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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Quantification of α-galactosidase activity in intact leukocytes

BackgroundMutations of the α-galactosidase (α-Gal) A gene in Fabry disease lead to a severe disturbance in glycosphingolipid catabolism. The atypical clinical picture of Fabry disease hampers diagnosis, resulting in a delayed start of therapy. Current tests utilize leukocyte lysates to evaluate the activity of α-Gal A. It has never been investigated whether cell homogenisation is necessary.MethodsIsolated leukocyte subsets were incubated with the α-Gal substrate methylumbelliferyl-α-d galactopyranosid (MU-Gal) and substrate conversion was measured by fluorimetry. Specificity of the reaction was evaluated using the α-Gal inhibitor deoxygalactonojirimycin (DGJ). The novel procedure was compared to the standard method. A reference population and Fabry patients were tested.ResultsSubstrate conversion in intact leukocytes was a function of substrate concentration, cell number and time and could be inhibited by DGJ. Monocytes showed the highest enzyme activity among leukocyte populations. The novel procedure highly correlated with the standard method. Both Fabry hemizygotes and heterozygotes showed reduced substrate conversion.ConclusionWe here present a novel sensitive, fast and simple procedure for the evaluation of α-Gal activity suitable to identify enzyme deficiencies in Fabry patients. Furthermore, we show for the first time that leukocyte subtypes have different α-Gal activities.     

Heart Valve Involvement in Fabry Cardiomyopathy

Fabry disease is a rare X-linked lysosomal storage disorder leading to an accumulation of glycosphingolipids in all tissues and organs including the heart. Among the pathologies of myocardial involvement, reviews and registry data list affection of heart valves and its hemodynamic significance as predominant alterations during progression of the disease. We thought to approach this uncertainty with a systematic observational study. In a single center study, 111 patients with genetically proven Fabry disease were systematically investigated by echocardiography for abnormalities of the valves in the left (aortic and mitral valve) and right heart (pulmonary and tricuspid valve). In addition, 60 patients were followed by echocardiography for 2.7 ± 1.5 y (range 1 to 6). Both valve stenosis and regurgitation were classified as mild, moderate or severe. Overall, no patient had severe heart valve abnormalities. The most frequent findings were mild aortic (n = 17), mitral (n = 57) and tricuspid (n = 38) valve regurgitation. Only two patients showed mild aortic valve stenosis. Moderate aortic (n = 1), mitral (n = 2) or tricuspid (n = 1) regurgitation were rarely detected. All Fabry patients in advanced stages (n = 9) had only mild mitral regurgitation and one of them had mild aortic and mitral regurgitation, moderate tricuspid regurgitation and mild aortic stenosis. Thirty patients had completely normal valve function. There was no significant change toward hemodynamic relevant heart valve abnormalities during follow-up. Mild left ventricular valve regurgitations are frequent in Fabry disease. However, these valve abnormalities are not the major limitations for the Fabry heart. (E-mail: weidemann_f@medizin.uni-wuerzburg.de)     

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.     

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.     

Urinary lipid profiling for the identification of fabry hemizygotes and heterozygotes

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the lysosomal hydrolase, alpha-galactosidase, for which enzyme replacement therapy is now available. In this study, we aimed to identify Fabry heterozygotes not only for genetic counseling of families but because it is becoming increasingly obvious that many heterozygous (carrier) females are symptomatic and should be considered for treatment. METHODS: We measured 29 individual lipid species, including ceramide, glucosylceramide, lactosylceramide, and ceramide trihexoside, in urine samples from Fabry hemizygotes and heterozygotes and from control individuals by electrospray ionization tandem mass spectrometry. Individual analyte species and analyte ratios were analyzed for their ability to differentiate the control and patient groups. RESULTS: The Fabry hemizygotes had increased concentrations of the substrate for the deficient enzyme, ceramide trihexoside, as well as lactosylceramide and ceramide, along with decreased concentrations of both glucosylceramide and sphingomyelin. Ratios of these analytes improved differentiation between the control and Fabry groups, with the Fabry heterozygotes generally falling between the Fabry hemizygotes and the control group. CONCLUSIONS: These lipid profiles hold particular promise for the identification of Fabry individuals, may aid in the prediction of phenotype, and are potentially useful for the monitoring of therapy in patients receiving enzyme replacement.     

