Postmortem Inductively Coupled Plasma Mass Spectrometry Analysis Reveals Elevated Heavy Metal Concentrations in Coronary Arteries: A Comparative Autopsy Study Supporting a Toxic Inflammatory Hypothesis for Atherosclerosis Open Access

Cardiovascular diseases (CVDs) continue to be the leading cause of mortality and morbidity today. The prevention and treatment of CVD begin with the struggle against atherosclerosis, which is accepted as the main cause of the CVD. Many studies have been conducted on the etiology, pathophysiology, and treatment of atherosclerosis so far and most of them have focused on lipid metabolism, inflammatory processes, and atherothrombosis [1, 2]. Conventional risk factors are insufficient to elucidate the causes of the disease and other factors are thought to be effective in many patients. Environmental factors and chemicals are thought to take part in pathogenesis of coronary artery disease (CAD) [3, 4].

Heavy metal accumulation is among the newly described risk factors for CVD. Recent studies demonstrated that intense exposure to toxic metals such as arsenic, cadmium, mercury, and lead increases the risk of CVD [5]. Metals are common in the living environment and chronic exposure occurs in people in daily life through drinking water, cigarettes, food, and inhaled air. Minerals and trace elements play an important role in the physiology of the cardiovascular system, along with their oxidative stress, antioxidant properties, and their involvement in enzymatic reactions [6]. The change in trace element concentrations will affect the antioxidant enzyme activity and directly on the susceptibility of the tissue to oxidative stress. In studies, Cu and Fe concentrations were higher in the CVD patient group [7]. Trace elements such as selenium and iodine participate in cellular oxidation-reduction reactions and take part in important metabolic activities, especially in thyroid hormone synthesis [8]. Several trace elements constitute the structure of metalloenzymes. Various trace elements such as Mn, Zn, Cu are found in the structure of superoxide dismutase that defend against free radicals, which are known to promote atherosclerosis [9]. The majority of electrolytes and trace elements such as Na, Ca, K, Mg, Cr, and Cd participate in blood pressure regulation. Ba, Li, Cu, Th, Na, and K promote catecholamine release [10]. Based on recent studies, heavy metals contribute to atherosclerosis via multiple interrelated molecular and cellular pathways. Oxidative stress induction: metals such as Fe, Hg, and As catalyze the generation of reactive oxygen species, which damage endothelial cells and promote lipid oxidation. Inflammatory signaling activation: exposure to metals triggers upregulation of pro-inflammatory cytokines (e.g., TNF-α, IL-6) and adhesion molecules (e.g., VCAM-1), enhancing leukocyte recruitment to the endothelium. Endothelial dysfunction: trace elements like Cd and Pb impair nitric oxide bioavailability and alter vasomotor tone, contributing to vascular stiffness and plaque development. Disruption of antioxidant enzymes: essential elements like Cu, Zn, and Se, when dysregulated, interfere with enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), tipping the redox balance toward oxidative injury. Vascular calcification and metabolic dysregulation: elevated Ca and P levels promote arterial wall calcification, while metals such as Hg contribute to insulin resistance and dyslipidemia, indirectly worsening vascular pathology.

Studies evaluating the association of metals with subclinical atherosclerosis have the potential to help identify individuals at risk before the onset of clinical symptoms. Although immunotoxic and carcinogenic properties of metals have been demonstrated, their clinical and CVD effects are less well characterized.

Contemporarily, the vast majority of elements can be measured precisely and accurately with technology of inductively coupled plasma optical emission spectrometry (ICP-OES). The ICP-OES method has made trace element measurement a feasible and convenient instrument in studies [11]. In this study, we aimed to determine and compare the complex chemical elements in non-atherosclerotic and atherosclerotic samples of coronary arteries obtained by autopsy studies.

