Potential conflict of interest: Nothing to report.
See Editorial on Page 1684
Project funding (to P.I. M.T.C.) was provided by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. A training scholarship (to J.E.L.) was provided by the Canadian Institutes of Health Research.
Hepatitis C virus (HCV) exerts a profound influence on host lipid metabolism. It has been suggested that the synthesis of both fatty acids (FA) and cholesterol is dysregulated in HCV but this has not been directly quantified in humans. The purpose of this study was to measure lipogenesis and cholesterol synthesis using stable isotopes in patients with HCV (n = 5) and healthy control (n = 9) subjects recruited from the University of Alberta hospital. Blood samples were taken at fasting (0 and 24 hours) and after meals over the day to mimic typical food consumption and postprandial metabolism. Isolation of free cholesterol (FC), cholesteryl ester (CE), and triglyceride (TG) from plasma and very low-density lipoproteins (VLDL) was used to measure FA and cholesterol synthesis using deuterium uptake and isotope ratio mass spectrometry. FA composition was analyzed by gas chromatography. VLDL-TG levels of polyunsaturated fatty acids (PUFA), including linoleic and linolenic acid, were lower in HCV compared to control (P < 0.05 for both). Fasting hepatic lipogenesis was significantly higher in HCV (2.80 ± 0.55%) compared to control (1.19 ± 0.27%; P = 0.03). Conversely, fasting whole-body synthesis of FC (HCV 1.64 ± 0.28% versus control 8.78 ± 1.59%) and CE (HCV 0.26 ± 0.08% versus control 1.92 ± 0.25%), as well as hepatic FC synthesis (HCV 1.68 ± 0.26% versus control 8.12 ± 0.77%) was lower in HCV (P < 0.001 for all). Conclusion: These data provide evidence that lipogenesis is elevated while cholesterol synthesis is impaired in HCV, supporting previous findings from cellular and animal models. Low PUFA levels combined with elevated lipogenesis suggests a role for dietary PUFA supplementation in HCV patients. (HEPATOLOGY 2013)
Hepatits C viral (HCV) infection is the leading cause of liver transplantation in the United States.1 Lipid metabolism, particularly very low-density lipoprotein (VLDL) machinery,2 is commandeered by HCV and is tightly linked with HCV progression.3 Discovery of the precise and multiple mechanisms by which HCV interferes with lipid metabolism is still unfolding.
Both fatty acids (FA) and cholesterol are implicated in HCV progression. De novo lipogenesis may contribute to HCV-induced damage by involvement in the viral replication process, and by contributing FAs to lipid storage in the liver, leading to steatosis. HCV is proposed to stimulate hepatic FA synthesis, as evidenced by up-regulation of enzymes involved in the lipogenic pathway such as sterol regulatory-element binding protein 1c and fatty acid synthase4-6 in cellular and animal models of HCV. However, a quantitation of lipogenesis in human patients with HCV has not been performed. The role of cholesterol in HCV is also unclear. Patients with HCV frequently have low plasma cholesterol levels,7 but cellular models suggest that HCV is associated with an elevation of enzymes regulating cholesterol synthesis and may be related to viral replication.6
The deuterium incorporation method is ideal for studying in vivo synthetic pathways and allows for estimation of FA and cholesterol synthesis simultaneously.8 Deuterium is safe, can be used with a short measurement period (≤24 hours), rapidly equilibrates across body pools, and provides a highly sensitive and precise measurement of lipid synthesis.9 The objective of this study was to investigate if synthesis of FA and cholesterol are enhanced in individuals with HCV compared to healthy subjects.
CE, cholesteryl ester; CRP, C-reactive protein; DNFA, de novo synthesized fatty acids; FA, fatty acid; FC, free cholesterol; FSR, fractional synthesis rate; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDL-c, high-density lipoprotein cholesterol; HOMA, homeostatic model assessment; IL, interleukin; IRMS, isotope ratio mass spectrometry; LDL-c, low-density lipoprotein cholesterol; PUFA, polyunsaturated fatty acids; PW, plasma water; TC, total cholesterol; TG, triglyceride; TPL, total phospholipid; VLDL, very low-density lipoprotein.
