Introduction

The body imbalances of trace elements have been increasingly accepted as an essential mediator for the pathogenesis of numerous diseases such as valvular heart disease (VHD) [1]. The human heart consists of four valves which technically characterized the heart as left- (mitral and aortic) and right- (tricuspid and pulmonary) sided. The critical function of each heart valve is to ensure the blood flow through the cardiovascular system and any malfunction in opening and closing cycles of one or more than one valve may result in valvular heart disease [2]. Types of the heart valve disease depend on two main ways that can affect any of the four valves; it may be stenosis (narrowing) and/or regurgitation (leaking). The major risk factors for VHD are age, gender, smoking, hypertension, diabetes mellitus, hypercholesterolemia and elemental imbalances [3, 4]. Essential/trace elements (such as Cu, Zn, and Fe) are indispensable in the sustenance of life because they are involved in variety of biochemical processes and regulate numerous enzymatic and hormonal activities [5]. It has also been recognized that the elemental contamination is involved in plethora of diseases, such as role of some elements in the development of VHD. Toxic metals are mostly accumulated in different environmental segments as they are not decomposed/decayed like organic pollutants [6, 7].

There always exists dynamic balance among various elements in the biological systems, which is responsible for many metabolic and physiological processes. Any disorder in the elemental balance is often related to some pathological conditions, resulting in the ailments [8]. Factors such as diet, absorption ability, toxicities and drug-nutrient interactions play a vital role in maintaining a balance of the elements in the body [9]. Trace elements have been proposed to have diagnostic and prognostic value in cardiovascular disease although there is no concrete evidence for the direct relationship between the development of cardiovascular disease and prevailing elements status [10]. Epidemiological studies support the possibility that imbalances in trace elements levels may increase the risk of cardiovascular disease; for example, Fe, Pb and Cd showed elevated levels in myocardial infarction patients than those in controls [11, 12].

Among the biological specimens, in the present study, scalp hair sample is selected for elemental analyses because it is an interesting tool in the assessment of health/nutritional status and in the biomonitoring of exposure to toxic elements [13]. Moreover, the hair is a potential repository of all the elements that enter the body and give information about the short- and long-term exposure. It is a stable matrix because elements are permanently incorporated in keratinous structure of hair fibre during its formation and are consequently excluded from metabolic processes. Other attributes of hair samples are easy collection, low cost, easy transport, storage and least probability of contamination although some limitations such as absence of well-defined reference concentration ranges and washing efficiency are also associated with hair samples [7, 13, 14]. Some elemental levels in the hair can be affected by colour, diameter, growth rate, geographic location, age, gender, dietary habits, hair care and lifestyle. There is no standard methodology related to the length of the hair sample to be collected, or the amount, or the position on the scalp [15, 16].

Literature survey showed the variations in the trace and toxic element concentrations in the blood and cardiac tissues of VHD patients [1, 2, 17,18,19,20], but according to best of our knowledge, there are no available reports related to the present elemental levels in the scalp hair of VHD patients. Therefore, the current study was designed to evaluate the distribution, correlation and multivariate apportionment of 14 elements (Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Pb, Sr and Zn) in the scalp hair of valvular heart disease patients in comparison with those of matching healthy subjects. Plausible differences in the elemental levels with respect to gender, abode, dietary/smoking habits and occupations were also assessed. It is anticipated that the study would provide comparative appraisal of the elemental imbalances in VHD patients and would help to understand the role of selected elements in the onset/progression of the disease.

Materials and Methods

Study Population

In the present investigation, scalp hair samples were collected from the patients (n = 52) admitted in the Punjab Institute of Cardiology, Lahore, Pakistan, on volunteer basis. Prior to the sample collection, the protocol of the study was approved by the human ethical committee of the institute. The diagnosis of valvular heart disease (VHD) in the patients had previously been established by a specialist cardiologist, by performing the echocardiography before the sample collection [17, 21]. The control subjects (n = 52) were also selected on volunteer basis from the same localities matched with the patient’s demographic characteristics [7]. Pertinent information related to the subjects such as age, gender, ailment nature/duration, abode, diet, smoking habits and occupation were recorded on a proforma at the time of sample collection.

