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Distribution of zinc, copper and iron in biological samples of Pakistani myocardial infarction (1st, 2nd and 3rd heart attack) patients and controls
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Clinica Chimica Acta 389 (2008) 114 – 119 www.elsevier.com/locate/clinchim
Distribution of zinc, copper and iron in biological samples of Pakistani myocardial infarction (1st, 2nd and 3rd heart attack) patients and controls Tasneem Gul Kazi a,⁎, Hassan Imran Afridi a , Naveed Kazi b , Mohammad Khan Jamali a , Mohammad Bilal Arain a , Raja Adil Sarfraz a , Nusrat Jalbani a , Rehana Ansari a , Abdul Qadir Shah a , Ateeq-ur- Rehman Memon a , Ghulam Abbass Khandhro a a
National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, 76080, Pakistan b Liaqat University of Medical and Health Sciences, Jamshoro, Pakistan Received 29 September 2007; received in revised form 1 December 2007; accepted 3 December 2007 Available online 14 December 2007
Abstract Background: The pathogenesis of some heart diseases has been associated with changes in the balance of certain trace elements. We examined the association of iron, copper and zinc between biological samples (scalp hair, whole blood and urine) and mortality from myocardial infarction (MI) patients of (first, second and third heart attack). Methods: The biological samples were from 130 MI patients (77 male and 53 female, age range 45–60 years) and 61 healthy age-matched controls (33 male and 28 female). The metals in the biological samples were measured by the flame atomic absorption spectrophotometry, prior to microwave assisted acid digestion. The validity of the methodology was checked by the biological certified reference materials. Results: During this study, 78% of the 32 patients aged N 50 years, registered after the third MI attack died. In these subjects the concentration of Fe and Cu were increased by 0.83% and 3.12% in the scalp hair while in blood samples 9.7% and 22.5% were enhanced respectively, as compared to those who tolerated 3rd MI attack (p = 0.072). The concentrations of Zn in whole blood and scalp hair samples were lower in MI patients as compared to normal subjects. Conclusion: Deficiency of zinc and high concentration of copper and iron may play a role in the development of heart disease. © 2007 Elsevier B.V. All rights reserved. Keywords: Zinc; Iron; Copper; Myocardial infarction; Atomic absorption spectrophotometer
1. Introduction Atherosclerosis is a well-known precursor of ischemic heart disease due to accumulation of lipids and fibrous elements in arteries [1]. The development of atherosclerosis depends on a balance between pro-inflammatory stimuli, anti-inflammatory and antioxidant defence mechanisms [2]. Trace elements are being increasingly recognized as essential mediators of the development and progression of cardiovascular disease (CVD). Although there is not necessarily a direct cause–effect relationship between the development of CVD and trace elements status, it is generally believed that some of them (Cu and Fe) are CVD risk factors [3]. ⁎ Corresponding author. Tel.: +92 022 2771379; fax: +92 022 2771560. E-mail address: [email protected] (T.G. Kazi). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.12.004
Fortunately, the cell possesses highly efficient protective mechanisms, including antioxidants such as α-tocopherol, ascorbate, β-carotene, glutathione, and metal-binding proteins such as transferrin, ceruloplasmin and enzymes such as manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (Cu–ZnSOD), selenoenzyme glutathione peroxidase (GSH-Px) and iron-containing enzyme catalase [4]. All these metal binding antioxidants are designed to prevent the occurrence of free radical-induced injury under normal conditions. However, it has been argued that these protective mechanisms may be overwhelmed then severe free radicalmediated injury may occur [5]. When a free radical comes in contact with the inner lining of the arteries, microscopic injuries result. Eventually, the build up of fat, cholesterol, toxic metals and other substance at the site of injury narrows the arteries, which lead to cardiovascular disease [6].
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Fe and Cu are essential for blood formation, Cu is a component of superoxide dismutase (SOD), which also requires Zn and Mn to function. Deficiency of any of these elements reduced the activity of SOD which has been linked to atherosclerosis and lung injury, especially in the elderly [7]. Numerous studies have assessed the association between the Fe status and risk of coronary heart disease [8]. A hereditary haemochromatosis or Fe overload condition has been identified as an independent risk factor for myocardial infarction and cardiovascular mortality. Sullivan [9] reported that the difference in heart disease risk between the sexes might be due to differences in body Fe stores. Cu is a powerful catalyst of LDL oxidation in vitro, often more effective than Fe [10]. When Cu ions are added to LDL, they can react with the lipid hydroperoxides within the LDL to generate peroxyl and alkoxyl radicals that continue LDL oxidation. The study of Zhang et al. [11] showed that the high concentrations of Cu might inhibit phosphodiesterase activity, thus exerting an influence on the contractibility of cardiomyocytes and of smooth muscle cells in coronary arteries. Zinc (Zn) is an important component of biomembranes and an essential cofactor in a variety of enzymes [12]. Zn has antioxidant-like properties; thus, it can stabilize macromolecules against radical-induced oxidation in vitro as well as limit excess radical production [13]. Zn deficiency is associated with an increase of Cu and Fe due to the antagonistic relationships between these metals. Indeed, the oxidative damage which occurred in Zn-deficient rats could be due to increases of tissue Fe. It has been suggested that an imbalance between Cu and Zn may be a factor in the etiology of CVD [14]. Determinations of trace metals in human tissues and fluids were used to obtain information on the nutritional status for diagnosis of diseases, indication of systemic intoxication and to obtain information on environmental exposure. In the majority of cases, whole blood, serum, plasma and urine were analyzed [15]. The importance of exploring the depot-storage capacities of various elements, particularly the toxic ones, remains a vital aspect in elemental analysis largely met by urine and hair testing [16]. The determination of trace metals in biological samples requires the use of sensitive and selective techniques such atomic absorption spectrometer (AAS). This technique has need for solubilization of the analyte and complete or partial decomposition of the matrix using either convective systems or microwave ovens and dry ashing. The main advantage of microwave assisted samples pretreatment is its requirement of a small amount of mineral acids and a reduction in the production of nitrous vapors [17]. 2. Materials and methods 2.1. Apparatus A Perkin-Elmer model A. Analyst 700 (Norwalk, CT) atomic absorption spectrometer equipped with deuterium background correction was used in the study. The hollow cathode lamps of Cu, Fe and Zn were run under the conditions suggested by the manufacturer. A single element hollow cathode lamp was operated at 7.5 mA for Fe and Zn while at 7.0 for Cu with a spectral bandwidth of 0.7 nm. The analytical wavelengths were set at 214.0, 248.5 and 324.8 nm for Zn, Fe and Cu respectively. The flow rate of air (oxidant) was 17.0 l/min, while 2.0 l/min for acetylene. Integrated absorbance signals computed by the AA spectrometer were employed throughout. A Pel (PMO23) domestic microwave
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oven (maximum heating power of 900 W) was used for digestion of the biological samples. Acid washed polytetrafluoroethylene (PTFE) vessels and flasks were used for preparing and storing solutions.
2.2. Reagents and glass wares Ultrapure water obtained from ELGA labwater system (Bucks, UK), was used throughout the work. Concentrated nitric acid 65% and 30% hydrogen peroxide, from Merck (Darmstadt, Germany) were checked for possible trace metal contamination. Standard solutions of Zn, Cu and Fe were prepared by dilution of certified standard solutions (1000 ppm) from Fluka Kamica (Bush, Switzerland). Dilute working standard solutions were prepared immediately prior to their use, by stepwise dilution of the stock standard solution with 0.2 mol/l HNO3. All solutions were stored in polyethylene bottles at 4 °C. For the accuracy of methodology, certified samples of human hair BCR 397 (Brussels, Belgium), Clincheck control-lyophilized human urine and human whole blood (Recipe, Munich, Germany) were used as certified reference materials (CRMs). All glassware and plastic materials used were previously soaked for 24 h in 2 mol/l nitric acid, washed with distilled water and finally rinsed with deionized water, dried and stored in a class 100 laminar flow hood.
