Pakistan J. Med. Res.

Vol. 43 No.2, 2004

 

Essential trace metals in human whole blood in relation to environment

Suhaila Rahman, Nasir Khalid , Shujaat Ahmad, Nasim Ullah, Muhammad Zafar Iqbal

Nuclear Chemistry Division, Pakistan Institute of Nuclear Science and Technology, Nilore, Islamabad, Department of Medicine, Rawalpindi Medical College, Rawalpindi, Institute of Chemistry, University of the Punjab Lahore, Pakistan.

 

SUMMARY

 

Levels of essential trace metals Cu, Zn, and Mg were determined in human whole blood samples. A simple, rapid and accurate method for the determination at trace levels based on Zeeman effect flame atomic absorption spectrometry (FAAS) is described by optimizing various instrumental parameters such as fuel pressure and burner height. Except dilution with 0.02 N HNO3, no sample preparation is required thus permitting direct determination of metals with less chances of contamination. The method is quick and only 0.5 ml of sample is required for the determination of all the above mentioned elements so the method is applicable at diagnostic stage. The precision and accuracy of the method was evaluated by analyzing the same sample with different procedures as well as by the standard addition method. The method was successfully applied to one hundred and three blood samples of healthy volunteers of Rawalpindi / Islamabad area. Determined average concentration of Cu, Zn, and Mg was found to be 1.13, 8.36 and 38.2 mg l-1 respectively. The results obtained were compared with the reported values for other countries. Levels of these metals in air water and integrated human diet samples of the area were also determined and their contribution in whole blood was evaluated.

 

Key Words:Trace Metals, Whole Blood, Atomic Absorption Spectrometer, Environmental Contribution.

 

INTRODUCTION

 

The essential metals at trace levels play vital role when present in human body and may cause some diseases when present beyond specific concentrations1-2. The spectacular advances in trace elements analytical techniques have led to the re-examination of many published data for trace elements in food, air, water and body fluids especially blood, since blood is a good indicator of the current body burden of metals. It is, therefore, important to monitor the exact levels of metals in blood using a reliable analytical technique.

                Determination of trace metals in human whole blood have been reported by spectrophotometry3, inductively coupled plasma mass spectrometry4, anodic stripping voltammetry5, neutron activation analysis6, proton induced X-ray emission7 and atomic absorption spectrophotometry8,9,10. The atomic absorption spectrophotometry (AAS) is one of the preferred techniques due to its rapidness, specificity and comparatively inexpensive. Although different procedures are available to simplify the matrix and minimize the background absorption for the determination of trace metals in whole blood using AAS, which include sample dilution with butanol or Triton X-100, decomposition of the organic matrix with acid mixture, addition of trichloroacetic acid, precipitation of blood cells11 and the extraction of metals using some chelating agents3. All these methods are either time consuming, expensive or ineffective in overcoming the physical interferences and required large volume of blood which is not available always. The excessive dilution will minimize the matrix effect but the analyte of low concentration will be diluted to either below or near the detection limits of the analyte. Recent advancements in instrumentation results in the improved sensitivities of metals by reducing background with Zeeman effect has drawn the attention of the analysts to check the sensitivities of metals in various matrices. Keeping in view of these facts it was considered important to look into a simple, rapid, accurate and inexpensive method for the determination of metals at trace levels in samples of whole blood, which requires minimum quantity of sample for the analysis of maximum number of elements.

                Copper, Magnesium and Zinc are essential trace metals and are involved in various metabolic functions. The deficiency symptoms of Copper are hypo chronic anemia, bones disorder ness, brain and nerve tissue degeneration, of Magnesium are vasodilatation, cardiac arrhythmia, Tetany, Muscle tremor and convulsions, and of Zinc are post  alcoholic cirrhosis, diabetes and Sickle cell anemia2. The daily requirements of Cu, Mg and Zn are 2-5, 200-400 and8-15 mg per person, respectively2.

                Therefore, in the present study a method based upon simple dilution has been described to determine trace metals in whole blood samples of the inhabitants of Rawalpindi/Islamabad area. As no such data is yet available for this area so this study will help to establish the base line levels of essential trace metals in whole blood of “normal” adult population of the area and to check the adequacy of these metals for human health. Samples of air, water and integrated human diet of this area were also analyzed for these metals to see the environmental contribution into human whole blood.