Sample Collection and Washing

The hair sample (~3.0 g) was cut from the sub-occipital region of the scalp using a pair of plastic scissors and stored in zip-locked polythene bag, along with attached proforma [21,22,23]. Before analysis, the hair sample was soaked in (5% w/v) detergent solution for at least 3 h and then washed with plentiful tap water and finally with doubly distilled water until all the detergent was removed. Afterwards, 25 mL of 0.5% v/v Triton X-100 solution was added and then placed the contents on auto-shaker for 25-min shaking. The sample was then washed with excess of doubly distilled water in order to remove the non-ionic detergent. Finally, the hair sample was dried in an electric oven for overnight at 70 °C [7, 23, 24].

Sample Digestion

An accurately weighed portion of washed hair sample (~1.0 g) was taken in an Erlenmeyer flask, followed by the addition of 10 mL of HNO3, and the contents were left for 30 min at room temperature. Afterwards, the sample contents were heated at 70–80 °C for 30 min and later cooled to room temperature. It was followed by the addition of 5.0 mL of HClO4 in the Erlenmeyer flask with subsequent heating to a soft boil until white dense fumes evolved. The clear solution thus obtained was transferred to 50-mL volumetric flask, and the final volume was adjusted by doubly distilled water [7, 24,25,26]. Every care was taken to eliminate the chances of contamination of the samples during processing and preparation. A blank, without hair sample, was also prepared in the same way along with each batch.

Quantification of the Metals

The selected essential and toxic elements (Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Pb, Sr and Zn) in the digested samples were measured by an atomic absorption spectrophotometer (Shimadzu AA-670, Japan) equipped with automatic background correction facility under optimum analytical conditions (Table 1). Three sub-samples of each sample were treated and run separately onto the spectrophotometer to pool the mean concentrations. Parallel routine check on the accuracy of quantified results was ensured through the use of standard reference material (human hair, batch GBW-07601) as presented in Table 1. The samples were also analysed at an independent laboratory for comparison of the results which were in good agreement (±2.5% difference) with each other. All reagents used were of ultrahigh purity (certified >99.99%) procured from E-Merck or BDH. Working solutions were prepared by serial dilutions of 1000-ppm stock standard solutions, just before the analysis of the elements on the instrument [7, 15, 24, 25].

Table 1 Optimum analytical conditions for the elemental analyses along with their detection/quantification limits and certified vs. measured (±SD) concentrations (μg/g) of the metals in standard reference material
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Statistical Analysis

Statistical analyses were carried out using the STATISTICA software [27]. Univariate analyses of the data provided basic statistical parameters, such as range, mean, median and quartiles, along with Spearman correlation. The elemental data for the patients and controls were compared by applying the non-parametric ‘Kolmogorov-Smirnov test’ to identify any significant differences between the two population groups. Additionally, principal component analysis (PCA) and cluster analysis (CA) were employed in the present study to investigate the multivariate apportionment of the essential and toxic elements in the scalp hair in order to assess the prognosis/progression of the disease [7, 15, 24].

Results and Discussion

Demographic Characteristics

The demographic data related to the valvular heart disease patients and healthy donors/controls are shown in Table 2. The subjects in both donor groups were closely matched for their age (about 39 years on the median basis) in each case. Majority of the subjects in both donor groups (>70%) were residing in urban areas and having same gender characteristics (58% female and 42% male). Eighty-one percent (81%) of the patients belonged to vegetarian class, while 63% of the controls were non-vegetarian; therefore, the subjects in the two donor groups differ significantly for their dietary habits (Table 2). Significant number of the patients and controls (81–88%) were not addicted to smoking. Seventy-five percent (75%) patients and 67% healthy donors were indoor workers. More than half of the patients suffered from aortic valve disease (56%) compared with the mitral valve disease (44%).

Table 2 Characteristics of the subjects
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Distribution of the Elements

Comparative distribution of the selected elements in the scalp hair of the valvular heart disease patients and healthy donors are shown in Fig. 1. Among the selected elements, Li, Cd and Cr showed the fairly lower concentrations in the scalp hair of the VHD patients (Fig. 1a). Relative variations of the elemental levels in the scalp hair of the VHD patients manifested appreciably broad distribution for most of the elements as shown by their minimum, maximum, median, 25th percentile and 75th percentile levels. Predominantly random variations were observed for Na, Mn, Zn, Cr, Co and Fe which showed varying concentrations of these elements in the scalp hair of the patients. However, mostly asymmetric and relatively narrow variations were noted for Sr, Mg, Cu and Ca while moderately symmetrical distribution was shown by Cd, Li, Pb and K in the scalp hair of the patients (Fig. 1a).

