Smoking and low serum testosterone associates with high concentration of oxidized LDL

Abstract

Background. The interplay between smoking, oxidized low-density lipoprotein cholesterol (ox-LDL) and gonadal hormones has been scarcely investigated.

Aim. To investigate associations in ox-LDL and gonadal hormones in smokers and non-smokers

Methods. Participants (n=164) were obtained from a population cohort of Finnish men aged 40–70 years. The subjects answered a detailed questionnaire on their health behaviour, medication, diseases, and different symptoms, and the hormonal and lipid profiles were measured.

Results. Smokers (n=33) had higher levels of ox-LDL (21%) and more free testosterone (12%) (P<0.01 for all) than non-smokers (n=131). The difference between smokers and non-smokers in ox-LDL persisted after controlling for possible confounding factors. When the smokers were divided into two subgroups (n=16 and n=17) according to total testosterone (≤15 and >15 nmol/L), the ox-LDL in the low-testosterone subgroup was significantly higher (30%) than in the high-testosterone group (P=0.006). Similarly in the corresponding non-smoking subgroups (n=72 and n=59), ox-LDL was significantly higher (11%) in the low-testosterone subgroup than in the high-testosterone subgroup (P=0.012).

Conclusions. Smoking men have significantly more ox-LDL than non-smoking men. Furthermore, if smoking is combined with a low serum testosterone, ox-LDL is even higher. This may suggest a higher risk for atherosclerosis.

• Atherosclerosis
• men
• oxidized LDL
• smoking
• testosterone

Introduction

Smoking substantially accelerates the progression of atherosclerotic cardiovascular diseases 1. Even among smokers without other cardiovascular disease risk factors, intima-medias are significantly thicker than in non-smokers, indicating early appearance of atherosclerosis as a consequence of smoking 2. Also the prevalence of smoking increases along with the degree of carotid artery stenosis 3. It is noteworthy that already an involuntary exposure to second-hand smoke causes atherogenic changes 4. A meta-analytical approach suggests that passive smoking has also been shown to increase the risk of coronary heart disease 5.

Low-density lipoprotein cholesterol (LDL) has been shown to have an essential role in the development of atherosclerosis 6. It is believed that this atherogenic nature of LDL is to a great extent a result of its modification. One form of modification is oxidation, of which a large body of evidence exists 7. The role of oxidized LDL (ox-LDL) in atherosclerosis was strengthened by Juul et al. 8 as they indicated that ox-LDL particles accumulate excessively into arterial walls in comparison to the native LDL particles. Furthermore, they established that the circulating ox-LDL is in fact minimally oxidized LDL. The importance of lipid oxidation in atherosclerosis was further verified by Holvoet and Collen 9. Vasankari et al. 10 observed that the ox-LDL/LDL ratio was independently associated to the extent of coronary atherosclerotic disease.

Several studies have showed that smoking increases peroxidation events, both in vitro and in vivo. Jensen et al. 11 proposed that nicotine in cigarettes might enhance fat oxidation. The deleterious consequences of smoking on the cardiovascular system are assumably, at least in part, due to generation of free radicals. Even after controlling for diet, smokers have increased levels of lipid peroxidation products in comparison to non-smokers 12. Additionally, abstinence from smoking leads to a significant alleviation of the oxidative stress 13.

Key messages

• Smoking men have significantly more oxidized low-density lipoprotein cholesterol (ox-LDL) than non-smokers.

• When the smokers are divided into two groups by serum testosterone, the ox-LDL of the lower-testosterone group is significantly higher than ox-LDL in the higher-testosterone group.

• There may exist an interaction between smoking, oxidation of LDL, and serum testosterone. Thus, smoking men with low serum testosterone may have a higher risk of atherosclerosis than those with high serum testosterone.

Low testosterone level is associated with severe coronary artery disease 14, atherogenic lipid profile (low high-density lipoprotein (HDL), high triglycerides)) 15, increased intima-media thickness 16, and high levels of antibodies against ox-LDL 17. Although smokers generally exhibit higher levels of sex hormones than non-smokers 15, this feature, as noted above, indeed does not provide shelter from atherosclerotic cardiovascular diseases. In this study we investigated whether smoking is associated with elevated concentration of circulating ox-LDL and whether the sex hormone status tempers this association. As a marker of circulating ox-LDL we measured oxidized LDL lipids, which have been shown to be associated with clinical atherosclerosis 10, 18 and arterial distensibility 19. Furthermore, a favourable response to statin therapy has also been observed in oxidized LDL lipids 20.

