International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa SOCIAL AND BEHAVIORAL SCIENCES. Health Care Sciences ORIGINAL RESEARCH Role of Lipid Profile, Apolipoproteins, and Their Ratio for Prediction of Cardiovascular Disease in Essential Hypertension Authors’ Contribution: A – Study design; Dubey R. 1 ABEFG , Baghel D. S. 1 ABCDE , Gaikwad K. 1 ABCDEF , B – Data collection; Rathore V. 1 ABCDEF , Saxena R. 1 BEF , Ansari Y. M. 1 BEF C – Statistical analysis; 1 Shyam Shah Medical College, Rewa, India D – Data interpretation; E – Manuscript preparation; F – Literature search; Received: 06.05.2023; Accepted: 07.06.2023; Published: 30.06.2023 G – Funds collection Abstract Background and Dyslipidemia is a risk factor for cardiovascular disease, and lipid metabolism Aim of Study: changes are linked to essential hypertension. The aim of the study: to investigate the significance of lipid parameters, apolipoproteins, and their ratio in predicting cardiovascular disease among individuals with essential hypertension. Material and Methods: 250 patients with essential hypertension and 250 healthy control subjects were enrolled in this case-control study and their serum lipids and apolipoproteins were analyzed. Differences between cases and controls were examined using independent sample t-test and, a p-value <0.05 was considered significant. Results: In the essential hypertensive group, fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), very low- density lipoprotein cholesterol (VLDL-C), apolipoprotein B100 (Apo B100) and Apo B100/Apo A1 ratio were increased significantly compared to control subjects. Essential hypertensive patients had significantly decreased levels of apolipoprotein A1 (Apo A1) and high-density lipoprotein cholesterol (HDL-C) compared to controls. Moreover, age, body mass index (BMI), FBG, TC, TG, LDL-C, and VLDL- C, as well as the Apo B100/Apo A1 ratio, were significantly positively correlated with both systolic blood pressure (SBP) and diastolic blood pressure (DBP), but HDL-C and Apo A1 were significantly negatively correlated in essential hypertensive subjects. There was a significant positive correlation between apo B100 and SBP in people with essential hypertension. Apo B100 and DBP showed a positive association, however, it was not statistically significant. Conclusions: Essential hypertensive people with dyslipidemia and an elevated Apo B100/Apo A1 ratio are at an increased risk for the development of cardiovascular disease. Keywords: essential hypertension, dyslipidemia, apolipoproteins, cardiovascular disease, systolic blood pressure, diastolic blood pressure Copyright: © 2023 Dubey R., Baghel D. S., Gaikwad K., Rathore V., Saxena R., Ansari Y. M. Published by Archives of International Journal of Science Annals DOI and UDC DOI https://doi.org/10.26697/ijsa.2023.1.3 UDC 616.411:616-079 Conflict of interests: The authors declare that there is no conflict of interests Peer review: Double-blind review Source of support: This research did not receive any outside funding or support Information about Dubey Rahul – https://orcid.org/0009-0001-2123-9470; Postgraduate Student, the authors: Department of Biochemistry, Shyam Shah Medical College, Rewa, India. Baghel Drutpal Singh – https://orcid.org/0009-0007-8078-7454; Doctor of Philosophy in Medical Biochemistry, Professor, Shyam Shah Medical College, India. Gaikwad Kapila – https://orcid.org/0000-0002-4265-936X; MD, Associate Professor, Department of Biochemistry, Shyam Shah Medical College, Rewa, India. Rathore Vedika (Corresponding Author) – https://orcid.org/0000-0001-9803-7052; ved_sin26@rediffmail.com; Doctor of Philosophy in Medical Biochemistry, Associate Professor (Designated), Shyam Shah Medical College, Rewa, India. Saxena Ravindra – https://orcid.org/0000-0003-2655-8531; Postgraduate Student, Department of Biochemistry, Shyam Shah Medical College, Rewa, India. Ansari Yar Mohammad – https://orcid.org/0009-0004-6371-9984; Postgraduate Student, Department of Biochemistry, Shyam Shah Medical College, Rewa, India. 