Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer

Our results confirm that depletion of vitamin A, lycopene, and β-carotene is widespread among patients with chronic HCV infection. This phenomenon seems to occur early in the disease process, even before fibrosis is apparent, and cannot be explained, based on our results, by diet, obesity, alcohol intake, smoking, or insulin resistance. Inverse associations with fibrosis progression were more apparent for serum as opposed to hepatic levels of retinoids and carotenoids, and were especially clear for serum retinol and RBP4. While we found relatively strong correlations between serum and liver tissue for lycopene and β-carotene, hepatic retinyl palmitate was poorly correlated with serum retinol, suggesting differential factors modulating these levels [17, 18]. It is also possible that declines in serum retinoids appear earlier in the disease process than declines in hepatic stores. We further observed that depletion of serum antioxidants is linked to increasing levels of urinary isoprostanes, which are reflective of systemic oxidative stress due to lipid peroxidation. An important finding was that hepatic retinyl palmitate levels were significantly and inversely associated with stellate cell activation, as measured by αSMA expression in liver biopsy specimens. Taken together, results from this cross-sectional analysis support the hypotheses that depletion of retinoid and carotenoid antioxidants occurs early in the disease process and that this depletion parallels an increase in oxidative stress and evidence of hepatic stellate cell activation.

In the present study, the reduced serum retinol levels associated with CLD progression were well above the widely accepted WHO cut-off point of 200 ng/mL for vitamin A deficiency. Moreover, only two of the HCV participants had a positive RDR test, indicating inadequate liver vitamin A reserve. Serum retinol and β-carotene levels in our HCV participants were also generally comparable to the NHANES III participants, a nationally representative sample of the US population [30]. Reduced dietary intake of retinoids and carotenoids do not appear to be responsible for the observed associations with fibrosis stage, which could be the result of diminished storage capacity, increased metabolism or defective mobilization of retinol due to impaired RBP4 synthesis [15, 31, 32]. In any event, a cross-sectional study such as this is unable to determine if antioxidant depletion is a causal factor in fibrosis progression, or simply an epiphenomenon that accompanies progression.

The current study supports prior evidence that serum RBP4 concentrations are inversely related to disease severity in HCV patients [33, 34]. We observed a high correlation between serum RBP4 and retinol (r = 0.78, P < 0.001). Serum RBP4 measured by an enzyme immunoassay might be a feasible and cost-effective alternative for assessing vitamin A status. No association was detected between RBP4 levels and albumin, suggesting that generally decreased hepatic protein synthesis did not contribute to the reduction in RBP4. However, impaired mobilization of hepatic stores could explain the decrease of RBP4 seen in CLD. Increased serum RBP4 has been reported to contribute to insulin resistance associated with type 2 diabetes and obesity, which are possible risk factors for CLD progression [35]. We observed no correlation between RBP4 concentrations and BMI, glucose, or insulin levels. The possible effects of reduced RBP4 levels on insulin resistance among CLD patients might warrant further study.

A small number of studies have evaluated serum and tissue concentrations of dietary antioxidants in patients with CLD; although these have focused on more severe, later stage disease. The lack of correlation between serum retinol and tissue retinyl palmitate could be explained by impaired release of retinol from damaged hepatocytes or by the presence of homeostatic mechanisms that maintain hepatic retinol until vitamin A stores are severely depleted. We did not observe a definite decline in hepatic retinyl palmitate concentrations with increasing disease severity in earlier stages of CLD. Yadav et al. reported lower levels of retinyl esters in 20 HCV patients compared to controls [17]. In our study, however, the lower concentration in controls could have been caused by the degradation of retinyl palmitate into retinol in the cadaver tissue by endogenous esterase. Indeed, we observed higher levels of hepatic retinol in the control subjects (data not shown). It has been reported that patatin-like phospholipase domain-containing protein 3 (PNPLA3) regulates retinyl ester release. Specifically, carriers of the I148M mutation in PNPLA3 have intracellular hepatic retention of retinyl palmitate [36]. Subsequently, Mondul et al. found in NAFLD patients that carriers of the PNPLA3 I148M mutation have lower circulating levels of retinyl palmitate and RBP4 but not β-carotene concentrations [37]. PNPLA3 is also a strong determinant for HCC [38]. Taken together, it is possible that the intracellular retention of vitamin A is contributing to fibrosis progression and HCC. The observed reduction in serum Vitamin A levels may reflect a pseudo-deficiency. Future studies investigating the PNPLA3 genotype in this population may provide further insight. The reason for the positive relationship between hepatic retinyl palmitate and liver enzymes is unclear, but this relationship would also be consistent with mechanisms that favor retention of vitamin A in the presence of early but ongoing liver damage.