In this study, the heavy metal accumulation in the samples was compared by taking samples from the coronary arteries of the cadavers with or without coronary atherosclerosis in autopsies of cadavers. The autopsy series included in this study were obtained from cases examined at the Council of Forensic Medicine in Istanbul, Turkey, between 2019 and 2021. All subjects were over 50 years of age, with a time of death to autopsy interval not exceeding 48 h, to preserve tissue integrity for elemental analysis. The cases represent an urban population residing in Istanbul and its surrounding regions. While detailed occupational and environmental exposure histories were not available due to the retrospective and postmortem nature of the study, it is important to note that Istanbul is a highly industrialized metropolis with known exposure risks to airborne and environmental pollutants, including heavy metals from traffic, industrial emissions, and water sources. Thus, the population studied likely reflects a moderate-to-high environmental burden of heavy metal exposure typical of large urban centers in developing or transitioning economies.

Overall, 2 cm samples were taken from the left anterior descending artery, circumflex artery, and right coronary artery from each cadaver. Sixteen autopsy specimens with CAD and 14 autopsy specimens without CAD were obtained. Heavy metal analysis was performed in 12 of 16 autopsy samples with CAD and in all 14 cases without CAD. Phosphatidylcholine/deoxycholate was applied to the remaining 4 samples with CAD. Each of the tissue samples taken from the autopsy room were named separately and transferred to the study center in isotonic solution and ice container.

The presence or absence of coronary atherosclerosis was determined by macroscopic and histopathological evaluation during autopsy, conducted by forensic pathologists in accordance with national standards. Coronary arteries were dissected longitudinally and visually assessed for the presence of atherosclerotic plaques, stenosis, fibrous cap formation, lipid cores, and calcifications. Histopathological confirmation was performed on paraffin-embedded tissue sections stained with hematoxylin-eosin in ambiguous cases.

Samples were categorized as non-atherosclerotic only when no visible or histological evidence of intimal thickening, lipid deposition, or calcification was present. Age alone was not used to assign groups; inclusion in the non-atherosclerotic group required completely normal coronary morphology under both gross and microscopic examination.

Although calcium levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and showed significantly higher concentrations in the atherosclerosis group, quantitative imaging or histological scoring of calcification was not performed in this study. Future studies incorporating advanced imaging (e.g., micro-CT or von Kossa staining) are warranted to objectively quantify calcium burden.

Tissue samples were dried in an acid solution with the Berghof microwave digestion system. The samples were dried first in a sealed Teflon container for 105 min and then in a microwave oven (Berghof Speedwave, Germany). Before analysis, samples were isolated from metallic materials and dust to avoid contamination. A 250 mg sample was placed in a digestion container and after 5 mL of nitric acid (65%) was added, the mixture was carefully shaken. After waiting for at least 20 min, the containers were closed and the samples were heated in accordance with the microwave oven program [12]. Sample solutions were diluted and analyzed on an Agilent 7500a series ICP/MS. For quality control, the concentration of internal standards is 200 ppb (9Be, 45Sc, 103Rh, 208Bi); reference materials were studied with samples and setting parameters were checked before analysis. The axis breakpoint of the calibration line was used to obtain the detection limit for each element. Linear ordering of the methodology by analyzing different standards with known lower and higher concentrations of each element (0, 1, 5, 10, 20, 30, 40, and 50 ppb) was ensured. At least five different reference materials were used to cover all the elements used. Duplicate samples were also used to determine the accuracy of the analysis. For each element, at least three standards were used to account for the analytical working range of the instrument. Ultrapure water was used to prepare calibration standards and blanks, and three replicates were determined for each sample. A reagent blank was subtracted from all sample results and detection limits were calculated as three times the standard deviation for reagent blanks [13]. In the study, As, Ca, Cd, Cl, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Rb, Sb, Se, Zn elements from tissues were determined by the method described by Berstörm et al. [14]. The method was developed on the Spectro ICP-OES device with the samples taken.

The present study was approved by the Local Ethics Committee of Istanbul Aydın University and the Council of Forensic Medicine (Approval Nos. 2019/212 and 2019/590). All the steps of this study met the standards of Declaration of Helsinki and Good Clinical Practice Guidelines. The written informed consent to participate in the study has been obtained from all adult participants and all vulnerable participants’ parent/legal guardian/next of kin. All cadavers used in this study were those whose legal guardians gave permission for research in autopsy cases supervised by the Council of Forensic Medicine Education and Scientific Research Commission.

