Abstract: Background and Objective: The elemental composition of the blood is significant to the health status of humans. In current study, whole blood of neurology patients, cardiology patients and healthy subjects were analyzed for elemental composition to determine the concentration levels in relation to age and gender. Materials and Methods: In this study, whole blood samples were freeze dried and screened for elemental concentration levels. Particle Induced X-ray Emission (PIXE) was used for elemental determination and quantification. Results: The results of analysis using ANOVA showed that Zn and Ca concentrations were low in the cardiology and neurology patients compared to those found in the control. Copper (Cu) concentrations were higher in the blood of cardio and in neuro-patients compared to the controls level. Selenium (Se) mean concentration was significantly higher in neurology patients (1436.21 ppm) compared with cardiology (633.57 ppm) and control (487.93 ppm). Potassium (K) was significantly high in cardiology patients compared to others. Na/K ratios were generally higher in females, Se/Zn ratio was significantly higher in female neurology and Cu/Zn ratio was significantly higher in male neurology subjects at 95% probability level. Conclusion: The study concluded that Se in neurology patients, K in cardiology patients and the metal-metal ratio values might be useful in the individual treatment of these patients.
INTRODUCTION
Metals ions affect human health in many ways. Some metals (lead, cadmium and mercury) show only deleterious effects on human health, some (selenium and manganese) play an essential role in the human biological system at certain concentration ranges, while negative health implications are observed when concentrations in biological systems are in deficit or excess. Biomonitoring then becomes an important tool to quantify human exposure to contaminants and their concentrations in the blood as biomarkers1-3. Biomonitoring minimizes the uncertainty of elemental composition by determining levels of exposure and making these determinations through calculations of estimated quantities based on analysis of environmental samples and postulations about exposure pathways4,5. Human are easily exposed to metals due to their wide presence in the industry and long-term environmental persistence. Among the general population, exposure to a number of metals is widespread but generally at substantially lower levels than have been found in industry. Accumulation of metal ions in fatty tissues and circulatory system, negative effects on central nervous system and functioning of internal organs as well as acting as triggers of several serious diseases including tumors can be listed as adverse effects of metal ions on humans6,7. Metals share certain physical and chemical features and it is reasonable to speculate that common mechanisms for carcinogenicity may take place. Specific carcinogenic pathways, however are determined by numerous factors including metal type, speciation, solubility, possible metal-metal interactions and others8,9. The carcinogenicity of arsenic (III), chromium (VI) and nickel (II) has been confirmed in humans8. Although epidemiologic data are less extensive for beryllium (II) and cadmium (II). Findings in humans of excess cancer risk are supported by the clear demonstration of carcinogenicity in experimental studies10. Other metals including antimony and cobalt may be carcinogenic in humans but the experimental and epidemiologic data are limited8. Heavy metal adsorptions from the digestive system and haemoglobin sensitivity to these metals are much higher in children compared to adults11-14. The health risk is especially on the high side for children because their tolerance to poisons is very low15. Furthermore, the chronic effects of metals that might not be immediately apparent represent an important issue that needs to be taken into consideration. Due to the negative characteristics attributable to metals, it is necessary to often monitor their levels in the body16-19. Based on these, it is very important to analyze complex biological matrices such as; blood, serum, tissue and urine.
There are nutrient elements needed for metabolism and proper functioning of the entire body. Minerals such as; Cu, Zn, Se, Ca, Mg K and Na are essential at concentrations for body system to function well and for prevention of diseases, excessive or low concentrations are detrimental to the body and can lead to diseases. Magnesium (Mg) plays an essential role in nerve transmission, neuromuscular conduction and for regulation of muscles contraction (including that of the heart), blood pressure and insulin metabolism and in the synthesis of DNA and RNA. It also functions in prevention of excessive excitation that can lead to neuronal cell death. It has been implicated in multiple neurological disorders20. These roles within the nervous system make Mg a mineral of intense interest for the potential prevention and treatment of neurological disorder15. Grober et al.21 suggested that there was a lot of study to be done to further clarify its role in Alzheimer’s and Parkinson’s diseases. Generally, the mechanistic attributes of Mg in neurological diseases connote the macro mineral as a potential neurological disease prevention and treatment element.
