1. Introduction
While non-alcoholic fatty liver disease (NAFLD) is a wide-spread liver disease, only some patients have progression towards cirrhosis and end stage liver disease (steatohepatitis), while others have a stable non-progressing form (simple steatosis). The risk of progression towards different stages of liver fibrosis is linked to the level of hepatocyte inflammation. Thus, differentiation between simple steatosis (mild/no portal or lobular inflammation) and NASH (ballooning degeneration and lobular inflammation) has been the goal in addressing this risk [
1]. The prevalence of NAFLD is estimated at one third of the general population [
2].
The current gold standard is the liver biopsy, which can identify the fast progressors and differentiate between NASH and NAFLD [
3]. This procedure is, however, an invasive procedure, with significant risks, including bleeding, organ perforation, sepsis, and death. A large retrospective study of 15.181 percutaneous liver biopsies at the Mayo Clinic in 2010 reported 70 cases of bleeding (0.5%) [
4]. The largest retrospective European study by Piccinino et al. in 1986 on 68.276 liver biopsies reported cases of bleeding (hemoperitoneum—0.032%, hematoma—0.006%, hemobilia—0.006%, hemothorax—0.022%), infection (0.01%) and perforation (pneumothorax—0.35%, followed by colon, kidney, and gall bladder puncture. Mortality was evaluated at 0.009%, with six total deaths, all in patients with cirrhosis or neoplasia [
5]. The procedure is also subject to a number of errors, including sampling and interpreting errors, as well as interobserver variability [
6]. The procedure is also rarely accepted by the patient, due to its invasive nature and associated risks.
Numerous tests that identify patients at risk for fast progression were proposed as alternatives to liver biopsy. Two such tests are the methacetin breath test (MBT), which analyzes the microsomal function of the liver, and the octanoate breath test (OBT), which analyzes the mitochondrial activity.
A key component in the fast progression of fibrosis in NASH is the modified hepatic beta-oxidation. Accumulation of fat in the liver results in an increased hepatic beta-oxidation and ketogenesis process [
7]. Sodium octanoate is the sodium salt of octanoic acid. It is a medium-chain fatty acid that was initially validated to be used in a non-invasive breath test to measure gastric emptying of solids [
8]. Octanoate is absorbed in the digestive tract and transported through the portal system to the liver, where it is metabolized by beta-oxidation to acetyl-CoA and CO
2 [
9].
Methacetin, a derivative of phenacetin, goes through the hepatic oxidase system in the liver mitochondria, where it is metabolized to acetaminophen and carbon dioxide [
10]. Previous studies have shown that methacetin can reliably differentiate between healthy controls and patients with cirrhosis [
11,
12,
13,
14,
15]. Some studies have also previously tried to assess the changes in mitochondrial activity with changes in NASH [
16].
Both substances are metabolized by the liver—tied to mitochondrial activity (in the case of methacetin) or tied to beta-oxidation (in the case of sodium octanoate). Both metabolisms have CO2 as an end result, and by marking the CO2 with an isotope (13CO2), we can measure the exact amount resulting from the metabolism we are trying to evaluate and estimate the mitochondrial activity and beta-oxidation of the liver.
Aim
The aim of our study was to comparatively analyze the two liver assessment methods and determine which is more accurate in detecting patients with steatohepatitis and estimating fibrosis.
2. Materials and Methods
2.1. Study Design
We performed a prospective study on patients with non-alcoholic steatohepatitis, between October 2014 and September 2018. A study protocol was applied for each patient, which included a standard clinical exam, blood tests (including hematology, liver function tests, coagulation tests), abdominal ultrasound, 13C-Methacetin Breath test, 13C-Octanaote Breath test, and liver biopsy. All tests were performed within a maximum of 1 week within each other (usually within 2–3 days), except for liver biopsy, where a recent biopsy within a maximum of 1 year was accepted.
