Biol Trace Elem Res (2008) 125:203–212 DOI 10.1007/s12011-008-8174-z
Evaluation of Iron in Serum and Urine and their Relation with Thyroid Function in Female Goitrous Patients Ghulam Abbas Kandhro & Tasneem Gul Kazi & Hassan Imran Afridi & Naveed Kazi & Mohammad Balal Arain & Raja Adil Sarfraz & Sirajuddin & Nasreen Syed & Jameel Ahmed Baig & Abdul Qadir Shah
Received: 16 April 2008 / Accepted: 30 May 2008 / Published online: 21 June 2008 # Humana Press Inc. 2008
Abstract In many developing countries, women are at high risk of goiter and iron deficiency anemia (IDA). Iron deficiency adversely affects thyroid metabolism and may decrease the efficiency of thyroid hormones in areas of endemic goiter. The aim of the present study was to compare the level of iron (Fe) in biological samples (serum and urine) and serum thyroid hormones, thyroid stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxin (FT4) of goitrous female patients (GFPs; n=69) with those of G. A. Kandhro (*) : T. G. Kazi : H. I. Afridi : M. B. Arain : R. A. Sarfraz : Sirajuddin : J. A. Baig : A. Q. Shah Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan e-mail:
[email protected] T. G. Kazi e-mail:
[email protected] H. I. Afridi e-mail:
[email protected] M. B. Arain e-mail:
[email protected] R. A. Sarfraz e-mail:
[email protected] Sirajuddin e-mail:
[email protected] J. A. Baig e-mail:
[email protected] A. Q. Shah e-mail:
[email protected] N. Kazi Liaqut University of Medical and Health Sciences, Jamshoro, Pakistan e-mail:
[email protected] N. Syed Nuclear Institute of Medical Radiotherapy (NIMRA), Jamshoro 76080, Pakistan
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nongoitrous women as control subjects (n=117), age range 21–45 years. The biological samples were analyzed for Fe concentration using flame atomic absorption spectrophotometer, prior to microwave-assisted wet acid digestion. The validity and accuracy of the method was checked by the certified sample and with those obtained by conventional wet acid digestion method on the same CRM and real samples. The overall recoveries of Fe in serum and urine were found in the range of 97.2–98.6% of certified values. The results of this study showed that the mean values of Fe in serum and urine samples of GFPs were significantly reduced as compared to control subjects (p=0.002 and p=0.015, respectively). The mean values of FT3 and FT4 were found to be lower in GFPs than in the age-matched healthy control women; in contrast, high mean values of TSH were detected in GFPs (p= 0.003). There was a positive correlation between serum Fe concentration and TSH (r=0.85, p=0.01), FT3 (r=0.95, p=0.003), and FT4 levels (r=0.98, p=0.007) in GFPs. It was observed that iron deficiency is prevalent in GFPs, so the need of Fe supplementation will be required to improve the efficacy of thyroid metabolism in goitrous women. Keywords Iron deficiency . Goiter . Female . Serum . Urine
Introduction It was extensively investigated that deficiencies of iron (Fe) and iodine remain major public health problems affecting greater than or equal to 30% of the global population [1]. Iron is present in all cells and has several vital functions. Iron deficiency has therefore a wide range of adverse health effects. It is not the only cause of anemia, but where anemia is prevalent, Fe deficiency is usually the most common cause [2]. A low level of dietary Fe or poor biological availability of Fe causes reduced absorption. In underdeveloped countries, a predominantly cereal-based diet rich in phytate, oxalate, phosphate, fiber, and other inhibitors of Fe absorption was found to be the main cause of iron deficiency disease [3]. Iron deficiency anemia (IDA) could impair thyroid metabolism through anemia and lowered oxygen transport [4]. The two initial steps of thyroid hormone synthesis are catalyzed by thyroid peroxidase and are dependent on Fe. The effects of Fe deficiency on the thyroid-stimulating hormone (TSH) axis are equivocal. Although IDA appears to blunt the TSH response to thyrotropin-releasing hormone (TRH), some studies have found an increase in circulating TSH concentrations in IDA [5], while others have reported no change or a decrease in TSH [6]. In regions of West and North Africa, 23% to 26% women suffer from both goiter and IDA [7, 8]. IDA, the most severe form of Fe deficiency, has important functional consequences for women of childbearing age. Iron deficiency reduces work efficiency in pregnant women [9] and may impair memory in adolescent girls [10]. Iron status is a function of Fe intake, storage, and loss [11]. Females of childbearing age require additional Fe because of the loss of Fe in menstrual blood and because of the demands of pregnancy [12]. Determinations of Fe in human tissues and fluids are used to obtain information on nutritional status for diagnosis of diseases, to indicate systemic intoxication, and to obtain information on environmental exposure [13]. In the majority of cases, serum, plasma, and urine are analyzed. The ability of the serum to counter changes in element presentation keeps nutritional and many toxic levels within a narrow range, unless under heavy exposure. This homeostatic response illustrates the effective clearance mechanisms in the serum and largely explains the short-term utility of serum analysis. The importance of
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exploring the depot storage capacities of various elements, particularly the toxic ones, remains a vital aspect in elemental analysis largely met by urine testing [14]. The aim of this study was to evaluate the concentration of Fe in biological samples (serum and urine) and other biochemical parameters such as TSH, free triiodothyronine (FT3), and free thyroxin (FT4) among female goitrous patients (GFPs) and compared them to age-matched healthy control women residing in the same area (Hyderabad), Sindh, Pakistan.
