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.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

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.     

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.     

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.     

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.     

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.     

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.     

The use of galactosylceramides with uniform fatty acids as substrates in the diagnosis and carrier detection of Krabbe disease

Cerebroside-β-galactosidase (galactosylceramidase EC 3.2.1.4.6) activity was studied using galactosylceramides of uniform fatty acid composition. The highest activity and the best discrimination between patients with Krabbe disease and controls were found with N-nervonoylgalactosylsphingosine (C 24: 1-cerebroside). As a general rule cerebrosides with a monoenoic fatty acid gave higher activity and better discrimination than the corresponding cerebroside with a saturated fatty acid, the differences being largest for the cerebrosides with the longest fatty acids. In two methods the C 24: 1 cerebroside was used as substrate in the assay of the cerebroside-β-galactosidase activity in leukocytes from 12 Krabbe patients, 14 parents and 22 controls. In a third method lactosylceramide prepared from mammalian brain gangliosides was used as substrate. With all three methods the residual activity in the leukocytes of the Krabbe patients did not exceed 5%, there was no tendency for overlap between the activities of the patients and those of the obligate carriers, and the values of half the carriers fell within the range for the controls.     

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.     

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.     

N-Acetyl β-d-glucosaminidase and α-l-fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes

N-Acetyl β-d-glucosaminidase and α-l-fucosidase were determined in human sera from 25 control subjects, in 23 diabetic patients without retinopathy and in 22 diabetic patients with retinopathy.The results show significantly higher N-acetyl β-d-glucosaminidase activity in diabetic patients independently of the development of retinopathy and also independently of the length of diabetes. No correlation was found between either serum enzymes and serum glucose concentration and glycosylated hemoglobin (HbA,).     

Atypical GM1 ganglioside accumulation in a case of juvenile amaurotic idiocy

The brain and liver from a 7-year-old Japanese girl with juvenile amaurotic idiocy were examined neuropathologically and biochemically. Visceromegaly and skeletal abnormalities were absent. Nerve cells in the central nervous system were swollen and contained fine fat granules. Electronmicroscopically, there were large numbers of irregular bodies in the perikarya and these corresponded to the curvilinear and membranous cytoplasmic bodies.Lipid analysis of the brain revealed that GM₁ ganglioside was increased in the parietal and occipital areas, while the frontal lobe showed a normal ganglioside pattern. N-Acetyl neuraminic acid (NANA) content in all areas was not elevated. Determinations of β-galactosidase activity were within normal ranges. The liver had no accumulation of GM₁ ganglioside and showed a normal β-galactosidase activity. These unusual findings in GM₁ gangliosidosis were discussed.     

Heterogeneity of acid phosphatase activity in human polymorphonuclear leukocytes

Evidence is presented indicating the existence of two acid phosphatase activities in human polymorphonuclear leukocytes. There is an acid phosphatase activity, acting preferentially on β-glycerophosphate. This enzyme has a typical lysosomal localization (p = 1.24) and is strongly inhibited by 1.5 mM (+)-tartrate and 15 mM fluoride, being insensitive to 45 mM alloxan and 0.3 M citrate. The second acid phosphatase acts preferentially on phenyl phosphate and is bound to structures of low density (p = 1.18). It is strongly activated by 0.3 M citrate and dl-tartrate, and insensitive to 15 mM fluoride. It is also extremely sensitive to 45 mM alloxan and to thiol-blocking agents, and to denaturation by preincubation under varying conditions.It was not possible to demonstrate any difference in K_m values, pH effect or response to different substances or ions between the β-glycerophospliatase or phenylphosphatase localized in the granular or supernatant fractions. It is concluded that the soluble acid phosphatase activities are similar to that of the granular fraction and may be derived from ruptured lysosomes or solubilization from other subcellular structures.     