2.2. Clinical Exam
The clinical exam consisted of a comprehensive analysis, including medical history, current symptoms, current treatments and allergies, alcohol consumption and abuse assessment (CAGE questionnaire). The physical exam included measurements of height, weight, waist circumference, blood pressure, heart rate, cardiac rhythm regularity, respiratory rate. Measurements were performed with the patient at rest for at least 10 min. Body mass index (BMI) was calculated (kg/m2) as weight (kg) divided by height (m2), and waist circumference (WC) was measured at the midpoint between the lower border of the rib cage and the iliac crest.
2.3. Blood Tests
A standard hemogram was assessed in all patients, including evaluation of thrombocytes, leukocytes, hemoglobin, red blood cell characteristics (mean corpuscular volume and mean hemoglobin concentration).
Biochemistry tests included both transaminases (alanine aminotransferase—ALT, aspartate aminotransferase—AST), serum albumin and serum total proteins, cholestasis enzymes (γ-glutamyl transpeptidase—GGT, total bilirubin, direct bilirubin, alkaline phosphatase), lipid profile (including triglycerides, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol), uric acid, renal function tests (urea, creatinine).
Coagulation tests included prothrombin time (PT), international normalized ration (INR), activated partial thromboplastin clotting time (APTT).
Other tests performed included screening for chronic viral hepatitis (HBs antigen, HCV antibodies), liver carcinoma (alfa-fetoprotein), and other types of chronic hepatitis (anti-mitochondrial antibodies, serum ceruloplasmin, antinuclear antibody, alfa1-antitripsin).
Devices used to analyze the samples included CELL-DYN 370 (Abbot Diagnostics, Chicago, IL, USA), ARCHITECT c 8000 (Abbot Diagnostics, Chicago, IL, USA), ACL TOP 500 (Instrumentation Laboratory, Bedford, MA, USA), Access 2 Immunoassay System (Beckman Coulter, Brea, CA, USA), Dimension RXL analyzer (Siemens, Erlangen, Germany).
2.4. Abdominal Ultrasound
Hepatic steatosis was assessed using the four-point scale of hyperechogenicity: 0 = absent, 1 = light, 2 = moderate, 3 = severe, according to the difference in density between the liver and the right kidney. Spleen volume was estimated by measuring the spleen longitudinal diameter (SLD) corresponding to the maximum length between the two poles of the spleen. The device used was Acuson S2000 (Siemens AG).
2.5. Liver Biopsy
Liver biopsy was performed in all patients, within a maximum of 1 year of the other tests. The procedure was performed by senior physicians, using the Menghini technique with a 1.4 mm diameter needle (Hepafix; Braun, Germany).
Histopathological analysis was performed by an expert (20-year experience) blinded to the patients’ clinical results. Liver biopsy samples accepted for histological assessment had a minimum length of 20 mm and 8 portal tracts.
The SAF classification and FLIP Diagnosis Algorithm were used to establish positive diagnosis [
17].
2.6. Breath Testing Protocols
All breath tests were performed after an overnight fast. During the test, any food consumption and physical activity were prohibited. To avoid cross-contamination, the two breath tests were performed on different days, but no longer than 1 week apart.
For each test, a substrate was administered orally (for the MBT test—100 mg methacetin labeled with stable, non-radioactive isotope 13C solved in 200 mL water, for the OBT test—100 mg octanoate labeled with stable, non-radioactive isotope 13C solved in 200 mL water). A timer was started at the moment of substrate administration, and each patient was asked to exhale at regular intervals into specific collection bags. Each bag was labeled with the patient study ID number, substrate, and time from administration.
In both cases, the first bag was collected before substrate administration (labeled as 0 min), to serve as control. Subsequently, samples were collected at regular intervals: 10, 20, 30, 40, 50, and 60 min from substrate administration for MBT and 15, 30, 45, 60, 120 min from substrate administration for OBT.
