Low Adenosine Deaminase Activity Research Articles

Gene Therapy for ADA-Deficient Severe Combined Immunodeficiency

Source: Reinhardt B, Habib O, Shaw KL, et al. Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency. Blood. 2021 Oct 14;138(15):1304-1316; doi:10.1182/blood.2020010260Investigators from multiple institutions conducted a study to evaluate long-term outcomes in children with severe combined immunodeficiency (SCID) caused by lack of adenosine deaminase activity (ADA) who were treated with autologous hematopoietic stem cell transplant (HSCT) of gene-corrected cells (gene therapy [GT]). Participants were children diagnosed with SCID with absence of ADA activity, an absolute lymphocyte count of <200 cells/mm3, and no suitable sibling donor for allogeneic HSCT who were being treated with ADA enzyme replacement therapy (ERT). These patients had CD34+ cells harvested by bone marrow aspirate that were transduced with a gamma-retroviral vector encoding for a functional human ADA cDNA sequence. The transduced cells were infused into the patient. Study patients were followed at annual visits at which interval histories were obtained along with blood specimens for multiple laboratory assays. Previously, short-term outcomes (up to 2 years post-GT treatment) were reported. For the current study, long-term outcomes, including occurrence of severe or unusual infections, leukoproliferative events or malignancies, ADA enzyme activity, need for ERT, lymphocyte counts, and immunoglobulin levels were assessed 8-11 years after treatment.Data were analyzed on 10 children with ADA SCID who received GT treatment between 2009 and 2012. At the time of treatment, the study patients ranged in age from 3 months to 15 years. At follow-up, no severe, persistent, or opportunistic infections, or leukoproliferative events or malignancies, occurred in any of the study patients. In 8 of the 10 study participants, peripheral blood mononuclear cell (PBMC) ADA activity was maintained in the normal range. In 1 child, with low PBMC ADA activity, ERT was resumed 6 months after GT; no other patient required ERT following treatment with GT. Patients who received GT at the youngest ages (3 months) had the highest PBMC ADA activity. Absolute lymphocyte counts remained relatively stable in study patients over the follow-up period, although levels were ≤ the 10th percentile for healthy children. Lymphocyte counts were higher in children receiving GT at 3 months of age and, overall, counts were highly correlated with ADA activity. Four patients had Immunoglobulin G (IgM) levels in the normal range. These children were taken off immunoglobulin replacement therapy, and 2 of these responded to vaccines. The other patients had IgM levels near or below the lower limit of normal.The authors conclude that in this group of children with ADA SCID, the long-term outcomes demonstrate enduring efficacy of GT.Dr Hogan has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device.ADA deficiency results from an autosomal recessive inheritance of mutations in the ADA gene, accounting for up to 15% of severe combined immunodeficiency cases and affecting 1:200,000 to 1:1,000,000 live births worldwide.1 Lack of ADA results in buildup of adenosine, 2’-deoxyadenosine, and deoxyadenosine, which is toxic to lymphocytes, bone, brain, lungs, and other organs. Within 6-12 months of age, infants develop life-threatening sinopulmonary and gastrointestinal infections with failure to thrive.2Abnormal newborn screening with reduced T cell receptor excision circles should prompt immediate testing of serum T, B, and NK lymphocytes and immunoglobulins.1 Referral to pediatric immunology, genetics, and a pediatric HSCT center will help expedite confirmatory enzyme and genetic testing while providing prophylactic antimicrobials and intravenous immunoglobulin.2 When an ideal HLA-matched healthy sibling HSCT donor is not available, enzyme replacement therapy (ERT) with intramuscular injections of polyethylene glycol-conjugated ADA helps provide some lymphocyte recovery until a possible unrelated donor or GT of autologous CD34+ HSCT.3The current study’s strengths include a standard replicable therapy protocol with consistent long-term follow-up of clinical and microscopic characteristics.3 Since the initial treatment of these individuals, advances in GT include improvements in vectors, transduction, and cryopreservation.3 Thus far, more than 100 affected individuals have undergone GT on 11 different clinical trials worldwide with excellent immunocompetent results.4 However, randomized controlled trials comparing ideal sibling HSCT or variations in GT have not been done.4 Long-term safety monitoring for malignant transformation or loss of immunocompetence can be difficult due to the diversity of treatment sites, data collection, and patient retention.5Infusion of autologous hematopoietic stem cells transduced with ADA-gammaretroviral vector gene therapy has provided long-term immune reconstitution in children with ADA deficiency.