Patients and Methods
Individuals with decompensated cirrhosis from HCV infection were recruited from waiting lists for the University of Alberta Clinical Liver Transplant program. This transplant center does not obtain donor organs from executed prisoners or other institutionalized persons. Patients with familial hypercholesterolemia and Type 2 diabetes were excluded to control for states where lipogenesis and cholesterol synthesis would be influenced, resulting in ∼50% of potential participants excluded. Of the remaining potential subjects, a further 50% were too ill or declined participation. Clinical characteristics of HCV subjects recruited (n = 5; four male, one female) are described in Table 1. All HCV patients also had hepatocellular carcinoma (HCC). HCV subjects were taking hypertensive medications (n = 1), furosemide and spironolactone (n = 4), pantoprazole (n = 3), and lactulose (n = 3) but were not currently undergoing treatment for HCV. Transplant center protocol dictated that subjects had abstained from alcohol for >6 months, monitored by random alcohol screening. Available imaging data from patient histories indicated the absence of steatosis (data not shown). Healthy participants (control; n = 9; three male, six female) were recruited by advertisements. Subjects were nonsmokers and normotensive with no history of cardiovascular disease, diabetes, or other metabolic disorders, and were not taking lipid-lowering medication. This study was approved by the Health Research Ethics Board of the University of Alberta (ClinicalTrials.gov NCT00947635).
Table 1. Anthropometric, Fasting Biochemical, and Dietary Characteristics of Control and Hepatitis C Viral (HCV) Subjects
Control (n = 9)
HCV (n = 5)
Data expressed mean ± SEM.
54 ± 4
56 ± 1
Body mass index (kg/m2)
25 ± 0.8
27 ± 2
Waist circumference (cm)
87 ± 4
104 ± 5
Body fat (%)
33 ± 3
30 ± 3
Resting metabolic rate (kcal/day)
1366 ± 58
1764 ± 117
Systolic blood pressure (mmHg)
119 ± 7
116 ± 5
Diastolic blood pressure (mmHg)
74 ± 6
78 ± 7
Total cholesterol (mmol/L)
5.1 ± 0.1
3.6 ± 0.5
3.1 ± 0.2
1.9 ± 0.4
1.5 ± 0.1
1.2 ± 0.1
0.8 ± 0.1
1.1 ± 0.2
5.0 ± 0.1
5.7 ± 0.8
5.7 ± 1.5
19.2 ± 3.6
1.3 ± 0.4
5.0 ± 1.3
0.7 ± 0.2
0.5 ± 0.1
1.9 ± 0.7
2.6 ± 0.8
3.5 ± 1.7
23 ± 13
0.3 ± 0.1
1.1 ± 0.5
84 ± 8
21 ± 2
1.16 ± 0.03
32 ± 2
137 ± 52
92 ± 33
138 ± 16
MELD – calculated
9.6 ± 0.2
MELD - assigned
23 ± 1
1607 ± 126
1750 ± 167
55 ± 3
54 ± 3
19 ± 1
19 ± 1
24 ± 2
29 ± 2
Saturated fat (%)
7.6 ± 0.8
11 ± 1
Monounsaturated fat (%)
7.6 ± 0.9
8.4 ± 0.6
Polyunsaturated fat (%)
4.4 ± 0.6
3.9 ± 0.8
201 ± 44
406 ± 77
22 ± 4
20 ± 3
Total sugars (g)
90 ± 7
111 ± 14
Prior to testing day, subjects were given food scales and completed a 3-day food record to estimate dietary intake, analyzed by Food Processor (ESHA Research, v. 10.0-10.4; Salem, OR). On testing days, subjects arrived at the Human Nutrition Research Unit after an overnight fast. Blood samples were drawn and an intravenous catheter inserted for subsequent draws. Subjects consumed a loading dose of deuterium-labeled water (2H2O; D-175; 99.9 atom%; CDN Isotopes, Pointe-Claire, QC) of 1 g/kg body water estimated at 60% body weight. Subjects were given the same dose of 2H2O in 1.5 L water to consume throughout the day to maintain plasma deuterium levels at plateau. 2H2O was prepared by slow filtration through a sterile and pyrogenic filter (0.22 μm, 25 mm Cameo 25ES Syringe Cellulosic; GE Water & Process Technologies, Fisher Scientific, Ottawa, ON) to remove potential contaminants. Subjects consumed breakfast within 30 minutes and timing of postprandial sampling was initiated at the completion, allowing consistent length of time between blood draws across subjects. Subjects underwent measurement of height and weight using electronic scales; blood pressure, waist circumference, resting metabolic rate assessed by metabolic cart (SensorMedics Vmax Spectra 29n Nutritional Assessment Instrument; Viasys Healthcare, Pointe-Claire, QC), and body fat by DEXA (GE Lunar Prodigy High Speed Digital Fan Beam X-Ray-Based Densitometer; GE Healthcare, Waukesha, WI) or BodPod (Life Measurement, Concord, CA). Blood samples were drawn at 2, 4, 6, and 8 hours following breakfast. Meals were provided in sequential fashion to represent free-living food consumption and elicit typical postprandial responses. Lunch was provided after the 4-hour blood sample, afternoon snack after the 8-hour sample, and supper and evening snack for subjects to consume at home. Subjects were instructed to consume only foods provided, fast after 8 PM, and arrive the next morning fasted for a 24-hour blood sample.
