Lysosomal enzymes in serum and synovial fluid in patients with osteoarthritis.

OBJECTIVE: To assess the activity of arylsulfatase (AS), acid phosphatase (ACP), cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood serum and synovial fluid (SF) of patients with hip or knee osteoarthritis (OA). METHODS: The study included 43 subjects with OA (35 hip OA, 8 knee OA), submitted total joint replacement. The control group consisted of 58 subjects with no past history of musculoskeletal disorders. RESULTS: The OA blood serum samples showed a significantly higher level of lysosomal enzymes activity than in the control group (AS by 17.8%, AAT by 42.4%); only the CAT-D activity decreased by 50%). AS, ACP and CAT-D activities were about two-fold higher in SF when compared with blood of OA patients. The differences between the genders were visible in the SF: Total protein concentration, activity of ACP (both higher in OA men) and activity of CAT-D (higher in OA women). Between the involved hip and knee, there were no significant differences in all estimated parameters in the blood serum of the OA group. In regard to the SF, only ACP activity was significantly higher in patients with a hip involved. CONCLUSIONS: The osteoarthritic SF enzymatic profile differs from that in normal joints. The OA in joints is not reflected in the systemic response. Our preliminary results suggest further studies on role of lysosomal enzymes (ACP and AS) as biomarkers for the detection of osteoarthritis.

[Arylsulfatase activity in the blood of men with malignat melanoma. Preliminary reports]. / Aktywnosc arylosulfatazy we krwi mezczyzn z czerniakiem skóry.

UNLABELLED: High arylsulfatase activity occurs in the course of cutaneous melanoma. There is a correlation between the activity of the enzyme, and the susceptibility to metastatic cancer, including metastatic melanoma. Melanoma of the skin were 1.4% of all cancers in men and the number of deaths from this cancer in Poland in 2002 was the 424th. The aim of the study was decided to determine the usefulness of arylsulfatase activity in patients with melanoma of men for diagnostic purposes and for monitoring of the disease course. MATERIAL AND METHODS: The study included 25 patients diagnosed men with melanoma (mean age 55.5 years). Blood samples were collected before excision of melanoma of the skin and in the period from 2 to 3 months after surgery. To donate blood after resection of malignant melanoma reported only 13 men (mean age 57.4 years). Arylsulfatase activity was determined by Roy the modified Bleszczynski method. RESULTS: Arylsulfatase activity before surgery to remove the changes was higher in comparison to the control group and the patients after surgery. CONCLUSIONS: Of men patients with malignat melanoma arylsulfatase activity was high. Arylsulfatase mark may be useful in diagnosing and monitoring the course of the disease.

Effect of cadmium injected in ovo on hatching results and the activity of plasma hydrolytic enzymes in newly hatched chicks.

The aim of the study was to determine the toxicity of cadmium ions in chick embryos, using plasma hydrolytic enzyme as its biomarker. Hatching eggs (n = 300) from Ross 308 broilers were incubated under standard conditions. On day 4 of incubation, 50 µl of saline solution, containing Cd ions at a concentration from 0 (control group) to 24 µg, was injected in ovo into the egg albumen. The results indicate that the administration of cadmium at doses exceeding 1 µg/egg caused a gradual decrease in hatchability, with an LD50 of 3.9 µg/egg. The greatest differences between the groups in the enzymatic activities studied were found for N-acetyl-ß-D-glucosaminidase (NAG), ß-D-mannosidase (ß-MAN) and arylsulphatase (ARYL). Compared to the control group, in the blood serum of chicks from the groups receiving 3, 6 and 12 µg Cd/egg the NAG activity increased by 79, 108 and 54% and ß-MAN activity by 33, 119 and 108%, respectively. Exposure to cadmium at a dose of 1 to 6 µg per egg caused an about 60% increase in ARYL activity while a dose of 12 µg decreased the activity by about 35% below the level observed in the control group. These findings show that cadmium has a similar toxicity mechanism in mammals and birds, which opens the possibility of using NAG activity as a biomarker of the cytotoxic effect of cadmium in birds.

Serum Arylsulfatase and Acid Phosphatase Activity in Patients with Metabolic Syndrome as a Result of Oxidative Damage to Lysosomes.