Materials and methods

Subjects

The present investigation is a nested substudy of a larger population-based study on middle-aged to aged men in south-western Finland. Originally, 600 men (100 men/each 5-year age group from 40 to 70 years) were randomly chosen from a population registry and then invited to the health behaviour questionnaire (see below). From these subjects, one-third was randomly chosen to come to the laboratory to provide a venous blood sample. A total of 165 men visited the laboratory. The study protocol was approved by the joint Ethical Committee of Turku University and Turku University Central Hospital. The study subjects gave their written informed consent.

Blood pressure and anthropometrical measures

Blood pressure was measured twice in the sitting position with a standard mercury sphygmomanometer (Omron HEM-705CP, Japan). The arithmetic mean of the two measurements of systolic and diastolic blood pressures (SBP, DBP) was used in the analyses. Weight was measured by a high-precision scale (Seca, Germany) in light underwear clothing.

The ageing male symptom questionnaire

A recently developed and validated questionnaire, which is aimed at detecting symptoms of ageing and androgen deficiency in men, served as the source of data of physical activity and contained data of former and current smoking status 21. A questionnaire about current smoking habits was applied. Metabolic equivalents (MET), coefficients describing the metabolic activity compared to resting state, were calculated by means of a questionnaire covering the frequency and intensity of exercise (like walking, fast walking, jogging, running) 22. There were no significant differences between smokers and non-smokers in prevalence of diseases. However, alcohol consumption was more frequent among smokers than non-smokers. The proportion of men drinking once per week or more often was 71.9% in smokers and 51.2% in non-smokers (P = 0.012).

Laboratory analyses

Lipid analyses took place in the Turku University Central Hospital. All serum lipids were determined from fresh samples with a Hitachi 717 chemistry analyser. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL), and triglycerides (TG) were measured enzymatically with the use of Boehringer (triglyceride) and Bio Merieux (TC and HDL) reagents. Analysis of oxidized LDL lipids was based on determination of the base-line level of conjugated dienes in LDL lipids 23. Appearance of conjugated diene double bonds is characteristic to peroxidation of all polyunsaturated fatty acids, and in the vast majority of all in-vitro and ex-vivo studies on LDL oxidation diene conjugation has been used as the index of LDL oxidation. The assay method consists of precipitation of LDL, extraction of LDL lipids, and spectrophotometric analysis of conjugated dienes in LDL lipids. Briefly, serum LDL was isolated by precipitation with buffered heparin. Lipids were extracted from isolated LDL by chloroform-methanol (2:1), dried under nitrogen, and redissolved in cyclohexane. The amount of peroxidized lipids in LDL was assessed spectrophotometrically as the amount of diene conjugation (234 nm). The coefficient of variation (CV) for within-assay precision of the LDL precipitation (12 determinations of the same serum) was 3.6%. The CV for within-assay precision for determination of oxidized LDL lipids (20 determinations from the same sample) was 4.4%, and the CV for the between-assay precision over a 3-month period was 4.5%.

Luteinizing hormone (LH) and sex hormone binding globulin (SHBG) was analysed by time-resolved immunofluorometric assay (Wallac PerkinElmer, Turku, Finland). Testosterone and estradiol concentrations were analysed by Spectria radioimmunoassays (Orion Diagnostica, Helsinki, Finland). Free testosterone (fT) concentration was calculated using the Anderson equation 24. Non-SHBG-bound was calculated as follows: proportion (%) of fT (fT%) = 2.28–1.38×log(SHBG nmol/L/10), and serum fT (pmol/L) = fT%×T (nmol/L)×10. The inter-assay precision of the assays were: for testosterone: 6.9% (21.6 nmol/L), for estradiol: 5.3% (0.136 nmol/L), for LH: 4.5% (0.4 U/L), and for SHBG: 7.0% (10 nmol/L), respectively.

Statistical analyses

After testing the normality, all variables were transformed by natural logarithm in order to reduce their skewed distribution. To reveal associations between ox-LDL and sex hormones, we used all participants of the study as one group.

