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.     

Adenosine deaminase activity of chicken erythrocyte and heart cytosols

1. The adenosine deaminase (ADA) activities of chicken erythrocyte and heart cytosols had pH optima of 6.5. The temperature optima for erythrocyte and heart ADA were 30 and 35 degrees C, respectively. 2. The deoxyadenosine/adenosine deamination ratios ranged from 0.75 to 0.84 for both ADA activities. 3. For erythrocyte ADA, Km values were 8.9-12.9 microM adenosine (range) and 8.3 microM 2'-deoxyadenosine. For heart ADA, Km values were 6.7-12.0 microM adenosine (range) and 5.3 microM 2'-deoxyadenosine. 4. Inosine was a competitive inhibitor of both erythrocyte (Ki = 73 microM) and heart (Ki = 109 microM) ADA.     

Separation of human from mouse and monkey adenosine deaminase by ion-exchange chromatography following retroviral-mediated gene transfer

A method for the chromatographic separation of human adenosine deaminase (ADA) from murine and monkey ADA is described. This procedure was developed in order to detect the expression of low or moderate levels of human ADA following retroviral-mediated gene transfer of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation was achieved on a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein liquid chromatography system and was found to be a highly reproducible method yielding enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KCl), while murine ADA elutes at 0.12 M KCl and monkey ADA at 0.15 M KCl. The column fractions were assayed for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and reproducible separation of human ADA from that of other species and yields partially purified enzymatically active protein.     

Molecular basis for paradoxical carriers of adenosine deaminase (ADA) deficiency that show extremely low levels of ADA activity in peripheral blood cells without immunodeficiency

Adenosine deaminase (ADA) deficiency causes an autosomal recessive form of severe combined immunodeficiency and also less severe phenotypes, depending to a large degree on genotype. In general, ADA activity in cells of carriers is approximately half-normal. Unexpectedly, healthy first-degree relatives of two unrelated ADA-deficient severe combined immunodeficient patients (mother and brother in family I; mother in family II) had only 1-2% of normal ADA activity in PBMC, lower than has previously been found in PBMC of healthy individuals with so-called "partial ADA deficiency." The level of deoxyadenosine nucleotides in erythrocytes of these paradoxical carriers was slightly elevated, but much lower than levels found in immunodeficient patients with ADA deficiency. ADA activity in EBV-lymphoblastoid cell lines (LCL) and T cell lines established from these carriers was 10-20% of normal. Each of these carriers possessed two mutated ADA alleles. Expression of cloned mutant ADA cDNAs in an ADA-deletion strain of Escherichia coli indicated that the novel mutations G239S and M310T were responsible for the residual ADA activity. ADA activity in EBV-LCL extracts of the paradoxical carriers was much more labile than ADA from normal EBV-LCL. Immunoblotting suggested that this lability was due to denaturation rather than to degradation of the mutant protein. These results further define the threshold level of ADA activity necessary for sustaining immune function.     

Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (-)-erythro-9-(2-hydroxy-3-nonyl)adenine.

The enantiomers of erythro-9-(2-hydroxy-3-nonyl)adenine [(+)- and (-)-EHNA) bound to adenosine deaminase (ADA) at pH 7 with concomitant changes in the optical properties of the enzyme. The association rate constant for (+)-EHNA was 2.9 x 10(6) M-1 s-1 and that for (-)-EHNA was 6.4 x 10(6) M-1 s-1. The dissociation of (-)-EHNA.ADA or (+)-EHNA.ADA in the presence of excess coformycin was monitored by the quenching of enzyme fluorescence as coformycin.ADA was formed. The dissociation rate constants of (+)- and (-)-EHNA.ADA were 0.0054 s-1 and 2.7 s-1, respectively. A similar value for the dissociation rate constant (0.005 s-1) for (+)-EHNA.ADA was calculated from the time course for the appearance of catalytic activity after dilution of (+)-EHNA.ADA into 100 microM adenosine. The Ki values of ADA for (+)- and (-)-EHNA were similar to the dissociation constants calculated from the ratio of the respective dissociation and association rate constants. The biphasic time-dependent inhibition of the catalytic activity of ADA by (+/- )-EHNA [Frieden, C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] was confirmed. However, the catalytic activity of ADA was inhibited monophasically by (+)-EHNA. Thus, the biphasic nature of the time course for inhibition of ADA by (+/- )-EHNA was the result of the presence of both enantiomers of the inhibitor in this assay. These kinetic data were interpreted in terms of single-step mechanisms for binding of (+)- and (-)-EHNA.     