2.3. Sample collection and pre-treatment The study protocol was approved by the local ethics committee of higher education commission of Pakistan. This was a hospital based study. The history of MI patients admitted in Dewan-e-Mushtaque, Civil Hospital Hyderabad, Pakistan in (2006–2007) was collected. Patients admitted to the emergency department of hospital within 12 h of the onset of clinical symptoms suggestive of myocardial ischemia were randomly chosen, while we sampled the scalp hair and blood samples of third attack patients within 15–30 min, admitted in hospital. The study was carried out on a sequential sampling of 130 patients, (35 males and 23 females of 1st MI attack, 25 males and 15 females of 2nd MI attack, 17 males and 15 females of 3rd MI attack patients), age range between 45 to 60 years, who were undergoing routine coronary angiography, mainly for stable angina, and who had at least one positive test of MI including (exercise stress test and Dobutamin stress echocardiography). These tests and the angiogram were performed at the cardiac ward. During the study period, 32 patients of 3rd MI attack, 13 males and 12 females expired; therefore we could not collect their urine samples. The major criteria of inclusion for the present study were skeletal muscle damage or trauma, cardiac resuscitation, and infectious or inflammatory diseases. Patients who were on lipid-lowering
Table 1 Clinical and biochemical characteristics of patients with and without CAD Parameters Male Height (cm) Weight (kg) Waist circumference (cm) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Dyslipidemia, n (%) Female Height (cm) Weight (kg) Waist circumference (cm) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Dyslipidemia, n (%)
Controls
1st MCI
2nd MCI
3rd MCI
183.2 ± 12.3 180.2 ± 8.7 82.5 ± 12.5 90.5 ± 9.5 88.4 ± 12.0 95.5 ± 10.1
186.0 ± 10.5 177.4 ± 7.6 93.5 ± 8.1 97.39 ± 6.3 102.4 ± 13.2 104.1 ± 10.1
24.58 ± 5.1 139.2 ± 6.8 81.7 ± 3.6 0%
27.02 ± 6.9 151.0 ± 22.4 98.6 ± 7.6 23 (92)
27.87 ± 5.8 149.0 ± 18.2 93.2 ± 10.6 18 (80)
30.95 ± 5.8 156.6 ± 20.1 103.2 ± 9.7 17 (100)
149.67 ± 7.8 152.72 ± 11.5 148.14 ± 8.9 150.59 ± 9.3 67.8 ± 9.2 70.5 ± 10.1 72.5 ± 10.2 76.4 ± 8.4 72.5 ± 12.0 81.7 ± 10.1 85.7 ± 7.9 90.2 ± 10.1 30.26 ± 4.4 132.3 ± 6.5 79.2 ± 2.3 0%
BMI = body mass index.
30.23 ± 4.7 139.9 ± 15.5 89.2 ± 8.9 30 (86)
33.04 ± 5.2 143.8 ± 8.6 93.5 ± 6.7 14 (91)
33.69 ± 4.5 146.9 ± 13.2 98.9 ± 7.8 15 (100)
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medication, oral contraceptives, or hormone replacement therapy were excluded from the study. Exclusion criteria included: established renal or hepatic disease, vascular disease (i.e., peripheral vascular disease, cerebrovascular disease), malignancy or those on treatment with antioxidants or aspirin. None of our subjects had a prior history of coronary angioplasty or coronary artery bypass graft (CABG). However, 59 (46%) subjects were taking anti-hypertensive medication and 20 (15%) of the subjects used anti-diabetic drugs. For all patients, anthropometric parameters including weight, height, and waist circumference were measured using standard protocols. Blood pressure, height, and weight were measured using standard methods (Table 1). Sixty one healthy subjects (33 male and 28 female), were recruited from the residence of the same city, matched age group and socioeconomic status. The criteria of healthy subjects included no history of symptoms of CVD and acute coronary syndrome documented in their medical notes, and no family history of heart disease was defined by a first degree relative with a myocardial infarction, or cardiac death before 55 years. All control subjects underwent a routine medical examination including MI test. All patients and controls were requested to complete an interviewer administered questionnaire, concerning their demographic characteristics, age, health history, lifestyle habits and diet. They gave written consent to participate in the study.
The % recoveries varied in the range of 97.2–98.7 for all 3 analytes under study. Non significant differences were observed (p N 0.05) when comparing the values obtained by (MDM and CDM), (paired t-test) (Table 2). The overall recoveries of Fe, Cu and Zn in certified biological samples, by using the microwave digestion method as compared to certified values are (97.64, 98.70 and 97.77%), (97.2, 98.65 and 98.05%) and (98.40, 97.60 and 98.63%) in CRM whole blood, CRM human urine and CRM BCR human hair respectively. Mean values for all the elements differed by b1–2% from the certified values.
2.7. Analytical figures of merit Statistical analyses were performed using the computer programs Excel XL State (Microsoft Corp., Redmond, WA) and Minitab 13.2 (Minitab Inc., State College, PA). Calibration was performed with a series of Cu, Fe and Zn standards. Sensitivity (m) was the slope value obtained by least-square regression analysis of calibration curves based on peak area measurements. The linear range of the calibration curve ranged from the quantification limit up to 100 µg/l was used for all metals. The limit of detection (LOD), equal to 0.17 pg/g, 0.45 ng/g and 0.065 ng/g for Cu, Fe and Zn respectively, was defined as 3 s/m, s being the standard deviation corresponding to 10 blank injections and mol/l the slope of the calibration graph. The quantification limits (LOQ), defined
2.4. Angiographic assessment Coronary angiograms were performed using routine procedures. Analysis of the angiograms was performed offline by a specialist cardiologist. The presence of ≥ 1 stenoses (50% in diameter) of at least 1 major coronary artery, (left main, right coronary artery, left anterior descending and circumflex) was considered as the evidence of significant CVD [18].
2.5. Collection of biological samples Venous blood was drawn from MI patients, immediately on arrival at the emergency cardiac ward of the hospital, before administration of any pharmaceutical treatment, within 6–12 h. Venous blood samples (3–5 ml) were collected using metal-free vacutainer tubes (Becton Dickinson, Rutherford NJ) containing N1.5 μg K2EDTA/ml, and were stored at − 20 °C until analysis. Urine was voided directly into acid-washed 100 ml polyethylene tubes, Kartell (Milan, Italy) which were decontaminated before handling. Between sampling sessions, the container was wrapped in a clean polyethylene bag. Urine samples were acidified with 1% ultrapure HNO3. Prior to sub-sampling for analysis, the samples were shaken vigorously for 1 min to ensure a homogeneous suspension. Hair samples of controls and patients were collected from the nape of the neck. The scalp hair samples were washed as reported in our previous study [19]. After washing, hair samples were dried at 80 °C for 6 h. Hair samples were kept into separate plastic envelopes with an identification (ID) number for each participant.
2.6. Microwave assisted acid digestion (MWD) A microwave assisted digestion procedure was carried out, in order to achieve a shorter digestion time. Duplicate samples of scalp hair (200 mg) and 0.5 ml of blood and urine samples, of each MI patients and control individuals were directly placed into Teflon PFA flasks. 2 ml of a freshly prepared mixture of concentrated HNO3–H2O2 (2:1, v/v) was added to each flask and kept for 10 min at room temperature, then the flasks were placed in a covered PTFE container . This was then heated following a one-stage digestion program at 80% of total power (900 W). Complete digestion of blood and urine samples required 2–4 min, while 5–8 min was necessary for scalp hair samples. After the digestion, the flasks were left to cool and the resulting solution was evaporated to semidried mass to remove excess acid. About 5 ml of 0.1 mol/l nitric acid was added to the residue and filtered through a Whatman no. 42 filter paper and diluted with deionized water up to 10.0 ml in volumetric flasks. Blank extractions were carried out through the complete procedure. Blanks and standard solutions were prepared in a similar acid matrix. The validity and efficiency of the microwave assisted digestion method was checked with certified values of CRMs of all 3 biological samples and with those obtained from conventional wet acid digestion method (CDM) [20].
