1. IntroductionChronic infection with hepatitis C virus (HCV) is a common cause of liver fibrosis, which may progress to cirrhosis [1]. Metabolic diseases such as liver steatosis or diabetes mellitus occur more often in association with chronic HCV infection. Fatty liver and diabetes can be direct effects of viral infection or a secondary event of insulin resistance due to viral infection. HCV patients may also suffer from the metabolic syndrome, which affects about 30% of the normal population. Thus, it is difficult to distinguish between these different causes of liver steatosis and insulin resistance. Hypertension and obesity are indicators of metabolic diseases, and HCV genotype 3 infection in patients with normal body weight is suggestive of viral fatty liver [2,3]. Effective eradication of HCV improves insulin resistance and liver steatosis related to viral infection but cannot resolve fatty liver disease and impaired insulin response as a component of the metabolic syndrome. Metabolic liver steatosis and insulin resistance contribute to the progression of liver fibrosis and patients are at a higher risk for secondary complications such as cardiovascular diseases [3].Direct-acting antivirals (DAAs) have emerged as highly efficient therapeutics, which eliminate HCV within a short time, and a sustained virologic response (SVR) of up to 100% can be achieved [4,5,6]. The model for end-stage liver disease (MELD) score is widely used for the assessment of liver disease severity [7]. Most of the patients followed for up to four years after treatment with DAAs had only marginal improvements of the MELD score, showing that liver dysfunction persists [8,9]. It was also reported that about 30% of patients had an improved MELD score ≥ 3 at sustained virologic response rates at off-treatment week 12 when treated with sofosbuvir/velpatasvir plus ribavirin [10]. Notably, DAA therapy is similarly effective in HCV patients with and without liver cirrhosis [10,11]. The diagnosis of liver cirrhosis in clinical practice is based on physical examination, laboratory parameters and ultrasound. Liver biopsy is usually not necessary to confirm liver cirrhosis [12]. The histological evaluation of a liver biopsy is regarded as gold standard for the staging of fibrosis. However, liver biopsy has several limitations and potential adverse events [12,13]. Therefore, non-invasive methods have been established for the assessment of liver fibrosis. In general, these methods display a high diagnostic accuracy for the exclusion of liver fibrosis and for the diagnosis of advanced liver fibrosis [13]. The fibrosis-4 (FIB-4) score is calculated from age, aspartate aminotransferase (AST; U/L), alanine aminotransferase (ALT; U/L) and platelet count (×109/L) [14]. A high FIB-4 score was validated as an accurate marker of advanced liver fibrosis in HCV patients [15]. Chronic HCV infection is associated with low levels of low-density lipoprotein (LDL) and serum cholesterol. Most, if not all, studies on DAA treatment so far have consistently shown that serum total cholesterol and LDL levels are induced early after the start of treatment [8,16,17,18]. Blood cholesterol exists as free cholesterol (FC) and different cholesteryl ester (CE) species [19,20]. Whether FC and the different CE species equally increase during therapy has not been studied in great detail so far. CE fatty acid composition is of importance for its biological functions. In human plasma, most of the CE species are produced by the activity of lecithin-cholesterol acyltransferase (LCAT) [21]. By esterifying FC derived from peripheral tissues, LCAT promotes reverse cholesterol transport, and thus, has an essential role in whole body cholesterol homeostasis [22]. LCAT forms CE 20:4, 22:5 and 22:6, whereas CE 16:0, 18:1 and 18:3 are derived from liver acyl CoA: cholesterol acyltransferase (ACAT) [23,24,25]. ACAT2 is expressed in hepatocytes, and ACAT2-deficient mice had low serum cholesterol and were protected from atherosclerosis and hepatic lipid accumulation [26]. Further studies showed that ACAT2-derived CE species are predominantly atherogenic blood lipids [27]. LCAT overexpression resulted in higher HDL levels and prevented the development of diet-induced atherosclerosis [24]. The higher unsaturation of LDL-carried CEs was supposed to have beneficial cardiovascular effects [28]. LCAT is expressed in the liver and its activity is decreased in patients with liver cirrhosis [29]. Whether ACAT2 activity is also modified in the cirrhotic liver has not been studied as far as we know. HCV infection induces the hepatic ACAT2 mRNA expression of patients, and upregulation is more prominent in genotype 3- compared to genotype 1-infected patients [30]. CEs of hepatocytes are used for the production of infectious HCV particles, and this is impaired by the inhibition of ACAT [30]. LCAT activity was higher in HCV patients who did not achieve SVR in comparison to those with efficient clearance of the virus, suggesting that LCAT activity increases by HCV infection [31]. Patients with liver cirrhosis have low serum cholesterol, and LDL as well as high-density lipoprotein (HDL) are reduced [32,33,34]. The cholesterol esterification fraction, which was defined as level of esterified cholesterol vs. total cholesterol, is closely related to liver function [35]. In a cohort of patients with mixed disease aetiologies, and a median MELD score of 12, a low cholesterol esterification ratio was a good predictor of mortality [35]. A separate study calculated the CE/FC ratio but could not identify a difference between patients with predominantly alcoholic liver cirrhosis and liver-healthy controls [36].Liver cirrhosis as well as HCV infection are characterized by low serum cholesterol [16,30,31,32]. Whether FC and the different CE species are all similarly reduced has not been analyzed in great detail so far. FC has unique properties and high levels are cytotoxic [37]. An association of serum FC with bilirubin has been described in patients with alcoholic liver cirrhosis [38]. Notably, serum CE levels were not associated with markers of liver cirrhosis in this cohort [38].The effective therapy of HCV elevates serum LDL and cholesterol levels, which are risk factors for cardiovascular diseases [39,40]. The elimination of HCV is, however, associated with a lower risk for cardiovascular diseases [3]. Not all of the CE species seem to contribute to an increased risk, and a higher polyunsaturated-to-saturated CE ratio protected from atherosclerosis [39]. Among the lipids with a strong predictive value for cardiovascular diseases were CE species with a low carbon number and a low double-bond content [41]. The main aim of the present analysis was to study the effect of DAA therapy on serum CE composition. It was also analyzed whether the CE profile differs between HCV patients with and without liver cirrhosis. Therefore, FC and 15 CE species were measured in the serum of HCV patients before therapy, at 4 weeks after treatment start—the time point where LDL levels have been recovered [8,18]—and at therapy end. 4. Discussion

