[The clinical diagnostic value of a study of the activity of enzymes of the guanyl branch of purine metabolism in patients with ankylosing spondyloarthritis].

AIM: To assess the diagnosis of the activity of a pathological process in patients with ankylosing spondyloarthritis (AS) and to reveal purine metabolic (PM) changes in relation to the clinical features of AS. MATERIALS AND METHODS: The serum activities of the PM enzymes: xanthine oxidase (XO), guanine deaminase (GDA), guanosine deaminase (GSDA), purine nucleoside phosphorylase (PNP), guanosine phosphorylase (GP), and adenosine deaminase (ADA) were determined in 55 patients (51 males and 4 females) aged 36.0 +/- 1.4 years). A control group comprised 30 apparently age- and gender-matched healthy individuals, as in the study group. RESULTS: On admission, the patients were found to have increased XO, GDA, PNP, and GD activities and decreased GSDA activity. The higher activity of the process was observed, the greater activities of GDA, XO, PNP, GP and the lower activity of GSDA and ADA were. There were the enzymatic activity differences that depended on the degree of pathological process activity, clinical form, the magnitude of X-ray changes, and the degree of joint functional insufficiency. CONCLUSION: The findings suggest that there may be PM disturbances in the immunocompetent cells.

High expression of APOBEC3G in patients infected with hepatitis C virus.

APOBEC3G (an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a member of the APOBEC family, which possesses cytidine deaminase activity that causes C/G to T/A transition mutations in virus genomes such as human immunodeficiency virus 1 and hepatitis B virus, is reported to play an important role in host-defense mechanisms. However, APOBEC3G expression in patients infected with chronic hepatitis C virus (HCV), of which there are currently more than 170 million worldwide, has not yet been well studied. We investigated this issue herein, and demonstrated an increased expression of APOBEC3G in both hepatocytes and lymphocytes of chronic hepatitis patients infected with HCV. Transfection of the NS5A gene, but not any other non-structural protein genes of HCV tested, to the hepatocellular carcinoma cell line enhanced APOBEC3G expression. Incubation of the cells with interferon also resulted in the augmentation. These results may provide new insight into the pathogenesis of chronic HCV infection.

STZ-induced diabetes in mice and heme pathway enzymes. Effect of allylisopropylacetamide and alpha-tocopherol.

A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.

S-adenosylhomocysteine hydrolase and adenosine deaminase activities in human red cell ageing.

The enzyme activities of S-adenosylhomocysteine hydrolase, adenosine deaminase and pyruvate kinase were determined in normal human erythrocytes subpopulations of different ages separated by centrifugation on a discontinuous Percoll:NaCl density gradient. The levels of S-adenosylhomocysteine hydrolase activity were found to undergo a sharp decrease with red cell ageing. Adenosine deaminase activities were, however, less critically dependent on erythrocyte age.

[Combination assay of IAP and ADA in hematologic malignancies].

We measured the levels of adenosine deaminase (ADA) and immunosuppressive acid protein (IAP) in 10 patients with acute myeloid leukemia (AML), 5 with acute lymphoblastic leukemia (ALL), 8 with chronic myeloid leukemia (CML), 7 with myelodysplastic syndrome (MDS), 5 with malignant lymphoma (ML), 3 with multiple myeloma (MM) and one with adult T cell leukemia. On admission, the level of IAP was abnormally high in all cases of AML and ALL 50% of CML cases, 71.4% of MDS cases, 60% of ML cases and none of MM cases. ADA was elevated in all cases of ALL, 77.8% of AML and CML cases, 57.1% of MDS cases, 60% of ML cases and 33.3% of MM cases. In 7 patients with AML, the level of IAP returned to normal when they achieved complete remission. On the other hand, the level of ADA had already returned to normal even during induction therapy. ADA showed a positive correlation with the absolute number of peripheral blasts and lactic dehydrogenase both in AML and ALL. These results suggest that ADA indicates the activity of leukemia and IAP indicates the immunocompetence of the host.

Automated enzymatic measurement of adenosine deaminase isoenzyme activities in serum.

