blood trace metals and hypertension: a cohort study … · 65% among the elderly masses in india...

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ABSTRACT

OBJECTIVES: To establish the baseline levels of trace metals (Cu, Zn, Mg, Pb and Cd) in whole blood of normal subjects and its correlation with age and sex as well as in hypertensive subjects.STUDY DESIGN: A cohort study.PLACE AND DURATION: The study was conducted from 1st March 2014 to 15th July 2015 and study was carried out at Pakistan Institute of Nuclear Science and Technology, Islamabad in collaboration with Federal Govt Services Hospital Islamabad.METHODOLOGY: A simple random sampling was done for 445 volunteered normal subjects donating blood in blood bank Federal Govt Services Hospital Islamabad. Similarly 252 hypertensive subjects' whole blood samples were collected from cardiology out patient department of above mentioned hospital. Data was collected from all volunteered normal subjects as well as hypertensive subjects irrespective of age and gender. All the blood samples were analyzed for trace metals Cu, Zn, Mg, Pb and Cd concentration by Atomic Absorption Spectrophotometer and whole blood of normal as well as hypertensive subjects with respect to age and sex was evaluated. Data was analyzed by SPSS version 17. The concentration obtained for Cu, Zn, Mg, Pb and Cd in 445 whole blood samples of normal subjects including 230 males and 215 females of 23-62 ages was analyzed using various statistical tools to establish some patterns and correlations.RESULTS: In this study 252 whole blood samples of hypertensive subject of which Males 135(53.57%) and Females 117(46.43%).The mean concentrate of Cu 0.5 - 2.9 mg L - 1, Zn 3.5 - 12.3 mg L - 1, Mg 17.3 - 39.8 mg L - 1 , Pb 201-499 ug L -1 and Cd7.5-16.6 ug L -1, in Males hypertensive subjects 135 (53.57%) while mean Concentration of Cu 0.4 - 2.3 mg L - 1, Zn 2.9 - 10.5 mg L - 1, Mg 9.3 - 30.3 mg L - 1 , Pb 152-421 ug L -1 and Cd 6.1-13.6 ug L -1 in Females hypertensive subjects 117 (46.43%). CONCLUSION: Concentration of trace metals baseline (Cu, Zn, Mg, Pb and Cd) of normal subjects were assessed in area of study and found higher level trace metals with the advancing age. Males adults have higher levels of trace metals than females' adults. The deficiency of Magnesium and the excess of Pb levels in whole blood in hypertensive subjects and is one of the risk factor in development of hypertension.KEY WORDS: Hypertension, Trace Metals, Analytical Technique Atomic Absorption Spectrophotometer, Whole Blood

ORIGINAL ARTICLE ISRA MEDICAL JOURNAL | Volume 8 - Issue 1 | Jan - Mar 2016

INTRODUCTION

Hypertension is one of the most prevalent diseases both in developed and developing countries. It has become an

1increasing concern worldwide . As there is radical decrease in infectious diseases and sharp decline of infant's mortality, the average life span of human has increased. Diet may also play important role in the development of hypertension along with

2stress, old age, obesity and environment . Hypertension is more common in Negro community as

3compared to whites in United States . Studies reported that hypertension is prevalent in 21% to 37%, adult population of Asia whereas its occurrence in rural populations is two to three

4times lower . It has been reported that about 29-45% of male adult and 25-38% female adult population suffering

5, 6hypertension in urban India . Its overall prevalence is about

765% among the elderly masses in India and Bangladesh . Accurate analysis of trace metals in human blood or serum was not always so easy. After the development of atomic absorption spectroscopic (AAS) technique, the clinical community realized that they had a highly sensitive and diverse trace metals detection technique that could be used for clinical research. In this study Atomic absorption Spectrometer is used for the determination of trace metals in whole blood.Study objective was to establish the baseline levels of trace metals (Cu, Zn, Mg, Pb and Cd) in whole blood of normal subjects and its correlation with age and sex as well as in hypertensive subjects in order to identify some of the risk factors for hypertension development.