Total quantity of foods for testing day meals was scaled for each subject to mimic normal total caloric intake, estimated from the food records. Meals provided 20% of calories from protein, 45% carbohydrates, and 35% fat, with 240 mg dietary cholesterol per 1,000 kcal. Meals consisted of typical foods (scrambled eggs, whole wheat toast with margarine, fruit, and milk for breakfast; pasta with chicken and vegetables in a marinara sauce and milk for lunch; vegetables with low-fat dressing for snack; turkey, cheese, and tomato sandwich and fruit for dinner; and hummus with whole-wheat pita bread for evening snack). Noncaffeinated beverages with no added sugar or dairy foods were permitted.
Assessment of Plasma Lipids and Inflammatory Markers.
Blood was drawn into SST or K2EDTA Vacutainer tubes and centrifuged at 4°C for 10 minutes at 3,000 RPM in a Jouan CR4.22 centrifuge with a Jouan M4.4 rotor. Plasma and serum were stored at −80°C or sent to the University of Alberta Hospital laboratory for analysis of plasma glucose, insulin, and lipids. Insulin was analyzed by Elecsys 2010 immunoassay. Total cholesterol (TC) and HDL-cholesterol (HDL-c), triglyceride (TG), and glucose were determined by SYNCHRON-LX System using standard kits. LDL-cholesterol (LDL-c) was calculated by the Friedewald equation. Serum cytokines including tumor necrosis factor alpha (TNF-α), interleukin-6 and -10 (IL-6, IL-10), and C-reactive protein (CRP) were measured from fasting samples using custom multiplex kits (Invitrogen Canada; Burlington, ON) and the Luminex 100 IS Total System (Luminex, Austin, TX). Additional patient data, including liver enzymes, were collected from the Clinical Liver Transplant program.
Lipid Extraction, Separation, and Analysis.
Lipoproteins obtained from 2 mL plasma were separated by sequential nonequilibrium density-gradient ultracentrifugation10 within 24 hours of collection. Briefly, plasma was ultracentrifuged twice using a 0.196M NaCl solution with a Beckman Optima Centrifuge with MLS 50 rotor for 25-30 minutes at 25,000 RPM and 20°C to remove the Sf >400 fraction. The Sf 60-400 fraction (VLDL) was isolated using 0.196M NaCl solution and centrifuged for 3 hours at 100,000 RPM and 20°C with a TL-100 centrifuge and Beckman TLA 100.2 100K rotor. Samples were stored at −80°C.
Plasma and VLDL lipids, including TG, total phospholipids (TPL), free cholesterol (FC), and cholesteryl ester (CE), were extracted and isolated using a modified Folch procedure and TLC.10 CE fractions underwent saponification and TLC to yield FC for analysis. Plasma- and VLDL-TG and TPL underwent saponification and methylation to FA methyl esters and analyzed using a Varian Star 3600 or Varian 3900 gas chromatograph with a SGE BP-20 column (25 m or 30 m, respectively; 0.22 mm i.d., 0.25 μm film). Addition of internal (15:0 or 17:0) and external (GLC 461; Nu-chek Prep, Elysian, MN) standards to TG and TPL were used to quantify content and composition of major FA, considered to be 14-18 carbons in length and 20:4n6, 20:5n3, and 22:6n3.