We developed a simple, rapid, and automated method for simultaneous measurement of adenosine deaminase (ADA, EC 3.5.4.4) isoenzymes in human serum, based on their apparent difference in Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) as inhibitor. Serum ADA was partially purified by CM-Sephadex, gel-filtration, and affinity chromatography into two types of isoenzymes, designated ADA1 (300 kDa) and ADA2 (120 kDa). Because ADA2 has a higher Km for adenosine and higher Ki values for EHNA than does ADA1, the activity of ADA1 is almost completely inhibited by EHNA at 0.1 mM (analytical recovery 4.1%), whereas ADA2 is practically unaffected (analytical recovery 94.8%) by that concentration of EHNA. We measured the activities of ADA2 and total ADA in the presence and absence of 0.1 mM EHNA. ADA1 activities were calculated by subtracting the activity of ADA2 from that of total ADA. The mean within-assay CV was 5.7% for ADA1 and 2.7% for ADA2. The interassay CV was 2.8% for ADA1 and 3.1% for ADA2. Results of the present method correlated well (r = 0.9026 for ADA1, 0.9438 for ADA2) with those of the ion-exchange chromatography method. The upper limits of the reference intervals, as calculated from data for 320 healthy donors, are 7.2 U/liter for ADA1, and 14.6 U/liter for ADA2. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the activities of ADA isoenzymes in serum.

Serum and pleural adenosine deaminase. Correlation with lymphocytic populations.

This study attempts to correlate levels of ADA in tuberculous and neoplastic pleural exudates with the different immunologic cellular expressions that follow these clinical situations. Seventy-three patients with pleural effusion were studied in order to assess ADA activity (pleural and serum); in 25 of them, a study of delayed cellular immunity (pleural and sanguineous) was performed through B, CD3, CD4, and CD8 lymphocytic populations. The activity of ADA was determined, and the study of lymphocytic populations was made through the use of monoclonal antibodies. The data obtained showed the following: levels of ADA were significantly (p less than 0.0005) higher in the pleural fluid and the serum of tuberculous effusions compared to neoplastic effusions; percentages of CD3 and CD4 T-cells were significantly (p less than 0.05 and p less than 0.0005, respectively) greater in tuberculous effusions. The statistical study of the levels of ADA activity and the percentage of CD4 T-cells in pleural exudates produced a significant regression curve (r = 0.612 and p less than 0.0001) which showed a positive correlation between these two parameters. The pathogenic implications of these results suggest the possibility that ADA could be a new marker of cell-mediated immune activity.

Evaluation of a nonequilibrium isoelectric focusing (IEF) method for the simultaneous typing of esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA).

A nonequilibrium isoelectric focusing method incorporating the chemical spacers MOPS and HEPES was developed and subsequently evaluated for its ability to reliably discriminate common and rare phenotypes in the esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA) isoenzyme systems. The validation procedures used were blind testing, comparison of results to conventional methods, and evaluation of known rare variant phenotypes. This method proved to be a quick and reliable method for typing all five isoenzyme systems, while providing an excellent probability of discrimination (PD = 0.96).

[Adenosine deaminase (ADA)--biologic and medical-diagnostic significance]. / Die Adenosindesaminase (ADA)--biologische und medizinisch-diagnostische Bedeutung.

The determination of the ADA activity was only of importance in special affections, as for instance typhus, epidemic jaundice, active liver cirrhosis till now, where high enzyme activities are measured. Nevertheless this enzyme is also useful for the pathogenetic classification of hyperuricemia, the elucidation of immunoaffections of obscure etiology, and, above all, of the genetic ADA defect. Our results show that the determination of the ADA activity enables some declaration for the therapy and the control of progress of the acute and the chronic leukemia. Investigations of the distribution in the organs and age-dependence in animals emphasize the great biological importance of this enzyme in the metabolism, where much questions are open till now.

Bioluminescent assay for serum adenosine deaminase with immobilized bacterial luciferase.

We describe a bioluminescence method for measuring adenosine deaminase activity in serum. The method involves use of batchwise enzyme reaction containing adenosine, alpha-ketoglutarate, glutamic dehydrogenase and NADH. The resulting solution is injected to the continuous-flow bioluminescence system. In the system, a bacterial luciferase and NAD(P)H:FMN oxidoreductase are covalently co-immobilized on Sepharose 4B. Carrier solution (pH 6.8) for bioluminescence reaction contains FMN and decanal. The continuous-flow light-emitting system, in which the reactor (flow cell packed with immobilized enzyme) is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. Concentration and response are linearly related from 1.2 to 92.5 pmol per injection of ammonia. The precision of the method is satisfactory (coefficient of variation 3.9-6.8%). We validated the technique by comparing results with conventional assay method (UV method). Normal values for adenosine deaminase activity of serum ranged from 7.0 to 22.0 U/l in agreement with those obtained by other method. The Sepharose 4B-immobilized enzymes are stable for more than one year. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage.

Determination of serum cytidine deaminase activity using ion-pair reversed-phase liquid chromatography.