 

MATERIALS AND METHODS

 

Instrumentation

                All the absorption measurements were made with Hitachi model Z-8000 polarized Zeeman atomic absorption spectrophotometer, which was coupled with a microprocessor-based data-handling facility. A water-cooled premix, fishtail type burner having a 10 x 0.05 cm2 slot was used for the air-acetylene flame. Hollow cathode lamps of Cu, Zn and Mg were used as radiation sources. Concentration mode of (Flame Atomic Absorption Spectrometer) was used for the measurements of all the metals.

 

Reagent

                Stock solutions of 1000 mg l-1 of Cu, Zn and Mg were prepared separately by dissolving appropriate amounts of specpure metals or their oxides (Johnson Matthey Chemicals Ltd.,) in minimum amount of nitric acid and the volume was made up to 100 ml with water. For the preparation of calibration curve fresh working standards were made by appropriate dilution of stock solution in 0.02N HNO3 immediately before use. Glassware was cleaned by overnight soaking in HNO3 (1:1) followed by repeated rinsing with water. Distilled and deionized water was used throughout this work.

 

Sampling and sample preparation

                Blood samples of normal healthy persons were drawn by vein puncture under contamination controlled conditions12 and various blood samples were collected from the Federal Government Services Hospital and Shifa International, Hospital, Islamabad, in 4 ml VACUETTES (Grainer labortechnik) which contained EDTA as an anticoagulant. Samples were analyzed within twelve hours after collection. Pooled blood sample was obtained by thorough mixing of 5.0 ml of blood sample from ten healthy volunteers after the addition of EDTA.

                Integrated human diet of Islamabad region was prepared by taking into consideration the nutritional habits and specific requirement of the residents of the city. The integrated diet was composed of wheat & wheat products, rice, egg, milk, fats and oils, pulses, sugar, vegetable, fruits, meat and spices. Sampling and diet preparation procedure was similar as reported by Quershi et al13.

                Air sampling was performed at a residential site of Islamabad using Gent stacked filter unit (SFU). The polycarbonate membranes filters were used for air collection. On weekly basis and average of two samples were collected for forty-eight hours. After sampling all exposed filters were stored in plastic bags till analysis. Samples of mixed human diet and filter paper used for air collection were digested by method14

                Drinking water samples were collected from the different localities of Islamabad and were analyzed either by direct aspiration or after preconcentration of metals.

 

Procedure

                Calibration standard solutions were prepared for each element separately from stock solution in 0.02 N HNO3. Blood samples were diluted accordingly with 0.02 HNO3. The absorbance values of a specific metal were measured by aspirating the solutions in the order of blank, standards, sample blank and samples into the air-acetylene flame employing the optimized conditions given in Table-1. A minimum of three absorbance values were recorded for each solution and the mean value of the absorption signal was used for subsequent calculations. The absorption signals were evaluated by subtracting the value of blank from the signal of the sample.

 

RESULTS AND DISCUSSION

 

                For the direct determination of trace metals in human whole blood a simple, rapid and accurate method has been described for atomic absorption measurements using an air acetylene flame. In order to attain sufficient thermal energy to decompose the organic matrix within the flame and to dissociate the atoms from their chemical bonds specified instrumental parameters such as fuel pressure, burner height etc were optimized. Pooled human blood was used for this purpose after appropriate dilution with 0.02N HNO3. The absorbance of the existing physiological concentrations of metals in pooled whole blood was recorded after diluting with 0.02N HNO3. The criteria for the optimization were the selection of instrumental parameters that show maximum, reproducible and stable absorbance signal with low background. All the reported results are average of at least triplicate independent determinations with relative standard deviation less than ± 3.0 % unless otherwise mentioned in the text. The optimized parameters (Table-1) were successfully applied for the determination of Cu, Zn and Mg.

 

Table 1: Optimized instrumental parameters used for the determination of trace metals in whole blood

 

Parameter

Cu

Zn

Mg

 

Lamp current (mA)

7.5

10.0

7.5

 

Resonance line  (nm)

324.8

213.8

285.2

 

Width of slit  (nm)

1.3

1.3

1.3

 

 

Burner Type

Standard*

Burner height

7.5

7.5

7.5

 

Fuel (C2H2) pressure (kg cm-2)

0.3

0.3

0.4

 

Oxidant (air) pressure (kg cm-2)

1.6

1.6

1.6

 

 

 

 

 

 

* See Experimental Section.