BACKGROUND: Metabolic and clinical disorders forming the complex of interrelated abnormalities is known as metabolic syndrome (METs). OBJECTIVE: Our goal was to assess the dependence of serum arylsulfatase (AS) and acid phosphatase (ACP) activities on anthropometric and biochemical parameters in patients with METs. METHODS: In 142 patients with METs (IDF criteria), consisting of different components in different sequences (hypertension, diabetes, lipid disorders), and in 65 healthy participants, basic biochemical parameters were determined in laboratory tests. The activity of serum hydrolases was determined using Bessey's (ACP) and Roy's (AS) methods. RESULTS: The AS activity is correlated with waist-to-hip ratio (WHR) (more strongly in women and in most advanced METs), BMI (in men), and triglycerides (TG) (in women, participants with I degree obesity, and those with three METs components). The ACP activity correlated with the WHR of patients with II degree obesity, TG in those with III degree of obesity, and total cholesterol (TC) in those with four METs components. CONCLUSION: Increased AS activity in patients with METs compared to lower AS activity in the control group may be due to decreased lysosomal function and related to the amount of adipose tissue. Low activity of ACP in the blood serum of patients with METs compared to high activity of ACP in the control group may indicate exhaustion of the lysosomal apparatus and loss of hydrolytic activity. The increase in TG and TC in groups with an increasing number of METs-defining components may be due to the abnormal lysosomal degradation of these compounds.

Co-occurrence of Metachromatic Leukodystrophy in Phelan-McDermid Syndrome.

Phelan-McDermid syndrome or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder characterized by neonatal hypotonia, severe speech delay, moderate to profound intellectual disability, and minor dysmorphic features. Regression of developmental milestones is often recognized as characteristic of this syndrome. We report a 6-year-old patient with Phelan-McDermid syndrome who presented with rapid neurologic deterioration secondary to metachromatic leukodystrophy due to a mutation of the arylsulfatase A gene (ARSA) on the other allele of 22q13.3. Metachromatic leukodystrophy was diagnosed later after clinical deterioration. Currently, there are no guidelines for screening Phelan-McDermid syndrome patients for metachromatic leukodystrophy. We propose screening for urine sulfatides at the time of Phelan-McDermid syndrome diagnosis to identify patients with pre-symptomatic or early symptomatic metachromatic leukodystrophy as it is important to facilitate discussion of treatment options and prognosis and provide medical surveillance for associated complications.

Activity of α1-antitrypsin and some lysosomal enzymes in the blood serum of patients with chronic obstructive pulmonary disease after smoking cessation.

The activity of α1-antitrypsin (AAT) and the lysosomal enzymes, cathepsin D (CTS D), arylsulfatase (ASA), and acid phosphatase, (AcP) was determined in patients with COPD (GOLD category A). Moreover, the diagnostic usefulness of these parameters in blood serum was assessed along with establishing whether smoking cessation affects these parameters. The study included 70 patients with COPD who ceased smoking (study group) and two control groups of 33 subjects each: nonsmokers without COPD (control I) and patients with COPD who continued smoking (control II). In control I, blood was taken once and in control II, at the start of the experiment and after the 1st, 2nd, and 3rd months. AAT in the patients exhibited higher activity than in the healthy subjects at all time points. AAT activity in the patients before the start of the experiment was ~80% higher (P < 0.001) than in control I. No statistically significant differences in CTS D, ASA, and AcP activity were found. COPD involves increased AAT activity and unchanged activities of the assessed lysosomal enzymes. Three-month tobacco abstinence does not affect these parameters in peripheral blood. Determining the AAT levels in blood serum can be used in the diagnostics of COPD.

Study of influence of sex and age on human serum lysosomal enzymes by using 4-methylumbelliferyl substrates.

The sex and age dependence of activity of eight glycosidases and acid phosphatase was assayed in serum samples using the 4-methylumbelliferyl substrates. The activity of these enzymes does not change in relation to sex and to age except for acid phosphatase and beta-galactosidase which show significantly higher values in children as compared to adults. The usefulness of the 4-MU substrates for the detection of homozygotes for those lysosomal diseases involving one of the glycosidases studied is discussed.

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.

Enzymatic diagnosis of steroid sulfatase deficiency by high performance liquid chromatography.

We established a reversed phase high performance liquid chromatographic (HPLC) method of assaying lymphocyte steroid sulfatase activity using estrone sulfate as the substrate. Application of this method for diagnosis of 8 patients allowed us to clearly distinguish the patients from the normal controls. This method is simpler and less expensive than the method previously reported, since neither radioisotope labeled substrates nor radioisotope facilities are required. We consider it to be easily used and widely available in most clinical laboratories.

Human leukocyte acid hydrolases: characterization of eleven lysosomal enzymes and study of reaction conditions for their automated analysis.