Table 2 Determination of Fe, Cu and Zn in certified samples by CDM and MWD (n = 10) Elements Conventional digestion method CDM
Microwave digestion method MWD
T value a % recovery b Certified values
Certified sample of whole blood (mg/l) Fe 14.4 ± 0.98 14.06 ± 1.02 0.037 (6.80) (7.25) Cu 0.01387 ± 0.01369 ± 0.72 0.00102 0.0089 (7.35) (6.5) Zn 2.25 ± 0.09 2.20 ± 0.08 0.091 (4.00) (3.64) Certified sample of urine (mg/l) Fe 0.0355 ± 0.0345 ± 0.0006 0.0004 (1.69) (1.59) Cu 0.054 ± 0.05327 ± 0.0007 0.0006 (1.29) (1.13) Zn 0.205 ± 0.201 ± 0.006 0.007 (2.93) (3.48)
97.64 98.70
14.2 ± 3.24 0.0139 ± 0.0027
97.77
2.27 ± 0.06
0.997
97.2
0.039 ± 0.01
0.41
98.65
0.056 ± 0.01
0.47
98.05
0.210 ± 0.05
Certified sample of human hair (μg/g) Fe 575.0 ± 22.0 565.8 ± 24.0 0.00112 98.40 (3.8) (4.2) Cu 108.4 ± 6.7 105.8 ± 7.2 0.00049 97.60 (6.1) (7.3) Zn 197.2 ± 12.8 194.5 ± 11.3 0.000034 98.63 (6.2) (5.7)
580.0 ± 10 c 110 ± 5 d 199 ± 5
Values in ( ) are RSD. a Paired t-test between CDM and MWD DF = 9, T (critical) at 95% C.L = 2.262, p = 0.05. b % recovery was calculated according to : ½MDM 100: ½CDM c d
Informative value. Indicative value.
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Table 3 Trace element concentrations in biological samples (scalp hair, blood and urine) of normal and myocardial infarction subjects Control subjects
1st heart attack
2nd heart attack
3rd heart attack
Male n = 110 Iron Scalp hair (μg/g) 31.9 ± 6.2 Blood (mg/l) 708.4 ± 78.4 Urine (mg/l) 2.4 ± 0.90 Zinc Scalp hair (μg/g) 212.1 ± 16.5 Blood (mg/l) 10.4 ± 1.7 Urine (mg/l) 0.8 ± 0.2 Copper Scalp hair (μg/g) 11.5 ± 2.9 Blood (mg/l) 1.4 ± 0.5 Urine (mg/l) 0.15 ± 0.11
Normal
1st heart attack
2nd heart attack
3rd heart attack
Female n = 81
40.9 ± 7.3
42.6 ± 5.7
36.4 ± 3.4
798.8 ± 69.8
769.9 ± 49.2
3.21 ± 1.05
2.98 ± 0.78
162.4 ± 14.5
145.9 ± 10.7
8.32 ± 1.55
7.2 ± 1.11
1.27 ± 0.18
1.43 ± 0.13
–
13.9 ± 2.7
13.6 ± 2.1
12.4 ± 1.9
722.5 ± 53.6 –
120.0 ± 11.1 6.4 ± 1.13
2.1 ± 0.42
2.4 ± 0.46
1.9 ± 0.33
1.9 ± 0.6
2.5 ± 0.10
–
as 10 s/m were calculated as: 0. 51 pg/g, 1.35 ng/g and 0.19 ng/g for Cu, Fe and Zn respectively.
3. Results The present hospital based study on patients suffering from myocardial infarction (MI), was carried out to determine the concentrations of Cu, Fe and Zn in biological samples (Scalp hair, blood and urine). It was found that the MI was associated with a pronounced imbalance in analytes under study (Table 3). The analyzed biological samples were categorized according to the MI patients, controls and gender. The MI patients were further divided into three subgroups according to first, second and third MI attack. The mean value of Zn in the scalp hair samples of male MI patients of 1st, 2nd and 3rd attack were found in the range of (120.0–173.6) μg/g, which were significantly lower than control subjects of the same age group (196.7–228.6) μg/g (p b 0.001), while the same trend was observed in females. The range of Zn concentrations in blood samples of controls of both genders (8.5–13.3) mg/l was significantly high as compared to the range of Zn concentrations observed in blood samples of 1st, 2nd and 3rd attack male and females patients (6.2–8.69) mg/l (p N 0.002). The excretion of Zn was high in the 1st and 2nd MI attack patients of both genders. The increased concentration of Fe was observed in scalp hair of 1st and 2nd MI attack patients of both genders (Table 3). The range of Fe in blood samples of male and female MI patients of 1st and 2nd attack (702.5–795.8) mg/l, was found to be higher as compared to values of Fe obtained in blood samples of normal subjects (p N 0.011), while the mean values of Fe in
28.0 ± 6.5
36.68 ± 6.98
35.3 ± 4.3
702.5 ± 83.6
784.4 ± 82.9
766.8 ± 51.3
2.6 ± 0.7
3.29 ± 0.46
2.85 ± 0.33
232.3 ± 9.3
173.6 ± 8.7
151.6 ± 5.8
32.0 ± 4.2 716.2 ± 55.9 –
128.0 ± 6.3
10.9 ± 2.4
8.69 ± 2.34
6.82 ± 1.52
6.2 ± 1.7
0.8 ± 0.2
1.2 ± 0.14
1.16 ± 0.14
–
13.2 ± 2.4
12.7 ± 2.5
11.9 ± 3.6
14.2 ± 2.5
1.5 ± 0.49
2.3 ± 0.32
2.5 ± 0.35
2.05 ± 0.44
0.14 ± 0.09
1.7 ± 0.09
2.4 ± 0.067
–
biological samples of 3rd MI attack of both genders were lower as compared to 1st and 2nd attack patients. The excretion of Fe was found to be higher in 1st and 2nd male and female MI patients than controls via urine (p N 0.01). Cu concentrations in hair revealed no significant difference between controls and patients, and were found in the range of (11.5–13.8) μg/g and (12.4–14.2) μg/g respectively (p = 0.05). In blood samples, the mean concentration of Cu in controls subjects of the (45–60) age group was found to be lower (0.9– 1.99) mg/l than those obtained in blood samples of 1st, 2nd and 3rd male and females MI attack patients (1.9–2.4) and (2.05– 2.5) mg/l respectively (Table 3), while the excretion of Cu was found to be significantly higher in 1st and 2nd MI attack patients of both genders than age-matched controls (p N 0.001). The unpaired student t-test at different degrees of freedom between MI and controls of both genders were calculated at different probabilities. Our calculated t value exceeds that of t critical value at 95% confidence intervals, which indicated that the difference between means values of all three metals in normal and MI patients showed significant differences (p b 0.001). 4. Discussion Despite the lack of accurate mortality data, there is enough evidence to indicate that CVD is increasing tremendously in Pakistan. The MI patients who died during the study predominantly belonged to 3rd attack, with the age of b 50 years. The mean systolic blood pressure and blood cholesterol were higher, while the mean high density lipoprotein cholesterol was found to be lower among participants dying in 3rd MI attack than among those who survived after 1st and 2nd MI attack (Table 1).
