Frequency of an allele for low activity of alpha-L-fucosidase in sera: possible increase in epithelial ovarian cancer patients.

Sera from a group of patients with ovarian cancer had a statistically significant deficiency of alpha-L-fucosidase activity compared with sera from healthy females or female patients with cervical or breast cancer. Mixing experiments did not identify an inhibitor of alpha-L-fucosidase activity in the sera of ovarian cancer patients. Decreased activity of alpha-L-fucosidases was not associated with stage of disease, tumor burden, histologic type, or grade of differentiation. Unlike alpha-L-fucosidase, beta-man-nosidase and beta-N-acetylglucosamindase in sera of ovarian cancer patients were not deficient in activity. Examination of population data of healthy females and of pedigrees of ovarian cancer patients suggested that the quantitative activity alpha-L-fucosidase in serum was genetically determined. Of 60 healthy females, 4 had low enzyme activity (less than 100 U alpha-L-fucosidase/ml serum), 26 had intermediate activity (100-274 U alpha-L-fucosidase/ml), and 30 had high activity (275 U/ml), whereas of 44 ovarian cancer patients, 11 had low, 29 had intermediate, and 4 had high activity. Application of the Hardy-Weinberg law to these data revealed that low enzyme activity in sera was three times more prevalent in the ovarian cancer group, the allele for this low enzyme activity being two times more common. These observations suggested that deficiency of alpha-L-fucosidase activity in sera of females may be a hereditary condition associated with increased risk for development of ovarian cancer.

Plasma tumor necrosis factor-a (TNF-a) levels in Gaucher disease.

Tumor necrosis factor-a (TNF-a) levels were measured in the plasma of patients with different types of Gaucher disease (GD) and patients with other lysosomal storage diseases. The highest TNF-a levels were observed in the most severe neuronopathic type of GD, exceeding those found in healthy individuals as well as patients with other lysosomal disorders. Type I GD cases showed a wide range of TNF-a levels ranging from normal to 2.5 x the highest control value. TNF-a is a pleiotropic cytokine produced mainly by activated macrophages. Our data suggest that it may play a role in the pathophysiology of GD disease.

Neutral alpha-mannosidase activity in human serum.

Two types of alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) with neutral pH optima exist in serum. The activity with an optimum between pH 6.0 and 6.4 is similar to alpha-mannosidase C, described earlier in tissues. The second activity, with a pH optimum between pH 5.2 and 5.8 is the dominant form in serum. These two forms can be differentiated from each other by gel-filtration, chromatography on DEAE-cellulose or chromatography on Concanavalin-A Sepharose. Using the chromatographic techniques, the serum type neutral activity co-elutes with the acidic forms of the enzyme. However, these two forms can be easily distinguished by effect of pH, heating or inhibition by the substrate methyl-alpha-D-mannopyranoside. The presence of the serum type alpha-mannosidase activity is discussed with respect to mannosidosis, a lysosomal storage disease.

Estimation of genotype distributions and posterior genotype probabilities for beta-mannosidosis in Salers cattle.

beta-Mannosidosis is a lethal lysosomal storage disease inherited as an autosomal recessive in man, cattle and goats. Laboratory assay data of plasma beta-mannosidase activity represent a mixture of homozygous normal and carrier genotype distributions in a proportion determined by genotype frequency. A maximum likelihood approach employing data transformations for each genotype distribution and assuming a diallelic model of inheritance is described. Estimates of the transformation and genotype distribution parameters, gene frequency, genotype fitness and carrier probability were obtained simultaneously from a sample of 2,812 observations on U.S. purebred Salers cattle with enzyme activity, age, gender, month of pregnancy, month of testing, and parents identified. Transformations to normality were not required, estimated gene and carrier genotype frequencies of 0.074 and 0.148 were high, and the estimated relative fitness of heterozygotes was 1.36. The apparent overdominance in fitness may be due to a nonrandom sampling of progeny genotypes within families. The mean of plasma enzyme activity was higher for males than females, higher in winter months, lower in summer months and decreased with increased age. Estimates of carrier probabilities indicate that the test is most effective when animals are sampled as calves, although effectiveness of the plasma assay was less for males than females. Test effectiveness was enhanced through averaging repeated assays of enzyme activity on each animal. Our approach contributes to medical diagnostics in several ways. Rather than assume underlying normality for the distributions comprising the mixture, we estimate transformations to normality for each genotype distribution simultaneously with all other model parameters. This process also excludes potential biases due to data preadjustment for systematic effects. We also provide a method for partitioning phenotypic variation within each genotypic distribution which allows an assessment of the value of repeat measurements of the predictive variable for genotype assignment.