The analysis was performed using a 13C/12C Infrared Spectrometer (IRIS Doc, Wagner), within a maximum of 60 min from the last sample being collected. Parameters measured by the device included 13CO2 recovered as a function of time (PDR [%/h]) and cumulative exhaled 13CO2 (cPDR [%]). The analyzer performed all the measurements for a single patient and a single substrate simultaneously and calculated the PDR and cPDR automatically for each point in time.
2.7. Inclusion Criteria
The main inclusion criteria consisted of patients of at least 18 years of age, willing to participate in the study (signed informed consent), with newly established or previously established (within the last 12 months) diagnosis of non-alcoholic fatty liver disease (by histological analysis of liver biopsy sample).
2.8. Exclusion Criteria
Patients unwilling to participate in the study at any point in time were excluded from the study.
An unclear or uncertain liver biopsy result was also considered an exclusion criterion. Other causes of chronic liver disease with necroinflammatory activity also led to the exclusion of the patient from the study: chronic viral hepatitis, alcohol induced liver disease, high alcohol consumption or alcohol abuse (CAGE questionnaire, alcohol consumption estimation of more than 20 mg/day for women and 30 mg/day for men, indirect markers—increased mean corpuscular volume, isolated increase in gamma-glutamyl transpeptidase), hemochromatosis, autoimmune hepatitis, primary biliary cirrhosis, Wilson’s disease, hepatocarcinoma.
The patient’s chronic drug administration was given special interest, and drugs that could cause drug-induced liver disease (highly liver toxic drugs), or that interfered with either the methacetin or octanoate metabolisms, or that could independently cause NAFLD, were carefully analyzed. Patients who required chronic administration of these drugs were excluded from the study. Such drugs included: amiodarone, corticosteroids, methotrexate, stavudine, tetracycline, valproic acid, zidovudine.
Comorbidities that excluded patients from the study included severe COPD (GOLD C or above) and severe asthma as these could potentially interfere with the elimination of the
13CO
2 as well as any type of malabsorption syndrome as these could potentially interfere with the bioavailability of the substrate. Other comorbidities that were considered exclusion criteria included: uncontrolled diabetes (HbA1C > 7%), severe congestive class failure (NYHA class 3 or above)—
Appendix A.
Other exclusion criteria included pregnancy, participating in other clinical trials, hypersensitivity to any of the substrates, significant weight change during the study protocol (defined as >10%), and recent acute disease that required medical or surgical treatment (past 3 months).
2.9. Study Groups
Patients were divided into three groups: group 1 included patients with simple steatosis (liver biopsy result negative for steatohepatitis), group 2 included patients with steatohepatitis but low-grade fibrosis (liver biopsy result positive for steatohepatitis, with F score 0 or 1), and group 3 included steatohepatitis with significant fibrosis (liver biopsy result positive for steatohepatitis, with F score of at least 2). For diagnosis accuracy analysis, group 1 was compared with both groups 2 and 3 together, while for significant fibrosis, diagnosis group 3 was compared with groups 1 and 2 together.
2.10. Statistical Analysis
The results were entered into a table using Microsoft Excel (Microsoft corporation) and analyzed using Excel (Microsoft Corporation), Python with the Pandas, Statsmodels and Scipy libraries, and SPSS version 23 (IBM corporation). Normality of the variables was assessed using histograms and the Shapiro–Wilk test. In univariate analysis, the independent samples t test was used to assess statistical difference. Crosstabulation analysis for sex was performed using the chi-square test. Bivariate analysis was performed to assess the strength of the association between breath test results and SAF score inflammation using Spearman’s rho test. The receiver operating characteristic (ROC curve) was constructed for the best parameters and an optimal cutoff was chosen using the Youden j statistic method. Sensitivity, specificity, positive predictive, and negative predictive values were calculated. The overall accuracy was calculated using the area under the ROC curve (AUROC).
4. Discussion
NAFLD is a continuum of fatty liver injuries characterized by interconnected histological, functional, and metabolic derangements of the mitochondria consisting of ultrastructural lesions, crystalline mitochondrial inclusions, increased mitochondrial volume, reduction of the mitochondrial cristae [
16].