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The findings of the present study clearly demonstrate that the concentration of metals varied in the biological samples of MI patients as compared to controls i.e., Cu and Fe concentrations increased both in blood and scalp hair; whereas for Zn, the reverse pattern was found as shown in Table 3. An increase in Cu and decrease in Zn concentrations were observed, these results are consistent with other study [21]. Moreover, a decrease of Zn/Cu ratio in biological samples of MI patients as compared to controls was also observed (Table 4). Our findings are consistent with another study, who reported that Zn appears to function in cardiovascular diseases primarily via its antagonism with Cu [22]. The observed results of Zn in the scalp hair and blood samples of subjects understudy are consistent with investigation of Tang et al., who reported that hair and fingernail concentration of Zn was significantly lower in the aged subjects with hypertension and CVD than the aged healthy controls [23]. The low Zn concentrations in blood and scalp hair correlate with intake of cardiovascular medication and also with reduced protein intake [24]. Several epidemiological studies reported that low concentrations of Zn in serum and high urinary Zn concentrations were found in patients of cardiovascular diseases (CVD), possibly due to diuretic medicines [25]. The mean values of Cu and Fe in the scalp hair and blood samples of MI patients of both genders were higher than those observed from corresponding controls of matched age groups of both genders (p N 0.001 for Cu) and (p N 0.011 for Fe) (Table 3). The high concentration of Cu in hair may be indicative of excess Cu in the body. However, it is important first to rule out exogenous contamination sources: dyes, bleaches, swimming pool/hot tub water and washing hair in acidic water carried through Cu pipes [26]. Both Fe and Cu are transition elements that fuel generation of damaging reactive oxygen species (ROS) if present in “normal” amounts, and this oxidant damage takes its toll in the later years of life [27]. The catalytic role of the transition metals, Fe and Cu is considered an important factor in Table 4 Zn/Cu and Zn/Fe mole ratio in normal subjects vs MCI patients of both genders in the age group (45–60) Specimens
Scalp hair Blood Urine
Gender
Male Female Male Female Male Female
Zn/Fe Normal
1st heart attack
2nd heart attack
3rd heart attack
5.68 7.08 0.0125 0.0132 0.29 0.26
3.39 4.04 0.0089 0.0097 0.34 0.31
2.99 3.67 0.0081 0.0080 0.44 0.37
Normal
1st heart attack
2nd heart attack
3rd heart attack
17.9 18.9 7.22 7.06 5.18 5.55
11.3 11.9 3.85 3.67 0.65 0.57
10.7 11.2 2.92 2.68 0.56 0.47
8.44 9.79 3.27 2.94 – –
2.53 3.42 0.0076 0.0074 – –
Zn/Cu
Scalp hair Blood Urine
Male Female Male Female Male Female
oxidative stress, which plays an important role in the pathogenesis of CVD [28]. Some of the investigations reported that the role of Cu in atherosclerosis is controversial and a higher cardiovascular risk has been associated with low as well as high plasma Cu concentrations [29]. It has been proposed that Cu deficiency rather than Cu excess is a risk factor for ischemic heart disease since it is part of antioxidant enzymes. With respect to the latter, they emphasize the role of Cu in oxidizing LDL, which they point out is important in the early phases of atherogenesis. It was also reported that homocysteine, a risk factor for cardiovascular disease, interacts with Cu to produce oxidant stress [30]. Oxidized low density lipoprotein cholesterol facilitates the evolution of early arterial wall lesions into atherosclerotic plaques by promoting the formation of foam cells from macrophages as well as the recruitment and retention of monocytes in the arterial wall, may contribute to atherogenesis by damaging arterial endothelium, promoting thrombosis and interfering with normal vasomotor regulation [31]. The mean values of Cu in scalp hair and blood were higher in females, which is in accordance with the findings of several other reports [32]. These findings may be due to differences in diet between males and females as well as the differences in Cu absorption between the genders [33]. Moreover, it has been proposed that Fe availability contributes to the impaired action of endothelium-derived nitric oxide in patients with atherosclerosis [34]. There has been some disagreement as to whether Fe from all sources is detrimental or whether it is just Fe from meat products that increases the risk of heart attack [35]. The evidence that relative Fe availability contributes to diseases of aging is strongest with atherosclerotic disease. The concept was first proposed by Sullivan, and his major rationale, that the much lower risk of atherosclerotic cardiovascular disease in menstruating women than in men of the same ages was due to the reduced Fe stores in the women. When women stop menstruating they begin to lose this protective effect [9]. This hypothesis was also supported by another investigation, that mean serum ferritin concentrations were higher in men and postmenopausal women who had a higher risk of atherosclerosis than did the corresponding controls [36]. The results of this investigation demonstrated that hypozincemia and hypercupremia were consistent with other studies on serum and urine samples of human subjects [37]. In this study, the concentrations of Fe and Cu as well as Zn at the onset of the disease were found to be different between patients of MI grouped according to the different attacks. Especially, in the third group of patients, the differences were much more prominent. The changes in trace elements concentrations observed between the different groups as well as the significant correlations between trace metals and cardiac markers suggest that their status is related to the clinical outcome. Furthermore, these changes point out a role of metals in the pathological mechanisms responsible for MI and their progression. Acknowledgment The authors thank the Higher Education Commission of Pakistan for sponsoring this project.
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References [1] Lusis AJ. Atherosclerosis. Nature 2000;407:233–41. [2] Stocker R, Keaney Jr JF. New insights on oxidative stress in the artery wall. J Thromb Haemost 2005;3:1825–34. [3] Harris ED. Copper. In: O'Dell BL, Sunde RA, editors. Handbook of nutritionally essential minerals. New York: Marcel Dekker, Inc.; 1997. p. 231–73. [4] Rubbo H, O'Donnell V. Nitric oxide, peroxynitrite and lipoxygenase in atherogenesis:mechanistic insights. Toxicology 2005;208:305–17. [5] Halliwell B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radic Res 1996;25:57–74. [6] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Fourth ed. Oxford: Oxford University Press; 2006. [7] Leone N, Courbon D, Ducimetiere P, Zureik M. Zinc, copper, and magnesium and risks for all-cause, cancer, and cardiovascular mortality. Epidemiology 2006;17:308–14. [8] Tuomainen TP, Punnonen K, Nyyssonen K, Salonen JT. Association between body iron stores and the risk of acute myocardial infarction in men. Circulation 1998;97:1461–6. [9] Sullivan JL. Iron and the sex difference in heart disease risk. Lancet 1981;1:1293–4. [10] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med 1992;13:341–90. [11] Zhang ZW, Shimbo S, Ochi N, Watanabe M. Relationship between trace elements and atherosclerosis. Sci Total Environ 1995;205(2–3):179–87. [12] Bray T, Bettger W. The physiological role of zinc as an antioxidant. Free Radic Biol Med 1990;8:281–91. [13] Hennig B, Meerarani P, Toborek M, Mc-Clain C. Antioxidant-like properties of zinc in activated endothelial cells. J Am Coll Nutr 1999;18:152–8. [14] DiSilvestro RA, Jones AA. High ceruloplasmin concentrations in rats without high lipoprotein oxidation rates. Biochim Biophys Acta 1996;1317:81–3. [15] Brettell TA, Butler JM, Saferstein R. Forensic science. Anal Chem 2005;77: 3839–60. [16] Polkowska Z, Kozlowska K, Namiesnik J, Przyjazny A. Biological fluids as a source of information on the exposure of man to environmental chemical agents. Crit Rev Anal Chem 2004;34:105–19. [17] Mesko MF, De Moraes DP, Barin JS, Dressler VL, Knappet G. Digestion of biological materials using the microwave-assisted sample combustion technique. Microchem J 2006;82:183–8. [18] Marroquin O, Kip K, Kelley DE, Johnson D, Shaw LJ, Merz N. Metabolic syndrome modifies the cardiovascular risk associated with angiographic coronary artery disease in women: a report from the women's ischemia syndrome evaluation. Circulation 2004;109:714–21. [19] Afridi HI, Kazi TG, Kazi GH. Analysis of heavy metals in scalp hair samples of hypertensive patients by conventional and microwave digestion methods. Spectrosc Lett 2006;39:203–14.