To indicate differences between smokers and non-smokers, subjects were divided into two separate groups based on current smoking status—information which was obtained from the life-style questionnaire (see above). As a result, the 34 men that were current smokers formed the smokers group and the rest of the men the non-smokers group (n=131). To be able to investigate the level of testosterone as a confounding factor in the association of smoking and oxidation, we divided both the smoking and non-smoking groups into two categories: those with serum testosterone equal to or less than 15 nmol/L and those with concentration greater than 15 nmol/L. However, one smoking subject was excluded because of the high concentration of his serum triglycerides (4.9 mmol/L), which rendered the precipitation outcome irrepresentative. Thus 33 smoking men formed the smokers group. In order to reveal possible statistical differences between smokers and non-smokers, analyses of Student's t test (of independent samples) or Mann-Whitney U tests for non-parametric variables were conducted. To establish the effects of possible confounding factors, an analysis of covariance (ANCOVA) was produced. Analyses were performed using SPSS (Statistical Package for Social Sciences) for windows, version 11.0. The level of statistical significance was defined as P < 0.05 throughout the study.

Results

Descriptive parameters before and after dividing the population into smokers and non-smokers, and level of significance, are listed in Table I. Smokers had higher concentrations of ox-LDL (Figure 1) (49.9 versus 39.3 µmol/L), TG (2.2 versus 1.4 mmol/L), and free testosterone (229.7 versus 202.9 pmol/L) than non-smokers, respectively (P≤0.01 for all). HDL in smokers was lower than in non-smokers (1.32 versus 1.46 mmol/L) (P < 0.05). Ratios of TC/HDL and LDL/HDL were significantly higher (4.8 versus 4.1 and 3.2 versus 2.6, respectively) in smokers than in non-smokers (P < 0.05). In addition, the mean age of smokers was about 5 years lower (54.4 versus 59.5 years) than that of non-smokers (P = 0.001). The results from ANCOVA analysis showed that the difference in ox-LDL between smokers and non-smokers remained statistically significant (P < 0.001) even after controlling for possible confounding factors attained from the medical history records. Cardiovascular diseases, related symptoms and medication, age, and alcohol consumption were used as covariates. Additionally, when HDL, TC, SHBG, and free testosterone were also included in the list of covariates (those being variables differing significantly between smokers and non-smokers in previously conducted independent samples t tests) the difference in ox-LDL between smokers and non-smokers still remained significant (P = 0.007).

Figure 1.  Means of oxidized LDL lipids in smokers and non-smokers. (**P=0.009.)

Table I.  Descriptive statistics (mean±SD) among all subjects and statistical significance indicating difference between smokers and non-smokers.

Variable	Descriptives all	Smokers	Non-smokers	Sig.
Ox-LDL (µmol/L)^a	41.39±13.7	49.88±19.6	39.28±10.9	0.009
Total cholesterol (mmol/L)	5.75±0.9	5.82±0.8	5.73±0.9	0.497, ns
LDL cholesterol (mmol/L)^b	3.66±0.7	3.81±0.6	3.63±0.7	0.125, ns
HDL cholesterol (mmol/L)^c	1.43±0.4	1.32±0.4	1.46±0.3	0.020
Ox-LDL/LDL ratio	11.40±3.3	12.95±4.3	11.01±3.0	0.010
Total cholesterol/HDL ratio	4.23±1.1	4.75±1.4	4.09±1.0	0.037
LDL/HDL ratio	2.73±0.9	3.17±1.1	2.62±0.8	0.012
Triglycerides (mmol/L)	1.58±0.9	2.19±1.3	1.43±0.7	0.010
Age (years)	58.42±8.0	54.36±7.4	59.45±7.8	0.001
SHBG (nmol/L)^d	45.79±15.6	43.97±15.2	46.24±15.8	0.492, ns
LH (U/L)^e	4.77±2.33	4.30±1.5	4.89±2.5	0.442, ns
Testosterone (nmol/L)	15.22±4.4	16.44±4.5	14.92±4.3	0.090, ns
Free testosterone (pmol/L)	208.29±48.3	229.65±51.1	202.91±46.2	0.007
Estradiol (pmol/L)	99.08±29.2	94.00±30.3	100.36±28.8	0.221, ns
Weight (kg)	84.94±11.8	83.48±11.8	85.32±11.9	0.416, ns
BMI (kg/m^2)^f	26.97±3.5	26.52±3.9	27.08±3.4	0.347, ns
Metabolic equivalents^g	37.01±21.9	38.58±24.2	36.62±21.4	0.927, ns
Systolic blood pressure (mmHg)	148.39±18.0	145.52±18.1	149.18±18.1	0.334, ns
Diastolic blood pressure (mmHg)	90.23±11.5	89.21±9.2	90.25±12.0	0.729, ns

