To the best of our knowledge, this is the first study that investigated a mutual involvement of a cluster of redox balance homeostasis parameters, lipid parameters/indexes (i.e., CRI-I and CRI-II) and inflammation in NAFLD.
Even though oxidative stress represents the underlying feature of NAFLD, there is no any single biomarker that could be regarded as a gold standard for redox status determination of this metabolic disorder so far (2).
We have shown increased levels of pro-oxidants (i.e., AOPP, TOS and OSI) in subjects with NAFLD. However, the increased levels of antioxidants (i.e., TAS) observed in this study might be in part related to enhanced compensatory mechanism to increased production of ROS.
Since controversial results were obtained by different studies regarding antioxidant levels/activities (4, 14, 15), we have decided to measure the TAS levels which detects simultaneously all antioxidants without elimination of their mutual interactions (16). Similarly, TOS reflects the measure of the overall pro-oxidants, whereas their ratio (i.e. TOS/TAS, as called OSI) represents the overall oxidative stress status. In addition, beside AOPP which reflects the measure of oxidative damage of proteins (9), we have examined pro-oxidant-antioxidant balance (PAB) as a potentially better representative of simultaneous antioxidants and pro-oxidants in the same assay than each pro-oxidant determined separately. It was obtained by the ratio of uric acid and hydrogen peroxide and increased serum values are indicative of higher production of ROS/RNS (10).
Our results are in accordance with Başkol et al. who also reported higher AOPP, TOS and OSI in patients with NASH, although they included a smaller number of participants than our study did (i.e., a total of 28 patients with NASH and 19 healthy controls). Contrary to the results of the current study, they did not observe the difference in TAS levels between examined groups (17).
In addition to oxidative stress, increased CRI-I, CRI-II and hsCRP that are demonstrated in this study are in line with our previous reports of involvement of dyslipidemia and inflammation in NAFLD (18, 19, 20). The current findings are further confirmed in multivariate binary logistic regression analysis thus extending our previous results, since we were limited earlier to confirm the diagnosis of NAFLD by abdominal ultrasound, but only with fatty liver index (FLI), and hepatic steatosis index (HIS), as proxy of NAFLD, both in diabetic and non-diabetic subjects (18, 19, 20).
We have also shown higher HbA1c levels in NAFLD patients which might be explained by higher number of participants with T2DM in this group, compared to non-NAFLD counterparts. Previous cross-sectional and longitudinal studies have confirmed an independent relationship between HbA1c and NAFLD (21, 22). This might explain the tight connection between NAFLD and T2DM since insulin resistance mediated by oxidative stress and inflammation represent the common soil of both disorders. In line with this, we have recently shown an independent association between HbA1c and comprehensive DOI score (i.e. dyslipidemia, oxidative stress and inflammation score) in patients with prediabetes and T2DM (23).
Insulin resistance is regarded to be the hallmark of NAFLD, leading to increased FFA flux in liver. This process originates from increased visceral adipose compartments that secrete a variety of proinflammatory adipokines and cytokines, as well as higher levels od FFA which compromise signalling pathways of insulin (24). Furthermore, mitochondrial dysfuction characterized by increased ROS generation during the process of oxidative phosphorylation and liver fat peroxidation, represent the typical pathophysiological trait in NAFLD (5, 24). Since the insulin's anti-lipolytic effects are attenuated, enhanced lipolysis of TG, increased secretion of FFA and overwhelmed production of ROS lead to structural and functional hepatic changes (5, 24).
Another consequence of insulin resistance status is redistribution of HDL particles to smaller HDL3 ones which exhibit proatherogenic properties. Also, increased synthesis of sdLDL occurs (25). The smaller HDL3 and sdLDL particles are prone to oxidative modifications and can favour atherosclerosis development (26), as well as other organ dysfunctions as a consequence of cytotoxic effects of FFA.
To further explore the interrelationship between oxidative stress, inflammation and metabolic disturbances, respectively and NAFLD, the PCA was applied. This analysis extracted 3 different factors explaining 68% of variance of the examined biomarkers. Oxidative stress-cardiometabolic related factor showed the highest percentage of variance (36%) with positive loadings of AOPP, TG, and HbA1c and with negative loading of HDL-c. The second factor explained 17% of the variance and consisted of Pro-oxidants related factor (i.e., TOS with negative loading and PAB with positive loading), whereas Antioxidants related factor explained 15% of the variance (i.e., TAS with negative loading). Furthermore, Oxidative stress-cardiometabolic related factor (i.e., TG, AOPP, HDL-c and HbA1c) and Pro-oxidants related factor (i.e., TOS and PAB) confirmed significant predictive ability towards NAFLD status, whereas Antioxidants related factor (i.e., TAS) lost its prediction. The obtained results further support the notion that oxidative stress-induced cardiometabolic disturbances are the key components of NAFLD.
Beside the cross-sectional design of the current research which limits us to conclude the causality between examined biomarkers and NAFLD, the single-center study is another limitation since it does not allow us to generalize these results, given the fact that ethnicity and race may also influence the prevalence of NAFLD (3). Thus, the obtained results can not be applied to non-Caucasian population.
Also, enviromental factors such as nutritional habits and/or regular physical activity were not taken into account since both of them may lead to induction of antioxidative enzymes activity, as well to increase in antioxidant molecules synthesis (27).
Nevertheless, the strengths of our study should also be emphasized. Beside the relatively larger sample of participants included as compared to other studies (17), we have examined a variety of parameters of redox homeostasis. Moreover, the current study is the first one that performed PCA to evaluate the mutual involvement of a cluster of redox balance homeostasis and cardiometabolic parameters to gain deeper knowledge into the pathological traits of NAFLD. Finally, lipid indexes, such as CRI-I and CRI-II can add significant contribution in discrimination between NAFLD and non-NAFLD patients.