32 International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa Introduction The prevalence of hypertension has increased to the B100/Apo A1 ratio) for the prediction of cardiovascular point where it is now a serious public health issue disease in essential hypertension. worldwide. It is the leading cause of chronic illness The aim of the study. To investigate the significance of treated in primary care clinics and the most common lipid parameters, apolipoproteins, and their ratio in type of non-communicable disease (Kossaify et al., predicting cardiovascular disease among individuals 2014). In 1975, there were 590 million people (14.5%) with essential hypertension. with high blood pressure around the world. This number went up to 1.13 billion (15.3%) in 2015. The number of Materials and Methods people with high blood pressure is expected to rise to This case-control study was conducted in the 1.56 billion by 2025 (Forouzanfar et al., 2017). In India, Department of Biochemistry, Shyam Shah Medical the prevalence of hypertension among adults is College, Rewa, Madhya Pradesh, India over 12 months generally over 30%, with 34% in urban areas and 28% after receiving ethical clearance from Institutional in rural areas (Anchala et al., 2014). Ethical Committee. A total of 500 subjects of either sex Ninety percent of those who are diagnosed with were selected for the present study. Of these, 250 were hypertension have essential hypertension, which is patients with essential hypertension and 250 were defined as having high blood pressure for no obvious healthy control subjects. 250 patients of essential reason. Idiopathic persistent elevation of the systemic hypertension (of either sex) of the age group 35-75 years arterial pressure characterizes essential hypertension were selected from the OPD of Medicine ward of Shyam (Kossaify et al., 2013). In addition to dietary patterns, Shah Medical College and associated Hospital, Rewa, variables such as obesity, smoking, alcohol intake, and Madhya Pradesh, India. The JNC 7 (Joint National dyslipidemia are key contributors to the development of Committee on Prevention, Detection, Evaluation, and hypertension (Bhavani et al., 2003). Treatment of High Blood Pressure) criteria were used to Dyslipidemia arises as a result of alteration in lipid determine which cases of essential hypertension should metabolism and is considered a risk factor for be included (Chobanian et al., 2003). 250 normal atherosclerotic cardiovascular disease (Hussain et al., healthy subjects of the same age group with no 2019). It is well-established that hypertension is linked symptoms and signs suggestive of hypertension and no to disturbances in lipid metabolism, which in turn lead family history of the hypertensive disease were selected to abnormalities in blood lipid and lipoprotein levels. from in and around the hospital as controls. Informed The prognosis of hypertensive patients is also written consent was obtained from each participant once significantly hampered by the presence of they had been fully explained about the study. hyperlipidemia, as has been well reported (Harvey et al., Patients with the following disease/condition were 1990). A tight relationship between dyslipidemia and excluded from the present study: secondary hypertension has been suggested by several researchers hypertension, severe hepatic failure, renal failure, (Nayak et al., 2016; Osuji et al., 2012). However, unstable cardiovascular condition, past incidences of conventional lipid biomarkers do not provide cerebrovascular conditions, collagenous tissue disease, sufficiently reliable measurements of dyslipidemia. malignancy, thyroid disease, severe depression, Low-density lipoprotein cholesterol (LDL-C), very low- dementia, and diabetes mellitus. Pregnant women were density lipoprotein cholesterol (VLDL-C), and also excluded from the present study. intermediate-density lipoproteins (IDL) all contain The standard apparatus was used to take measurements transporting molecules like apolipoprotein B (Apo B), of both height and weight with the subjects dressed in so determining these molecules allows for a more minimal clothing and barefoot. Calibrated electronic accurate estimation of atherogenicity than the traditional weighing scales were used for the measurement of lipid parameters. Apolipoprotein A1 (Apo A1) is a key weight whereas height was measured to the nearest component of HDL-C, the antiatherogenic lipoprotein. centimeter using a portable stadiometer. Body mass Apo A1 is favored over high-density lipoprotein index (BMI) was calculated as weight in kilograms, cholesterol in predicting cardiovascular diseases (HDL- divided by height in meters squared (kg/m2). The same C). The Apo B/Apo A1 ratio thus appears to be a more person took all of the anthropometric measurements. precise and