Characterization of a soluble class I alpha-mannosidase in human serum.

Class I alpha-mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N-glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble alpha-mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man(5-8)GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+-dependent, sensitive to 1-deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi-mannosidase IA and IB and Man9-mannosidase.

alpha-D-mannosidases in serum of patients with I-cell disease (ICD).

The specific activity of alpha-D-mannosidase in serum of ICD-patients is considerably increased due to increased amounts of the component with optimal activity at pH 4.6 (acidic form). The intermediate form with pH optimum of 6.0 remains unaltered. These conclusions were reached by using optimal conditions for differential assay of the alpha-D-mannosidases checked by partial separation of the components in serum by sucrose gradient centrifugation.

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I.

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.

Plasma alpha-D-mannosidase in mucolipidosis II and mucolipidosis III.

There is a marked increase in the acidic alpha-D-mannosidase in the plasma of a patient with mucolipidosis II and of a patient with mucolipidosis III. A small proportion (3--4%) of this acidic alpha-D-mannosidase does not bind to concanavalin A-Sepharose, suggesting an alteration in the glycosylation and some of the enzyme in these disorders. A slight elevation in intermediate alpha-D-mannosidase was also demonstrated in these samples by using a differential assay for the acidic and intermediate alpha-D-mannosidase activities. A combination of chromatography on concanavalin A-Sepharose and Sephadex G-200 showed that intermediate alpha-D-mannosidase components I2 and I4, which account for approximately 80% of the intermediate activity in normal plasma, were also present in ML II and ML III plasma. The minor intermediate alpha-D-mannosidase components in normal plasma, I1 and I3 were either present in small amounts or not detected. These results suggest that a defect in intermediate alpha-D-mannosidase is unlikely to be the primary defect in these disorders.

Changes in human plasma alpha-D-mannosidase activity during pregnancy.

There are significant and progressive increases in plasma acidic (pH optimum 4.2) and intermediate (pH optimum 5.6) alpha-mannosidase during pregnancy. The acidic alpha-mannosidase in plasma from pregnant women binds to concanavalin A and has the same apparent molecular weight as the acidic alpha-mannosidase in control plasma. The 2-3-fold increase in acidic alpha-mannosidase in pregnancy is due to an increase in the most negatively charged form of acidic alpha-mannosidase, B2, which is slightly more negatively charged than its counterpart in the control plasma. The intermediate alpha-mannosidase, which increases by approximately 50% during pregnancy, can be resolved by a combination of chromatography on concanavalin A and gel filtration into the same forms found in control plasma.

Acid hydrolases in sera and plasma from patients with cystic fibrosis.

The enzyme activities of alpha-fucosidase (pH 4.0 and pH 5.5), alpha-galactosidase, beta-galactosidase, alpha-glucosidase (pH 4.5 and pH 6.0), beta-glucosidase, beta-glucuronidase, beta-hexosaminidase, and alpha-mannosidase (pH 4.5 and pH 5.5) were investigated in sera from cystic fibrosis (CF) patients. Several of these activities were significantly increased in sera from patients compared to age-matched control children. CF-patients in a more advanced stage of the disease had a tendency to higher values of some of these hydrolases than those in better condition. No new isoenzymes of these hydrolases were found. Only minor differences could be detected in the pH-profiles of alpha-mannosidase and acid phosphatase from age-matched normal controls, heterozygotes and homozygotes for CF. With our technique, alpha-mannosidase and acid phosphatase showed the same thermostability in CF-patients. CF-heterozygotes and age-matched controls, except at 56 degrees C, when the activity of acid-phosphatase in the plasma from adult CF-heterozygotes decreased more than that from adult controls

Glycosidases in serum of cystic fibrosis patients.