Comparison of malondialdehyde, nitric oxide, adenosine deaminase and glutathione levels in patients with Entamoeba coli, Enterobius vermicularis, Giardia intestinalis, Demodex spp. positive, hydatid cyst and Toxoplasma gondii serum positive

In this study we aimed to compare glutathione (GSH), adenosine deaminase(ADA), nitric oxide (NO) and malondialdehyde (MDA) values between the patients with and without different parasitic infections. Material and Methods: MDA, NO, ADA and GSH levels were studied in the serums of the patients group with T. gondii and cyst hydatid seropositivity and E. vermicularis and E. coli positive and the control group, while ADA levels alone were studied in G. intestinalis and Demodex spp. Results: There was a statistically significant difference between the groups in terms of the amounts of ADA, GSH, MDA, and NO according to results of the variance analysis (p0.001). Conclusion: Given the change in the levels of GSH and ADA activities and MDA and NO levels observed in patients with parasitic infection, over production of active neutrophils, macrophages, reactive oxygen radicals and reactive nitrogen species may be an indicator of accelerated oxidative stress and lipid peroxidation in these patients. Increased serum ADA activity in the group infected by E. coli might be resulted from fight of the immune system with parasites. Low serum ADA activity in the other parasitic infection groups may be explained by suppression of lymphocyte proliferation by macrophages that were activated by sporozoite antigens in the late period of the infection. Accordingly, determination of the levels of MDA, NO, ADA and GSH may be important in treatment follow up and control of parasitic infections.

Changes of adenosinergic system in piglets fed a diet co-contaminated by mycotoxin and their effects on the regulation of adenosine

The effects of diets co-contaminated with 300 μg/kg of aflatoxins and 8000 μg/kg of fumonisins on adenosinergic system of the pigs weaned at 15 days of age were studied. Piglets were inspected daily, and body weight measurement and blood collections were performed at every five days. Piglets intoxicated by mycotoxins presented lower weight gain (p < 0.001) in comparison to control. Intoxicated piglets also showed a reduction in the serum levels of zinc and adenosine and in adenosine deaminase (ADA) activity (p < 0.001). Positive correlations between zinc levels and ADA activity (p < 0.001) and between adenosine levels and ADA activity (p < 0.05) were observed. Ternary plot shows the influence of zinc levels on ADA activity and on adenosine levels, suggesting that low zinc levels, caused by subclinical mycotoxin intoxication, can cause immunomodulatory effects in piglets. We conclude that piglets intoxicated by fumonisins and aflatoxins have low ADA activity and adenosine levels in serum. This can be directly related to zinc reduction, which is a cofactor for ADA. The co-contamination by these mycotoxins in piglet feed impairs growth and immune defenses of the animals, adversely affecting animal health and production. Therefore, changes in the purinergic pathway may affect the pathogenesis of the disease.

Cattle naturally infected by Eurytrema coelomaticum: Relation between adenosine deaminase activity and zinc levels

The enzyme adenosine deaminase (ADA) is critical for modulating the immune system, and in the presence of zinc, its activity is catalyzed. The aim of this study was to evaluate the ADA activity in pancreas of cattle naturally infected by Eurytrema coelomaticum in relation to the results of zinc levels, pathological findings and parasite load. For this study 51 slaughtered cattle were used. The animals were divided into two groups: Group A consisting of animals naturally infected by E. coelomaticum (n=33) and Group B of uninfected animals (n=18). Blood and pancreas were collected of each animal for analysis of zinc and ADA, respectively. Infected cattle showed a reduction on seric levels of zinc, and decreased ADA activity in the pancreas (P>0.05). A positive correlation between zinc levels and ADA activity was observed. Thus, high parasite load and severity of histopathologic lesions affect the ADA activity in pancreas, as well as the zinc levels in serum of infected animals (negative correlation between these variables). Therefore, we can conclude that cattle infected by E. coelomaticum have low ADA activity in pancreas, which can be directly related to zinc reduction, responsible for ADA activation and catalyzes.