This study shows that DAA therapy increases serum CE levels and leads to a more favorable CE profile. Patients with liver cirrhosis display an adverse CE profile, the relative content of saturated and monounsaturated CEs with short acyl chains is high and that of polyunsaturated CE species with 20 or 22 carbon atoms is low, and this does not change by DAA therapy.

Nowadays, it is well accepted that the effective elimination of HCV causes a rise in serum LDL and cholesterol [17,47,48,49,50]. All but CE 14:0, CE 14:1, CE 15:1 and CE 16:1 were significantly increased at therapy end.LCAT forms CE 20:4, 22:5 and 22:6, whereas CE 16:0, 18:1 and 18:3 are derived from liver ACAT [23,24,25] (Figure 9). This might indicate that ACAT as well as LCAT activity are higher at therapy end—a hypothesis that has to be yet confirmed. However, various other enzymes and pathways have a role in CE composition. Cholesterol-esterifying activity in serum was found to be decreased in patients with acute hepatitis and chronic alcoholic liver disease, and besides LCAT, CE hydrolase seems to have a function herein [51]. Thus, it is currently unclear which pathways are involved. Notably, % CE 16:0 was reduced and % CE 18:3 was induced at therapy end, suggesting that a more favorable CE profile exists when HCV is efficiently cleared.HCV patients have a higher incidence of metabolic diseases, including atherosclerosis [2]. There is evidence that cardiovascular diseases and insulin resistance improve after SVR by DAA therapy [52]. Future studies have to evaluate whether the decline of % CE 16:0 and the higher abundance of % CE 18:3 contribute to these beneficial effects.

The recovery of the CE species in the cirrhosis group was not significant. Total CE levels similarly increased during therapy in cirrhosis and non-cirrhosis patients, and were 119% and 116% higher at therapy end, respectively. This suggests that the elimination of HCV changes the CE levels of both groups. Because of the lower number of patients with cirrhosis, this was not significant in this subgroup.