^aOxidized LDL; ^bLow Density Lipoprotein; ^cHigh Density Lipoprotein; ^dSex Hormone Binding Globulin; ^eLuteinizing Hormone; ^fBody Mass Index. ^g‘Metabolic equivalent’ stands for a coefficient that describes the metabolic activity compared to resting state. Values listed here illustrate the activity of a participant during one week.

Smokers with serum testosterone concentration ≤15 nmol/L have more ox-LDL than smokers with serum testosterone >15 nmol/L (59.1 versus 41.2 µmol/L) (P = 0.006) (Figure 2). Descriptives of other variables between smokers in these subgroups of different total testosterone concentration are listed in Table II. Among non-smokers, when divided into two subgroups in a similar manner, the lower-testosterone group had significantly higher levels of ox-LDL (41.5 versus 36.8 µmol/L, Figure 3), ox-LDL/LDL ratio (11.6 versus 10.4), and TG (1.6 versus 1.2 mmol/L), and lower levels of HDL (1.4 versus 1.6 mmol/L) (P < 0.05 for all). Differences in LDL and TC were non-significant.

Figure 2.  Oxidized LDL lipids in smokers according to serum testosterone status. Dots express mean values of each participant. P=0.006, difference between the low and high testosterone groups (n=16 and 17, respectively) in oxidized LDL lipids (ox-LDL).

Figure 3.  Oxidized LDL lipids in non-smokers according to serum testosterone concentration. Dots express mean values of each participant. P = 0.012, difference between the low and high testosterone groups (n=72 and 59, respectively) in oxidized LDL lipids (ox-LDL).

Table II.  Descriptives of smokers according to total testosterone concentration.

Variable	Total testosterone ≤15 nmol/L	Total testosterone >15 nmol/L	Sig.
Ox-LDL (µmol/L)^a	59.07±20.2	41.22±14.9	0.006
Total cholesterol (mmol/L)	6.10±0.8	5.56±0.7	0.049
LDL cholesterol (mmol/L)^b	4.03±0.6	3.61±0.6	0.043
HDL cholesterol (mmol/L)^c	1.22±0.3	1.42±0.4	0.145, ns
Ox-LDL/LDL ratio	14.64±4.5	11.35±3.4	0.024
Total cholesterol/HDL ratio	5.29±1.3	4.24±1.3	0.032
LDL/HDL ratio	3.55±1.1	2.81±1.1	0.060, ns
Triglycerides (mmol/L)	2.82±1.3	1.61±1.0	0.004
SHBG (nmol/L)^d	34.75±13.2	52.65±11.6	0.001
LH (U/L)^e	3.93±1.6	4.62±1.4	0.133, ns
Total testosterone (nmol/L)	12.84±2.5	19.82±3.1	<0.001
Free testosterone (pmol/L)	201.55±48.3	256.09±38.8	0.001
Estradiol (pmol/L)	91.94±31.3	95.94±30.21	0.722, ns
BMI (kg/m^2)^f	27.91±4.6	25.22±2.6	0.047

^aOxidized LDL; ^bLow Density Lipoprotein; ^cHigh Density Lipoprotein; ^dSex Hormone Binding Globulin; ^eLuteinizing Hormone; ^fBody Mass Index.

Discussion

In this study we showed that low serum testosterone was associated with significantly higher (30%) concentration of ox-LDL in smoking men (Table I). With regard to other lipids (TC, LDL, HDL, TC/HDL ratio, LDL/HDL ratio, TG), smokers with testosterone ≤15 nmol/L had worse lipid profiles than smokers with testosterone concentration >15 nmol/L. Of these, only the differences in HDL and LDL/HDL ratio were non-significant. The difference of ox-LDL was significant also among non-smoking testosterone groups, but in magnitude was only approximately one-third (11%) of that observed in smokers. The significantly higher levels of serum total testosterone in smokers have been observed in several studies 15, 25–28, but not all 29. Even though the difference of serum total testosterone did not reach statistical significance in our study, we found that free testosterone was significantly higher (12%) among smokers than non-smokers (P = 0.007) (Table I).

