Adenosine Deaminase Enzyme Activity Research Articles

Targeting ADA and HIV-1 Nef Protein with Protease Inhibitors: A Repurposing Strategy through Molecular Networking and an In silico Approach for Integrated Management of HIV Co-infected with Abdominal Tuberculosis

Background: Background: The co-infection of HIV and abdominal TB poses a worldwide danger to humanity. This is because there are more strains of bacteria that are resistant to many classes of currently available medications. According to current findings, repurposing existing available medications will result in more effective functioning than using newly designed medications. Objective: Based on this fact, we hypothesised that the PI could be repurposed; we used Food and Drug Administration (FDA)-approved PI drugs to treat HIV co-infected patients with abdominal TB, and a computational study has been conducted. Methods: This comprises network analysis models to find their protein drug interaction (PDI) through a search tool for interacting chemicals (STICH) module of Cytoscape network analysis model followed by the screening of these drugs for their ADMET prediction and binding affinity with adenosine deaminase (ADA), a protein responsible for abdominal TB, and the HIV-1 Nef protein, responsible for the regulation of immune function (CD4+). Results: The network analysis showed 13 nearest binding drugs of these proteins of interest. The ADMET study result showed the pharmacologically relevant parameters that have a significant effect on the binding affinity, bioavailability, and toxicity of PI. The top three scores achieved by PI against adenosine deaminase enzyme activity (PDB ID: 1A4M) are viz., -23.7919, -23.3529, and -22.6773 for Ritonavir, Tipranavir, and Atazanavir, respectively. The top three scores achieved by PI against HIV-1 Nef protein activity (PDB ID: 6URI) are viz., -28.7321, -28.4987, and -28.3155 for Atazanavir, Tipranavir, and Simeprevir, respectively. The active site of ADA and HIV-1 Nef proteins comprises amino acid residues such as for Tipranavir: Arene-Cation interaction (Phenyl and Pyridine)- Arg B1081, and Lys B1033 (1A4M) and Arene-Cation interaction (Pyridine and Phenyl)- Lys D11 and Arg D33; Sidechain acceptor Thr B40; Sidechain donor- Asp D30; Backbone donor- Ala B37 (6URI). Atazanavir: Arene-Cation interaction (Phenyl)- Lys A254 and Lys B1033; Sidechain acceptor - Arg A251 (1A4M). Conclusion: Thus, from the computational studies carried out, we could obtain hints for optimising the molecular selectivity of the PI to provide help in the design of new compounds via the repurposing strategy of the FDA-approved PI for effective treatment of co-morbidity with HIV and abdominal TB. However, further pharmacokinetics, pharmacodynamics, preclinical, and clinical studies permit the design of the new agents without undesirable interactions.

Investigation of serum neopterin levels and adenosine deaminase enzymatic activity in measles infection

The aim of this study was to investigate the serum neopterin level and ADA activity in acute measles infection and determine whether there is a correlation between the measles optical density values, and the neopterin and ADA levels. The neopterin level and ADA activities were investigated in the samples of 136 measles IgM-positive patients along with those of 40 measles IgM-negative patients as the control group. The most important findings of the study was the determination of significantly higher neopterin levels and ADA activity in the measles IgM-positive group when compared to the measles IgM-negative group. These findings have shown that the neopterin level and ADA activity can assist in the diagnosis of measles in the acute period as biomarkers.

Adenosine Deaminase as Inflammatory Marker in Type II Diabetes Mellitus

Objective: To evaluate the enzymatic activity of Adenosine Deaminase in type II diabetes mellitus (T2DM).Methods: This study was conducted on 60 clinically diagnosed type II diabetes mellitus patients, with 60 healthy subjects as the control group. Subjects were enrolled in the study only after their written consent was obtained. The inclusion of diabetes mellitus cases (DM) was conducted as per the WHO guidelines. Estimation of enzymatic activity of serum ADA was performed by Kinetic method using a commercial kit.Result: The observed serum ADA activity in DM patients was 48.34 ± 21.05 U/L, which was significantly higher in comparison to healthy controls (25.02 ± 5.78 U/L). The serum activity raised in about 80% of patients and they had higher values above the reference activity of 30 U/L. The increased activity of ADA among the diabetic subjects indicates inflammatory changes in these individuals.Conclusion: It is possible that in the coming years, a new therapeutic strategy based on anti-inflammatory properties with beneficial effects on diabetic complications can be translated into real clinical treatments.

