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.     

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.     

An improved procedure for diagnosis of Gaucher disease using cultured skin fibroblasts and the chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-β-d-glucopyranoside

A procedure has been developed for the determination of glucocerebrosidase activity using the substrate analogue, $\text{2-N-}\text{hexadecanoylamino}\text{-4-}\text{nitrophenyl}\text{-β-}\text{d}\text{-glucopyranoside}$ (HNGlu) with sodium taurocholate and oleic acid as activators. Cultured skin fibroblasts and amniotic fluid cells have been used as the enzyme source. It has been used successfully to confirm the diagnosis of two Type I and two Type II Gaucher patients.The procedure shows approximately a 15-fold increase in sensitivity over other procedures using HNGlu as substrate. Compared with 4-methylumbelliferyl-β-d-glucoside, HNGlu proves to be a highly specific substrate for glucocerebrosidase with little or no hydrolysis by the other β-glucosidases present in fibroblast extracts. It is therefore the chromogenic substrate of choice for determining a glucocerebrosidase deficiency.     

Stability of enzymes in urine at 37°C

The activity of alanine aminopeptidase, alkaline phosphatase, γ-gluta-myltransferase, lactate dehydrogenase and β-$\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosaminidase}$ in urine at 37°C was investigated by a model simulating in vivo conditions.The stability of these urinary enzymes is influenced particularly by pH. At low pH values in urine (about pH 5.0) the four first-mentioned enzymes rapidly lose a considerable part of their activity, whereas β-$\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosaminidase}$ is inactivated at higher pH values in urine (about at pH 8.0).This inactivation effect is also time-dependent and can be modified by urinary substances such as creatinine, urea and electrolytes. To avoid misinterpretation of enzyme activity determinations in urine, the simultaneous measurement of urinary pH should be performed.     

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.     

Differential assay of human pancreatic and salivary α-amylases in serum using a new fluorogenic substrate

The difference in the mode action of human pancreatic and salivary α-amylases on $\text{O-6-}\text{deoxy}\text{-6-[(2-}\text{pyridyl)amino}\text{]-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-}\text{d}\text{-glucitol}$ (FG6R), a fluorogenic derivative of maltohexaitol, was found. The products of the enzymatic hydrolysis were analyzed by high-performance liquid chromatography (HPLC) in 8 min. FG6R was hydrolyzed by these enzymes to $\text{O-6-}\text{deoxy}\text{-6-[(2-}\text{pyridyl)amino}\text{]-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-}\text{d}\text{-glucose}$ (FG3) and maltotriitol, or $\text{O-6-}\text{deoxy}\text{-6-[(2-}\text{pyridyl)amino}\text{]-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-O-α-}\text{d}\text{-glucopyranosyl}\text{-(1 → 4)-}\text{d}\text{-glucose}$ (FG4) and maltitol.Pancreatic α-amylase produced more FG4 than salivary α-amylase. Taking advantage of the differences in action of the two amylases, a differential α-amylase assay in serum was performed. The method is simple and rapid and can be used for routine clinical assays of α-amylases.     

A comparative study of recent assays for the determination of cystine aminopeptidase (oxytocinase)

A simple and rapid colorimetric determination for oxytocinase in pregnancy serum is described. The assay which uses $\text{Sf-}\text{benzyl}\text{-}\text{l}\text{-}\text{cysteine}\text{-p-}\text{nitroanilide}$ as substrate is based on the formation of an azo-dye. The method requires 5 min incubation at 25°, followed by 6 min colour development.Optimum conditions, K_m determination, the relationship between enzyme activity and enzyme concentration, and the effect of time on enzymatic activity are described.The coefficient of variation of the proposed colorimetric method is 1.3%. The assay is compared with two colorimetric methods using $\text{l}\text{-}\text{cystine-bis}\text{-p-}\text{nitroanilide}$ as substrate and with the kinetic method using $\text{S-}\text{benzyl}\text{-}\text{l}\text{-}\text{cysteine}\text{-p-}\text{nitroanilide}$ as substrate.     

Substrates for the assay of α-l-iduronidase

Procedures are described for the preparation of two disaccharides, 4-O -α -L-iduronosyl-2, 5-anhydro[³H]mannitol and $\text{3-O-α-}\text{l}\text{-iduronosyl}\text{-2,5--}\text{anhydro}\text{[}{}^3\text{H]-talitol}$, from heparin and dermatan sulfate, respectively. These disaccharides lend themselves to an easy assay of α-L-iduronidase which is based on the fractionation of the liberated neutral anhydro[³H]mannitol or anhydro[³H]talitol from the unreacted substrate by adsorption of the latter to Dowex 1.Investigation of the reaction conditions showed that the α-L-iduronidase activity (enzyme from human fibroblasts and Helix pomatia) was optimal at pH 3.6 in acetate buffer containing 0.01 M NaCl with iduronosyl-2,5-anhydro[³H]mannitol as substrate. For iduronosyl-2,5-anhydro[³H]talitol the pH optimum was 4.0 with the H. pomatia enzyme.The K_m for iduronosyl-2,5-anhydro[³H]mannitol was 0.23 mM with human fibroblasts and 0.04 mM with Helix enzyme; a KM value of 0.02 mM was determined for iduronosyl-2,5-anhydro[³H]talitol with the Helix α-l-iduronidase.     

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.     

Polyamines in human semen: presence of S-adenosyl-L-methionine decarboxylase

The presence of $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ decarboxylase in human semen has been demonstrated. The activity of the $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ decarboxylase was high in the semen samples with less than 30 million sperm count per ml. In semen with sperm counts between 30 to 60 million/ml, activity of the enzyme was low and increased at high concentrations of spermatozoa (above 60 million/ml).Studies were also carried out to determine the possible site of origin of this enzyme in the semen. This investigation on the split ejaculates of the semen indicated that the seminal vesicles contribute the major amount of this enzyme to the seminal plasma pool.     