These evolutive and dynamic changes are translated into disfunction of the respiratory chain, followed by reduced energy production and increased oxidative stress [
18].
The aim of the present study is to provide a comparison in terms of significance and accuracy of two noninvasive metabolic tests, used to stratify the patients with fatty liver disease. Moreover, these tests were considered useful tools for the stratification of the prognosis in NAFL patients, being surpassed only by liver biopsy. A supplementary advantage of these tests are the real-time evaluation results, allowing a fast clinical intervention when necessary. Both
13C MBT and OCT belong to the category of dynamic tests able to assess the function of the liver. As long as the
13C MBT evaluates the microsomal function, it is considered a functional mass of the liver and proved to be the highest accuracy on long-term evaluation of the liver disease prognosis [
12].
In the present study, we demonstrated that both OBT and MBT were able to differentiate patients with simple steatosis from those with NASH.
We identified the OBT PDR at 30 min as the best parameter for NASH diagnosis—average values 22.08 (NASH) vs. 17.45 (steatosis) p < 0.001, even though there were significant values of the PDR (%/h) at 15 min, 30 min, 45 min, and 60 min from substrate administration, and of the cPDR at most timepoints after OCT ingestion (p < 0.05).
Our results confirm previous data where we showed that
13C-OBT can identify the patients with NASH through their ability to better/faster metabolize octanoic acid [
19].
This increase of the octanoate metabolization, followed by the
13CO
2 elimination in the exhaled air which is expressed as cumulative percentage of recovery—cPDR, was also confirmed in a previous study of our group [
20].
Similar results regarding the higher terminal oxidation of the
13C-octanoate were reported in patients with the NASH versus control group [
18,
21].
To evaluate the efficacy of OBT in differentiating between patients with NASH and patients with NAFL, we analyzed the area under the ROC curve, which revealed that the PDR at 30 min has the best diagnostic power for NASH—0.855 (95%CI 0.766–0.943 for a cutoff value of 19, with a sensitivity of 80%, a specificity of 83%, a positive predictive value of 85%, and negative predictive value of 77%.
13C-methacetin suffers the process of O-demethylation in the liver’s microsome, resulting in
13CO
2.
13C-MBT was reliable for the discrimination between patients with and without significant fibrosis and cirrhosis [
22].
In our study, when we talked about the significant fibrosis, MBT PDR and OBT were correlated across most timepoints (p < 0.05); however, the most accurate test was MBT cumulative dose at 30 min, with an average of 7.55 in significant fibrosis vs. 12.64 in non-significant fibrosis, p < 0.001. The area under the ROC was best for MBT cPDR at 30 min, with an overall area under the curve of 0.891 (95%CI 0.798–0.984).
13C MBT was better than 13C OCT in stratifying patients with significant fibrosis and cirrhosis (p-values < 0.001 for most timepoints analyzed).
Recent data correlate the excessive fat storage to increased liver stiffness and subclinical microsomal liver dysfunction, which can be detected by
13C MBT. The importance of this finding is the potential identification of increased portal resistance in the absence of fibrosis, due to the higher stiffness of the lipid droplets in comparison to hepatocytes’ cytoplasm [
23], however, this will need to be investigated in future studies. Other potential new directions for research could include the application of artificial intelligence (AI) to these parameters, as some AI algorithms have already been applied to the histopathological exam [
24].
Our study has a few limitations. The exclusion criteria were chosen to, as accurately as possible, assess the underlying metabolic process, with as little interference as possible from other pathologies or complications. Unfortunately, in practice, this means the study has not assessed patients that have common comorbidities with steatohepatitis, including diabetes and other elements of metabolic syndrome. Another significant limitation of the study was the relatively small number of patients included in the study. While for most patients, the biopsy was performed within a few weeks of the other tests, for some, the difference between the biopsy and the other tests was as long as one year.