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


CONCLUSIONS
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.
Kitano
Soichi
S
Development Planning Section, Technology Development Department, SRL Inc., 5-6-50 Shinmachi, Hino-shi, Tokyo, 191-0002, Japan. kitano@srl.srl-inc.co.jp
Hisatomi
Hisashi
H
Hibi
Nozomu
N
Kawano
Katsumi
K
Harada
Shoji
S
D000328:Adult; D000428:Alcohol Drinking / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D012680:Sensitivity and Specificity; D012907:Smoking / Q000097:blood*; D052616:Solid Phase Extraction / Q000379:methods*
D015415:Biomarkers; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost