**2. Methods**

702 Lipoproteins – Role in Health and Diseases

for predicting therapeutic efficacy [28].

In addition, there has been accumulating evidence that the HCV core protein is induced upon the redistribution of lipid droplets, affecting the assembly of both HCV and VLDL [13, 14]. The function of core protein in lipid metabolism has been widely examined including in models of steatosis involving HCV core protein transgenic mice [15]. Findings include reduction in activity of MTP [9] and Tyr164Phe substitution in relation to marked steatosis in HCV-G3 infection [16]. Although the participation of Arg70Gln/His substitution in steatosis of hepatocytes has been proposed [17], the precise connection between hepatic

As mentioned earlier, HCV particles in peripheral blood may associate not only with VLDL, but also with other lipoproteins, especially LDL, since circulating LVPs span a wide range of buoyant gravity and physicochemical characteristics [8, 18]. The association of lipoproteins with HCV particles may be beneficial for HCV through protection against anti-HCV neutralizing antibodies, as the antigenicity of HCV surface proteins is hidden beneath the associated lipoprotein particle [18]. Lipoprotein particles isolated from sera of HCV patients displayed differentially modulated lipid synthesis in human monocyte-derived macrophages in comparison to lipoproteins obtained from normal subjects, suggesting that HCV infection influences the biochemical composition of lipoproteins, thus revealing an alternative influence on lipid metabolism [19]. HCV entry into liver cells may occur through many receptors, including CD81 (direct binding to HCV E2 protein) and claudin-1, both of which act during the later steps of HCV entry [20]. The predominant role of LDL-receptors or remnant receptors is to catch VLDL-derived lipoprotein particles. Meanwhile, SR-BI (a receptor for HDL and oxidized LDL) directly binds HCV E2 protein [21]. Very recently, Nieman-Pick C1-like 1 cholesterol absorption receptor has been reported as a new factor for HCV entry to hepatic cells [22]. Interestingly, lipoprotein lipase, which hydrolyzes VLDL, is reported to increase the binding of LVP to hepatic cells while simultaneously decreasing infection levels of hepatic cells [23].

These findings suggest that examination of serum lipid profiles in chronic HCV infection may be important for understanding the biological features of HCV infection. Compared to normal subjects, low levels of TC, high-density lipoprotein cholesterol (HDL-C) and LDL-C was reported in chronic HCV-G3a infection [24]. However, lipoprotein profiles in infections of genotypes other than HCV-G3 have not been fully described and the data are somewhat conflicted. Moriya et al. indicated that TC levels and apoB, CII and CIII were reduced in HCV-G1b compared with chronic HCV-G2a or hepatitis B virus (HBV) infection [25], while others have not reported such a distinction. In addition, distortion of serum lipid levels has been widely observed in connection with virological outcome of IFN-based antiviral therapy, especially in HCV-G1 infection. Lower LDL-C, HDL-C, TC and/or TG was reported to be a possible predictor for unfavorable response to IFN-based therapy [24, 26, 27]. However, after the discovery of a genetic polymorphism near the human IL28B gene as the most potent predictor of the outcome of IFN-based therapy, the distortion of serum lipid levels is no longer thought to be an independent factor, but rather a confounding variable

In this chapter, we described the lipoprotein profiles in chronic HCV-G1b infection (the most common genotype in Japan) compared with that in chronic HCV-G2 infection (the second most common genotype in Japan). In addition, the influence of the genetic

steatosis and HCV-G1b and/or HCV-G2 infection remains unclear.

#### **2.1. Patients and materials**

Fasting sera of patients who were diagnosed as having chronic HCV infection were collected and stored at lower than -30 degrees centigrade until examination of apolipoproteins and/or lipoproteins. At the time of serum collection, TC, TG and HDL-C were measured using a routine laboratory kit. Serum LDL-C was measured by a direct assay using an LDL-cholesterol kit (Sekisui Medical, Japan). A good correlation between the direct assay and indirect measurement using the Friedwald equation (r=0.96) in 96 healthy adults whose TG level was lower than 400 mg/dl was observed.