Selenium (Se) is an antioxidant trace element and an important regulator of the brain function. Humans suffer from health problems due to Se deficiency22. Se is consumed mainly via food in oxidative state of II. The total Se content in adults varies between 10-20 mg depending on place of living or health23,24. This oxidative state enables Se to be incorporated into selenoproteins as an amino acid. Several selenoproteins are expressed in the brain25, some of them are glutathione peroxidases and thioredoxin reductases. Selenoproteins are strongly involved in antioxidant defense and in maintaining intercellular reducing conditions26. Increased oxidative stress has been implicated in neurological disorder, including Parkinson’s diseases, Alzheimer’s Diseases (AD), stroke, epilepsy, cognitive decline and others. Growing evidences suggested that Se depletion followed by decrease activity of Se-dependent enzymes may be important factors connected with those symptoms27-34. Some research showed a direct correlation between Se and the cognitive decline in AD patients when compared with healthy individual35. Se is crucial for preserving brain function and preventing age-related neurodegenerative disorders27.
There are many methods that have been reported in literature for biomonitoring, including; Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and other spectroscopic and electrochemical methods36,37. It is clear that the monitoring of metals in biological samples is given great attention17,38,39. Blood as a biological material is collected most frequently for laboratory testing. The way of sampling is given by methodology of examination in the laboratory and its technical equipment.
Cardiology diseases refer to diseases and abnormalities of the heart as well as some parts of the circulatory system. Neurology diseases are those that seriously affect the nervous system, structural, biochemical or electrical abnormalities in brain, spinal cord and nerve. Symptoms may include paralysis, seizures, confusion, loss of sensorium and many others. Since metals, PAHs had been implicated in reducing IQ40,41 and neurological diseases affect the brain and the nervous system. It is necessary to investigate whether there is a link between the elements and the diseases41.
In this study, whole blood of neurology, cardiology and healthy subjects were analyzed for elemental composition to determine the concentration levels in relation to age and gender and study the metal-metal ratio to arrive at possible implicated elements.
MATERIALS AND METHODS
The research was carried out between January, 2014- December, 2018 and sampling were taken from patients visiting the consultant outpatient neurology and cardiology clinics of the Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria. Medical and Postgraduate students of the Obafemi Awolowo University were the control subjects for this study.
Sampling: Ten milliliters of whole blood was collected via the vein using syringe and needles in heparinized bottles and transferred to the laboratory for freeze drying prior to analysis. After drying the samples were pulverized and stored in clean and well labeled zips bag prior to analysis.
Sample analysis: The freeze dried blood samples were ground in agate mortar and prepared into thick pellets 13 mm in diameter without a binder.
The pellets formed were then analyzed with Particle Induced X-ray Emission (PIXE) for elemental determination and quantification.
Statistical analysis: The elemental composition and concentrations of the cardiology and neurology patients as well as the controls were statistically determined using descriptive and inferential analysis by IBM SPSS statistics (version 21) at 95% confidence interval.
Validation of equipment: The PIXE experiments were performed at the Ion Beam Analysis (IBA) facility of the Centre for Energy Research and Development, Obafemi Awolowo University, Ile-Ife, Nigeria using a 2.5 MeV proton beam source. The facility is centered on a NEC5SDH 1.7 MV Pelletron Accelerator, equipped with a radio frequency charge exchange ion source.
IAEA-A-13 (Animal blood) standard was used for the determination of the H-value which was subsequently used for analyzing the samples and to assure the accuracy of the experimental procedure. The PIXE set up was calibrated using some pure element standards and NIST geological standard (NBS278).
RESULTS
Mean concentration of elements and the significance difference: Twenty elements were determined using PIXE at varying concentrations as expressed in Fig. 1a-e. The concentrations of the dominant elements (Na, K, Fe, Cl, S, Pb) were above 5,000 ppm. There was no significant difference in the levels of Cl, Fe, Na and Pb in the patients (Cardiology, Neurology) and the control subjects. There was a significant difference in K levels of the cardiology and neurology patients but no difference compared to the control. Potassium (K) was significantly higher in the cardiology than in the neurology patients.
Selenium (Se) was significantly higher in neurology patients than cardiology and control subjects. There was also a significant difference between the sulphur concentrations in cardiology patients and the control subjects but no significant difference was observed between the neurology patients and the control subjects at 95% probability level. Copper (Cu) was higher in the neurology patients than cardiology but not at significant level.
Gender and age difference in elemental composition: The elemental compositions were considered in terms of gender and age difference.
The elemental distribution according to age did not follow a particular trend among the subjects (patients and controls) presented in Fig. 2a-f. Elements such as Zn, Mg, Ca, Mn and Cr were significantly higher in the control females than in the female patients between the ages of 20-50 years (Fig. 2b, c, e).
