea) of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52) was determined with 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide as substrate, with a much higher value being found for the Hex B isoenzyme (Ea = 75.1 kJ/mol) than for the Hex A isoenzyme (Ea = 41.8 kJ/mol). This fact allowed for the development of a fast and reliable thermodynamic method to determine the isoenzyme composition of Hex in different biological specimens (serum/plasma, saliva, cerebrospinal fluid, seminal plasma, urine, and leukocyte lysates). The results in serum given by the proposed method may be superimposed upon those obtained by the heat inactivation assay of O'Brien et al. (N Engl J Med 1970;273:15-20), and the catalytic activity calculated for Hex A offers a good correlation with that obtained by using the specific substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6 sulfate (n = 25, r = 0.953).

Activity of serum/plasma beta-N-acetylhexosaminidase (EC 3.2.1.52) was determined by means of a continuous spectrophotometric method using 3,3'-dichlorophenylsulphonphthaleinyl-N-acetyl-beta-D-glucosaminid e as substrate, with very satisfactory results. Incubation of an undiluted aliquot (1 ml) of samples at 52 degrees C for 8 hours with an adjusted pH 5.5-6.0 provoked only the inactivation of isoenzyme A, thus allowing the evaluation of beta-N-acetylhexosaminidase isoenzyme composition. In 25 serum samples from control subjects and pregnant women, a good correlation between the percentage of isoenzyme B obtained by this procedure and the fluorimetric assay of O'Brien et al. (New Engl J Med 1970; 273:15-20) was found (r = 0.983, S(yx) = 1.51), with no statistically significant difference between the means (43.2 vs 42.8%). In 84 healthy adult subjects, an average value of 30.3% for the proportion of isoenzyme B was obtained, with an interval of 25.4-35.0%, in agreement with results reported by other authors.

Using an electrophoretic technique on cellulose acetate, three multiple forms of the 5'-nucleotidase (5'NU) with mobilities alpha 1, alpha 2, and beta appear in the serum of healthy subjects. The difference between the alpha 1 and alpha 2 isoforms lies in their sialylation degree, the alpha 2-5'NU being a de-sialylated form. In 147 patients with different hepatobiliary diseases the alpha 2 isoform was present in only 19% of cases, and there was no significant difference in the activity of total 5'NU, alpha 1-5'NU and beta-5'NU between patients with or without alpha 2-5'NU, alpha 1-5'NU and beta-5'NU (P < 0.001), as with other biochemical indicators of liver damage. It is suggested that in hepatobiliary diseases an increase of the sialylation of serum 5'NU occurs, which would explain the absence of the desialylated alpha 2 isoform in the majority of cases. However, the decrease of hepatic receptors of asialoglycoproteins would lead to an increase of this de-sialylated isoform in the serum of certain patients.

Serial measurements of alkaline phosphatase and 5'-nucleotidase multiple forms in two patients undergoing surgical procedures to release biliary obstruction suggested an inverse relationship between high-M(r) isoforms and serum bile acids concentrations. Furthermore, the study of several groups of patients with cholestatic disorders confirmed this inverse correlation. Mechanisms responsible for these observations are discussed.