METHODOLOGY

This cohort study was conducted from 1st March 2014 to 15th July 2015 and study was carried out at Pakistan Institute of Nuclear Science and Technology, Islamabad in collaboration with Federal Govt Services Hospital Islamabad.A simple random sampling was done for 445 volunteered normal subjects donating blood in blood bank Federal Government Services Hospital Islamabad. Similarly 252 hypertensive subjects' whole blood samples were collected from cardiology out patient department of above mentioned hospital. Data was collected from all volunteered normal subjects as well as hypertensive subjects irrespective of age and

1 2SOHAILA REHMAN , MOHAMMAD ARSHAD

BLOOD TRACE METALS AND HYPERTENSION:A COHORT STUDY ON HUMAN SUBJECTS

1. Deputy Chief Scientist Pakistan Institute of Nuclear Science and Technology (PINSTECH)2. Assistant Professor of Forensic Medicine Al-Nafees Medical College, Isra University, Islamabad Campus, Pakistan

Correspondence to:Sohaila RehmanDeputy Chief ScientistPakistan Institute of Nuclear Science and Technology (PINSTECH)P. O. Nilore, Islamabad.E. Mail: [email protected]

ISRA MEDICAL JOURNAL | Volume 8 - Issue 1 | Jan - Mar 2016Sohaila Rehman et al.

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respectively.Group1V males were 60 (13.48%) and female were 25(5.62%) with mean concentration of Zinc6.3±1.11 mg L-1 and 5.3±1.09 mg L-1 respectively

Magnesium in normal subjectsGroup1 males were 50(11.23%) and female were70 (15.73%) with mean concentration Mg of 26.9±2.4 mg L-1 and 19.1±2.7 mg L-1 respectively. Group11 males were 45 (10.11%) and female were 55 (12.35%) with mean concentration of Mg 32.1±3.5 mg L-1 and 23.1±2.8 mg L-1 respectively Group111 males were 75(16.85%) and female were 65(14.60%) with mean concentration of Mg 33.2±4.1 mg L-1 and 29.7±3.8 mg L-1 respectively.Group1V males were 60 (13.48%) and female were 25(5.62%) with mean concentration of Mg 39.8±4.1 mg L-1 and 30.3±2.3 mg L-1 respectively.

Lead in normal subjectsGroup1 males were 50 (11.23%) and female were70 (15.73%) with mean con of Pb 119±21and 94±14 ug L-1 respectively.Group11 males were 45 (10.11%) and female were 55 (12.35%) with mean concentration of Pb 129±23 ug L-1 and 106±16 ug L-1 in female respectively. Group111 males were 75(16.85%) and female were 65(14.60%) with mean concentration of Pb 157±19 ug L-1 and 120±10 ug L-1 respectively. Group1V males were 60 (13.48%) and female were 25(5.62%) with mean concentration of Pb 169±17 ug L-1 and 129±10 ug L-1 respectively.

Cadmium in normal subjectsGroup1 males were 50(11.23%) and female were70 (15.73%) with mean con of Cd 5.5±1.0 ug L-1 and 4.1±1.0 ug L-1 respectively. Group11 males 45 (10.11%) and female were 55 (12.35%) with mean concentration of Cd 5.8±1.2 ug L-1 and 4.8±0.92 ug L-1 respectively. Group111 males were 75(16.85%) and female were 65(14.60%) with mean concentration of Cd 6.9±1.01 ug L-1 and 5.6±1.13 ug L-1 respectively. Group1V males were 60 (13.48%) and female were 25(5.62%) with mean concentration of Cd 6.7±1.01 ug L-1 and 5.9±1.03 ug L-1 respectivelyCu, Zn, Mg, Pb & Cd in Hypertensive SubjectsIn this study 252 whole blood samples of hypertensive subject of which Males 135(53.57%) and Females 117(46.43%).The mean concentration of Cu 0.5 - 2.9 mg L-1, Zn 3.5 - 12.3 mg L-1, Mg 17.3 - 39.8 mg L-1 , Pb 201-499 ug L-1 and Cd 7.5-16.6 ug L-1, in males hypertensive subjects 135(53.57%) while mean concentration of Cu 0.4 - 2.3 mg L-1, Zn 2.9 - 10.5 mg L-1,, Mg 9.3 - 30.3 mg L-1, Pb 152-421 ug L-1 and Cd 6.1-13.6 ug L-1 in Females hypertensive subjects117 (46.43%). The Z-score method, which indicates the difference between the mean of the laboratory and the assigned value, is a standard procedure for ISO/ IUPAC (International Standard Organization and International Union of Pure and Applied Chemists) is applied to studied data. Fig-1 represents the calculated Z-scores values plotted against each element for IAEA (Reference Material blood, A-13) determined by AAS. The positive scores indicate higher value and negative scores depicts lower value than the certified. The plots in Fig-1 indicate that elements

