Extraction of plasma 8-isoprostane
Using 3H-labeled 8-isoprostane and its related compounds as spiked tracers, optimal conditions for the two-step solid-phase extraction of plasma 8-isoprostane were carefully determined. The method was based on a procedure used in a radioimmunoassay for serum TXA2. First, the plasma samples were treated with reverse-phase ODS gel to remove proteins and lipids. The 8-isoprostane bound to the gel was then eluted and separated from its related compounds on a Sep-Pac NH2 column by stepwise elution with increasing concentrations of 2-propanol in the eluent. As shown in Figure 2, most of the 8-isoprostane eluted from the column at 55% 2-propanol, whereas the other compounds eluted at 10%-25% 2-propanol. The average yield of plasma 8-isoprostane in the overall extraction was estimated to be 67.1% by counting the 3H-labeled 8-isoprostane in 5 spiked plasma samples.
Quantification of extracted 8-isoprostane by ELISA
Since it has been shown that plasma 8-isoprostane can be accurately measured using a commercially available ELISA kit after the two-step extraction, we next evaluated the analytical performance and accuracy of the overall ELISA for plasma 8-isoprostane. The detection limit of the ELISA kit was 2.2 ng/L, and this was defined as the concentration corresponding to the optical density of the zero calibrator plus 2SD. The reproducibility of this method estimated using the plasma samples from 3 control subjects was 4.2%-6.3% for the within-run (8 repeats) and 0.8%-8.1% for the between-run (5 repeats) , respectively. To assess the linearity of the assay, the above mentioned plasma samples were diluted serially, extracted and measured for their 8-isoprostane levels. A good dilution linearity was obtained for the assay, as shown in Figure 3. Analytical recovery studies were carried out using 3 plasma samples containing 3 different concentrations of 8-isoprostane, revealing that the recovery rate ranged from 95.9%-97.8%. There were no significant differences between the 8-isoprostane levels in serum and those of the plasma collected with heparin, EDTA or EDTA + ｔrasirol + indomethacin. All of the samples were freshly prepared and subjected to assay. No interference in the assay was observed for hemoglobin (up to 5 g/L), free bilirubin (up to 342 mmol/L), conjugated bilirubin (up to 342 mmol/L) or triacylglycerol (up to 55 mmol/L).
Storage stability of plasma 8-isoprostane
The 8-isoprostane in the plasma samples was stable for at least 120 d at -80℃, as long as freezing and thawing were avoided. However, 2 cycles of freezing at -80℃ and thawing at room temperature decreased the apparent 8-isoprostane levels by 30%. It was previously reported that plasma 8-isoprostane was stable for 6 mo at -80℃, and increased approximately 1.4-fold after 3 cycles of freeze and thawing. Although the reason for this disagreement is not clear, it may be attributed to the method of blood collection, since we used blood collection tubes containing 10 mmol/L EDTA・3Na, 20 kU/L trasylol and 0.1 mmol/L indomethacin, while they used common evacuated tubes containing EDTA・2Na (2.5 mmol/L). The EDTA and indomethacin could function to prevent the induction of new synthesis of 8-isoprostane in plasma samples during storage. When the plasma samples were stored at 4℃, 8-isoprostane levels increased rapidly after 1 wk and were 3-4-fold higher after 28 d. When the samples were stored at 25℃, 8-isoprostane levels reached 15-50-fold the original values after 28 d.
Association of plasma 8-isoprostane levels with drinking habits
The mean level of the plasma 8-isoprostane in 157 healthy subjects using our method was 20.9 ± 9.3 ng/L and no age or gender differences were observed. For the drinking habits obtained by questionnaire, the subjects of each gender were divided into three groups according to their alcohol consumption, namely non-habitual drinkers (nondrinker, rare drinking ,Group I, n = 64), moderate drinkers (1-2 times drinking/wk Group II, n = 56), and habitual drinkers (3-5 times drinking /wk, Group III, n = 37), and the plasma 8-isoprostane levels were compared among these groups. In females, the plasma 8-isoprostane levels were significantly higher in Group Ⅲ (30.0 ± 10.3 ng/L) than in Group I (18.1 ± 5.0 ng/L, P Ⅱ (20.8 ± 6.6 ng/L, P Ⅲ were elevated in both genders (males: 488 ± 335 nkat/L at 37℃; females: 298 ± 185 nkat/L at 37℃) compared with those in Group I (males: 343 ± 253 nkat/L at 37℃; females: 220 ± 70 nkat/L at 37℃; P g-GTP and various lipid parameters (TC, TG, LDL-C, HDL-C, ApoA-I, apoB and Lp(a)) in the subjects (Table 2).
Next, the subjects in each group were further divided into 3 groups according to their ALDH2 genotypes, and the plasma 8-isoprostane levels were compared along with those of AST, ALT and g-GTP. For both the ALDH2*1/1 and ALDH2*2/1 genotypes, the plasma 8-isoprostane level was significantly higher in Group Ⅲ than that in Groups I and Ⅱ (Table 3) in both genders. This tendency was more prominent in females with the ALDH2*21/1 genotype. Especially, the 8-isoprostane level was significantly higher in female habitual drinkers with the ALDH2*2/1 than those with the ALDH2*1/1 genotype (41.2 ± 12.3 vs 26.9 ± 7.7 ng/L, P ALDH2*1/2 genotype was significantly higher in Group Ⅲ than in Group Ⅱ (22.3 ± 9.2 vs 17.1 ± 2.5 nkat/L at 37℃, P g-GTP level was significantly higher in subjects with the ALDH2*1/1 genotype than those with the ALDH2*1/2 genotype (478 ± 396 vs 303 ± 198 nkat/L at 37℃, P g-GTP. Plasma 8-isoprostane significantly increased on d 1, and returned to its original level on d 2 (Figure 4).
Association of plasma 8-isoprostane with smoking habits
The same population of 157 healthy subjects were divided into two groups: non-smokers (n = 96; age 36.7 ± 8.2 years) and smokers (n = 61; age 35.3 ± 8.7 years), and their plasma 8-isoprostane levels were compared. As shown in Figure 5, no significant difference of plasma 8-isoprostane level was observed between these two groups (21.5 ± 7.3 vs 22.8 ± 7.4 pg/mg)