Determination of FA and Cholesterol Synthesis.
FA was isolated from plasma- and VLDL-TG to describe whole-body and hepatic de novo fatty acid synthesis (DNFA; %), respectively. Deuterium enrichment of FA was determined using a Delta PlusXL isotope ratio mass spectrometer (IRMS) (Finnigan Mat) with HP 6890 GLC (Agilent Technologies, Mississauga, ON) and PAL-GC1 autosampler or Delta V Plus IRMS with Trace GLC Ultra and Triplus Autosampler (Thermo Scientific, Mississauga, ON) with an SGE BP-20 column (30 m, 0.22 mm i.d., 0.25 μm film). Oven temperature was 90°C and increased to 230°C. Sample injection was 1.3 μL, helium flow 1.0 mL/min, injector temperature 240°C, and D/H reactor temperature 1420°C. Samples were analyzed for enrichment (delta; δ) at all timepoints in duplicate and averaged for all calculations to represent fasting and postprandial synthesis.
Fractional synthesis rate (FSR; % pool/day) of both FC and CE were determined to capture total cholesterol synthesis. FC was isolated from plasma and VLDL to describe whole-body and hepatic cholesterol synthesis, respectively, and CE was isolated from plasma only. FC and CE was measured at 0, 4, 8, and 24 hours for estimation of morning (0-4 hours), afternoon (4-8 hours), and total 24-hour synthesis. VLDL-FC was measured at 24 hours only. FC and CE were derivatized by addition of 150 μL acetic anhydride and 40 μL pyridine.11 Samples were analyzed using the Delta V Plus and a DB-5 column (30 m, 0.25 mm i.d., 0.25 μm film) (J&W Scientific). Oven temperature was 140°C, and was increased to 310°C, and 320°C for the post-run. Sample injection size was 1.0 μL, helium flow 1.3 mL/min, and injector temperature 280°C. Samples were analyzed in duplicate, and enrichment (δ) averaged for calculations.
Undiluted plasma for deuterium analysis was filtered by centrifugation in 10K centrifugal filters (VWR International, Mississauga, ON) for 1 hour at 14,000 RPM on a Jouan A-14 centrifuge for isolation of plasma water (PW). Samples were injected on a High Temperature Conversion-Elemental Analyzer (Finnigan TC/EA) with AI/AS 3000 autosampler (Thermo Scientific) coupled to the Delta V Plus. Sample injection was 0.3 μL, oven temperature 90°C, pyrolysis reactor temperature 1400°C, and helium flow 2.0 mL/min. Samples were analyzed at all timepoints and introduced in sequential order of expected enrichment to reduce memory effect. Each sample was injected 4-6 times and the last 3 values averaged for calculations.
ΔδFA and ΔδPW represent changes in FA or PW 2H-enrichment (measured-background). A different constant (C) was used for each individual FA based on 2H incorporation (0.487 for 14:0, 0.449 for 16:0, 0.480 for 16:1, 0.477 for 18:0, and 0.447 for 18:1).10, 12
ΔδFC and ΔδPW represent changes in 2H-enrichment in FC, CE, or PW compared to baseline (0 hours). The constant 0.478 represents maximal ratio 2H incorporation.9
Use of Deuterium Oxide in Subjects With Fluid Overload.
Individuals with cirrhosis often have ascites and peripheral edema, which could affect deuterium equilibration. Nonhepatic edema (e.g., cardiac failure) delays isotopic equilibration13 but evidence in edematous cirrhotic patients suggests that 2H2O equilibration with body fluids occurs as early as 2.5 hours after administration of a single dose.13, 14 It was predicted that the loading plus maintenance doses given here would be sufficient to raise plasma deuterium in a short period of time for reliable calculation of lipid synthesis. In support of this prediction, total 24-hour and change in PW 2H-enrichment from baseline was not different between HCV and control (P = 0.36 both) or postprandial timepoints (P > 0.05 both) (data not shown).
Statistical analysis was performed using GraphPad Prism (v. 5.0c; GraphPad Software). Groups were compared by two-tailed Mann-Whitney nonparametric t tests or by two-way analysis of variance (ANOVA) for postprandial data (significance set at P < 0.05). Correlations between variables were calculated using Spearman's rank test. Data are presented as mean ± standard error of the mean (SEM).
There were no significant differences between groups in most anthropometric markers (Table 1). Resting metabolic rate and waist circumference was higher in HCV subjects, likely because of the greater number of men in the HCV group. Fasting plasma TC and LDL-C concentrations were lower in HCV compared to control, but HDL-C and TG levels were not different (Table 1). Postprandial plasma-TG level was similar between HCV and control subjects (Fig. 1A; P = 0.16). Fasting blood glucose levels were similar between groups, but insulin and homeostatic model assessment (HOMA) index were higher in the HCV subjects (Table 1). Plasma IL-6 was higher in HCV subjects (Table 1) but there were no differences in other inflammatory markers. Total caloric intake was not different between groups (Table 1), but intake of cholesterol and fat was greater in HCV compared to control, and intake of saturated fat tended to be greater in HCV subjects.
Fatty Acid Composition.
Fasting VLDL-TG FA concentration was similar between HCV and control (105 ± 32 μg/mL versus 151 ± 34 μg/mL respectively; P = 0.24). Postprandial VLDL-TG FA concentration was lower in HCV compared to control but not significantly different (Fig. 1B; P = 0.25), whereas plasma-TG levels tended to be higher (Fig. 1A). The difference in VLDL-TG FA concentration between groups despite similar plasma-TG levels is supported by evidence suggesting that in hepatitis there is a reduction in VLDL-TG levels but an increase in non-VLDL-TG, resulting in similar total plasma-TG levels.15 This elevation in non-VLDL-TG may be associated with the association of highly infectious HCV particles with LDL and VLDL.16
Plasma- and VLDL-TG FA composition was similar in both groups except for contribution from polyunsaturated FA (PUFA). Plasma-TG 16:0 was higher in HCV compared to control (33 ± 1% versus 27 ± 1%, respectively; P = 0.02) while 18:2n6 was lower in HCV (11 ± 1% versus 16 ± 1%; P = 0.03), and 18:3n3 tended to be lower (0.62 ± 0.06% versus 1.23 ± 0.22%; P = 0.05). VLDL-TG composition was similar between groups except for 18:2n6 (P = 0.04) and 18:3n3 (P = 0.01), which were lower in HCV compared to control (Fig. 1C; P > 0.05).
Concentration of fasting TPL-FA was lower in HCV compared to control (366 ± 45 μg/mL and 732 ± 101 μg/mL, respectively; P = 0.003). There was no significant difference between groups in individual TPL-FA (data not shown), but 18:1 was higher in HCV (17 ± 1% versus 12 ± 1%, respectively; P = 0.003), while 16:1 tended to be higher (1.0 ± 0.2% versus 0.4 ± 0.1%, respectively; P = 0.07) and 18:2n6 tended to be lower (26 ± 2% versus 21 ± 1%; P = 0.08).
De Novo Fatty Acid Synthesis.
Fasting whole-body DNFA estimated from plasma-TG was significantly higher in HCV compared to control (Fig. 2A; P = 0.03), and ranged from 0.47%-3.4% in HCV and from 0%-2.7% in control. Fasting synthesis of 14:0, 18:0, and 18:1 was not different between groups (P > 0.05), whereas synthesis of 16:0 (4.7 ± 0.8% versus 2.2 ± 0.6%; P = 0.03) and 16:1 (2.3 ± 0.5% versus 0.8 ± 0.2%; P = 0.01) was higher in the HCV group. Postprandial whole-body synthesis of total FA was not different between groups (P > 0.05; data not shown). Rates of lipogenesis for each subject are provided in the Supporting Material for reference. Fasting hepatic DNFA estimated from VLDL-TG FA was significantly higher in HCV compared to control (Fig. 2B; P = 0.03) and ranged 1.35%-4.25% in HCV and 0.43%-2.82% in control. Fasting hepatic synthesis of 14:0 and 18:0 was not different between groups (P > 0.05), but synthesis of 16:0 (P = 0.01), 16:1 (P = 0.006) and 18:1 (P = 0.02) was higher in the HCV group (Fig. 2C). Postprandial hepatic synthesis of total (P = 0.73; Fig. 2D) and individual FA (P > 0.05; data not shown) did not differ between groups. Fasting hepatic DNFA was negatively correlated to dietary fiber intake (r = −0.77; P = 0.02) in the control group. In the HCV group, fasting hepatic DNFA was correlated with HDL-C (r = −1.00, P = 0.02), dietary PUFA intake (r = −1.00; P = 0.02), VLDL-TG 18:3n3 (%) (r = −1.00; P = 0.02), and marginally with fasting plasma-TG (r = 0.90; P = 0.08) and dietary carbohydrate (r = 0.90; P = 0.08).