Fig. 1
figure 1

Distribution of the concentrations of selected elements (μg/g) in the scalp hair of VHD patients (a) and healthy donors (b) (statistically significant differences in the median levels are shown by asterisks)

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Likewise in the case of the healthy donors, least contributions were noted for Li and Cd (Fig. 1b). Comparative variations of the selected elements as shown in Fig. 1b exhibited moderately narrow distribution for Ca, Cu, K, Mg and Zn in the scalp hair of the healthy donors, while considerably large variations were noted for Pb and Cr, followed by Mn, Na, Fe, Co and Cd which manifested significant variations in their concentrations. Moderately asymmetrical distribution was observed for Mn, Cu, Co, K, Pb and Zn as manifested by their minimum, maximum, median, 25th percentile and 75th percentile levels (Fig. 1b). In the case of the healthy donors, most of the elements depicted rather symmetrical distribution compared to those of the VHD patients.

Comparison of the median elemental levels in the scalp hair of the patients and healthy donors revealed that there were insignificant differences between the measured concentrations of Ca, Cd, Co, Cr, Li, Pb and Zn in the scalp hair of the VHD patients and healthy donors; however, statistically significant differences were observed in the concentrations of the rest of the elements as shown in Fig. 1. Scalp hair of the patients showed significantly higher concentrations of Cu (34%), Fe (26%), Mg (25%), Mn (40%) and Sr (46%) than that of the controls at p < 0.05. On the other hand, scalp hair of the healthy donors demonstrated significantly elevated levels of K (69%) and Na (79%) compared with that of the patients (p < 0.05).

Some past studies clearly demonstrated the significant disparities in the concentrations of selected elements in biological samples of VHD patients compared to those of the healthy donors [18]. Al-Younes et al. [28] described the supporting role of Fe in the inflammation of heart valves. In addition, Fe is involved in increased body oxidative stress by generating reactive oxygen species (ROS) via Fenton reaction; thus, Fe may participate in the progression of VHD [29]. In the present study, increased Fe levels in scalp hair of the patients supported the above hypothesis (Fig. 1). The trace element Cu is a cofactor of many enzymes, and it is involved in redox reactions such as superoxide dismutase and ascorbate oxidase. Due to the redox-active nature of Cu, it helps in the production of ROS which oxidizes the low-density lipoprotein (LDL). The oxidized LDL subsequently induces the major risk factors (hypertension and coronary artery disease) of VHD [30, 31]. In the current study, the Cu levels were found to be significantly higher in the patients than those in the controls, which corroborates the above arguments (Fig. 1).

Zinc is an antioxidant and anti-inflammatory agent, which controls the enzymatic metabolism and immune activities of the body [32]. Because of these characteristics of Zn, some past studies reported the inverse relationship in the plasma Zn level and the progression of VHD [19]. But in present study no significant differences were observed in the hair Zn levels of the VHD patients and healthy donors. In the present study, the Cu/Zn ratio in the scalp hair of the VHD patients was found at 0.060 which was significantly higher (28%) in comparison with the Cu/Zn ratio of the healthy donors (0.043) at p < 0.05. This increased Cu/Zn ratio in the patients evidenced various inflammatory activities inside the body which later on caused VHD, thereby indicating its progression [19]. One of the earlier studies pointed out the involvement of free-radical processes, as well as imbalances in several trace elements especially Cu, Fe and Zn in atherogenesis which potentially encourages the development of VHD [33].

Lead is notorious element for human, and its toxic role at molecular level in the advancement of VHD is not clear yet but study of Nyström-Rosander et al. [19] exhibited the significant positive correlation between elevated Pb level in heart valve tissue and prevalence of valvular heart disease. In the present study, Pb showed more or less comparable levels in both patients and controls (Fig. 1). Strontium was noticeably linked with increased events of cardiovascular diseases. The role of Sr in arterial stiffness may be explained by its potential role in increasing oxidative stress [24, 34]. The above statement about the Sr is in good agreement with the present study (Fig. 1). Potassium in optimum concentration is an important electrolyte which is essential for a healthy nervous system and a regular heart rhythm, while hypokalaemia contributes to the pathogenesis of cardiovascular disease [35]. The present K levels were found to be lower in scalp hair of the patients compared to those of the healthy donors (Fig. 1). Similarly, Mn is regarded as an antioxidant element in trace amount but considerable increase in its concentration may adversely affect the normal cardiovascular activities [36]. Study of Nyström-Rosander et al. [19] revealed significantly elevated level of Mn in blood plasma of VHD patients compared to that of the controls; similar findings for the VHD patients were noted in the present study (Fig. 1).

