Association between Serum Liver Enzymes and Cardiovascular Diseases: A Case-control Study among Adults with Cardiovascular Disease

1. PhD Research Scholar, Department of School of Life and Allied Health Sciences, Glocal University, Saharanpur, Uttar Pradesh, India. 2. Professor and Head, Department of Biochemistry, Manipal College of Medical Sciences, Pokhara, Gandaki , Nepal. 3. Postgraduate Student, Department of School of Health & Allied Sciences, Pokhara University, Pokhara, Gandaki, Nepal. 4. Lecturer, Department of Biochemistry, Manipal College of Medical Sciences, Pokhara, Gandaki, Nepal. 5. Assistant Professor, Department of Biochemistry, Manipal College of Medical Sciences, Pokhara, Gandaki, Nepal. 6. Assistant Professor, Department of School of Life and Allied Health Sciences, Glocal University, Saharanpur, Uttar Pradesh, India.

Abstract

Introduction: Cardiovascular Diseases (CVDs) are the leading cause of global morbidity and mortality. However, their prevalence is disproportionately distributed, with the highest rate in low-income countries, moderate in middle-income countries, and the lowest in high-income countries. Several studies conducted elsewhere have identified that certain liver enzymes in serum could predict incident CVDs in adults.

Aim: To explore the association between liver enzymes and CVDs in adults.

Materials and Methods: This was a hospital-based case-control study conducted at Manipal Teaching Hospital, Pokhara, Kaski, Nepal, from January 2018 to December 2021among 400 adult subjects (200 healthy controls and 200 CVD patients). Socio-demographic, physiological, and biochemical variables were collected with structured questionnaires and appropriate standardised and validated measurement methods. The p-value (two-tailed) <0.05 was considered statistically significant.

Results: The frequency of smoking (p<0.001 and drinking habits (p<0.001), and mean values of Body Mass Index (BMI) (p<0.001), and Systolic Blood Pressure (SBP) (p=0.006), Wait Hip Ratio (WHR), Aspartate aminotransferases (AST), Alanine aminotransferases (ALT), Alkaline phosphatase (ALP), Fasting Blood Sugar (FBS), Glycated Haemoglobin (HbA1C), Creatine Kinase MB (CK-MB), Troponin I (TpI), Total Cholesterol (TC), and Triglyceride (TG) were significantly higher (p<0.001) in CVD patients than in healthy controls. The High Density Lipoprotein Cholesterol (HDL-C), on the other hand, was significantly lower in CVD patients. Only the AST showed significant correlation with the cardiac markers CK-MB and TpI. Logistic regression analysis revealed that aminotransferases was the best predictor (due to differences in odd’s ratio) than ALP for incident CVDs in the adult population.

Conclusion: Present study verifies that aminotransferases, particularly AST, was significantly associated with the incident CVD and thus could potentially be measured together with established cardiac biomarkers for the staging and differential diagnosis of CVDs in Nepali adults.

Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Adult population

The CVDs are the major cause of death globally (1). However, their prevalence is disproportionately distributed, with the highest rate in low-income countries, moderate in middle-income countries, and the lowest in high-income countries (2). Of all CVDs deaths, 43% of deaths occur in low-income countries, 41% in middle-income countries, and 23% in high-income countries (3). The South Asian region including Nepal is considered the hotbed of CVDs in the world (4) and therefore causing considerable economic loss and draining a large chunk of their national GDPs in their health services. Therefore, measures leading to prevention, early detection, and proper treatment of CVDs save the economy of the country and increase the quality and lifespan of the people (5),(6). The risk factors of CVDs include dietary habits, physical inactivity, tobacco use, diabetes mellitus, hypertension, obesity, and high blood lipids (7),(8). Despite early identification, prevention, and treatment of these risk factors, the overall mortality associated with CVDs is still increasing (9).

The liver plays an important role in the metabolism and homeostasis of multiple biomolecules such as amino acids, proteins, carbohydrates, fatty acids, lipids, and lipoproteins (10). For this purpose, the liver utilises, myriads of enzyme systems including AST, ALT and ALP. The serum levels of liver enzymes, particularly AST, ALT, and ALP, have been shown to elevate in many types of liver diseases and thus have been used as markers of hepatic dysfunctions in clinical settings. Many studies have shown that the plasma levels of these enzymes are significantly associated with incident CVDs and therefore could be used as possible risk markers for CVDs (11),(12). However, past studies have shown that there is no proportionate relationship between individual liver enzymes and the risk of CVDs (13),(14). A stratified analysis has shown a positive association between ALT and stroke but a negative association with coronary heart disease (15), while another study has shown that both elevated ALT and AST were associated with CVD mortality (16). Likewise, a population-based study conducted in Iran showed that ALP was positively associated, and ALT was negatively associated with 10-year CVD risk in both genders (17). However, there have been no such previous studies conducted in Nepal that validate the findings of these external studies in Nepali adults with incident CVDs. Therefore, the main aim of this study was to explore the association between liver enzymes and CVDs among the adult from Kaski district, Nepal.

