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Biology Articles » Methods & Techniques » Improved method of plasma 8-Isoprostane measurement and association analyses with habitual drinking and smoking » Discussion

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


We have developed a simple and accurate method for quantifying plasma 8-isoprostane by employing a combination of two-step solid-phase extraction of samples and a commercially available ELISA kit.

When the plasma 8-isoprostane level was measured by using our method, the mean value of 157 healthy volunteers was 20.9 ± 9.3 ng/L, which is almost equal to the reported values using GC/MS or LC/MS[5,24,25]. The level of plasma 8-isoprostane in 4 healthy subjects among these samples measured directly by ELISA without extraction was more than 20-fold higher than those obtained by the combination of the two-step extraction and ELISA (651.5 ± 149.2 vs 24.4 ± 4.1 ng/L). According to 8-Isoprostane EIA kit booklet, although the cross-reactivity of the anti 8-isoprostane antibody employed in the ELISA kit is reported to be low, the extraction of plasma 8-isoprostane from plasma prior to the assay is indispensable. In fact, it has been reported that various 8-isoprostane analogues and related compounds are present at dozens to hundreds of times than the concentration of 8-isoprostane in biological fluids[26].

In an association study of plasma 8-isoprostane with drinking habits, heavy drinkers were higher than non-habitual drinkers and moderate drinkers. However, the plasma 8-isoprostane level was not significantly different when compared among the 3 male groups. Also, the plasma 8-isoprostane levels were significantly higher in heavy drinkers than in non-habitual and moderate drinkers. This tendency was more prominent in females, and with the ALDH2*21/1 genotype. In other words, the 8-isoprostane level was significantly higher in female habitual drinkers with the ALDH2*2/1 than with the ALDH2*1/1 genotype. These results suggest that excessive drinking may increase oxidative stress, especially in females with the ALDH2*2/1 genotype. When the same amount of alcohol is ingested, females tend to show a higher concentration of blood alcohol than males due to their lower body weight and higher ratio of adipose tissue into which alcohol shows poor penetration. In addition, female hormones, such as estradiol, inhibit the activity of ADH, contributing to the increased concentration of blood alcohol[27].

Changes in plasma 8-isoprostane levels after alcohol (0.5-1.3 g/kg) intake for 3 d, showed 8-isoprostane significantly increased on d 1, and returned to its original level on d 2. It was previously reported that alcohol consumption induced lipid peroxidation in healthy volunteers, and urinary 8-isoprostane increased in a dose- and time-dependent manner reaching its peak at 0-6 h after ingestion[9], and through induction of the CYP450 2E1 isozyme, alcohol intake may increase the generation of reactive oxygen intermediates that have the potential to peroxidize lipids[28,29].Similarly, our results may suggest that alcohol ingestion induced oxidative stress in a relatively short time after drinking.

It has been reported that smoking induces oxidative stress and increases urinary 8-isoprostane[30]. Therefore, we compared plasma 8-isoprostane levels in non-smokers and smokers. In contrast to urinary 8-isoprostane, no difference in plasma 8-isoprostane was observed between these 2 groups This is consistent with a previous report measured by LC/MS[5]. The plasma 8-isoprostane may be rapidly metabolized since 75% of plasma 8-isoprostane is excreted in the urine within 4.5 h[31].
In conclusion, we have developed a simple and accurate method for quantifying plasma 8-isoprostane by employing a combination of two-step solid-phase extraction of samples and a commercially available ELISA kit. 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. However, the clinical utility of plasma 8-isoprostane for drinking and smoking habits was limited. Further studies using a large population is required for a final conclusion for an association of plasma 8-isoprostane with drinking and smoking habits.


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