Determination of some trace elements Zn , Cu and Fe in the blood serum for patients with thyroid disease in middle and south of Iraq using atomic absorption spectrophotometer .

This investigation included the determination of some trace elements Zn, Cu, and Fe in the blood serum by flame-atomic absorption spectrometry after finding the optimum conditions for the estimation which involve the wave length, spectral band, lamp current, the burner width and the burner height.The concentration of zinc in the blood serum of the patients with hyperthyroidism was in the range of (0.596-0.681mg.l 1 ) which was lower than that of control concentration range (0.801-0.850mg.l -1 ) whereas for the patients with hypothyroidism was (0.601-0.748mg.l -1 ).The concentration of cupper in the patients with hyperthyroidism was higher range (0.93-1.12mg.l -1 ) than that of average concentrations in control (0.85-0.86mg.l -1 ) but decreased in the patients with hypothyroidism in the range of (0.77-0.83mg.l -1 ).The concentration of iron in the serum was increased in the patients with hyperthyroidism (0.97-1.03mg.l -1 ) and in the patients with Hypothyroidism (0.80-0.91mg.l -1 ) comparing with control concentrations (0.900.95mg.l -1 ).The concentration linear range was (4-0.5) mg.l -1 and correlation coefficient ( r ) was not less than 0.984. The statistical and other analytical results show that the relative standard deviation RSD% for trace elements (Zn, Cu, Fe) were (2.4, 1.5, 2.3) and detection limits were (0.0063, 0.0087, 0.0093)mg.l -1 respectively with percent recovery ranged between (95.0-100)%. The results obtained by this research give a good explanation for the spread of thyroid disease in middle and south of Iraq. Introduction It will be known that the decrease in Zn amounts in human serum leads to a growth stopping(Perrone et al, 1999). Also, zinc has a direct role in the formation of thyroid gland hormones and some enzymes and the decrease of this element will cause troubles in the function of these hormones and other enzymes (Karlik et al, 1996).On the other hand, it has been suggested that the increase of Zn concentration will cause troubles in the defensive systems, the function of antioxidant in the thyroid cells and leads to changing in red blood cells characteristics(Shopsis,1994). Zinc is regarded as an essential element in testosterone hormone formation which is necessary for women more than men especially in the polluted regions with


Introduction
It will be known that the decrease in Zn amounts in human serum leads to a growth stopping (Perrone et al, 1999).Also, zinc has a direct role in the formation of thyroid gland hormones and some enzymes and the decrease of this element will cause troubles in the function of these hormones and other enzymes (Karlik et al, 1996).On the other hand, it has been suggested that the increase of Zn concentration will cause troubles in the defensive systems, the function of antioxidant in the thyroid cells and leads to changing in red blood cells characteristics (Shopsis,1994).Zinc is regarded as an essential element in testosterone hormone formation which is necessary for women more than men especially in the polluted regions with ,200 3 , No. 2 Scientific Studies , vol.-Journal of Kirkuk University heavy metals (Shimada et al,1997).Furthermore, Zn enters as an active site in the work of super oxide dimustase enzyme which is acting as an anti free radical enzyme converting the free radicals to hydrogen peroxide and oxygen which protect the sensitive tissues from the destruction by oxidation (Hawk et al, 1998).As for Iron element, it is indispensable for the normal function of hemoglobin in the red cells, myogloben in the heart muscle and other function in calactase and peroxidase enzyme (Seymen et al, 1997).Also, Fe affect the activity of both glutathion peroxidase and super oxide dimustase enzymes (Zimmermann et al,2000).The scarcity of iron concentration makes the exposure to thyroid deficiency most possible (Rosenzweig et al, 1999), in addition to physiological changes in the body (Beard et al, 1998).There is a relationship between the patients with iron deficiency and the decrease of (T 3 ,T 4 ) hormones (Iwase et al,1993).Copper has an essential role in the growth and multiplication processes in addition to its role in the protection of thyroid tissues from free radical, also enters in the components of many enzymes as a catalyst especially in antioxidant enzymes such as super oxide dimustase and terosetase (Esipenko and Marsakova 1990).Moreover, it has an essential role in thyroid gland function (Karlik et al,1996), in which the studies indicate that the concentration of thyroid hormone in the serum and the activity of T 3 hormone were affected with the decrease of this element ratio (Oliver, 1975) (Alar et al,2000)and this will lead to thyroid deficiency disease (Kajil et al, 1992).Also, Cu affect the system work and reduce its activity while the increase of this element will lead to troubles in this system efficiency(1976, ‫.)أنيس‬ Kajil and his colleagues indicated in a study that the presence of cupper in the cells may has a defensive role in protection the body cells from the poisoning risks with high concentration of heavy metals such as Cd (Brian, 1983) .In addition to what was mentioned above, cupper enters as a catalyst in red blood cells building and its scarcity is leading to anemia and shortening the life of red blood cells (Perrone et al, 1999).There are a number of studies and researches concern with the determination of (Fe, Zn, Cu) in the biological fluids.In these studies, Fe, Cu and Zn have been determined in the blood serum of patients with nerves diseases (Eberk et al,1999) using solution of 10% Trichloroacetic acid.Brian have determined (Patricia et al, 1999) Zn and Cu in the hair using HNO 3 as a digester material through the dipping of hair in the acid for 24 hour.In the human bone zinc was determined through the ,200 3 , No. 2 Scientific Studies , vol.-Journal of Kirkuk University incineration of the bone at 48C 0 for 24 hour then determine it by the flame atomic absorption spectroscopy (Zaichich et al, 1995).
Moreover, there are another studies to determine Fe, Zn and Cu in the thyroid cells and its effect on the antioxidant enzymes in these cells especially in the patients animals with thyroid deficiency (Brunetto et al, 1999).Also, these studies have concerned with the effect of Fe, Cu and Zn on thyroid gland cancer by determination of these elements in the blood serum of the patients with this disease (Kelson & Shamberger, 1978).In addition, these elements have been determined in the blood serum of children (Bialy et al,1980) and their effects on the children.Our study has focused on determination of these trace elements in the serum of the patients with thyroid disease (hyper or hypo) and their effect on the function of thyroid hormones especially in south and middle of Iraq.