The definition of androgen deficiency includes low serum total testosterone (<10 nmol/L), and/or a high LH concentration (>8 U/L). In our study, none of the participants satisfied both of these criteria. Consequently, the cut-off point of 15 nmol/L of total serum testosterone was regarded as convenient, because it roughly equalled the median of total testosterone in smoking men. Furthermore, only 37 men would have met either of the two androgen deficiency criteria, whereas the total number of subjects with serum testosterone ≤15 nmol/L was 88. In smokers, only four subjects would have met the either of the androgen deficiency criteria (versus our 16 with serum total testosterone ≤15 nmol/L). This less aggressive criterion does not bias the results by artificially enhancing differences; on the contrary, it might hide a stronger association. Of curiosity, the four smoking men in our study, who met the above criteria of a possible androgen deficiency, had extremely high mean ox-LDL (72.9 µmol/L) when compared to that of the rest of smokers (46.7 µmol/L).

When compared to non-smokers, HDL was significantly lower (10%) and ratios of TC/HDL and LDL/HDL significantly higher (14% and 17%, respectively) in smokers. Of sex hormones only the difference in free testosterone (12%) reached statistical significance between smokers and non-smokers. Apparently, the higher level of testosterone in smokers is not capable of compensating for the increase in cardiovascular disease risk related to smoking. The significantly higher level of ox-LDL (21%) among smokers in our study supports this finding, since ox-LDL has been observed to correlate with brachial, carotid 30 and coronary artery atherosclerosis 10, and arterial functions 19, 31, 32.

It has been previously shown that smoking exerts a blocking effect on enzymes (21- or 11β-hydroxylase) of the adrenal cortex, possibly accounting for the increased levels of adrenal androgens in smokers 29. It has also been hypothesized that the higher levels of testosterone in smokers, observed in some 15, 25 but not all 29 studies, might be explained through changes in SHBG, initiated by smoking 28. However, it is also noteworthy that despite these observed differences in testosterone and SHBG in smokers, some of these studies showed no significant differences in free testosterone 25, 28 or free androgen index 15, whereas others did 26, 27. In our study, smokers did not significantly differ in levels of SHBG and total testosterone from non-smokers, but did have significantly higher concentrations of free testosterone. Gyllenborg et al. 15 found that smokers had significantly more estradiol than non-smokers. Our results do not support this observation. It seems that the pathway of interaction between smoking and sex hormones is inconsistent.

Even though the connection between cigarette smoking and increased cardiovascular disease (CVD) risk is supported by several investigations 1, 33, the exact pathophysiology accounting for this observation has largely remained an enigma. A theory suggesting a potential involvement of nicotine and its immuno-active properties has been proposed 34. Cigarette smoke has also been shown to modify lipoproteins, e.g. increasing the electronegativity of LDL, thus accelerating their uptake by macrophages and, in essence, the progression of atherosclerosis 35. However, as cigarette smoke contains thousands of free radicals, the theory placing oxidative stress into the core of the multi-faceted pathogenesis of CVD can also be easily adopted 36, 37. With these views, the result of this present study seem acceptable, as smokers turned out to have significantly higher concentrations of ox-LDL and generally worse lipoprotein profiles than non-smokers.

In conclusion, this study provides evidence that smoking middle-aged men have significantly impaired lipoprotein profile with higher levels of ox-LDL, TC/HDL ratio, LDL/HDL ratio, TG, and lower levels of HDL than non-smokers. Smokers also have higher free testosterone concentration. Moreover, among smokers with low testosterone, ox-LDL is significantly even higher than among those with high/normal testosterone. Thus, coexistence of smoking, low testosterone, and high ox-LDL may indicate a higher risk of atherosclerosis.

Acknowledgements

The corresponding author has received a research grant for a doctoral thesis from Aarne Koskelo Foundation and Juho Vainio Foundation.