Statistical analyses were performed with the SPSS 23.0 (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, version 23.0. Armonk, NY: IBM Corp.) software package. Continuous variables were presented as mean ± standard deviation, and categorical variables were presented as percentages. Non-normally distributed variables were presented as median (minimum–maximum) value. The normality of continuous variables was determined by the Shapiro-Wilk test. Continuous variables were compared by nonparametric Mann-Whitney U test; categorical variables were compared by chi-square tests. Some elemental values were below the detection limit and their values were entered into calculation as lowest detection value of ICP/OES system provided by laboratory. Lowest detection value of each element is provided in Table 2 in ppm equivalent. Probability (p) values of <0.05 were considered to indicate statistical significance.

There was no statistically significant difference in age between groups (60.6 ± 12.3 vs. 67.2 ± 12.4 years; p = 0.593), nor in gender distribution (66.7% male in normal vs. 71.4% in atherosclerosis group; p = 0.069). Similarly, hypertension (25.0% vs. 21.4%; p = 0.829), diabetes mellitus (8.3% vs. 7.1%; p = 0.910), and previous cerebrovascular accident (0% vs. 21.4%; p = 0.088) did not differ significantly. However, a history of CAD was significantly more frequent in the atherosclerosis group (35.7% vs. 0%; p = 0.021). No significant differences were observed in heart weight (453.8 ± 167.1 g vs. 471.4 ± 146.3 g; p = 0.734), left ventricular wall thickness (1.29 ± 0.21 cm vs. 1.32 ± 0.31 cm; p = 0.364), or right ventricular wall thickness (0.31 ± 0.10 cm vs. 0.36 ± 0.13 cm; p = 0.482) (Table 1). Although Table 1 is referred to as a demographic comparison, the data primarily reflect clinical history and echocardiographic parameters, including comorbidities (e.g., hypertension, diabetes), history of cardiovascular and cerebrovascular disease, and basic anthropometric measurements. Apart from gender, detailed sociodemographic data such as occupation, socioeconomic status, or environmental exposure history could not be retrieved due to the autopsy-based nature of the study.

As a result of the analyses made from the samples with and without coronary atherosclerosis, Na, Cu, B, Mn, Bi, Co, Cr, Mo, Ni, Pb, Sb, Se, Sn, Ti, W levels were not significantly different between the groups. Bi, Co, Mo, Pb, Ti, W levels were below the detection limit of ICP-OES in both groups. Mg, K, Ca, P, Fe, Zn, Al, S, As, Pt, Sb, Hg concentrations of the group with coronary atherosclerosis were significantly higher than the group with normal coronary arteries (Table 2).

ND symbolizes the values below the detection limit in both two groups. In case an element level was below only in one group, lowest limit of detection was used for statistical analysis. Ppm was used to describe the quality of performance of concentration normalized to the weight of the tissues entered into the process.

This study aimed to investigate the association between heavy metal accumulation and the presence of coronary atherosclerosis by analyzing trace element concentrations in coronary artery tissues obtained postmortem. Using ICP-MS, we sought to determine whether specific elements are elevated in atherosclerotic coronary tissues and to evaluate their potential contribution to the pathophysiology of atherosclerosis through oxidative, inflammatory, and metabolic pathways.

Our results demonstrate that coronary arteries with histologically confirmed atherosclerosis contain significantly higher levels of several trace elements, including Ca, P, Mg, Fe, K, Zn, Al, S, As, Hg, Pt, and Sb. These findings suggest an active role for heavy metals in plaque biology rather than incidental accumulation.