Molecular forms of adenosine deaminase in pleural effusions

The molecular forms of adenosine deaminase (ADA) were studied in pleural effusions with high ADA activity. The molecular forms were separated by polyacrylamide gel electrophoresis (PAGE), and the molecular masses estimated by gel filtration. Effusions investigated were: tuberculosis (TB) (20 cases), lymphoma (3 cases), chronic myelogenous leukemia (1 case) and empyema (6 cases). Two ADA forms were identified, a small form (Smf-ADA) and a large form (Lmf-ADA). Without exception, the tuberculous effusions have shown only Lmf-ADA. All the other effusions contained both forms, the Smf-ADA being predominant. This was also the ADA pattern seen in normal lymphocytes. These findings may indicate different mechanisms of ADA release or origins of ADA in the various effusions. The Lmf-ADA may be secreted by activated T cells in TB, which would confirm the notion that ADA activity reflects cellular immunity. In contrast, in the nontuberculous cases the ADA probably leaked from damaged lymphocytes or neutrophils, hence the reflection of the cellular ADA pattern. The PAGE pattern may also be of value in distinguishing between TB and these other causes of high pleural fluid ADA.     

腺苷脱氨酶活性对自身免疫性肝病的临床意义研究

摘要 目的：探讨腺苷脱氨酶（ADA）在自身免疫性肝病患者血清中的变化及其临床意义。方法：利用酶法试剂盒检测自身免疫性肝病患者血清中的总ADA（tADA）及其同工酶ADA1和ADA2的活性变化,利用受试者工作曲线（ROC）分析总ADA、ADA1及ADA2活性的诊断价值。利用Spearman相关性分析自身免疫性肝病患者各指标之间的相关性。结果：与对照组相比,tADA和ADA2活性在自身免疫性肝病患者血清中均极显著升高（P<0.001）,ADA1活性有一定程度升高（P=0.035）。不同自身免疫性肝病亚型患者之间的血清tADA、ADA1及ADA2活性均无显著差异。ROC分析显示,血清tADA和ADA2活性具有诊断价值,ADA1活性无诊断价值。tADA活性截断值取13.5 U/L时,诊断特异性为93.3%,敏感性为81.2%。血清ADA2活性截断值取9.5 U/L时,诊断特异性为85.0%,敏感性为83.3%。而ADA1无显著的诊断价值,ADA活性与白蛋白球蛋白比值呈显著负相关（r=-0.41,P=0.004）,与球蛋白水平具有一定程度的正相关（r=0.34,P=0.018）,与ALT呈弱正相关（r=0.29,P=0.042）,与其他指标的相关性均无统计学显著性。结论：血清tADA及ADA2活性在自身免疫性肝病患者血清中显著升高,并且具有一定的诊断价值。     

The immunogenicity to the first anti-TNF therapy determines the outcome of switching to a second anti-TNF therapy in spondyloarthritis patients

Introduction

Anti-TNF drugs have proven to be effective against spondyloarthritis (SpA), although 30% of patients fail to respond or experience adverse events leading to treatment discontinuation. In rheumatoid arthritis, the presence of anti-drug antibodies (ADA) against the first TNF inhibitor influences the outcome after switching. Our aim was to assess whether the response to a second anti-TNF drug is related to the previous development of ADA to the first anti-TNF drug SpA patients.

Methods

Forty-two SpA patients began a second anti-TNF drug after failing to respond to the first anti-TNF therapy. Clinical activity was assessed by the Ankylosing Spondylitis Disease Activity Score (ASDAS) at baseline (at the beginning of the first and second anti-TNF therapy) and at 6 months after switching. The drug and ADA levels were measured by ELISA before each administration.

