Lysosomal enzyme variations in thirteen cell strains cultured from one amniotic fluid

Fifteen primary amniotic fluid cultures were established from a single sample of amniotic fluid. Three different methods were used to set up these cultures which yielded 13 cell strains. Nine lysosomal enzymes (acid phosphatase, β-glucuronidase, β-galactosidase, α-galactosidase, α-glucosidase, α-mannosidase, α-arabinosidase, $\text{N-}\text{acetyl}\text{-ß-}\text{d}\text{-}\text{glucosaminidase}$ and arylsulphatase A) were assayed in these 13 cell strains. The coefficients of variation of these enzyme levels were less than the coefficients for enzyme levels in cell strains grown from different samples of amniotic fluid but greater than those for the combined culture and assay system used. No assay values were found which could have suggested a possible enzyme deficiency disease.     

Isoenzymes of four acid hydrolases in human kidney and urine

The occurrence of different isoenzymes of β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-mannosidase in human urine and kidney tissue was studied by isoelectric focusing. Artificial substrates were used for the enzymatic assays. There was a predominance of isoenzymes with a low isoelectric point in the urine. In the kidney tissue isoenzymes with higher isoelectric point predominated. This difference may be due to a higher proportion of N-acetylneuraminic acid-containing enzymes in the urine than in the kidney tissue.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Urinary lysosomal glycosidases after renal allotransplantation: correlation of enzyme excretion with allograft rejection and ischemia

Urinary β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase were measured in patients with renal allotransplants and compared with normal controls. Increased excretion of all three enzymes was noted in the transplant patients resulting possibly from mild chronic rejection.A second part of the investigation correlated renal function with daily N-acetyl-β-glucosaminidase excretion by the patients. In acute rejection, enzyme levels rose sharply from a baseline then decreased following successful treatment. With cadaveric grafts and initially good urinary flow, N-acetyl-β-glucosaminidase levels were high and decreased as creatinine clearance improved; however, with initial oliguria, levels were low and rose as diuresis began then decreased to a baseline. This was attributed to a washing out of enzyme released during the unavoidable ischemic period involved in handling cadaver kidneys.Because it reflects physiological changes in the kidney, daily monitoring of urinary N-acetyl-β-glucosaminidase should be helpful in the diagnosis of renal damage caused by rejection and ischemia.     

Lysosomal enzyme activities in cultured lymphoid cell lines

Several lysosomal enzyme activities in cultured lymphoid cell lines were studied during 3 phases of cell culture; logarithmic growth phase, stationary phase and decline phase.Enzyme induction during cell growth was found in N-acetyl-hexosaminidase, β-galactosidase and α-l-fucosidase, but no induction in a-d-mannosidase, α-glucosidase and β-glucuronidase. The latter two enzymes were unchanged during all cell culture phases.A drop in α-l-fucosidase and α-d-mannosidase activity was found during the stationary and decline phases of cell culture.     

Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases—β-N-acetylglucosaminidase; β-galactosidase; β-glucuronidase; α-mannosidase; α-fucosidase—in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible.Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected healthy subjects were studied. No sex differences were observed for all the enzymes studied with the exception of β-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10–14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by β-galactosidase and by β-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.     

LYSOSOMES OF THE ARTERIAL WALL : I. ISOLATION AND SUBCELLULAR FRACTIONATION OF CELLS FROM NORMAL RABBIT AORTA

Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks'' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, β-galactosidase, β-glucuronidase, α-mannosidase, β-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-β-glucosaminidase and β-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5''-nucleotidase and leucyl-β-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.     

Activity of beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in human rectal mucosa

Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents.The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent K_m values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM.The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.     

Detection of Fabry hemizygotes and heterozygotes by measurement of -galactosidase in urine

The determination of α-galactosidase in urine can be used as a simple method for the diagnosis of Fabry hemizygotes. The activity of this enzyme was related to that of N-acetyl-β-glucosaminidase. The ratio N-acetyl-β-glucosaminidase/α-galactosidase in urine was relatively constant in any one individual. In the control group, the mean value of this ratio was 7.4 (range 1.2–20.5). In Fabry hemizygotes (n = 6) the ratio was 50 or higher. Three types of carriers could be recognized, with high (n = 1), intermediate (n = 2) and normal (n = 3) values, so that with this procedure some of the carriers are detected.     