gender.All whole blood samples were analyzed for trace metals Cu, Zn, Mg, Pb and Cd concentration by Atomic Absorption Spectrophotometer. Stock solutions of 1000 ppm of Cu, Zn, Mg, Pb and Cd were prepared by taking appropriate known weights of spec pure metal or its compounds and were dissolved separately in minimum amount of nitric acid. The solutions were heated near to dryness and the volumes were made up with 0.02 N HNO3 to known volumes with de-ionized water. Standards were made by appropriate dilution of the stock solution in 0.02N HNO3 immediately before use. All the measurements were made with Hitachi model Z-2000 polarized Zeeman AAS, which was coupled with a microprocessor-based data-handling facility and a printer. A water-cooled premix, fishtail type burner having a 10 x 0.05 cm2 slot was used for the air-acetylene flame in flame atomic absorption spectrometer (FAAS) for Cu, Zn and Mg. Graphite furnace atomic absorption spectrometer (GFAAS) was used for the analysis of Pb and Cd. Data was analyzed by SPSS version 17. The concentration obtained for Cu, Zn, Mg, Pb and Cd in 445 whole blood samples of normal subjects including 230 males and 215 females of 23-62 ages was analyzed using various statistical tools to establish patterns and correlations. For quality assurance purpose certified reference material (CRM) from IAEA was analyzed under optimized experimental conditions, identical to those used for analysis of whole blood samples of the said study

RESULTS

Data of trace metals concentration in 445 Normal subjects as a whole is depicted in Table-1 with concentration ranges with mean values.Normal subjects whole blood samples were further divided into four groups' age wise as underGroup 1 23-30 years Group 11 31-40 years Group 111 41-50 years Group 1V 51-62 years

Copper in normal subjectsGroup1 males were 50 (11.23%) and female were 70 (15.73%) with mean concentration of Cu 0.83±0.20 mg L-1 and 0.68±0.22 respectively.Group11 males were 45 (10.11%) and female were 55 (12.35%) with mean concentration of Cu 0.89±0.33 mg L-1 and 0.77±0.16 mg L-1 respectively. Group111 males were 75(16.85%) and female were 65(14.60%) with mean concentration of Cu 1.0±0.26 mg L-1 and 0.92±0.13 mg L-1 respectively.Group1V males were 60 (13.48%) and female were 25(5.62%) with mean concentration of Cu 1.33±0.30 mg L-1 and 1.17±0.25 mg L-1 respectively this is shown in Table-II

Zinc in normal subjectsGroup1 males were 50 (11.23%) and female were70 (15.73%) with mean concentration of Zinc 7.4±1.05 mg L-1 and 6.9±1.13 mg L-1 respectively. Group11 males were 45 (10.11%) and female were 55 (12.35%) with mean concentration of Zinc 7.8±1.07 mg L-1 and6.2±0.97 mg L-1 respectively.Group111 males were 75(16.85%) and female were 65(14.60%) with mean concentration of Zinc 1.0±0.26 mg L-1 and 0.92±0.13 mg L-1


