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.     

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.     

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.     

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.     

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.     

AN ENZYME IN MAST CELLS WITH PROPERTIES LIKE CHYMOTRYPSIN

Mast cells contain an enzyme which hydrolyzes 3-chloroacetoxy-2-naphthoic acid anilide. By using highly purified mast cells isolated by differential centrifugation in high density sucrose solutions we have been able to study this enzymatic activity in more detail. The enzyme has properties similar to those of chymotrypsin: Chymotrypsin will hydrolyze the histochemical substrate, and the chymotrypsin and mast cell activities with this substrate are similarly inhibited by diisopropylfluorophosphate. The mast cell enzyme is capable of hydrolyzing the N-acetyl esters of tryptophan, tyrosine, and phenylalanine, the relative rates of hydrolysis being similar to those seen with chymotrypsin. A characteristic trypsin substrate, p-toluenesulfonyl arginine methyl ester, is not acted upon by the mast cell enzyme or chymotrypsin. The pH activity curve of the new cell enzyme is similar to that of chymotrypsin as determined with N-acetyl-L-tryptophan ethyl ester as substrate.     

Isotope dilution analysis of methylcitric acid in amniotic fluid for the prenatal diagnosis of propionic and methylmalonic acidemia

A stable isotope dilution assay for methylcitric acid in amniotic fluid was developed to provide rapid prenatal diagnosis of the inherited disorders propionic acidemia and methylmalonic acidemia. The method utilizes two ²H₃-labeled diastereoisomers of methylcitric acid as internal standards, isolation by liquid partition chromatography and quantitation of the trimethyl esters by chemical ionization selected ion monitoring gas chromatography-mass spectrometry. Methylcitric acid at a concentration of 0.38 ± 0.10 μmol/1 was detected in normal amniotic fluid. Highly elevated levels of 7.87 and 9.16 μmol were found in the fluids surrounding fetuses affected with propionic acidemia and levels of 1.79, 2.72 and 12.27 μmol were found for fetuses with methylmalonic acidemia. Methylcitric acid was not elevated in the amniotic fluid of a fetus heterozygous for propionic acidemia. In the five pregnancies at risk for propionic acidemia, and three pregnancies at risk for methylmalonic acidemia, the levels of methylcitric acid in amniotic fluid gave the diagnosis in all cases. Measurement of methylcitric acid in amniotic fluid therefore provides a rapid and reliable method for the prenatal diagnosis of these genetic disorders.     

Comprehensive quali-quantitative profiling of neutral and sialylated O-glycome by mass spectrometry based on oligosaccharide metabolic engineering and isotopic labeling

Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for quantitatively profiling abnormal sialylated O-glycans associated with disease development, but technically hindered by the poor releasing efficiency of O-glycans from glycoprotein or the labile nature of sialic acid residues at glycans. Herein, we developed an isotopic precursor based metabolic amplification and labeling (IPMAL) technique for relative quantitative profiling of the repertoire O-glycans between normal and tumor cells by ESI-MS. Two groups of cells were incubated with peracetylated benzyl-α-N-acetylgalactosamine (Ac₃GalNAc-α-Bn^d0) or a heavy labeled peracetylated benzyl-α-N-acetylgalactosamine (Ac₃GalNAc-α-Bn^d5) precursor respectively to amplify the repertoire of O-glycans as Bn^d0/d5-O-glycans which could achieve the quantitative O-glycome analysis by ESI-MS after derivatization. The established method demonstrates desirable feasibility, accuracy (relative error (RE) ≤ 4.20%), reproducibility (coefficient of variation (CV) ≤ 7.61%, n = 3) and good quantitation linearity (R² > 0.99, n = 3) for five Bn-O-glycans with 2 orders of magnitude. Finally, the method has been successfully applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell line L02 and human hepatoma cell line SMMC-7721. Moreover, the α-2,3/2,6 sialic acid isomers of Bn-O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid specific derivatization procedure.

An isotopic precursor based metabolic amplification and labeling (IPMAL) strategy using the Ac₃GalNAc-α-Bn precursor to simultaneously quantify neutral and sialylated O-glycans.     