Elevated ADA2 Enzyme Activity at the Onset of Chronic Graft-versus-Host Disease in Children

Adenosinergic signaling has potent, context-specific effects on immune cells, particularly on the dysregulation of lymphocytes. This in turn may have a role in immune activation and loss of tolerance in such diseases as chronic graft-versus-host disease (chronic GVHD). We assessed whether changes in the enzymatic activity of adenosine deaminase 2 (ADA2), an enzyme that depletes adenosine in the extracellular space via conversion to inosine, may be associated with the onset of chronic GVHD. ADA2-specific enzyme activity was measured in plasma samples from 230 pediatric hematopoietic stem cell transplantation (HSCT) recipients enrolled on the Applied Biomarkers of Late Effects of Childhood Cancer (ABLE)/Pediatric Blood and Marrow Transplant Consortium (PBMTC) 1202 study and compared between patients developing chronic GVHD and those not developing chronic GVHD within 12 months of transplantation. ADA2 and its relationships with 219 previously measured plasma-soluble proteins, metabolites, and immune cell populations were evaluated as well. Plasma ADA2 enzyme activity was significantly elevated in pediatric HSCT recipients at the onset of chronic GVHD compared to patients without chronic GVHD and was not associated with prior history of acute GVHD or generalized inflammation as measured by C-reactive protein concentration. ADA2-specific enzyme activity met our criteria as a potential diagnostic biomarker of chronic GVHD (effect ratio ≥1.30 or ≤.75; area under the receiver operating characteristic curve ≥.60; P < .05) and was positively associated with markers of immune activation previously identified in pediatric chronic GVHD patients. These results support the potential of ADA2 enzyme activity, in combination with other biomarkers and subject to future validation, to aid the diagnosis of chronic GVHD in children post-HSCT.

Nanomolar clodronate induces adenosine accumulation in the perfused rat mesenteric bed and mesentery-derived endothelial cells.

The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5'-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01-1,000nM clodronate, 1-1,000nM Evans blue (EB), and 1-10µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1-1,000nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1-1,000nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion.

Abstract C062: Metabolic rewiring: A role for adenosine-inosine axis in African-American prostate cancer

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer (PCa) compared to European-American (EA) men. An understanding of the altered biological responses and the factors that lead to health disparity in PCa is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be crucial for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across PCa/benign adjacent tissue pairs from ancestry typed AA and EA men done in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio was observed in AA men compared to EA men. In line with this, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. This study will address the knowledge gap on the effects adenosine-inosine alterations in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in AA(MDA-PCa-2A) and EA PCa (LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed high ADA enzyme activity decreases the cell-ECM adhesion. Molecular analyses revealed that upon ADA OE there is a reduction in Tenascin C(TNC), a matrix protein known for its anti-adhesive properties. TNC-rich microenvironment facilitates reactive stromal induction and high motility. This TNC-mediated reprogramming enhances the metastatic ability and PCa progression in bone. TNC induction was observed in stromal cells (DPT44) upon treatment with ADA OE conditioned media and inosine addition. PCa progression on bone was assessed using a cancer cell-bone in vitro co-culture system. High inosine and adenosine levels enabled better attachment of PCa cells to the bone. These findings suggest that an altered adenosine/inosine axis could facilitate adhesion decrease and modulate the stromal microenvironment to aid in the metastatic dissemination and migration towards the bone. Adenosine-inosine alterations could serve as a biomarker for metastatic PCa. We will further analyze the inosine and adenosine levels in patient samples and correlate them with various clinical outcomes in PCa. Our study so far shows high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the metabolite levels with (i)Biochemical recurrence (BCr) (ii)Time to attaining castration resistance (iii)Time to metastasis (iv)Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect the inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, the enzyme ADA could serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles. Metabolic rewiring: A role for adenosine-inosine axis in African-American prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C062.

Adenosine deaminase, not immune to a mechanistic rethink in central nervous system disorders?