Epidemiologic and prospective studies have reported that dietary Mg intake and an increase in serum Mg levels reduce the risk of CVD with its inhibitory effects on endothelial dysfunction, insulin resistance, and vascular calcification [15]. In the CORDIOPREV study, which included 939 patients, a statistically significant relationship was shown between Mg levels and carotid intima-media thickness [16]. Although Mg has positive effects on CVD, in our study it was observed at a higher rate on coronary artery plaques in patients who developed atherosclerosis compared to the other group. Likewise, when sclerotic aortic valves changed by aortic valve surgery were examined, Mg was found to be higher than the control group [17]. Another earlier postmortem study showed higher Mg levels in fibrous plaques when compared to normal aortic tissue [18]. Although the effects of Mg on atherosclerosis are not yet clear, it is found at a higher rate in plaque tissue than in normal tissue in autopsy studies. On the other hand, clinical studies have reported the anti-atherogenic effects of Mg [15]. This apparent paradox may reflect the differential roles of magnesium in circulation versus within plaque microenvironments. In early stages, magnesium may exert anti-inflammatory and endothelial-protective effects. However, in advanced plaques, local mineral accumulation may reflect cellular necrosis, altered ion transport, or osteogenic transformation of vascular smooth muscle cells, similar to vascular calcification mechanisms seen with calcium. Indeed, both calcium and magnesium are actively involved in hydroxyapatite formation, and their intraplaque presence may represent a terminal pathologic endpoint rather than a protective function. Thus, tissue-level accumulation in late-stage lesions should not be interpreted as a direct contradiction to the protective role of systemic magnesium but rather as an indicator of complex remodeling processes in vascular pathology.

A multicenter meta-analysis of more than 350,000 participants showed that exposure to As, Pb, Cd, and Cu was directly associated with exposure dose and increased CVD risk; however, there was not statistically significant relationship between Hg and CVD. An inverse correlation was observed between Se and Zn and CVD risk [5]. Cd exposure closely linked to atherosclerosis and hypertension. Cd induces oxidative stress and alters the endothelial function by decreasing nitric oxide level. Acute exposure of Cd was investigated revealing that release of endothelium-derived vasoconstrictor prostanoids and angiotensin II increased phenylephrine levels in circulation [19]. Previous epidemiological studies demonstrated that the association with carotid artery intima-media thickness and As was consistently positive. Also the findings of experimental studies were proving the tendency of atherosclerosis even in low concentrations [20]. Lead exposure is associated with increased risk of CVD and blood pressure; however, the mechanism is not only arised from hypertension; nevertheless, experimental studies suggested increased incidence of atherosclerosis [21]. Most common mentioned pathogenesis in previous studies were oxidative stress and lipid peroxidation stimulates endothelial injury and smooth muscle cell proliferation, which is the initial phase of atherosclerosis. Increased collagen levels and impaired elastin is related with arterial stiffness that leads to hypertension [22, 23]. Another significant association between heavy metal and atherosclerosis derives from Hg. The pathogenesis of Hg on the path of atherosclerosis, similar to that of others, is oxidative stress and inflammation, but apart from the other poisonous metals Hg induces hyperlipidemia and impaired fasting glucose level [24]. In our study, As, Pb, and Hg were found significantly higher in atherosclerotic tissue samples, confirming the literature. Since Cd could not be isolated in atherosclerotic patients group, a difference occurred between them, but it could not reach statistical significance.

Increased serum Ca and P levels lead to vascular calcification and consequently increased cardiovascular mortality, especially in patients with chronic renal failure [25]. Studies have shown the correlation between serum Ca and P levels and arterial stiffness, carotid intima-media thickness [26]. Calcification and increased coronary atherosclerosis were detected in the autopsy series of Robert et al. [27] with chronic hypercalcemia. Although several observational studies and meta-analyses have suggested a potential association between calcium supplementation and increased cardiovascular risk, the evidence remains controversial and far from definitive. Some studies have linked calcium supplements – especially without concurrent vitamin D – to elevated coronary artery calcification and increased myocardial infarction risk, while others have failed to demonstrate a causal relationship. Importantly, our findings do not directly evaluate supplement use but rather identify tissue-level calcium accumulation within atherosclerotic plaques. This supports the role of vascular calcification as a hallmark of advanced atherogenesis, irrespective of systemic intake. Therefore, while our study adds to the growing concern about calcium’s role in plaque biology, it should not be misinterpreted as evidence against dietary or therapeutic calcium use without consideration of individual cardiovascular risk profiles and clinical context [28‒30]. Increased serum P levels were also associated with an increased incidence of CVD. Although the mechanism is not clear, it is thought that increased P level decreases 1,25-dihydroxyvitamin D synthesis and also directly leads to vascular calcification [31]. In our study, the most prominent differences were observed in calcium and phosphorus levels (Ca: 51,384 vs. 1,723 ppm; P: 30,791 vs. 3,443 ppm; p < 0.005), supporting the notion that vascular calcification is a hallmark of advanced atherosclerosis. These minerals, in excess, contribute to arterial stiffness and plaque destabilization. The observed Ca/P ratio shift in atherosclerotic plaques (1.67 vs. 0.5) aligns with previous autopsy and imaging studies of microcalcifications in early plaque development.