Materials and Methods Studied Population An epidemiological study was conducted among GFPs (n=69) of age group 21–45 years recruited from the outpatient clinic of the Nuclear Institute of Medicine and Radiotherapy Jamshoro (NIMRA), and age-matched nongoitrous women (n=117) were informed by administration through a consent about the aim of the study, and all agreed to participate and signed the form. A questionnaire was also administered to them in order to collect details concerning physical data, ethnic origin, health, dietary habit, age, and consent. Physical examinations were performed in the basic health unit of NIMRA, to measure the participant’s weight, height, blood pressure, and biochemical data. The criteria for the collection of biological samples (serum and urine) of GFPs set was that, prior to any treatment, they were not taking any mineral supplement during last 3 months. The normal women, belonging to same socio-economic status and diet habits, who were mostly the healthy family members of the patients not suffering from any goitrous problem, were selected as healthy control subjects. The preliminary exclusion criteria for patients and controls were hypertension, alcoholism, smoking, diabetes, cardiovascular disease, and taking of any vitamin and minerals that could affect oxidative parameters. Reagents and Standard Solutions All the chemicals were of analytical reagent grade and were supplied by Merck (Darmstadt, Germany). Ultrapure water was prepared by passing deionized water from a Milli-Q system (Bedford, USA) and was used throughout the study. Standard solution of Fe was prepared by dilution of certified standard solution (1,000 mg/L, Fluka Kamica). The certified reference samples of human serum (SERO-M10181, Billingstad, Norway) and Clincheck control-lyophilized human urine (Recipe, Munich, Germany) were used for validating methodology. All glassware and polyethylene bottles were thoroughly washed and then soaked overnight in 2 mol/L nitric acid and rinsed with ultrapure water before use. Apparatus Perkin-Elmer model A Analyst 700 (Norwalk, CT, USA) flame atomic absorption spectrophotometer (FAAS) with deuterium background correction equipped was used. The hollow cathode lamp of Fe was run under the conditions suggested by the manufacturer. A single-element hollow cathode lamp was operated at 7.5 mA and spectral bandwidth of 0.7 nm. The analytical wavelength was set at 248.5 nm. A Pel (PMO 23) domestic microwave oven (900 W maximum heating powers) was used for the digestion of
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the samples. Centrifugation was carried out to separate the supernatant from the sample extracts with a WIROWKA Laboratoryjna type WE-1, nr-6933 centrifuge, with speed range 0–6,000 rpm. Sampling All the venous blood samples (3–5 mL; including the control group) were collected after 12-h fasting, using metal-free Safety Vacutainer blood-collecting tubes (Becton Dickinson, Rutherford, USA) between 9:30 and 11:00 A.M. The blood samples were left standing for 1 h; sera were separated at 2,500 rpm centrifugation and preserved at −20°C until analysis. While for the analysis of other biochemical parameters, up to 5 mL blood samples from same subjects were sampled by using metal-free Safety Vacutainer blood-collecting tubes containing greater than 1.5 mg K2EDTA and sent to the pathological laboratories of NIMRA. Morning urine samples were collected in acid-washed, decontaminated 100-mL polyethylene tubes (Kartell1, Milan, Italy). In between sampling sessions, the container was wrapped in a clean polyethylene bag. Urine samples were acidified with ultrapure concentrated HNO3 (1% v/v) and kept at −4°C. Prior to subsampling for analysis, the sample should be shaken vigorously for 1 min to ensure a homogeneous suspension. Biochemical Analyses To assess thyroid function, FT4 and FT3 were measured by using radioimmunoassay (Gamma counter, Oakfield, England). TSH was measured by using immunoradiometric assay (Gamma counter). Zinc protoporphyrin (ZnPP) was measured in washed red blood cells (RBCs) by using a hematofluorometer (Aviv Biomedical, Lakewood, NJ, USA). The urinary thiocyanate ratio was analyzed by a colorimetric method [15]. Serum ferritin (SF) and transferrin receptor were measured by using commercial kits (RAMCO, Houston, TX, USA), and other hematological