Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-sachs heterozygotes during pregnancy

Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEXA) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I dose not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays we examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassay of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power.     

Separation and comparison of isoenzymes of N-acetyl-beta-D-hexosaminidase 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 $\text{α-}\text{naphthol-AS-BI}\text{-N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminide}$ as substrate.Elevation of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ (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.     

N-acetyl-beta-glucosaminidase isoenzymes in serum and urine of patients with diabetes mellitus

The isoenzyme pattern of N-acetyl-beta-glucosaminidase (NAG) in serum and urine was studied in two groups of patients with diabetes mellitus and in 30 control subjects. Total NAG activity was significantly (P less than 0.001) increased in the serum and urine of the 20 diabetics with vascular complications, but was insignificantly increased in the 20 diabetics without vascular complications. Ion-exchange chromatography demonstrated the presence of two major isoenzymes of NAG, A and B. The proportion of isoenzyme A activity always exceeded that of isoenzyme B. The proportion of isoenzyme B in serum of diabetics was lower than in controls; the reverse was true for urine of diabetics. The NAG isoenzymes pattern may provide additional diagnostic information regarding diabetic status and complications of diabetes.     

Human hexosaminidase isozymes: Chromatographic separation as an aid to heterozygote identification

The correct identification of Tay-Sachs heterozygotes requires a reliable procedure for separation and quantitation of the hexosaminidase isozymes. The most commonly employed method involves thermal inactivation of the heat labile hexosaminidase A and assay of residual enzyme activity. This procedure, however, consistenly yields a significantly lower absolute and relative activity of hexosaminidase A and a higher activity of the thermostable components (B and I) in comparison with the results obtained by DEAE-cellulose chromatography. DEAE-cellulose chromatographic separation of the hexosaminidase isozymes in serum following thermal inactivation reveals the presence of relative and absolute increase in the activity of the B and I components in addition to loss of the heat-labile A isozyme. Because the conversion of hexosaminidase A into thermostable forms by heating may vary according to the conditions employed, the thermal inactivation procedure may lead to ambiguity in heterozygote identification. This difficulty can be minimized by fractionation of the hexosaminidase isozymes by DEAE-cellulose chromatography followed by assay of the individual components.In addition to the Tay-Sachs carrier state, other conditions can alter the distribution of the hexosaminidase isozymes in tissues and body fluids. For example in serum of patients with juvenile diabetes mellitus there is a characteristic elevation of hexosaminidase B and less consistently, of hexosaminidase A. Since the activity of hexosaminidase A in serum of diabetics fractionated by ion exchange chromatography is at least as high as the activity in serum of healthy non-carriers, patients with diabetes can be easily differentiated from Tay-Sachs heterozygotes. Similarly, the distribution of the hexosaminidase isozymes in serum is altered during pregnancy, where there is usually a significant rise in hexosaminidase A and I (P). However, during pregnancy activities of hexosaminidase A and I in serum of obligate Tay-Sachs carriers are only 50% of the values observed in non-carriers at comparable gestational periods. Since the absolute activities of hexosaminidase A in serum of pregnant carriers obtained by ion exchange chromatography do not overlap with the activities in serum of non-carrier pregnant women at comparable gestational periods, this method has obvious advantages for identification of pregnancies where the fetus may be at risk for Tay-Sachs disease.     

茶多酚对硝酸羟胺染毒小鼠血常规和血清生化值的影响

目的　观察绿茶提取物茶多酚对硝酸羟胺染毒小鼠血常规、血清生化值的影响。方法　采用腹腔注射硝酸羟胺建立中毒模型。雄性ICR小鼠随机分为正常组、硝酸羟胺染毒组、茶多酚预防组、茶多酚治疗组 ,常规测量各组外周血细胞和生化指标。结果　经过统计学分析 ,茶多酚可明显促进硝酸羟胺中毒导致的小鼠血常规的WBC、RBC、HGB、PLT计数改变的恢复 ,部分血清生化值AST/ALT、ChE、ALB、LDH、UA、Fe病理性改变的恢复。结论　茶多酚灌胃给药对硝酸羟胺中毒引起的氧化损伤具有一定的保护作用     

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.     

Isoenzymes of class I and II alcohol dehydrogenase in chronic hepatitis.

Using class-specific fluorogenic substrates, the activities of class I and II alcohol dehydrogenase (ADH) isoenzymes were determined in the sera of patients with chronic hepatitis. The activity of the total alcohol dehydrogenase and indicator enzymes of liver damage were also investigated. We found a statistically significant increase of class I alcohol dehydrogenase isoenzymes in the total tested group which included those with the viral hepatitis. The (2-fold) increase in the activity of class I isoenzymes was similar to the increase of aminotransferases. The serum activity of class II isoenzymes was unchanged. Here an increase in total enzyme activity was not statistically significant. Class I isoenzymes and total enzyme activity correlated well with aminotransferases. These results demonstrate that serum activity of class I ADH measured with fluorogenic substrates confirms liver cell damage and may be useful in the diagnosis of chronic hepatitis.     

