1. Introduction
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are increasingly recognized as common clinicopathological entities that occur in individuals without significant alcohol use [1]. The former is believed to have a benign clinical course, whereas the latter represents a form of liver injury that carries a risk for progressive fibrosis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) [2,3]. Due to the obesity epidemic and the increasing prevalence of metabolic syndrome, NAFLD and its progressive form, NASH, are seen more commonly in different parts of the world [4,5]. NAFLD has become a serious public health issue not only in Western countries, but also in many Asian countries, including Japan [6,7,8]. NASH is characterized by parenchymal injuries, including macrovesicular steatosis, ballooning degeneration, Mallory-Denk bodies, and inflammation in hepatic lobes [9]. On the other hand, chronic hepatitis C (CHC) is characterized by portal tract infiltration of dense aggregates of lymphocytes with follicle formation, and mild macrovesicular steatosis can be seen in lobules, particular in periportal hepatocytes [10,11]. Thus, the manner of fibrosis progression in NASH is different from that in CHC. Although there is currently no validated test involving serum biomarkers available to diagnose NASH, and histologic evaluation with a liver biopsy remains the gold standard, and screening for fibrosis is recommended in patients with suspected NASH. However, liver biopsy has some clinical problems related to its invasiveness and complications. On the other hand, there are validated tests with serum biomarkers available to diagnose the stage of hepatic fibrosis (e.g., Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA+-M2BP), hyaluronic acid (HA), 7S domain of type IV collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), type III procollagen N peptide (PIIIP), FIB4-index, etc.). Recently, WFA+-M2BP has been reported to be a useful marker for staging in patients with NAFLD [12] and CHC [13,14]. Especially in CHC patients, WFA+-M2BP can be a useful surrogate marker not only as a fibrotic marker, but also for the risk of HCC development [13,14]. However, there are few reports about WFA+-M2BP on the basis of the etiology of chronic liver disease (CLD).
Since the liver receives blood flow from both the portal vein and hepatic artery, which account for 70% and 30%, respectively, this double blood supply mechanism is a specific characteristic of the liver. The portal vein receives the blood supply from the intestine, which engages in metabolism as a functional vessel. Xenon-CT has been established as a non-invasive technique to visualize tissue blood flow (TBF) in the neurosurgical field [15,16]. Xe-CT can also be applied to obtain separate measurements of hepatic arterial and venous blood flow to detect changes in hepatic blood flow (HBF). We previously reported that PVTBF and total hepatic TBF (THTBF) decrease with the progression of liver fibrosis in patients with CHC [17,18] and NAFLD [19,20]. However, few reports have addressed the association between HBF and liver function; no report has examined the progression of liver function according to the etiology of CLD. Moreover we previously reported that hepatic TBF in patients with liver cirrhosis varied according to the etiology of the disease and there is a close correlation between liver function and hepatic blood flow in patients with alcoholic liver cirrhosis [21,22]. In the present study, we investigated the difference in the fibrosis markers between patients with initial chronic hepatitis and those with advanced chronic hepatitis in NASH and CH-C. Furthermore, we attempted to clarify the relationship between hepatic TBF and liver function. It is extremely important to understand the characteristics of CLD progression for the management and treatment of CLD. The aim of this study was to elucidate the difference in disease progression between CHC and NAFLD in CLD by comparing the cutoff values for fibrosis markers and the associations of liver function and HBF.
2. Results
2.1. The Cutoff Value and Diagnostic Ability of Each Fibrosis Marker in NAFLD Patients
The receiver operating characteristic (ROC) curve and the area under the ROC (AUC) for each fibrosis marker predict definitive advanced fibrosis. The AUC values of WFA+-M2BP, TIMP-1, HA, PIIIP, platelet count (Plt), FIB4-index, aspartate aminotransferase-platelet index (APRI), AST/ALT ratio, and ICG-R15 were 0.70, 0.50, 0.87, 0.58, 0.74, 0.77, 0.62, 0.75, and 0.74, respectively (Table 1).
