Lysosomal Enzymes In Plasma Research Articles

Multiplex tandem mass spectrometry enzymatic activity assay for the screening and diagnosis of Mucolipidosis type II and III

Mucolipidosis type II and III (MLII/III) is caused by defects in the mannose-6-phosphate system, which is essential to target most of the lysosomal hydrolases to the lysosome. MLII/III patients present with marked elevations in the activities of most lysosomal enzymes in plasma, but their profiles in dried blood spots (DBS) have not been well described. In the current study, we measured the activities of 12 lysosomal enzymes in DBS, among which acid sphingomyelinase, iduronate-2-sulfatase, and alpha-N-acetylglucosaminidase were significantly elevated in MLII/III patients when compared to random newborns. This sets the stage for using DBS to diagnose MLII/III. Furthermore, given an increasing number of lysosomal storage disorders are being included in the recommended uniform screening panel, our results also indicate that population-based newborn screening for MLII/III can be implemented with minimal efforts.

Analysis of long-term observations of the large group of Russian patients with Hunter syndrome (mucopolysaccharidosis type II)

BackgroundThis article presents the results of long-term observations and comparative analysis of genotype–phenotype features in a large group of patients (227 males and one female) with a severe, intermediate and mild form of Hunter syndrome, evaluating the quality and span of their lives, as well as their ability to social adaptation.MethodsWe used electrophoresis of glycosaminoglycans of urine, determination of the activity of lysosomal enzymes in plasma, in dried blood spots according to the generally accepted method and DNA analysis.ResultsThe clinical symptomatology of 228 patients with Hunter syndrome was characterized by growth retardation, lesions of the bronchopulmonary, cardiovascular, nervous systems, etc. Thirty-five patients had an attenuated form of the disease. DNA was available from all patients. 19 patients from 10 families had a mild form of the disease. 42 patients from 41 families had an intermediate form of the disease. All other patients had a severe form of the disease. We provide brief clinical examples of some patients with a mild form of Hunter syndrome. Currently, 113 patients with Hunter syndrome receive enzyme replacement therapy (idursulfase or idursulfase beta).ConclusionThe long-term study of the large number of patients with Hunter syndrome helped identify disease-associated variants leading to severe and mild forms of the disease. The treatment effect and successful social adaptation of patients with a mild form of Hunter syndrome were revealed.

CLINICAL AND GENETIC CHARACTERISTICS OF MUCOLIPIDOSIS II AND IIIA TYPES IN CHILDREN

The article is devoted to a rare pathology from a group of accumulation diseases with an autosomal recessive type of inheritance – mucolipidosis II and IIIA types. The disease is characterized by a greater phenotypic similarity to mucopolysaccharidosis. Objective: analysis of genophenotypic parameters in Russian patients with mucolipidosis II and IIIA types. The activity of lysosomal enzymes in plasma (β-glucuronidase, total hexosaminidase and N-acetyl-α-D-glucosaminidase) was measured using a standard technique using chromogenic and fluorogenic substrates. Genomic DNA of peripheral blood leukocytes was isolated using a set of reagents Preb 100 (DIAtomTM). Amplification of all exons of the GNPTAB gene was carried out by polymerase chain reaction (PCR) followed by direct non-radioactive sequencing by Sanger. 50 patients aged from 1.5 to 10 years were examined. The clinical symptoms of the disease included: a Hurler-like phenotype, growth retardation, skeletal, cardiac and vascular damage, and CNS. Mucolipidosis type II (I-cell disease) was characterized by a more severe course. The clinical diagnosis was confirmed by the results of laboratory methods of investigation: normal parameters of renal excretion of glycosiminoglycans (GAG), high (5-15 times higher than normal) activity of lysosomal hydrolases in blood plasma and detection of mutations in the GNPTAB gene. 35 probands are completely genotyped. In 8 patients only 8 mutant alleles were detected; 7 mutations were not detected. Six new mutations in exons 1 (p.I31N; p.Q36P), 10 (p.L398P), 11 (p.W446X) and 13 (p.S738X; c.2250delT) were found, including a frequent mutation for Russian patients P.S738X (21% alleles). The most common (31.4% alleles) in the Russian cohort of patients was a known small deletion c. 3503_3504delTC, leading to a reading frameshift. A clinical observation of a child with type 2 mucolipidosis (I-cell disease) with typical symptomatology of the disease caused by two nonsense mutations of the GNPTAB gene is presented: p.S738X / p.R375X. In conclusion, it is emphasized that the identification of mutations of the GNPTAB gene provides prediction of the severity of the disease course, an understanding of the mechanisms of its development that will contribute to the development of pathogenetic treatment methods, improving the quality of life of patients and effective medical and genetic counseling.