comprehensive biomarker of lipid Following a resting period of 10 minutes, both the metabolism and cardiovascular disease prediction systolic and diastolic blood pressures were measured (Nurtazina et al., 2020). using a mercury sphygmomanometer following an Multiple studies have elucidated that lipid markers play accepted medical practice. role in essential hypertension (Bhavani et al., 2003; Under aseptic conditions, about 05 ml of fasting venous Osuji et al., 2012). However, the predictive value of blood was drawn from both patients with essential apolipoproteins and their ratio (Apo B100/Apo A1 ratio) hypertension and control participants, and the sample in cardiovascular disease risk assessment is still not well was distributed into two tubes based on the analysis to recognized. Because of the paucity of information on be done. Approximately 2 ml of blood was drawn into a apolipoproteins in essential hypertension, we undertook fluoride bulb to determine fasting plasma glucose and this study to better understand the role of lipid the remaining 03 ml blood sample was dispensed into parameters, apolipoproteins, and their ratio (Apo the plain tube for analysis of lipid parameters and apolipoproteins. After drawing blood, the samples were 33 International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa centrifuged for 10 minutes at 3000 rpm to get sample t-test”. The chi-squared test (χ2 test) was applied serum/plasma. The routine biochemical parameters to the categorical information. To ascertain the were analyzed by standard methods using a Biosystem correlation between important parameters, Pearson’s BA-400 chemistry analyzer. Apo B100 and Apo A1 correlation coefficient was determined. Significant was were measured by turbidimetric immunoassay, endpoint defined as the p-value is less than 0.05. method. Low-density lipoprotein and very low-density lipoprotein cholesterol were calculated using Results Friedewald’s equation (Friedewald et al., 1972). Table 1 shows the baseline characteristics of the studied Statistical analysis subjects. Patients with essential hypertension and those The data were analyzed using Statistical Package for serving as controls were statistically indistinguishable Social Science version 20 (IBM, SPSS Statistics 20, from one another in terms of age and gender. Body mass Armonk, NY, USA) and results were presented as index, SBP, and DBP were statistically significantly mean±SD values. GraphPad Prism 5 was used to create increased in essential hypertension cases compared to the graph. Statistical differences between cases and controls. controls were examined using the “student independent Table 1 Baseline Characteristics of Studied Subjects Variables Controls Cases Age (years) 48.24±11.23 48.70±11.82NS Sex (M/F) 113/137 114/136NS BMI (Kg/m2) 21.89±1.00 27.60±1.68** SBP (mmHg) 115.32±3.95 155.35±7.40** DBP (mmHg) 76.12±4.95 96.03±5.57** Note. NSNot significant (p>0.05); ** Significant at p<0.001; BMI=Body mass index; SBP=Systolic blood pressure; DBP=Diastolic blood pressure. Table 2 shows fasting blood glucose and lipid profiles in significantly reduced in essential hypertension subjects. the studied subjects. In essential hypertension subjects, Apolipoprotein B100 was increased in essential fasting blood glucose (FBG) and all the lipid parameters hypertension subjects whereas Apo A1 was reduced i.e. TC, TG, LDL, and VLDL except HDL were compared to normal healthy control subjects and these increased compared to control subjects and were differences were statistically significant. statistically significant whereas HDL was statistically Table 2 Fasting Blood Sugar and Lipid Profile in Studied Subjects Variables Controls Cases FBG (mg/dl) 85.08±6.23 95.01±7.22** TC (mg/dl) 170.74±12.40 220.57±11.48** TG (mg/dl) 135.37±7.77 236.32±47.10** HDL-C (mg/dl) 52.83±4.67 37.34±3.98** LDL-C (mg/dl) 90.83±13.35 135.97±11.63** VLDL-C (mg/dl) 27.07±1.55 47.26±9.42** Apo A1(mg/dl) 128.96±25.73 87.83±4.14** Apo B100 (mg/dl) 99.50±22.44 149.87±11.30** Note. **Significant at p<0.001; FBG= Fasting blood glucose; TC=Total cholesterol; TG=Triglyceride; HDL-C=High density lipoprotein cholesterol; LDL-C=Low density lipoprotein cholesterol; VLDL-C=Very low density lipoprotein cholesterol. Figure 1 Comparison of Apo B100/Apo A1 Ratio Figure 1 shows the comparison of the Apo B100/Apo between Cases and Controls A1 ratio between essential hypertension subjects and 2.0 ** controls. Essential hypertension patients had Controls significantly increased levels of Apo B100/A1 ratio 1.5 Cases Mean values compared to control subjects. **Significant at p<0.001 Table 3 shows the correlation of studied parameters with 1.0 systolic blood pressure and diastolic blood pressure in 0.5 essential hypertension subjects. 