Adenosine deaminase (ADA) is a purine metabolism enzyme that catalyses the breakdown of adenosine and deoxyadenosine. The enzyme is important in several cellular processes, including the innate immune response and cellular differentiation, and it is also an important enzyme for the maintenance of brain homeostasis, in part due to its regulation of adenosine. Aberrant regulation of ADA enzyme activity has been linked to several neurodegenerative diseases and diseases that can result in neurological impairment. However, the mechanisms behind altered ADA regulation and how this leads to the development of neurological dysfunction are poorly characterised. This review summarises the current research on ADA and its role and regulation in disease pathology, with a focus on the central nervous system (CNS) and the neurodegenerative disease, amyotrophic lateral sclerosis (ALS).

Quercetin mitigates rheumatoid arthritis by inhibiting adenosine deaminase in rats

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and bone destruction. Adenosine deaminase (ADA) is a key inflammatory enzyme that increases joint stiffness and pain in RA. In this study, we evaluated the in-silico, and in vivo inhibitory effect of quercetin isolated from Egyptian Fenugreek on ADA enzyme activity. We also determined the combinatorial effect of quercetin on methotrexate mediated anti-inflammatory efficacy and toxicity. In-silico molecular docking was conducted and confirmed in an in vivo RA rat model. The results showed that the inhibition constant of quercetin on joint ADA by docking and in-vitro was 61.9 and 55.5 mM, respectively. Therefore, quercetin exhibits anti-inflammatory effect in a rat RA model as evidenced by reducing the specific activity of ADA in joint tissues, lower jaw volume, enhance body weight, downregulate ADA gene expression, reduce levels of RA cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, also, rheumatoid factor, C-reactive protein, and anti-cyclic citrullinated peptide RA biomarker levels. These findings demonstrate that the purified quercetin has a promising anti-inflammatory effect against RA disease through its inhibitory effects on the ADA enzyme. Furthermore, isolated quercetin improved the anti-inflammatory efficacy of methotrexate, reduced its toxic effects by increasing antioxidant enzymes and reducing oxidative stress.

Adenosine deaminase gene variant in diabetes and obesity.

Personal medicine is a new notion for individualizing treatment in the future. Studying pathogenic markers including genetic variants would be beneficial in better diagnosis and management of complex diseases such as diabetes and obesity. Adenosine deaminase (ADA) is a purine metabolic enzyme and modulates insulin activity in various tissues through several different mechanisms. Increased ADA activity is associated with decreased glucose uptake. A significant increase in serum deaminase activity has been reported in patients with T2DM and obesity. ADA gene polymorphisms seem to affect ADA enzymatic activity and a polymorphism at the position 4223 in the first intron of ADA gene (ADA 4223 A/C) has been previously associated with obesity. The aim of this study was to explore ADA gene 4223 A/C polymorphism and its association with obesity in patients with Type 2 diabetes. Obese patients (N = 133: 64 diabetic +69 non-diabetic) with BMI ≥ 30 and subjects with BMI < 30 (N = 152: 83 diabetics +69 non-diabetic) were recruited into a case-control association study. Blood samples were collected and after DNA extraction, the allele and genotype frequency for ADA gene polymorphism was determined using PCR-RFLP technique. We observed a significant increase for the frequency of AA+CA genotype in non-obese patients with diabetes compared to obese patients with diabetes (P = 0.04, OR = 2.1, 95%CI; 0.93-4.9). The higher frequency of AA+CA genotype in none obese diabetes individuals and lower frequency of this genotype in obese diabetes subjects indicates an important role for ADA gene polymorphism in diabetes subjects without obesity.

Abstract PO-152: Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The effects of these metabolic alterations on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. Molecular analyses revealed a significant reduction in Tenascin C(TNC), a matrix protein known for its anti-adhesive properties. TNC is critical for modulating cell motility and adhesion. TNC is also a marker for the reactive stromal microenvironment. Therefore, we evaluated the effects of altered adenosine-inosine levels on stromal cells. TNC induction was observed in stromal cells upon treatment with ADA OE conditioned media and inosine addition. These molecular findings suggest that high ADA activity could facilitate adhesion decrease and modulate the stromal microenvironment to aid in the metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i)Biochemical Recurrence(BCr) (ii)Time to attaining castration resistance in castration-sensitive patients (iii)Time to metastasis (iv)Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect the inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles. Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-152.

Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells.