Material and Methods

This was a case-control hospital-based study conducted at Manipal Teaching Hospital, Pokhara, Kaski, Nepal, from January 2018 to December 2021. Before embarking the present study, ethical clearance was obtained from the Institutional Review Committee (IRC), (MEMG/ IRC/ 279/GA), of Manipal College of Medical Sciences. A total of 400 subjects were enrolled, out of which 200 were healthy controls and the other 200 were subjects with incident CVDs.

Sample size calculation:

In present study the prevalence for sample size calculation i.e. prevalence of 5.7% was taken (4).

N=Z² ×p×q / e²

Where:
Z=1.96 (for 95% confidence interval)
p=0.057
q=1 – p
e=0.05 (allowable error of 5%)

Hence, n=1.96² ×0.057×(1-0.057) / 0.05²

=83 in each group

Therefore, minimum sample size=166 but total 400 samples were taken.

Inclusion criteria: The patients who were admitted to emergency department and cardiology unit with the complain of chest pain and history of heart problem were included in this study. The presence of CVDs in these admitted subjects were diagnosed by the treating cardiologists based on complains and history of disease, serum creatine kinase MB (CK-MB), troponin I (TpI) and electrocardiogram findings.

Exclusion criteria: Patients who had CVDs together with acute or chronic liver disease (liver damage, jaundice, hepatitis, cirrhosis of liver), intestinal disease (inflammatory bowel disease), and musculoskeletal diseases (myopathies, muscular dystrophies, and celiac disease) were excluded from the study.

Written informed consent was obtained from each enrolled study participant for interviews, questionnaires, and sample collection.

Baseline variables: Data were collected, regarding age, gender and smoking and drinking habits. WHR, BMI, was calculated (18). The blood pressures of all patients were measured using sphygmomanometer in sitting position.

Biochemical Analysis

Five mL of venous blood samples was collected from each study subject in a plain gel tube with clot activator (micronised silica particles). The collected blood samples were then allowed to clot at room temperature, centrifuged at 4000 rpm for about 10 minutes, and the serum was collected in clean dry serum tubes. The sera obtained were analysed immediately whenever possible or stored at -20°C in case of delay. The serum levels of AST, ALT, ALP, CK-MB, glucose, TC, TG, and HDL-C were measured using a fully automated dry chemistry-based analyser (VITROS^® 350 chemistry system, Ortho clinical diagnostics, UK) while serum TpI was measured using VITROS EciQ Immunoassay analyzer (Ortho clinical diagnostics, UK)-according to the protocols provided by the manufacturer. HbA1c was assayed by using a fully automated Biorad D-10 HbA1c analyser. The processed sera were assayed in a single batch after proper standardisation and quality control of the analysers and using the same lots of reagents. Internal Quality Control (QC) was run every day and the observed values were within ±2SD.

Diagnostic Criteria

CVD was defined based on one or more time findings of elevated cardiac markers, abnormal ECG findings, and echocardiogram. The serum liver enzymes were classified as high when their reported values were above their suggested reference ranges. The diagnostic reference ranges used were as follows: AST >59 U/L in males and >36 U/L in females, ALT >50U/L in males and >35U/L in females, ALP >126 U/L in both males and females (19).

Statistical Analysis

All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 21.0 (SPSS, IBM, Chicago, IL). Continuous data were expressed as mean ±Standard Deviation (SD and 95% CI, whereas categorical data were presented as numbers and percentages. The statistical differences in mean values of continuous variables between healthy controls and CVD subjects were assessed by the Student’s t-test. On the other hand, Chi-square test was used to compare the difference in the frequency of categorical variables between the two study groups. Pearson’s correlation analysis was carried out to estimate the level of correlation between liver enzymes and other baseline variables. Association between liver enzymes and CVDs was examined by multinomial logistic regression analysis. The p-value (two-tailed) <0.05 was considered statistically significant.

Results

A total of 400 subjects were enrolled in the present study, out of which 200 were healthy controls and 200 subjects with CVDs. There were 125 males and 75 females in control group and 130 males and 70 females in the patient group. The details of baseline socio-demographic and biochemical variables are presented in (Table/Fig 1).

The mean ages of healthy controls and CVDs subjects were 53.0±9.7 and 61.3±12.7 years, respectively. The frequency of smoking and drinking habits (p<0.001) and mean values of BMI, SBP, WHR, serum AST, ALT, ALP, glucose, HbA1C, CK-MB, troponin-I, TC and TG were significantly higher in CVD patients while HDL-C (p<0.001) was significantly lower in them. The frequency of elevated liver enzymes was higher in males than females in CVDs, although the difference was not statistically significant (Table/Fig 2).

The results of Pearson’s correlation analysis are presented in (Table/Fig 3). Among the three liver enzymes analysed, it was only AST that showed a significant (p<0.001), and moderate correlation with CK-MB (r=0.448) and troponin-I (r=0.397). The correlations with other baseline variables were weak and not statistically significant (p>0.05).

The different models of adjusted multinomial logistic regression analysis were generated to identify the best predictors of incident CVDs. Present study results showed that liver aminotransferases (AST and ALT) and ALP were statistically significant (p=0.001) in all the models with slightly differences in adjusted odd ratio. Based on the differences in odd ratio, liver aminotransferases (AST and ALT) predict the CVDs better than ALP.

