| BackgroundStatins are most effective lipid-modulating drugs in the world. Japanese studieshave proved that pitavastatin has great efficacy in reducing serum cholesterol andlow-density lipoprotein cholesterol. Lovastatin/niacin is a broad-spectrum agent,which can improve the lipids profile as a whole. However, there is no data of usingthese drugs in Chinese population up to now.Since plasma protein biomarkers may be used as sensors of drug therapy, wehypothesize that lipid-modulating drugs may influence the plasma proteomicexpression. Therefore, the plasma protein biomarkers may be helpful to assess theeffects of the lipid-modulating drugs. Proteomics technique has been a prospectingtechnology in screening biomarkers. However,the proteomic studies inhyperlipidemia and its drug-therapy are very limited.Objectives:1. In order to explore the hyperlipidemia-related differentially-expressed proteins in the plasma by comparing the proteomics profile of hyperlipidemia patients and healthy volunteers.2. To explore the differentially-expressed proteins in the plasma related to lipid-modulating drug therapy by comparing the proteomics profile of hyperlipidemia patients before and after lipid-modulating therapy.3. To provide clues and evidence for biomarkers research in lipid modulating therapy. Study populationsThe hyperlipidemia populations were obtained from two clinical trials. The firstwas pitavastatin phaselI clinical trial and the second was lovastatin/niacin phaselIclinical trial. A total of 15 and 36 hyperlipidemia patients were randomizedly enrolledin the pitavastatin trial and lovastatin/niacin trial respectively during August 2005 toDecember 2006. Inclusion criteria of the studies: 1. Men or women aged at 18～70years old who had primary hyperlipidemia. 2. Patient with serum LDL-C(?)140～160mg/dl(3.6～4.13mmol/dL) and TG(?)400mg/dl(4.52mmol/dL). The studies wereimproved by the Independent ethics committee of Fuwai Hospital. Written informedconsent was obtained from each participant before the study.Lipid-modulating therapy: The patients of pitavastatin trial received 2mgpitavastin and the patients of lovastatin/niacin trial received 10mg/500mg every nightfor 8 weeks.Healthy control population was obtained from Fuwai Hospital. 13 healthyvolunteers were included by matching the sex, age.Methods of proteomic study:Blood samplingHyperlipidemia patients: Peripheral venous blood samples for proteomic studywere collected at before (0 week) and after lipid-modulating therapy (eighth week) inthe morning after an overnight fast. Routine blood biochemistry was measured bothbefore and after the therapy. Serum concentrations of total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol(LDL-C) and triglycerides (TG) were measured using a Beckman Synchrom CX5(Fullerton, California,USA).Healthy control: Peripheral venous blood samples for proteomic study werecollected in the morning after an overnight fast. Routine blood biochemistry wasmeasured in the healthy population. Sampling divisionThe blood samples were divided into 6 groups: groups before and afterpitavastin treatment, groups before and after lovastatin/niacin treatment, patients withboth hyperlipidemia and hypertension, healthy control group.Procedure of proteomic studyEach EDTA-anticoagulated blood sample(4ml) was centrifuged at 4℃(3000rpmfor 10 minutes) and the supernatant was collected. The plasma samples of each groupwere pooled and then the albumin and IgG were removed using the ProteoExtractAlbumin/IgG Removal Kit (CALBIOCHEM). Prior to 2-DE, samples were labeledwith fluorescent cyanine dyes for 2-D DIGE, Cy2, Cy3, and Cy5(CyDye DIGE FluorLabeling Kit, Minimal Dye) following the manufacturer's protocols. The internalstandard was prepared by combining equal quantities of each of all the tested samples.Six DIGE gels were performed to analyze hypercholesterolemia samples (before andafter treatment) or control samples. The first-dimension isoelectric focusing wasperformed using an immobilized drystrips (24 cm, pH 3-10 NL, Bio-Rad) andPROTEAN IEF CELL (Bio-Rad).The Cy2, Cy3, and Cy5 components of each gelwere individually imaged with a Typhoon 9410 scanner (Amersham Biosciences).DeCyder v. 5.02 (GE Healthcare) was used for a differential gel analysis. A totalof 950～1050 protein spots were analyzed. Protein relative abundance ratios largerthan +1.5 or smaller than—1.5 were set as a threshold indicating significantchanges. The Student's t-test was used to calculate significant differences in therelative abundance of individual protein spot between different groups.A preparative gels was run for MS identification. The proteins of interest, asdefined by the 2-D DIGE/DeCyder analysis, were excised from the CBB-stained gelfor an in-gel tryptic digestion. Each gel-piece was incubated with the digestion andextraction solutions and then evaporated to dryness for MS analysis. Peptides fromeach gel were mixed with MALDI matrix and its MALDI PMF was analyzed using a4800 MALDI-TOF/TOF Analyzer (Applied Biosystems). Protein identification byPMF was performed using the MASCOT search engine (version 1.9, Matrix Science).The IPI human database v3.23 (http://www.ebi.ac. uk/IP/IPIhelp.html) was used for the protein searching.Results1. There was not dignificant difference of baseline levels of plasma TC,TG,LDL-C and HDL-C between the two groups of hyperlipidemia patients.2. Compared to heslthy population, the plasma fibrinogen was raised expression in hyperlipidemia patients. Both pitavastatin and lovastatin/niacin therapy decreased the plasma fibrinogen level.3. The expression of plasma alpha-2-glycoprotein, zinc, a fat mobilization factor, was lower in hyperlipidemia patients than that in healthy population.4. The expression of plasma haptoglobin was lower in hyperlipidemia patients than that in healthy population. However, both pitavastatin and lovastatin/niacin therapy increased the plasma haptoglobin level.5. Lovastatin/niacin therapy increased the plasma transferrin level of hyperlipidemia patients.Conclusion1. In Chinese population, both pitavastatin and lovastatin/niacin therapy can decrease the plasma fibrinogen level in hyperlipidemia patients, which indicated that the drugs had anticoagulation effect.2. The expression of plasma haptoglobin was lower in hyperlipidemia patients than that in healthy population. It indicated that plasma anti-oxidative ability was depressed in hyperlipidemia patients. However, both pitavastatin and lovastatin/niacin therapy can increase the plasma haptoglobin level, indicating that the drugs had anti-oxidative effectrs.3. Lovastatin/niacin therapy can increase the plasma transferrin level of hyperlipidemia patients.4. Therefore, plasma fibrinogen, haptoglobin and transferrin may be therapy biomarkers of these lipid-modulating drugs. These proteins are all acute-phase proteins, indicating that the effects on acute-phase proteins may be the pleitropic effects of the drugs.5. Proteomic technology is helpful for exploring the biomarkers of drug-therapy.
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