Aluminium (Al) and Si concentrations were significantly higher in the female control than cardiology and neurology patients and male control (Fig. 2d). Comparison between the male patients (cardio and neuro) showed no significance in the Al and Si concentrations, but Cu was significantly higher in the blood of cardio-female subjects than the male counterparts (Fig. 2f), while Mn was higher in all female subjects (Fig. 2e).
| Fig. 1(a-e): | Mass concentrations of the elements in the blood of cardiology, neurology patient and the controls |
| Error base: ±2SE | |
| Fig. 2(a-f): | Gender and age difference in elemental composition of blood of the patients and controls |
| Table 1: | Gender and disease metal-metal ratio |
| Cont: Control, Card: Cardiology, Neu: Neurology | |
| Appendix 1: | Descriptive concentration of the elements in the blood samples |
| Appendix 2: | Comparison of Se mean values in females |
Metal-metal ratio: Table 1 showed that Ca/Zn (18.87), K/Zn (536.47) and Cu/Zn (69.49) ratio were highly elevated in female cardiology patients than the male patients. The total elemental ratio (T) calculated for K/Zn (1031.21) was significantly higher in cardiology than in neurology and the control.
It was also discovered that Cu/Zn ratio in male neurology patients was significantly higher (115.77) above all the male and female in cardio and control. The total ratio (T) for the neurology (138.71) was above those of cardiology (112.22) and control (16.94). Se/Zn ratio was found to be significantly high in the female neurology (101.44) compared with cardiology and control, respectively.
The metal-metal ratio was analyzed in the patients’ whole blood to discover the enrichment of these metal in the whole blood as presented in Appendix 1. The value above one (>1) in cardio/neuro (P/Cl, Ca/Zn, K/Zn), cardio/control (P/Cl, Ca/Zn, K/Zn, Cu/Zn and Se/Zn) and neuro/control (Na/K, P/Cl, Cu/Zn and Se/Zn) ratios showed that numerators were more enriched than the denominators in the elements involved. Selenium was significantly higher at 95% probability level with value 0.005 in neurology female than female cardiology and 0.003 confidence level above the control subjects as presented in Appendix 2.
DISCUSSION
Sulphur was among the elements with mean concentration above 5000 ppm (8945.35, 8026.95 and 8334.35 ppm) in cardiology, neurology and control subjects (Appendix 1). Seneff42 had explained the role of sulphur as a healing agent in joint diseases and possible contributor in heart related diseases, especially as Vitamin D3 sulfate and cholesterol sulfate are found in the blood stream. Sulphur is part of methionine and cysteine which are amino acids needed in protein synthesis. Though these elements are essential, there is a need for regulation in the body. Nimni et al.43 prescribed minimum requirement for all essential amino acids as being estimated in terms of their ability to maintain nitrogen balance.
Other mineral elements such as Ca, Mg, Zn, Cu and Mn were compared at different associations. Calcium and Magnesium were significantly higher in the blood of the controls than in the patients at 95% confident interval. The significantly lower concentration of Mg compared with those of controls might have contributed to neurological diseases as claimed by Kirkland et al.20 and Grober et al.21. Selenium was significantly higher in the neurology patients (1436.21 ppm) than cardiology patients (633.57 ppm) and the controls (690.60 ppm) (Appendix 1). Selenium high concentration has been implicated in neurological diseases26,27. Other mineral elements Cu, Zn and Mn showed no significant difference at 95% confidence interval in the patients and the controls, Zn in the cardiology and neurology were lower in concentrations (25.61, 33.25 ppm) compared to the controls but no difference was highlighted statistically in the patients’ blood concentrations. These were within the literature values of normal subjects as indicated in the study of El-Amri et al.44, Zhuk et al.45 and Iyengar et al.46. Copper concentration was low in the controls’ blood (1496.95 ppm in cardio and 1864.08 ppm in neuro patients) as compared to the controls level of 1230.83 ppm. The values of Cu in the subjects of this research were comparable and within the range of 6.02-3525 μg g–1 as found in normal subjects44.
Figure 2a-e described the gender distributions of the elemental mean concentration in the patients and controls. While, Se was significantly higher in the female neuro-patients (Appendix 2). Copper was only significantly higher in female cardio-patients. Al and Si concentrations were significantly higher in the female controls than other female counterpart. Selenium depletion has been found to decrease activity of Se-dependent enzymes, which has been an important factor connected with the symptoms such as neurological disorder, epilepsy and cognitive decline27,30,32. Se as an antioxidant trace element regulates the brain function, if found in excess it is possible to increase activities of Se-dependent enzymes that could increase oxidative stress or neurology disorder. Copper is a trace element present in all tissues and is required for cellular respiration, peptide amidation, neurotransmitter biosynthesis and connective tissue strength47. Copper is a cofactor for numerous enzymes and plays an important role in central nervous system development, low concentrations of copper may result in incomplete development, whereas excess copper may be injurious48. Copper may be involved in free radical production that results in mitochondrial damage, DNA breakage and neuronal injury. Evidence of abnormal copper transport and aberrant copper-protein interactions in numerous human neurological disorders supports the critical importance of this trace metal for proper neuro development and neurological function. While, Al and Si were found to be significantly lower in the blood of male-cardiology patients, these 2 elements could exist as aluminosilicates and main component of antacids (MgSi3O3) and anti-diarrhoeal (Al and Mg silicates) and as excipients in proprietary analgesics48.