Ultrasound Diagnosis of Carotid Artery Stiffness in Patients with Ischemic Leukoaraiosis

The pathophysiology of ischemic leukoaraiosis (ILA) is unknown. It was recently found that ILA patients have increased aortic stiffness. Carotid stiffness is a more specific parameter and could have value as a non-invasive diagnostic value for ILA. Therefore, using color-coded duplex sonography, we compared local carotid stiffness parameters of 59 patients with ILA with those of 45 well-matched controls. The diagnosis of ILA was based on exclusion of other causes of white matter changes seen on magnetic resonance imaging. Pulse wave velocity β (PWVβ, m/s), pressure–strain elasticity modulus (E_p, kPa), β index and augmentation index (A_ix, %) values were higher and arterial compliance (AC, mm²/kPa) values were lower in the ILA group; however, only E_p and PWVβ reached statistical significance (p ≤ 0.05). β, E_p and PWVβ exhibited an increasing trend with higher Fazekas score, though only E_p reached significance (p = 0.05). The main conclusion was that E_p and PWVβ could have a diagnostic role in patients with ILA.     

Lysosomal enzymes in cerebral atrophy

In seven patients with cerebral atrophy due to pre-senile dementia and/or cerebrovascular disease, the activity of acid phosphatase in lumbar cerebrospinal fluid (CSF) was higher (p < 0.05) than in six controls. The activity of arylsulphatase and β-galactosidase in CSF was the same in the two groups. In the serum, the activities of acid phosphatase and arylsulphatase were the same in the two groups but the activity of β-galactosidase was lower (p < 0.02) in patients with cerebral atrophy.     

Longitudinal Analysis of Parent Communication Behaviors and Child Distress during Cancer Port Start Procedures

Background: The roles parents play in supporting their child during painful cancer procedures have been studied as communication strategies versus a broader caring framework and from a cross-sectional versus longitudinal perspective. Objectives: To examine the longitudinal change in parent communication behaviors over repeated cancer port start procedures experienced by their children. Methods: This study used a longitudinal design. Two trained raters coded 104 recorded videos of port starts from 43 children being treated for cancer. This included 25 children with two video-recorded port starts and 18 children with three (T1, T2, T3). The Parent Caring Response Scoring System derived from Swanson's Caring Theory was used to code parent communication behaviors as caring responses during their children's port starts. Three 3- to 5-minute slices (pre–port start, during, and post–port start) were coded for each video. Mixed modeling with generalized estimating equations and Friedman test were used to analyze longitudinal change in parent behaviors. Results: Significant differences were found between T1 versus T3 in eye contact (β = −1.05, p = .02), distance-close-enough-to-touch (β = −0.81, p = .03), nonverbal comforting (β = −1.34, p = .04), and availability (β = −0.92, p = .036), suggesting that more parents used communication behaviors at T3 compared with T1. Parent burdensome or intrusive questions (e.g., Why do you cry? β = −1.11, p = .03) and nonverbal comforting (β = −1.52, p = .047) increased from T2 to T3. The median values of parent communication behaviors overall had no significant changes from T1 to T3. Conclusion: Parents adjusted to use more nonverbal caring behaviors as their child experienced additional port starts. Experimental studies should be designed to help parents use caring behaviors to better support their children during cancer procedures.     

beta-Mercaptolactate cysteine disulfiduria in two normal sisters. Isolation and characterization of beta-mercaptolactate cysteine disulfide

β-Mercaptolactate cysteine disulfide (βMLCD) was detected in two mentally normal sisters, 11 and 13 years old. After isolation by ion-exchange chromatography, high-voltage electrophoresis, and adsorption chromatography on Porapak Q, βMLCD was identified by mass spectrometry of its following methyl ester derivatives: O, N-di-trifluoracetyl, O-trifluracetyl-N-dinitrophenyl, O, N-diacetyl, and O-acetyl-N-dinitrophenyl, with acetyl groups 50% perdeuterated, as well as by gas chromatography-mass spectrometry combination of the desulfuration products alanine (as N-trifluotacetyl-methy; ester) and lactic acid (as methyl ester). The isolated βMLCD contained no sulfoxide nor sulfone and exhibited an UV spectrum closely related to cystine. In urine the concentration range of βMLCD was 345–751 μmole/1 (n=5) and 54–133 mg/g creatinine; in plasma a trace of βMLCD could be detected only in one case. Oral administration of L-cysteine or L-methionine elevated the concentration of βMLCD in urine and in plasma. A further disulfide accompanying βMLCD in urine was isolated and identified as thioglycolate cysteine disulfide.