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[20] Kazi TG, Afridi HI, Kazi GH, Jamali MK, Arain MB. Evaluation of essential and toxic metals by ultrasound-assisted acid leaching from scalp hair samples of children with macular degeneration patients. Clin Chim Acta 2006;369:52–60. [21] Arnaud J, Faure H, Bourlard P, Denis B, Favier AE. Longitudinal changes in serum zinc concentration and distribution after acute myocardial infarction. Clin Chim Acta 1994:30147–56. [22] Wood RJ. Assessment of marginal zinc status in human. J Nutr 2000;130: 135S–1354S. [23] Tang YR, Zhang SQ, Xiong Y, et al. Studies of five microelement contents in human serum, hair, and fingernails correlated with aged hypertension and coronary heart disease. Biol Trace Elem Res 2003;92:97–104. [24] Eltayeb MA, Van Grieken RE. Iron, copper, zinc and lead in hair from Sudanese populations of different age groups. Sci Total Environ 1990;95: 157–65. [25] Wastney ME, Ahmed S, Henkin RI. Changes in regulation of human zinc metabolism with age. Am J Physiol 1992;263:R1 162–8. [26] Sukumar A, Subramanian R. Relative element concentrations in the paired samples of scalp hair and fingernails of patients from New Delhi. Sci Total Environ 2007;372:474–9. [27] Zacharski LR, Ornstein DL, Woloshin S, Schwartz LM. Association of age, sex, and race with body iron stores in adults: analysis of NHANES III data. Am Heart J 2000;140:98–104. [28] Giles WH, Anda RF, Williamson DF, Yip R, Marks J. Iron and ischemic heart disease. Circulation 1993;87:2065–6. [29] Klevay LM, Salonen JT, Salonen R, Korpela H, Suntioinen S, Tuomilehto J. Serum copper and the risk of acute myocardial infarction: a prospective population study in men in eastern Finland. Am J Epidemiol 1992;135: 832–4. [30] Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr 1996;63:797S–811S. [31] Jain VK, Mohan G. Serum zinc and copper in myocardial infarction with particular reference to prognosis. Biol Trace Elem Res 1991;31:317–22. [32] Ghayour Mobarhan M, Taylor A, New S, Lamb DJ, Ferns G. Determinants of serum copper, zinc and selenium in healthy subjects. Ann Clin Biochem 2005;42:364–75. [33] Ferns GA, Lamb DJ, Taylor A. Copper and atherosclerosis. Metal Ions Biol Med 2000;6:622–4. [34] Duffy SJ, Biegelsen ES, Holbrook M, Russell JD, Gokceet N. Iron chelation improves endothelial function in patients with coronary artery disease. Circulation 2001;103:2799–804. [35] Klipstein-Grobusch. Dietary iron and risk of myocardial infarction in the Rotterdam Study. Am J Epidemiol 1999;149:421–8. [36] Kraml P, Andel M, Malkova J. Serum ferritin concentrations in subjects at high risk of atherosclerosis. Cent Eur J Public Health 2000;8:4–9. [37] Ripa S, Ripa R, Giustiniani S. Are failured cardiomyopathies a zinc-deficit related disease? A study on Zn and Cu in patients with chronic failured dilated and hypertrophic cardiomyopathies. Minerva Med 1998;89:397–403.
Clinica Chimica Acta 389 (2008) 114 – 119 www.elsevier.com/locate/clinchim
Distribution of zinc, copper and iron in biological samples of Pakistani myocardial infarction (1st, 2nd and 3rd heart attack) patients and controls Tasneem Gul Kazi a,⁎, Hassan Imran Afridi a , Naveed Kazi b , Mohammad Khan Jamali a , Mohammad Bilal Arain a , Raja Adil Sarfraz a , Nusrat Jalbani a , Rehana Ansari a , Abdul Qadir Shah a , Ateeq-ur- Rehman Memon a , Ghulam Abbass Khandhro a a
National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, 76080, Pakistan b Liaqat University of Medical and Health Sciences, Jamshoro, Pakistan Received 29 September 2007; received in revised form 1 December 2007; accepted 3 December 2007 Available online 14 December 2007
Abstract Background: The pathogenesis of some heart diseases has been associated with changes in the balance of certain trace elements. We examined the association of iron, copper and zinc between biological samples (scalp hair, whole blood and urine) and mortality from myocardial infarction (MI) patients of (first, second and third heart attack). Methods: The biological samples were from 130 MI patients (77 male and 53 female, age range 45–60 years) and 61 healthy age-matched controls (33 male and 28 female). The metals in the biological samples were measured by the flame atomic absorption spectrophotometry, prior to microwave assisted acid digestion. The validity of the methodology was checked by the biological certified reference materials. Results: During this study, 78% of the 32 patients aged N 50 years, registered after the third MI attack died. In these subjects the concentration of Fe and Cu were increased by 0.83% and 3.12% in the scalp hair while in blood samples 9.7% and 22.5% were enhanced respectively, as compared to those who tolerated 3rd MI attack (p = 0.072). The concentrations of Zn in whole blood and scalp hair samples were lower in MI patients as compared to normal subjects. Conclusion: Deficiency of zinc and high concentration of copper and iron may play a role in the development of heart disease. © 2007 Elsevier B.V. All rights reserved. Keywords: Zinc; Iron; Copper; Myocardial infarction; Atomic absorption spectrophotometer
1. Introduction Atherosclerosis is a well-known precursor of ischemic heart disease due to accumulation of lipids and fibrous elements in arteries [1]. The development of atherosclerosis depends on a balance between pro-inflammatory stimuli, anti-inflammatory and antioxidant defence mechanisms [2]. Trace elements are being increasingly recognized as essential mediators of the development and progression of cardiovascular disease (CVD). Although there is not necessarily a direct cause–effect relationship between the development of CVD and trace elements status, it is generally believed that some of them (Cu and Fe) are CVD risk factors [3]. ⁎ Corresponding author. Tel.: +92 022 2771379; fax: +92 022 2771560. E-mail address: [email protected] (T.G. Kazi). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.12.004
Fortunately, the cell possesses highly efficient protective mechanisms, including antioxidants such as α-tocopherol, ascorbate, β-carotene, glutathione, and metal-binding proteins such as transferrin, ceruloplasmin and enzymes such as manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (Cu–ZnSOD), selenoenzyme glutathione peroxidase (GSH-Px) and iron-containing enzyme catalase [4]. All these metal binding antioxidants are designed to prevent the occurrence of free radical-induced injury under normal conditions. However, it has been argued that these protective mechanisms may be overwhelmed then severe free radicalmediated injury may occur [5]. When a free radical comes in contact with the inner lining of the arteries, microscopic injuries result. Eventually, the build up of fat, cholesterol, toxic metals and other substance at the site of injury narrows the arteries, which lead to cardiovascular disease [6].