Electrophoretic pattern of amylase isoenzymes in serum and urine of normal persons.

We separated and measured amylase isoenzymes in the serum and urine of 3036 normal persons by electrophoresis on a thin layer of polyacrylamide gel. We wished to establish the normal pattern of these isoenzymes and to evaluate the usefulness of this method of electrophoresis in clinical diagnosis. Results for patients with hyper- or hypofunctioning pancreas and salivary glands suggested that essentially all the isoamylases in human serum and urine are derived from the salivary glands and the pancreas, and revealed that isoamylases of more than 98% of normal persons consisted of two major isoenzymes and two to three minor ones. Although these observations indicate that data on changes in the proportion of amylase activity of each isoenzyme can be useful in clinical medicine, the following points should be remembered: (a) quantitative differences in the isoenzyme pattern were observed, depending upon the condition of the samples; (b) because the proportion of isoamylase activity in serum of different normal persons differs, seriatim determination of amylase isoenzymes is necessary; and (c) because five different genetically controlled types of isoamylases were observed in normal persons, genetic investigations are also necessary.     

慢性酒精中毒患者心肌酶活性变化及临床意义

目的探讨慢性酒精中毒患者心肌酶活性的变化情况和临床意义。方法选择我院入院时心肌酶各项指标异常增高的慢性酒精中毒256例作为研究组,同期入院但心肌酶各项指标在正常范围内的慢性酒精中毒128例作为对照组,于治疗前及治疗后2、4周进行心肌酶、肌钙蛋白检测,同时使用阳性与阴性症状量表(PANSS)评定病情。结果研究组入院时及治疗后2周心肌酶及PANSS中有关兴奋行为条目的评分与对照组比较差异均有统计学意义(P<0.01);治疗后4周上述各项指标与对照组比较差异无统计学意义(P>0.05)。研究组治疗后2、4周上述各项指标与入院时比较差异亦均有统计学意义(P<0.01)。治疗前及治疗后2、4周两组肌钙蛋白始终为阴性。结论慢性酒精中毒患者心肌酶升高并非由心肌细胞受损引起,其兴奋谵妄状态与心肌酶活性密切相关。     

Lymphoblastoid cell lines, transformed by Epstein-Barr virus, in the enzymatic study of hereditary lysosomal storage diseases

Assay conditions were studied for eight lysosomal enzymes in lymphoblastoid cell lines transformed by Epstein-Barr virus. The transformed lymphoblastoid cells retained all eight enzyme activities, though the levels sometimes differed from those in the peripheral lymphocytes or granulocytes. The levels of these eight lysosomal enzymes were measured in lymphoblastoid cells from 11 patients with hereditary lysosomal storage diseases--GMI-gangliosidosis, a variant of beta-galactosidase deficiency (sialidase deficiency with a partial beta-galactosidase deficiency), Tay-Sachs disease, Gaucher disease, Hurler syndrome, Scheie syndrome and I-cell disease--and from 20 of their obligate heterozygotes. No activity of enzymes that were deficient in the respective disease, except I-cell disease, was detected in the lymphoblastoid cells from the patient. In I-cell disease, the cells showed lower levels of some enzyme activities. beta-D-Galactosidase activity from heterozygotes of the patient with GMI-gangliosidosis and alpha-L-iduronidase activity from heterozygotes of the patient with Hurler syndrome were in carrier range. On sephadex G-150 gel filtration, beta-D-galactosidase in control material gave two peaks (I and II). In GMI-gangliosidosis, peak II was absent and peak I was markedly diminished. Peak II in the heterozygotes was smaller than that of control. On DEAE cellulose column chromatography of hexosaminidase, two major isoenzymes (hexosaminidase A and B) were detected in control. However, hexosaminidase A was not detected in Tay-Sachs disease, and the ratios of hexosaminidase (Hex) A/Hex B in the parents were lower than those in control.     