Variation of the Elemental Levels with Age

Median levels of the selected elements in the scalp hair of valvular heart disease patients and healthy donors/controls were compared for different age groups as shown in Table 3. The median concentrations of K and Na for the controls of all age groups were significantly elevated than those for the patients (p < 0.05), while the median levels of Ca, Cd and Li showed inconsequential differences for both donor groups. However, the median concentrations of Cu, Fe, Mn and Sr for the patients belonging to age groups ≤40 and 41–50 years were significantly higher than those of the counterpart age groups of the controls. The median concentrations of Pb in the patients and those of Co and Cr in the controls of age group ≥51 years were significantly higher than those in the counterpart subjects (p < 0.05). Similarly, median level of Mg in the patients for age groups ≤40 and ≥51 years while that of Zn in age group 41–50 years was significantly higher than that in the counterpart controls (p < 0.05).

Table 3 Comparison of the median concentrations (μg/g) of the selected elements in the scalp hair of the VHD patients and controls for different age groups
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Variations of the Elemental Levels with Demographic Characteristics

The presents study showed that the median levels of Cu, Fe, K, Mg, Mn, Na and Sr in the scalp hair of the VHD patients and healthy donors were significantly divergent; therefore, these levels were further analysed to explore the demographic-based disparities in both donor groups. The gender-based comparison of the elemental levels (median ± 25th–75th%, logarithmic scale) in the scalp hair of the patients and controls is shown in Fig. 2a. Median levels of Fe depicted significant increase in the hair tissues of female patients and male controls while Na showed significant accumulation in the male patients and female controls than that in the counterpart groups (p < 0.05). The median level of K was found significantly higher in the male patients than that in the female patients, while median contents of Sr and Mn were noted at considerably higher levels in the male and female controls, respectively. Nevertheless, the median concentrations of Cu and Mg exhibited insignificant differences in the male and female subjects of both donor groups. The measured levels of Mn and Sr were almost comparable in male and female patients, whereas no significant difference was observed for K in the male and female healthy donors. Such elemental dispersals indicated the effect of environmental contamination on the body elemental burden [7, 37].

Fig. 2
figure 2

Comparison of the median levels of selected elements (μg/g ± 25th–75th%) in the scalp hair of VHD patients and healthy donors based on gender (a), habitat (b), diet (c), smoking/non-smoking habits (d) and occupation (e) (statistically significant differences in the median levels are shown by asterisks)

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The habitat-based comparison (Fig. 2b) indicated that the median levels of K, Na and Sr showed significant increase for the rural subjects compared to those for the urban subjects while the median contents of Fe were significantly higher for the urban subjects than those for the rural subjects of both the patients and controls. However, Mn were found at significantly higher levels for the rural patients and urban controls (p < 0.05). The median concentration of Mg was found to be considerably higher for the rural controls than that for the urban controls, whereas no significant disparity was observed for the rural/urban patients. Nonetheless, the median levels of Cu revealed more or less comparable contributions for the patients/controls living in the urban and rural habitat.

The dietary-based comparison (Fig. 2c) showed that the median concentrations of Fe, K and Na exhibited considerably higher accumulation in the hair tissues of the non-vegetarian subjects for both the patients and controls, whereas noticeably elevated levels of K and Na were observed in the healthy donors irrespective of the food habits. These differences may be partially attributed to the significant differences in number of the samples of the two groups based on the dietary habits. The median levels of Sr were found to be significantly higher in the scalp hair of non-vegetarian patients and vegetarian controls which manifested considerably higher levels for Mn and Mg, respectively. Similarly, the median level of Cu was noticeably higher in the hair of non-vegetarian controls than that of the vegetarian controls. In the case of patients, Cu and Mg demonstrated insignificant differences while Mn showed almost similar contributions for the controls with vegetarian and non-vegetarian food habits.

