Data The Thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃) are tyrosine-based hormone, produced by thyroid gland [1]. Thyroid gland is regulating the body’s metabolism [2]. It is the largest gland in our neck it is located in the front neck below skin and muscle [2,3]. Triiodothyronine (T₃) is about ten times more active than T₄ [4]. This hormones contain four Iodine atoms Triiodothyronine is identical to T4 but it has one less Iodine atome per molecule [5]. Thyroid hormones play a particular role in brain development during pregnancy [6]. These are essential for normal growth and development of the sex organs, and control the rate of metabolism and the function of every organ in the body [7]. Hypothyroidism can be defined as a syndrome characterized by deficient thyroid gland production of thyroid hormone [8,9]. The various causes of hypothyroidism can be classified according to their site and the nature as table shows [10]. Hypothyroidism causes sluggishness and low energy; body temperature goes down; weight gain is common [11]. Hypothyroidism is relatively common with prevalence of 2- 3% of general population [12]. Clinical diagnosis of Hypothyroidism is based on the symptoms and signs in patients like being sensitive to cold, unexplained weight gain, constipation, dry skin, floky inelastic skin [13]. Levothyrotine is generally considered to be the treatment of choice for patients with hypothyroidism has long half-life 7- 10 days, may be administrated P0/IV/IM [14,15]. The BMI is a convenient rule of thumb used to broadly categories a person as underweight, normal weight, overweight, obese based on tissue mass and height [16]. Adolphe Quetelet, a Belgian astronomer mathematician, Statistician and Sociologist devised the basis of BMI between 1830 and 1850 as he developed what he called social physics [17].

From this point, this study aimed to evaluate the BMI in hypothyroid Iraqi patients and its response to thyroxin therapy.

Subjects and Materials 

This study was performed during the period from November 2018 to May 2019. One hundred and five hypothyroid subjects were selected from people attending the specialized Centre for Endocrinology and Diabetes. Their ages range from 10-70 years. For each patient the following tests were carried out: Thyroid Function tests which include T3, T4 and TSH using special kits of ELISA. Careful history was obtained from patients including age, sex, height, and weight to estimate body mass index. For blood sampling, five ml of blood sample has been collected from one of the big veins in the ante-cubital fossa of each hypothyroid patient and control group which were undergone sera is isolated for subsequently T3, T4, TSH tests (Table 1).

Table 1: Details of the kits used

For BMI is calculated for a person weight and height (Table 2), it’s used as a screening tool to identify possible weight problems for adult; BMI was calculated according to the following formula [18,19].

BMI= weight in kg/height in cm * 10000

BMI= <18.5 = people considered under weight

BMI= 18.5-24.9 = normal weight

BMI=25-29.9 = overweight 

BMI= ≥ 30 =obese

Table 2: Equipment and instruments

Methods

Total Thyroxin (T4) Estimation

Principle: The assay principle combines an enzyme immunoassay competition method with a final fluorescent detection (ELFA). The solid phase receptacle (SPR) serves as the solid phase as well as the pipetting device for the assay. Reagents for the assay are ready to use and predisposed in the sealed reagent strips. All the assay steps are performed automatically by the instrument. The reaction medium is cycled in and out of SPR several times. The sample is taken and transferred into the well containing T4 antigen labeled with alkaline-phosphates’ (Conjugate). Competition occurs between the antigen present in the sample and the labeled antigen for the specific anti-T4 antibodies coated on the interior of the SPR. During final detection step, the substrate (4-methylumbelliferyl phosphate) is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-methyl-umbelliferone) the fluorescence of which is measured at 450 nm. The intensity of the fluorescence is inversely proportional to the concentration of the antigen present in the sample. At the end of the assay, results are automatically calculated by the instrument in relation to calibration curve stored in memory and then printed out [20].

Test Procedure

The required reagents were removed from the refrigerator and allowed to reach the room temperature for at least 30 minutes. Only one T4 strip and one SPR were used for each sample, control or calibrator to be tested. T4 was selected on the instrument to enter the test code. The calibrator must be identified by "S1" and tested in triplicate if the control was tested, it should be identified by "C1". The calibrator, Control and sample were mixed using vortex type mixer.

Two hundred µL of calibrator, sample, or control was added into the sample well. SPRs and strips were inserted into the instrument provided the colour labels with the assay code on the SPR and the reagent strips were matched. The assay was initiated as directed in the operator manual. All the assay steps were performed automatically by the instrument. The assay was completed within approximately 40 minutes. After the assay was completed, The SPR and strips were removed from the instrument. The used SPRs and Strips were disposed into an appropriate recipient.