Diagnostic and pathogenetic significance of determining lactate dehydrogenase isoenzyme activity in various biological media in patients with viral hepatitis

The activity of lactate dehydrogenase (LDH) and its isoenzymes was studied in various biological media in 850 patients with viral hepatitides (VH). The activity of LDH and its isoenzyme LDH5 in the blood serum, formed elements and in the urine of VH patients changed more noticeably in a grave form of disease and in antigen positive VH. A sharp rise of LDH and LDH5 activity in the blood serum of patients with a severe form of VH with its subsequent considerable decrease was indicative of the development of acute liver necrosis. Changes in the activity of LDH and its isoenzymes in the formed blood elements of VH patients pointed out to disturbance in the energetic system influencing their morphological and functional condition. Shifts of LDH and its isoenzymes in the urine of patients indicated renal lesion which could be used for the detection of latent disorders of renal function in VH. A study of the activity of LDH and its isoenzymes in the blood formed elements and in the urine of VH patients was of certain diagnostic value but inferior in many respects to its determination in the blood serum.     

Elevated serum levels of S-100B reflect the extent of brain injury in alcohol intoxicated patients after mild head trauma

Elevated systemic levels of S-100B are proposed as a potential indicator of brain damage in identifying high-risk patients after mild head trauma (MHT). Although incidence of alcohol intoxication is high in these patients, the influence of alcohol intoxication on S-100B levels is unclear. Therefore, the aim of our study was to investigate serum concentrations of S-100B in intoxicated (group 1) and sober (group 2) patients after MHT in comparison with those of mild (group 3) or severely intoxicated (group 4) individuals without trauma. S-100B was significantly increased in MHT patients exhibiting posttraumatic lesions in initial cranial computed tomography scan. Alcohol intoxication did not elevate S-100B levels in group 3 or 4 subjects. Our data indicate for the first time that alcohol intoxication does not influence the diagnostic value of S-100B measurements in patients after MHT.     

Isoenzyme pattern and partial characterization of hexosaminidases in the membrane and cytosol of human erythrocytes

OBJECTIVES: Hexosaminidase activity is present in lysosomes, plasma membrane and cytosol of many human cells. Plasma membrane and cytosolic hexosaminidase is not well characterized, particularly as regards their isoenzyme forms and their relationship with the lysosomal ones. DESIGN AND METHODS: Erythrocyte hexosaminidase isoforms were chromatographically separated, characterized and compared to those in the plasma of healthy individuals and in the erythrocytes of a Tay-Sachs patient. RESULTS: Hexosaminidase isoenzymes were found in plasma membrane and cytosol and were composed of the same alpha- and beta-subunits as the lysosomal and plasma hexosaminidase A and B isoenzymes, though with some structural and kinetic differences. In addition, the cytosol contained a hexosaminidase that is a specific N-acetyl-beta-D-glucosaminidase, the one involved in the removal of N-acetylglucosamine residues O-linked to proteins, named O-GlcNAcase. CONCLUSIONS: This work provides an additional step in the characterization of hexosaminidases helping better understand their role in non-lysosomal compartments and their involvement in physiological or pathological situations.     

Nucleotide pyrophosphatase and phosphodiesterase. I. Organ distribution and activities in body fluids.

We estimated nucleotide pyrophosphatase and phosphodiesterase I activities in human and rat organs and in body fluids from man and dog. The highest organ activities were found in epididymis, kidney, liver, and intestine. In body fluids, the activity was highest in seminal plasma, followed by intestinal lymph, serum, heart lymph, cerebrospinal fluid, milk, and urine. The ratio nucleotide pyrophosphatase/phosphodiesterase I and the urea resistance of phosphodiesterase I differed among human organs, body fluids, and blood cells. Different isoenzymes probably exist. The activities in serum share several properties with those in several organs--e.g. pH-optimum 9.6-9.8, dependency on Zn2+, and the effects of inhibitors. Phosphodiesterase I in erythrocytes, which has not been described previously, differs from enzyme from other sources by lower pH optimum (8.5), dependency on Mg2+, inhibition by Zn2+, and stimulation by dithiothreitol.     

角鲨烯对硝酸羟胺中毒小鼠网织红细胞计数和脾脏含铁血黄素沉积的影响

目的 观察角鲨烯对硝酸羟胺中毒小鼠网织红细胞、脾脏含铁血黄素沉积的影响。方法 ICR雄性小鼠硝酸羟胺1／2LD50染毒，每天1次，连续染毒3d，角鲨烯130mg／k灌胃给药治疗1周，染毒后第2、4、6、8天小鼠尾静脉取血煌焦油染色计数网织红细胞，第8天处死小鼠观察脾脏中含铁血黄素沉积情况。结果 角鲨烯可抑制硝酸羟胺染毒后网织红细胞以及脾脏含铁血黄素沉积的显著增多。结论 角鲨烯对硝酸羟胺中毒小鼠具有一定的治疗作用。     

Presence of serum and tissue forms of N-acetyl-beta-glucosaminidase in urine from patients with renal disease.