2.2. The Cutoff Value and Diagnostic Ability of Each Fibrosis Marker in CHC Patients
The ROC curve and the area under the ROC (AUC) for each fibrosis marker predict definitive advanced fibrosis. The AUC values of WFA+-M2BP, TIMP-1, HA, PIIIP, Plt, FIB4-index, APRI, AST/ALT ratio, and ICG-R15 were 0.89, 0.84, 0.87, 0.71, 0.82, 0.87, 0.82, 0.62, and 0.86, respectively (Table 2).
2.3. Liver Function and Hepatic TBF in Each Stage of NAFLD Patients
Liver function and hepatic TBF in each stage of NAFLD patients are shown in Table 3. With fibrosis progression, Alb, ChE, TC, PT, Plt, PVTBF, and THTBF decreased significantly (p < 0.001, r = −0.47; p < 0.001, r = −0.52; p < 0.01, r = −0.26; p < 0.001, r = −0.69; p < 0.001, r = −0.66; p < 0.001, r = −0.32; p < 0.01, r = −0.22, respectively). On the other hand, with fibrosis progression, ICG-R15, HA, and WFA+-M2BP increased significantly (p < 0.001, r = 0.58; p < 0.001, r = 0.78; p < 0.001, r = 0.50, respectively).
2.4. Liver Function and Hepatic TBF in Each Stage of CHC Patients
Liver function and hepatic TBF in each stage of CHC patients are shown in Table 4. With fibrosis progression, Alb, Ch-E, TC, PT, Plt, PVTBF, and THTBF decreased significantly (p < 0.001, r = −0.67; p < 0.001, r = −0.65; p < 0.001, r = −0.64; p < 0.001, r = −0.69; p < 0.001, r = −0.74; p < 0.001, r = −0.56; p < 0.001, r = −0.48, respectively). On the other hand, with fibrosis progression, ICG-R15, HA, and WFA+-M2BP increased significantly (p < 0.001, r = 0.39; p < 0.001, r = 0.76; p < 0.001, r = 0.62, respectively).
2.5. Correlation between Hepatic TBF and Liver Function in NAFLD Patients
Correlations between hepatic TBF, as measured by Xe-CT and liver function in NAFLD patients, are shown in Table 5. There were significant correlations between PVTBF and Alb, ChE, TC, PT, ICG-R15, HA, and Plt (p < 0.001, r = 0.53; p < 0.001, r = 0.46; p < 0.001, r = 0.29; p < 0.001, r = 0.40; p < 0.001, r = −0.25; p < 0.05, r = −0.17; p < 0.01, r = 0.25, respectively). There were also significant correlations between HATBF and ChE and HA (p < 0.05, r = 0.21; p < 0.05, r = 0.21, respectively). There were significant correlations between THTBF and ChE, TC, and ICG-R15 (p < 0.001, r = 0.39; p < 0.001, r = 0.34; p < 0.05, r = 0.21, respectively). There were significant correlations between the P/A ratio and ChE, TC, and PT (p < 0.001, r = 0.37; p < 0.001, r = 0.42; p < 0.05, r = 0.17, respectively) (Table 5).
2.6. Correlation between Hepatic Blood Flow and Liver Function in CHC Patients
Correlations between hepatic blood flow and liver function in CHC patients are shown in Table 6. There were significant correlations between PVTBF and Alb, ChE, TC, PT, ICG-R15, HA, and Plt (p < 0.001, r = 0.50; p < 0.001, r = 0.66; p < 0.001, r = 0.66; p < 0.001, r = 0.70; p < 0.001, r = −0.36; p < 0.001, r = 0.37; p < 0.001, r = 0.37, respectively). There was also a significant correlation between HATBF and PT (p < 0.05, r = 0.18). There were significant correlations between THTBF and Alb, ChE, TC, PT, and Plt (p < 0.001, r = 0.42; p < 0.001, r = 0.55; p < 0.001, r = 0.67; p < 0.001, r = 0.37; p < 0.001, r = 0.35, respectively). There were significant correlations between the P/A ratio and Alb and ChE (p < 0.01, r = 0.21; p < 0.001, r = 0.34; p < 0.01, r = 0.27, respectively) (Table 6).
