THE LEVEL OF ENDOGENOUS TESTOSTERONE AND CORRELATION WITH LIPID PROFILE IN MEN WITH ACUTE MYOCARDIAL INFARCTION OLDER THAN 40 YEARS NIVO ENDOGENOG TESTOSTERONA I NJEGOVA KORELACIJA SA LIPIDNIM PROFILOM KOD MUŠKARACA SA AKUTNIM INFARKTOM MIOKARDA STARIJIH OD 40 GODINA Authors

Introduction. The effect of lipid profile on AMI is well known. On the other hand, the role of testosterone (T) as one of the possible predictive factors in men with acute myocardial infarction and his interplay with lipid profile in AMI is still controversial. The aim of the study was to measure the level of testosterone in men with AMI and in the same patients 6 months after AMI, and to compare with testosterone levels of healthy control.Also we correlate testosterone levels with lipid profile in AMI and 6 months after AIM. Methods. The study was designed as prospective study. Patients were divided into III groups: Group I included 35 men, aged value (55.00±3.00 years) with AMI. Group II included the same 35 males, analyzed 6 months after the AMI. The Group III, control group consists of 20 healthy men aged value( 57±2.12 years). The blood samples for the Group I (AMI) were taken in the first 12 hours of AIM, and also 6 months after the AIM (Group II). Following analyses were performed: total cholesterol, triglycerides, low density lipoproteins, high density lipoprotein, lipoprotein, apolipoprotein A-1, apolipoprotein B and testosterone level. Results. By comparing the values of testosterone (T) in patients with acute myocardial infarction ,Group I (16,86±7.18nmol/l) and the values of testosterone in the same patient 6 months after the AMI, Group II (18,12±7.96nmol/l), with testosterone values in control, Group III (27.11±10.48nmol/), a high statistically significant difference was obtained, p<0.001. Testosterone values in a patient with AIM as well as 6 months after AIM were lower than those in a healthy population of the same age (p <0.001). In the Group I, statisticaly significant positive correlation were found for tetsosterone and HDL (r 0.403, p<0.05), and high statistical significant positive correlation of T with ApoA1(r 0.747, p<0.01). In Group II, statistical significant positive correlation was found for T and


Klučne reči: testosteron, lipidni profil, akutni infark mioakrda
The testosterone level and correlation with lipid profile in men with acute myocardial infarction older than 40 years

Abstract
Introduction.The effect of lipid profile on AMI is well known.On the other hand, the role of testosterone (T) as one of the possible predictive factors in men with acute myocardial infarction and his interplay with lipid profile in AMI is still controversial.The aim of the study was to measure the level of testosterone in men with AMI and in the same patients months after AMI, and to compare with testosterone levels of healthy control.Also we correlate testosterone levels with lipid profile in AMI and 6 months after AIM.Methods.
The study was designed as prospective study.Patients were divided into III groups: Group I included 35 men, aged value (55.00±3.00years) with AMI.Group II included the same males, analyzed 6 months after the AMI.The Group III, control group consists of healthy men aged value( 57±2.12 years).The blood samples for the Group I (AMI) were taken in the first 12 hours of AIM, and also 6 months after the AIM (Group II).Following analyses were performed: total cholesterol, triglycerides, low density lipoproteins, high density lipoprotein, lipoprotein, apolipoprotein A-1, apolipoprotein B and testosterone level.Results.By comparing the values of testosterone (T) in patients with acute myocardial infarction ,Group I (16,86±7.18nmol/l)and the values of testosterone in the same patient 6 months after the AMI, Group II (18,12±7.96nmol/l),with testosterone values in control, Group III (27.11±10.48nmol/),a high statistically significant difference was obtained, p<0.001.Testosterone values in a patient with AIM as well as 6 months after AIM were lower than those in a healthy population of the same age (p <0.001).In the Group I, statisticaly significant positive correlation were found for tetsosterone and HDL (r 0.403, p<0.05), and high statistical significant positive correlation of T with ApoA1(r 0.747, p<0.01).In Group II, statistical significant positive correlation was found for T and