Hypothermia and Infection I. Influence of Hypothermia on Antibody Formation in Mice in the Secondary Response to Typhoid-H-Antigen

Öckerman, P. A. Lysosomal Acid Hydrolases in the Liver in Gargoylism. Deficiency of 4-methylumbelliferyl-β-galactosidase. Scand. J. clin. Lab. Invest. 22, 142-146, 1968.

The activites of six lysosomal acid hydrolases were measured in liver biopsy specimens from six patients with gargoylism and in post-mortem tissue from two patients. A marked diminution was noted for the activity of 4-methylumbelliferyl-β-glactosidase in all patients with gargoylism. The deficiency of β-galactosidase did not seem to be due to the presence of an inhibitor in the liver.

The activities of β-glucuronidase, β-acetylglucosaminidase, acid phosphatase and α-fucosidase were significantly increased in some of the patients. The activity of α-mannosidase was not significantly different from the values in the controls.     

Human amniotic fluid α-glucosidase

Amniotic fluid in midpregnancy contains significant α-glucosidase activity. This enzyme is distinguishable from the lysosomal acid α-glucosidase, deficiency of which is associated with Pompe's disease.The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic α-glucosidase is also different from the classical neutral form.Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that α-glucosidase in amniotic fluid is linked to lipids.     

A new variant mucolipidosis: Biochemical investigations on two siblings

Biochemical studies are presented on two siblings with some features of Mucolipidosis III, but with distinctive clinical findings. Levels of β-galactosidase, -mannosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and -fucosidase found in serum from these patients ranged from 10 to 100 times higher than normal. The ratio of heat stable to heat labile serum isoenzymes of N-acetyl-β-glucosaminidase is considerably greater than normal.

An extremely low activity of β-galactosidase was found in fibroblasts cultured from one patient. Levels of the remaining enzymes were in the low normal range. Similarly, β-galactosidase levels were low in heart, kidney, liver, spleen and lung of one patient who died during the course of the study. Activities of the remaining enzymes were close to normal.

No excessive excretion of mucopolysaccharide was noted, however, changes in distribution of several fractions were found. Mucopolysaccharide labeled with radioactive sulfate was degraded by cultured fibroblasts at a normal rate.

In addition to clinical differences, the biochemical studies further demonstrate the uniqueness of these patients.     

Mucolipidosis I: Studies of sialidase activity and a prenatal diagnosis

The characteristics of the sialidase (N-acetyl-α-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[³H]actitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3′-methoxyphenyl)-N-acetyl-α-neuraminic acid, or 2′-(4-methylumbelliferyl)-Nacetyl-α-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and β-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[³H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell culture.     

Automated assay of N-acetyl-β-GLucosaminidase in normal and pathological human urine

An automated method for the assay of N-acetyl-β-glucosaminidase in human urine is described and a normal range of urinary N-acetyl-β-glucosaminidase activity is reported. The automated method has been used routinely over a twelve month period in two London hospitals for monitoring the excretion of N-acetyl-β-glucosaminidase in renal transplant patients. The abnormal elevation of urinary N-acetyl-β-glucosaminidase provides an early warning of rejection by indicating the presence of renal parenchymal damage. The automated assay of urinary N-acetyl-β-glucosaminidase may also be used in screening for the presence of renal disease.     

A Comparison of the β-Glycosidase Excretion during Kidney Damage Induced by 4-Nitrophenylarsonic Acid and by Rabbit Anti-Rat Kidney Antibodies

Abstract. In a study of the value of urinary enzyme activities as an indication of glomerular or tubular damage, the effects of two nephrotoxic agents (4-nitrophenylarsonic acid and rabbit anti-rat kidney serum) on the urinary excretion of four β-glycosidases were compared using fluorimetric assays with 4-methylumbelliferyl substrates. The electrophoretic mobilities on starch gel of urinary β-galactosidase, β-glucosidase, β-glucosaminidase and β-glucuronidase, at various times up to 26 days after the injection of the nephrotoxins into the rat, were compared with those of the corresponding enzymes present in normal rat urine, kidney and serum. 4-Nitrophenylarsonic acid causes tubular damage characterised by an immediate rise in the rates of excretion of the four β-glycosidases, followed by a return to normal values. In Masugi glomerulonephritis the immediate rise in enzyme excretion is much less marked but greater increases occur after 10 to 12 days. The increases in excretion rate correlate roughly with the stages of the disease. Both glomerular and tubular damage produce characteristic changes in the excretion of all of the enzymes studied, but β-glucosidase appears to be the most useful indicator of kidney tubular damage, as it is the least widely distributed of these enzymes and is not normally found in urine or serum at significant levels of activity.     