Results

All patients were treated with anti-TNF drugs and mainly due to inefficacy were switched to a second anti-TNF drug. Eleven of 42 (26.2%) developed ADA during the first biologic treatment. At baseline, no differences in ASDAS were found in patients with or without ADA to the first anti-TNF drug (3.52 ± 1.03 without ADA vs. 3.14 ± 0.95 with ADA, p = 0.399) and to the second anti-TNF drug (3.36 ± 0.94 without ADA vs. 3.09 ± 0.91 with ADA, p = 0.466). At 6 months after switching, patients with previous ADA had lower disease activity (1.62 ± 0.93 with ADA vs. 2.79 ± 1.01 without ADA, p = 0.002) and most patients without ADA had high disease activity state by the ASDAS (25 out of 31 (80.6%) without ADA vs. 3 out of 11 (27.3%) with ADA, p = 0.002).

Conclusions

In SpA the failure to respond to the first anti-TNF drug due to the presence of ADA predicts a better clinical response to a second anti-TNF drug.     

Dietary regulation of adenosine deaminase activity in stomach, small intestine and spleen of mice

Activity of adenosine deaminase (ADA) and its regulation by dietary restriction were studied in the stomach, small intestine and spleen of mice. ADA activity (U/mg protein) was highest in the stomach, followed by small intestine and spleen of mice on normal diet. The activity decreased significantly in the stomach (41%) and small intestine (45%) of 24 hr fasted mice, when compared to mice fed ad-libitum. However, ADA activity in spleen did not show any change by dietary intervention. Refeeding of fasted mice for 24 hr restored the activity of ADA in tissues. In addition, dietary restriction (alternate days of feeding for three months) had a cumulative effect, whereby ADA activity decreased significantly in the stomach (53% on the day of feeding and 60% on the day of fasting) and small intestine (50% and 54% on the day of feeding and fasting, respectively) without any change in activity in spleen. These findings indicate that dietary restriction reduces ADA activity in a tissue-specific manner. Long-term dietary restriction leads to a cumulative adaptation in lowering the ADA activity of GIT, but not in spleen.     

ADA2 isoform of adenosine deaminase from pleural fluid

Adenosine deaminase isoenzyme 2 (ADA2) was isolated from human pleural fluid for the first time. Molecular and kinetic properties were characterized. It was shown that the inhibitors of adenosine deaminase isoenzyme 1 (ADA1), adenosine, and erithro-9-(2-hydroxy-3-nonyl)adenine (EHNA) derivatives are poor inhibitors of ADA2. Comparison of the interaction of ADA2 and ADA1 with adenosine and its derivative, 1-deazaadenosine, indicates that the isoenzymes have similar active centers. The absence of ADA2 inhibition by EHNA is evidence of a difference of these active centers in a close environment. The possible role of Zn2+ ions and the participation of acidic amino acids Glu and Asp in adenosine deamination catalyzed by ADA2 were shown.     

Rat Brain Adenosine Deaminase After 2''-Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2''-[3H]Deoxycoformycin Ligand Binding

Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of benzene on the activity of adenosine deaminase in tissues of rats

Swiss Albino (Rat rattus norvegicus) rats were intraperitoneally injected with a 100 mg kg(-1) dosage of benzene, a toxic and carcinogenic agent widely used for industrial purposes. Changes in the adenosine deaminase (ADA) activity in the liver, kidney and serum of rats were investigated at 0, 2, 4, 8, 16, 32 and 64 h following injection. Serum physiological was administered to each control group. Enzyme activities were measured spectrophotometrically. Our purpose was to further investigations of some diseases caused by benzene, and present evidence of variations in the activity of ADA enzyme effected by benzene. While benzene caused significant inhibitions in ADA activity in the liver at 16 and 32 h and at 0.05 probability level, no significant inhibition or activation occurred at other test periods (hours). ADA activity did not present any significant variation in the kidneys. It was observed that ADA activity displayed similar patterns in the control groups. Comparisons of ADA activities in the two groups showed a statistically significant decrease between 4(th) and 64(th) hours (p< 0.05), demonstrating a direct correlation between benzene and its effects on ADA enzymes.     