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.     

Neuraminidase activity in the mucolipidoses (types I,II and III) and the cherry-red spot myoclonus syndrome

Two neuraminidase (EC 3.2.1.18) components, A and B, were distinguished in cultured skin fibroblasts on the basis of thermolability at 37°C. The more labile component (A) $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.7–5.3}\text{min}$ at 37°C, comprises 66–90% of total neuraminidase activity when determined using sodium ($\text{4-}\text{methylumbelliferyl}\text{-α-}\text{d}\text{-N-}\text{acetylneuraminate}$) (MU-α-N) as substrate. Activity was assayed at 0°C for 18 h instead of 37°C to fully determine both thermolabile and thermostable components. Diminished activity was noted in cultured fibroblasts from mucolipidoses I, II and III (MLI, MLII, MLIII) and the cherry-red spot myoclonus syndrome (CRSM) patients when assayed at both 0 and 37°C with either MU-α-N or each of a series of α(2 → 3)- and α(2 → 6)-linked N-acetylneuraminyl-oligosaccharides. Increased sensitivity and rapidity of analyses were achieved using MU-α-N as substrate in determining neuraminidase activity. Results from two obligate heterozygote MLI cell lines (14.5 and 8.0% of control activity) indicate that the MU-α-N substrate could be useful for heterozygote detection.     

Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica

The possession of a 42- to 48-megadalton plasmid alone does not appear to be predictive of virulence in $\text{Yersinia}$ species. Twelve of 100 $\text{Yersinia enterocolitica}$ strains contained a 42 to 48-megadalton plasmid, and 4 of 30 $\text{Y. enterocolitica}$-like strains contained a 42- to 48-megadalton plasmid. Seven strains of $\text{Y. enterocolitica}$ contained the 42- to 48-megadalton plasmid plus an 82-megadalton plasmid, and these were the only study strains lethal for mice. Based on restriction endonuclease digestion, the 42- to 48-megadalton plasmid DNA from these seven strains were similar and were not similar to the 42- to 48-megadalton plasmids present in the other nine strains. The ability to invade guinea pig eye tissues, calcium dependency, autoagglutination, and colonial morphology at 37°C were also associated with plasmid DNA, but the relationships were either variable or not reciprocal. Neither tissue culture invasiveness nor heat-stable toxin production was associated with plasmid DNA. It was concluded that biochemical speciation and a total plasmid profile in combination with enzyme digests are predictive of virulence in $\text{Y. enterocolitica}$ as it is measured by mouse lethality.     

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.     

Temocillin: In vitro activity against 734 selected clinical isolates,including β-lactamase-producing strains

The in vitro activity of temocillin against 734 clinical isolates was tested by broth microdilution. Good activity was demonstrated against Enterobacteriaceae and both β-lactamase-positive and -negative strains of $\text{Haemophilus influenzae}$ and $\text{Neisseria gonorrhoeae}$. There was little to no activity against gram-positive cocci and nonfermenting gram-negative bacilli. Bactericidal activity and effect of inoculum size on temocillin activity were comparable to that of ticarcillin. Temocillin was stable to commonly encountered β-lactamases and significantly inhibited Richmond-Sykes type 1 enzymes of $\text{Enterobacter cloacae}$.     

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.     

Ureaplasma urealyticum: Subcultures invalid for antibiotic susceptibility tests

We tested the antibiotic susceptibilities of 100 $\text{Ureaplasma}$$\text{urealyticum}$ strains from 99 patients using a broth-disk method and two types of inocula: urine sediments and overnight broth subcultures of the sediments. Of the 100 ureaplasma-positive urine sediments tested, nine (9%) of the ureaplasmas were found resistant to all four tetracyclines. When overnight broth cultures were used as the inoculum, 54 (54%) were found to be resistant to all four tetracyclines, an increase in resistance of 45 (45%). Thirty-seven susceptible strains remained susceptible upon subculture. The nine resistant strains remained resistant. Loss of susceptibility was not related to the pH or titer of ureaplasmas in the urine sediment inoculum but was related to the pH and titers when subcultures were used as the inoculum. Results of cultures following treatment, available for 53 patients, showed that treatment successes and treatment failures were significantly related to antibiotic susceptibility tests done with urine sediments but not to those done with broth subcultures as the inocula. Because reliable susceptibility testing is essential for appropriate therapy for $\text{U.}$$\text{urealyticum}$ infections, all factors influencing this test need to be recognized and defined.     

A new procedure for the visualization of multiple forms of gamma-glutamyl transferase (GGT) results in normals, patients receiving enzyme-inducing drugs and patients having liver parenchymal lesions

The activity of gamma-glutamyl transferase (GGT, EC 2.3.2.2) in sera of 68 healthy individuals, of 38 patients receiving anti-epileptic drugs, and of 27 patients having liver parenchymal lesions, was visualized after electrophoresis on cellulose-acetate gel using the $\text{substrate}\text{γ-}\text{l}\text{-}\text{glutamyl}\text{-p-}\text{nitroanilide}$ as a new colour reagent.The liberated p-nitroaniline was converted in situ into a lilac-coloured product using the Bratton-Marshall reaction.In most samples two bands were demonstrated, one located between albumin and α₁-globulin, called GGT 1, the other located in the α₂-globulin region, GGT 2. In a few samples a third band, GGT 3, with far less activity than the other bands, occurred in the β-globulin region.When it occurred, an increase in total GGT activity was mainly due to an increase of GGT-1 activity.The possible role of the determination of GGT-1 activity as a monitor of microsomal enzyme induction is discussed.