Triiodothyronine (T3) Estimation

In this test, a second antibody (goat anti-mouse IgG) is coated on the microtiter wells. A measured amount of patient’s serum, a certain amount of mouse monoclonal antibody (mAb) anti-T3 antibody, and a constant amount of T3 conjugated with horse radish Peroxidase are added to the microtiter wells. During the incubation period, the mouse anti-T3 Ab is bound to the second Ab on the wells; and T3 & conjugated T3 compete for the limited binding sites on the anti-T3 Ab. After 60 minutes of incubation at room temperature, wells are washed 5 times by water to remove unbound T3 conjugate. A solution of TMB reagent is then added and incubated for 20 minutes, resulting in the development of blue colour. The colour development is stopped with addition of the stop solution, and the absorbance is measured spectrophotometrically at 450 nm. The intensity of the colour is proportionated with the amount of unlabeled T3 standards assayed in the same manner, and the concentration of T3 in the unknown sample is then calculated [21].

Procedure 

The desired No. of coated wells was secured in the holder. A data sheet was made for sample identification. Fifty μl of Standard, samples and Controls were pipetted into appropriate wells. Fifty μl of the Ab reagent was dispensed into each well, mixed thoroughly for 30 sec.

One hundred μl of working conjugate reagent was added into each well and mixed thoroughly for 30 seconds. The plate was incubated at room temperature for 60 minutes and then the incubated mixture was removed by flicking the plate contents into a waste container. The microtiter wells were rinsed and flicked 5 times with DW or deionized water. The wells were stroked sharply onto absorbent paper to eliminate the residual water droplets. One hundred μl of TMB reagent was added to each well and mixed gently for 10 seconds. The plate was incubated at room temperature in the dark for 20 minutes without shaking. The reaction was stopped by the addition of 100 μl of Stop solution to each well and mixed gently for 30 seconds. The absorbency was read at 450 nm using the ELISA reader and within 15 minutes.

Thyroid Stimulating Hormone (TSH) Estimation

This test is based on the principle of a solid phase enzyme-linked immunosorbent assay ELISA [22]. The assay system utilized a unique monoclonal antibody directed against a distinct antigenic determinant on the intact molecule. Mouse monoclonal anti-TSH antibody is used for solid phase immobilization (on the microtiter wells). A goat anti-TSH antibody is the antibody enzyme (Horse Radish Peroxidase)-conjugate solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the RSH molecule being sandwiched between the solid phase and enzyme-linked antibodies, then after two hours of incubation at room temperature, and wells are washed with water to remove unbound labelled antibodies. A Solution of TMB reagent is added and incubated for 20 minutes, resulting in the development of blue colour. The colour development is stopped with the addition of stop solution, changing the blue colour to yellow. The concentration of TSH is directly proportionated to the colour intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.

Assay procedures

The desired number of coated wells is secured in the holder. One hundred μL of standards, specimens, and controls were dispensed into appropriate wells .one hundred μL of enzyme conjugate reagent were dispensed into each well and mixed thoroughly for 30 seconds. The plate was incubated at room temperature (18- 25⁰C) with shaking at 175 RPM, for 120 minutes. The incubated mixture was removed by flicking plate contents into a waste container. The microtiter wells were rinsed and flicked 5 times with distilled or deionized water. The wells were stroke sharply onto absorbent paper to remove residual water droplets. One hundred μL of TMB reagent are dispensed in to each well Mix gently for 10 seconds. The plate was incubated at room temperature for 20 minutes. The reaction was stopped by adding 100 μL of stop solution to each well and then mixed gently for 30 seconds. The absorbance was read at 450 nm within 15 minutes.

Statistical Analysis

The suitable statistical methods were used in order to analyse and assess the results; they include the statistical tables including observed frequencies with their percentages. Summary statistic of the reading distribution (mean, SD& SEM). Graphical presentation by (bar charts). Also, Chi-square (χ²), Kruskal Wallis test, Matched paired t-test for repeated measurements, Person correlation coefficient (r).

Hypothyroidism results from the failure to maintain adequate tissue levels of thyroid hormones, and if thyroid hormone T3, T4 decreased it would cause an increase in secretion of TSH by pituitary gland, this case is called hypothyroidism [14] (Table 3).