In the study, most traces were observed in metal plaque at a higher rate than in normal vessel structure since postmortem coronary artery samples and metal precipitated in the plaque formed on the artery wall were measured. Calcium deposit accumulation, which lies in the basic mechanism of atherosclerosis, has been associated with calcification regulatory proteins and colocalization of trace metals such as Zn and Fe [32]. The Ca/P ratio, which is above 2.2 in hydroxyapatite crystals, is observed between 1.4 and 2 in microcalcifications. In our study, the Ca/P ratio was 0.5 in normal coronary artery cases and 1.67 in atherosclerotic coronary arteries [32]. There is a statistically significant increase in both Ca and P ratios in atherosclerotic plaques compared to the other group.

Certain trace elements participate in the structure of enzymes and take part in hormone synthesis. The first line of defense consists of antioxidant enzymes such as SOD, catalase, and GPx, which suppress the formation of free radicals. GPx is present in the cytoplasm of cells and protects cells against oxidative damage caused by H₂O₂. It contains a high amount of Se in its GPx structure [33]. Evidence obtained so far is that superoxide radicals have a serious impact on the pathogenesis of hypertension and atherosclerosis. SOD derivatives prevent endothelial and mitochondrial dysfunction by inhibiting intracellular peroxynitrite formation and oxidation products that reduce nitric oxide levels [34‒36]. Cu and Mn in the vascular tissue have a catalytic effect on the intracellular SOD activation reaction, while Zn takes part in the stabilization of the reaction [34]. Cu is involved in the structure of SOD. Cu is involved in the SOD structure as well as in enzymes such as tyrosinase, ascorbic acid oxidase, and cytochrome oxidase [37]. The trace element Cu is essential for enzymes; however, it has a negative effect by inhibiting at a certain elevated concentration. Similarly, Cd produced dramatic effects on malondialdehyde, which serves as lipid peroxidation marker [38]. In our study, since the concentration levels of Se and Cd were relatively low, there was not any statistically significant difference between the groups.

Elements like platinum (Pt) and antimony (Sb), which were significantly elevated in the atherosclerosis group, are less frequently studied but may reflect exposure to industrial pollutants. Their roles in vascular biology are poorly understood but warrant further investigation given their cytotoxic potential.

It has been shown in the literature that heavy metals increase both the tendency to atherosclerosis and the incidence of CVD [5, 24, 39]. Heavy metal exposure is as effective as modifiable risk factors in increasing cardiovascular risk. In our study, plaque structures that developed atherosclerosis were examined regardless of the cause of death, and almost all trace metals were detected more in the coronary atherosclerosis group than in the other group. While all cadavers were examined under standardized forensic protocols, the absence of cause-specific categorization prevents us from evaluating whether the observed heavy metal accumulation may have contributed to fatal cardiovascular events. Future studies incorporating cause-of-death data could help elucidate the relationship between trace element burden and acute clinical outcomes such as myocardial infarction or sudden cardiac death. To completely prevent heavy metal exposure is almost impossible in this era. Heavy metals are also included in the etiology of CVD, apart from oncological and neurological diseases. Before the primary percutaneous coronary intervention era, heavy metal chelation therapy studies were conducted and some positive results were obtained [40]. Also, a contemporary study showed reduction of cardiovascular events after myocardial infarction with the edetate disodium chelation therapy significantly [41]. While chelation therapy has shown promise in reducing cardiovascular events in high-risk populations, it remains a secondary or adjunctive approach. Our findings support the need for a broader preventive strategy focused on reducing environmental and occupational exposure to toxic metals such as arsenic, mercury, lead, and platinum. Public health policies should prioritize monitoring industrial emissions, regulating contaminants in water and food, and implementing exposure surveillance in vulnerable populations. Given that heavy metals may act as upstream triggers in the atherosclerotic cascade, mitigating exposure at the population level could represent a cost-effective and scalable preventive measure in reducing CVD burden globally.