Analysis of urinary albumin,transferrin, N-acetylβ-D-glucosaminidase and β2-microglobulin in patients with impaired glucose tolerance

We investigated the changes in urinary albumin and urinary transferrin as glomerular proteins, and in urinary N-acetyl-β-D -glucosaminidase and urinary β₂-microglobulin as tubular proteins, in patients with impaired glucose tolerance. We attempted to compare the proteins of normal subjects to those of diabetics with pre-nephropathy. Transferrin and N-acetyl-β-D -glucosaminidase levels were significantly increased in patients with impaired glucose tolerance, while albumin and β₂-microglobulin levels were only slightly increased. In addition, there was no significant difference in transferrin levels between patients with impaired glucose tolerance and type 2 diabetics with pre-nephropathy. In our observation, although albumin levels were only slightly increased in patients with impaired glucose tolerance, a sharp increase in transferrin levels was reflected in patients with glomerular disorders. In addition, since N-acetyl-β-D -glucosaminidase levels varied markedly, tubular disorders were suspected. It should be stressed that increased parameters for both glomerular and tubular disorders in group C—patients who showed abnormal levels in three proteins—had already been observed in some patients with impaired glucose tolerance. Therefore, the evaluation of the mutual relationships between various urinary protein components in patients with impaired glucose tolerance will become a more important assessment tool than that of single urinary protein components. J. Clin. Lab. Anal. 12:351–355, 1998. © 1998 Wiley-Liss, Inc.     

Alkaline phosphatase and acid lysosomal hydrolases in pancreatic juice and fibroblast cell cultures of patients with chronic calcifying pancreatitis

Abstract. Nine lysosomal enzymes and alkaline phosphatase have been assayed in human pancreatic juice from controls and patients with chronic calcifying pancreatitis. Specific activities were evaluated by a non parametric test (Wilcoxon) with a probability of 2 P .0.5. The values of acid phosphatase, α -glucosidase, β -glucosidase and α -galactosidase are significantly higher in pathological juices; the values of α -mannosidase and β -glucuronidase are also increased in the same patients but at the limit of significance. Alkaline phosphatase, β -hexosaminidase and α -fucosidase follow the same trend but the values are not statistically significant between the two groups of patients. Studies on skin cultures of four patients with chronic calcifying pancreatitis demonstrate that the increased specific activities of lysosomal enzymes in the pathological juices do not correspond to a leakage of these enzymes into the extracellular space as described for cystic fibrosis.     

Impaired degradation of keratan sulfate in GM1-gangliosidosis

We have prepared a new radiolabeled substrate (galactose-N-acetylglucosamine 6-sulfate-[1-³H]galactitol), from shark cartilage keratan sulfate, for an assay of acid β-galactosidase activity. Using this substrate, we found that there was a striking deficiency of β-galactosidase activity in the cultured skin fibroblasts of patients with GM₁-gangliosidosis. However, there seemed to be no quantitative differences in residual enzyme activity between type 1 and type 2 GM₁-gangliosidosis.     

Debranching enzyme in fibroblasts, amniotic fluid cells and chorionic villi: pre- and postnatal diagnosis of glycogenosis type III

Glycogenosis type III is characterized by a deficiency of debranching enzyme in most tissues, and it can be detected by the inability to liberate glucose from limit dextrin. However, using this assay, the deficiency is not expressed in cultured fibroblasts from patients with glycogenosis type III. We have demonstrated that the failure to detect debranching enzyme deficiency in fibroblasts is entirely due to interference of acid alpha-glucosidase, which can also hydrolyse limit dextrin. A method is described to remove specifically acid alpha-glucosidase allowing clear discrimination between fibroblasts from patients and controls, whereas heterozygotes showed intermediate values. The results with amniotic fluid cells and chorionic villi suggest the feasibility of first- and second-trimester prenatal diagnosis of glycogenosis III.