Lysosomal enzymes activity in patients with pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) is a complex systemic disorder of the extracellular matrix affecting the eye and visceral organs. Pseudoexfoliation material deposits on various structures of the anterior segment of the eye and around blood vessels of connective tissue. Animal studies have suggested that the lysosomal trafficking regulator (LYST) gene is potentially important in PEX (Liton et al. 2009). Lysosomes are the cellular recycling centres responsible for the physiologic turnover of cell constituents. Inadequate enzyme activity results in disruption of the degradation process and accumulation of substrates for that specific enzyme, leading to variety of pathological changes. Increasing evidence suggests that the oxidative–antioxidative balance is disturbed in patients with PEX (Uçakhan et al. 2006; Lesiewska-Junk et al. 2013). The lysosomal proteolytic activity is compromised under oxidative stress conditions (Wenger et al. 2013). We studied 52 consecutive patients with PEX (16 males and 36 females), who presented for senile cataract surgery, median 75 years, range 59–89. The reference group consisted of 30 individuals (10 males and 20 females), matched for age and gender, with senile cataract without PEX, median 72 years, range 61–90. Both groups did not differ in stage of cataract nor body mass index and smoking habits. PEX was recognized based on typical material in the anterior segment seen after pupil dilation. The exclusion criteria were systemic and local diseases except cataract and PEX (including diabetes, rheumatoid arthritis, lipid profile disorders, intraocular surgery in history). The activity of four lysosomal enzymes in plasma was assessed as follows: acid phosphatase (AcP) with the use of Bessey method, alpha-1-antitrypsin (AAT) using Eriksson method, cathepsin D (CAT D) using Anson method and arylsulphatase (AS) using Robinson method. Kolmogorov–Smirnov test was used to assess normality of the data. Mann-Whitney test was used for comparison of data from small size independent samples. P-value of <0.05 was considered statistically significant. The values of activities of assessed enzymes are shown in Table 1. Reduced activity of proteolytic enzymes and subtle inflammatory processes may trigger the PEX-specific fibrotic matrix process (Schlötzer-Schrehardt 2012). Although Mizuno et al. (1980) found acid phosphatase activity significantly higher in the aqueous humour of eyes with PEX than in that of cataractous eyes without PEX, in our group, these values in blood plasma appeared to be lower in PEX patients. According to Cumurcu et al. (2008) serum A-1-AT activity was increased in patients with PEX, which is in accordance with our results. It may indicate the role of inflammation in this group of patients. To our best knowledge, this is the first study on AS and CAT D activities in PEX patients. Our observations may shed light on the complex metabolic contribution and molecular pathway of the disease.

Microbial immunostimulants reduce mortality in whiteleg shrimp ( Litopenaeus vannamei) challenged with Vibrio sinaloensis strains

The effect of microbial immunostimulants on the survival and immune response of juvenile Litopenaeus vannamei challenged with Vibrio sinaloensis strains was evaluated. Dead microorganisms were added to feed with the attractant Dry Oil ® and consisted of four lactic acid bacteria (Lta2, Lta6, Lta8, and Lta10) and one yeast (Lt6). V. sinaloensis strains or saline solution were inoculated to shrimp by injection. The bioassay was conducted for 21 days with five treatments in triplicate: (I) shrimp fed with commercial feed + sterile saline solution at 2.5% NaCl (control group I); (II) shrimp feed with commercial feed + LD 50 Vibrio (control group II); (III) shrimp fed daily with experimental diet + LD 50 Vibrio; (IV) shrimp fed every 3 days with experimental diet + LD 50 Vibrio; and (V) shrimp fed every 6 days with experimental diet + LD 50 Vibrio. Shrimp (8.1 ± 1.4 g) were cultured in 120-L plastic tanks and fed twice a day. The activity of lysosomal enzymes in plasma and hemocytes were determined with the API ZYM kit and lysoplate assay. Survival of shrimp in treatment IV was significantly higher than those of control II. Total hemocyte count in treatment III was significantly higher than control II. The activity of nine hydrolytic enzymes was found in plasma and six in the hemocyte lysate supernatant (HLS). Shrimp fed with immunostimulants every six days were not protected against V. sinaloensis. The results indicate that these microbial immunostimulants administered every three days is a good feed additive against Vibrio spp. in shrimp culture.

Clinical and biochemical presentation of siblings with COG‐7 deficiency, a lethal multiple O‐ and N‐glycosylation disorder

Congenital disorders of glycosylation (CDG) represent a group of inherited multiorgan diseases caused by defects in the biosynthesis of glycoproteins. We report on two dysmorphic siblings with severe liver disease who died at the age of a few weeks. Increased activities of lysosomal enzymes in plasma were found, though total sialic acid in plasma was strongly decreased. Isoelectric focusing of serum sialotransferrins showed a type 2-like CDG pattern. Some of the known CDG subtypes were excluded. O-Glycosylation was investigated by isoelectric focusing of apolipoprotein C-III, which showed increased fractions of hyposialylated isoforms. In a consecutive study a defect in the conserved oligomeric Golgi complex was established at the level of subunit COG-7, leading to disruption of multiple glycosylation functions of the Golgi. This report on patients with a new variant of CDG, due to a multiple Golgi defect, emphasizes in addition to sialotransferrins the importance of analysis of a serum O-linked glycoprotein, e.g. apolipoprotein C-III, in unclassified CDG-X cases.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Preparation of a stable liquid material for calibration and quality control for lysosomal enzymes in plasma. Assay of enzymes of lysosomal origin in plasma, I.