A rapid and sensitive assay for serum cytidine deaminase has been developed utilising ion-pair reversed-phase high-performance liquid chromatography. The addition of 1-octanesulphonic acid (OSA) caused the retention of cytidine and uridine to reverse and uridine, the minor component in the assay, to elute first. Cytidine, uridine and allopurinol (internal standard) were separated on a 5-micron Hypersil ODS column using 100 mM ammonium acetate with 1% (v/v) methanol and 1 mM OSA adjusted to pH 5.0. Detection was at 262 nm. Peak areas were linear from 7 pmol to 6 nmol injected (r = 0.99). Intra-assay variation was 7.8% (n = 10) and the correlation with a colorimetric assay was r = 0.78 (p less than 0.001).

Serum adenosine deaminase activity in HIV positive subjects. A hypothesis on the significance of ADA2.

Adenosine deaminase activity (ADA) was assayed in the serum of 137 HIV positive subjects: 131 intravenous drug abusers, 3 female partners of HIV positive abusers, 1 son of HIV positive abuser and 2 blood-transfused patients, subdivided into the following groups: 73 asymptomatic, 15 with ARC, 37 with LAS, 5 with L-AIDS and 7 with AIDS. Results show an increase of ADA activity in 100% of L-AIDS group, in 64% of AIDS group, in 62% and 42% of LAS and ARC groups, respectively, and in 36% of the asymptomatic groups. These findings must be confirmed with wider series in order to be further and better evaluated. According to relative substrate specificities, optimal pH and Km, enzymatic activity can be attributed principally to the ADA2 isoenzyme. The probability that ADA2 originates exclusively from the Monocyte-Macrophage cell system (MoMaCS) which actively releases this enzyme in the presence of live parasites in the cells' interior, is discussed. Moreover, it was hypothesized that in the MoMaCS the enzyme constitutes a microbicidal mechanism independent of the respiratory burst.

Erythrocyte adenosine deaminase overproduction in hereditary hemolytic anemia.

A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5'- and 3'-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.

Depressed activities of purine enzymes in lymphocytes of patients infected with human immunodeficiency virus.

Enzyme activities were studied in peripheral blood lymphocytes from patients infected with, or at risk for, infection with human immunodeficiency virus (HIV). No significant differences were observed in the HIV-infected and HIV-seronegative high-risk patients with regard to enzyme activities of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and purine nucleoside phosphorylase (EC 2.4.2.1) in peripheral blood. Adenosine deaminase (EC 3.5.4.4) was significantly (P less than 0.02) depressed in asymptomatic HIV-seropositive patients and HIV-seronegative patients at high risk of HIV infection as compared with a healthy HIV-seronegative population. Adenosine kinase (AK, EC 2.7.1.20) was significantly increased in the asymptomatic seropositive (P less than 0.02) and also in the HIV-seronegative high-risk groups (P = 0.01) compared with the normal controls. AK activity was significantly lower in subjects with AIDS than in the asymptomatic (P less than 0.002) and high-risk groups (P less than 0.01). Taken together, these results indicate that adenosine deaminase and AK activities are influenced by the health of the patient, and that measurement of AK activity may prove useful in monitoring the clinical progress of patients with HIV infection.

Circadian rhythm of serum cytidine deaminase in patients with rheumatoid arthritis during rest and exercise.

Circadian rhythm of serum cytidine deaminase and C reactive protein was assessed in 11 inpatients with rheumatoid arthritis who were crossed between 24 hours of bed rest and 24 hours of normal ward activity. Blood was taken at six hourly intervals and the results analysed by fitting sine waves with an assumed period of 24 hours to the measured concentrations. Cytidine deaminase after activity, but not at rest, showed circadian variation, with a 24 hour mean level of 17.4 units (normal 3-13 units) and an amplitude of 1.1 units. The circadian variation, defined as the curve's peak to trough difference as a percentage of the 24 hour mean, was 12.3% and occurred at 1208 hours. C reactive protein showed no significant circadian rhythm, in keeping with published findings. The timing of the peak in serum cytidine deaminase concentrations after a period of morning physiotherapy, but not during the bedrest morning, suggests that exercise accounts for the circadian rhythm, probably by increasing the lymphatic clearance from inflamed joints.

[Changes in the activities of enzymes of metabolism of DNA precursors in peptic ulcer and cancer of the stomach]. / Izmenenie aktivnosti fermentov metabolizma predshestvennikov DNK pri iazvennoi bolezni i rake zheludka.

Comparative assessment of the remote results of Hoffmeister-Finsterer gastrectomy with creation of valve anastomoses has demonstrated rare occurrence of postoperative complications owing to anastomoses. The operation is simple as concerns operative techniques and can be recommended as operation of choice in the treatment of peptic ulcer.

Ecto-enzyme activity of human erythrocyte adenosine deaminase.

Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl-, which is precipitated at its locus of formation by added Ag+, and the precipitated AgCl converted into the electron dense Ag0 upon exposure to light. From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane. The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.