0.0 Apo B100/Apo A1 34 International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa Table 3 Correlation of Studied Parameters with Systolic Blood Pressure and Diastolic Blood Pressure in Essential Hypertension Subjects With SBP With DBP Variables r-value p-value r-value p-value Age 0.299 0.000** 0.225 0.000** BMI 0.635 0.000** 0.527 0.000** FBG 0.393 0.000** 0.286 0.000** TC 0.540 0.000** 0.388 0.000** TG 0.623 0.000** 0.462 0.000** HDL -0.449 0.000** -0.350 0.000** LDL 0.183 0.004** 0.129 0.042* VLDL-C 0.623 0.000** 0.462 0.000** Apo A1 -0.401 0.000** -0.301 0.000** Apo B100 0.148 0.019* 0.111 0.080NS Apo B100/Apo A1 0.306 0.000** 0.232 0.000** Note. NSNot significant (p>0.05); **Correlation is significant at the 0.01 level (2-tailed); *Correlation is significant at the 0.05 level (2-tailed); BMI=Body mass index; SBP=Systolic blood pressure; DBP=Diastolic blood pressure; FBG= Fasting blood glucose; TC=Total cholesterol; TG=Triglyceride; HDL-C=High density lipoprotein cholesterol; LDL-C=Low density lipoprotein cholesterol; VLDL-C=Very low-density lipoprotein cholesterol. In essential hypertension participants, age, BMI, FBG, other cardiovascular illnesses in older individuals as lipid measures such as TC, TG, LDL, and VLDL, as well compared to younger people. There is an upward trend in as the Apo B100/Apo A1 ratio, were significantly the prevalence of hypertension and vascular stiffness as positively associated with both SBP and DBP, but HDL people age (AlGhatrif et al., 2013; Ferreira et al., 2012). and Apo A1 were significantly negatively correlated. In Additionally, compared to controls, the hypertension persons with essential hypertension, there was a participants in this study had statistically significantly significant positive correlation between Apo B100 and higher BMI, which is consistent with studies done by SBP. Despite a favorable correlation between Apo B100 Nayak et al. (2016); Osuji et al. (2012); Sur et al. (2015). and DBP, it was not statistically significant. Moreover, BMI was positively related to both SBP and DBP in essential hypertension subjects. This is due to the Discussion association between a greater BMI and a higher plasma This case-control study took place in a hospital setting volume and cardiac output. Therefore, obesity is a risk and included patients with essential hypertension. In the factor for hypertension. Losing weight has a significant current study, an attempt was made to describe the influence on reducing cardiovascular morbidity and abnormality of lipid parameters, apolipoproteins, and mortality in hypertensive people, including stroke, heart their ratio (Apo B/Apo A1 ratio) among patients with attack, and heart failure (Linderman et al., 2018). essential hypertension in a central Indian setting. In this study, essential hypertension participants had The present study found that both systolic and diastolic significantly higher fasting blood sugar levels than blood pressures were significantly higher in participants control subjects. This is following the study carried out with essential hypertension compared to controls. This is by Nayak et al. (2016), who reported statistically in line with previous studies (Mahapatro et al., 2020; significant increased levels of fasting blood sugar in both Nayak et al., 2016; Osuji et al., 2012; Pyadala et al., 2017; stage I and stage II hypertensive subjects. Furthermore, Sur et al., 2015). As blood pressure rises, so does the in our study, we found a significant and positive chance of cardiovascular events; the more hypertensive a correlation of fasting blood sugar with both systolic and person is, the greater the likelihood that he/she may suffer diastolic blood pressure in essential hypertension from cardiovascular disease. Since there was no subjects. As the fasting blood glucose level increases as statistically significant age difference between the people a result of metabolic disorders, obesity, and with essential hypertension and the controls, it can be hyperglycemia with insulin resistance, the renin- concluded that the study participants were age-matched. angiotensin system (RAS) may undergo alterations. This In hypertensive participants, however, age was found to may have an effect on the patient’s blood pressure (Jia et have a positive and statistically significant relationship al., 2016; Zhou et al., 2015). with both systolic and diastolic blood pressures. Systolic Alterations in lipid metabolism leading to abnormalities blood pressure increases with age, which may be caused in blood lipid and lipoprotein levels have been related to by an increase in artery stiffness brought on by hypertension. It has also been demonstrated that atherosclerotic changes to the arterial wall. Numerous hyperlipidemia dramatically worsens the prognosis in epidemiological studies have emphasized the link hypertensive individuals (Harvey & Beevers, 1990). An between arterial stiffness in hypertension patients and abnormality in blood lipid and lipoprotein levels (also 35 International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa known as dyslipidemia) is a major, controllable risk A 7-year follow-up study on Finnish males revealed that factor for cardiovascular disease (CVD) (Kannel et al., dyslipidemia, a component of the metabolic syndrome, 1971). Abnormal serum lipid and lipoprotein levels have foreshadowed the onset of hypertension (Laaksonen et been identified as independent risk factors for essential al., 2008). Furthermore, Halperin et al. (2006) have hypertension, giving rise to the term “dyslipidemic shown that hypertension is brought on by dyslipidemia in hypertension” (Williams et al., 1988). In subjects with people who appear to be in good health. According to essential hypertension, we found that all of the lipid Hausmann et al., people who have elevated TG levels parameters (total cholesterol, triglycerides, low-density along with low HDL-C have more widespread coronary lipoprotein cholesterol and very low-density lipoprotein atheromas than people who only have elevated LDL-C cholesterol) were significantly higher than in controls, (Hausmann et al., 1996). except for HDL, which was significantly lower. These Numerous studies have demonstrated the significance of results are consistent with the studies done by Mahapatro apolipoproteins- Apo A1 & Apo B100, the two main et al. (2020) and Nayak et al. (2016). Osuji et al. (2012) apolipoproteins for lipid transport in the processes of also found such a type of dyslipidemic pattern in atherosclerosis and its consequences (Luc et al., 2002; hypertensive subjects compared to control subjects. In Meisinger et al., 2005; Walldius et al., 2001; Yusuf et al., addition, we found a significant positive association of 2004). However, the association between apolipoprotein TC, TG, LDL-C, and VLDL-C with both systolic and levels and the risk of hypertension has only been diastolic blood pressures in essential hypertension discovered in a few studies. Nayak et al. (2016) observed subjects whereas HDL-C was significantly negatively a non-significant fall in the value of serum Apo A1 in the associated with SBP and DBP in cases of essential hypertensive patients when compared to controls hypertension. Hypertension and dyslipidemia are both whereas a significant increasing trend was observed in well-known contributors to the development of the levels of Apo B100 from the control group to Stage I cardiovascular disease (Carmena et al., 2004; Gotto, and Stage II hypertensive patients reflecting its 2005). Endothelial dysfunction, which is exacerbated by contributing role as a cardiovascular risk marker. dyslipidemia, is a major contributor to the etiology of Consistent with these results, we found that people with several cardiovascular disease risk factors, including essential hypertension had significantly higher levels of atherosclerosis, thrombosis, insulin resistance, and Apo B100 and lower levels of Apo A1 than controls. In hypertension. Endothelial cells are known to be addition, apo A1 was significantly inversely correlated negatively affected by low-density lipoprotein (LDL) with both SBP and DBP in essential hypertensive cholesterol and lipoproteins high in triglycerides, while individuals, while Apo B 100 was positively correlated high-density lipoprotein (HDL) cholesterol may have with both. In our study, the Apo B100/Apo A1 ratio was protective effects (O’Connell & Genest, 2001). also increased significantly in essential hypertension Macrovascular problems like coronary heart disease subjects compared to controls