Nicotine metabolite ratio (3-hydroxycotinine/cotinine) in plasma and urine by different analytical methods and laboratories: implications for clinical implementation

Abstract

Background: The highly genetically variable enzyme CYP2A6 metabolizes nicotine to cotinine (COT) and COT to trans-3'-hydroxycotinine (3HC). The nicotine metabolite ratio (NMR, 3HC/COT) is commonly used as a biomarker of CYP2A6 enzymatic activity, rate of nicotine metabolism, and total nicotine clearance; NMR is associated with numerous smoking phenotypes, including smoking cessation. Our objective was to investigate the impact of different measurement methods, at different sites, on plasma and urinary NMR measures from ad libitum smokers.

Methods: Plasma (n = 35) and urine (n = 35) samples were sent to eight different laboratories, which used similar and different methods of COT and 3HC measurements to derive the NMR. We used Bland-Altman analysis to assess agreement, and Pearson correlations to evaluate associations, between NMR measured by different methods.

Results: Measures of plasma NMR were in strong agreement between methods according to Bland-Altman analysis (ratios, 0.82-1.16) and were highly correlated (all Pearson r > 0.96, P < 0.0001). Measures of urinary NMR were in relatively weaker agreement (ratios 0.62-1.71) and less strongly correlated (Pearson r values of 0.66-0.98, P < 0.0001) between different methods. Plasma and urinary COT and 3HC concentrations, while weaker than NMR, also showed good agreement in plasma, which was better than that in urine, as was observed for NMR.

Conclusions: Plasma is a very reliable biologic source for the determination of NMR, robust to differences in these analytical protocols or assessment site.

Impact: Together this indicates a reduced need for differential interpretation of plasma NMR results based on the approach used, allowing for direct comparison of different studies.

Figure 1

Correlation and linear regression of measures of metabolites from plasma and urine (n = 35) from 8 methodological approaches (Methods 1A–C, 2A–C, 3, 4A–B). Each point on the plot represents an individual measurement of a sample by one test method compared to a reference method (Method 1A). Plasma log-corrected (A) 3-hydroxycotinine over cotinine ratio (log3HC/COT, logNMR), (B) cotinine (logCOT) and (C) 3-hydroxycotinine, measured by methods 1–3. Urine log-corrected (D) 3-hydroxycotinine over cotinine ratio (log3HC/COT, logNMR), (E) cotinine (logCOT) and (F) 3-hydroxycotinine (log3HC), measured by methods 1A, 1B, 4A, and 4B. Pearson correlations were similar among logged and non-logged NMR, COT, and 3HC (Pearson r values for plasma NMR, COT, and 3HC concentrations ranged from 0.92–0.99, all P<0.0001; Pearson r values for urine NMR, COT, and 3HC measurements ranged from 0.55–0.95, all P<0.0009).