 

Effect of Fuel Pressure

                The temperature of flame changes with the variation of fuel and oxidant ratio. Effect of fuel pressure was, therefore, checked on the absorbance values of Cu, Zn and Mg for the existing physiological concentration in the pooled blood sample, using a fixed oxidant pressure of 1.6 kg cm-2 and burner height of 7.5 (arbitrary units). The absorbance of all the metal increases gradually with the increase in fuel pressure attains a maximal and than decreases with furthers increase in fuel pressure Fig.1. The maximum absorbance was observed at a fuel pressure of 0.3 kg cm-2 for Cu and Zn whereas for Mg it was 0.4 kg cm-2. Therefore, these fuel pressures were used in all the subsequent measurements.

 

Variation in the Burner Height

                The effect of burner height on the absorption signals of metals in diluted pooled blood sample was also studied using the optimized fuel pressures for the respective metals and a fixed oxidant pressure of 1.6 kg cm-2. The maximum absorption of Cu, Zn and Mg was observed at burner height of 7.5 Fig. 2. Relatively small variation in the absorption signals of Cu and Zn with the change in burner height indicates that the atomic species formed are relatively uniformly distributed within the flame. 

Limits of Detection and Sensitivities

Limits of detection (LOD) and sensitivities were determined for the elements of interest in the diluted pooled blood sample by using the optimized conditions given in Table 1. The LOD was calculated as the concentration of analyte required to give a signal equal to twice the standard deviation of ten replicates of the blank measurements whereas the sensitivities were computed as the characteristic mass of analyte, which corresponds to 0.0044 absorbance units. The determined values of LODs and sensitivities are given in Table 2, along with the reported values from literature, which shows that the determined LOD of Cu is exactly the same and that of Zn is significantly lower than the reported values (15). The detection limits of all the metals found by the present method are low enough to determine the concentration of metals in the whole blood of healthy individuals with adequate precision and accuracy.

 

Table  2: Comparison of the determined and reported lod and sensitivities of trace metals in whole blood using aas.

 

 

S. NO

 

Metal

Proposed method

Reported Values

Sensitivity

(mgl-1)

LOD

(mg l-1)

LOD

(mg l-1)

1.

Cu

0.23

0.02

0.02

2.

Zn

0.03

0.008

0.04

3.

Mg

0.015

0.002

_

 

Analytical Performance

                The human blood is relatively a complex matrix in which physical or chemical interferences might occur during the determination of metals at trace levels, therefore, the precision of the method was checked by replicate analysis of unspiked pooled blood sample for its copper, magnesium and zinc contents. The results are shown in Table 3. The relative standard deviation at the prevailing concentrations of Cu, Mg and Zn was found to be 2.67%, 1.29% and 3.02% respectively.

                As no reference material is available for blood matrix in liquid state the accuracy of the method for the analysis of whole blood sample was checked by standard addition method using the optimized parameters. The results obtained were compared with the direct method of analysis, which are comparable with each other (Table 4). The data show that although the results of standard addition method are comparable with the proposed method the latter is preferred since the former method requires more portions of sample resulting in the consumption of more sample volume for the analysis, which is not always available.

                The accuracy of the method was cross checked by comparing the results of direct analysis of blood with the method of sample digestion using a mixture of nitric acid and perchloric acid14 prior to the determination of metals by AAS using aqueous standard solutions and routine analytical conditions. The results are shown in Table 4. It is seen that the determined concentration of all the metals in the digested sample was lower as compared to the concentration observed by direct method especially Zn, that is lower, by 21 %. Significantly lower concentration of Zn determined by the acid digestion method could probably be explained on the basis of reduction in nebulizing efficiency due to high acid concentration in the final analyte solution, thus resulting in lower number of atomic species in the flame. Similar observation has been reported during the AAS measurements of lead and cadmium in acidic solutions 16.

 

Table  3: Replicate determination of copper, zinc and magnesium in unspiked pooled blood by the proposed method

 

 

S.NO:

Metal Concentration (mgl-1)

 

Cu

Zn

Mg

 

 

1.

0.62

7.7

33.6

 

 

2.

0.60

8.2

32.7

 

 

3.

0.63

7.7

33.2

 

 

4.

0.61

7.7

32.4

 

 

5.

0.64

7.5

32.8

 

 

6.

0.59

7.7

33.2

 

 

7.

0.64

7.5

32.8

 

 

8.

0.63

7.7

32.4

 

 

9.

0.62

7.7

32.4

 

 

Mean ± SD

0.62 ± 0.017

7.71 ± 0.20

32.83 ± 0.42

 

                   

 

Table 4:Comparison of results of trace metal determination in whole blood by different methods

 

S. No:

Concentration (mg l-1)

Elements

Direct analysis

Standard addition method

Acid digestion Method

1.

Copper

0.62 ? 0.06

0.65 ? 0.02

0.6 ? 0.2

2.