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2′-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Abstract PR17: Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. This adhesion decrease is facilitated by a decrease in cell adhesion protein integrin β1 (ITGB1). Elevated ADA enzyme activity decreases cAMP which mediates this adhesion decrease by disrupting the Epac-Rap1 pathway. It is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes this decrease in cAMP levels. These molecular findings suggest that ADA-mediated adhesion decrease could facilitate metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels (ADA enzyme activity) in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i) Biochemical Recurrence (ii) Time to attaining castration resistance in castration sensitive patients (iii) Time to metastasis (iv) Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Brian Simons, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR17.

Adenosine deaminase 2 activity negatively correlates with age during childhood

BackgroundHuman adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered ADA2 enzyme activity has been associated with some viral infections and rheumatic diseases. The potential utility of ADA2 as a biomarker is, however, limited by the absence of established ranges of ADA2 concentration and enzyme activity in the healthy population. It is known that ADA2 enzyme activity is lower in adults, but when (and why) this decline happens is not known. The purpose of this study was to establish normative ranges of ADA2 enzyme activity and protein concentration in the healthy pediatric population.MethodsWe modified a commercially available ADA2 enzyme activity assay to enable higher throughput analysis of fresh, frozen and hemolyzed blood samples. With this assay and ADA2 protein immunoblotting, we analyzed ADA2 enzyme activity and protein concentration in blood plasma from a cohort of children and adolescents (n = 94) aged 5 months to 18 years. One-way ANOVA with subsequent Tukey multiple comparison test was used to analyze group differences. Reference intervals were generated using the central 95% of the population (2–97.5 percentiles).ResultsADA2 enzyme activity was consistent in fresh, frozen, and hemolyzed sera and plasma as measured by our modified assay. Analysis of plasma samples from the healthy pediatric cohort revealed that ADA2 enzyme activity is significantly lower in older children than in younger children (p < 0.0001). In contrast, there was no significant correlation between ADA2 protein concentration and either age or ADA2 enzyme activity.ConclusionWe observed that ADA2 enzyme activity, but not ADA2 protein concentration, negatively correlates with age in a cohort of children and adolescents. Our findings stress the importance of appropriate age-matched controls for assessing ADA2 enzyme activity in the clinical setting.

Evaluation of adenosine deaminase activity in serum of cattle and buffaloes in the diagnosis of bovine tuberculosis

Background and Aim:Bovine tuberculosis (bTB) is a chronic bacterial disease of cattle caused by Mycobacterium bovis. bTB causes severe economic losses resulting from livestock deaths, chronic disease, and trade restrictions. Determination of serum levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human TB. This study aimed to evaluate the role of ADA enzyme activity in the diagnosis of bTB.Materials and Methods:In this study, a total of 100 animals (cattle and buffaloes) were screened for bTB by comparative intradermal tuberculin test (CITT) and interferon-γ (IFN-γ) test and in serum samples obtained from 100 screened animals, ADA seric activity was evaluated using ADA-MTB kit procured from Tulip Diagnostics.Results:A total of 18 animals were positive TB reactors by CITT, 8 were positive by IFN-γ, and 4 animals were positive by both CITT and IFN-γ. The average ADA value of bTB-positive animals either by CITT, IFN-γ, or both CITT and IFN-γ was 12.55 U/L, 14.8 U/L, and 18.36 U/L, respectively, in CID negative, it was 10.57 U/L and in IFN-γ negative, it was 10.59 U/L.Conclusion:The average ADA value of bTB-positive animals positive either by CITT, IFN-γ, or both CITT and IFN-γ was more than the average ADA value in animals negative for bTB by either of the tests.

ADA Deficiency: Evaluation of the Clinical and Laboratory Features and the Outcome