Effects of reduction of adenosine deaminase activity in the serum of dogs naturally infected by Ehrlichia canis and Hepatozoon canis

Ehrlichia canis and Hepatozoon canis have been reported infecting a variety of Carnivora species worldwide, including domestic dogs and wild canids. Since both diseases can follow their course while the infected animals remain asymptomatic, assessments of different parameters that may help toward reaching a more accurate diagnosis are welcome. In this regard, the aim of the present study was to assess the activity of adenosine deaminase (ADA) in serum from naturally infected dogs. Forty-nine samples from dogs were used in our study: 15 positive for E. canis (8 asymptomatic and 7 symptomatic animals); 8 positive for H. canis; 14 with mixed infection; and 12 uninfected (used with control). Our results showed that the ADA activity was lower (P < 0.001) in serum from dogs with both infections singly and with mixed infection, in comparison with the uninfected animals (P < 0.001). The symptomatic dogs for E. canis had lower ADA activity when compared with asymptomatic dogs by H. canis (P < 0.01). Therefore, ADA activity reduces in positive dogs for E. canis and H. canis, which can be related to an inflammatory response against infection.

Further observations on associations between the ADA gene and past malaria morbidity in Sardinia

Adenosine Deaminase (ADA) contributes to the regulation of adenosine concentration and in turn to T cell activation. Genetic variability of ADA activity may have, therefore, an important role in resistance to malaria. Indeed, previous studies in Sardinia have shown a lower frequency of ADA1 *2 allele (associated with low ADA activity) in areas, where malaria was heavily endemic compared to areas where malaria was not endemic. We have now studied the ADA2 locus, another polymorphic site with two alleles ADA2 *1 and ADA2 *2 within the ADA gene. In the area of Oristano (where malaria was endemic in the past) 51 consecutive newborns and in the area of Nuoro (where malaria was not as endemic) 48 consecutive newborns were examined. ADA1 and ADA2 genotypes were determined by DNA analysis. The low frequency of the ADA1 *2 allele in the area where malaria was endemic is confirmed. The frequency of the ADA2 *2 allele is higher in Oristano than in Nuoro resulting in a higher frequency of the ADA1 *1/ADA2 *2 haplotype in Oristano as compared to Nuoro. This suggests a selective advantage of this haplotype in a malarial environment. The ADA gene shows other polymorphic sites further studies on their role in human adaptation to malaria could be rewarding.

ADENOSINE DEAMINASE ACTIVITY IN HIV POSITIVE CASES

Adenosine deaminase (ADA) converts adenosine and deoxyadenosine to toxic metabolites inosine and 2’deoxyinosine. ADA is responsible for the maturation and proper function of cell mediated immunity. Depletion of CD4 T-lymphocytes in HIV infection which is a hallmark makes a pavement for a variety of diseases and malignancies. To ascertain the ADA activity in before ART and after ART treated HIV-positive patients and its variation in comparison to controls within this locality. Total 50 HIV positive subjects and 27 HIV Seronegative healthy individuals were recruited into the study. CD4 counts were estimated to the HIV positive subjects and ADA activity was assessed to both the group individuals. In general the study ‘p’ value of ADA activity demonstrated highly significant when controls vs before treatment (ART) HIV positive subjects and controls vs after treatment (ART) HIV positive subjects were compared. Among untreated HIV positive subjects had significantly higher ADA activity [males (27.7±5.5 U/L vs females (14.6±4.2 U/L), P < 0.0001] than ART treated [males (14.3±3.4 U/L vs females (10.4±2.5 U/L), P < 0.0001]. In HIV infected individuals low ADA activity with low CD4 count is dangerous. HIV Seropositive subjects should be constantly assessed the ADA activity to know the prognosis of the disease and also the status of their immune status more prominently males. Nevertheless ADA activity also guides when to start the ART treatment regimen. Hence helpful in extending the life span of HIV infected individuals to some extent.

Adenosine deaminase activity in tuberculous peritonitis among patients with underlying liver cirrhosis.

To investigate the value of adenosine deaminase (ADA) for early detection of tuberculous peritonitis (TBP) among cirrhotic patients. We retrospectively analyzed 22 patients with TBP from July 1990 to June 2010. Twenty-five cirrhotic patients with uninfected ascites were prospectively enrolled as the cirrhosis control group from July 2010 to June 2011. An additional group of 217 patients whose ascites ADA levels were checked in various clinical conditions were reviewed from July 2008 to June 2010 as the validation group. The mean ascites ADA value of cirrhotic patients with TBP (cirrhotic TBP group, n = 8) was not significantly different from that of non-cirrhotic patients (non-cirrhotic TBP group, n = 14; 58.1 ± 18.8 U/L vs. 70.6 ± 29.8 U/L, P = 0.29), but the mean ascites ADA value of the cirrhotic TBP group was significantly higher than that of the cirrhosis control group (58.1 ± 18.8 U/L vs. 7.0 ± 3.7 U/L, P < 0.001). ADA values were correlated with total protein values (r = 0.909, P < 0.001). Using 27 U/L as the cut-off value of ADA, the sensitivity and specificity were 100% and 93.3%, respectively, for detecting TBP in the validation group. Even with lower ADA activity in ascites among cirrhotic patients, ADA values were significantly elevated during TBP, indicating that ADA can still be a valuable diagnostic tool.