Discussion

The present study revealed that liver enzymes were positively associated with CVDs among Nepali adults. This was the first study from Nepal that provides information on the association between liver enzymes and CVDs. All socio-demographic variables and biochemical parameters were statistically significant (p<0.05) in CVDs compared to healthy controls, except DBP. The levels of liver enzymes were found to be higher in CVDs. Present findings were supported by previous studies (13),(17).

In this study, elevated liver enzymes were statistically higher (p<0.05) in patients with CVDs. The serum levels of AST, ALT, and ALP were about five, two and a half, and one and a half time higher in patients with CVDs than in the healthy control, respectively. Higher levels of liver enzymes (AST, ALT, and ALP) were found in male than female patients with CVDs. Present study findings showed AST were significantly associated with CVDs and showed a positive correlation with age, BMI, CK-MB, troponin-I, FBS, and HbA1C. Present findings were supported by a number of cohort and population-based studies. A cohort study conducted in Korea showed a strong association between AST activity and all causes of mortality (20). Arndt V et al., showed an association between liver enzymes and all-cause mortality in southern Germany (21). A population-based, nationwide cohort study in Korean adults investigated a significant association between AST and Myocadial Infarction (MI) and ischaemic stroke risk (13). A study conducted by Rehman H et al., showed a positive association between AST and CVDs was more common in the Asian population (22). The basic mechanism for AST and CVDs association is mainly hypothetical. The AST elevation is commonly due to liver diseases and Non Alcoholic Fatty Liver Disease (NAFLD) is predominantly associated with CVDs (23). The elevated AST is also associated with cardiovascular risk factors, like obesity, metabolic syndrome, and diabetes mellitus (24).

In present study, ALT showed a significant association with CVDs after adjustment for age, sex, BMI, WHR, FBS, HbA1C, SBP, DBP, TC, TG, smoking habits, and drinking habits. Similarly, ALT showed a positive correlation with age, BMI, CK-MB, troponin-I and FBS. The agreements were found in several studies (21),(25). Choi KM et al., also reported a significant (p<0.001) association between ALT and ischaemic stroke, MI, and total mortality after adjustment for age, sex, alcohol, BMI, exercise, dyslipidemia, hypertension, and diabetes mellitus (13). Ndrepepa G and Kastrati A found, the association between elevated ALT and CVD or mortality was stronger in the Asian population than US or European populations (12). According to a study by Goessling W et al., after adjustment for age, sex, alcohol-intake, smoking, physical activity, waist circumference, triglycerides, SBP, fasting glucose and HDL-C, the association with all-cause mortality and CVD events was attenuated, whereas the association with IHD remained significant (26). Similarly, according to the Fragmingham offspring heart study, there was an increased risk of CVD in age-sex adjusted models HR=1.23 (1.12–1.34); p<0.01 which was attenuated after multivariable adjustment (27). The underlying mechanism for elevated ALT and CVDs mainly due to clinical or subclinical liver diseases (12).

In the present study, ALP also showed a positive significant association with CVDs after adjustment for age, sex, BMI, WHR, FBS, HbA1C, SBP, DBP, TC, TG, smoking habits, and drinking habits. ALP showed a positive correlation with SBP, DBP, troponin-I, and FBS. Kunutsor SK et al., reported that ALP was correlated with several risk factors of CVDs but strongly with age (28). Several studies have shown an association between elevated ALP and CVDs in different populations (15),(29),(30). Many underlying mechanisms are explained for the elevated ALP and CVD association. Inflammation is a common connection between elevated ALP and CVDs. The elevated ALP level may be associated with liver disease (NAFLD) (31). Another possible mechanism, ALP catalyses the hydrolysis of inorganic pyrophosphate, an inhibitor of vascular calcification, causes vascular hardening and enhances the process of atherosclerosis (30),(32).

The strength of this study included an adjustment for well-defined cardiovascular risk factors including age, BMI, lipids, drinking habits, and smoking habits to investigate the relationships. However, some limitations should be considered in the present study.

Limitation(s)

The limitations of this study were a small sample size and that the study was conducted at single centre. Therefore, prospective large-scale studies in the general population are needed to investigate the possible mechanisms between liver enzymes and the incident of CVDs.

Conclusion

Elevated serum aminotransferease, in particular AST, was found to be the strongest predictor of incident CVDs in Nepali adults. Therefore, their measurement together with the established cardiac biomarkers could potentially help in the differential diagnosis of CVDs and ascertain the liver damage which may appear at the last stage of the disease.

Acknowledgement

The authors wish to thank all participants, laboratory staffs, nurses, and faculty members of the cardiology department who were closely associated and contributed in one or the other way to this study.

DOI and Others

DOI: 10.7860/JCDR/2022/54831.16581

Date of Submission: Jan 12, 2022
Date of Peer Review: Feb 06, 2022
Date of Acceptance: Apr 27, 2022
Date of Publishing: Jul 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: No
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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