Reduced adenosine deaminase activity in the CNS of spontaneously-hypertensive rats

The activity of adenosine deaminase (ADA) has been measured in the hypothalamus, pons medulla and cerebral cortex from 30-day-old and 100-day-old spontaneously-hypertensive rats (SHR) and age-matched WKY controls. At 100 days there was a significant reduction in ADA activity in the hypothalamus (18.0%), pons medulla (20.6%) and cerebral cortex (14.7%). In 30-day-old SHR animals (prior to the development of significant hypertension) no significant changes were seen in the cerebral cortex or pons medulla but there was a small but significant reduction in ADA activity in the hypothalamus (9.2%). There was no significant reduction in the ADA activity in heart or kidney. Extracts of 100-day-old pons medulla which had been briefly heated to destroy endogenous ADA activity did not differentially affect the activity of exogenous purified ADA.     

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.     

腺苷脱氨酶及同工酶对自身免疫病诊断和病情监测的作用

摘要 目的：检测腺苷脱氨酶(ADA)及其同工酶在自身免疫病患者血清中的变化,探讨其诊断和病情监测作用。方法：收集70例系统性红斑狼疮（SLE）、114例类风湿性关节炎(RA)、55例强直性脊柱炎(AS)、及其年龄性别对应的健康血清标本。测定血清总ADA（tADA）、ADA1及ADA2活性。ROC曲线分析其诊断价值。结果：与健康对照相比,ADA活性在SLE患者血清中显著升高[tADA：15（12,20）vs 8（7,10）U/L;ADA1：3.5（2,5）vs 3（2,3）U/L;ADA2：11（8,15）vs 6（5,7）U/L;P<0.01）]。与健康对照相比,RA患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性水平升高[8（5.25,10）vs 7（5,9）U/L,P<0.05）];与健康对照相比,AS患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性升高[7（5,9）vs 6（5,7）U/L,P<0.05）]。ROC分析显示tADA及ADA2对SLE具有较好诊断价值（tADA：88.6%特异性、77.1%敏感性;ADA2：92.9%特异性、68.6%敏感性）。ADA活性对RA及AS患者无诊断价值。spearman相关性分析显示,tADA活性与SLE患者疾病活动度有一定正相关性（r=0.303,P=0.011)。结论：血清tADA活性检测可作为SLE辅助诊断和病情监测指标。     

Developmental expression of adenosine deaminase in placental tissues of the early postimplantation mouse embryo and uterine stroma

In this study, we have investigated the distribution of adenosine deaminase (ADA) in embryonic, extra-embryonic, and decidual tissues of the developing mouse embryo. ADA catalyzes a key step in purine metabolism converting adenosine to inosine. ADA specific activity (nmol/min/micrograms protein) was present at low levels in the embryo-decidual unit during the first 2 days of postimplantation development but then increased starting late on Day 6 of gestation (Day 0 plug). By Day 9, ADA specific activity was 80-fold higher than on Day 6. A histochemical staining method for ADA activity was applied to cryostat sections of the implantation site. The developmental increase localized primarily to the trophoblast/antimesometrial decidua interface between Days 7 and 9 of gestation, and decidua basalis and the metrial gland by Day 11. Immunofluorescent staining with sheep anti-mouse ADA antiserum confirmed the presence of ADA antigenicity in tissues forming the maternal/fetal interface. ADA specific activity was 19-fold higher in homogenates of the Day 11 decidua/parietal yolk sac than in the thymus, a tissue generally thought of as ADA-rich. High levels of ADA activity and immunoreactivity were also detected in the embryonal plasma during organogenesis, but the embryo proper showed only low levels. These results indicate that ADA is tightly regulated within tissues forming the maternal/fetal interface during early postimplantation stages of development.