Lycopene cannot be converted to vitamin A and thus it presents an attractive potential alternative for antioxidant supplementation in CLD. Adverse effects have not been reported with consuming lycopene supplements or high amounts of lycopene-rich foods [39, 40]. We observed an inverse relationship of serum lycopene levels with fibrosis stage. Moreover, unlike vitamin A, there was also a strong correlation between serum and hepatic lycopene levels, thus indicating that dietary supplementation could result in higher hepatic lycopene concentrations. Yuan, et al. demonstrated an inverse association between lycopene concentration in baseline serum and risk of developing HCC in China. Lycopene is a potent carotenoid antioxidant that is also thought to affect processes related to mutagenesis, carcinogenesis, cell differentiation, and proliferation [41, 42]. Epidemiological data suggest that lycopene may act as a chemopreventive agent for many cancer types such as prostate, breast, and lung [43]. In vitro studies have observed that lycopene can inhibit mouse and human hepatocytes by inducing cell cycle arrest [44, 45]. However, dietary lycopene did not prevent liver cancer in an in vivo model of spontaneous hepatocarcinogenesis [46]. These discrepant results warrant further investigation.

Serum hs-CRP is synthesized by hepatocytes in response to inflammation and is regulated by pro-inflammatory cytokines such as IL-6. In a recent cohort analysis in China, higher serum CRP levels at baseline were associated with liver cancer incidence and death from CLD [47]. As such, we hypothesized that higher levels of hs-CRP would occur with increasing fibrosis stage. However, in this study, hs-CRP concentrations decreased with increasing fibrosis stage. While these findings are counterintuitive, a few smaller studies have suggested similar results [48‐50]. Nasciemento, et al. observed lower hs-CRP to IL-6 ratio compared to controls, suggesting that IL-6 stimulation of CRP production in the liver might be mitigated by HCV [48]. It appears that CRP production is affected early during the natural history of HCV infection, thus negating its utility as a biomarker of inflammation in this population.

F₂-isoprostanes are a sensitive and validated urinary marker of systemic oxidative stress due to lipid peroxidation. In our study, smokers had higher isoprostane levels compared to non-smokers. Our data indicate a positive association between urinary isoprostane concentrations and fibrosis stage. To our knowledge, this extends beyond previous data that have shown patients with CLD and cirrhosis have increased isoprostane levels compared to controls [16]. As hypothesized, we observed a significant inverse association of F₂-isoprostanes with serum retinol, β-carotene, and RBP4 levels. However, tissue retinoid and carotenoid levels were not associated with urinary isoprostane levels with the possible exception of lycopene. Urinary isoprostanes are a measure of total body lipid peroxidation, and therefore do not reliably reflect oxidative stress in the target organ.

As we expected, subjects with moderate to severe fibrosis had higher levels of αSMA expression in hepatic tissue compared to subjects with mild fibrosis or none at all. Thus, it appears that HSC activation might not be apparent, at least by immunohistochemical technique, until substantial fibrosis is present. Our data indicate that vitamin A depletion could occur prior to development of significant fibrosis and evidence of HSC activation. The hepatic vitamin A depletion might be attributable to either loss of storage capacity or increased consumption via autophagy. Increased metabolism of vitamin A droplets through autophagic mechanism generates substrates needed for energy intensive pathways to meet the metabolic demands of proliferation, fibrogenesis, and contractility [51].

This is the first comprehensive analysis to examine the interrelationships among dietary, serum, and hepatic levels of retinoids and carotenoids with early progression of disease in HCV patients. The present study, to our knowledge, is the largest to date to quantitate hepatic retinoids and carotenoids. In addition, the study benefitted from quantitative image analysis that provided objective and reproducible measures of αSMA expression in liver biopsy specimens. However, certain limitations are acknowledged including, a limited sample size for specific subgroups and a cross sectional design that cannot be used to infer causal direction. Unfortunately, we were unable to administer the RDR to more patients due to the additional blood sample required five hours after the initial retinyl ester dose. Additionally, the post-mortem tissue samples of normal liver were not ideal as they could have undergone artifactual changes in retinoid and carotenoid concentrations.

Findings from this study could be applicable to the emerging epidemic of obesity-related non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) [52]. Oxidative stress is believed to play a key role in these disease processes, similar to HCV-related liver disease. Currently, there are no specific therapies for NAFLD beyond dietary modifications and exercise. Lower levels of retinoids have been implicated in NAFLD. Transgenic mice with impaired retinoid signaling develop steatohepatits and eventually HCC [23]. It has also been found that NAFLD patients have higher levels of oxidative stress and lower vitamin A intake, independent of metabolic syndrome status, suggesting that adequate vitamin A intake is important in protecting against oxidative stress in NAFLD patients [53].