2-Chemicals & Reagents
The chemicals which were used in this study were at high purity and equipped from the International companies as shown in table(1).

Reagents: Preparation of the standard solutions
A) Preparation of zinc standard solution (1000mg.l - ).
It was prepared by dissolving of 1.2446gm from zinc oxide (ZnO) in an amount of deionized water, followed by addition of (5M HCl) to complete the dissolving and completing the volume to (1000ml) by ion free water.Diluted concentrations (0.5, 1, 1.5, 2, 2.5) were prepared from zinc standard solution by diluting with deionized water.B) Preparation of cupper standard solution (1000mg.l - ).
It was prepared by dissolving (4.3700)gm from hydrous Cupper nitrate Cu(NO 3 ) 2 .5H 2 O in a few amount of deionized water then completing the volume to(1000ml) by deionized water.Diluted concentrations (0.5, 1, 1.5,2, 2.5) were prepared from the Cu standard solution by diluting with deionized water.C) Preparation of Iron standard solution (1000mg.l - ).
It was prepared by dissolving (1.4297)gm from ferric oxide Fe 2 O 3 in 33ml of concentrated hydrochloric acid (HCl) then heating the solution for 10 minutes and completing the volume to (1000ml) with deionized water.Diluted concentrations (0.5, 1, 1.5, 2, 2.5) were prepared from the Fe standard solution by diluting with deionized water.

3-Optimum experimental conditions
Zn, Cu and Fe were determined after fixing the optimum experimental conditions using flame atomic absorption which its details were shown in instrument manual.

4-General procedure for the flame atomic absorption
A) Determination of Zn in the blood serum (Oliver et al, 1987).Zn element content in the blood serum was determined by diluting(0.5ml) of the serum sample with eight volumes of 1% V/V HNO 3 .After that, the atomic absorption signal of the resulted solution was registered under the optimum conditions as shown in table(2).B) Determination of Cu in the blood serum (Ohta et al, 1988).
Cu content in the blood serum was determined by diluting (5ml) from the serum sample with (5ml) from (W/V 0.03% polyvinylalcohol).After that, the atomic absorption signal of the resulted solution was registered under the optimum conditions as shown in table(2).C) Determination of Fe in the blood serum (Ohta et al, 1988).
Fe content in the blood serum was determined by mixing(5ml) from the serum sample with (25ml) from HCl and keeping the mixture in an incubator for 30 minutes then adding 250ml from Trichloroacetic acid to the mixture.After that, 50ml from the resulted solution will be injected to the atomic absorption spectrophotometer to register the signal of atomic absorption under the optimum conditions.