The optimal reaction conditions and kinetic properties of eleven leukocyte acid hydrolases determined with the use of fluorigenic derivatives of 4-methyl-umbelliferone are described. The enzymes studied were acid phosphatase, aryl sulfatase, alpha- and beta-glucosidase, alpha- and beta-galactosidase, alpha-mannosidase, N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-glucuronidase and alpha-fucosidase. More than 90% of the activity of each enzyme was released into a 27,000 X g supernatant by a double sonication procedure employing 0.9% sodium chloride and 0.1% Triton X-100. The Km values obtained were similar to those previously reported for chromogenic subtrates. A single Km value could not be derived for beta-galactosidase because its double reciprocal plot was not linear. All enzymes could be measured with less than 10 mug of protein within 15 min. Activators and inhibitors studied included the chloride salts of Na+, K+, Zn2+, Ca2+, Mg2+, Hg2+, and Fe2+ as well as p-chloromercuriphenysulfonate, glutathione, BAL, EDTA, EGTA, Triton X-100 and sodium taurocholate. The reaction conditions described in this report can be used for the diagnosis of various lysosomal storage diseases and should facilitate the development of automated procedures for the analysis of these eleven enzyme activities with small quantities of blood.

Serum levels of steroid sulfatase protein in gynecologic carcinomas.

Steroid sulfatase (STS) desulfates a number of 3 beta-hydroxysteroid sulfates, converting inactive steroid hormone to the active form. We have established an enzyme-linked immunosorbent assay (ELISA) of STS by using polyclonal antibody against STS purified from human placenta to measure the amount of the enzyme protein in sera. ELISA was performed by a 'Sandwich' method using a peroxidase conjugated anti-STS IgG Fab' fragment. A range of STS of 10-1,500 ng/ml in serum was assayed by this method. When the serum STS from the patients with gynecologic carcinomas was assayed by the ELISA, the level was significantly elevated in endometrial carcinoma (P < 0.05) and ovarian carcinoma (P < 0.01), respectively, as compared with that of normal healthy women.

Lysosomal enzymes and severity of saphen varicosis.

The severity of the varicosis estimated clinically and histologically is well correlated with the seric levels of beta-acetylglucosaminidase and beta-glucuronidase. No correlations are observed with the arylsulfatase level in serum nor with the levels in venous tissue for the three enzymes.

Corneal manifestations of X-linked ichthyosis in two brothers.

PURPOSE: To report the unusual manifestation of X-linked ichthyosis in two brothers. METHODS: Leukocyte separation and sterylsulfatase assay are performed to show the deficiency of sterylsulfatase. RESULTS: Two brothers presented in our clinic with cutaneous alterations consistent with X-linked ichthyosis. Ocular examination disclosed fine, flour-like, punctate, evenly, and diffusely distributed opacities of the posterior corneal stroma, close to Descemet membrane in both patients. In one patient, superficial, small, granular opacities were detected. They were gray in color and seemed to involve the epithelium and the subepithelial and anterior stromal layers. In both patients, the deficiency of sterylsulfatase could be shown and confirmed the diagnosis. CONCLUSIONS: Flour-like opacities in the posterior stroma have been shown to be a common manifestation of X-linked ichthyosis. Though the underlying biochemical defect in X-linked ichthyosis has been discovered, the question of how these opacities develop is still a subject of debate. Subepithelial stromal keratopathies or epithelial irregularities are uncommon and are rarely described in the literature. The superficial corneal changes seen in one of our patients are unusual and are not similar to those reported by other authors.

Prevention of deterioration in metachromatic leukodystrophy by bone marrow transplantation.

The first girl in a family was affected with late infantile metachromatic leukodystrophy (MLD) and had the expected characteristic central nervous system progressive deterioration, which resulted in decerebration and death. The second girl (propositus) demonstrated similar symptoms and signs at the same age. Both girls had characteristically low arylsulfatase A levels. The propositus underwent allogeneic bone marrow transplantation (BMT) from a normal histocompatible sibling. Two and a half years later, the propositus has not developed the intellectual and neurologic impairment demonstrated by the first sibling, although nerve conduction has continued to worsen. These results suggest that the induction of normal enzyme levels by BMT may be retarding or inhibiting CNS deterioration. These results, confirming earlier results of others, are sufficiently promising to warrant a larger scale critical trial of BMT early in the course of MLD.

Activity of some lysosomal enzymes in serum and in tumors of patients with squamous cell lung carcinoma.

The aim of the study was an assessment of some lysosomal enzymes activity in serum and in tumors of patients with lung cancer histopathologically confirmed as squamous cell lung carcinoma. The first group constisted of 10 patients with stage II of the disease and the second group consisted of 11 patients with stage III of the disease. Lysosomal enzymes activities were assayed in serum before surgery and on the 10th day after surgery in serum and in tumors. Arylsuphatase, cathepsin D and acid phosphatase activities were higher in the patients serum than in that of the control group. The decrease of arylsulphatase and cathepsin D activities after surgery was statistically significant in both groups of patients, but the cathepsin D activity was still 3 times higher in patients than in those from the control group. The decrease of acid phosphatase activity after surgery was about 50% in both groups of patients and this decrease was statistically significant. The arylsulphatase and acid phosphatase activity in tumors was nearly 3 times higher in stage III patients than it was in stage II patients, but the cathepsin D activity was nearly the same in both patient groups. Higher lysosomal enzyme activity may be a useful factor in diagnosing and monitoring of lung cancer. However, further investigations are needed.