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Fe and Cu are essential for blood formation, Cu is a component of superoxide dismutase (SOD), which also requires Zn and Mn to function. Deficiency of any of these elements reduced the activity of SOD which has been linked to atherosclerosis and lung injury, especially in the elderly [7]. Numerous studies have assessed the association between the Fe status and risk of coronary heart disease [8]. A hereditary haemochromatosis or Fe overload condition has been identified as an independent risk factor for myocardial infarction and cardiovascular mortality. Sullivan [9] reported that the difference in heart disease risk between the sexes might be due to differences in body Fe stores. Cu is a powerful catalyst of LDL oxidation in vitro, often more effective than Fe [10]. When Cu ions are added to LDL, they can react with the lipid hydroperoxides within the LDL to generate peroxyl and alkoxyl radicals that continue LDL oxidation. The study of Zhang et al. [11] showed that the high concentrations of Cu might inhibit phosphodiesterase activity, thus exerting an influence on the contractibility of cardiomyocytes and of smooth muscle cells in coronary arteries. Zinc (Zn) is an important component of biomembranes and an essential cofactor in a variety of enzymes [12]. Zn has antioxidant-like properties; thus, it can stabilize macromolecules against radical-induced oxidation in vitro as well as limit excess radical production [13]. Zn deficiency is associated with an increase of Cu and Fe due to the antagonistic relationships between these metals. Indeed, the oxidative damage which occurred in Zn-deficient rats could be due to increases of tissue Fe. It has been suggested that an imbalance between Cu and Zn may be a factor in the etiology of CVD [14]. Determinations of trace metals in human tissues and fluids were used to obtain information on the nutritional status for diagnosis of diseases, indication of systemic intoxication and to obtain information on environmental exposure. In the majority of cases, whole blood, serum, plasma and urine were analyzed [15]. The importance of exploring the depot-storage capacities of various elements, particularly the toxic ones, remains a vital aspect in elemental analysis largely met by urine and hair testing [16]. The determination of trace metals in biological samples requires the use of sensitive and selective techniques such atomic absorption spectrometer (AAS). This technique has need for solubilization of the analyte and complete or partial decomposition of the matrix using either convective systems or microwave ovens and dry ashing. The main advantage of microwave assisted samples pretreatment is its requirement of a small amount of mineral acids and a reduction in the production of nitrous vapors [17]. 2. Materials and methods 2.1. Apparatus A Perkin-Elmer model A. Analyst 700 (Norwalk, CT) atomic absorption spectrometer equipped with deuterium background correction was used in the study. The hollow cathode lamps of Cu, Fe and Zn were run under the conditions suggested by the manufacturer. A single element hollow cathode lamp was operated at 7.5 mA for Fe and Zn while at 7.0 for Cu with a spectral bandwidth of 0.7 nm. The analytical wavelengths were set at 214.0, 248.5 and 324.8 nm for Zn, Fe and Cu respectively. The flow rate of air (oxidant) was 17.0 l/min, while 2.0 l/min for acetylene. Integrated absorbance signals computed by the AA spectrometer were employed throughout. A Pel (PMO23) domestic microwave
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oven (maximum heating power of 900 W) was used for digestion of the biological samples. Acid washed polytetrafluoroethylene (PTFE) vessels and flasks were used for preparing and storing solutions.
2.2. Reagents and glass wares Ultrapure water obtained from ELGA labwater system (Bucks, UK), was used throughout the work. Concentrated nitric acid 65% and 30% hydrogen peroxide, from Merck (Darmstadt, Germany) were checked for possible trace metal contamination. Standard solutions of Zn, Cu and Fe were prepared by dilution of certified standard solutions (1000 ppm) from Fluka Kamica (Bush, Switzerland). Dilute working standard solutions were prepared immediately prior to their use, by stepwise dilution of the stock standard solution with 0.2 mol/l HNO3. All solutions were stored in polyethylene bottles at 4 °C. For the accuracy of methodology, certified samples of human hair BCR 397 (Brussels, Belgium), Clincheck control-lyophilized human urine and human whole blood (Recipe, Munich, Germany) were used as certified reference materials (CRMs). All glassware and plastic materials used were previously soaked for 24 h in 2 mol/l nitric acid, washed with distilled water and finally rinsed with deionized water, dried and stored in a class 100 laminar flow hood.
2.3. Sample collection and pre-treatment The study protocol was approved by the local ethics committee of higher education commission of Pakistan. This was a hospital based study. The history of MI patients admitted in Dewan-e-Mushtaque, Civil Hospital Hyderabad, Pakistan in (2006–2007) was collected. Patients admitted to the emergency department of hospital within 12 h of the onset of clinical symptoms suggestive of myocardial ischemia were randomly chosen, while we sampled the scalp hair and blood samples of third attack patients within 15–30 min, admitted in hospital. The study was carried out on a sequential sampling of 130 patients, (35 males and 23 females of 1st MI attack, 25 males and 15 females of 2nd MI attack, 17 males and 15 females of 3rd MI attack patients), age range between 45 to 60 years, who were undergoing routine coronary angiography, mainly for stable angina, and who had at least one positive test of MI including (exercise stress test and Dobutamin stress echocardiography). These tests and the angiogram were performed at the cardiac ward. During the study period, 32 patients of 3rd MI attack, 13 males and 12 females expired; therefore we could not collect their urine samples. The major criteria of inclusion for the present study were skeletal muscle damage or trauma, cardiac resuscitation, and infectious or inflammatory diseases. Patients who were on lipid-lowering
Table 1 Clinical and biochemical characteristics of patients with and without CAD Parameters Male Height (cm) Weight (kg) Waist circumference (cm) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Dyslipidemia, n (%) Female Height (cm) Weight (kg) Waist circumference (cm) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Dyslipidemia, n (%)
Controls
1st MCI
2nd MCI
3rd MCI
183.2 ± 12.3 180.2 ± 8.7 82.5 ± 12.5 90.5 ± 9.5 88.4 ± 12.0 95.5 ± 10.1
186.0 ± 10.5 177.4 ± 7.6 93.5 ± 8.1 97.39 ± 6.3 102.4 ± 13.2 104.1 ± 10.1
24.58 ± 5.1 139.2 ± 6.8 81.7 ± 3.6 0%
27.02 ± 6.9 151.0 ± 22.4 98.6 ± 7.6 23 (92)
27.87 ± 5.8 149.0 ± 18.2 93.2 ± 10.6 18 (80)
30.95 ± 5.8 156.6 ± 20.1 103.2 ± 9.7 17 (100)
149.67 ± 7.8 152.72 ± 11.5 148.14 ± 8.9 150.59 ± 9.3 67.8 ± 9.2 70.5 ± 10.1 72.5 ± 10.2 76.4 ± 8.4 72.5 ± 12.0 81.7 ± 10.1 85.7 ± 7.9 90.2 ± 10.1 30.26 ± 4.4 132.3 ± 6.5 79.2 ± 2.3 0%
BMI = body mass index.
30.23 ± 4.7 139.9 ± 15.5 89.2 ± 8.9 30 (86)
33.04 ± 5.2 143.8 ± 8.6 93.5 ± 6.7 14 (91)
33.69 ± 4.5 146.9 ± 13.2 98.9 ± 7.8 15 (100)
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medication, oral contraceptives, or hormone replacement therapy were excluded from the study. Exclusion criteria included: established renal or hepatic disease, vascular disease (i.e., peripheral vascular disease, cerebrovascular disease), malignancy or those on treatment with antioxidants or aspirin. None of our subjects had a prior history of coronary angioplasty or coronary artery bypass graft (CABG). However, 59 (46%) subjects were taking anti-hypertensive medication and 20 (15%) of the subjects used anti-diabetic drugs. For all patients, anthropometric parameters including weight, height, and waist circumference were measured using standard protocols. Blood pressure, height, and weight were measured using standard methods (Table 1). Sixty one healthy subjects (33 male and 28 female), were recruited from the residence of the same city, matched age group and socioeconomic status. The criteria of healthy subjects included no history of symptoms of CVD and acute coronary syndrome documented in their medical notes, and no family history of heart disease was defined by a first degree relative with a myocardial infarction, or cardiac death before 55 years. All control subjects underwent a routine medical examination including MI test. All patients and controls were requested to complete an interviewer administered questionnaire, concerning their demographic characteristics, age, health history, lifestyle habits and diet. They gave written consent to participate in the study.
The % recoveries varied in the range of 97.2–98.7 for all 3 analytes under study. Non significant differences were observed (p N 0.05) when comparing the values obtained by (MDM and CDM), (paired t-test) (Table 2). The overall recoveries of Fe, Cu and Zn in certified biological samples, by using the microwave digestion method as compared to certified values are (97.64, 98.70 and 97.77%), (97.2, 98.65 and 98.05%) and (98.40, 97.60 and 98.63%) in CRM whole blood, CRM human urine and CRM BCR human hair respectively. Mean values for all the elements differed by b1–2% from the certified values.