Adenosine deaminase (ADA) deficiency is an autosomal recessive primary immunodeficiency. It results in the intracellular accumulation of toxic metabolites which have effects particularly on lymphocytes and the brain. The aim of this study was to evaluate the outcome of 13 ADA-deficient patients. We planned to evaluate their clinical and laboratory findings before and after enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (aHSCT), and hematopoietic stem cell gene therapy (HSCGT). Measurement of ADA enzyme activity and metabolites and sequencing of the ADA gene were performed in most of the patients with ADA deficiency. One of the patients with late-onset ADA deficiency was diagnosed by the help of primary immunodeficiency panel screening. Ten out of 13 patients were diagnosed as SCID, while 3 out of 13 were diagnosed as delayed-/late-onset ADA deficiency. Late-onset ADA deficiency patients had clinical and laboratory findings of combined immunodeficiency (CID). Eight patients with ADA-SCID were found to have higher levels of ADA metabolite (dAXP%) (62.1% (34.6-71.9)) than 3 patients with delayed-/late-onset ADA deficiency (6.9% (2.1-8.9). All but one patient with SCID had T-B-NK- phenotype, one had T-B-NK+ phenotype. Genetic defect was documented in 11 patients. Four out of 11 patients had compound heterozygous defects. Three out of 4 patients with compound heterozygous defects had delayed-onset/late-onset ADA deficiency. Seven out of 11 patients with SCID had homozygous defects. Five out of 7 had the same homozygous indel frameshift mutation (c.955-959delGAAGA) showing a founder effect. There were two novel splice site defects: one (IVS10+2T>C) was heterozygous in a patient with late-onset ADA deficiency, and the other was homozygous (IVS2delT+2) in a SCID patient. Other defects were missense defects. Nine out of 13 patients were put on pegylated ADA ERT. Four out of six patients were transplanted without using a conditioning regimen. HSCGT was performed to one of the patients. The genetic diagnosis of SCID is utmost important. There is a chance to give ERT before the definitive therapy if the patient with SCID/CID has ADA deficiency. Although ERT was insufficient to restore a normal immune function in ADA-SCID patients, it was useful to improve and stabilize the clinical status before curative therapy (aHSCT/HSCGT). Enzyme replacement therapy was successful in patients with late-/delayed-onset ADA deficiency who presented with the features of combined immunodeficiency. Gastrointestinal polyposis in a patient with late-onset ADA deficiency may be an association or a coincidental finding. Intermittent neurodevelopmental evaluation especially for hearing impairment should be performed in most of the ADA-deficient patients. This may alleviate the speech delay and cognitive abnormalities which may be observed in the follow-up.

Adenosine deaminase (ADA) activity and isozymes in the serum of patients with hepatitis B compared with healthy people: a useful method in diagnosis clinical status

Background: Adenosine deaminase (ADA) specifications (EC3.5.4.4) is an enzyme is involved in purine metabolism that breaks adenosine and deoxy-adenosine and produce inosine and deoxy-inosine and ammonia. The highest levels are of adenosine deaminase activity in monocytes and lymphocytes. High serum ADA activity with increased serum levels of AST, ALT and immunoglobulins were reported in variety of diseases including hepatitis. We aimed to investigate the activity of total ADA in serum of patients with hepatitis B and were determined molecular weight ADA1 and ADA2 isozymes in serum and RBC of hepatitis patients. Materials and Methods: We were defining experiments by electrophoresis on SDS-PAGE, for isozymes of ADA; ADA1 and ADA2 in serum and red blood cells. ADA1 molecular weight was estimated at about 35 KDa and ADA2 about 110 KDa. The total ADA activity was measured by the modified Ellis method in 37 patients with hepatitis B and 40 healthy controls in the age range (20-60 years). Results: The normal values for serum ADA in humans have been studied by various workers and found in serum samples to be in the range of 0-15 U/L, whiles in our analysis total ADA (tADA) enzyme activity is in controls and patients with hepatitis B, respectively, 13.35 ± 1.62 and 27.05 ± 8.49. Our results indicated that tADA level was higher in patients with hepatitis B than those of corresponding controls (P < 0.05).Total ADA enzyme activity shows a significant increase compared to the control group in all age groups tested. Conclusion: Therefore, the serum ADA level could be used as an index along with other parameters in follow up of patients with hepatitis B.

