An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma.

BACKGROUND: Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs) could be a sensitive marker to predict future cardiovascular events. However, a method to evaluate oxidized forms of LDLs systemically in human plasma is not yet established. In this study, we developed a novel and convenient high-performance liquid chromatography (HPLC) method for measuring ox-LDL levels in humans. METHODS: Human plasma lipoproteins were separated by a modified HPLC method using a diethylaminoethyl-type anion-exchange gel column with stepwise elution. Ox-LDLs were detected by postcolumn reaction with a reagent containing cholesterol esterase and cholesterol oxidase. Particle size of each LDL fraction separated by HPLC was determined in 61 healthy subjects. RESULTS: Our HPLC method separated LDLs into three fractions, which were designated as LDL-1, LDL-2 and LDL-3, on the basis of their negative charges, with LDL-3 the most strongly retained fraction migrating fastest in the anodic direction, a property that reflects the net negative charge of the molecule. Western blot analysis revealed that apolipoprotein B100 in LDL-3 fraction was the most fragmented and oxidatively modified. When LDLs were oxidized in vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modified by various aldehydes, all of the LDL fractions migrated at the position of LDL-3. Further, among three fractions, particle size was smallest in LDL-3 fraction. CONCLUSION: Here, we developed a convenient HPLC method and identified LDL-3 as oxidized LDL fractions, although ox-LDLs were present in LDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction. Measuring ox-LDL levels in human plasma by this method may be useful to evaluate atherosclerotic disorders.

Oxidative status of human low density lipoprotein isolated by anion-exchange high-performance liquid chromatography--assessment by total hydroxyoctadecadienoic acid, 7-hydroxycholesterol, and 8-iso-prostaglandin F(2alpha).

This study aims to measure the oxidative status of LDL from human plasma (n=26) as assessed by biomarkers for lipid peroxidation, total hydroxyoctadecadienoic acid (tHODE), 7alpha- and 7beta-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)) after subfractionation of LDL with an anion-exchange HPLC (AE-HPLC). LDL was separated and quantified by AE-HPLC as LDL-1, LDL-2, and LDL-3 in the order of the anionic charge of the LDL particles. The concentrations of tHODE, t7-OHCh, and t8-iso-PGF(2alpha) in both plasma and LDL subfractions were assessed after reduction and saponification. In this method, the free and ester forms of hydroperoxides, ketones, and hydroxides of linoleic acid and cholesterol are measured as tHODE and t7-OHCh, respectively. It was found that tHODE significantly correlated with the proportion of LDL-2 and LDL-3 as well as with the concentration of malondialdehyde-modified LDL in plasma. Further, by the analyses of LDL subfractions, the concentrations of tHODE, t8-iso-PGF(2alpha), and t7-OHCh in LDL-3 were found to be significantly higher than those in LDL-1 and LDL-2. These results clearly indicate that the extent of oxidation increases in the order of LDL-1

Elevated circulating oxidized LDL levels in Japanese subjects with the metabolic syndrome.

In the present study, we examined the relationship between circulating oxidized low-density lipoprotein (LDL) and the metabolic syndrome in Japanese patients. Subjects who had no histories of coronary or peripheral artery disease and were taking no medications (n=119; age 57+/-10 years; male/female, 90:29) underwent a complete history and physical examination, determination of blood chemistries and oxidized LDL levels. In stepwise regression analysis, triglycerides (p=0.0001) and HDL-cholesterol (p=0.0493, inversely) were independently correlated to oxidized LDL levels. Furthermore, a significant association (p<0.0001) was found between circulating oxidized LDL levels and the accumulation of the number of the components of the metabolic syndrome. Oxidized LDL levels were one of the independent determinants of intima-media thickness of the common carotid artery, a surrogate marker of atherosclerosis. The present study reveals that circulating oxidized LDL levels are strongly associated with the metabolic syndrome. Our results suggest that elevation of oxidized LDL may be a possible molecular link between accelerated atherosclerosis and the metabolic syndrome in Japanese subjects.

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

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.

Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity.

