**3. Results**

704 Lipoproteins – Role in Health and Diseases

resolving overlapping peaks by mathematical treatment.

of Skylight Biotech (http://www.lipo-search.com).

LipoSEARCH was illustrated.

(LipoSEARCH).

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

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

Figure 1 illustrates chromatographic pattern of cholesterol and triglycerides derived

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

**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.

Difference in lipoprotein profiles between patients with chronic HCV-G2 and HCV-G1b infection, between HCV-G1b patients with favorable and unfavorable response to PEG-IFN plus RBV combination therapy, and between minor (non-responder) genotypes and major (responder) genotypes of IL28B (rs8099917)

Serum lipoprotein profiles are clearly distorted in patients with chronic HCV-G3 infection and this is characterized by a decrease in apoB-100-related cholesterol [24]. However, serum lipoprotein disturbances in other genotypes are an issue that has been under discussion. In addition, disturbances in lipoprotein profiles have been reported in patients who were not responsive to IFN-based antiviral therapy in comparison to responsive patients with chronic HCV-G1 infection [26, 27]. However, whether or not dyslipoproteinemia is an independent factor affecting the efficacy of IFN-based therapy remains controversial. The latest interpretation seems to be that dyslipoproteinemia in HCV-G1 patients may be a confounding factor of the host genotype of IL28B that is the strongest predictor for virological outcome following PEG-IFN plus RBV therapy [28].

Initially, we compared serum levels of apolipoproteins in chronically HCV-G2- and G1binfected patients paying special attention to virological outcome of PEG-IFN plus RBV therapy in the HCV-G1b patients. Of the pre-treatment fasting sera taken from 42 HCV-G1b patients, 23 achieved a sustained viral response (SVR; negative for HCV RNA at 6 months after standard 48 weeks of therapy); 8 had a transient viral response (TVR; HCV RNAnegative during therapy, but reappearing after therapy); and 11 had a non-viral response (NVR; HCV RNA-positive during therapy). 24 HCV-G2 patients were also examined.

There were no differences in the concentration of apoAI, apoAII, apoCIII, apoE and apoB-48 (138.2±27.4 mg/dl vs. 142.4±32.4 mg/dl, 30.4±6.5 mg/dl vs. 29.9±4.8 mg/dl, 5.7±2.0 mg/dl vs. 6.2±2.4 mg/dl, 4.4±1.1 mg/dl vs. 4.3±1.0 mg/dl and 3.3±4.2 mg/dl vs. 3.1±1.4 mg/dl, respectively) between HCV-G1b and G2 patients. In addition, no significant changes were observed among patients with HCV-G1b showing different outcome of PEG-IFN plus RBV therapy. However, there were substantial differences in apoB and apoCII levels according to the response to PEG-

IFN plus RBV therapy in HCV-G1b patients. In HCV-G1b infection, apoB and apoCII levels were significantly higher in SVR patients than in NVR patients. The levels of apoB and apoCII in HCV-G2 patients were similar to those in HCV-G1b patients who achieved SVR (Figure 2).

These data suggest that the apolipoprotein profile in HCV-G1b patients is basically indistinguishable from that in HCV-G2 patients. Meanwhile, the profile differed in relation to different outcomes of PEG-IFN plus RBV therapy among chronic HCV-G1b infection. From these observations, we presumed that a decrease in LDL and/or VLDL may be a feature of dyslipoproteinemia in HCV-G1b patients who failed to respond to PEG-IFN plus RBV therapy. ApoB-100 (about 95% of apoB is apoB-100 in fasting sera) is an indicator of total VLDL, intermediate-density lipoprotein (IDL) and LDL particles in the blood because each particle of VLDL, IDL or LDL is composed of one molecule of apoB-100. Moreover, the majority of apoCII, which activates the enzyme lipoprotein lipase in capillaries, is associated with VLDL. Therefore, a decrease in apoCII could be related to a decrease in VLDL. Taking into consideration these observations, we concluded that a characteristic of the lipoprotein profile during the pre-treatment period of chronic HCV-G1b patients who subsequently fail antiviral IFN-based therapy is a decline in VLDL levels. Although lower serum apoE has recently been reported to be related to a favorable response to IFN-based therapy [35], we did not find any differences in serum apoE levels.