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in both male and female subjects. This is shown in Table-11The concentrations obtained for Cu, Zn, Mg, Pb and Cd were analyzed using various statistical techniques to establish some patterns or/and correlations in the data studied. Statistical data analysis tool was used to get correlation coefficient for the determined elements. Table-111 represents correlation coefficient for (Cu, Zn, Mg, Pb and Cd) in whole blood of normal subjects with age, indicating positive correlation of age with Pb, Cd and Cu where as negative correlation with age is evident for Zn and Mg in healthy adults.

analyzed in IAEA Animal Blood (A-13) by AAS fall between +1 to -1. Z-scores values thus not only validating our method of analysis but also certifying the authenticity of data reported for concentration of metals (Cu, Zn, Mg, Pb and Cd) in whole blood samples. The whole blood samples of 445 normal subjects of Islamabad were analyzed for the establishment of baseline levels of trace metals (Cu, Zn, Mg, Pb and Cd) contents in their blood. Table-11 depicts the concentration of metals determined with respect to age in both normal male and female subjects. Data was divided in three age groups. Study found that the concentration of all the determined metals increased with age

-1TABLE – I: MEAN BLOOD METAL (Cu, Zn, Mg, Pb and Cd) CONCENTRATION (mg L ) IN NORMAL MALEAND FEMALE SUBJECTS OF DIFFERENT AGE GROUPS (n=445)

-1TABLE – II: MEAN BLOOD METAL (Cu, Zn, Mg, Pb and Cd) CONCENTRATION (MG L )IN NORMAL MALE AND FEMALE SUBJECTS OF DIFFERENT AGE GROUPS (n=445)

TABLE – III: CORRELATION COEFFICIENT FOR (Cu, Zn, Mg, Pb and Cd)IN WHOLE BLOOD OF NORMAL SUBJECTS WITH AGE (n=445)


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-1TABLE - IV: COMPARISON OF TRACE METALS (mg L ) IN WHOLE BLOOD SAMPLES OFNORMAL SUBJECTS FROM DIFFERENT POPULATIONS (REF, 8-12) (n=445)

-1TABLE - V: BLOOD METAL (Cu, Zn and Mg) LEVELS (mg L ) IN HYPERTENSIVE PATIENTS (n=445)

-1TABLE – VI: BLOOD METAL (PB AND CD) LEVELS (µG L ) IN HYPERTENSIVE PATIENTS (n=445)


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TABLE – VII: ORRELATION COEFFICIENT FOR (Cu, Zn, Mg, Pb and Cd) IN WHOLE BLOOD OF HYPERTENSIVE PATIENTS (n=445)

mean, median and standard deviation of Cu, Zn and Mg in whole blood samples of hypertensive patients are depicted in Table-V and Pb and Cd in whole blood samples of hypertensive patients are depicted in Table-VI. The perusal of data in Table-V indicates that concentration of Cu in whole blood samples of hypertensive patients was significantly greater than normotensives. It may be due to the reason that copper is known to be essential for the enzyme activity and it forms the cross-linkages of normal elastin in vessels. The probable reason of raised Cu levels could be due to enhanced copper binding capacity of ceruloplasmin. A high copper intake may lead to abnormal vessel wall formation resulting to pathological changes, which may lead to hypertension and also

13cardiovascular diseases .A study carried out in University of Surrey, UK, concluded that serum copper and zinc levels have correlation to physiological

10conditions such as age, diet and gender . In that study it was also reported that male subjects had significantly lower serum copper (P<0.001) and higher serum zinc (P<0.05) and zinc:

14copper ratio (P<0.001) than female subjects . In another study of the Rania M. Al-Groom in Jordan reported that serum-Zn