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.

Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2) in schizophrenic patients

Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A) (ADA1*2) exhibits 20-30% lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2) in schizophrenic patients and healthy controls. The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6%) relative to controls (13/111 - 11.7%, p = 0.032, OR = 2.6). These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.

Caspase-4 Activation in Association with Decreased Adenosine Deaminase Activity May Be a Factor for Gastric Ulcer

Background/Aims: Adenosine deaminase (ADA)-deficient disease, a severe combined immunodeficiency, is most commonly associated with gastrointestinal disorders such as ulcer. The present study investigated the role of ADA in the pathogenesis of gastric ulcer. Methods: ADA activity was measured in a variety of organs and tissues from rats and in human gastric biopsy samples from patients who underwent gastrofiberscope. An MTT assay and TUNEL staining were carried out, and activity of caspase-3 and -4 was enzymatically measured in MKN45 human gastric cancer cells. In the Western blot analysis, caspase-4 activation was identified in gastric biopsy samples. Results: In rat organs and tissues, the epithelium of the gastrointestine exhibited higher ADA activity. The ADA inhibitor EHNA reduced cell viability, increased TUNEL-positive cells, and activated caspase-3 and -4 in MKN45 cells. For gastric biopsy samples, much lower ADA activity was found in gastric ulcer tissues, with a tendency of caspase-4 activation. Conclusion: A decline in ADA activity and the ensuing increase in intracellular adenosine concentrations for the stomach could induce gastric epithelial cell apoptosis by activating caspase-4 and the effector caspase-3. This may represent a fresh pathogenetical pathway for gastric ulcer relevant to ADA activity and caspase-4 activation.

Adenosine deaminase and 5′‐nucleotidase activities in saliva from patients with oral and laryngeal cancer

The aim of this study was to evaluate saliva's activities of adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) enzymes and their utility as diagnostic and therapeutic markers in oral and laryngeal cancer. Pre- and post-operative saliva's activities of ADA and 5'-NT enzymes were measured in patients with squamous cell oral (n = 10) and laryngeal cancer (n = 17) and compared with control saliva samples (n = 19). The ADA was found to be lower in saliva of the patients with oral cancer compared with the laryngeal cancer and controls. However, no significant differences were found between pre- and post-operative values for both enzymes in the patient groups. We also could not find statistically significant differences between saliva's activities of 5'-NT in patients and control subjects. Low activity of ADA observed in saliva of the patients with oral cancer has been suggested as a compensatory mechanism against rapid purine and DNA metabolism in cancer cells. The current study does not support the hypothesis that saliva's activities of these enzymes may be used as additional diagnostic and prognostic cancer markers.

Comparative hydrolysis of extracellular adenine nucleotides and adenosine in synaptic membranes from porcine brain cortex, hippocampus, cerebellum and medulla oblongata

We have investigated the metabolism of extracellular adenine nucleotides and adenosine in porcine brain. The cortex synaptic plasma membranes hydrolyzed ATP to ADP, AMP and adenosine. We also observed a slow hydrolysis of adenosine with the concomitant accumulation of inosine. These results indicate that NTPDase1, NTPDase2, ecto-5′-nucleotidase, and adenosine deaminase are present in cortex synaptic membranes from porcine brain. We further showed that all these enzymes are also abundant in synaptic membranes from hippocampus, cerebellum, and medulla oblongata and compared their specific activities. Brain cortex and hippocampus exhibited higher activities of NTPDase1 and NTPDase2 than cerebellum and medulla oblongata. It was consistent with the high level of the expression of NTPDases in the two first structures. Adenosine deaminase activity was found in all brain structures analyzed; however, it was lower than the activity of ecto-nucleotidases. Taken together, our data suggest that investigated enzymes have a ubiquitous abundance in porcine brain, and observed differences in their activities in cortex, hippocampus, cerebellum, and medulla oblongata may correlate with the pattern of P2 receptor expression in these brain areas. In addition, low activity of adenosine deaminase may indicate that nonenzymatic mechanism(s) are responsible for the termination of P1 receptor signaling in porcine brain.