1. The A, B, I1 and I2 forms of N-acetyl-beta-glucosaminidase present in urine, serum, kidney, liver and cerebral spinal fluid were separated on DEAE-cellulose and their presence confirmed by cellogel electrophoresis. The relative activities of each enzyme were determined by integrating the area under the elution peaks. 2. Serum A-form was eluted at a lower molarity of chloride than liver A-form and this was designated the As-form to distinguish it from the A-form of N-acetyl-beta-glucosaminidase found in liver and kidney. 3. The P-form of N-acetyl-beta-glucosaminidase present in the serum of a group of pregnant women was not detectable in urine samples from the same women. 4. Urinary NAG activities were found to be abnormally high in patients with impaired renal function. 5. The activity of both N-acetyl-beta-glucosaminidases A and B increased in pathological urines. The higher the total N-acetyl-beta-glucosaminidase activity excreted the higher the % of activity of the B-form present. 6. In a number of patients with haematuria an A-form similar to the serum As-form was present in the urine.     

Octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride

We examined whether octacosanol, the main component of policosanol, attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride (CCl(4)). In rats intoxicated with CCl(4) (1 ml/kg, i.p.), the activities of serum transaminases increased 6 h after intoxication and further increased at 24 h. In the liver of CCl(4)-intoxicated rats, increases in lipid peroxide (LPO) concentration and myeloperoxidase activity and decreases in superoxixde dismutase activity and reduced glutathione (GSH) concentration occurred 6 h after intoxication and these changes were enhanced with an increase in xanthine oxidase activity and a decrease in catalase activity at 24 h. Octacosanol (10, 50 or 100 mg/kg) administered orally to CCl(4)-intoxicated rats at 6 h after intoxication attenuated the increased activities of serum transaminases and the increased hepatic myeloperoxidase and xanthine oxidase activities and LPO concentration and the decreased hepatic superoxide dismutase and catalase activities and GSH concentration found at 24 h after intoxication dose-dependently. Octacosanol (50 or 100 mg/kg) administered to untreated rats decreased the hepatic LPO concentration and increased the hepatic GSH concentration. These results indicate that octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in CCl(4)-intoxicated rats.     

Development of a long-term ascending urinary tract infection mouse model for antibiotic treatment studies

A model of ascending unobstructed urinary tract infection (UTI) in mice was developed to study the significance of the antibiotic concentration in urine, serum, and kidney tissue for efficacy of treatment of UTI in general and pyelonephritis in particular. Outbred Ssc-CF1 female mice were used throughout the study, and Escherichia coli was used as the pathogen. The virulence of 11 uropathogenic E. coli isolates and 1 nonpathogenic laboratory E. coli strain was examined. Strain C175-94 achieved the highest counts in the kidneys, and this strain was subsequently used as the infecting organism. The model gave reproducible bladder infections, i.e., bacteria were recovered from 22 of 23 control mice after 3 days, and histological examination of kidney tissue showed that of 14 infected kidneys, 7 (50%) showed major histological changes, whereas 3 of 36 uninfected kidneys showed major histological changes (P = 0.018). Once the model was established, the efficacies of different doses of cefuroxime and gentamicin, corresponding to active concentrations in urine only or in urine, serum, and kidney tissue simultaneously, were examined. All cefuroxime doses resulted in significantly lower counts in urine than control treatments, but the dose which produced concentrations of cefuroxime only in urine and not in serum or kidney tissue had no effect on kidney infection. Even low doses of gentamicin (0.05 mg/mouse) resulted in concentrations in renal tissue for prolonged times due to accumulation. All gentamicin doses had a significant effect (compared to the effect of the control treatment) on bacterial counts in urine and kidneys. The antibiotic effect on bacterial counts in bladders was negligible for unknown reasons. Use of the mouse UTI model is feasible for study of the effect of an antibiotic in the urinary system, although the missing antibacterial effect in the bladder needs further evaluation.     

Human hexosaminidase isozymes. Assay of platelet activity for heterozygote identification during pregnancy

Identification of carriers of the Tay-Sachs gene during pregnancy is difficult because of the increase in serum of a heat stable hexosaminidase isozyme I (or P) as well as changes in the relative and absolute activities of the various molecular forms of the enzyme with advancing pregnancy. In contrast, isolation of blood platelets followed by ion exchange Chromatographic separation and assay of the hexosaminidase isozymes in platelet extracts by an automated method provides a sensitive and reliable method for heterozygote identification during pregnancy. This method appears superior to procedures involving thermal inactivation of extracts of peripheral blood leukocytes because of significant differences in the content of the hexosaminidase isozymes in granulocytes, lymphocyte and other cell types, as well as variations in the proportion of these cell types in samples of peripheral blood. It also alleviates the problem inherent in any method involving thermal inactivation of hexosaminidase A by avoiding possible interconversion of the various molecular forms of the enzyme associated with heating.     

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.