Diagnostic significance of alanine aminotransferase isoenzymes in alcoholic and non-alcoholic fatty liver cancers Open Access

Alanine aminotransaminase (ALT) is an enzyme involved in amino acid metabolism and gluconeogenesis. ALT has two isoforms, namely ALT1 and ALT2, which are encoded by different genes and expressed in different tissues [1]. ALT1 is a cytoplasmic protein, and ALT2 is a mitochondrial protein [2]. ALT1 is found only in the cytosol and endoplasmic reticulum, whereas ALT2 is mainly localized in the mitochondrial fraction [3]. In a previous systematic and comprehensive study of ALT isoenzymes, we found that ALT1 mRNA was mainly expressed in the liver, kidney, skeletal muscle, myocardium, fat, colon, rectum, small intestine, pancreas, and adrenal gland in the order from high to low expression, while ALT2 mRNA was expressed in the fat, liver, kidney, skeletal muscle, myocardium, adrenal gland, pancreas, lung, and gallbladder in the order of high to low expression. ALT1 protein was highly expressed in the liver, skeletal muscle, kidney, and gastrointestinal tract, moderately expressed in the myocardium, fat, and pancreas, and weakly expressed in the adrenal gland, thyroid, lung, and gallbladder; ALT2 protein level was higher mainly in the fat, kidney, skeletal muscle, myocardium, and pancreas, and lower in the liver and gastrointestinal tissues [4]. The differences in structure, subcellular, and tissue-specific distribution of the ALT isoenzymes suggest that the two isoenzymes may contribute differently to the total ALT activity [2].

The incidence of liver cancer is increasing worldwide and it is now the second leading cause of cancer-related mortality [5–7]. Among the causes of liver cancer, hepatitis B virus is the major cause of liver cancer. However, with the use of hepatitis B vaccine, antiviral drugs, improved living conditions and the development of testing techniques, the spectrum of liver diseases has also undergone significant changes [8]. The proportion of viral liver diseases is decreasing year by year, while the proportion of non-viral liver diseases, including autoimmune liver diseases, drug-induced liver injury, alcoholic liver disease (ALD), and non-alcoholic fatty liver disease (NAFLD), is increasing year by year [9,10]. In most cases, serum ALT activity is used as a sensitive marker for assessing the liver injury in humans [11–13]. ALT is used in combination with AST, ALP, bilirubin (total, conjugated, and unconjugated), γ-glutamyl transferase (GGT), prothrombin time (PT), and international normalized ratio (INR) to determine liver injury (quality and quantity). However, elevated serum ALT activity is also found in patients with non-hepatic damage such as polymyositis, obesity, metabolic diseases, and in healthy populations [14–16]. Notably, ALT2 shares 69% identity and 78% similarity with ALT1 at the protein level [17], resulting in two isoenzymes with similar enzymatic activities. Since the measurement of ALT activity alone does not accurately reflect the true levels of serum ALT1 and ALT2, the measurement of serum ALT isoform expression may be more clinically useful for the diagnosis of the disease than the measurement of total ALT activity. Serum ALT protein concentration has been reported to increase with the severity of liver disease [2], and the determination of serum ALT1 protein concentration may be an effective method for differential diagnosis of inflammation grading and fibrosis staging in patients with chronic hepatitis B [18]. Currently, the use of ALT in both alcoholic and non-alcoholic fatty liver disease is limited to total ALT activity [19], which does not accurately reflect the true changes in serum ALT1 and ALT2 levels. Therefore, the aim of the present study was to investigate the diagnostic value of ALT1 and ALT2 in alcoholic and non-alcoholic fatty liver disease.

Liver cancer and corresponding paracancerous tissue specimens were collected from the general surgery and hepatobiliary surgery departments of the Second Affiliated Hospital of Chongqing Medical University from January 2012 to September 2018. During the data collection process, we were able to simultaneously identify the information about the medical history of individual participants. A total of 25 primary liver cancer cases were confirmed by liver imaging examination, including 9 cases of alcoholic fatty liver and 16 cases of non-alcoholic fatty liver. The following criteria were used for collecting these specimens: no history of diabetes mellitus, and the exclusion of other medical history such as hepatitis B, hepatitis C, myocardial infarction, and skeletal muscle injury. None of the patients received radiotherapy and chemotherapy prior to the surgery, and the stage of the disease was confirmed based on postoperative pathological examination. This study was approved by the Second Affiliated Hospital of Chongqing Medical University ethics committee and all the patients were well informed and have signed the informed consent. Serum samples were collected from the abovementioned patients prior to the surgery, and nine randomly collected serum samples from healthy subjects were used as controls. The samples were stored at −80°C.

