Total Hexosaminidase Activity Research Articles

Age- and sex-associated differences in the relationship of serum hexosaminidase and T4 levels in human neonates.

N -Acetyl-β-glucosaminidase (HEX) activity (total, A and B) and serum thyroxine (T 4 ) were determined in 22 cord serum samples, 240 serum samples from newborns up to 7 days of life, and 18 samples from infants one month of age, approximately equally divided by sex. Normal values for HEX activity in the neonatal period are given for both sexes. Cord serum values showed no significant sex differences in T 4 levels, total HEX activity, and ratio of A:B isozymes. During the first week of life, highest levels of HEX activity (males: 1378 ± 337 U/ml; females: 1149 ± 178 U/ml; P < 0.01) occur between the first and second day, and coincide with peak serum T 4 levels (males: 15.5 ± 2.9 μg/dl; females: 18.3 ± 3.3 μg/dl; P < 0.025). Although serum HEX activities in males are significantly elevated above female values throughout the first 7 days of life, no sex difference is seen in the (A:B) isozyme ratio (males = 51%:49%; females = 52%:48%). Sera from male infants at one month of age have a mean HEX level of 1734 ± 306 U/ml, which is significantly greater than that at one week of life (1231 ± 190 U/ml) (P < 0.0005). The ratio of A:B, however, remains constant (51%:49% at 1 week; 53%:47% at 1 month). Females show less-pronounced changes in total HEX values between one week (966 ± 155 U/ml) and one month (1174 ± 163 U/ml) (P < 0.005), but exhibit a marked alteration in the (A:B) isozyme ratio (52%:48% at 1 week to 61%:39% at 1 month, P < 0.0005). The percentage of HEX A in females at one month thus approaches prepubertal and adult values. The observed sex differences in HEX activity during the first month of life suggest that not only T 4 but other sex-and/or developmentally related factors as well influence total HEX activity by specific regulation of its isozymes.

Relationship of thyroid status and serum N-acetyl- β-glucosaminidase isoenzyme activities in humans

Serum from 28 hyperthyroid, hypothyroid, and euthyroid pre- or early puberty females was examined for N-acetyl- β-glucosaminidase (HEX) activity. Total, isoenzyme A (labile), and isoenzyme B (stable) levels were determined for this enzyme. A high degree of correlation ( r = 0.76; p < 0.001) exists between total hexosaminidase activity and thyroid hormone levels (as reflected by the Free Thyroxine Index). Examining each isoenzyme individually, A is selectively enhanced ( r = 0.84; p < 0.0005) whereas B displays no significant change regardless of thyroid activity. In hyperthyroid individuals, levels of total hexosaminidase (730 ± 67 units) (mean ± S.D.) and isoenzyme A (516 ± 46) were significantly higher than levels found in either the hypothyroid (total: 547 ± 30; isoenzyme A: 352 ± 31) or euthyroid (total: 620 ± 81 A; isoenzyme A: 423 ± 45) groups. However, no change was observed in levels of isoenzyme B among hypothyroid (195 ± 19), euthyroid (197 ± 43) and hyperthyroid (215 ± 32) groups. These data substantiate our earlier findings in the rat, wherein thyroxine administration evoked a similar response in the liver. They are of particular interest in light of the deficiency of HEX A in variants of GM 2 gangliosidosis.