2.7. Analytical figures of merit Statistical analyses were performed using the computer programs Excel XL State (Microsoft Corp., Redmond, WA) and Minitab 13.2 (Minitab Inc., State College, PA). Calibration was performed with a series of Cu, Fe and Zn standards. Sensitivity (m) was the slope value obtained by least-square regression analysis of calibration curves based on peak area measurements. The linear range of the calibration curve ranged from the quantification limit up to 100 µg/l was used for all metals. The limit of detection (LOD), equal to 0.17 pg/g, 0.45 ng/g and 0.065 ng/g for Cu, Fe and Zn respectively, was defined as 3 s/m, s being the standard deviation corresponding to 10 blank injections and mol/l the slope of the calibration graph. The quantification limits (LOQ), defined
2.4. Angiographic assessment Coronary angiograms were performed using routine procedures. Analysis of the angiograms was performed offline by a specialist cardiologist. The presence of ≥ 1 stenoses (50% in diameter) of at least 1 major coronary artery, (left main, right coronary artery, left anterior descending and circumflex) was considered as the evidence of significant CVD [18].
2.5. Collection of biological samples Venous blood was drawn from MI patients, immediately on arrival at the emergency cardiac ward of the hospital, before administration of any pharmaceutical treatment, within 6–12 h. Venous blood samples (3–5 ml) were collected using metal-free vacutainer tubes (Becton Dickinson, Rutherford NJ) containing N1.5 μg K2EDTA/ml, and were stored at − 20 °C until analysis. Urine was voided directly into acid-washed 100 ml polyethylene tubes, Kartell (Milan, Italy) which were decontaminated before handling. Between sampling sessions, the container was wrapped in a clean polyethylene bag. Urine samples were acidified with 1% ultrapure HNO3. Prior to sub-sampling for analysis, the samples were shaken vigorously for 1 min to ensure a homogeneous suspension. Hair samples of controls and patients were collected from the nape of the neck. The scalp hair samples were washed as reported in our previous study [19]. After washing, hair samples were dried at 80 °C for 6 h. Hair samples were kept into separate plastic envelopes with an identification (ID) number for each participant.
2.6. Microwave assisted acid digestion (MWD) A microwave assisted digestion procedure was carried out, in order to achieve a shorter digestion time. Duplicate samples of scalp hair (200 mg) and 0.5 ml of blood and urine samples, of each MI patients and control individuals were directly placed into Teflon PFA flasks. 2 ml of a freshly prepared mixture of concentrated HNO3–H2O2 (2:1, v/v) was added to each flask and kept for 10 min at room temperature, then the flasks were placed in a covered PTFE container . This was then heated following a one-stage digestion program at 80% of total power (900 W). Complete digestion of blood and urine samples required 2–4 min, while 5–8 min was necessary for scalp hair samples. After the digestion, the flasks were left to cool and the resulting solution was evaporated to semidried mass to remove excess acid. About 5 ml of 0.1 mol/l nitric acid was added to the residue and filtered through a Whatman no. 42 filter paper and diluted with deionized water up to 10.0 ml in volumetric flasks. Blank extractions were carried out through the complete procedure. Blanks and standard solutions were prepared in a similar acid matrix. The validity and efficiency of the microwave assisted digestion method was checked with certified values of CRMs of all 3 biological samples and with those obtained from conventional wet acid digestion method (CDM) [20].
Table 2 Determination of Fe, Cu and Zn in certified samples by CDM and MWD (n = 10) Elements Conventional digestion method CDM
Microwave digestion method MWD
T value a % recovery b Certified values
Certified sample of whole blood (mg/l) Fe 14.4 ± 0.98 14.06 ± 1.02 0.037 (6.80) (7.25) Cu 0.01387 ± 0.01369 ± 0.72 0.00102 0.0089 (7.35) (6.5) Zn 2.25 ± 0.09 2.20 ± 0.08 0.091 (4.00) (3.64) Certified sample of urine (mg/l) Fe 0.0355 ± 0.0345 ± 0.0006 0.0004 (1.69) (1.59) Cu 0.054 ± 0.05327 ± 0.0007 0.0006 (1.29) (1.13) Zn 0.205 ± 0.201 ± 0.006 0.007 (2.93) (3.48)
97.64 98.70
14.2 ± 3.24 0.0139 ± 0.0027
97.77
2.27 ± 0.06
0.997
97.2
0.039 ± 0.01
0.41
98.65
0.056 ± 0.01
0.47
98.05
0.210 ± 0.05
Certified sample of human hair (μg/g) Fe 575.0 ± 22.0 565.8 ± 24.0 0.00112 98.40 (3.8) (4.2) Cu 108.4 ± 6.7 105.8 ± 7.2 0.00049 97.60 (6.1) (7.3) Zn 197.2 ± 12.8 194.5 ± 11.3 0.000034 98.63 (6.2) (5.7)
580.0 ± 10 c 110 ± 5 d 199 ± 5
Values in ( ) are RSD. a Paired t-test between CDM and MWD DF = 9, T (critical) at 95% C.L = 2.262, p = 0.05. b % recovery was calculated according to : ½MDM 100: ½CDM c d
Informative value. Indicative value.
T.G. Kazi et al. / Clinica Chimica Acta 389 (2008) 114–119
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Table 3 Trace element concentrations in biological samples (scalp hair, blood and urine) of normal and myocardial infarction subjects Control subjects
1st heart attack
2nd heart attack
3rd heart attack
Male n = 110 Iron Scalp hair (μg/g) 31.9 ± 6.2 Blood (mg/l) 708.4 ± 78.4 Urine (mg/l) 2.4 ± 0.90 Zinc Scalp hair (μg/g) 212.1 ± 16.5 Blood (mg/l) 10.4 ± 1.7 Urine (mg/l) 0.8 ± 0.2 Copper Scalp hair (μg/g) 11.5 ± 2.9 Blood (mg/l) 1.4 ± 0.5 Urine (mg/l) 0.15 ± 0.11
Normal
1st heart attack
2nd heart attack
3rd heart attack
Female n = 81
40.9 ± 7.3
42.6 ± 5.7
36.4 ± 3.4
798.8 ± 69.8
769.9 ± 49.2
3.21 ± 1.05
2.98 ± 0.78
162.4 ± 14.5
145.9 ± 10.7
8.32 ± 1.55
7.2 ± 1.11
1.27 ± 0.18
1.43 ± 0.13
–
13.9 ± 2.7
13.6 ± 2.1
12.4 ± 1.9
722.5 ± 53.6 –
120.0 ± 11.1 6.4 ± 1.13
2.1 ± 0.42
2.4 ± 0.46
1.9 ± 0.33
1.9 ± 0.6
2.5 ± 0.10
–
as 10 s/m were calculated as: 0. 51 pg/g, 1.35 ng/g and 0.19 ng/g for Cu, Fe and Zn respectively.