2.7. Comparison of Each TBF at Initial LC (Child-Pugh A) in NASH-LC and C-LC
PVTBF and THTBF were significantly lower in NASH-LC than in C-LC (p = 0.014, p = 0.048, respectively). Hepatic arterial TBF (HATBF) did not differ significantly between the groups (Figure 1).
2.8. Comparison of Each Liver Function Test at Initial LC (Child-Pugh A) in NASH-LC and C-LC
Alb, Ch-E, TC, and Plt were significantly higher in NASH-LC than in C-LC (p = 0.016, p = 0.016, p = 0.004, p = 0.021, respectively). PT and ICG-R15 were not significantly different between the groups (Figure 2).
2.9. Comparison of Typical Cases at Initial LC (Child-Pugh A) in NASH-LC and C-LC
Figure 3 shows cases of the advanced fibrosis stage in NASH and CHC. An 85-year-old Japanese man (case 1) was pathologically diagnosed with Stage 4 NASH (Brunt’s classification [23]). His clinical features were also obviously LC-like (e.g., thrombocytopenia, HCC, and esophagogastric varices). His fibrosis markers were increased, reflecting advanced liver fibrosis. A 75-year-old Japanese man (case 2) was pathologically diagnosed with stage 3 CHC (Desmet’s classification [24]). The WFA+-M2BP was significantly lower in NASH-LC than in CHC (Figure 3). In this way, the cutoff values of fibrosis markers, including WFA+-M2BP, might differ by the etiology of liver disease. The present results showed that the cutoff values (WFA+-M2BP, TIMP-1, HA, and FIB-4 index) to detect the advanced fibrosis stage were lower in NAFLD than in CHC (Table 1 and Table 2). Furthermore, the diagnostic reliability to detect the advanced fibrosis stage was lower in NAFLD than in CHC (Table 1 and Table 2). The reason for this is that the manner of fibrosis progression differs between NASH and CHC. With fibrosis progression, PVTBF gradually decreases in both CHC and NASH. However, PVTBF decreases at an earlier stage in NAFLD than in CHC. This might be attributed to the different manner of fibrosis between NASH and CHC.
3. Discussion
A definite diagnosis of NASH requires liver biopsy, though various non-invasive measures are under development [6]. NASH is characterized by parenchymal injury, including macrovesicular steatosis, ballooning degeneration, Mallory-Denk bodies, and inflammation in hepatic lobes [9]. Fibrosis begins in zone 3 or the centrilobular area of the hepatic lobule. Periportal and bridging fibrosis develop with progression of the disease, and once cirrhosis is established, features of steatohepatitis and perisinusoidal fibrosis may be obscured. It is well known that exercise, itself, is an important factor to treat NASH and, therefore, the role of exercise should be emphasized. Exercise, in fact, improves NASH-related fibrosis markers (collagen 1α1 mRNA, p < 0.05 and fibrosis score, p < 0.01) and the inflammation score; exercise increases the hepatic stellate cell senescence marker CCN1 [25,26].