Zinc

6.98 ? 0.3

7.2 ? 0.10

5.2 ? 0.14

3.

Magnesium

36.0 ? 0.2

37.6 ? 0.8

35.9 ? 2.2

 

Application of the method

                The optimized procedure was successfully applied to the determination of Cu, Zn and Mg in whole blood samples of one hundred and three healthy volunteers. The volunteers include fifty-two male and fifty-one female subjects. All the volunteers were adult without any symptoms of any type of exposure. These volunteers were chosen statistically because the objective of this work is two fold i.e., development of the method and the establishment of reference ranges for the area. The results obtained are summarized in Table 5, which show that the average determined concentrations of Cu, Zn, and Mg for both male and female were 1.13, 8.36 and 38.2 mg l-1 respectively. The determined average levels of Cu, Zn and Mg in whole blood for male and female subjects Table-5 depict that although the determined concentration of all the metals are higher in male subjects as compared to female subjects but this difference is not significant.

                The concentration of these metals in whole blood from the present study were compared with the reported values for different countries (Table-6), which shows that the concentration of copper is comparable to the reported values for Spain10 and China10, greater than Bangladesh7 and Japan17. It is lower than those of Canada and Italy18. The determined concentration of zinc is comparable to the reported value for China10, while greater to those of Canada and Italy18 Bangladesh7, Japan17 and Spain19. The concentration of magnesium is comparable to those of Canada18 and China10. These variations could probably be due to the different geographical conditions.

 

Table 5: Determined concentration (mg l-1) of metals in whole blood samplesof healthy volunteers

 

S.NO:

Elements

Range

Average

S.D

Male + Female (103)

1

Copper

0.6 - 1.91

1.13

0.30

2

Zinc

4.0 - 11.4

8.36

2.24

3

Magnesium

31.1 - 49.1

38.2

4.4

Male (52)

1

Copper

0.63 - 1.91

1.18

0.33

2

Zinc

6.7-11.4

8.83

1.61

3

Magnesium

31.2 - 49.1

40.2

4.9

Female (51)

1

Copper

0.6 - 1.4

0.88

0.23

2

Zinc

4.0-10.6

7.17

1.70

3

Magnesium

31.1 - 47.9

36.4

3.3

 

Table 6: Comparison of determined concentration of trace metals (mgl-1) in whole  blood with reported values.

 

Countries

Cu

Zn

Mg

Present study

1.13

8.36

38.2

Japan

0.82 - 0.86

5.8 - 6.2

-

Canada

1.22

6.59

37.7

Spain

1.05 - 1.17

5.85 - 6.07

-

Bangladesh

0.98

4.39

-

Italy

1.22

6.34

-

China

1.09

7.83

38.9

 

Environmental Contribution

In trace metal studies of essential elements in whole blood the requirement have to be defined by taking into the consideration all the possible contributing factors including nutrition and environment. In order to evaluate the possible environmental factors with the levels of essential trace metals in blood, the concentration of copper, zinc and magnesium has been determined in air, water and integrated diet samples of the area. The results have been reported in Table 7 along with daily intake amounts (bracketed quanta ties) of metals per person. The intake values have been calculated by using the average determined concentration of the metals on the basis of assumption of inhalation of 8.64 m3 air and consumption of three liter of water per person per day. Where as the intake of metals through integrated diet has been estimated on the basis of 537 gm consumption per person 13. All the reported values have been calculated on dry weight basis unless otherwise specified in the text. The perusal of the intake data of metals (Table 7), indicates that the percentage contribution through air and water is upto 1.59 % only as compared to the intake amount through integrated diet. It is, therefore, concluded that the integrated diet is the main contribution for the levels of Cu, Zn and Mg in human whole blood, whereas, the contribution of air and water with respect to these metals is insignificant.

 

Table 7: Determined Concentration and daily intake of trace metals (mg) through air, water and integrated diet samples of Islamabad Region

 

Type of samples

 

Copper

Zinc

Magnesium

 

Integrated diet

 

 

2.83 ± 0.52

(1528)

 

25.65 ± 4.2

(1385)

7.32 ± 0.81

(3952)

Water

 

 

4.4 ± 0.87

(13.2)

 

0.20 ± 0.001

(0.6)

21 ± 0.4

(63.0)

Air

 

 

0.14 ± 0.002

(0.050)

0.93 ± 0.03

(0.20)

15.8 ± 0.93

(2.20)

Determined concentration: Integrated diet (mg/gm)

Water                      (mg/l)

Air                           (mg/ m3)

 

CONCLUSIONS

 

The method described is simple, rapid and economical. Only 0.5 ml blood sample is required for the determination of Cu, Zn and Mg by applying the proposed method. The required sample treatment is minimum and there is no need to use matrix-matched standards. The method can be applied as routine diagnostic procedure in any pathological laboratory. Integrated diet was found to be the main source of these metals in human whole blood.