RNA was extracted from tissue samples using E.Z.N.A.® Total RNA Kit I (Omega Bio-tek Inc., Norcross, GA, U.S.A.), and cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, Wisconsin, U.S.A.). The quality and quantity of RNA and cDNA were assessed using gel electrophoresis and spectrophotometer (Beijing Haixinrui Technology Co., Ltd., Beijing, China). The mRNA expression levels of ALT1, ALT2, and GAPDH were measured using TB Green™Premix Ex Taq™ Kit (Takara Bio Inc., Japan) and Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, U.S.A.). The following primer sequences were used: ALT1: forward primer, 5′-CGCAGTTCCACTCATTCA-3′ and reverse primer, 5′-GTTCACCACCTCCACATAG-3′ [20]; ALT2: forward primer, 5′-AGACTTCCACATCAACTTCC-3′ and reverse primer, 5′-TCTCAACATCAAGGCACAA-3′ [4], and GAPDH: forward primer, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse primer, 5′-GGGGTCGTTGATGGCAACA-3′ [21]. GAPDH was used as the internal reference control.

High-quality monoclonal antibodies against ALT1 and ALT2 were successfully obtained by our team in previous studies [4,20]. RIPA lysis buffer (Beyotime, Shanghai, China) and phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China) were used to extract the total protein from 50 mg tissue samples, and the protein concentration was assessed using the BCA protein concentration assay kit (Beyotime, Shanghai, China). Serum samples were diluted 20 times with phosphate buffer saline (PBS) and boiled at 100°C for 5–10 min directly for Western blot analysis. For Western blot analysis, samples were separated by electrophoresis on 10% SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes, and incubated with primary ALT1 or ALT2 monoclonal antibodies and secondary antibody (Sigma Aldrich, St. Louis, MO, U.S.A.), and developed using TMB Chromogen Solution (Beyotime, Shanghai, China). The membranes were then exposed to ECL Western Blotting Substrate Reagent (Tsea biotech Co., Shanghai, China). β-Actin was used as the internal control. Western blot images were quantified using ImageJ software.

The paraffin-embedded samples were mounted on slides, fixed at 60°C for 2 h, and then deparaffinized in xylene, and hydrated in a decreasing series of alcohols. Subsequently, the endogenous peroxidase was blocked by washing with 3% hydrogen peroxide for 10 min. Antigen retrieval was performed in a microwave oven for 15 min at 800 W power with the slides immersed in Tris-HCl (pH 9.0). Slides were incubated overnight at 4–8°C in a humidity chamber with ALT1 or ALT2 primary antibody (1:400 dilution) for 12 h. This was followed by three 5-min washes in PBS buffer, incubation with goat anti-mouse IgG conjugated with horseradish peroxidase (1:5000 dilution) for 30 min; and subsequent three 5-min washes in PBS buffer. After that, the slides were incubated with horseradish peroxidase (Beyotime, Shanghai, China) for 30 min and rinsed twice in PBS buffer. The slides were developed with a solution containing PBS and diaminobenzidine (Beyotime, Shanghai, China). The slides were counter-stained with hematoxylin, dehydrated in a gradient of ethanol, and rinsed three times with xylene. The slides were observed under a microscope (Olympus, Japan) to determine the expression levels and distribution of ALT1 and ALT2 in the tissues. Scoring criteria for staining intensity: tissue with almost no yellow color is worth 0 points, light yellow staining is worth 1 points, brownish yellow staining is worth 2 points, brownish staining is worth 3 points. Positive cell ratio score: 0 points for positive cells <5%, 1 point for positive cells <25%, 2 points for positive cells <50%, 3 points for positive cells <75%, and 4 points for positive cells ≥75%. The final score is determined by multiplying the staining intensity and positive cell ratio values: 0 points were scored as (-), 1–4 points as (1+), 4–6 points as (2+), and >7 points as (3+).

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, U.S.A.). The measurement data were first examined for normal distribution. Comparisons between two groups were performed using paired two-tailed t-test if they were normally distributed, and the Mann–Whitney U-test was used for comparison between two groups if they were not normally distributed. The calculation and evaluation of RT-qPCR results were performed using the 2^−ΔΔCt method. P<0.05 was considered statistically significant.

We determined the mRNA expression levels of ALT1 and ALT2 in liver cancer and paracancerous tissues (Figure 1). The results showed that the relative mRNA expression of ALT1 and ALT2 were significantly lower in both alcoholic and non-alcoholic fatty liver compared with paracancerous tissues (P<0.01). However, there was no significant difference in the mRNA expression of ALT2/ALT1 ratio between alcoholic and non-alcoholic fatty liver (P>0.05).