On the other hand, fibrosis begins in zone 1 or the periportal area of the hepatic lobule in patients with CHC. CHC is characterized by a portal tract that is infiltrated by dense aggregates of lymphocytes with follicle formation, and mild macrovesicular steatosis can be seen in lobules, particularly in periportal hepatocytes [10,11]. Moreover, it has been reported that daily use of recreational drugs, in particular cannabis, has a deleterious effect on the speed of progression of fibrosis and steatosis in patients suffering from chronic hepatitis C [27]. There are other differences between NAFLD and CHC. Previous reports indicated that at the early stages of CLD the numbers of liver monocytes/macrophages were elevated without the evidence of local proliferation, supporting a role for infiltrating monocyte-derived macrophages in disease progression in patients with both CHC and NAFLD. However, CHC and NAFLD differentially affected the circulating monocyte phenotype, suggesting that unique injury-induced signals may contribute to the intrahepatic monocyte recruitment and the systemic activation state. Moreover, it was also shown that monocyte function was similarly impaired in patients with both CHC and NAFLD, particularly in advanced disease [28]. Thus, the manner of fibrosis progression resulting from inflammation could be different between NASH and CHC.
The results of present study showed the relationship between liver function and PVTBF (Table 5 and Table 6). PVTBF was well correlated with hepatic synthesis capacity, which included Alb, ChE, TC, and PT. The reason why liver function tests in NASH was better than that in CHC is suggested the excess energy intake and lipid hypermetabolism [29]. ICG-R15 is the indicator which reflects liver function [30] and the presence of portal hypertension. Furthermore ICG-R15 is well correlated to the hepatic tissue blood flow [31]. Lisotti et al. reported that the ICG-R15 test is an effective tool for assessment of portal hypertension in patients with compensated cirrhosis [30]. We confirmed that the hemodynamic changes occurred earlier in NAFLD relative to CHC. For example, 15% of ICG-R15 correspond to the stage 3 in NASH and LC in CHC (Table 3 and Table 4). Yamazaki reported that the average of ICG-R15 was 15.4% in which the presence of esophageal varices cases [32], and their data, supported our results.
Alteration in hepatic microcirculation in human donor livers with steatosis was first reported during organ retrieval before mobilization by Seifalian et al. [33] using laser Doppler flowmetry. A significant decrease in hepatic microcirculation in liver donors with steatosis was observed in comparison with that in normal liver donors [34]. Experimental studies in animal models with fatty liver showed that steatosis led to reduce hepatic blood flow and microcirculation, and that there was an inverse correlation between the degree of steatosis and both total hepatic blood flow and flow in the microcirculation [30]. The severity of fatty infiltration has a greater effect on the microcirculation than on total hepatic blood flow [35,36]. In spite of steatosis alone, hepatic blood flow reduced. Moreover, hepatic blood flow reduced with fibrosis development, in addition to steatosis [37]. Fat-laden hepatocytes are swollen, and in steatohepatitis, further swelling occurs due to the ballooning of hepatocytes, causing sinusoidal distortion, as visualized by in vivo microscopy, reducing intrasinusoidal volume and microcirculation [38].
In addition to steatosis, a mechanism of decreasing portal blood flow other than steatosis has been reported in NAFLD. In livers with perfusion from cafeteria diet-fed rats, there was increased portal pressure and decreased endothelium-dependent vasodilation. This was associated with decreased Akt-dependent endothelial nitric-oxide synthase (eNOS) phosphorylation and NOS activity. They demonstrated in a rat model of the metabolic syndrome that hepatic endothelial dysfunction occurs before the development of fibrosis and inflammation [39]. Ying-Ying Yang et al. reported that hyperleptinemia increases hepatic endocannabinoid production, promotes hepatic fibrogenesis, enhances the hepatic vasoconstrictive response to endothelin-1, and aggravates hepatic microcirculatory dysfunction. These events subsequently increase intrahepatic resistance and portal hypertension in NASH cirrhotic rats [40].