15showed a positive correlation with age . Almost all the metals analyzed are present in lesser concentration in female subject. This could be because of smaller duration of exposure to environment of females and because of their intake of more controlled diet as compared to male subjects (as interpreted from the personal information gathered before taking the sample). Therefore, it can be asserted that environment and diet are the probable factors for elevated levels in whole blood.Data in the present study do not reflect any correlation of metals in blood with occupation of the normal subjects. However, the socioeconomic background of the subjects has a mild effect on metal concentration in whole blood especially for Pb and Mg. The blood lead levels were found to be lower in high socioeconomic status personals whereas the Mg levels were found to be enhanced in them. The higher levels of Pb in blood of lower socioeconomic status personals might be due to observing improper hygiene. The depleted concentration of Mg in the blood samples of hypertensive patients is supported by a study carried out in USA suggesting that African-Americans are known to have many chronic diseases such as diabetes, hypertension, cardiovascular and renal diseases. Therefore, lower levels of serum /blood Mg have been associated with an increased prevalence of

16hypertension . In our study an inverse association between blood Mg and hypertension is in accordance with the study

17carried out in Canada thus describing a negative relation

DISCUSSION

Table-I depicts the concentration range and mean of Cu, Zn, Mg, Pb and Cd in 445 male and female normal subjects. The age range of normal subjects falls between 23-62 years. The data of normal subjects was further splits into male and females and four age groups were formed as depicted in Table-II. A comparison of the determined metals concentration in the blood of normal subjects with reported values for different countries was done. Table-III depicts the comparison of these metals in blood samples of normal subjects with other

(8-12)populations in the world . The determined concentration of -1Cu in present study (0.89 mg L ) is comparable to China (1.09 mg

-1L ) Japan (0.82-0.86) whereas these are lower than Canada -1 -1 -1(1.22 mg L ) Spain (1.05-1.17 mg L ), Italy (1.22 mg L ), UK (1.11

-1 -1mg L ) and in the range of Nigeria (0.8-1.5 mg L ). The determined mean concentration of Zn in whole blood of normal subjects is (6.2 mg L -1) that is almost similar to the reported levels for Canada (6.59 mg L -1), comparable to those of Italy

-1(6.4 mg L -1) and Spain (5.85-6.7 mg L ). Whereas these are -1 -1higher than those UK (4.30 mg L ) FRG (5.7 mg L ) and are lower

-1 -1than those of China (7.83 mg L ) and India (12 mg L ). The mean -1concentration level of Mg in the present study is (24.5 mg L )

-1that are lower than the reported values for Canada (37.7 mg L ), -1and China (38.9 mg L ). The determined levels of Pb in whole

-1blood samples of normal subjects are 119 ug L that are lower than the reported normal concentration levels for India (212 ug

-1 -1L ), in the range of FRG (82-148 ug L ) and are higher than those -1 -1of Japan (49 ug L ) and UK (112 ug L ). The determined levels for

-1)cadmium in the present work are (5.3 ug L that are comparable -1to UK (5.0 ug L ), but lower than that of Nigerian population (9-

-1 -1140 ug L ) and are higher than those of Japan (1.2 ug L ), Italy -1 -1(3.4 ug L ) and FRG (1.6 ug L ).

Correlation coefficient for (Cu, Zn, Mg, Pb and Cd) in whole blood of normal subjects with Age is depicted in table IV. These variations in the concentration levels of trace metals in blood samples among different countries could probably be due to the different geographical and environmental conditions. This could also depend upon the adequacy of essential and extent of toxic metal contents in the food articles being consumed by individuals in a specific environment/ location. Since all the hypertensive patients fall in a narrow range of age group i.e. 45-68 years, no further sub groups of patients with respect to age were formed and only a single group of hypertensive patients was discussed and for interpretation of results. In this regard 252-whole blood samples 135 males and 117 females of hypertensive patients of age 45-68 years were analyzed for Cu, Zn, Mg, Pb and Cd. The concentration range,


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5. Hypertension Study Group, Prevalence, awareness, treatment and control of hypertension among the elderly in Bangladesh and India: A multicentre study. Bull. World Health Organ.2001; 79(6):490–500.

6. Sushil KB, Vartika S, Sunil DK, William KG, Richard WW, Deepak G. The prevalence of hypertension and hypertension risk factors in a rural Indian community: A prospective door-to-door study. J Cardiovasc Dis Res. 2012; 3(2):117–23.