Effects of Centella asiatica extract on mucopolysaccharide metabolism in subjects with varicose veins.

The effects were studied of the total triterpenic fraction of Centella asiatica on serum levels of the uronic acids and lysosomal enzymes involved in mucopolysaccharide metabolism (beta-glycuronidase, beta-N-acetylglucosaminidase, arylsulfatase) in patients with varicose veins. The basal levels of uronic acids (467.7 +/- 69.3 micrograms/ml) and of lysosomal enzymes (beta-glycuronidase 1.8 +/- 0.4 microM/min/l, beta-N-acetylglucosaminidase 23.1 +/- 0.4 microM/min/l, arysulfatase 0.078 +/- 0.003 microM/min/l) were elevated, indicating an increased mucopolysaccharide turnover in subjects with varicose veins. During treatment with Centella asiatica extract (60 mg/day for three months), these levels fell progressively. At the end of treatment the serum uronic acid (231.8 +/- 51.5 micrograms/ml), beta-glycuronidase (1.2 +/- 0.05 microM/min/l), beta-N-acetylglucosaminidase (17.7 +/- 0.7 microM/min/l) and arysulfatase (0.042 +/- 0.003 microM/min/l) levels were highly significantly lower than the basal levels (p less than 0.01). The results of this trial provide an indirect confirmation of regulatory effects of the extract of Centella asiatica on metabolism in the connective tissue of the vascular wall.

The effect of Escherichia coli endotoxin on lysosomal enzymes in the domestic fowl.

During the 2 h following the injection of chickens (aged nine to 11 weeks) with endotoxin isolated from a pathogenic strain of Escherichia coli (O78) there was a transient rise in the activity of acid phosphatase, aryl sulphatase and beta-glucuronidase in the plasma, suggesting increased release of these enzymes from lysosomes. This was followed by a fall in their activity which may have been due largely to stabilisation of the lysosomal membrane brought about by increased secretion of adrenocortical hormones and/or accelerated removal of the enzymes from circulation by the reticuloendothelial system.

Modified granulocyte test for determination of mannosidosis genotype of cattle.

Recovery of alpha-mannosidase, beta-N-acetylglucosaminidase and arylsulphatase activity from granulocytes is increased by inclusion of 154 mM sodium chloride, in place of 1 mM zinc chloride, in aqueous Triton X-100 used to extract the cells. Calculation of the ratio of alpha-mannosidase to beta-N-acetylglucosaminidase and to arylsulphatase permits diagnosis of the mannosidosis genotype without recourse to examination of smears of the granulocyte preparations.

Granulocyte plasma membrane damage by leukotoxic supernatant from Pasteurella haemolytica A1 and protection by immune serum.

Bovine respiratory disease caused by Pasteurella haemolytica may be partially mediated by a leukotoxin secreted by the microorganism. We examined the effect of leukotoxic Pasteurella supernatants on leakage of the cytosol enzyme lactate dehydrogenase and the lysosomal enzyme arylsulfatase from bovine granulocytes. Lactate dehydrogenase release (94%) was much higher than arylsulfatase release (38%) over 30 minutes of incubation. The Pasteurella supernatants inhibited superoxide production by stimulated granulocytes at concentrations which also caused substantial cell death as measured by failure to exclude trypan blue. Both toxic effects were prevented by serum from aerosol-immunized calves, and protection appeared to be antibody-specific by comparison with fetal bovine serum or with serum absorbed against intact P. haemolytica. These findings suggest that the leukotoxin may selectively disrupt the granulocyte plasma membrane, and that antibody directed against a surface component of the microorganism is also capable of protecting against the leukotoxin effect.

Degranulation of peripheral blood neutrophils during haemoperfusion in chronic uraemics.

In 10 chronic uraemics activities of some lysosomal enzymes were determined in peripheral blood neutrophils and plasma during haemoperfusion with charcoal cartridge Adsorba 300 C (Gambro). Blood plasma activities of arylsulfatases, beta-N-acetylglucosaminidase and beta-glucuronidase did not significantly change during haemoperfusion, while lysozyme activity was significantly increasing. Neutrophil contents of these enzymes decreased. The determinations of these enzyme activities revealed a positive outlet-inlet difference in blood plasma and a negative outlet-inlet difference in neutrophils during the first 20 minutes of the procedure. The results suggest that during haemoperfusion degranulation of the peripheral blood neutrophils occurs.