Adenosine deaminase 2 activity negatively correlates with age during childhood

Abstract

Background: Human 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.

Methods: We 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).

Results: ADA2 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.

Conclusion: We 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.

Keywords: Adenosine; Adenosine deaminase 2; Inflammation; Pediatrics.

Fig. 1

Assay optimization for analysis of ADA2 activity in plasma and serum a Total ADA activity (ADA1 and ADA2) (y-axis: Total ADA activity (U/L)) in serial dilutions of rhADA1 in plasma (x-axis: rhADA1 added to plasma (0–1 μg/mL), n = 3) and b ADA2 activity (y-axis: ADA2 activity (U/L)) in serial dilutions of rhADA2 in PBS (x-axis: rhADA2 added to PBS (0–1 μg/mL), n = 3) in the presence of 5 μL of increasing concentrations (0, 1, 10, 100, 1000 μM) of EHNA per 10 μL of sample in an assay volume of 285 μL. c Total ADA activity (y-axis: ADA (U/L)) in plasma samples from non-hemolyzed whole blood (x-axis: < 1% hemolysis, n = 3) and hemolyzed whole blood (x-axis: > 95% hemolysis, n = 3) in the absence (grey bars) and presence (black bars) of 5 μL of 1000 μM EHNA. d Percent of total ADA activity (y-axis: % Total ADA activity, n = 3) in plasma samples from hemolyzed whole blood in the absence (x-axis: 0 EHNA) and presence of EHNA (x-axis: 5 μL of 1000 μM EHNA). Significance only shown for the highest concentration of EHNA. e–f ADA enzyme activity (y-axis: ADA activity (U/L)) measured in the absence (x-axis: PBS: total ADA1 and ADA2 activity) and presence of ADA1 inhibitor, EHNA (x-axis: EHNA: only ADA2 activity) in e fresh plasma and serum (n = 3 adults) and f plasma subjected to freeze thaw (FT) cycles (n = 3 adults). Bars show mean + SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 2

Plasma ADA2 enzyme activity and concentration in a healthy pediatric cohort. Plasma samples from otherwise healthy children and adolescents (x-axis, unless otherwise noted) aged 0–2 yrs (n = 16), 3–5 yrs (n = 19), 6–8 yrs (n = 18), 9–11 yrs (n = 16), 12–14 yrs (n = 13), 15–18 yrs (n = 12) and healthy adults (> 18 yrs, n = 5) were analyzed. a–b ADA2 enzyme activity (y-axis: ADA2 activity (U/L)). c–d ADA2 concentration (y-axis: ADA2 concentration (μg/mL)). e–f ADA2 activity normalized to ADA2 concentration (y-axis: ADA2 activity (mU/mg). Trend lines show linear regression of data. Outliers are denoted by open shapes and were excluded from statistical analyses. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

Quantitation of plasma ADA2 protein. a Representative western blot of ADA2 in plasma (Control 1–8) and recombinant human (rh) ADA2 (5, 10, 20 ng rhADA2). b Representative standard curve of rhADA2 (y-axis: Area (square units); x-axis: ADA2 (ng)). c ADA2 activity (y-axis: ADA2 activity (U/L)) compared to ADA2 protein concentration (x-axis: ADA2 concentration (μg/mL)) for each adult and pediatric healthy control (n = 99, age = 5 months–45 yrs) and d for controls > 5 yrs of age (n = 64). Outliers are denoted by open circles and were excluded from statistical analyses