Normal human liver cells have a limited capacity for proliferation due to telomere shortening, whereas immortalized cells prevent shortening of the 3' single strand telomeric repeat by expressing telomerases, including human telomerase reverse transcriptase (hTERT). The hTERT transcript contains three deletion sites that give rise to alternatively spliced variants (ASVs). Recently, hTERT expression was observed in cycling primary presenescent human fibroblasts, which were believed to lack hTERT expression and telomerase activity. hTERT mRNA was expressed in the synthesis (S) phase of the cell cycle. Although hTERT mRNA has eight isoforms, it is not known which of the hTERT ASVs are expressed in S phase. In order to determine the possible relationships between the cell cycle and ASV expressions, we measured the full-length isoform and ASVs of hTERT mRNA in a mortal liver cell line and immortal cell lines that were synchronized in S phase of the cell cycle. Using RT nested-PCR analysis, the full-length isoform and alpha-deletion ASV of hTERT were detected in the LI90 mortal liver cell line at points when cells in S phase represented >48% of the cell population without detectable telomerase activity. hTERT was always expressed in the HLE and Huh-7 hepatocellular carcinoma cell lines, regardless of the cell cycle. Our results suggest the possibility that telomerase is regulated in a cell cycle-dependent manner in normal liver cells.

Identification of an alternative splicing transcript for the resistin gene and distribution of its mRNA in human tissue.

OBJECTIVE: Adipocytes secrete a number of molecules such as tumor necrosis factor-alpha, leptin and free fatty acids that can influence the ability of the body to metabolize glucose. Recently, a novel 12.5 kDa cysteine-rich protein, termed resistin, was shown to be secreted by adipocytes. Resistin expression was markedly induced during the conversion of 3T3-L1 cells to mature adipocytes. Expression of resistin has been studied in human, mouse and rat; however, sequence information about an alternative splicing variant (ASV) of resistin mRNA has not been reported. In the present study, we investigated the occurrence of a novel ASV of the resistin gene in human normal tissues. DESIGN AND METHODS: We identified a novel ASV of resistin mRNA in human lung tissue by RT-PCR analysis in human lung tissue. We then investigated a novel ASV of resistin mRNA by real-time PCR analysis in 26 different types of normal human tissues. RESULTS AND CONCLUSIONS: We identified a novel deletion variant of the resistin transcript in the normal human tissues. The deleted transcript of resistin was characterized by an in-frame deletion of 78 bp, corresponding to the complete loss of exon 2 (resistin delta2 ASV). Thus, resistin delta2 ASV causes protein truncation. Our results provide the basis for more detailed studies on the regulation of resistin activity, and should assist in the development of clinical trials with resistin for the central regulation of adipogenesis and adipocyte metabolism.

[An association between plasma levels of 8-isoprostane and alcohol drinking habits in Japanese volunteers].

Lipid peroxidation and oxidative stress may play a certain role in the pathogenesis of pressure-induced atherosclerosis, and alcohol related diseases. Recently, 8-isoprostane in biological fluids has been reported to be a reliable marker for lipid peroxidation and oxidative stress in vivo. In the present study, we developed an ELISA method for 8-isoprostane which has high sensitivity, intra- and inter-assay reproducibility and wide dynamic assay range. Using this method, we examined the effects of drinking and smoking habits on plasma levels of 8-isoprostane in healthy subjects. A total of 157 apparently healthy volunteers was assayed for plasma 8-isoprostane. Subjects were divided into three groups according to their alcohol consumption. Group I is non- or few-drinkers, Group II includes subjects who drink once or twice a week, and subjects of Group III intake 3 to 5 times a week or almost every day. In addition, the same population was divided into two groups, 96 non-smokers and 61 smokers. Plasma 8-isoprostane was extracted with ODS gel followed by NH2 Sep-Pak column. The 8-isoprostane fractions thus separated were assayed by a commercial ELISA kit (Cayman Chemical). The plasma 8-isoprostane was estimated to be 20.9 +/- 93 pg/ml in a total of 157 volunteers (83 male, 74 female). The plasma 8-isoprostane levels were elevated in the Group III (26.6 +/- 9.5 pg/ml) compared with Group I (20.3 +/- 6.1 pg/mL, p<0.0001) and Group II (20.9 +/- 5.7 pg/ml, p<0.001). Significant increase of the plasma 8-isoprostane was observed only in habitual drinkers of females, but not in those of males. On the other hand, no significant difference of the plasma 8-isoprostane levels were observed between non-smokers (21.5 +/- 7.3 pg/ml) and smokers (22.8 +/- 7.4 pg/ml, p>0.05). We suppose that plasma 8-isoprostane may increase in the habitual drinkers due to the oxidization stress induced by alcohol intake, and it may become a useful marker to estimate drinking habit