Several lysosomal enzymes present in human plasma (N-acetyl-beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-galactosidase, alpha-L-fucosidase, alpha-mannosidase, beta-glucosidase) were maintained in a fully active state for at least 8 months by the addition of ethylene glycol (300 milligrams final concentration) to freshly prepared plasma and storage at -20 degrees C. Pools of human plasma from healthy humans, stabilized and stored as above, and containing a low, medium or high content of the above enzymes, were used to establish the analytical imprecision (within-run, day-to-day and total imprecision) of the fluorimetric assay. Ten replicates in ten different analytical series, covering a period of two months, were performed. The total imprecision (expressed as coefficient of variation) was in general lower than 10%; in a few cases, particularly plasma samples with a low enzyme content, the total imprecision was 18%. The isozymes A, B, I1, and I2 of N-acetyl-beta-glucosaminidase displayed the same stability upon storage as the unfractionated enzyme. It is concluded that pools of human plasma containing known amounts of lysosomal enzymes, stabilized by the addition of 300 micrograms ethylene glycol and stored at -20 degrees C, are suitable liquid materials for calibration and quality control for the assay of the same enzymes.

Activity of some lysosomal enzymes in plasma and leucocytes of rabbits exposed to effect of retinol and hydrocortisone.

Observing activity of some lysosomal enzymes in blood serum and leucocytes of rabbits subjected to injection of 200,000 units of retinol and 25 mg of hydrocortisone/kg of body weight it was found that: 1. In the effect of retinol administration there was an increase in the activity AP, BGAL, BGLU, AspAT and lipase in blood serum after 72 hours and NAGL after 168 hours while in leucocytes BGAL and NAGL after 72 hours and AGAL after 168 hours. 2. As a result of hydrocortisone injection the activity of all the enzymes examined (except Ala-Na) in blood serum increased markedly already after 24-48 hours. 3. In leucocytes hydrocortisone caused a significant increase in the activity of AP, BGRD, NAGL, BGAL, AGAL and cathepsin D. 4. The glucose level in blood plasma decreased after 48 hours and 120 hours after hydrocortisone injection and 168 hours after retinol injection.

Affinity chromatography of lysosomal enzymes in plasma, urine and fibroblasts of patients with mucolipidosis (ML) II and III

Using Concanavalin A Sepharose (Con A Sepharose), the elution pattern of plasma α- d-mannosidase, N- acetyl-β- d-glucosaminidase , acid phosphatase and α- l-fucosidase from patients with ML II and III was comparable to that of plasma of a normal individual. The elution of urinary N- acetyl-β- d-glucosaminidase and α- d-galactosidase was the same in mucolipidosis II as in normal controls. ML II patients' fibro blasts have a somewhat lower percentage elution of N- acetyl-β- d-glucosaminidase and acid phosphatase than do normal controls. Using a column of fucose-binding lectin, no activity could be found for eight lysosomal enzymes which were tested in the elution fractions of plasma from patients with mucolipidosis II and normal controls. Elution of seven plasma lysosomal enzymes was obtained using a galactosebinding lectin. No significant differences were noted between plasma of normal persons and patients with mucolipidosis II. Our results indicate that the lysosomal enzymes tested do not adsorb to fucose-binding lectin and that there are no profound differences in the elution of lysosomal enzymes from Con A Sepharose and galactose-binding lectin between normal persons and patients with ML II and ML III.

Elevation of renal cyclic GMP concentrations and plasma lysosomal enzyme activity following cobalt treatment in rats

Summary Following an erythropoietic dose of cobalt in rats, elevations in renal guanosine 3′,5′-monophosphate (cyclic GMP) concentrations occurred within 10 minutes reaching peak levels within 40 minutes of injection. Subsequent to the cyclic CMP changes, activities of lysosomal enzymes in plasma (β-glucuronidase, acid phosphatase) increased markedly, with maximal elevations of both enzyme activities occurring 2 hours after cobalt treatment. Bilateral nephrectomy abolishes the elevations in plasma lysosomal enzyme activity following cobalt treatment, suggesting that the kidney is the source of these enzymes. It is proposed that a cyclic GMP-mediated release of lysosomal enzymes from the kidney may be an early effect of cobalt leading to the generation of renal erythropoietin.

Changes in the level of lysosomal enzymes in plasma and lymph in hemorrhagic shock.

Changes in the level of lysosomal enzymes in plasma and lymph in hemorrhagic shock.