Introduction
Androgens as well as estrogens, show influence on many risk factors related to cardiovascular diseases.The basic triage of risk factors for cardiovascular disease includes: hypercholesterolemia, high density lipoprotein (LDL), low density lipoprotein (HDL), hypertension and cigarette consumption.Epidemiological studies have shown that each of these factors is of high importance depending on the degree of exposure concerning prediction of cardiovascular disease (CVD) and acute myocardial infarction (AMI).The common feature of these factors is their ability to damage the arterial endothelium.
Hypertension produces an increased mechanical stress on blood vessels.Cigarette smoking causes transient but intensified release of free radicals into the arterial system, and oxidized cholesterol can act as endothelial toxin. 2 Besides conventional risk factors including: diabetes mellitus, positive family history and age, the additional factors such as abdominal obesity, alcohol consumption and physical inactivity could be added; those risk factors represented the focus of research in many studies e.g.(INTERHART, a global risk factors study for acute myocardial infarction). 3Major studies concerning risk factors for CVD (INTERHART, AMORIS, MONICA/CORA), focused special attention to the role of apolipoproteins as informative indicators for CVD and AMI, primarily apolipoprotein B (Apo B) and apolipoprotein AI (Apo AI). 4 The influence of androgens on lipid status and interpretation of the results are extremely controversial.The fact that androgens usually reduce the level of HDL (plasma high density lipoprotein) was used throughout history to characterize these steroids as harmful to blood vessel health. 5But, along with these changes, it is noticed that reduction of lipoprotein a (Lp (a)) level and plasma triglycerides could lead to a reduction of CVD risk. 1 Although risk factors for CVD do not appear to be isolated, and cholesterol metabolism and triglycerides is highly interconnected, the fact that triglycerides concentrations vary day by day in the individual to a much greater extent compared to cholesterol concentration, that is why in multivariate statistical analyses, cholesterol level was marked as stronger predictor for CVD. 6 puberty boys, the increase in testosterone concentrations was followed by a decrease in the HDL cholesterol concentration, probably as a result of hepatic lipase induction, a sensitive enzyme of sex hormones of the lipoprotein metabolism.This decline in HDL levels represents the basic difference and a higher risk for early CVD in males compared to women.Unlike the puberty period in men in the later years, there is a positive correlation between the concentration of testosterone and HDL, which is due to the influence of testosterone on the hepatic synthesis of Apo AI. 7 The effect of androgen on the level of LDL in plasma, which represents a classic clinique metabolic risk factor in men, is difficult to be interpreted and analyzed.In some men who abused anabolic -androgenic steroids (AAS), extremely high values of LDL were observed, indicating an elevated risk for CVD.In contrast, an increase in LDL did not occur in patients who used androgens for the purpose of contraception or substitution therapy, whereas in one of the studies, decline in LDL levels was observed in patients who abused the AAS. 6though LDL is known as the primary lipid risk factor for CVD, there are several limiting factors for using only LDL as a risk factor.Recent data suggest that apolipoproteins are important indicators and predictors for CVD primarily Apo A-I, which represents antiatherogenic high-density lipoprotein.Several studies, including two major (AMORIS) 7 and (INTERHART) 8 , as well as (MONICA/KORA STUDY) 9 , showed strong direct relationship between the high levels of the Apo B/Apo A-I ratio and the increased risk of fatal acute myocardial infarction.Apo B is found in low-density lipoprotein (VLDL), medium-density lipoproteins (IDL), as well as in large boyant LDL and sd-LDL, with one molecule of Apo B in each of these atherogenic particles.Therefore, the total number of Apo B reflects the total number of atherogenic particles.Apo B also plays role in the "capture" of these lipoproteins in the walls of the blood vessels.Apo B synthesized in the liver also stabilizes and allows the transport of cholesterol and triglycerides in VLDL, IDL to large boyant LDL and plasma sd-LDL.Apo B serves as a ligand for Apo B and Apo E rectore and thus facilitates cholesterol intake in peripheral tissues and liver.Apo A-I is the main protein of HDL particles and is the major initiator of reverse cholesterol transport.
The balance between Apo B and Apo A-I, as well as Apo B / Apo A-I ratio, induces the risk of CVD, the higher ratio the higher is risk factor.
In addition to the standard lipid profile as well as the above-mentioned apolipoprotein, Lp (a), which originates from LDL modification, may also be one of the predictors of CVD and AMI.Due to its structural similarity to plasminogen, Lp (a) impairs plasma synthesis and the fibrinolysis process. 10Lp (a) also plays a role in macrophage binding through high affinity receptors, which leads to the formation of foam cells and discharge of cholesterol into atheromatous plaques. 11The correlation between Lp (a) and risk for CVD and AMI was first suggested in some cross-sectional and prospective studies, while in some studies were contradictory results were obtained.In a prospective PROCAM study of 788 men aged 35-65, with follow-up period of 10 years, the risk of acute coronary events was 2.7 times higher in patients with Lp (a) levels > 20 mg/dl. 12e aim of the study was to measure the level of testosterone in men older than 40 years in AMI and 6 months after AMI, and to compare with testosterone levels of healthy male of the same age.Another aim was to examine the correlation of testosterone levels with lipid profile in males over 40 years of age in AMI and 6 months after AIM.