Table 3: Aetiology of hypothyroidism [10]

In Table 4 represented the distribution of patients according to age group. The table shows that the majority of patients were between 31-50 years (51.4 %), also the age's frequency of patients at 51-70 years was (26.7%). Moreover, the table showed that the number of hypothyroid patients 10-30 years was (21.9%) only. The data analysis revealed a highly significant difference between the number of patients in the different age groups with p<0.01. The above observation somewhat disagreed with the result of Morganti et al. [23], who stated an increased prevalence of hypothyroidism demonstrated in the elder population, while increased incidence of primary hypothyroidism shown in Table 4, in age group 31-50 years has no interpretation in available references, but may be due to older people come for checking up less frequently than younger people. 

Table 4: Characteristics of studied samples according to some variables

All forms of thyroid disease were four to five times more common in females than in males. The reason was not clear [24]. The distribution of patients was according to gender. It was obvious that women were at high risk for developing disease (84%) in comparison with men (16%) with highly significant difference between number of females to males p<0.01. These results were in agreement with the observation of Bjoro et al. [25] and the study performed in West Indians, Jamaica in which it was represented that females were more suspected to have hypothyroid disease than males [26]. Also, in Table 4, revealed that there was an increase of hypothyroidism's incidence among obese patients (48.6%), while those who were overweight form (29.5%) in comparison with (21.9%) who was normal and under-weight 18-19 with a highly significant difference p< 0.01. These results were in agreement with the observation of Zulewski et al. [27]. In spite of data supporting the use of serum TSH concentration as the best test to detect abnormal thyroid function, measurement of circulating thyroid hormones with or without serum TSH continued to be frequently requested to evaluate thyroid function [28]. Highly significant difference p<0.01 was observed in mean of T3 and T4 (0.914±0.216, 35.155±26.846) respectively in the first visit in comparison with control group (2.213±0.249, 80.202±10.431) respectively, with highly significant difference p<0.01 in the mean of them in the second visit (1.219±0.387, 49.774±32.401) respectively in comparison with control group. While non-significant differences in the mean of T3 and T4 were shown between the first and second visit p>0.05, p>0.05 respectively. There was a highly significant difference in the mean of TSH level in the first visit in comparison with control group (17.490±10.614) (2.883±1.935) respectively p<0.01. On the contrary, there was no significant difference in TSH level (7.121±5.864) at the second visit in comparison with control group p>0.05. On the other hand, there is a highly significant difference between TSH level at first and second visit p<0.01. These facts are presented in Table 5 and Figure 1. 

Table 5: Mean distribution of serum thyroid hormone levels among studied groups

The current results were in agreement with the study of Hind [29] who found that TSH is a good marker for laboratory diagnosis and patients' follow up with primary hypothyroidism [30].

Figure 1: Mean distribution of serum thyroid hormone levels among studied groups

Data in table 6, showed the correlation between T3 and T4 in the first visit which revealed that there was strong positive correlation (r=0.542) with a highly significant difference p=0.00. Most patients under treatment with thyroxin are converted to the active T3 within the cell 1.

Table 6: Correlation between serum chemicals in 1^st visit patients

*Significant Correlation (p<0.05) **Highly Significant Correlation p<0.01

Correlation between T3 and TSH in this table showed that there was a weak negative relationship (r = -0.116) with anon significant difference p=0.236. These results provide that normal thyroid secretion depends on (TSH) [31], and this was the basis of mechanism between these two variables. T3 and BMI had a weak Negative relationship (r = -0.146) with anon significant difference p=0.134. This result was in agreement with Zulewski et al. [27], who found greatly decreased thyroid hormone almost increases the body weight. 

In correlation between T4 and TSH there was a weak negative correlation (r = -0.033) with a highly significant difference p=0.734. This study is in agreement with [31], who found that T4 was low and TSH level high in patients with hypothyroidism and this was the basis of mechanism between these two variables and this was considered as final diagnosis of primary hypothyroidism. Between T4 and BMI there was a weakly negative correlation (r = -0.018) with a non-significant difference p=0.856. Patient with hypothyroidism have an increase in body weight and this study is in agreement with Zulewski et al. [27]. The relationship between TSH and BMI revealed that there was a weakly Negative correlation (r = - 0.025) with a non-significant difference p=0.803. this result is in agreement with worker in WHO [32]. Who found that the correlation between TSH and BMI was really contradictory and has been documented by other workers as hypothyroid patients' usually over weight but this correlation was non-significantly different.

We concluded that the hypothyroidism is more common in females than males. The patients aged (31-50) years showed a high prevalence of hypothyroidism when compared with other age groups. The number of patients who had increased in BMI (Obese) is a highly significant difference with the others. It was recognized that TSH is a good marker for laboratory diagnosis and follow up of patients with hypothyroidism. There was a role for thyroxin in decreased BMI. It is important to discover hypothyroid patients as early as possible.

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