Type 2 diabetes and the genetics of signal transduction: A study of interaction between adenosine deaminase and acid phosphatase locus 1 polymorphisms

Acid phosphatase locus 1 (ACP1) is a highly polymorphic enzyme that has an important role in flavoenzyme activity and in the control of insulin receptor activity and band 3 protein phosphorylation status. Adenosine deaminase (ADA) is a polymorphic enzyme that catalyses the irreversible deamination of adenosine to inosine and has an important role in regulating adenosine concentration. Based on the hypothesis that ACP1 counteracts insulin signaling by dephosphorylating the insulin receptor and that adenosine has an anti-insulin action, we reasoned that low ACP1 activity (low dephosphorylating action on insulin receptor) when associated with high ADA activity (low adenosine concentration) would result in a cumulative effect towards an increased glucose tolerance. On the contrary, high ACP1 activity when associated with low ADA activity would result in a cumulative effect towards a decreased glucose tolerance. A total of 280 adult subjects with type 2 diabetes from the population of Penne (Italy) were studied. There was a nonsignificant trend toward an increase in the proportion of subjects with the complex type with high ACP1 activity and low ADA activity (ie, ∗B/∗B; ∗A/∗C; ∗B/∗C; ∗C/∗C//ADA∗1/∗2 and ∗2/∗2) in type 2 diabetes relative to that observed in newborn infants from the same population. High ACP1 activity/low ADA activity joint genotype was positively associated with high glycemic levels and with high body mass index (BMI) values. Low ACP1 activity/high ADA activity joint genotype was also positively associated with dyslipidemia. These findings suggest that both ACP1 and ADA contribute to the clinical manifestations of type 2 diabetes and probably also have a marginal influence on susceptibility to the disease. Both additive and epistatic interactions between the 2 systems seem to be operative.

Determination of adenosine and deoxyadenosine in urine by high-performance liquid chromatography with column switching

The means of measurement of adenosine and deoxyadenosine in urine was developed by separating adenosine and deoxyadenosine from other compounds using high-performance liquid chromatography with column switchings. This method is simple and convenient since no pretreatment of the urine is needed. Using this method, it could be demonstrated that urinary adenosine was higher in an adenosine deaminase (ADA) deficient patient who had a bone marrow transplant treatment (1.97 μmol/mmol creatinine) and in a heterozygote who had a markedly low erythrocyte ADA activity (1% of control ADA activity) (1.33 μmol/mmol creatinine) as compared to normal subjects (0.22±0.09 μmol/mmol creatinine, n=11). It was also noted that urinary deoxyadenosine was below the detection limits in the ADA-deficient bone marrow transplant patient, but it was detected in the heterozygote (3.7 μmol/mmol creatinine). Furthermore, it was also demonstrated that a fructose infusion increased the urinary concentration of adenosine from 0.21±0.03 to 2.66±1.21 μmol/mmol creatinine in five normal subjects.

Full genetic rescue of adenosine deaminase-deficient mice through introduction of the human gene.

We have shown recently that adenosine deaminase (ADA)-deficient mice die perinatally with severe liver cell degeneration. In addition to enzyme substitution, we report the restoration of viability through introduction of the human ADA gene. The ADA gene is subject to complex developmental and tissue-specific regulation. To include the cis-regulatory elements necessary for correct regulation of the human ADA gene, a large transgenic locus constituting the human ADA gene with 10 kb of 5' and 4 kb of 3' flanking sequences was generated by co-injection of two overlapping DNA fragments into murine zygotes. Probably as a result of extrachromosomal (homologous) recombination between the fragments, one of the two transgenic lines contained a reconstituted, functional human ADA gene. As in man, human ADA expression generally was low in these transgenic mice, but high in the thymus, spleen and gastro-duodenal part of the gut. Apparently, all cis-regulatory elements essential for a human expression pattern were incorporated in the transgene and were functional in the murine background. Similarly to man, the upper alimentary tract of the transgenic mice revealed low human ADA activity in contrast to extremely high levels of murine ADA. The human gene probably lacks the cis-regulatory elements that target high level murine ADA expression to the murine upper alimentary tract. ADA-deficient mice rescued by introduction of the human ADA transgene appeared histologically and immunologically normal. Apparently, human ADA can complement murine ADA in all tissues, even in the epithelium of the upper alimentary tract where human ADA activity is as much as 70-fold lower than murine ADA activity in wild-type mice. Clearly, the lethal phenotype of ADA-deficient mice is due to the absence of ADA.