Methods
The study was designed as prospective clinical study.Clinical examination and recruitment of participants were conducted at the Clinic for endocrinology, diabetes and metabolic All patients were informed concerning the method and methodology of the study and all of them voluntarily filled out the information consent.The study was approved by Ethical Committee of Clinical Center of Serbia.

Biochemical analysis was performed by chromatography, and tetsosteron analysis by RIA method respectively
Results were reported as mean ± standard deviation and presented in tables.Differences between groups were assessed by Student's t test and t par test.Correlations between parameters were analyzed with Spearman's correlation test.Differences were considered statistically significant at p<0.05.SPSS 20.0 software was used for the statistical analysis.
All three groups were homogenous in BMI and age.No statistical significant differences were find between all three groups for BMI, and age, p>0.05.By comparing the values of testosterone in patients with acute myocardial infarction, Group I (16,86±7.18nmol/)with control, Group III (27.11±10.48nmol/),ahigh statistically significant difference was obtained.Testosterone values in a patient with AIM were statistically significantly lower than those in a healthy population of the same age p<0.001.Also, high statistically significant difference was obtained by comparing testosterone values in patients 6 months after AIM Group II (18.12±7.96nmol/l),with control, Group III (27.11±10.48nmol/).The values of testosterone in patients 6 months after the AIM were statistically significantly lower in relation to the control group, p<0.001.
No statistically significant difference was obtained p>0.05 for testosterone values in patients with AIM and 6 months after AMI.
Correlations of testosterone levels and lipid profile in patient with AMI and 6 months after AMI were also performed.Table 4 In the Group I, statisticaly significant positive correlation was found for tetsosterone and HDL, ApoB and choletsterol, p<0.05 and high statistical significant positive correlation with ApoA1, p<0.01.
In Group II, statistical significant positive correlation was found for for testosterone and HDL and also for tetsoterone and ApoA1, p<0.05.

Discussion
Men, unlike women, do not experience a sudden decrease in concentration and production of sex hormones in middle age, but a gradual decline of endogenous testosterone has been present since 30 years of every man's life.As a new field of endocrinology its premiere occurred in 1998.Since the first world congress " Aging Male", during the past 20 years, the decrease in testosterone levels has gone from "Andropause" to "late onset hypogonadism LOH ," and ultimately, the involutive hypoandrogenism as the most acceptable definition of changes in the concentration of endogenous testosterone in the aging process in men.The problem with the name and nomenclature is only part of the controversy associated with testosterone and its role in the development of various pathological processes in men.
Many large-scale studies with a large number of participants tried and partly managed to give an answer on the role of sex hormones and their impact on the cardiovascular system in women, but in men mostly this was not the case.For this reason, and especially because of the diametrically different results obtained in animal models and in some smaller studies, over the past 10 years, increasing attention has been paid to the role of sex hormones in the prevention, treatment and occurrence of CVD and AMI in men. 13

