27J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 27–30 Research Objective This study focuses on the relationship between calcium ion concentration in human body and thyroid hormones with liver enzymes to determine the effect of some physiological and biochemical parameter of liver [glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), Bilirubin] and thyroid stimulating hormone (TSH; T3, T4), on the levels of calcium ion in the blood. Methods The current study included 40 patients from two different hospitals in Karbala city. This study has been investigated to find the effect of thyroid hormones (T3, T4, TSH) and liver enzymes (GOT, GPT, Bilirubin) on calcium ion concentration. Results The results revealed there is no significant difference (P > 0.01) between calcium ion concentration and thyroid hormones (T4, T3, TSH) in each groups of different ages, respectively. Therefore, weak correlation between calcium and hormones pointed to an increase or decrease of calcium ion concentration effect by these hormones. Conclusion There is no significant difference (P > 0.01) between liver enzymes (GOT, GPT, Bilirubin) and calcium ion concentration in each groups of different ages respectively, although weak correlation between calcium and hormones that pointed to an increase or decrease of calcium ion concentration is also affected by these enzymes. Keywords calcium ion, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, Bilirubin, T3, T4, TSH Study of the relationship between calcium ion and thyroid hormones, liver enzymes in some patients of hypocalcaemia and hypercalcaemia Jasim A Abdullah, Hanen Abdulkalek, Sura Haider & Aliaa Aamer Introduction Calcium is a body’s abundant divalent cation and more than 99% of the body calcium is concentrated in the skeletal system and approximately 1% is rapidly exchangeable with blood cal- cium. Small amounts of calcium located outside the bone cir- culates in the serum partly bound to protein and partly ionized. Calcium has a major role in the transmission of nerve that include normal heart beat and has vital role in cardiac action potential and is essential for cardiac pacemaker auto- maticity, and this ion is also involved in blood clotting and hormone secretion.1,2 Intracellular calcium is a key of intracel- lular second messenger that play a pivotal role in controlling various cellular processes such secretion, differentiation, pro- liferation, motility and cell death. In contrast, extracellular cal- cium is crucial for a spectrum of physiological phenomena including blood coagulation, muscle function and mainte- nance of skeletal integrity.3 Calcium balance is tightly regu- lated by the interplay between gastrointestinal absorption, renal excretion, bone reabsorption, vitamin D and parathyroid hormone (PTH) system.4 The thyroid gland plays a role in cal- cium metabolism and regulation, by secreting when needed and the thyroid stimulating hormones (TSHs) are key to regu- late the metabolism of thyroid gland to produce both T4 (thy- roxine) and T3 (tri-iodothyronine). T4 is the main product and is converted in the periphery via de iodination to T3 which is the main biological active of thyroid hormones. The production of TSHs is regulated by thyrotrophic; therefore, thyroid stimulating hormone in response is regulated by a negative feedback mechanism related to serum level of free T3 and T4.5 The liver enzyme has an important role in amino acid metabolism. Aspartate aminotransferase (AST) and ala- nine aminotransferase (ALT) are enzymes found mainly in the liver. They are also found in red blood cells, heart muscle tis- sues and other organs such as the pancreas and kidney.6 AST and ALT formerly was called serum glutamic oxaloacetate transaminase (SGOT) and serum glutamic pyruvic transami- nase (SGPT), respectively. AST or ALT levels are valuable aid primarily in the diagnosis of liver diseases but not specific for diagnosis of liver disease.7 Materials and Methods 1. Collection of sample The specimens were collected from two hospitals, Al-Hussainy hospital and Al-Hindia hospital in Karbala city. Venous bloods were collected in a laboratory, as specimens, from 40 patients. The average age was 10–45 years. 