Date Published: April 25, 2019
Publisher: Public Library of Science
Author(s): Steven G. Carmella, Alisa K. Heskin, Mei Kuen Tang, Joni Jensen, Xianghua Luo, Chap T. Le, Sharon E. Murphy, Neal L. Benowitz, F. Joseph McClernon, Ryan Vandrey, Sharon S. Allen, Rachel Denlinger-Apte, Paul M. Cinciripini, Andrew A. Strasser, Mustafa al’Absi, Jason D. Robinson, Eric C. Donny, Dorothy K. Hatsukami, Stephen S. Hecht, Yu Ru Kou.
The urinary metabolites (Z)-7-[1R,2R,3R,5S)-3,5-dihydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid (8-iso-PGF2α), an F2-isoprostane and biomarker of oxidative damage, and “prostaglandin E2 metabolite” (PGE-M), a biomarker of inflammation, are elevated in cigarette smokers. However, there is little information in the literature on the longitudinal stability of these widely used biomarkers. In a large clinical trial involving 10 institutional sites, smokers were given, free of charge over a period of 20 weeks, Spectrum NRC600/601 research cigarettes containing 15.5 mg nicotine/g tobacco. All participants were instructed to smoke these cigarettes for the duration of the study. At weeks 4, 8, 12, 16, and 20, first morning urine voids were collected and analyzed for 8-iso-PGF2α and PGE-M using validated liquid chromatography-electrospray ionization-tandem mass spectrometry methods. The mean level of 8-iso-PGF2α at Week 4 was 1.34 ± 1.08 (S.D.) pmol/mg creatinine (N = 226) while that of PGE-M was 73.7 ± 113 (S.D.) pmol/mg creatinine (N = 232). The corresponding levels at Week 20 were 1.35 ± 0.93 (S.D.) pmol/mg creatinine (N = 209) for 8-iso-PGF2α and 74.2 ± 142 (S.D.) pmol/mg creatinine (N = 210) for PGE-M. There was variation in these values in the intervening weeks. The intra-class correlation coefficients (ICC) were 0.51 (95% CI, 0.45, 0.57) and 0.36 (0.30, 0.43), for 8-iso-PGF2α and PGE-M, respectively, indicating fair longitudinal stability for 8-iso-PGF2α and poorer longitudinal stability for PGE-M in cigarette smokers. Males had higher ICC values than females for both 8-iso-PGF2α and PGE-M. These results indicate that, in addition to cigarette smoking, endogenous processes of oxidative damage and inflammation influence the levels of these biomarkers over time among current smokers.
The closely linked phenomena of oxidative damage and inflammation play a significant role in diseases caused by cigarette smoking including cancer, cardiovascular disease, and chronic obstructive pulmonary disease [1, 2]. As an example, extensive research has clearly demonstrated that cigarette smoke and its condensate have tumor promoting and co-carcinogenic activity, properties that are associated with oxidative damage and inflammation and are critical in cancer induction [1, 2]. As noted below, the urinary metabolites (Z)-7-[1R,2R,3R,5S)-3,5-dihydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid (8-iso-PGF2α), an F2-isoprostane and biomarker of oxidative damage, and “prostaglandin E2 metabolite” (PGE-M)(Fig 1), a biomarker of inflammation, are elevated in smokers. Furthermore, a recent nested case-control study within the prospective Shanghai Cohort Study demonstrated a significant relationship of 8-iso-PGF2α to lung cancer incidence in cigarette smokers . Thus, 8-iso-PGF2α and PGE-M are considered “biomarkers of potential harm” reflecting early biological effects strongly associated with disease .
We assessed the biomarkers in the smokers of Spectrum cigarettes as summarized in Table 1. The mean age of the smokers was 45.0 ± 13.4 years. They were 61% white, 30% African American, and 9% other races. They smoked a mean of 20.6 ± 11.4 cigarettes per day, 97.7% of which were the Spectrum research cigarettes. Further characteristics of the group have been reported . The methods for 8-iso-PGF2α and PGE-M were validated in our laboratory. For 8-iso-PGF2α, the average value obtained upon analysis of 290 pooled smokers’ urine control samples distributed in 97 plates throughout the course of a recent study was 1.03 ± 0.072 pmol/mL urine giving an overall inter-assay precision (%CV) of 7.0%. The intra-assay precision from the assay of 8 replicates was 2.9%. To assess accuracy, three levels of 8-iso-PGF2α were added in triplicate to samples of pooled smokers’ urine and assayed. The added levels were 0.564, 1.41 and 14.1 pmol/mL. This was repeated on two additional days. The average inter-assay accuracies for each level were 104%, 98% and 102% respectively. The calculated assay limit of detection (LOD) was 0.03 pmol/mL.
The analytical chemistry validation data reported here were important for our study of the longitudinal stability of 8-iso-PGF2α and PGE-M. Although these LC-MS/MS methods are well described and validated in the literature [7, 23], there are some differences between the methods we used and the originally described versions. For 8-iso-PGF2α, we used one tenth the amount of urine and a different solid-phase extraction and high throughput 96-well plate protocol. For both 8-iso-PGF2α and PGE-M, the LC-MS/MS conditions were also somewhat different from those described previously. The validation parameters reported here support the accuracy and precision of our methods.