Testosterone in acute myocardial infarction
In the last few years, several studies, trials, and case studies 14 have been associated with an increased risk of developing AMI in men who received testosterone injection therapy.
Thus, in a study of Layton and associates published in 2018, 2898 patients with coronary events demonstrated an increased risk of AMI, CVI and unstable angina pectoris in patients who were immediately treated with T injection. 15What has always provoked controversy concerning the level of testosterone is the question what are the appropriate, "normal" values, of testosterone levels depending on the age.Avoiding supraphysiological doses and maintaining a physiological balance potentially unwanted effects of testosterone are omitted.For this reason, in recent years one of the largest studies, a retrospective cohort study of Li HI and collaborators, was conducted on 200000 participants, where the effect of testosterone therapy on the development of AMI was compared to 200,000 hypogonadic patients who did not receive testosterone therapy over a one year period and no association between testosterone and AMI therapy was found. 16In favor of the positive effect of substitution therapy with testosterone in hypogonadal males, a large cohort Chettham CT and associates study conducted on 8808 individuals reported smaller risk of developing AMI in the follow-up period of 3.4 years. 17at differentiated our study from recent trials is that we monitored the level of testosterone in patients with acute myocardial infarction and after 6 months, compared with a healthy population which represented homogenous group concerning the age and body mass index.The obtained values indicated high-significant lower values of testosterone not only in the ischemic period of 12 months from the onset of AMI but 6 months after the acute phase as well ,compared to a healthy control group.