In the control subjects, lipogenesis measured from plasma-TG was consistently lower than measured in VLDL-TG (Supporting Material), which is expected due to dilution from TG in other lipoproteins. However, in the HCV patients lipogenesis observed from plasma-TG was similar to VLDL-TG. This suggests that there is newly synthesized FA in other lipoproteins contributing to the observed whole-body lipogenesis, supported by the observation of greater non-VLDL-TG concentrations in HCV.15 Future studies of lipogenesis in HCV may need to measure both plasma- and VLDL-TG to capture all newly made FA.
Cholesterol synthesis was significantly lower in HCV compared to control for all fractions measured (P < 0.001 for all) (Fig. 3A). Postprandial FC-FSR was also lower in HCV at 8 hours (P = 0.01) but not 4 hours (P = 0.44) compared to control (Fig. 3B). Postprandial CE-FSR was marginally lower in HCV at 8 hours (0.10 ± 0.05% versus 0.35 ± 0.08%; P = 0.06) but not 4 hours (0.46 ± 0.4% versus 0.11 ± 0.04%; P = 0.42) compared to control. Rates of cholesterol synthesis for each subject are provided in the Supporting Material. Fasting FC-FSR was significantly correlated with dietary PUFA intake (r = −1.00; P = 0.02) and creatinine (r = −1.00; P = 0.02) in HCV subjects, and with fasting TC level (r = −0.72; P = 0.04) in control subjects. Hepatic cholesterol synthesis was not significantly correlated with any variables in control or HCV subjects.
The current work provides evidence that lipogenesis is elevated and cholesterol synthesis is impaired in HCV infection. Although the sample size is limited, the data are the first evidence in humans with HCV and provides support for previous evidence from cellular and animal models of HCV.
Evidence is mounting from multiple sources to suggest lipogenesis is up-regulated in HCV and tied to viral progression.4-6, 17-19 Interestingly, lipogenesis was elevated in these HCV patients despite greater dietary fat intake, which normally suppresses lipogenesis.20 High carbohydrate intake stimulates lipogenesis,21 and in the present study lipogenesis was marginally correlated with dietary carbohydrate in the HCV subjects. FA from dietary fat and lipogenesis can contribute to steatosis, but the deleterious effects of lipogenesis may be particularly critical in the context of HCV, as lipogenesis may induce greater endoplasmic reticulum stress than dietary saturated fat.22 HCV is also associated with insulin resistance, which may independently stimulate lipogenesis.20 It is not possible to determine whether the elevated lipogenesis in the present subjects is due to elevated insulin, HCV, or a combination, but ample evidence from cellular models exists to suggest a primary role for HCV on lipogenesis. Insulin resistance likely exacerbates the HCV-induced influence on lipogenesis, and may explain the association between insulin resistance, disease progression, and nonresponse to therapy in HCV.23
Lipogenesis is also up-regulated in HCC24 and is gaining recognition as a general hallmark of cancer pathogenesis,25 which may lend insight as to the mechanism for enhanced lipogenesis in HCV. The main FA products of lipogenesis are saturated and therefore elevated lipogenesis has the potential to increase the availability of saturated FA for cell membranes, which consequently reduces plasma membrane PUFA content.26 Membrane lipid saturation is observed in cancer cells and is hypothesized to provide protection from chemotherapy agents and cell death by reducing peroxidation, due to replacement of PUFA with saturated FA.27 Enhanced lipogenesis may also contribute to hepatic lipid droplets, which are necessary for HCV assembly.28