In a study of eight glycosidases in serum samples from 72 cystic fibrosis patients, 85 cystic fibrosis parents and 34 healthy and diseased controls, significant elevations of mean alpha-glucosidase levels were found in cystic fibrosis patients. All other glycosidases did not show any significant change. Mean alpha-glucosidase levels in obligate heterozygotes were the same as in control individuals. Moreover, alpha-glucosidase levels in cystic fibrosis patients correlated with the degree of clinical impairment as measured by the Schwachman score.

Enzymes of lysosomal origin in human plasma and serum: assay conditions and parameters influencing the assay.

The condition for maximal activity (pH, buffer, saturating substrate concentration, range of linear relationships between enzyme activity versus incubation time, and versus enzyme concentration) in the fluorimetric assay of several glycohydrolases of lysosomal origin in human plasma and serum have been established. The following enzymes were studied: alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-glucuronidase, alpha-mannosidase, alpha-fucosidase. All examined enzymes turned out to be more or less unstable upon storage at 37 degrees C, 4 degrees c, and -20 degrees C in both serum and plasma. The only exceptions were beta-glucuronidase, which was stable in plasma and serum, and alpha-fucosidase which was stable only in plasma. Generally the degree of instability was greater in serum than in plasma. The levels of some enzymes (alpha-galactosidase, beta-galactosidase, beta-N-acetyl glucosaminidase, beta=glucuronidase) were markedly higher in serum than in plasma; conversely the levels of the same enzymes in "platelet free" serum equalled those in plasma. This stresses the necessity to use freshly prepared plasma for lysosomal glycohydrolase assay. Under the procedural conditions recommended for the assay the methods for the determination of lysosomal glycohydrolases in plasma appeared to be simple, sensitive and reproducible.

[A study of lysosomal enzyme activities in serum and leukocytes in chronic hepatic disease (author's transl)]. / Etude des activités enzymatiques lysosomiques sériques et leucocytaires au cours des hepatopathies chroniques.

Five lysosomal enzyme activities (arylsulfatase A, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase and beta-D-galactosidase) were determined in serum and leucocytes of 30 controls and 114 patients suffering from various liver diseases, including 30 with idiopathic hemochromatosis and 34 with alcoholic cirrhosis. The results show (1) a decrease of the serum arylsulfatase A activity in idiopathic hemochromatosis, (2) an increase of this activity in cholestatic jaundice, and (3) mainly, a sharp rise of the leucocyte lysosomal enzyme activities in the liver diseases studied (chiefly idiopathic hemochromatosis and alcoholic cirrhosis). The mechanism and the meaning of these disturbances are discussed.

Increased serum alpha-L-fucosidase and beta-N-acetylglucosaminidase activities in diabetic, cirrhotic and gastric cancer patients.

The activities of several glycosidases (alpha-L-fucosidase, alpha-D-mannosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-N-acetylglucosaminidase and beta-D-glucuronidase) were determined in human sera from 10 normal subjects and in three groups each of 10 patients with diabetes mellitus, hepatic cirrhosis and gastric carcinoma. The results show significantly higher activities in the patients for alpha-L-fucosidase (p less than 0.001) and for beta-N-acetylglucosaminidase (p less than 0.1, p less than 0.001 and p less than 0.05, respectively), and smaller or not significantly greater values for the other glycosidases.

Characterization of alpha-L-fucosidase from two different families with fucosidosis.