530 GENE FREQUENCIES FOR TAY-SACHS (TSD) AND SANDHOFF'S DIS-530 EASE (SD) IN JEWISH AND NONJEWISH POPULATIONS

Since 1974, 40,012 individuals have volunteered for TSD heterozygote screening in Ca.. Of these, 6,795 are “nonJewish”. With an automated serum hexosaminidase (HEX) assay, a tri-variable analysis is possible: Total HEX activity (TA) ; % HEX A (56°); and nanomoles HEX A (nM): TA × %A. Genotype assignment is based on: Suspect SD carriers (serum TA < 450; %A > 75) and TSD heterozygotes (%A < 50; nM A <420) are confirmed by WBC testing and family studies. The SD gene has been identified in 20 families in this way (4 Jewish). In Jews the gene frequency for TSD is 0.0167 (1:30 carriers) and for SD is 6.0 × 10−5 (1:8304 carriers). In “nonJews” the TSD gene frequency is 0.0035 (1:141 carriers) and for SD, 0.0012 (1:425 carriers). These data provide the first direct approximations of the gene frequencies for TSD and SD in the Jewish and nonJewish populations. Because of possible nonrandomness in the sample and ancestral uncertainties, these figures should be viewed as reasonable estimates only. Similar analyses with sera from obligate heterozygotes for I-Cell Disease and Mucolipidosis III may allow carrier frequency estimates for these recessive alleles in these populations as well.

A new variant of Sandhoff's disease.

Extract: A 28-month-old Negro male with atypical Sandhoff's disease (GM2 gangliosidosis, type 2) is described. Clinical presentation closely resembled Sandhoff's disease. The appendiceal neuron cytoplasm was distended with periodic acid-Schiff (PAS)-positive material. Hepatic glycolipid N-acetylneuraminic acid was elevated 1.5-fold, chiefly because of increased GM2. Neutral glycolipid hexose was elevated twofold, mainly because of a component with globoside-like chromatographic properties. Unlike patients with Sandhoff's disease, the child had total β-D-N-acetylhexosaminidase activity 20–24% of normal in serum and plasma and 7–11% of normal in leukocytes and cultured fibroblasts, but activity in liver (<2% of normal) was similar to that in Sandhoff's disease. In all tissues examined, >95% of the activity was heat denaturable, corresponding in electrophoretic mobility to the heat-denaturable component of normal tissues. The decrease in heat-denaturable activity with time and the pH optimum were similar in patient and control crude tissue preparations and partially purified preparations of plasma hexosaminidase A. In the child's mother, total hexosaminidase activity was approximately 50% of mean control values in serum, plasma, and leukocytes, and serum hexosaminidase A was relatively increased. This finding, together with the death of at least three other children in the kindred from an apparently phenotypically similar illness, indicates the inherited nature of the disorder.Speculation: Appreciable hexosaminidase activity in serum and plasma and its marked deficiency in liver suggest that the active form (or forms) in serum and plasma differ from those in solid tissues, and that the mutation in the case described has altered the hexosaminidases in a manner that virtually abolishes their activity in solid tissues but permits their partial activity in serum and plasma.

HEXOSAMINIDASE A IN TEARS AND SALIVA FOR RAPID IDENTIFICATION OF TAY-SACHS DISEASE AND ITS CARRIERS

Homozygotes and heterozygotes for the Tay-Sachs gene can be identified by hexosaminidase A determinations of tear fluid collected in filter-paper strips. Total hexosaminidase activity in tears was 4576 ± 665 nmol per ml. per hour, and the A fraction comprised from 38 to 72% of the total. The hexosaminidase A levels in tears are six to ten times higher than in serum. Only Tay-Sachs homozygotes can be identified by hexosaminidase A determinations in saliva. In tears, hexosaminidase A migrates with the α-globulin fraction and is not associated with lysozyme. The simple method of tear collection on filter-paper strips and subsequent storage in screw-cap vials with no loss of hexosaminidase A activity would ease considerably the dispatch of samples from one city to another. The method is of value for screening other inborn errors of metabolism and carriers.

Sandhoff disease: Diagnosis of heterozygous carriers by serum hexosaminidase assay

1. (1) A batch-wise fractionation of hexosaminidases with DEAE-cellulose gave consistent and reproducible results. The ratio of the A and B enzymes by this method was slightly higher than that by the heat inactivation method. 2. (2) Investigations on obligate and possible carriers of Sandhoff disease revealed a characteristic serum hexosaminidase pattern; low total hexosaminidase activity with relative preservation of component A. This was well contrasted with low activity of component A and normal total activity in Tay-Sachs disease heterozygotes.

Separation and comparison of isoenzymes of [formula omitted] 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 α- naphthol-AS-BI-N- acetyl-β- D-glucosaminide as substrate. Elevation of N- acetyl-β- D-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.