and the ratio of Apo (CHD) and stroke have been linked to elevated blood B100/Apo A1 was significantly and positively associated cholesterol levels (Albuche et al., 2000). Lewis suggested with both SBP and DBP, which is in accordance with that blood TC in the range of 5.0-6.5mmol/L may be Nayak et al. (2016). Similarly, Lee et al. (1986), found regarded as undesirable due to the increased risk of higher levels of Apo B100/Apo A1 ratio in hypertensive coronary heart disease when the cholesterol level reaches subjects compared to controls but was statistically 5.0 mmol, as observed in various epidemiological studies insignificant. Even, the severity of atherosclerosis was (Lewis, 1986; McGill, 1968). Low HDL levels are found to have a strong correlation with serum Apo B in a associated with an increased risk of cardiovascular normolipidemic subgroup, according to the findings of disease, albeit the reasons why are not well understood. Haidari et al. (2001). After controlling for the impact of According to experiments, HDL-C helps increase the gender, age, smoking, and hypertension, Sabino et al. outflow of cholesterol from foam cells in vascular (2008), observed that both the Apo B level and the Apo atherosclerotic plaque depots to the liver, where it may B/A1 ratio independently linked with peripheral be broken down and eliminated. HDL-C also possesses atherosclerosis and brain stroke. The ApoB/ApoA-I ratio powerful anti-inflammatory and antioxidant is widely acknowledged as a more accurate predictor of characteristics, which lower the likelihood of developing cardiovascular risk than other conventional lipid markers atherosclerosis (Barter et al., 2004; Mackness et al., (Montali et al., 2015). Therefore, essential hypertensive 2000). It has also been shown that the existence of people with dyslipidemia and an elevated Apo B100/Apo additional atherogenic risk factors is linked to a low A1 ratio are at an increased risk for the development of HDL-C level. According to Pavithran et al. (2007), cardiovascular disease. modifications in lipid metabolism, including a drop in HDL-C, might result in endothelial damage and Conclusions increased blood pressure, which may help to partially The present study demonstrated that essential explain why it has such a high predictive value for hypertension is characterized by dyslipidemia (increased coronary heart disease. It is well known that dyslipidemia total cholesterol, TG, LDL, and decreased HDL), and hypertension frequently coexist. This relationship alteration in apolipoproteins levels (increased Apo B100 has been attributed to underlying central obesity and the and decreased Apo A1), and their ratio (increased Apo accompanying insulin resistance, which are crucial to the B100/Apo A1 ratio), suggesting that high blood pressure pathophysiology of both hypertension and dyslipidemia. may be responsible for disturbances in lipoprotein 36 International Journal of Science Annals, Vol. 6, No. 1, 2023 рrint ISSN: 2617-2682; online ISSN: 2707-3637; DOI:10.26697/ijsa metabolism. Furthermore, the Apo B100/ Apo A1 ratio Chobanian, A. V., Bakris, G. L., Black, H. R., may be used as a complementary marker for the Cushman, W. C., Green, L. A., Izzo, J. L., prediction of the risk of cardiovascular disease in Jones, D. W., Materson, B. J., Oparil, S., essential hypertension subjects. Wright, J. 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(2015). https://doi.org/10.1016/S0140-6736(04)17018-9 Evaluation of role of serum lipoprotein and lipid Zhou, M. S., Liu, C., Tian, R., Nishiyama, A., & Raij, L. profile in essential hypertension patients in a (2015). Skeletal muscle insulin resistance in salt- tertiary care hospital. Journal of Hypertension, 4, sensitive hypertension: role of angiotensin II 204. https://doi.org/10.4172/2167-1095.1000204 activation of NFκB. Cardiovascular Diabetology, 14, 45. https://doi.org/10.1186/s12933-015-0211-6 Cite this article as: Dubey, R., Baghel, D. S., Gaikwad, K., Rathore, V., Saxena, R., & Ansari, Y. M. (2023). Role of lipid profile, apolipoproteins, and their ratio for prediction of cardiovascular disease in essential hypertension. International Journal of Science Annals, 6(1), 32–39. https://doi.org/10.26697/ijsa.2023.1.3 The electronic version of this article is complete. It can be found online in the IJSA Archive https://ijsa.culturehealth.org/en/arhiv This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/deed.en). 39