Although previous studies have demonstrated elevated levels of certain metals – such as arsenic, cadmium, and lead – in the arterial tissues of patients with atherosclerosis, our study provides several novel contributions. First, the current investigation uniquely utilizes postmortem human coronary artery samples with standardized anatomical sampling from the left anterior descending artery, right coronary artery, and circumflex artery, allowing a more consistent and representative comparison between diseased and non-diseased vessels. Second, unlike many earlier studies that focused on serum or urine metal concentrations, we directly analyzed tissue-level elemental accumulation using ICP-MS, which reflects chronic exposure and local pathogenic relevance more accurately. Third, this study presents a comprehensive panel of 20 trace elements, offering a broader perspective than most prior reports. Notably, the significant elevation of platinum (Pt), antimony (Sb), and mercury (Hg) in atherosclerotic samples – elements not consistently emphasized in earlier literature – points to underrecognized contributors that may act synergistically in promoting oxidative stress and inflammation in arterial walls. Lastly, the study contextualizes these findings within a toxic inflammatory hypothesis, proposing that localized heavy metal accumulation may not only mark but actively drive atherosclerotic changes, laying the groundwork for potential new diagnostic or therapeutic pathways.

A major strength of this study is the use of standardized autopsy samples and precise ICP-MS quantification. However, several limitations exist: lack of detailed exposure history, small sample size, and the inability to assess cumulative systemic metal burden. Quantitative imaging or histological scoring of calcification was not performed, which may limit interpretation of Ca/P data beyond biochemical concentration. The materials obtained in the study protocol were examined within the first 48 h after death, and concentration changes that may occur during the 24-h period is the another limitation of the study.

In this autopsy-based study, we identified significantly higher concentrations of multiple heavy metals – including Ca, P, Mg, Fe, K, Zn, Al, S, As, Hg, Pt, and Sb in atherosclerotic coronary arteries compared to non-atherosclerotic vessels. These findings provide robust tissue-level evidence suggesting a potential role for heavy metal accumulation in the pathogenesis of atherosclerosis.

While oxidative stress and inflammation are established mechanisms through which heavy metals exert vascular toxicity, our study does not directly assess these pathways. Whether oxidative stress acts as a mediator in this context remains to be elucidated in future prospective or cross-sectional studies that jointly evaluate both heavy metal burden and oxidative stress markers.

Alternative explanations for the observed differences between groups may include confounding environmental factors such as air pollution or lifestyle-associated exposures like smoking, which are known sources of heavy metal accumulation and also contribute to cardiovascular risk. These hypotheses underscore the need for future investigations integrating environmental exposure histories, systemic biomarkers, and vascular outcomes.

The present study was approved by the Local Ethics Committee of Istanbul Aydın University and the Council of Forensic Medicine (Approval No. 2019/212 and 2019/590). All the steps of this study met the standards of Declaration of Helsinki and Good Clinical Practice Guidelines. All cadavers used in this study were those whose legal guardians gave permission for research in autopsy cases supervised by the Council of Forensic Medicine Education and Scientific Research Commission. The written informed consent to participate in the study has been obtained from all adult participants and all vulnerable participants’ parent/legal guardian/next of kin.

The authors declare that there is no conflict of interest regarding the publication of this article.

The authors have not declared financial support.

Onur Yolay, Emine Esra Kasapbasi, and Ayhan Olcay contributed to the study as design of study, sample collection, critical analysis, text writing, and histologic examination and contributed 25% each. Serdar Baki Albayrak and Erdem Tezcan contributed to the study as design of study and critical analysis and contributed 11%. Ceyhun Kucuk, Hasan Karaoglu, Emir Canturk, Bekir Inan, Dogac Oksen, Ozge Cetinarslan, and Fadil Umihanić contributed to the study as sample collection and text writing and contributed 2% each.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.