The present data show that the liver function was better in initial NASH-LC than in C-LC. However, because PVTBF was lower in NASH than in C-LC, portal hypertension might occur at an earlier stage in NASH than in CHC. In fact, portal hypertension occurs without LC in NASH [41,42,43]. Mendes et al. investigated the prevalence of portal hypertension in NAFLD patients, and found that clinical signs of portal hypertension, including esophageal varices, splenomegaly, portosystemic encephalopathy, and ascites, were present in 25% of patients at the time of diagnosis. Furthermore, portal hypertension can occur in a small proportion of patients with mild or no fibrosis and is associated with the extent of steatosis [44]. Brunt et al. reported that hepatic fibrosis in NAFLD patients was found in the pericellular space around the central vein and in the presinusoidal region in zone 3 in the early stage [23]. The pericellular fibrosis in the early stage of NAFLD patients may lead to an elevated portal vascular resistance and result in a change of hepatic blood flow [45]. Therefore, we considered that the hemodynamic changes occurred earlier in NAFLD relative to CHC.
In this study, there are two limitations, such as sampling error during liver biopsy and by permeation of Xe gas. Xe-CT cannot monitor the exact result in the patients with chronic lung disease (e.g., chronic obstructive pulmonary disease, lung cancer) and heart failure, because Xe gas is taken up by the lung via the respiratory tract. On the contrary, we believe that there are also many strong points of Xe-CT which objectively and repeatedly measure hepatic blood flow with reproducibility. Moreover, we have safely performed a Xe-CT for patients with acute or chronic renal failure because there are no complications associated with the contrast agent, such as allergic reactions and radiocontrast nephropathy.
Thus, in the present study, the difference between NAFLD and CHC was investigated based on TBF, fibrosis markers, and liver function. In conclusion, compared to C-LC, PVTBF decreased significantly in the Child-Pugh A stage of NASH-LC, indicating that portal hemodynamic changes could occur earlier in NASH-LC without impaired liver function. Therefore, patients with NASH should be monitored carefully for portal hypertensive complications in the early fibrosis stage.
4. Materials and Methods
4.1. Patients
Between October 2001 and March 2016, 730 patients underwent Xe-CT at the St. Marianna University School of Medicine Hospital. Of the 730 patients, 291 with NAFLD and CHC were enrolled in this study. Liver biopsy was performed for 118 of the 139 NAFLD patients and 106 of the 152 CHC patients. During hospitalization for three days, Xe-CT was performed before or after each liver biopsy. The NAFLD patients included 80 men and 59 women, with a mean age of 53.2 ± 11.2 years and a mean body mass index (BMI) of 28.5 ± 4.9 kg/m2. The CHC patients included 75 men and 77 women, with a mean age of 59.9 ± 11.2 years and a mean BMI of 23.2 ± 3.7 kg/m2 (Table 7).
The diagnosis of NAFLD was based on: (1) substantial alcohol consumption (>20 g/day for women or >30 g/day for men); (2) pathological findings showing characteristics of NAFLD (large-droplet fat deposits, hepatocyte ballooning, inflammatory cell infiltration, and fibrosis around the central vein); and (3) the exclusion of other liver diseases, such as viral hepatitis, autoimmune liver disease, and drug-induced liver injury. The diagnosis of CHC was based on anti-HCV antibodies and HCV-RNA. Patients were excluded for the presence of other causes of liver disease, acute illness, acute or chronic inflammatory or infective diseases, an end-stage malignant disease, or other confounding conditions. Liver biopsy was performed through the right intercostal space under ultrasonography-guided liver biopsy using a 16-gauge needle biopsy kit (Quick-Core® biopsy needle set; Cook Medical, Bloomington, IN, USA). The aims of liver biopsy were to assess fibrosis and steatosis and to exclude other liver disease. Histological diagnosis was confirmed by two experienced pathologists who were blinded to the clinical data. There were 15 patients with nonalcoholic fatty liver (NAFL) who had no fibrosis and inflammatory cell infiltration. The patients with NASH were evaluated on the basis of Brunt’s classification [21,46,47], while those with CHC were evaluated on the basis of Desmet’s classification [22]. Staging fibrosis in NASH based on Brunt’s classification: Stage 1: zone 3 perivenular perisinusoidal/pericellular fibrosis, focal or extensive; Stage 2: as above with focal or extensive periportal fibrosis; Stage 3: bridging fibrosis, focal or extensive; and Stage 4: cirrhosis. Staging fibrosis in CHC based on Desmet’s classification: Stage 0: lack of fibrosis; Stage 1: fibrosis confined to portal tract; Stage 2: bridging fibrosis; Stage 3: bridging fibrosis with structural distortion; and Stage 4: cirrhosis. Clinical liver cirrhosis was defined by the presence of a portosystemic shunt or ascites.