Curcumin's effects on sialic acid level and sialidase activity in Ehrlich ascites tumor bearing mice

In this study, we determined curcumin's anticancer and chemopreventive effects in mice bearing Ehrlich ascites tumor by evaluation of cancer biomarkers, sialic acid level and sialidase activity. Both plasma sialic acid level and sialidase activity increased significantly in the mice group with Ehrlich ascites tumor. When the tumor groups fed with curcumin and fed with sesame oil were compared, sialic acid level and sialidase activity in ascites fluid significantly reduced in the group fed with curcumin in addition to the increases of plasma sialic acid level and sialidase activity. The tumor group fed with curcumin lived twice longer than the one fed with sesame oil. Curcumin as a phenolic compound decreased all these parameters in Ehrlich ascites tumors and lengthened survival by 88% in the mice with tumor. We concluded that curcumin has anticancer activity.     

Microenzymatic assays for lysosomal enzymes in primary amniotic fluid cell cultures

A study of three lysosomal enzymes (hexosaminidase, beta-galactosidase and alpha-galactosidase) in normal primary amniotic fluid cell cultures using a microenzymatic assay is presented. No difference in enzyme activity was found between primary and amniotic cell cultures in passage number one. A progressive change in the proportions of hexosaminidase A and hexosaminidase B with time was demonstrated in culture. The feasibility of this procedure for the early prenatal diagnosis of disorders due to lysosomal enzyme deficiency is discussed.     

A sensitive procedure for the diagnosis of N-acetyl-galactosamine-6-sulfate sulfatase deficiency in classical Morquio's disease

The trisaccharide $\text{6-}\text{sulfo}\text{-N-}\text{acetylgalactosamine-glucuronic acid}\text{-6-}\text{sulfo}\text{-N-}\text{acetyl}\text{-[1-}{}^3\text{H]galactosaminitol}$ was used as a substrate for the determination of N-acetylgalactosamine-6-sulfate sulfatase activity. The amount of liberated sulfate was measured indirectly by separating monosulfated reaction products from the substrate on Dowex 1 × 2 microcolumns in a simple two step procedure.Fibroblast homogenates from patients with various genotypes, except classical Morquio's disease, released 410 ± 90 pmol sulfate/h/mg cell protein. The enzyme exhibited a pH optimum of pH 4.8 and a K^M of about 1 × 10^?4 mol/l. It was strongly inhibited by phosphate, sulfate and chloride ions.In three cell lines from patients with classical Morquio's disease a residual activity between 1 and 2% of the mean normal activity was found. All cell lines tested released sulfate from $\text{6-}\text{sulfo}\text{-N-}\text{acetylglucosamine}\text{-}\text{glucuronic acid}\text{-[1-}{}^3\text{H}\text{]-}\text{anhydromannitol}$.Cell extracts from cultured amniotic fluid cells exhibited a N-acetylgalactosamine-6-sulfate sulfatase activity between 120 and 320 pmol/h/mg protein.An enzyme activity of 370 ± 100 pmol sulfate/h/mg protein was found in peripheral leucocytes from healthy donors. The determination of N-acetylgalactosamine-6-sulfate sulfatase activity in one family with an affected patient indicated that the enzyme deficiency is also expressed in leucocytes.     

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.     

Polymorphonuclear leukocytes from asthmatics release more calcium from intracellular stores and have enhanced calcium increase after stimulation withN-formyl-methionyl-leucyl-phenylalanine