The smoking-based comparison (Fig. 2d) revealed that the median concentrations of Cu and K were significantly higher for the patients’ addicted to smoking than those for the non-smoker patients, whereas the median concentration of Sr was significantly higher for the smoking controls. Similarly, the median levels of Na were found to be markedly higher for the non-smokers compared to those for the smokers of both donor groups. The median contents of Fe were found to be significantly higher in the hair of the non-smoker patients and smoker controls whereas Mn showed considerable increase for the non-smoker controls. Afridi et al. [38] investigated the antagonistic effects of some metals in the scalp hair of smoking/non-smoking cardiovascular patients; it was concluded that accumulation of Cd may replace Zn in the arteries, and as a result, arteries become brittle and inflexible. Once it happened, the body may coat the inflamed and brittle arteries with Ca and fatty plaques. This plaque unfortunately reduces the interior diameter of the arteries, and as a consequence, different VHD risk factors prevailed like hypertension and coronary artery diseases (CAD). Several epidemiological studies reported the association between risk factors (smoking, hypertension and CAD) and development of VHD [39, 40].

Occupation-based comparison (Fig. 2e) revealed some statistically significant differences among the elemental levels for the indoor and outdoor patients; the median concentrations of K were found to be significantly higher for the outdoor workers than those for the indoor workers of both groups; nonetheless, the median levels of Fe and Sr exhibited comparatively higher contributions for the outdoor controls. The median levels of Na were found to be relatively higher for the outdoor patients and indoor controls while the rest of the elements (Cu, Mg and Mn) exhibited almost comparable concentrations for both indoor/outdoor workers.

Comparison of the Elemental Levels for Aortic vs. Mitral Valve Disease Patients

Comparison of the median elemental levels (±25th–75th%) for the aortic and mitral valve disease patients is shown in Fig. 3. Significantly higher median concentrations of Cd and Zn were noted for the patients suffering from aortic valve disease, while comparatively higher median levels of Co, K, Mn, Na and Pb were found for the mitral valve disease patients. In addition, the median levels of Ca, Cr, Cu, Fe and Li revealed almost comparable levels in both types of valvular heart disease patients while Mg and Sr exhibited slight disparities but the differences were insignificant. Study of Ohnishi et al. [20] revealed that in heart valve tissues, Fe and Mg showed significant relationship (p = 0.006) in mitral valve disease patients while no such association was observed in the case of aortic valve disease patients. Moreover, Zn exhibited non-significant relationships with Fe and Mg in the case of aortic valve disease patients, thus supporting the accumulation of some elements in both types of the VHD patients.

Fig. 3
figure 3

Comparison of the median levels of selected elements (μg/g ± 25th–75th%) in the scalp hair of aortic valve disease patients and mitral valve disease patients (statistically significant differences in the median levels are shown by asterisks)

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Correlation Study

The data related to the mutual correlations among selected elements in the scalp hair of valvular heart disease patients and healthy donors are shown in Table 4, wherein the significant r values are shown in bold (p < 0.05). In the case of the VHD patients, very strong positive relationships were noted between Ca and Sr (r = 0.786), Cu and Sr (r = 0.703), Mg and Sr (r = 0.666), Ca and Mn (r = 0.631), Na and K (r = 0.622), Mg and Mn (r = 0.622), Mn and Sr (r = 0.608), Ca and Cu (r = 0.536), and Ca and Mg (r = 0.535). In addition, some significant associations were observed among the following pair of the elements (0.500 < r > 0.350): Mn and Fe, Pb and Sr, Mn and Zn, Cu and Pb, Cd and Cr, Cu and Mn, Fe and Li, Cu and Mg, Fe and Mg, Co and K, Fe and Sr, Cd and Cu, Ca and Cd, and Ca and Zn, thus indicating their probable common variations/origin in the patients. The rest of the elements showed weak positive and negative correlation. Significant correlation between Ca and Cd in the present study highlighted their critical role in the onset and progression of heart disease, which were evidenced by several epidemiological studies [7, 41, 42].