Viral load did not correlate with FC or any of the CE species. Interestingly, genotype 3 infection reduced CE 15:0 and 16:0 in comparison to 1a infection, and CE 16:0, 18:2 and 20:4 in comparison to 1b infection. These differences did not persist at therapy end, showing that genotype 3 differently affects CE composition in comparison to genotype 1. Genotype 3-infected patients were described to have lower serum LDL cholesterol than genotype 1-infected patients [53]. Yet, CEs such as cholesteryl linoleate were higher in genotype 3 than genotype 1 [53]. The present analysis revealed lower levels of CE 18:2 in genotype 3- than 1b-infected patients, and currently, there is no explanation for these divergent results. The two studies agree that the genotype-related changes of the CE lipidome are not apparent in patients who achieved SVR [53].Besides having low levels of serum cholesterol, patients with liver cirrhosis have a reduced CE/FC ratio. This may be due to a lower activity of enzymes involved in FC esterification. Cholesterol esterification in plasma has been described as a marker for liver function in patients with advanced stages of liver disease [35]. Interestingly, the CE/FC ratio increased during DAA treatment in the cirrhosis and non-cirrhosis group, showing that the cholesterol esterification rate is higher in both cohorts. Liver function does not greatly improve during DAA therapy [7,8] and the MELD score of our patient group did not change [46], indicating that the cholesterol esterification rate is not solely affected by liver disease severity.Notably, serum FC levels did not differ between cirrhosis and non-cirrhosis patients before and after therapy. Pathways contributing to serum FC levels such as reverse cholesterol export [54] are, thus, not grossly impaired in cirrhosis.In particular, CEs with longer acyl chains declined in the serum of patients with liver cirrhosis. In patients with a high FIB-4 score, CE 16:0, 18:2, 18:3, 20:3, 20:4 and 20:5 were low (Figure 9). These species, and CE 18:1, 20:5, 22:5 and 22:6, were reduced in the serum of patients with ultrasound-diagnosed liver cirrhosis. Moreover, CE 18:2, 20:3, 20:4 and 22:6 negatively correlated with the MELD score in patients with liver cirrhosis, further indicating an association with liver disease severity.Shorter CEs are the product of ACAT and the longer CEs from LCAT. Because CE 18:1 as well as CE 22:5 were low in cirrhosis, both cholesterol esterification pathways may be impaired (Figure 9). Decreased LCAT activity has been described in patients with liver cirrhosis [29], but whether ACAT2 activity is also low has not been clarified yet. In addition, ELOVL6, which catalyzes the elongation of saturated and monounsaturated fatty acids with 12 to 16 carbon atoms, was found to be lower expressed in patients with advanced fibrosis [55] (Figure 9). Impaired activity of this enzyme may partly explain the depletion of longer chain fatty acids in CEs, and the CE 18-/C 16 ratio is low in our HCV patients with liver cirrhosis. Notably, the CE 18/CE 16 ratio was modestly higher at the end of DAA therapy, suggesting that HCV infection may also lower the activity of ELOVL6 (Figure 9). The different CE species are, however, not reduced to a similar extent in the serum of patients with liver cirrhosis. The proportion of CEs with short acyl chains and no or one double bond increased, whereas CEs with longer acyl chains and at least two double bonds declined. This did not change at therapy end, showing that liver cirrhosis is associated with a unfavorable CE profile. In patients with decompensated liver cirrhosis mostly because of alcohol abuse, the relative content of total plasma CE 14:0, 16:0 and 18:1 was higher and the relative content of CE 18:2 and 20:4 was lower compared to controls [56], and thus, were similarly altered, as observed in the HCV cohort studied herein. The activity of delta-6-desaturase, an enzyme involved in the synthesis of polyunsaturated fatty acids, was found to be reduced in liver cirrhosis [57], and this may also partly explain this observation.Liver cirrhosis is not associated with a higher risk for atherosclerosis [58] and the pathophysiological role of the altered CE profile has still to be evaluated. A frequent comorbidity of liver cirrhosis is type 2 diabetes [59], but the associations of serum CE species with glucose homeostasis have not been finally resolved.HCV-infected patients with diabetes studied herein had lower serum levels of CE 20:5, 22:5 and 22:6, and % CE 20:5 and 22:6 were reduced. In patients with type 2 diabetes, the proportions of CE 18:0 and CE 20:3n−6 were higher, and those of CE 18:1n−7 and C20:4n−6 were reduced compared to patients with normal glucose metabolism [60]. A further study identified protective associations of CE 18:1n−7 and 18:1n−9 and harmful associations of CE 18:3n−6 and 18:0 with insulin sensitivity and beta-cell function [61]. Current findings on the association of CE species with diabetes are inconsistent and further research is needed. It has to be noted that—in the current analysis—gender, BMI and age were not associated with FC levels or the change in any of the CE species analyzed.In serum, CE species are carried by LDL, HDL and VLDL. LDL cholesterol content is about 2-fold higher than that of VLDL and HDL [20]. CE levels in serum mainly correlated with LDL levels, and thus, serum CE content was mostly related to LDL rather than HDL levels.Interestingly, CE composition did not greatly differ between HDL, LDL and VLDL of healthy volunteers [20] and patients with liver cirrhosis [56]. This suggests that the CE profile of LDL, HDL and VLDL is changed in cirrhosis. How and whether an altered CE c influences the function of lipoproteins needs future investigations.This study has limitations. Total serum CE and FC levels were measured but the composition of individual lipoproteins and LCAT activity were not analyzed. Serum was not collected in the fasted state, and dietary habits of the patients were not documented. However, serum cholesterol levels do not greatly vary during the day [62]. It is, moreover, very unlikely that the identified changes in CE composition achieved by HCV therapy and in patients with liver cirrhosis are explained by different diets. A further limitation is that healthy controls and patients with liver diseases of distinct etiologies were not included.

In summary, the present analysis showed that viral genotype, DAA therapy and cirrhosis differentially affect serum CE species levels. Prospective studies have to evaluate the prognostic value of CE species for cardiovascular diseases, insulin resistance, morbidity and mortality in chronic HCV infection and liver cirrhosis.