Polymorphonuclear leukocytes isolated from peripheral blood of asthmatics appear to be primed to release more reactive oxygen species than cells of healthy subjects. The enhanced agonist-induced rise in the intracellular free calcium concentration may be responsible for this increased respiratory burst. To test this hypothesis we studied theN-formyl-methionyl-leucyl-phenylalanine- and cyclopiazonic acid-(an inhibitor of Ca²⁺-ATPase of intracellular calcium stores) induced calcium increase in the polymorphonuclear leukocytes of 28 subjects (16 with moderate asthma, 69.6% ±8.3% predicted normal peak expiratory flow and 12 normal controls) using a fluorescent probe Fura-2AM at 100 nM and 1 mM extracellular calcium concentrations. In 1 mM calcium, theN- formylmethionyl-leucyl-phenylalanine-induced calcium increase was 1.7-fold higher in asthmatics than in healthy subjects. Similarly, the contribution of calcium from intracellular stores to the calcium response toN-formyl-methionyl-leucyl-phenylalanine was higher in asthmatics (55% ±14% vs. 39%±14%, P<0.01). The pool of calcium released from intracellular stores byN-formyl-methionyl-leucyl-phenylalanine and cyclopiazonic acid was 2.3- and 2.2-fold larger than in control cells. There was a correlation between maximal intracellular calcium concentration related toN-formyl-methionyl-leucyl-phenylalanine-induced calcium release from intracellular stores and forced expiratory volume in 1 s expressed as percentage predicted and reversibility in asthmatics (r=0.63,r=?0.53,P<0.05). In conclusion, polymorphonuclear leukocytes of asthmatics exhibit an altered calcium response that is mainly dependent on increased calcium release from intracellular stores.     

INTRAPHAGOCYTIC β-N-ACETYLGLUCOSAMINIDASE : PROPERTIES OF THE ENZYME AND ITS ACTIVITY ON GROUP A STREPTOCOCCAL CARBOHYDRATE IN COMPARISON WITH A SOIL BACILLUS ENZYME

The β-N-acetylglucosaminidases of rabbit and human polymorphonuclear leukocytes and of rabbit alveolar macrophages have been studied in comparison with the β-N-acetylglucosaminidase derived from a soil bacillus which had previously been shown to hydrolyze the group-specific polysaccharide of Group A streptococci. The phagocytic enzymes are lysosome associated and have an acid pH optimum. In contrast, the soil bacillus enzyme is an extracellular product, has a higher pH optimum, and is probaby of smaller molecular size. When tested on p-nitrophenyl-βN-acetylglucosaminide as substrate, the K_m of the phagocytic enzymes is slightly higher than that of the soil bacillus. However, there were extreme differences in their effect on the Group A streptococcal polysaccharide. Thus, 5 x 10⁶ units of the alveolar macrophage enzyme were required to hydrolyze the available N-acetylglucosamine of 1 mg of polysaccharide in 18 hr, while 100 units of the soil bacillus enzyme were sufficient to achieve this hydrolysis. In both cases, the serological reactivity of the polysaccharide is altered with loss of Group A specificity and acquisition of a new specificity characteristic of A-variant streptococci. Possible explanations for differences in the activity of the enzymes are considered, and the role of the phagocytic enzymes in intracellular degradation of Group A streptococci is discussed.     

Enzymatic assay of serum sialic acid

Serum sialic acid has been assayed enzymatically. The reaction includes neuraminidase hydrolysis of glycoprotein, cleavage of sialic acid to pyruvate by N-acetyl neuraminic acid (NANA)-aldolase, oxidation of pyruvate by pyruvate oxidase which produces hydrogen peroxide, and colorimetry of hydrogen peroxide using the peroxidase-p-chlorophenol-4-amino-antipyrine method. This method showed good correlation between a chemical method (r = 0.984), good recovery (98.8%) and good reproducibility (within-run-precision : 1.0% C.V.; day-to-day precision: 1.9% C.V.). Intrinsic serum pyruvate produces an equi- molar positive effect. The normal value range is 1.94 ± 0.29 mmol/1 (mean ±S.D., n =24).     