Lipids as risk factors for CVD and AIM
Lipid status with all its components (cholesterol, HDL, LDL, Lp a, triglycerides, ApoAI, ApoB) was completely processed and statistically analyzed in order to determine its correlations with the concentration of endogenous testosterone level in patient with AIM and 6 months later.
Statistically significant higher values have been demonstrated for LDL and cholesterol in subjects with acute myocardial infarction compared to values found after 6 months in the same subjects.
High-density lipoproteins (HDL) with their anti-atherogenic effects mark one of the frequent controversies associated with the concentration of endogenous testosterone and its effect on HDL levels.The evident decline in HDL concentrations in puberty associated with testosterone jumping (negative correlation) due to the induction of sex hormone sensitive enzyme of lipoprotein metabolism, hepatic lipase, is one of the main causes of the early onset of CVD in men compared to women. 7Contrary to this in many studies, a positive correlation between levels of endogenous testosterone and HDL 19 has been demonstrated in older man, which we also confirmed in our study.In our study statistical significant positive correlation was observed for testosterone levels and HDL in AMI (group I), as well in second group, 6 months after AMI.This positive correlation can be explained by hepatic effect of testosterone on the production of ApoA-I.The "Massachusetts male aging study" (MMAS), showed a positive and highly statistically significant correlation of HDL levels and endogenous testosterone in males over 40 years of age with or without CVD, thus definitely confirming the fact that there is difference of endogenous T effect on HDL and risk factors in older men compared to men immediately after puberty. 20Similar results were obtained in the San Antonio Hearth Study, where a positive correlation between endogenous testosterone concentration and HDL in 178 men with normal glycemic values was demonstrated, and it was concluded that the less atherogenic lipid profile (lower triglyceride values and higher HDL values) is present in men with a higher concentration of endogenous testosterone, vs. women in whom the increased concentration of androgens is in a strong correlation with high levels of triglycerides and low HDL values. 21ere were no statistical significant correlation between testosterone levels with the level of triglycerides, a negative correlation was obtained, which, although not statistically significant, corresponded in many ways to the results of large studies.Tromso Study also dealt with the effect of endogenous testosterone on the level of triglycerides during the day in 1274 men who did not have a verified cardio vascular disease and who participated in the population study (Tromso Study from1994 to 95).Analyzing the triglyceride values taken during the day, a linear increase in triglyceride values has been demonstrated in subjects with endogenous testosterone levels below 50 percentile.On the contrary, in men with values of endogenous testosterone above 50th percentile there were no statistically significant changes in triglyceride values during the day.Also highly statistically negative correlation was found between triglycerides and endogenous testosterone levels, and it was highly positive related to HDL.Men with poor lipid profile (HDL<0.9 and TG>1.8) had significantly lower testosterone levels compared to men with normal lipid profile. 22The conclusion of this large study was that low level of endogenous testosterone correlates with the linear rise in triglycerides during the day, and that it is independently bound to a poor lipid profile indicating that low levels of testosterone affect the poor triglycerides metabolism.
Low-density lipoproteins (LDLs) representing one of the risk factors for the development of cardiovascular diseases and, unlike HDL, showed positive correlation with that risk. 23In addition, the role of Lp (a) as an important risk factor for the development of cardiovascular diseases has been highlighted over the past years.Due to the structural similarity with plasminogen, as well as its binding properties with high affinity macrophages and the formation of foam cells, Lp (a) directly affects the development of cardiovascular diseases. 24In our study, we have not found statistically significant correlation of testosterone levels with LDL and Lp (a) in patients with acute myocardial infarction as well as 6 month later.
In our study we found statistically significant, p<0.05 positive correlation between testosterone levels and ApoA1 in both AMI, Group 1 and 6 months after AMI in Group 2.
The role of ApoA-I, ApoB, as well as their quotient in the formation of CVD and AMI, is known from major studies such as AMORIS 7 , INTERHART 8 and MONICA/CORA 9 .
The AMORIS study showed that high levels of Apo B were highly correlated with an increased risk of developing CVD and AMI, while the level of Apo A-I had a protective role in both men and women.In this prospective study, more than 175000 men and women of the Swedish population were monitored, during 98 months, nearly 2000 of them died of myocardial infarction.In this study, ApoB was labeled as a stronger marker for CVD than LDL, and especially for subjects with normal / lower LDL values. 7A single variable representing the strongest indicator for the occurrence of a fatal myocardial infarction was the ApoB/ApoA-I ratio.This ratio was an indicator of the risk of fatal myocardial infarction, independent of lipid phenotype, especially when the other lipid levels were normal or low. 25This ratio was a stronger risk factor for CVD compared to all other TC / HDL, LDL / HDL or non HDL / HDL ratios. 26 impressive INTRHARTH study, based on 30000 patients from 52 countries all over the world, also showed that the ApoB/ApoA-I ratio is the strongest risk factor among the other conventional risk factors for incidence of acute myocardial infarction. 8veral other studies have confirmed that the ApoB/ApoA-I ratio is in a strong correlation with increased carotid artery intima-media thickness and that its ratio progressively increases in patients with metabolic syndrome.This ratio was in a positive correlation with the CVD risk, described as first or myocardial reinfarction. 27e MONICA / Cora Study included 1414 men and 1436 women aged 35-64 years without a previous history of myocardial infarction.The period of follow-up was an average of 13 years, during which 114 men and 31 women had a coronary event, of which 71 were fatal and 74 were not.The strongest correlation was demonstrated for high ApoB values and risk for myocardial infarction, as well as for the ApoB/ApoA-I ratio. 4The results of the MONIKA/Cora study are completely coherent with those obtained in the INTERHART study, based on 15000 AMI patients compared to 15000 healthy controls, and both studies have shown that ApoB/ApoA-I ratio is the most important of all risks factors besides: smoking, hypertension, abdominal obesity, diabetes, alcohol, psycho-social stress, vitamin intake, and physical inactivity.The results were independent concerning gender, age and ethnicity.The ApoB/ApoA ratio remained the strongest risk factor after the multivariate analyses were performed. 8In the last few years, numerous studies estimated the role as well as the significance of the ApoB / Apoa-I ratio concerning CVD and AMI incidence.
The study published in 2015 explored the predictive value of ApoB/Apoa-I ratio and non-HDL-C values and their effect on CVD incidence.The study was conducted on 826 patients, of whom 532 had CVD, 165 of them unipolar, 175 bipolar disorders, and 192 multipolar cardiovascular heart disorders vs. 294 healthy subjects.After a follow up period of 3 years, it has been confirmed, that there is a positive correlation among the high values of the ApoB/Apoa-I ratio and non-HDL-C with the most serious, multiply forms of coronary heart disorders and the increased risk of developing adverse events such as: angina pectoris, AMI, heart insufficiency, stroke and death caused by CVD. 28atistically significantly higher ApoB values we found in the patient group with AMI compared to the same subjects 6 months after myocardial infarction.
Negative but not statistically significant correlation of endogenous testosterone concentration and the ApoB/ApoA-I ratio were found in first and in the second group.
The analysis of the studies conducted so far as well as the results of our study suggest that natural endogenous testosterone has a positive or neutral effect on the development of cardiovascular diseases and AMI.The antiatherogenic mechanism of testosterone is unknown in general, but several solutions have been offered so far.Some data emphasize the modulating effect of endogenous testosterone on the risk factors: diabetes 29 , insulin resistance 30 , obesity 31 , hypercholesterolemia 32,33 , and hypertriglyceridemia 32 in CVD .It was assumed that increase of triglyceride levels was modified by changes in hepatic triglyceride lipase. 34On the contrary, endogenous testosterone can have a direct effect on HDL-cholesterol by increasing the hepatic production of apolipoprotein A-I as the main protein component of a nascent high-density lipoprotein 28 .

