Validation of PON1 enzyme activity assays for longitudinal studies

72 K. Huen et al. / Clinica Chimica Acta 402 (2009) 67–74assays). We found that PON1 enzyme activities were consistently higherin serum than in heparinized plasma both by paired t-test analysis(pb0.01) and Deming regression. The Deming regression equations forthe difference between heparin plasma (y)andserum(x, 95% confidenceinterval in parentheses) were: y=1.02(0.67,1.38)x−17.3(−65.1,30.6),y =1.12(0.66,1.59)x − 1.31(− 5.80,3.19), y =1.10(0.77,1.42)x − 1571.7(−4066.8,923.4), and y=1.25(1.04,1.46)x−1.79(−3.21,−0.36) for AREase,lnDZOase, CPOase, and lnPOase, respectively. The average percentdifference between PON1 activity measured in serum and plasma fromthe same individual was 21.6%, 22.2%, 23.5% and 26.1% for AREase,DZOase, CPOase, and POase, respectively. The absolute differences forAREase and ln POase are shown in the Bland–Altman plots (Fig. 2Band D).3.3. Intra- and inter-assay variability in plasmaLaboratory volunteer samples were collected in 2006 and multiplealiquots were prepared and stored at −80 °C for use as internalcontrol samples in N20 separate experiments over 2 y. In addition,within the same experiment, multiple aliquots of the same internalcontrols were prepared and each aliquot was assayed on a differentplate. Within-assay variability as characterized by the average CV forwithin experiment measurements of lab control samples wasrelatively low. The CV was 4.3%, 2.8%, 2.1%, and 2.8% for AREase,DZOase, CPOase, and POase, respectively. As expected, the inter-assayvariability was slightly higher than intra-assay variability. Theaverage CV between assays was 7.5%, 5.7%, 7.6%, and 9.0% for AREase,DZOase, CPOase, and POase, respectively (Fig. 3).3.4. Effects of temperature on AREase and DZOase activityLab volunteer samples were assayed multiple times at temperaturesranging from 24.7 °C to 29.3 °C. Enzyme activity in both assaysincreased with temperature (Fig. 4). Using linear regression analysis,we found that an increase of temperature by 1 °C was associated witha 4.5% (p b0.001) and a 2.6% (p b0.001) increase in AREase andDZOase, respectively. Since temperature affected the assay results,we used the equations generated by linear regressions (Fig. 4) topredict AREase and DZOase values for a constant temperature of 25 °Cin subsequent analyses. As an alternative to using these temperaturecorrection factors, we also found that including an assay temperaturevariable in the statistical model yielded similar results.3.5. Effect of storage durationTo examine the effects of storage duration in samples stored for b2y,we assayed multiple plasma aliquots from the same lab volunteersample at different lengths of storage time (−80 °C) ranging from 415 to631 days. All 4 PON1 assays were performed on these aliquots a total of17 times. We did not observe a significant association between samplestorage duration and AREase, ln DZOase, and CPOase activity. However,we did find a statistically significant decline in ln POase activity withstorage duration. The decrease was equivalent to 0.05% in POase activityper day of storage (p=0.027) and appeared linear for the particular rangeof time observed (days 415–631).Plasma samples from 95 CHAMACOS mothers were assayed in2002 at the UW laboratory (after 2 y of storage) [24,36] and thenseparate aliquots of the same samples that had not yet been thawedwere assayed in 2007 after 2 y of storage in the UCB laboratory todetermine the long term effects of storage duration. The measurementsmade after 2 y of storage were significantly correlated withmeasurements made after 7 y of storage for all 4 assays; however, thecorrelation was highest for POase activity. The Pearson's correlationcoefficients were 0.51, 0.64, 0.55, and 0.79 for AREase, ln DZOase,CPOase, and ln POase respectively (pb0.0005 for all 4 assays). Onaverage, enzyme activities were 17.1%, 39.4%, and 37.6% lower in theset of samples stored for 7 y as compared to those stored for 2 y(pb0.001 for all 3 assays) for AREase, CPOase, and POase respectively(Fig. 5). In contrast, DZOase activity was 22.9% higher (pb0.0005).3.6. Effect of freeze–thaw cyclesWe used multiple aliquots of plasma samples from 5 lab volunteersto examine the effects of freeze–thaw cycles on PON1 enzyme activity inspecimens stored at −80 °C for b2y.Aliquotsofeachsampleunderwentbetween 1 and 4 freeze–thaw cycles before PON1 assays wereperformed. We did not identify a significant change in PON1 activityfor any of the 4 assays in samples experiencing a greater number offreeze–thaw cycles (Fig. 6). The inter-sample CV (freeze–thaw cycles)was 6.6%, 4.9%, 5.3%, and 6.0% for AREase, DZOase, CPOase, and POase,respectively. Thus, freeze–thaw cycles did not appear to affect PON1enzyme activity assays in samples stored b2 y.In contrast, freeze–thaw cycles did affect PON1 enzyme activity insamples that had been stored for N2 y. We assayed a subset of 27CHAMACOS samples at years 2 and 7 (duration of storage at −80 °C).Between years 2 and 7, these samples had undergone one additionalfreeze–thaw cycle. On average, PON1 enzyme activity was 25.7% lower(pb0.0005) for AREase and 14.2% higher for DZOase activity (p=0.001)after adjusting for temperature (Fig. 5). In comparison to the 95CHAMACOS samples which had not been thawed between years 2 and 7,the thawed samples experienced a more dramatic decrease in AREaseactivity (17.1% for unthawed samples and 25.7% for thawed samples)that was statistically significant (pb0.0005). For ln DZOase, both thawedand unthawed samples had increased activity at year 7. For CPOase andln POase activity, the additional freeze–thaw cycle did not significantlyaffect PON1 enzyme activity differences between samples stored for 2Fig. 6. Effect of freeze–thaw cycles on PON1 enzyme activity. Five lab control samples were frozen at −80 °C (sample 1 ♦; sample 2 ●; sample 3 ▲; sample 4 ■; sample 5 ✱) andunderwent 1–4 freeze–thaw cycles over 2 weeks. Average within-sample CV (%) was 6.5, 4.9, 5.3, and 6.0 for AREase, DZOase, CPOase, and POase, respectively.