2. Separating serum from blood First, blood taken from patients by syringe was placed in the plane tubes (not anticoagulant tube). Then, these tubes were centrifuged at 5000 rpm for 5 min. The serum thus separated from blood was transferred to another tube and gave a special number and name to the tube to avoid its loss. 3. Measurement of calcium The first test is the measurement of calcium ion in the body via electrolytes instrument in both hospitals, Al-Hussainy and Al-Hindia. 4. Hormonal test procedure (TSH-T3-T4) by ELISA i. Prepare both the samples (serum) and reagents in room temperature. ii. Format the microplate wells for each serum reference, control and patient specimen to be assayed in duplicate. iii. Pipette 50 µl of the appropriate serum reference, con- trol or specimen into the assigned well. Department of Clinical Laboratories, College of Applied Medical Sciences, Karbala University, Iraq. Correspondence to Jasim A Abdullah (email: jasim.abdulabbas@gmail.com). (Submitted: 23 March 2015 – Revised version received: 26 May 2015 – Accepted: 2 June 2015 – Published online: Summer 2015) ISSN 2413-0516 28 J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 27–30 Calcium ion and thyroid hormones Research Jasim A Abdullah et al. iv. Add 100 µl of working reagent A (T3, T4, TSH), enzyme con- jugated solution to all the wells. v. Swirl the microplate gently for 20–30 sec to mix and cover it. vi. Incubate 60 min at room temperature. vii. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. viii. We used automatic plate washer and washed plates for 3 times. ix. Add 100 µl of working signal reagent solution to all wells. Don’t shake the plate after sub- strate addition. x. Incubate at room temperature for 15 min. xi. Add 50 µl of stop solution to each well and gently mix for 15–20 sec. xii. Read the absorbance in each well at specific wave length compatible with hormones that we tested (T3, T4, TSH). xiii. The result should be read within 30 min of adding the stop solution. 5. Biochemical test procedure (include GOT, GPT, bilirubin) We used Reflatrone device to determine this parameter. This procedure includes: i. Working the device (Reflatrone). ii. Take specific strip for test that you need (GOT, GPT, Bili- rubin) and add in the dedi- cated space one drop or 32 µl from the patient serum. iii. Put the strip in the device and wait for some minutes and read the result. 6. Statistical analysis We used the correlation test to deduce correlation factor between calcium ion concentration and thyroid hormones and liver enzymes at probability level 0.01 and degree freedom, n–2.8 Results and Discussion The Relationship between Calcium Ion Concentration and Thyroid Hormones The present study included 40 patients and tested the relationship between thy- roid hormones (T3, T4), TSH and cal- cium ion concentration; in other words, this experiment studied the effect of thyroid hormones on calcium ion con- centration. Through this study we observed the relationship between cal- cium ion concentration and thyroid hormones (T4, T3), TSH. There is no significant difference when P > 0.01 in all groups of different ages. Weak corre- lation between calcium ion concentra- tion and thyroid hormones (T4, T3, TSH) pointed to an increase of calcium ion concentration depending on an increase of these hormones, but it was weak. This result is shown in Table 1 and Fig. 1. The calcium sensing receptor (CaSR) represents the molecular mech- anism by which parathyroid cell detect change in blood ionized calcium con- centration and modulate parathyroid hormone (PTH) secretion to maintain serum calcium levels within a narrow physiological range.9 Depending on some studies that observed the blood ionized calcium concentration are remarkably stable in healthy individual because of the hemostatic system involving the action of the three calcio- tropic hormone on the organ of bone. Therefore, the secretion of PTH is highly dependent on the ionized calcium con- centration and represent a simple nega- tive feed-back loop. The serum PTH concentration decreases as the serum PTH secretion is not entirely suppress- ible; however, there is relatively narrow range of regulation of PTH secretion by extracellular calcium.10 The major effect of PTH is to maintain normal ionized serum calcium concentration. PTH stimulates the bone reabsorbing releasing calcium into extracellular fluid. The rise in calcium concentration is caused prin- cipally by two effects, first is the effect of PTH to increase calcium and phosphate absorption from the bone and second is a rapid effect of PTH to decrease the excretion of calcium by the kidneys.11 Effect of exercises on thyroid