**Figure 2.** Serum apoB and apoCII levels in patients with chronic HCV-G1b or HCV-G2 infection. All HCV-G1b patients were treated with standard PEG-IFN plus RBV combination therapy and clarified according to the virological response. (NVR; non-viral response, TVR; transient viral response, SVR; sustained viral response)

Next, we examined the profiles of serum lipoproteins in 32 patients who were chronically infected with HCV-G2 and 111 patients with HCV-G1b, along with genotyping of the region near the IL28B gene (SNP ID rs8099917), the strongest predictor of outcome to PEG-IFN plus RBV therapy. Recently, we reported that serum apoB-100 levels are prescribed by genetic polymorphism of rs8099917, and a minor genotype of rs8099917 (TG or GG; also known as non-responder genotype of PEG-IFN plus RBV combination therapy) may be related to a decrease in serum apoB-100 in chronic HCV-G1b infection [36]. In chronic HCV-G1b infections in Japan, the minor genotype was found in 25-35% of cases, whereas it was only 10-20% in chronic HCV-G2 infections. We examined SNP of rs8099917 and the lipoprotein profiles obtained by LipoSEARCH were compared among 29 HCV-G2 patients who had the major genotype of IL28B (rs8099917), 75 HCV-G1b patients who had the major IL28B genotype and 36 HCV-G1b patients who had the minor IL28B genotype.

706 Lipoproteins – Role in Health and Diseases

did not find any differences in serum apoE levels.

sustained viral response)

IFN plus RBV therapy in HCV-G1b patients. In HCV-G1b infection, apoB and apoCII levels were significantly higher in SVR patients than in NVR patients. The levels of apoB and apoCII in HCV-G2 patients were similar to those in HCV-G1b patients who achieved SVR (Figure 2). These data suggest that the apolipoprotein profile in HCV-G1b patients is basically indistinguishable from that in HCV-G2 patients. Meanwhile, the profile differed in relation to different outcomes of PEG-IFN plus RBV therapy among chronic HCV-G1b infection. From these observations, we presumed that a decrease in LDL and/or VLDL may be a feature of dyslipoproteinemia in HCV-G1b patients who failed to respond to PEG-IFN plus RBV therapy. ApoB-100 (about 95% of apoB is apoB-100 in fasting sera) is an indicator of total VLDL, intermediate-density lipoprotein (IDL) and LDL particles in the blood because each particle of VLDL, IDL or LDL is composed of one molecule of apoB-100. Moreover, the majority of apoCII, which activates the enzyme lipoprotein lipase in capillaries, is associated with VLDL. Therefore, a decrease in apoCII could be related to a decrease in VLDL. Taking into consideration these observations, we concluded that a characteristic of the lipoprotein profile during the pre-treatment period of chronic HCV-G1b patients who subsequently fail antiviral IFN-based therapy is a decline in VLDL levels. Although lower serum apoE has recently been reported to be related to a favorable response to IFN-based therapy [35], we

**Figure 2.** Serum apoB and apoCII levels in patients with chronic HCV-G1b or HCV-G2 infection. All HCV-G1b patients were treated with standard PEG-IFN plus RBV combination therapy and clarified according to the virological response. (NVR; non-viral response, TVR; transient viral response, SVR;

There were no differences related to gender, age, fibrosis score of liver biopsy, BMI, serum ALT level, viral load or platelet count among these three groups (data not shown). We did not cite the chylomicron fraction because this fraction was too small to analyze precisely.


(mg/dl)

\**P*>0.05 vs HCV-G2, \*\**P*>0.05 vs HCV-G1b with IL28B major, \*\*\**P*>0.01 vs HCV-G1b with IL28B major

**Table 1.** Differences in lipoprotein fractions among patients with chronic HCV-G1b and G2 infection with different IL28 genotype

In patients with HCV-G1b having the IL28B major (responder) genotype, the concentration of cholesterol in the LDL fraction (LDL-C) was significantly lower than that in patients with HCV-G2. In addition, the tendency of a reciprocal increase of VLDL-C (*P*=0.055) in HCV-G1b patients having the responder genotype was observed. As a result, the level of LDL-C plus VLDL-C was similar between these two groups. In contrast, a reciprocal increase of VLDL-C was not observed in G1b patients who had the non-responder genotype. Decrease of VLDL-C and increase of HDL-C is a feature of the non-responder genotype compared with the responder genotype in chronic HCV-G1b patients.