Conclusions
This study showed that men over 40 years of age with AMI have high statistically significant lower concentrations of endogenous testosterone compared to a healthy male population of the same age.The concentration of testosterone in same patient, after the AMI maintains statistically significant lower values, even after 6-month of rehabilitation period in comparison to a healthy male population over 40 years of age.High-density lipoproteins (HDL) and ApoA1, with their anti-atherogenic effects mark one of the frequent controversies associated with the concentration of endogenous testosterone and its effect on HDL and ApoA1 levels.In our study high statistically significant positive correlation was reported between the levels of endogenous testosterone and HDL and ApoA-I levels in men with acute myocardial infarction as well in the same patients 6 months after the AMI.Long-term, well designed prospective clinical trials are required to verify potential testosterone role, his interplay with lipid profile and possible predictive value in male with AMI.
Efekat lipidnog profila na AIM dobro je poznat.Nasuprot tome, uloga nivoa diseases of the Clinical Center of Serbia and the Clinic for Cardiology of the Emergency Center (coronary unit), laboratory test were performed in the Center for Medical biochemistry of the Clinical Center of Serbia.Patients were divided into III groups: Group I included 35 men aged 40-80 years with AMI.Group II included the same 35 males aged 40-80 years who were analyzed 6 months after the AMI.Group III, control group consists of 20 healthy men aged 40-80 years.All groups were homogeneous concerning body mass index (BMI) and age.Total ischemic time in the first group was shorter than 12 hours.All participants were taken blood samples early in the morning for the following analyses: a) lipid profile: total cholesterol, triglycerides, low density lipoproteins (LDL), high density lipoprotein (HDL), lipoprotein a (Lp (a)), apolipoprotein A-1 (ApoA1), apolipoprotein B. b) Hormone analysis: testosterone Hormone and biochemical values were measured directly in AMI event (for the first group of subjects), as well as six months after release from the hospital (for the second group of subjects).

Table All tested parameters in AIM (Group I ) and 6 months after AMI (Group II), averge values and correlation
MV-mean value; SDstandard deviation; Nnumber of patients;p<0.05*,p<0.01**