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots

BackgroundAs an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays.MethodsA streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene.ResultsThe HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification.ConclusionThe results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations.     

Fabry disease: diagnosis by alpha-galactosidase activities in tears.

The enzymatic diagnosis of hemizygotes with Fabry disease and heterozygous carriers was accomplished by the fluorometric determination of alpha-galactosidase activities in tears. Two components of total alpha-galactosidase activity were differentiated by their relative thermostabilities and by chromatography on DEAE-cellulose. The major component, alpha-galactosidase A, was thermolabile and represented approximately 90% of total activity; the remaining activity was thermostable, eluted at a slightly higher salt concentration and was designated alpha-galactosidase B. A single, symmetric pH optimum was observed for total alpha-galactosidase activities from heterozygotes and normal individuals, whereas the total activity from hemizgotes, which was about 10% of that in normal controls, had a broad pH profile, identical to those for alpha-galactosidase B activities from all individuals studied. The apparent Km values for total activities were 3.2, 4.0, and greater than 13 mM for normal individuals, heterozygotes, and hemizygotes, respectively. In contrast, apparent Km values for alpha-galactosidase B activities were greater than 13 mM for all individuals, further suggesteng that the residual activity in hemizygotes with Fabry disease represented the alpha-galactosidase B component. of the potential inhibitors studied, alpha-D-melibiose was found to competitively inhibit total alpha-galactosidase activity (Ki approximately 10 mM). These studies demonstrate that tears provide an easily obtainable source of freshly secreted enzyme for the diagnosis of hemizygotes and heterozygotes with Fabry disease and suggest that tears may be useful for the diagnosis of other inborn errors of metabolism.     

Adjunctive N-Acetyl-l-Cysteine in Treatment of Murine Pneumococcal Meningitis

Despite antibiotic therapy, acute and long-term complications are still frequent in pneumococcal meningitis. One important trigger of these complications is oxidative stress, and adjunctive antioxidant treatment with N-acetyl-l-cysteine was suggested to be protective in experimental pneumococcal meningitis. However, studies of effects on neurological long-term sequelae are limited. Here, we investigated the impact of adjunctive N-acetyl-l-cysteine on long-term neurological deficits in a mouse model of meningitis. C57BL/6 mice were intracisternally infected with Streptococcus pneumoniae. Eighteen hours after infection, mice were treated with a combination of ceftriaxone and placebo or ceftriaxone and N-acetyl-l-cysteine, respectively. Two weeks after infection, neurologic deficits were assessed using a clinical score, an open field test (explorative activity), a t-maze test (memory function), and auditory brain stem responses (hearing loss). Furthermore, cochlear histomorphological correlates of hearing loss were assessed. Adjunctive N-acetyl-l-cysteine reduced hearing loss after pneumococcal meningitis, but the effect was minor. There was no significant benefit of adjunctive N-acetyl-l-cysteine treatment in regard to other long-term complications of pneumococcal meningitis. Cochlear morphological correlates of meningitis-associated hearing loss were not reduced by adjunctive N-acetyl-l-cysteine. In conclusion, adjunctive therapy with N-acetyl-l-cysteine at a dosage of 300 mg/kg of body weight intraperitoneally for 4 days reduced hearing loss but not other neurologic deficits after pneumococcal meningitis in mice. These results make a clinical therapeutic benefit of N-acetyl-l-cysteine in the treatment of patients with pneumococcal meningitis questionable.     