Adenosine deaminase activity in gastric cancer

Adenosine deaminase activity, the key enzyme of adenosine inactivation, was studied in slices taken endoscopically from gastric cancer and macroscopically unchanged gastric mucosa surrounding the cancer. The activity of the enzyme was measured in mucosa homogenates by determination of ammonia liberated from substrate. It was found that adenosine deaminase activity in neoplastic lesions did not differ significantly from normal mucosa and that the gastric region studied (antrum, corpus) did not have an impact. A significant difference in enzyme activity was noticed between intestinal and diffuse-type gastric carcinoma (according to Lauren's classification); the intestinal type was characterized by lower adenosine deaminase activity than was the diffuse type. Since the activity of adenosine deaminase in gastric cancer did not exhibit significant differences from normal mucosa the diagnostic value of its determination is of less importance.

Adenine nucleotide degradation in cultured chick heart muscle cells

Cultured chick heart muscle cells degrade ATP during metabolic inhibition via ADP to AMP. Whether AMP is primarily deaminated to IMP or dephosphorylated to adenosine depends on the 'metabolic block' (glycolysis vs. oxidative phosphorylation). Inhibition of glycolysis (deoxyglucose) results in an inosine/adenosine ratio greater than 1 in the supernatant, whereas the nucleoside ratio is less than or equal to 1 during inhibition of oxidative phosphorylation (hypoxia, rotenone). EHNA, a blocker of adenosine deaminase, has little effect on inosine release during metabolic inhibition, consistent with the reported low activity of adenosine deaminase in cardiac muscle cells. The amount of adenosine and inosine released can be largely attenuated by two nucleoside carrier inhibitors, nitrobenzyl-thioinosine and dipyridamole, which suggests that nucleosides are produced intracellularly and subsequently released. These results indicate that the amount of inosine or adenosine released from the cardiomyocyte during impaired energy metabolism (e.g. ischemia) can be controlled by the metabolic state of the cell.

44 ADENOSINE ACCUMULATION IN HEMORRHAGIC CEREBROSPINAL FLUID

Although adenosine (Ado) may play a critical regulatory role in brain, little information is available with respect to its metabolism in cerebrospinal fluid (CSF). In normal, cell-free CSF from children of various ages, degradation of 1 μM Ado reached only 0.028 ± 0.010 nmol/h/ml at 25 °C (mean ± SEM for n = 9). Both intact and hemolysed red blood cells (RBC, ~ 50,000/mm3 of test volume) produced no or negligible Ado when incubated in the absence of CSF. Incubation of intact RBC with CSF similarly did not result in Ado accumulation. However, incubation of a hemolysate with CSF resulted in buildup of Ado at rates that were proportional to both amount of hemolysate and of CSF. Accumulation was enhanced in the presence of the adenosine deaminase inhibitor deoxycoformycin (1μM). Further studies showed that CSF contains a 5′-nucleotidase (activity : 7.4 ± 1.8 nmol/h/ml at 25 °C, n = 11) with the kinetic characteristics of an ecto-5′-nucleotidase (high affinity for AMP, inhibition by adenosine 5′-methylene diphosphonate, no stimulation by 2,3-bisphosphoglycerate). It is concluded that Ado may accumulate in hemorrhagic CSF as a result of the combination of red cell hemolysis providing AMP, the presence of a membrane-derived 5′-nucleotidase, and the low activity of adenosine deaminase.

Low value of adenosine deaminase in tuberculous pleural effusions

It has been claimed that a value of adenosine deaminase (ADA) activity lower than 43 U·/·1 excludes tuberculous aetiology of pleural effusion [1, 2]. We report nine cases of tuberculous pleurisy with at least one determination of ADA (by Giusti and Galanti) activity less than 43 U-/·1 , and two other cases with an ADA value below 44 U-/·1 , out of 114 ADA determinations (representing 8% or 10%). The tuberculosis diagnosis was confirmed by bacteriological cultures of pleural fluid or biopsy material and the pathological finding of a granulomatous lesion in the pleural tissue. In the literature we have found eighteen cases of tuberculous pleurisy with an ADA activity value <43 U·/·1 [35]; however, among the largest series of ADA and tuberculosis no cases with a value of ADA activity <43 U-/·1 were described [1, 2, 6] . In five of the nine cases reported with low ADA activity values, a second determination a few days later evidenced a rise in the ADA activity over 43 U·/'1 to values well within the range of tuberculous effusion (fig. 1). In another two cases a second ADA determination six and eight days later, respectively, still showed a value below 43 U·/·1. In the last two cases only one determination was obtained.