Total Hexosaminidase Research Articles

SANDHQFF DISEASE: INCREASED β-D-N-ACETYLGLUCOSAMINIDASE ACTIVITY AFTER CORTICOSTEROID EXPOSURE IN CULTIVATED SKIN FIBROBLASTS

We studied a 20 months old French-Canadian girl presenting the clinical picture of Tay-Sachs disease with progressive blindness, bilateral cherry red spots, hyperacusis, hepatosplenomegaly and hypotonia, with onset of symptoms by 9 months of life. Serum and WBC β-D-N-acetylglucosaminidase activities were reduced to levels less than 1% normal. Obligate heterozygotes showed reduced levels of total serum hexosaminidase activity and lower than normal % B form:A similar reduction of total hexosaminidase to 8% normal values was found in cultivated skin fibroblasts from our patient. Interestingly, levels of hexosaminidase were raised up to 25% normal value when fibroblasts were grown in nutritional medium supplemented with prednisolone or cortisone (1 μg/ml). This observation, if confirmed in other cases of Sandhoff disease might suggest a therapeutic approach in this rapidly progressive and always fatal disease.Supported by MRC grants MA-4767 and 5163.

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.

Gm2-gangliosidosis with total hexosaminidase deficiency.

Gm2-gangliosidosis with total hexosaminidase deficiency.

Screening for Tay-Sachs disease in utero using amniotic fluid.

SummaryThe amniotic fluid from 38 control and 5 heterozygote TSD mothers were analyzed by acrylamide gel electrophoresis and heat fractionation for the percent of hexosaminidase A activity. This study indicates that amniotic fluid, when analyzed by acrylamide gel electrophoresis, can be used for the prenatal diagnosis of TSD.ADDENDUMSince this paper was submitted for publication, the amniotic fluids of 35 additional normal (non-Tay-Sachs) pregnant women, 3 pregnant Tay-Sachs mothers (predicted normal) and 2 pregnant Tay-Sachs mothers (predicted Tay-Sachs) were analyzed for total hexosaminidase and hexosaminidase A content with both the heat fractionation and acrylamide gel procedures. The results obtained extend and confirm the findings reported in this paper.

Partial deficiency of hexosaminidase component a in juvenile gm2-gangliosidosis.

SUMMARYA previously reported case of juvenile G_M2-gangliosidosis was reinvestigated enzymatically with cellulose acetate electrophoresis. There was a partial deficiency of hexosaminidase component A, in contrast to the complete absence of this component in classical infantile G_M2-gangliosidosis (Tay-Sachs disease) and to the complete loss of both components A and B in a case of total hexosaminidase deficiency. Juvenile G_M2-gangliosidosis, therefore, may be tentatively characterized as partial hexosaminidase A deficiency, although this partial deficiency may be the result of complete deficiency of one or more specific subcomponents not yet identified within “component A” of hexosaminidase.

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.

Glycoprotein and collagen metabolism in the dermis of hypophysectomized rats.

The effect of hypophysectomy on the concentration of total, salt soluble (0.15 and 0.5M sodium chloride and 0.5M sodium citrate) and insoluble collagen, nitrogen and hexosamine (Hex) in the of 60-day-old male was investigated. The pituitary glands were ablated at the 30th day of age. The total hydroxyproline (Hyx) concentration in hypophysectomized animals was lowered by about 25 % and that of total Hex increased by 30% as compared to the controls. The Hex/Hyx ratio was 0.047 controls and 0.081 hypophysectomized rats. Since the mucopolysaccharide (MPS) concentration was not modified in hypophysectomized [Nature (London) 200: 784, 1965], the higher Hex concentration appears due to increased glycoprotein content. Only about one fourth of the total Hex in intact and one fifth in hypophysectomized animals can be accounted for as MPS. (Endocrinology 78:1255,1966) P hormones have been reported to be involved in mucopolysaccharide (1-3) and hexosamine (3) metabolism. Our studies on permeability suggested that the ablation of the pituitary gland increases the degree of polymerization of hyaluronic acid in connective tissue (4). In the present work we investigated the effect of hypophysectomy on the concentration of collagen, glycoproteins and mucopolysaccharides in the dermis. Materials and Methods The studies were performed on young male albino of the CFE strain. Animals in the experimental group were hypophysectomized when 30 days old; they weighed 95-100 g. Both hypophysectomized and control were purchased from Carworth Farms, Inc., New City, Rockland County, N. Y. They were fed Wayne Lab Blox mice and rats (Allied Milling Co.) diet and had free access to tap water. The of both groups were sacrificed when 60 days old by exsanguination under light ether anesthesia. At that time, those in the control group weighed an average of 240 g±30, whereas the hypophysectomized averaged only 100 g +10. The of the animals was shaved, freed from Received December 13, 1965. This work was aided by Grant A-4619 from the National Institute of Arthritis and Metabolic Diseases, USPHS. 1 Present address: Laboratory of Experimental Metabolic Diseases, Veterans Administration Hospital, Long Beach, California 90804. 2 Standard deviation. 3 The animals which increased their body weight after hypophysectomy were eliminated from the experiment. adherent muscles by scraping, ground into paste in a mill (Wiley Laboratory mill, intermediate size) and weighed. One portion of the wet skin was defatted with acetone in a Soxhlet extractor, dried at 80 C in a drying oven and used total hexosamine, collagen and mucopolysaccharide determinations. The other portion of wet skin was successively extracted with 0.15M, 0.5M sodium chloride and 0.5 sodium citrate, pH 3.6. These extracts, the insoluble residue, and the portions of dry defatted samples were then hydrolyzed with 6N hydrochloric acid in sealed glass tubes at 104 C 4 hr and analyzed hexosamine, collagen and nitrogen contents. The hexosamine content was determined by the Elson-Morgan method according to Blix (7). The collagen content was determined by the Stegemann method hydroxyproline (8). Nitrogen content was determined by the micro-Kjeldahl procedure using Nessler reagent. Acid mucopolysaccharides were extracted from the skin, fractionated and determined according to our previously described method (9). The isolated acid mucopolysaccharides (MPS) were precipitated with cetylpyridinium chloride (CPS) and CPCMPS complex was successively extracted with 0.4M, 1.2M and 2.1M sodium chloride in order to solubilize and separate the hyaluronic acid, chondroitin sulfate and heparin fractions, and then analyzed by the carbazole method according to Bitter and Muir (10) using glucurone as the standard. Results and Discussion The animals with an intact pituitary gland (control) manifested normal growth, whereas 4 Sorensen's citrate buffer (5, 6). 6 Since only small samples of could be obtained from hypophysectomized rats, the determination of acid mucopolysaccharides this group was performed on a pooled sample.