The distribution of essential elements were localized at age 31-40 especially in the female of control subjects. This may be attributed to the production age in which all these elements are needed for pregnancy, lactation and breast feeding.
The total K/Zn ratio of 1031.31 above neurology and control showed that K was elevated in the blood of cardiology patients. Potassium is a mineral that is essential for health but too much of sodium in the diet or certain medicine can increase the need for it49. Sun et al.49 concluded that high K reduces vascular calcification and artery stiffness, thus allows free flow of blood and lowers blood pressure. This also corroborate the advocacy of low dietary salt level by physicians. Na/K was linked to cardiovascular disease risk. Cook et al.50 reported that the higher the ratio, the higher the risk. In this study, it was found that the female stand the chances of having cardiovascular diseases than male as seen across the Na/K ratio, in cardiology (0.97), neurology (1.59) and the control (1.38). The low value of Na/K ratio in cardiology may be attributed to on-going treatment while neurology and control may be prone to or indication of on-set of cardiovascular condition. Ca/Zn ratios should be searched as major treatment factors in the management of female cardiology patients.
The high Se/Zn ratio in female neurology confirmed that Se may be one of causative agent of neurology illness as stated by Brigelius-Flohe and Kipp26 and Dominiak et al.27. The research of Osredkar and Sustar51 reported that Cu/Zn ratio Superoxide Dismutase (SOD1) generated during metabolism activities causes damage to cell membrane especially when Cu is not present in sufficient quantity, the activity of the superoxide dismutase is diminished. Copper enhances the functioning of Cu/Zn (SOD1) enzymes by working together with mineral Zn and it is actually the ratio of Cu/Zn rather than the absolute amount of Cu or Zn alone that helps the enzyme to function properly. Neurological diseases involving copper include Amyotrophic lateral sclerosis, Huntington disease and Menkes diseases; Deficiency and excess of copper on the brain and neural tissues as a result of these diseases could serve as biomarker of copper status.
The regulation of elemental ratio at 50% standard deviation according to Ojo et al.52 showed a similar trend (P/Cl, Ca/Zn, Na/Zn, Fe/Cu, Cu Zn) with the above metal ratios. More studies should be carried out on Na/K, Na/Cl ratios in cardiology patients and Cu/Zn, Se/Zn ratios in neurology patients.
CONCLUSION
This study highlights the importance and role of elemental concentrations and elemental concentration ratios in health and disease. Copper levels were significantly higher in the blood of female cardiology patients and Se was higher in female neurology patients as confirmed by Cu/Zn ratio and Se/Zn ratio in these subjects which supported previous literature findings. The low concentration of Mg and Ca across age boundaries in the neurology and cardiology patients is very vital as they were significantly higher in the 21-30 age in both male and female controls. Clinical studies are recommended on the possible inclusion of Se/Zn and Cu/Zn concentration ratios in the management and treatment of neurology patients with gender consideration. The K/Zn, K/Na, Ca/Zn and Cu/Zn synergy in the treatment of cardiac ailments showed that these elemental ratio were significantly higher at 95% confidence level.
SIGNIFICANCE STATEMENT
This study discover the synergistic effect of metals that can be beneficial and use as biomakers in the treatment of cardiology and neurology diseases. The Na/K ratio was more pronounced in female generally than in male both in the patients and controls, thus, mean concentrations of elements may not be enough in determining the cause of diseases, rather the metal -metal ratio as confirmed in the literature may be a better option. This study will help the researcher to uncover the critical areas of gender differences in elemental body burden that many researchers were not able to explore. Thus a new theory on importance of gender metal ratio may be arrived at.
ACKNOWLEDGMENTS
The authors wish to acknowledge the support of Obafemi Awolowo University and the Tertiary Education Fund (TETFUND) for sponsoring this project with grant number DVC/AC/37/TETFUND/DESS/RP/VOL.V/2014/4 and the staff and students of the Department of Medicine, Obafemi Awolowo University Teaching Hospital for assistance in sample collection.