Detection of HCV infection was made using a commercial real-time PCR Kit. Detection of HCV genotype was performed by a PCR method based on the 5' non-coding sequence. Some patients diagnosed as having HCV-G1b infection, aa substitutions at core 70/91, interferon sensitivity determined region (ISDR) and/or IFN RBV resistance determining region (IRRDR) were examined. All patients examined were Japanese without ongoing treatment of IFN-based antiviral therapy.

All patients were confirmed not to be co-infected with HIV, HTLV, tuberculosis or other chronic bacterial infections. In addition, patients who were diagnosed with hepatic cirrhosis were excluded from the study. Study protocols were approved by the review board of each institution and written informed consent was obtained prior to study enrollment.

#### **2.2. Detection of serum apolipoproteins**

Serum levels of apolipoproteins (apoA1, apoA11, apoB, apoCII, apoCIII and apoE) were analyzed by immunonephelometry using an apolipoprotein detection kit (Sekisui Medical, Japan). Serum apoB-48 was assayed by chemiluminescent enzyme immunoassay [29] using an apoB-48 CLEIA kit (Fujirebio, Japan). Serum apoB-100 level was determined as apoB minus apoB-48.

#### **2.3. Detection of lipoprotein fractions by HPLC based method**

Fasting serum lipoprotein profiles were analyzed using an HPLC system with on-line enzymatic dual detection of cholesterol and TG as described previously (LipoSEARCH, Skylight Biotech, Japan) [30]. Briefly, 10 l of whole serum sample was injected into two connected columns (300 7.8 mm) of TSKgel LipopropakXL (Tosoh, Japan) and eluted by TSKeluent Lp-1 (Tosoh). The eluent from the columns was divided by a micro splitter and continuously monitored at 550 nm after an online enzymatic reaction with a commercial kit, Determiner L TC (Kyowa Medex, Japan) and Determiner L TG (Kyowa Medex). Then, the cholesterol and TG concentrations were calculated by the computer program, which was designed to process complex chromatograms with a modified Gaussian curve fitting for resolving overlapping peaks by mathematical treatment.

Lipoprotein particles were fractionated into four major lipoproteins according to particle diameter as follows: >80 nm classified as chylomicrons; 30 to 80 nm as VLDL; 16 to 30 nm as LDL; and 8 to 16 nm as HDL. Next, the concentration of cholesterol and TG was measured in each major lipoprotein fraction and the ratio of cholesterol:TG concentration (C:T ratio) was calculated. This system has been successfully applied elsewhere in clinical research with excellent reproducibility [31]. Although freezing and thawing may affect the lipoprotein fraction, the influence of freezing and thawing on the measurement of cholesterol and TG concentration in healthy samples is fairly low, as described on homepage of Skylight Biotech (http://www.lipo-search.com).

Figure 1 illustrates chromatographic pattern of cholesterol and triglycerides derived LipoSEARCH was illustrated.

**Figure 1.** Chromatographic pattern of fasting serum cholesterol and TG derived HPLC-based method (LipoSEARCH).

#### **2.4. Detection of amino acid substitutions at aa 70/91 in core region of HCV-G1b**

HCV RNA was extracted from serum samples and reverse transcribed with random primers and MMLV reverse transcriptase (Takara Shuzo, Japan). Based on the method of Akuta et al. [32], nucleotide sequences of the core region were analyzed by direct sequencing after nested PCR. Subsequently, the aa substitutions at position 70 (arginine, Arg70; or glutamine/histidine, Gln70/His70) and at position 91 (leucine, Leu91; or methionine Met91) were determined.

## **2.5. Detection of amino acid substitutions related to ISDR in NS5A of HCV-G1b**

Nucleotide sequences of NS5A-ISDR were analyzed by direct sequencing after doubleround PCR according to Enomoto et al. [33]. Then, the aa sequence from 2209-2248, termed the ISDR region, in NS5A was determined and the numbers of aa substitutions defined.
