Correlations of Hepatic Hemodynamics, Liver Function, and Fibrosis Markers in Nonalcoholic Fatty Liver Disease: Comparison with Chronic Hepatitis Related to Hepatitis C Virus

The progression of chronic liver disease differs by etiology. The aim of this study was to elucidate the difference in disease progression between chronic hepatitis C (CHC) and nonalcoholic fatty liver disease (NAFLD) by means of fibrosis markers, liver function, and hepatic tissue blood flow (TBF). Xenon computed tomography (Xe-CT) was performed in 139 patients with NAFLD and 152 patients with CHC (including liver cirrhosis (LC)). The cutoff values for fibrosis markers were compared between NAFLD and CHC, and correlations between hepatic TBF and liver function tests were examined at each fibrosis stage. The cutoff values for detection of the advanced fibrosis stage were lower in NAFLD than in CHC. Although portal venous TBF (PVTBF) correlated with liver function tests, PVTBF in initial LC caused by nonalcoholic steatohepatitis (NASH-LC) was significantly lower than that in hepatitis C virus (C-LC) (p = 0.014). Conversely, the liver function tests in NASH-LC were higher than those in C-LC (p < 0.05). It is important to recognize the difference between NAFLD and CHC. We concluded that changes in hepatic blood flow occurred during the earliest stage of hepatic fibrosis in patients with NAFLD; therefore, patients with NAFLD need to be followed carefully.

Int. J. Mol. Sci. 2016, 17 Table 3. Liver function and hepatic tissue blood flow in each fibrosis stage in NAFLD.

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).

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).

Figure 1.
Comparison of each TBF at initial LC (Child-Pugh A) in NASH-LC and C-LC. PVTBF and THTBF are significantly lower in NASH-LC than in C-LC (p = 0.014, p = 0.048, respectively). HATBF is not significantly different between the LC groups. NS: not significant; PVTBF: portal venous tissue blood flow; HATBF: hepatic arterial tissue blood flow; THTBF: total hepatic tissue blood flow; NASH-LC: liver cirrhosis related to nonalcoholic steatohepatitis; C-LC: liver cirrhosis related to hepatitis C virus.

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). HATBF is not significantly different between the LC groups. NS: not significant; PVTBF: portal venous tissue blood flow; HATBF: hepatic arterial tissue blood flow; THTBF: total hepatic tissue blood flow; NASH-LC: liver cirrhosis related to nonalcoholic steatohepatitis; C-LC: liver cirrhosis related to hepatitis C virus.

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-R 15 were not significantly different between the groups ( Figure 2). hepatitis C virus.

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).   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 (Tables 1 and 2). Furthermore, the diagnostic reliability to detect the advanced fibrosis stage was lower in NAFLD than in CHC (Tables 1 and 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.

Comparison of Typical Cases at Initial LC (Child-Pugh A) in NASH-LC and C-LC
the advanced fibrosis stage were lower in NAFLD than in CHC (Tables 1 and 2). Furthermore, the diagnostic reliability to detect the advanced fibrosis stage was lower in NAFLD than in CHC (Tables 1 and 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.  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, hepatocellular carcinoma, and esophagogastric varices). His fibrosis markers were increased to reflect advanced liver fibrosis. A 75-year-old Japanese man (case 2) was pathologically diagnosed with stage 3 CHC (Desmet's classification [24]). In case 2, TBF was evaluated in the whole liver excluding the region of hepatocellular carcinoma. The WFA + -M2BP of the NASH-LC case was significantly lower than that of the CHC case. TBF was evaluated in both cases. PVTBF and the P/A ratio are lower in NASH-LC (case 1) than in CHC (case 2).  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, hepatocellular carcinoma, and esophagogastric varices). His fibrosis markers were increased to reflect advanced liver fibrosis. A 75-year-old Japanese man (case 2) was pathologically diagnosed with stage 3 CHC (Desmet's classification [24]). In case 2, TBF was evaluated in the whole liver excluding the region of hepatocellular carcinoma. The WFA + -M2BP of the NASH-LC case was significantly lower than that of the CHC case. TBF was evaluated in both cases. PVTBF and the P/A ratio are lower in NASH-LC (case 1) than in CHC (case 2).

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 (Tables 5 and 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-R 15 is the indicator which reflects liver function [30] and the presence of portal hypertension. Furthermore ICG-R 15 is well correlated to the hepatic tissue blood flow [31]. Lisotti et al. reported that the ICG-R 15 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-R 15 correspond to the stage 3 in NASH and LC in CHC (Tables 3 and 4). Yamazaki reported that the average of ICG-R 15 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.

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/m 2 . 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/m 2 (Table 7). * p < 0.05 (unpaired t-test); ** In this study, 58 samples of NAFLD and 72 samples of CH-C were enrolled. The blood sample was taken on the day of the liver biopsy.
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.

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).

Measurements of TIMP-1, HA, PIIIP, and WFA + -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].

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.

Conclusions
It is important to recognize the difference between NAFLD and CHC. We concluded that changes in hepatic blood flow occurred during the earliest stage of hepatic fibrosis in patients with NAFLD and, therefore, patients with NAFLD need to be followed carefully.