4.2. Xe-CT Theory and Imaging Protocol
As described in previous publications, 25% stable Xe gas was used in conjunction with an AZ-726 Xe gas inhalation system (Anzai Medical, Tokyo, Japan) [48,49]. The wash-in and wash-out periods were both 4 min. The entire liver was CT-scanned at 1-min intervals at four levels, including the porta hepatis (nine scans in total, including the baseline scan). Using an AZ-7000W image processing system (Anzai Medical), PVTBF and HATBF were calculated, and PVTBF and HATBF maps were created. THTBF was calculated as the sum of PVTBF and HATBF, and THTBF maps were also created. The time course change rate for the arterial Xe concentration, which was needed to calculate PVTBF and HATBF, was derived using the time course of the Xe concentration in splenic tissue. An Aquilion CT scanner (Toshiba Medical Systems, Tokyo, Japan) was used, with exposure factors of 120 kV, 150 mA, and 13.8 mGy. All examinations were performed with the patients in the fasting state. Informed consent was obtained from each patient. All study protocols were reviewed and approved by the ethics committee at our institution (approval No. 480, 18 September 2001), and conformed to the ethics guidelines of the 1975 Declaration of Helsinki (Allen, 1991).
4.3. Liver Function Tests and Fibrosis Markers
Liver function tests were measured on admission. Liver function tests included the following parameters: albumin (Alb) (g/dL), cholinesterase (ChE) (IU/L), total cholesterol (TC) (mg/dL), prothrombin time (PT) (%), Plt (×104 μL−1), hyaluronic acid (HA) (ng/mL), Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA+-M2BP) (C.O.I.), and the retention rate of indocyanine green 15 min after administration (ICG-R15) (%). ICG (Diagnogreen®, Daiichisankyo Pharmaceutical Co., Tokyo, Japan; 0.5 mg/kg body weight) was administered via a peripheral vein, and venous blood was sampled before and 15 min after injection. Specimens were analyzed for ICG concentrations on a spectrophotometer (HITACHI, Tokyo, Japan) at 805 nm.
4.4. Measurements of TIMP-1, HA, PIIIP, and WFA<sup>+</sup>-M2BP
For all patients in the cohort, the blood sample was taken on the day of the liver biopsy at the St. Marianna University School of Medicine Hospital. All samples were processed to separate serum and stored at −80 °C. At the time of blood withdrawal, all patients underwent liver biopsy. In this study, 58 samples of NAFLD and 72 samples of CHC were enrolled (Table 7). TIMP-1, HA, and PIIIP were measured using a fully automatic immunoanalyzer (Sysmex Co., Hyogo, Japan). WFA+-M2BP quantification was measured based on a lectin-Ab sandwich immunoassay using a fully automatic immunoanalyzer, HISCL-2000i (Sysmex Co., Hyogo, Japan) [50].
4.5. Statistical Analysis
Each parameter is expressed as the mean ± standard deviation (SD). Spearman’s rank correlation coefficient was used to examine correlations of TBF with progression of fibrosis. The Pearson product-moment correlation coefficient was used to examine correlations between TBF parameters and liver function tests. To assess the utility of each fibrosis marker to distinguish the advanced fibrosis stage, the sensitivity and specificity were calculated for each value, and then ROC curves were constructed by plotting the sensitivity against the reverse specificity (1-the specificity) for each value. We used Student’s t-test, which was two-tailed and performed by the statistical software GraphPad Prism (GraphPad Software, San Diego, CA, USA). p-Values of <0.05 were considered significant.