 

ACKNOWLEDGEMENT

 

                The authors would like to express their thanks to the medical staff of the Federal Government Services Hospital and Shifa International Hospital, Islamabad, for the valuable help in collection of blood samples.

 

References

 

1.        Trace element bioavailability and interactions Chapter-3, Trace elements in human nutrition and health, printed in Belgium,World Health Organization, Geneva, 1996, 22-41.

2.        Khurshid, S. J., Qureshi, I. H, The role of inorganic elements in the human body, The Nucleus, 1984, 21: 3-23.

3.        Leary, N. O., Pembroke, A., Duggan, P. F, Single stable reagent (Arsenazo-III) for optically robust measurement of calcium in serum and plasma. Clin. Chem., 1992. 38(6): 904-908.

4.        Nixon, D. E., Moyer, T. P., Routine clinical determination of lead, arsenic, cadmium and thallium in urine and whole blood by inductively coupled plasama-mass spectrometry, Spectrochim. Acta, 1996.  51B(1): 130-18.

5.        Brainina, K., Schafer, H., Ivanova, A., Khanina, R., Determination of Cu, Pb and Cd in whole blood by stripping voltammetry with the use of graphite electrodes, Anal. Chim. Acta, 1996. 330: 175-181.

6.        Xilei, L., Renterghem, D. V., Cornelis, R., Mess, L., Radiochemical neutron activation analysis for thirteen trace metals in human blood serum by using inorganic ion exchanger, Anal. Chim. Acta; 1988. 211: 231-241.

7.        Khan, A. H., Khaliquzzaman, M., Zaman, M. B., Husain, M. Abdullah, M. Akhter, S., Trace element composition of blood in adult population in Bangladesh, J. Radioanal. Chem., 1980. 57: 157-167.

8.        Stoeppler, M., Atomic absorption spectrometry – A valuable tool for trace and ultratrace determination for metals and metalloids in biological materials, Spectrochim. Acta, 1981. 38B: 1559–1568.

9.        Nygren, O., Nilsson, C. A., Gastavsson, A., Determination of lead in blood using flow injection and nebuliser interface for flame atomic absorption spectrometry, Analyst, 1988. 113: 591-594.

10.     Shang, S., Hang, W., 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 J. Anal. Chem., 1997. 357: 997-999.

11.     Haswell, S. J., Atomic Absorption Spectrometry, Analytical Spectroscopy Library. Vol. 5. Elsevier, Amsterdam, 1991. 359-361

12.     Behne, D., Sources of error in sampling and sample preparation for trace element analysis in medicine. J. Clin. Chem. Clin. Biochem., 1981. 19: 115-120.

13.     Qureshi. I. H,  Manan. A,  Zaidi. J. H,  Arif. M and Khalid. N., Determination of trace elements in integrated human diet for dietary assessment. Intern. J. Environ. Anal. Chem. 1990, 38: 565-577.

14.     Rahman, S., Yawar, W., Khalid, N., Chaudhri, S. A., Determination of lead and cadmium in blood by electrothermal atomic absorption spectrophotometry. Proc. Natl. Symp. Spectroscopy for material analysis, 1995. 431-434.

15.     Neil, I. W., Roger, S., Douglas, E. R., Comparison of three analytical methods for the determination of trace elements in whole blood, Anal. Chim. Acta, 1979. 110: 9-19.

16.     Khalid, N., Chaudhri, S. A., Effect of acids on determination of lead and cadmium by atomic absorption spectrometry, Anal. Chim. Acta, 1990. 233: 165-170.

17.     Satoh, Y., Yazawa, A., Contents of heavy metals in the blood of inhabitants in Yokohama city in Japan, Yokohama E., Ken Nenpo, 1978. 17: 63-66.

18.     Minoia, C., Sabbioni, E., Apostoli, P., Pietra, R., Pozzoli, L., Gallorini, M., Nicolaou, G., Alessio, L., Capodaglio, E., Trace element reference values in tissues from inhabitants of the European Community, Sci. Total Environ, 1990.  95: 89-105.

19.     Buxaderas, S. C., Farre-Rovira, R., Whole blood and serum copper levels in relation to sex and age, Rev. Esp. Fisiol., 1986. 42: 213.-216.