Results and discussions 1-Effect of Zn on hyperthyroidism and hypothyroidism disease
The results have shown that the Zn blood serum content for the normal men was (0.850mg.l -1 ) while for the patients men with hyperthyroidism was varying between (0.613-0.681mg.l - ).As for the women, the results have shown that Zn blood serum content for the normal women was (0.80mg.l -1 ) while for the patients women was (0.596-0.657mg.l - ) as shown in the table (3-1).It is clear from the results mentioned above that the decrease of Zn level in the blood serum leads to increase the activity (deiodination) through the increase of (Heptatic-5-deionase) enzyme activity which convert T 4 to T 3 and this means the increase of the active thyroid hormone T 3 (Iwase,K et al 1993) and the decrease of Zn will lead to oxidation risks for the Methalouonien protein (MT) which is acting as a carrier for Zn and other trace elements.Concerning the patients with hypothyroidism, the results have shown that the Zn blood serum content ratio for the normal men was (0.850mg.l -1 ) while for the patients men was varying between (0.665-0.748 mg.l -1 ).On the other hand, for the normal women Zn blood serum content ratio was (0.801 mg.l -1 ) while for the patients women was varying between (0.601-0.715mg.l - ) as shown in the table (3-2).The results have shown that the decrease of Zn concentration in the blood serum will lead to decrease the concentration of glutathion peroxidase enzyme and reduce the concentration of T 3 ,T 4 in the blood serum therefore the activity of (heptatic-1,5-deiodenase) will be reduced by 67% which means that the convertion of T 4 to the active T 3 will be decreased.For the patients with hyperthyroidism, it was found that if there is a scarcity in Zn in their body then its concentration will be increased in the urine while for the patients with hypothyroidism the concentration of Zn in the urine will be decreased (Muller et al, 1996).

Calibration graph of Zn determination:
After fixing the optimum conditions, the calibration graph was drawn up.A linear relationship has arisen for concentrations to range from 1 to 4 mg.l - and correlation coefficient 0.9896 which are shown in fig( 1).In addition, the accuracy of the flame atomic absorption method was measured using standard solutions from Zn solution then the recovery percentage and the relative standard deviation (RSD%) were calculated as shown in table(4).The method has shown a high recovery percentage and detection limit of (0.0063 mg.l -1 ) with high accuracy.Zn lower detection limit = 0.0063 mg.L -1 or 6.3µ.gL -1 =6.3ppb

2-Effect of Cu on hyperthyroidism and hypothyroidism disease
The results have shown that the Cu blood serum content for the normal men was (0.86 mg.l -1 ) while for the patient men with Hyperthyroidism was to range (0.93-1.03 mg.l -1 ).As for the normal women, the Cu blood serum content was (0.85 mg.l -1 ) while for the patient women with hyperthyroidism was to range (0.95-1.12 mg.l -1 ).The results of analysis gave a differences in Cu blood serum content for the normal and patients as shown in table (5-1).In view of Cu importance in the body where it has a role in the formation of many enzymes especially super oxide dimustase enzyme in addition to its role in Fe-metabolism in the body, therefore, in the case of hyperthyroidism, the oxidation of lipids and fatty acids will be increased (Karlik et al,1996).Also, there is a probability for destruction of the cell wall leading to troubles in the ion distribution in addition to destruction the proteins which is connecting with Cu then the increase in

Calibration graph of Cu determination:
After fixing the optimum conditions, the calibration graph was drawn up.A linear relationship has arisen for concentrations to range from 0.5 to 4 mg.l - and correlation coefficient 0.9962 which are shown in fig( 2).Also, the accuracy of the flame atomic absorption method was measured using standard solutions from Cu solution then the recovery percentage and the relative standard deviation (RSD%) were calculated as shown in table (6).The method has shown a high recovery percentage and low detection limit with high accuracy.accuracy of the used method was measured through the preparation of standard solutions from Fe solution and then the recovery percentage and the relative standard deviation (RSD%) were calculated as shown in table (8).The method was characterized with high accuracy, low detection limit and high recovery percentage.Fe lower detection limit = 0.0093 mg.L -1 =9.3µg.L -1 =9.3ppb

Conclusion
The results have shown that the trace elements which are studied have an essential role in the exposure to the thyroid gland disease.It was found that the concentration of Zn decrease in both (hyper and hypothyroidism).The concentration of Cu and Fe decrease in the patients with hypothyroidism but increase in the patients with hyperthyroidism.

Fig
Fig(2):Calibration graph for Cu determination

Table ( 1
):The chemicals purity and its sources.

Table ( 2
-1): The average concentration of Zn in the blood serum for both control group and patients groups.

Table ( 2
-2): The average concentration of Zn in the blood serum for both control group and patients groups.
* average of 20 samples