Table 4 Correlation coefficient (r) matrix of selected elements in the scalp hair of the VHD patients (below the diagonal) and healthy donors (above the diagonal)
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In the scalp hair of healthy donors (Table 4), strong associations were observed among Sr and Mg (r = 0.628) and Na and K (r = 0.584), while some significant relationships were noticed for Li and Cd (r = 0.490), Fe and Mn (r = 0.459), Sr and Cd (r = 0.423), Cd and Cr (r = 0.418), Cd and Pb (r = 0.380), and Co and Mg (r = 0.363). Some elemental pairs (Li and Na, Cd and Na, Li and Zn, K and Sr, and K and Cd) exhibited significant negative relationships as manifested by their correlation coefficient values. However, Ca and Cu were not significantly correlated with any other elements in the case of healthy donors, thus evidencing their independent character. Overall, the correlation outcome of the elements for the healthy donors remained significantly dissimilar compared to that for the heart valve disease patients, which may be ascribed to the imbalances of the nutrients and trace elements in the patients.

Multivariate Analyses

Multivariate statistical methods comprising of PCA and CA were employed for the apportionment and source identification of trace elements in the scalp hair of the two donor groups [7, 15, 24]. The PCA of the selected elements in the scalp hair of valvular heart disease patients and healthy donors is given in Table 5. In the case of the VHD patients, PCA yielded five significant principal components (PCs), with eigen values >1, commutatively explaining about 77% of the total variance of the data. The consequent CA in the scalp hair of the patients based on Ward’s method is portrayed in Fig. 4a, which revealed significantly strong clusters among Ca and Sr; Mg and Mn; Cd, Cu and Zn; Co and Pb; Cr, Fe and Li; and K and Na. In this case, PC 1 showed elevated loadings for Ca, Mg, Mn and Sr supported by similar strong clusters in CA, whereas PC 2 showed higher loadings for essential elements K, Na, Zn and Co. These elements loading are mainly associated with the nutritional habits of the donors. PC 3 revealed maximum loadings of Cd and Li whereas Cu and Pb were the major representative of the PC 4. These elements were mostly originating from anthropogenic activities (automobile emissions, burning fuels, mining and metal processing operations). In addition, Cu and Pb both increase the oxidative stress of the body which would believe to be a reason for the development of disease [28]. The last PC showed maximum build-up of Cr and Fe, which may be trace-originated from the mixed sources and controlled by the lifestyle of the donors.

Table 5 Principal component analysis for the selected elements in the scalp hair of the VHD patients and healthy donors
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Fig. 4
figure 4

Cluster analysis of selected elements in scalp hair of VHD patients (a) and healthy donors (b)

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The principal component loadings of the selected elements for the healthy donors are depicted in Table 5, which yielded five PCs with eigen values >1, commutatively exhibiting more than 85% of the total variance of the data, while the corresponding CA based on Ward’s method in the form of dendrogram is shown in Fig. 4b. The CA of the elemental data in the scalp hair of healthy subjects revealed very strong clusters of Ca, Sr, Li and K; Zn, Mg and Na; Pb, Fe and Cd; Cr and Cu; and Co and Mn. PC 1 explaining most of the variance of the data showed maximum loadings for essential elements (Ca, Cr, Mg and Zn) which indicated their common origin, but in the case of the patients, these elements exhibited loadings to different PCs. In the case of the controls, PC 2 showed elevated loadings for Cd, Li, Na and Sr, while PC 3 exhibited higher loadings of Co, Fe and Pb. These elements were mainly associated with anthropogenic pollutants. PC 5 indicated elevated loadings for Cu and Mn, which may be related to the dietary life style of the donors and regulated by body internal metabolism. However, PC 4 showed significant loading for one of the major electrolytes (K) which may be trace-originated from the independent sources. Thus, the PCA and CA showed significantly diverse apportionment and associations of trace elements for the VHD patients compared to those for the healthy donors. Consequently, the multivariate apportionment of the selected elements in the patients was considerably affected by the progression of the disease but its applications for diagnosis in the clinical investigations depend on the outcome of more studies with larger population groups from diverse geographical areas.

Conclusions

Comparative distributions of the selected essential and toxic elements in the scalp hair of the VHD patients and healthy donors were considerably different. Measured concentrations of Cu, Fe, Mg, Mn and Sr for the patients were significantly elevated than those for the healthy donors which exhibited markedly higher levels of K and Na. In addition, Cu/Zn ratio was found to be significantly higher in the patients compared to that in the controls. Median elemental levels revealed significant differences with respect to gender, habitat, dietary/smoking habits and occupations of the donors. The correlation study demonstrated noticeably diverse mutual associations of the essential and toxic elements in the two donor groups. Multivariate analyses by PCA and CA indicated significant disproportions among the selected elements in the VHD patients in comparison with those in healthy donors.