parameters were measured by Medonic CA 620, Haematological Counter (Stockholm, Sweden). The traditional diagnosis of goiter is based on palpation and World Health Organization classification [16], but palpation is subjective and reliable in adults, and recent developments in ultrasound technology have facilitated the accurate measurement of thyroid volume. The method used in the present study estimated the thyroid volume accurately, showing a correlation of 0.99 between the estimated volume and actual volume of surgical thyroid gland specimens [17]. The formula we used for the calculation of standard thyroid volume was based on a model describing the variation in individual thyroid volume as a function of several covariates such as age, height, and body weight [18]. Microwave-assisted Acid Digestion Method A microwave-assisted digestion procedure was carried out, in order to achieve a shorter digestion time. Certified samples of serum and urine were replicated six times, while duplicate samples of 0.5 mL of each goitrous and control individual were directly placed into Teflon PFA flasks. One milliliter of a freshly prepared mixture of concentrated HNO3– H2O2 (2:1, v/v) were added to each flask and placed in a covered poly(tetrafluoroethene) container, heated following a one-stage digestion program at 80% of total power (900 W), for 2–3 min for serum and urine. After the digestion, the flasks were left to cool and diluted to 10.0 mL in volumetric flasks with deionized water.
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Blank extractions (without sample) were carried out by performing the complete procedures without standard and sample. The resulted digested solutions were analyzed for Fe by FAAS. The concentrations were obtained directly from calibration graphs after correction of the absorbance for the signal from an appropriate reagent blank. Quality Control Calibration was performed with a series of Fe standards. The regression equation was: y = (0.0191) (Fe) + 0.0007, R2 = 0.9986, where y is integrated absorbance and the concentration is expressed as μg/L. The linear range of the calibration curve reached from the detection limit up to 200 μg/L. The limits of detection (LOD) and limits of quantification (LOQ) for Fe were calculated as under, LOD ¼ 3 s=m and LOQ ¼ 10 s= respectively, where s is the standard deviation of ten measurements of the blank and m m is the slope of the calibration graph. The LOD and LOQ of Fe were 64.5 and 215.2 μg/L, respectively. The validity and efficiency of the microwave-assisted digestion method was checked by human serum (SERO-M10181) and urine sample (Clincheck control-lyophilized human urine). The overall recoveries of Fe in certified biological samples by using the microwaveassisted acid digestion method were 97.2% and 98.6% in CRM of serum and urine, respectively. Data handling and Statistical Methods Data processing and statistical analysis were conducted by using computer program EXCEL (XP 2002; Microsoft, Redmond, WA, USA) and Minitab 13.2 (Minitab, State, College, PA, USA) software packages. Normally distributed data were expressed as means±SD. Student’s t test and Mann–Whitney test were used to assess the significance of the differences between the variables investigated in goitrous and nongoitrous female subjects.
Results The results of the biochemical parameters are shown in Table 1.There were no significant differences in weight and body mass index in GFPs and normal female subjects. The GFPs have lower hemoglobin (Hb) percent as compared to recommended range for normal persons (11.5–16.5 g/dL). The presence of anemia may act as an independent risk factor for thyroid problems. The hematocrit and SF levels were significantly lower in GFPs than normal female subjects (p0.05). The levels of thyroid volume in GFPs was significantly higher than control subjects (p=0.004) indicating the consequence of thyroid disorder. The results in Table 2 show that the concentration of Fe in serum samples of GFPs at 95% confidence interval (CI=0.51, 0.63 mg/L) was significantly lower than control subjects (p=0.002). The concentration of Fe in urine samples of controls was at 95% CI (2.50, 2.74 mg/L), which was higher as compared to GFPs (95% CI 1.74, 2.11 mg/L; p=0.015). Positive correlations were found between serum and urine Fe concentrations of normal control (p