To further verify the expression of ALT1 and ALT2 in liver cancer and paracancerous tissues, the protein levels of ALT1 and ALT2 were analyzed using Western blotting (Figure 2). For liver cancer with alcoholic fatty liver (Figure 2A), there was all no significant difference in the protein expression of ALT1and ALT2 compared with paracancerous tissues (P>0.05). In liver cancer without alcoholic fatty liver (Figure 2B), ALT2 protein expression in cancer tissues was lower than that in the paracancerous tissues (P<0.05), while there was no significant difference in the protein expression in ALT1 between these two tissues (P>0.05). Notably, there was a statistically significant difference in the protein ratio of ALT2/ALT1 between alcoholic and non-alcoholic liver cancer (Figure 2C).

ALT isoenzyme expression was measured by immunohistochemistry in 9 cases of alcoholic liver cancer and paired paraneoplastic tissues and 16 cases of nonalcoholic liver cancer and paired paraneoplastic tissues. The protein expression levels of ALT1 and ALT2 in liver cancer and paracancerous tissues are shown in Figure 3. In liver cancer with alcoholic fatty liver, almost no signal for ALT1 was observed in the carcinoma cells (Figure 3A: (-)), whereas a low-level signal for ALT2 was detected in the cytoplasm of the cancer cells (Figure 3B: (1+)). ALT1 and ALT2 were expressed at higher levels in the paracancerous tissues than in the cancerous tissues (Figure 3C: (3+); Figure 3D: (2+)). In non-alcoholic fatty liver cancer, ALT1 and ALT2 were mainly expressed in the cytoplasm, with moderate expression of ALT1 (Figure3E: (2+)) and strong expression of ALT2 (Figure 3F: (3+)). In contrast with alcoholic liver cancer (Figure 3G: (2+); Figure 3H: (3+)), the expression levels of ALT1 and ALT2 in cancerous tissues were similar to those in paraneoplastic tissues. We found that the relative protein expression of both ALT1 and ALT2 was increased in the tissues of non-alcoholic liver cancer compared with that of alcoholic liver cancer.

To measure the serum ALT levels under physiological and pathological conditions, samples were retrieved from healthy controls and patients with liver cancer prior to the last surgery. All the serum samples were analyzed by Western blotting using anti-ALT1 and ALT2 antibodies as the primary antibodies. The ALT activity in serum was detected using an automatic biochemical analyzer. The serum ALT activity in healthy controls was in the range of 15–45 U/L. Western blot analysis (Figure 4A) revealed that both ALT1 and ALT2 could be detected in the serum of healthy controls and the protein level of ALT1 was significantly higher than that of ALT2 (P<0.001) (Figure 4D). For patients with alcoholic fatty liver cancer (Figure 4B), the protein levels of ALT1 and ALT2 in the serum were increased with the serum ALT activity of 56–550 U/L. ALT1 protein was present in abundance in the serum and contributed greatly to the ALT activity (P<0.001) (Figure 4D). For patients with non-alcoholic fatty liver cancer (Figure 4C), ALT activity ranged from 26 to 545 U/L. Western blot analysis showed that ALT1 and ALT2 proteins were abundant in the serum samples, and both contributed significantly to the total ALT activity (P<0.001) (Figure 4D). Taken together, we found that serum ALT2 to ALT activity was higher in non-alcoholic fatty liver cancer than that in alcoholic fatty liver cancer.

Next, we further analyzed whether there were significant differences between ALT2/ALT1 ratio in normal control group, alcoholic liver cancer group and non-alcoholic liver cancer group. Figure 4D shows that ALT2/ALT1 ratio can well distinguish normal control group, alcoholic liver cancer group and non-alcoholic liver cancer group. Therefore, we believe that ALT2/ALT1 ratio can be used as a serum diagnostic marker for alcoholic and non-alcoholic liver cancer.