Plasma cholesterol concentration was lower in HCV, as is frequently observed,7 and cholesterol synthesis was also suppressed. Cholesterol synthesis may be impaired due to reduced hepatic function or HCV itself. HCV appears to up-regulate cholesterol synthetic genes6, 19 but may interrupt cholesterol synthesis by diverting the intermediate geranylpyrophosphate, which appears to be required for viral replication.17, 29 Therefore, HCV may induce a paradoxical state in which the process of cholesterogenesis is up-regulated but the actual synthesis of cholesterol is impaired. This is suggested by the present data, which clearly show that synthesis of cholesterol is significantly lower in HCV patients. Taken together, interruption of the cholesterol synthetic pathway may explain numerous observations surrounding cholesterol metabolism and HCV. First, it may explain why Fujino et al.6 observed a moderate but negative correlation between plasma LDL-C and expression of HMGCoA-reductase in liver biopsies taken from HCV-infected patients. Second, it may explain why statin therapy can reduce HCV viral RNA levels in patients30 even though synthesis of cholesterol might be suppressed. Finally, it may explain why cholesterol levels rise in patients who have a sustained virologic response to antiviral treatment,7 and why patients with higher cholesterol levels are more likely to respond to antiviral therapy.31
Similar to other patients with cirrhosis,32 plasma lipid PUFA concentration was significantly lower in these HCV subjects. The cause of reduced plasma PUFA levels is unclear but may be due to malnutrition, peroxidation, or incorporation into hepatic lipids.32, 33 Malnutrition is not suspected here because dietary PUFA intake was similar between groups, and a hypermetabolic state was not observed (Supporting Material). Cirrhotic patients have lower plasma vitamin E concentrations despite normal intake and greater susceptibility to plasma oxidation,34 suggesting a greater relative antioxidant requirement. Finally, higher intake of saturated fat in combination with elevated lipogenesis in HCV may increase competition between saturated and polyunsaturated FA for hepatic lipid incorporation. Reduced VLDL-TG 18:2n6 content occurs when saturated fat intake35 or lipogenesis36 is increased, and supported here by a negative correlation between lipogenesis and VLDL-TG 18:2n6 content in HCV subjects.
Regardless of the cause of reduced PUFA status, supplementation of PUFA, particularly fish oil, may be beneficial in HCV patients by way of direct and indirect mechanisms. Directly, PUFA supplementation inhibits progression of HCV17 and HCC.37 Indirectly, PUFA inhibits lipogenesis,38 alleviates impairments in insulin signaling,26 and is antiinflammatory.39 Finally, although not observed in the present study (Supporting Material), PUFA may protect muscle mass in cachexic states,40 which is clinically relevant given that sarcopenia occurs in patients with cirrhosis41 and is associated with mortality.42
The limitations of this study deserve acknowledgment. Although the study sample is small, this is the first quantitation of lipogenesis and cholesterol synthesis in humans with HCV. In addition, IRMS allows for sensitive and precise measurement of lipid synthesis, and the testing day was designed to mimic free-living food intake allowing for translation to clinical populations. Analysis of a larger group of subjects, ideally with a range of cirrhosis and steatosis, is required to further delineate the role of lipid synthesis across the HCV spectrum. Further, HCV genotyping was not performed and would be advantageous for future investigations because lipid aberrations may differ between genotypes. Finally, the patients described here also had HCC, which may influence lipogenesis. However, HCC is a secondary pathology and a focal process as opposed to a primary cause of hepatic damage; therefore, it is unlikely to exert an influence on lipogenesis independently from HCV.
In conclusion, this study provides direct evidence that lipogenesis is elevated but cholesterol synthesis is reduced in patients with HCV compared to healthy humans. Further study is required to determine if these aberrations are related to progression of cirrhosis, steatosis, and viral replication. The potential for targeting lipid metabolism in these patients is high, given the ample evidence of dietary-related methods to reduce lipogenesis. In particular, dietary PUFA supplementation as adjuvant therapy has potential for numerous positive effects for HCV patients but further study is required to delineate exactly what benefits this therapy may hold.
The authors thank the study participants, the assistance of the University of Alberta Clinical Liver Transplant Program, and the technical assistance of Dr. Y.K. Goh, K.A. Dribnenki, and O. Levner.