We also examined serum lipoprotein profiles of five patients who had been infected with HCV-G1b, but achieved SVR by PEG-IFN plus RBV therapy (HCV-free status continuing for longer than 6 months). In these cured patients, low levels of VLDL-C and high levels of LDL-C and HDL-C were observed (Table 2). The lipoprotein profiles of these cured patients were quite normal with a normal composition of cholesterol and TG in each lipoprotein fraction when assessed by C:T ratio. When compared with cured patients, a relative decrease of TG in the VLDL fraction and a relative increase of TG in the LDL and HDL fractions were noted in chronic HCV infection.

In chronic HCV infection, we unexpectedly found that the serum levels of LDL-C measured by an HPLC system (LipoSEARCH) were considerably lower than those measured directly using a conventional method (HCV-G1b: 93.8±26.76 mg/dl; HCV-G2: 101.21±34.19 mg/dl), or measured indirectly by the Friedwald equation. In place of the decreased LDL fraction, the VLDL fraction was increased. This pattern is somewhat reminiscent of hyperlipidemic (high TG) samples [31]. However, in chronic HCV infection, TG levels are not much different from those in cured patients. Therefore, this finding cannot be explained by an increase of TG.


(mg/dl)

**Table 2.** Serum lipid profiles of patients who achieved SVR by PEG-IFN plus RBV therapy for chronic HCV-G1b infection (sera were obtained at least 6 months after HCV was completely eradicated)

An increase of the VLDL fraction in chronic HCV infection could be explained by reduced enzymatic activity of lipoprotein lipase, which may facilitate HCV cell entry [23] while delaying the conversion of VLDL to LDL. Alternatively, discrepancy between chemically determined LDL (conventional measurement method) and levels determined by particle size (HPLC-based method) may be explained by the existence of LDL-associated LVPs in the blood during chronic HCV infection. These particles may have the physicochemical surface nature of LDLs, but particle sizes larger than 55 nm because the diameter of the HCV particle is about 55 nm, and hence must be eluted in the VLDL fraction. Although we must take into consideration that lipoprotein particles could theoretically become fused together during freezing and thawing, thus seriously distorting the lipoprotein fraction pattern determined by the HPLC-based method, freezing and thawing has reportedly been found not to seriously affect lipoprotein profiles (Skylight Biotech, http://www.lipo-search.com).

A recent study by Nishimura et al. [37] suggested that diminished VLDL-TG/non-VLDL-TG is a key feature of chronic HCV infection. They detected VLDL-TG based on the chemical nature of VLDL. Their findings do not conflict with our data. Our results indicate a relative decrease of TG in the VLDL fraction, but a relative increase of TG in the LDL and HDL fractions. Thus, their findings of decreased VLDL-TG/non-VLDL-TG appear to be consistent with our results.

As this kind of lipid abnormality is not easily determined by conventional methodology, the HPLC-based method is extraordinarily useful for the study of lipoprotein profiles in chronic HCV infection. However, close attention to sample handling is needed because lipoprotein particles are fairly unstable.

The tentative conclusions are as follows: 1. Decrease of apoB (apoB-100) and apoB-100 related cholesterol might be a main feature of dyslipoproteinemia in chronic HCV-G1b infection with unfavorable response to PEG-IFN plus RBV combination therapy; 2. Existence of abnormally large particles (LPVs) eluted in the VLDL fraction by HPLC, in spite of the chemical characteristics of LDL, and/or reduced activity of lipoprotein lipase with delayed VLDL dissociation, may be a feature of dyslipoproteinemia in chronic HCV infection; 3. Relative increase of TG in the LDL and HDL fractions with a relative decrease of TG in the VLDL fraction may be a feature of chronic HCV infection. These conclusions are partially in concordance with those reported by Mawatari et al. [38].