Gangliosidosis with total hexosaminidase deficiency: Clinical, biochemical and ultrastructural studies and comparison with conventional cases of Tay-Sachs disease

Biochemical studies were performed on brain biopsies, and ultrastructural studies on brain and liver biopsies from three children presenting clinically the infantile form of Tay-Sachs disease. Gangliosides were markedly increased both in grey and white matter, and consisted mainly of fraction GM2. In one case (patient 1), the amount of N-acetylaminotrihexosyl-ceramide, the asialo-residue of ganglioside GM2, was much larger than in the two other cases (patients 2 and 3). Enzyme studies performed on liver tissue disclosed, in patient 1, a nearly complete absence of the total hexosaminidase activity, which was within normal limits in the two other cases. Ultrastructural data in brain and liver were also different in the three cases. In patient 1, the lipid inclusions of the neurons, astrocytes and endothelial cells were markedly more pleiomorphic than in cases 2 and 3. In the liver of patient 1, hepatocytes and, in a lesser degree, Kupffer cells were full of lipid inclusions, whereas in the two other cases, only a small number of lipid lamellar elements were present in a few cells.

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.

Age-dependent variations of the human N-acetyl- -D-hexosaminidases.

AbstractThe total N‐acetyl‐β‐d‐hexosaminidase (hexosaminidase A plus B) activity of the human brain increases during life until the age of 70 years to about twice the activity found in the foetal brain. This activity roughly parallels the increasing level of brain ganglio‐sides. Simultaneously, the ratio between the hexosaminidases A and B changes from approx. 3.4 to 1.1 as established by the microscale isoelectric focussing of the enzymes from crude extracts. Age‐dependent variations can be also demonstrated for other glycosidase activities: The β‐d‐glucosidase is shown to increase, the α‐d‐mannosidase and α‐l‐fucosidase to decrease during life. The enzyme values are related to the protein content, the age‐dependent shift of which we have determined in the human brain. In different organs and body liquids distinct differences are found for the levels of the total hexosaminidase activity as well as for the hexosaminidase pattern. The relation of the hexosaminidase activity to the ganglioside degradation during the human development is discussed.

Juvenile GM2 gangliosidosis. Biochemical and ultrastructural studies on a new variant of Tay-Sachs disease.

Morphological and chemical studies were performed on a cerebral biopsy in a patient with the clinical course of late infantile amaurotic idiocy. Accumulation of the Tay-Sachs ganglioside, G M2 (G 5 ) was noted in both gray and white matter. Hexosaminidase A in serum and skin fibroblast cultures was reduced to 6.6% and 12% of total hexosaminidase activity, as contrasted with 57.3% and 49.0% in controls. On electron microscopic examination there was an accumulation of intraneuronal membranous cytoplasmic bodies, zebra bodies, other varieties of lipid bodies (including aggregates of the preceding types), and lipofuscin bodies. These findings indicate that the patient had juvenile G M2 gangliosidosis, a newly recognized G M2 ganglioside storage disease in which hexosaminidase A is partially active but apparently to an insufficient degree to prevent the slow evolution of neurological symptoms.

Hexosaminidase activities in a case of systemic GM2 gangliosidosis of late infantile type.

SummaryAn unusual systemic GM2 gangliosidosis of late infantile type was shown to have high levels of hexosaminidase activity in brain and liver. It, therefore, differs from another GM2 variant of late infantile type reported by Sandhoff et al. (2), in which hexosaminidase activity was absent.Addenum. Okada and O'Brien reported that the isoenzyme hexosaminidase A was absent in TSD tissue (6). The percentage of hexosaminidase A and B in brain extracts from the present case and from a TSD and control case were analyzed by acrylamide gel electrophoresis and quantitative fluorometric scanning using 4-methylumbelliferyl-2-acetamide-2-deoxy-β-D-glucopyranoside as substrate. In the present case the A fraction comprised 40% of the total hexosaminidase activity in contrast to 60% in a control and 0% in TSD brain.