Sialic acid in sickle cell disease

Neuraminic (sialic) acid concentrations in serum from normal and sickle cell (HbSS) subjects were determined for discrete age groups from childhood through adolescence. Values in sickle cell disease were consistently lower over the entire age range. We further investigated the effect of exogenous sialic acid on the rate of sickling reversion of HbSS erythrocytes and demonstrated that this compound in millimole per liter concentrations could revert pre-sickled erythrocytes to their normal morphology in a concentration-dependent manner. When subjected to partial de-sialation with sialidase (EC 3.2.1.18), the HbSS erythrocytes not only sickled faster upon deoxygenation, they also reverted more slowly on treatment with phenylalanine (a more efficient anti-sickling agent than sialic acid) than did untreated cells. We conclude that, in sickle cell disease, erythrocyte sialic acid content could play a significant role, not only in the control of the sickling rate in vivo, but also, after sickling has occurred, in the rate of recovery from a sickling crisis.     

Human diploid lung fibroblast cell lines WI 26 and WI 38 exhibit isozyme shift of alkaline phosphatase after viral transformation

The testing of the type of membrane associated alkaline phosphatase (EC 3.1.3.1) by immunotitration has revealed that there is a shift from a liver-like type of alkaline phosphatase in normal cell cultures of the human diploid fibroblast cell strains WI 26 and WI 38 to a placenta-like variant in cultures of the same cell strains after the transformation by the DNA-tumor virus SV 40, the WI 26 SV 40 and the WI 38 SV 40 cell lines. The immunologically detectable switch-over has been confirmed by measuring the apparent Michaelis constant and the heat stability of the AP from normal and transformed cells.Liver-like AP is heat labile and has an apparent Michaelis constant for p-nitro-phenylphosphate (about 4.0 1 10^?4 M). The placenta-like AP shows heat stability and a lower apparent K_M (about 2.2 1 10^?4 M). The appearance of the so-called Regan enzyme of AP in some human tumors in vivo is discussed in this connection.     

Hypersialyloligosacchariduria in mucolipidoses: a method for diagnosis

A method is described for the detection of abnormal oligosaccharides in a small (5 ml) volume of urine, employing filtration on a Bio Gel P-6 column, determination of neutral sugar and bound sialic acid, and determination of creatinine content. With this method increased urinary excretion of sialic acid-rich oligosaccharides has been detected in nine patients with mucolipidoses (five cases of mucolipidosis II and four patients of mucolipidosis, with beta-galactosidase deficiency). The filtration patterns of oligosaccharides in mucolipidoses were clearly distinguishable from those in other inborn errors of metabolism. Total excreted oligosaccharides were increased 5--30-fold in these patients; mucolipidosis II, 640--1350 microgram neutral sugar/mg creatinine; control 54 +/- 20 microgram neutral sugar/mg creatinine. The oligosaccharides consisted of three sialic acid-rich fractions and were common in both types of mucolipidosis. Our data indicate that hypersialyoligosacchariduria is the main biochemical feature of both types of mucolipidosis.     

Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5''-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity.

Newly synthesized acid hydrolases, destined for transport to lysosomes, acquire a phosphomannosyl targeting signal by the transfer of N-acetylglucosamine 1-phosphate from uridine 5'-diphosphate (UDP)-N-acetylglucosamine to a mannose residue of the acid hydrolase followed by removal of the outer, phosphodiester-linked N-acetylglucosamine to expose 6-phosphomannose. This study demonstrates that fibroblasts from patients with the lysosomal enzyme storage diseases, I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III), are severely deficient in UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase, the first enzyme of the sequence. The N-acetylglucosaminylphosphotransferase activity (assayed using endogenous acceptors) in cultures from six normal subjects ranged from 0.67 to 1.46 pmol N-acetylglucosamine-1-phosphate transferred/mg protein per h, whereas five pseudo-Hurler polydystrophy and five I-cell disease cultures transferred less than 0.02 pmol/mg protein per h. The activity in five other pseudo-Hurler cultures ranged from 0.02 to 0.27 pmol transferred/mg protein per h. The activity of alpha-N-acetylglucosaminyl phosphodiesterase, the enzyme responsible for phosphomonoester exposure, is normal or elevated in cultured fibroblasts from both I-cell disease and pseudo-Hurler polydystrophy patients. The deficiency of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase explains the biochemical abnormalities previously observed in I-cell disease and pseudo-Hurler polydystrophy.