hormone T3 demonstrated that this effect can lead to increase T3 hormone.12 Renal calcium influenced by thyroid hormone was decrease in hyperthyroidism and increase in hypothyroidism.3 Other studies showed the elevated level of total and free T4 hormone also have been reported in patient with acute psychi- atric illness, iodinated contrast agent also elevate T4 hormone level by inhib- iting peripheral conversion of T4 to T3. The decrease of T4 hormone occurs in patient with most severe monothyroid illness.13 The serum calcium level decreased significantly in patient with high TSH concentration in contrast with normal TSH. Also, renal calcium influenced by thyroid hormones have been decreased in hypothyroidism and increased in hypothyroidism.9 The inhibitory action of calcium on the Table 1. Relationship between calcium and TSH, T3 and T4 Parameters Age Ca+2 – TSH Ca+2 – T4 Ca+2 – T3 10–20 years (n = 12) 0.4117 0.4105 0.1566 20–30 years (n = 10) 0.10502 0.00604 0.20412 30–40 years (n = 18) 0.08956 0.17519 0.00602 *The number refers to correlation factor (r value). 0 5 10 15 20 25 30 10–20 year       Co nc en tr at io n m g/ dl a nd p g/ l Age Relationship between calcium and Hormones        Ca TSH T3 T4 20–30 year 30–40 year Fig. 1 Mean value of calcium and thyroid hormones. 29J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 27–30 Research Calcium ion and thyroid hormonesJasim A Abdullah et al. thyroid gland has been suspected since the last century but more recent studies have confirmed its effect. It is known that calcium decreases thyroid activity and that its absorption increases thyroid insufficiency.14 The thyroid gland nor- mally enlarge in response to an increase demand for thyroid hormone that occur in puberty, pregnancy, iodine deficiency and immunologic viral or genetic dis- order in this increase in thyroid follicles and thyroid hormone synthesis. There- fore, elevation of thyroid hormone leads to graves’ disease or other thyroid abnormalities. The primary characteris- tics of graves’ disease are diffuse thyroid enlargement (as much as two to three times the normal size, 40–60 grams), then the tumor release TSH that induce Table 2. Relationship between calcium and (GOT, GPT and Bilirubin) Parameters Age Ca+2 – GPT Ca+2 – GOT Ca+2 – Bilirubin 10–20 years (n = 12) 0.2273 0.5427 0.0660 20–30 years (n = 10) 0.2424 0.03136 0.02636 30–40 years (n = 18) 0.01288 0.0263 0.0942 *The number refers to correlation factor (r value). elevated thyroid synthesis and release and are not responsive to normal hormonal feedback control. Such patients present with many symptoms of hyperthyroidism.15 However, periph- eral metabolism of thyroid hormone can be changed significantly by condi- tion, which can alter the deionization pathway and lead to change in the circu- lating level of thyroid hormones. The biological effect of short-term change in the thyroid hormone level are not cur- rently completely understood but are potentially important in the body adjustment to stressful or catabolic state. Low dietary calcium intake and hypoc- alcaemia are major stimuli for PTH secretion leading to parathyroid gland hyperplasia.16 Relationship between Calcium Ion Concentration and the Liver Enzyme GOT, GPT and Bilirubin In the present study, we tested the rela- tionship between calcium ion concen- tration and liver enzymes (GOT, GPT and Bilirubin). We observed there is no significant difference (P ≥ 0.01) between calcium ion concentration and liver enzymes (GOT, GPT and Bilirubin) in all groups of different ages respectively. Weak correlation between calcium and liver enzyme pointed to the increase or decrease parameter effects on calcium ion concentration. This result is shown in Fig. 2 and Table 2. A significant decrease in total and ionized calcium phosphorus and chlo- ride and an increase in sodium were also observed. Significant increase was found in most enzymes such as AST, GK and GGT.17 A diet supplement by calcium leads to an increase in serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. Addition of calcium ion significantly reduced liver GST and elevated liver GPX activities.18 Human erythrocytes that incubated with different concentra- tion of calcium chloride (0.17–1.67 mm) showed hemolytic after addition of bil- irubin. Hemolysis was observed only when cell were incubated first with cal- cium followed by bilirubin and not vice versa. 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