Adenosine DeAminase (ADA) as an adjuvant molecule for human HIV-1 vaccine

Abstract Follicular helper T cells (Tfh) play critical role in shaping, instructing, and initiating T-cell dependent antibody responses. Understanding the underlying mechanisms that enhance their function is therefore critical for vaccine development. Using a unique gene array analysis, we have identified adenosine deaminase (ADA), as a novel key molecule that drives Tfh helper program in proliferating Germinal Centers Tfh (GC Tfh) and circulatory Tfh (cTfh) cells following their interactions with B cells. In fact, our gene array analysis showed that CD26 and ADA were exclusively up-regulated within the less efficient cTfh1 (CD4+CD45RA−CXCR5+CXCR3+) and cTfh2-17 (CD4+CD45RA−CXCR5+CXCR3+CCR6+/−) subsets, respectively. ADA enzymatic activity is significantly higher, as well, in cTfh2-17 than in the less-efficient cTfh1 cells. Exogenous ADA enhances the ability of Tfh cells to provide B cell help while inhibition of ADA activity by specific inhibitors impeded Tfh function and blunted antibody response. Our results further demonstrated that enhancement of Tfh function by ADA pathway could be due to increase in IL-6 and decrease in IL-2 production in the co-culture, and maintenance of low extracellular expression of CD26. Moreover, blocking IL-2 in cTfh2-17 co-culture from virally suppressed HIV subjects (ST) showed a significant decrease of CD26 expression associated with a rescued helper capacity. Finally, in vivo use of recombinant ADA as an adjuvant in a DNA based HIV vaccine enhanced Tfh cells differentiation and enhanced anti-Env humoral response and isotype class-switch. Thus, ADA activity fine-tunes Tfh helper program and deciphering how ADA regulate the function of Tfh subsets would benefit future vaccine adjuvant design.

The Organogermanium Compound Ge-132 Interacts with Nucleic Acid Components and Inhibits the Catalysis of Adenosine Substrate by Adenosine Deaminase.

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2'-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.

Effects of purine nucleotide administration on purine nucleotide metabolism in brains of heroin-dependent rats

Heroin is known to enhance catabolism and inhibit anabolism of purine nucleotides, leading to purine nucleotide deficiencies in rat brains. Here, we determined the effect of exogenous purine nucleotide administration on purine nucleotide metabolism in the brains of heroin-dependent rats. Heroin was administrated in increasing doses for 9 consecutive days to induce addiction, and the biochemical changes associated with heroin and purine nucleotide administration were compared among the treated groups. HPLC was performed to detect the absolute concentrations of purine nucleotides in the rat brain cortices. The enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in the treated rat cortices were analyzed, and qRT-PCR was performed to determine the relative expression of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT), and adenosine kinase (AK). Heroin increased the enzymatic activity of ADA and XO, and up-regulated the transcription of ADA and XO. Alternatively, heroin decreased the transcription of AK, APRT, and HGPRT in the rat cortices. Furthermore, purine nucleotide administration alleviated the effect of heroin on purine nucleotide content, activity of essential purine nucleotide metabolic enzymes, and transcript levels of these genes. Our findings therefore represent a novel, putative approach to the treatment of heroin addiction.

Chronic alcohol administration affects purine nucleotide catabolism in vivo

AimsTo investigate the relationship between chronic alcohol administration and purine nucleotide metabolism in vivo. Main methodsRat models of alcohol dependence and withdrawal were used. The concentrations of uric acid (UAC), urea nitrogen (UREA), creatinine (CREA), and beta-2-microglobulin (β2-M) and creatinine clearance rate (CCR) in plasma were measured. The PLC method was used to detect the absolute content of purine nucleotides in different tissues. Enzymatic activities of adenosine deaminase (ADA), xanthine oxidase (XO), ribose 5-phosphate pyrophosphokinase (RPPPK), glutamine phosphoribosylpyrophosphate amidotransferase (GPRPPAT), hypoxanthine-guanine phosphate ribose transferase (HGPRT), and adenine phosphoribosyltransferase (APRT) in the tissues were analyzed. Real-time PCR was used to determine the relative level of ADA and XO. Key findingsThe renal function of rats with alcohol dependence was normal. Further, the content of purine nucleotides (GMP, AMP, GTP, and ATP) in tissues of the rats was decreased, which indicated that the increased uric acid should be derived from the decomposition of nucleotides in vivo. The activity of XO and ADA increased, and their mRNA expression was enhanced in the alcohol dependence group, but there was no significant difference in the activity of RPPPK and GPRPPAT in the liver, small intestine, and muscle; furthermore, no significant difference in the activity of HGPRT and APRT was observed in the brain. SignificanceThese results indicate that chronic alcohol administration might enhance the catabolism of purine nucleotides in tissues by inducing gene expression of ADA and XO, leading to elevation of plasma uric acid levels.