7. Alvarez SF, Escanero JF (ed). Trace Elements: Action on Health and its role in the pathologies, Marcen Publisher:2012;1-36.

8. Sufen S, Wang H. Flame atomic absorption spectrometry using a microvolume injection technique for the determination of Cu, Zn, Ca, Mg and Fe in whole blood from healthy infant and mother ears. Fresenius' of Analytical Chemi. 1997; 357(7): 997-99.

9. Satoh, Y., Yazawa, A., Contents of heavy metals in the blood of inhabitants in Yokohama city in Japan. 1978;17 (3), 63-71.

10. Rahman S, Waheed S. Validation of INAA and AAS analysis protocols for whole blood: A study on cardiovascular and malignant hypertensive patients. Journal of Radioanal. and Nuclear Chemistry. 2009; 279:(3) 915-21.

11. Minoia C, Sabbioni E, Apostoli P, Pietra R, Pozzoli L, Trace element reference values in tissues from inhabitants of the European community. I. A study of 46 elements in urine, blood and serum of Italian subjects.The Sci of Total Environ. 1990; 95(6):89-105.

12. Akanle OA, Akintanmide A, Durosinmi MA, Oluwole AF, spyrou NM. Elemental analysis of blood of nigerian hypertensive subjects. Biological Trace Element Research. 1999; 71(1): 611-16.

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between serum Mg and blood pressure, so supporting the role of Mg in the pathogenesis of hypertension. A significant difference in concentration levels of Pb and Cd in hypertensive blood (Table-VI) was observed when compared with the blood levels of normal subjects. This observation is in accordance with the findings that relationship exists between

18the whole blood-Pb levels and hypertension . However, there is no extensive evidence available in literature that aging in human's per-se enhances the susceptibility of hypertension. To a certain extent, age provides the time that is essential for the cellular events to occur. And these events in turn are necessary for the initiation of hypertension as well. The higher levels of Pb and Cd in blood of hypertensive patients could be attributed to un intentionally higher intake of these metals through dietary items, environment or due to malfunction in the metabolism of these metals as well. In this study the levels of blood lead were found to be lower in females than in males. The data belongs to general population and higher levels of blood lead were found in patients not henceforth believed to have significant lead exposure as also described in a previous study conducted in

192006 . The cohort nature of this study regarding lead in human whole blood is making a major contribution to hypertension, which in turn is becoming a major risk factor for hypertension.The calculated mean concentrations of metals studied were used in statistical analysis. Statistical data analysis tool was used to get correlation coefficient for the determined elements. Table-VII depicts the correlation coefficient between elements in whole blood samples of hypertensive subjects. It shows positive correlations of 0.536 and 0.537 between Pb-Cd and Cu-Pb respectively. A relatively higher negative correlation of –0.800 exists between Cu and Zn in hypertensive subjects thus indicating the strong antagonistic behavior of these two elements in hypertensive subjects blood. In order to study whether the apparent difference between the determined mean blood concentration of metals in males and females is statistically significant, student t-test was applied at 95% confidence levels to the data set of hypertensive patients. The output of t-Test (p < 0.05) shows that for Cu, Zn, Pb and Cd the t-statistic (4.06, 4.25, 5.27 and 3.48) is greater than t critical (1.96), indicating significant differences between the two population groups. For Mg the t-statistical (1.946) is almost similar to t-critical (1.966) indicating no significant variation among the male and female population groups of hypertensive patients.

CONCLUSION

Concentration of trace metals (Cu, Zn, Mg, Pb and Cd) of normal subjects were assessed in area of study and found higher levels of these metals with the advancing age. Males adults have higher levels of trace metals than females' adults. The deficiency of Magnesium and the excess of Pb levels in whole blood of hypertensive subjects and is one of the risk factor in development of hypertension.


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children with high family risk of premature cardiovascular disease. Annals Saudi Med. 2002; 22(5-6):291-94.

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blood trace metals and hypertension: a cohort study … · 65% among the elderly masses in india...

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