Many studies have reported that ALT is significantly elevated in the serum of patients with liver cancer [22,23]; however, the level of serum ALT activity does not necessarily parallel the severity of liver injury [24]. In humans, serum ALT activity has a circadian rhythm and diurnal variation and is influenced by individual factors such as age, sex, body mass index, exercise, coenzyme levels, and medication use [25–28]. Elevated serum ALT activity can also occur in non-hepatic diseases such as muscle injury, thyroid disease, and hemolysis [16,29]. Thus, serum ALT activity may appear falsely high, thereby overestimating the severity of liver injury. In addition, plasma ALT from patients with hepatocellular carcinoma and cirrhosis is characterized by higher levels, higher immunoreactivity, but lower catalytic activity, mainly due to the presence of higher levels of ALT-immunoglobulin A complexes in the plasma [30]. Thus, serum ALT activity may be falsely low, thereby underestimating the severity of liver injury. In conclusion, the determination of serum ALT isoenzyme expression may be more clinically valuable for the diagnosis of disease than the measurement of serum ALT isoenzyme activity. In this study, we systematically investigated the contribution of ALT1 and ALT2 to the overall ALT activity in alcoholic and non-alcoholic fatty liver cancers. At the mRNA level, the expression of ALT1 and ALT2 was lower in both alcoholic and non-alcoholic fatty liver cancer tissues compared with paraneoplastic tissues. At the histone level, the expression of ALT1 and ALT2 was lower in alcoholic fatty liver cancer tissues compared with paraneoplastic tissues, whereas the expression of ALT1 and ALT2 was not significantly different in non-alcoholic fatty liver cancer tissues. At the serum level, total ALT activity was indeed significantly elevated in cancer patients compared with healthy controls. This is consistent with the results of numerous previous studies [31,32]. We found that ALT1 contributed chiefly to the total ALT activity in alcoholic fatty liver cancer, whereas ALT1 contributed only slightly more to the total ALT activity than ALT2 in non-alcoholic fatty liver cancer. Compared with alcoholic fatty liver cancer, ALT2 contributed relatively more to the total ALT activity in non-alcoholic fatty liver cancer. More importantly, ALT2/ALT1 ratio can well distinguish normal control group, alcoholic liver cancer group and non-alcoholic liver cancer group. This analysis would help us in distinguishing alcoholic fatty liver cancer from non-alcoholic fatty liver cancer more quickly and accurately.

Recent studies have shown that ALT is an independent risk factor for the development of hepatocellular carcinoma [33–35]. Kim et al. found that ALT1 could predict the grade of inflammation and stage of fibrosis in patients with chronic hepatitis B [18]. Notably, Fu et al. have reported that ALT1 protein was involved in the development of hepatocellular carcinoma through the p53 signaling pathway [36]. In previous studies regarding on the distribution of ALT2 in normal tissues, we have found that ALT2 protein was expressed at higher levels mainly in the adipose tissue, kidney, skeletal muscle, cardiac muscle, and pancreas [4]. In this study, the results of immunohistochemical analysis showed that ALT2 protein was highly expressed in the tissues of non-alcoholic fatty liver cancer. Moreover, serum ALT2 accounts for a high percentage of the total ALT activity. Therefore, we speculate that ALT2 may play an important role in the development of non-alcoholic liver cancer.

However, our study has some limitations. First, all patients in the validation cohort were Chinese. Second, the number of samples was rather small. Third, ALT isoenzyme activity still needs to be assessed in other cancer diseases.

In conclusion, we systematically elucidated for the first time the contribution of ALT1 and ALT2 to the total ALT activity in alcoholic and non-alcoholic fatty liver cancer. We found that ALT2 was strongly expressed in the non-alcoholic fatty liver cancer tissues and the percentage contribution of ALT2 to the total ALT activity was higher as assessed by serum samples. The ALT2/ALT1 ratio has some diagnostic significance for alcoholic and non-alcoholic fatty liver cancer; however, further studies with more cases are needed. In particular, the mechanism underlying ALT2 elevation in non-alcoholic fatty liver cancer still needs further investigation.

The data used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by National Natural Science Foundation of China [grant number 81101318] and Chongqing Science and Technology Commission [grant number cstc2021 jx j1130038].

Dandan Zhou: Resources, Data curation, Software, Formal analysis, Supervision, Validation, Writing—original draft, Writing—review & editing. Zuowei Yuan: Writing—original draft, Project administration, Writing—review & editing. Xiaoqin Shu: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision. Hejun Tang: Validation, Investigation, Visualization, Writing—original draft, Project administration. Jie Li: Data curation, Software. Yangmin Ye: Investigation, Visualization, Methodology, Writing—original draft. Nana Tao: Conceptualization, Supervision, Validation. Fangzhu Zhou: Validation, Writing—original draft. Jun Zhang: Methodology, Project administration. Jian Zheng: Writing—review & editing. Qian Wu: Resources, Data curation, Investigation. Juan Zhang: Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review & editing.

This study was approved by the Second Affiliated Hospital of Chongqing Medical University ethics committee.

All the patients were well informed and have signed the informed consent.

ALD

alcoholic liver disease

ALT

alanine aminotransferase

GGT

γ-glutamyl transferase

INR

international normalized ratio

NAFLD

non-alcoholic fatty liver disease

PBS

phosphate buffer saline

PT

prothrombin time

RT-qPCR

reverse Transcription quantitative PCR