1. Two different families with a different type of fucosidase deficiency are described. 2. In the first family the activity of alpha-L-fucosidase in leucocytes of two patients with fucosidosis type I was about 4 to 8% of the normal value. The activity of alpha-L-fucosidase in the leucocytes of the father and the mother are in the heterozygote range, while a sister of the propositus showed normal values. 3. The activity of alpha-L-fucosidase of the propositus from urine, serum and liver were also severely decreased. The activity of alpha-L-fucosidase in the urine of the parents and the healthy sister of thr propositus were about 5% of the mean normal value. However in the serum these values were above 50%. 4. The KM value for alpha-L-fucosidase from leucocytes of the patient was increased about 10 times and in serum this value was even higher. The KM values from the enzyme of the parents were in the normal range. 5. The abnormal enzyme from the propositus is unique in its thermal behaviour since after heating its activity increased. 6. In the second fanily the activity of alpha-L-fucosidase in the leucocytes of the patient is about 30% of the mean normal value, while the arylsulphatase A activity is also decreased (25% of the mean normal value). 7. The activity of alpha-L-fucosidase from the leucocytes of the father and the healthy brother are about 50% of the mean normal level, while the enzyme of the mother showed a normal activity. 8. The alpha-L-fucosidase activity in the urine and the liver of the propositus is also decreased. The serum enzyme activity however was in the normal range. 9. The KM value of alpha-L-fucosidase and heat stability of the enzyme of the patient were normal. In the leectrophoretic pattern of the whole family one bond was missing.

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-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-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 health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-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 beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

Measurement of the alpha-mannosidase activities in human plasma by a differential assay.

A differential assay based on their difference in thermal stability has been used to measure the acidic and true intermediate alpha-mannosidases in the plasma of cntrols and individuals homozygous or heterozygous for mannosidosis. The intermediate activity was found to be independent of age, sex or mannosidosis genotype. The acidic alpha-mannosidase did not vary significantly with age or between sexes for groups of the same age. The concentrations of acidic and intermediate alpha-mannosidase showed a positive correlation for adults but not for children. The ratio of acidic to true intermediate alpha-mannosidase might therefore be a useful secondary test for the detection of adult heterozygotes for mannosidosis.

A granulocyte test for detection of cattle heterozygous for mannosidosis.

alpha-Mannosidase and beta-N-acetylglucosaminidase activities were found to differ between lymphocytes and granulocytes isolated from bovine blood. Activities of both enzymes in granulocyte preparations were found to be related to the eosinophil content of the preparations. A procedure is described that allows definition of the mannosidosis genotype of adult cattle by giving consideration to the influence of eosinophil content of granulocyte preparations upon the activity of alpha-mannosidase relative to that of beta-N-acetylglucosaminidase.

Decreased serum beta-D-mannosidase activity in diabetic patients, in comparison with other glycosidases.

A study of four lysosomal glycosidases' activities was carried out on sera from 64 diabetic patients, which revealed important variations in comparison with the activities observed in sera of control subjects. Depending on the type of diabetes mellitus (I, insulin-dependent, or II, non-insulin-dependent), three activities were more or less increased: alpha-L-fucosidase, alpha-D-mannosidase, and N-acetyl-beta-D-glucosaminidase, in agreement with previously published results. Against that, the beta-D-mannosidase activity shows a highly significant decrease in sera from either diabetic type. Up to now, no suitable explanation has been found for these variations occurring in an unusual direction.

Circadian and circannual rhythms of several enzymes of lysosomal origin in human plasma.

The circadian and circannual group rhythms in the plasma concentrations of the following lysosomal enzymes were studied in women and men: beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, alpha-D-galactosidase, alpha-L-fucosidase and alpha-D-mannosidase. The circadian rhythm was detected in all the tested enzymes of women, and only in alpha-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase and beta-D-N-acetylglucosaminidase of men. A statistically significant difference between genders in the circadian rhythm was exhibited by beta-D-galactosidase, beta-D-glucosidase, beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase and alpha-L-fucosidase. A circannual rhythm was detected in all the tested enzymes, with the exception of beta-D-glucuronidase and beta-D-N-acetylglucosaminidase, without any statistically significant difference between genders. The group rhythms of some of the enzymes (alpha-D-galactosidase, beta-D-glucosidase, beta-D-galactosidase) showed similar values of both circadian and circannual acrophases, suggesting that they may be subjected as a group to the same chronobiological coordination. The chronobiological rhythms of lysosomal enzymes were different from those of lactate dehydrogenase and alpha 1-antitrypsin, indicating that these rhythms are not merely reflecting fluctuations of the water content of plasma. No in-phase relationship was observed between the circadian and circannual rhythms of plasma cortisol and those of the tested lysosomal enzymes, excluding a direct chronobiological relationship between this hormone and lysosomal enzymes.