Effect of agalsidase alfa replacement therapy on fabry disease—related hypertrophic cardiomyopathy: A 12- to 36-month,retrospective, blinded echocardiographic pooled analysis

Background: Fabry disease is an X-linked disease caused by a deficiency in the activity of the lysosomal enzyme α-galactosidase A. Affected individuals typically develop left ventricular hypertrophy (LVH) among other pathologies.Objective: The purpose of the present study was to investigate the effect of ≥12 months of enzyme replacement therapy (ERT) with agalsidase alfa on LV mass (LVM) in men and women with Fabry disease.Methods: This was a retrospective, blinded, pooled analysis of data from several studies assessing the effect of ERT with agalsidase alfa on LVM in patients with Fabry disease with baseline LVH. Men and women aged ≥18 years with a confirmed diagnosis of Fabry disease who had received ≥36 months of ERT with agalsidase alfa were eligible, provided they had a baseline echocardiogram and a follow-up echocardiogram at 12 and/or 36 months. Data from 4 studies were included in the present analysis. LVM was normalized to height (in meters) to the 2.7 power (LVM/h = LVM/m^2.7).Results: In total, 45 adult patients (34 men and 11 women) with a confirmed diagnosis of Fabry disease and serial echocardiograms obtained at baseline and after 12 and/or 36 months of treatment were included. The mean (SD) age of this cohort was 39.8 (10.4) years (range, 18.9–67.2 years), and the mean weight was 72.5 (13.4) kg (range, 46.7–102.9 kg). Forty-two patients were white, 2 were Hispanic, and 1 was classified as other. At baseline, 14 patients had LVH (mean LVM/h = 55.4 [5.7] g/m^2.7). After 12 months of ERT with agalsidase alfa, LVM/h decreased significantly by 9.2 (7.9) g/m^2.7 in 9 patients (P = 0.008), and after 36 months, LVM/h decreased significantly by 5.1 (7.5) g/m^2.7 in 10 patients (P = 0.037). In patients without baseline LVH (n = 31), a significant increase in LVM/h was observed after 12 months of treatment (3.6 [5.7] g/m^2.7; P = 0.002). After 36 months of treatment, however, there was no significant change from baseline in 10 patients (2.1 [7.9] g/m^2.7; P = NS).Conclusion: Treatment with agalsidase alfa for 12 or 36 months was associated with reduced LVM in these patients with Fabry disease with baseline LVH, and it appeared to stabilize LVM in these patients without baseline LVH.     

How well does urinary lyso-Gb3 function as a biomarker in Fabry disease?

BackgroundFabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb₃), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb₃), is increased in patients with the disease. Until now, lyso-Gb₃ was not detectable in urine, possibly because of the presence of interfering compounds.MethodsWe undertook to: 1) characterize lyso-Gb₃ in urine; 2) develop a method to quantitate urinary lyso-Gb₃ by mass spectrometry; 3) evaluate urinary lyso-Gb₃ as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb₃ is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb₃ from 83 Fabry patients and 77 healthy age-matched controls.ResultsThe intraday and interday bias and precision of the method were < 15%. Increases in lyso-Gb₃/creatinine correlated with the concentrations of Gb₃ (r² = 0.43), type of mutations (p = 0.0006), gender (p < 0.0001) and enzyme replacement therapy status (p = 0.0012). Urine from healthy controls contained no detectable lyso-Gb₃. Lyso-Gb₃ did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb₃ of Fabry patients correlated well with a number of indicators of disease severity.ConclusionLyso-Gb₃ is a reliable independent biomarker for clinically important characteristics of Fabry disease.     

N-Acetyl-L-aspartic acid, N-acetyl-α-l-aspartyl-l-glutamic acid and β-citryl-l-glutamic acid in human urine

$\text{N-}\text{Acetyl-}\text{l}\text{-aspartic}$ acid (NA-Asp), $\text{N-}\text{acetyl}\text{-α-}\text{l}\text{-aspartyl-L-glutamic}$ acid (NA-Asp-Glu) and β-citryl-l-glutamic acid (β-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and β-CG using electrophoretic and Chromatographie methods and by acid hydrolysis.A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and β-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and ν-CG were 41.2 ± 10.1 (n = 27), 20.8 ± 9.6 (n = 27) and 30.2 ± 13.2 (n = 21) μmol/g creatinine in adult males, and 62.2 ±16.3 (n = 27), 24.0 ±8.2 (n = 27) and 40.5 ± 21.1 (n = 24) μmol/g creatinine in adult females, respectively.