Improved method of plasma 8-Isoprostane measurement and association analyses with habitual drinking and smoking

Abstract

Aim: To develop a simple and accurate method for quantifying 8-isoprostane in plasma by employing a combination of two-step solid-phase extraction of samples and a commercially available ELISA kit, and by this method to examine the effects of drinking and smoking habits against the levels of plasma 8-isoprostane in healthy Japanese volunteers.

Methods: Plasma 8-isoprostane was extracted with ODS gel suspension followed by NH(2) Sep-Pak column. The 8-isoprostane fractions were assayed using a commercially available ELISA kit. We measured plasma 8-isoprostane levels in 157 healthy Japanese volunteers divided into three groups (64 non-habitual drinkers, 56 moderate drinkers and 37 habitual drinkers) according to their alcohol consumption per week. Genotypes of aldehyde dehydrogenase 2 (ALDH2) were also determined to investigate the plasma 8-isoprostane levels with reference to drinking habits. In addition, the plasma 8-isoprostane levels of 96 non-smokers and 61 smokers from the same subjects were compared.

Results: Our method fulfilled all the requirements for use in routine clinical assays with respect to sensitivity, intra- and inter-assay reproducibility, accuracy and dynamic assay range. Significant increases of plasma 8-isoprostane levels were observed in female habitual drinkers when compared with those of non-habitual drinkers (t = 5.494, P<0.0001) as well as moderate drinkers (t = 3.542, P<0.005), and 8-isoprostane levels were also significantly different between ALDH2*2/1 and ALDH2*1/1 in the female habitual drinkers (t = 6.930, P<0.0001), suggesting that excessive drinking of alcohol may increase oxidization stress, especially in females. On the contrary, no significant difference of the plasma 8-isoprostane levels was observed between non-smokers and smokers.

Conclusion: Our present method was proved to be a simple and accurate tool for measuring plasma 8-isoprostane. However, the clinical utility of plasma 8-isoprostane for drinking and smoking habits was limited since elevated 8-isoprostane levels were observed in female heavy drinkers, and no association was found between smokers and nonsmokers.

Figure 1

Flow chart of the improved ELISA for plasma 8-isoprostane.

Figure 2

Results of the second extraction step using an NH₂ Sep-Pac column to isolate the plasma 8-isoprostane. Spiked plasma samples containing ³H-labeled 8-isoprostane, PGF2α, TXB2, 6-keto-PGF1, PGE2 or PGD2 were used. Samples extracted with ODS gel were used to assess the absorption by, washing and elution from the NH₂ Sep-Pac column.

Figure 3

Dilution curves of plasma 8-isoprostane in 3 different plasma samples ( ▲ y = 227.6x-2.34, r = 0.999; ■ y = 126.3x + 0.06, r = 1.000; ◆ y = 21.8x-0.04, r = 0.999).

Figure 4

Changes in AST, ALT, γ-GTP and plasma 8-isoprostane after alcohol intake. The levels of plasma 8-isoprostane in the 3 individuals increase significantly on d 1 after drinking, but return to their original levels on d 2. No significant changes are observed in AST, ALT and γ-GTP.

Figure 5

Effect of smoking habit on plasma 8-isoprostane levels. The levels of plasma 8-isoprostane were not significantly different between non smokers and smokers (21.5 ± 7.3 vs 22.8 ± 7.4 ng/L).