3. Results The present hospital based study on patients suffering from myocardial infarction (MI), was carried out to determine the concentrations of Cu, Fe and Zn in biological samples (Scalp hair, blood and urine). It was found that the MI was associated with a pronounced imbalance in analytes under study (Table 3). The analyzed biological samples were categorized according to the MI patients, controls and gender. The MI patients were further divided into three subgroups according to first, second and third MI attack. The mean value of Zn in the scalp hair samples of male MI patients of 1st, 2nd and 3rd attack were found in the range of (120.0–173.6) μg/g, which were significantly lower than control subjects of the same age group (196.7–228.6) μg/g (p b 0.001), while the same trend was observed in females. The range of Zn concentrations in blood samples of controls of both genders (8.5–13.3) mg/l was significantly high as compared to the range of Zn concentrations observed in blood samples of 1st, 2nd and 3rd attack male and females patients (6.2–8.69) mg/l (p N 0.002). The excretion of Zn was high in the 1st and 2nd MI attack patients of both genders. The increased concentration of Fe was observed in scalp hair of 1st and 2nd MI attack patients of both genders (Table 3). The range of Fe in blood samples of male and female MI patients of 1st and 2nd attack (702.5–795.8) mg/l, was found to be higher as compared to values of Fe obtained in blood samples of normal subjects (p N 0.011), while the mean values of Fe in
28.0 ± 6.5
36.68 ± 6.98
35.3 ± 4.3
702.5 ± 83.6
784.4 ± 82.9
766.8 ± 51.3
2.6 ± 0.7
3.29 ± 0.46
2.85 ± 0.33
232.3 ± 9.3
173.6 ± 8.7
151.6 ± 5.8
32.0 ± 4.2 716.2 ± 55.9 –
128.0 ± 6.3
10.9 ± 2.4
8.69 ± 2.34
6.82 ± 1.52
6.2 ± 1.7
0.8 ± 0.2
1.2 ± 0.14
1.16 ± 0.14
–
13.2 ± 2.4
12.7 ± 2.5
11.9 ± 3.6
14.2 ± 2.5
1.5 ± 0.49
2.3 ± 0.32
2.5 ± 0.35
2.05 ± 0.44
0.14 ± 0.09
1.7 ± 0.09
2.4 ± 0.067
–
biological samples of 3rd MI attack of both genders were lower as compared to 1st and 2nd attack patients. The excretion of Fe was found to be higher in 1st and 2nd male and female MI patients than controls via urine (p N 0.01). Cu concentrations in hair revealed no significant difference between controls and patients, and were found in the range of (11.5–13.8) μg/g and (12.4–14.2) μg/g respectively (p = 0.05). In blood samples, the mean concentration of Cu in controls subjects of the (45–60) age group was found to be lower (0.9– 1.99) mg/l than those obtained in blood samples of 1st, 2nd and 3rd male and females MI attack patients (1.9–2.4) and (2.05– 2.5) mg/l respectively (Table 3), while the excretion of Cu was found to be significantly higher in 1st and 2nd MI attack patients of both genders than age-matched controls (p N 0.001). The unpaired student t-test at different degrees of freedom between MI and controls of both genders were calculated at different probabilities. Our calculated t value exceeds that of t critical value at 95% confidence intervals, which indicated that the difference between means values of all three metals in normal and MI patients showed significant differences (p b 0.001). 4. Discussion Despite the lack of accurate mortality data, there is enough evidence to indicate that CVD is increasing tremendously in Pakistan. The MI patients who died during the study predominantly belonged to 3rd attack, with the age of b 50 years. The mean systolic blood pressure and blood cholesterol were higher, while the mean high density lipoprotein cholesterol was found to be lower among participants dying in 3rd MI attack than among those who survived after 1st and 2nd MI attack (Table 1).
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The findings of the present study clearly demonstrate that the concentration of metals varied in the biological samples of MI patients as compared to controls i.e., Cu and Fe concentrations increased both in blood and scalp hair; whereas for Zn, the reverse pattern was found as shown in Table 3. An increase in Cu and decrease in Zn concentrations were observed, these results are consistent with other study [21]. Moreover, a decrease of Zn/Cu ratio in biological samples of MI patients as compared to controls was also observed (Table 4). Our findings are consistent with another study, who reported that Zn appears to function in cardiovascular diseases primarily via its antagonism with Cu [22]. The observed results of Zn in the scalp hair and blood samples of subjects understudy are consistent with investigation of Tang et al., who reported that hair and fingernail concentration of Zn was significantly lower in the aged subjects with hypertension and CVD than the aged healthy controls [23]. The low Zn concentrations in blood and scalp hair correlate with intake of cardiovascular medication and also with reduced protein intake [24]. Several epidemiological studies reported that low concentrations of Zn in serum and high urinary Zn concentrations were found in patients of cardiovascular diseases (CVD), possibly due to diuretic medicines [25]. The mean values of Cu and Fe in the scalp hair and blood samples of MI patients of both genders were higher than those observed from corresponding controls of matched age groups of both genders (p N 0.001 for Cu) and (p N 0.011 for Fe) (Table 3). The high concentration of Cu in hair may be indicative of excess Cu in the body. However, it is important first to rule out exogenous contamination sources: dyes, bleaches, swimming pool/hot tub water and washing hair in acidic water carried through Cu pipes [26]. Both Fe and Cu are transition elements that fuel generation of damaging reactive oxygen species (ROS) if present in “normal” amounts, and this oxidant damage takes its toll in the later years of life [27]. The catalytic role of the transition metals, Fe and Cu is considered an important factor in Table 4 Zn/Cu and Zn/Fe mole ratio in normal subjects vs MCI patients of both genders in the age group (45–60) Specimens
Scalp hair Blood Urine
Gender
Male Female Male Female Male Female
Zn/Fe Normal
1st heart attack
2nd heart attack
3rd heart attack
5.68 7.08 0.0125 0.0132 0.29 0.26
3.39 4.04 0.0089 0.0097 0.34 0.31
2.99 3.67 0.0081 0.0080 0.44 0.37
Normal
1st heart attack
2nd heart attack
3rd heart attack
17.9 18.9 7.22 7.06 5.18 5.55
11.3 11.9 3.85 3.67 0.65 0.57
10.7 11.2 2.92 2.68 0.56 0.47
8.44 9.79 3.27 2.94 – –
2.53 3.42 0.0076 0.0074 – –
Zn/Cu
Scalp hair Blood Urine
Male Female Male Female Male Female
oxidative stress, which plays an important role in the pathogenesis of CVD [28]. Some of the investigations reported that the role of Cu in atherosclerosis is controversial and a higher cardiovascular risk has been associated with low as well as high plasma Cu concentrations [29]. It has been proposed that Cu deficiency rather than Cu excess is a risk factor for ischemic heart disease since it is part of antioxidant enzymes. With respect to the latter, they emphasize the role of Cu in oxidizing LDL, which they point out is important in the early phases of atherogenesis. It was also reported that homocysteine, a risk factor for cardiovascular disease, interacts with Cu to produce oxidant stress [30]. Oxidized low density lipoprotein cholesterol facilitates the evolution of early arterial wall lesions into atherosclerotic plaques by promoting the formation of foam cells from macrophages as well as the recruitment and retention of monocytes in the arterial wall, may contribute to atherogenesis by damaging arterial endothelium, promoting thrombosis and interfering with normal vasomotor regulation [31]. The mean values of Cu in scalp hair and blood were higher in females, which is in accordance with the findings of several other reports [32]. These findings may be due to differences in diet between males and females as well as the differences in Cu absorption between the genders [33]. Moreover, it has been proposed that Fe availability contributes to the impaired action of endothelium-derived nitric oxide in patients with atherosclerosis [34]. There has been some disagreement as to whether Fe from all sources is detrimental or whether it is just Fe from meat products that increases the risk of heart attack [35]. The evidence that relative Fe availability contributes to diseases of aging is strongest with atherosclerotic disease. The concept was first proposed by Sullivan, and his major rationale, that the much lower risk of atherosclerotic cardiovascular disease in menstruating women than in men of the same ages was due to the reduced Fe stores in the women. When women stop menstruating they begin to lose this protective effect [9]. This hypothesis was also supported by another investigation, that mean serum ferritin concentrations were higher in men and postmenopausal women who had a higher risk of atherosclerosis than did the corresponding controls [36]. The results of this investigation demonstrated that hypozincemia and hypercupremia were consistent with other studies on serum and urine samples of human subjects [37]. In this study, the concentrations of Fe and Cu as well as Zn at the onset of the disease were found to be different between patients of MI grouped according to the different attacks. Especially, in the third group of patients, the differences were much more prominent. The changes in trace elements concentrations observed between the different groups as well as the significant correlations between trace metals and cardiac markers suggest that their status is related to the clinical outcome. Furthermore, these changes point out a role of metals in the pathological mechanisms responsible for MI and their progression. Acknowledgment The authors thank the Higher Education Commission of Pakistan for sponsoring this project.
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