Field survey and sampling
Naqu county and Chanang county were selected from the pastoral and agricultural areas, respectively, for the field survey. The residents in Naqu county primarily depend on livestock farming for living, and the residents in Chanang county primarily live from crop farming, and their lifestyle and food structure have remained unchanged for years. Both counties are self-sufficient in terms of economy and lifestyle. The geographical and climatic environments of the two counties are described in Table 2 [11].
Table 2 Geographical and climatic environment of the pastoral Naqu county and the agricultural Chanang county
|
Population
|
Area
(km2)
|
Altitude
(m)
|
Annual mean temperature
(℃)
|
Annual mean precipitation (mm)
|
Naqu
|
90,000
|
16,195
|
4,450
|
-2.2
|
400
|
Chanang
|
83,000
|
2,163
|
3,620
|
8.2
|
420
|
The sample size was determined using the formula: n = tɑ2(1 - p) p/d2, which is usually adopted to estimate the minimum sample size for cluster sampling or simple random sampling [12]. We considered the average goiter rate (11%) of Tibetan children in 2005 as the probability (p), 1.96 as the t-critical value (tɑ, ɑ = 0.05), and 10% as the allowable sampling error (d). Therefore, the sample size for each area should be 38 participants, at least [n = 1.962(1 - 0.11)0.11/0.12 = 37.6].
Two towns each in the pastoral Naqu county and the agricultural Chanang county were selected as the investigation sites for the dietary survey. The selected towns met the following requirements: the populations should be relatively concentrated (more than 2,000 habitants per town), have low mobility, a similar economic level, and stable living habits. One hundred households from each area were randomly sampled, according to the local household registration, and one adult aged 18–55 years was selected from each family to participate in the dietary interview. In total, there were 200 participants, with an approximately equal number of men and women.
Food frequency questionnaires (FFQs) and the 24 h history recall were adopted to assess the dietary habits of the residents over the current and the previous year [13–15], with emphasis on meat and dairy consumption. Based on the dietary habits of Tibetan people, questions were asked about the intake of meat (yak beef, dried yak beef, cattle beef, dried cattle beef, mutton, pork, and chicken), dairy (cow milk, sheep milk, butter, and yogurt), cereals (barley, wheat, and rice), potatoes, vegetables (Chinese cabbage, green pepper, tomato, cauliflower, eggplant, lettuce, radish, mushroom, pumpkin, carrot, and cucumber), and eggs. The response options were defined as follows: “never/rarely,” “1–5 times per six months,” “1–3 times per month,” “1 time per week,” “2–4 times per week,” “5–6 times per week,” “1 time per day,” and “2+ times per day.” Subsequently, the 24 h history recall (24 h recall) was conducted for three consecutive days to collect the dietary data. The seasonal food intake, such as lamb in the winter and autumn, was calculated according to the annual consumption. The daily food intake was estimated using a food map developed by Nanjing Medical University, China [16].
The survey group consisted of eight well-trained surveyors and one experienced epidemiologist. The unified FFQ and 24 h recall questionnaires were used to conduct the household surveys, which were carried out one by one in agricultural areas first, and then in the pastoral areas. The investigators participated together in the first five household surveys for each area, and then conducted separate investigations.
Dried beef, milk, mutton, and pork were sampled and stored in an ice box, and then transported to the laboratory to measure the iodine and thyroid hormone levels.
Method For Measuring The Thyroxine Levels In Milk
The milk samples were centrifuged at 3,000 rpm (r = 16 cm) for 10 min, and the intermediate layer was used to measure the thyroxine (T4) content using the chemiluminescence method. The analysis was performed using a Siemens Adivia Centaur system and the corresponding kits. The recovery, intra- and inter-assay variation coefficients of this method were 89.2–91.6%, 3.96%, and 5.83% (n = 6), respectively [17].
Method For Measuring The Thyroxine Levels In Meat
First, the thyroxine in beef jerky was determined using the method recommended by the American Thyroid Association (ATA), in which thyroxine was extracted using methanol: chloroform (1:2) and its level was determined using radioimmunoassay (RIA) [18]. However, the results exhibited poor repeatability. The intra-assay variation coefficient ranged from 9.3–25% among the six random samples. It is possible that the protein denaturation of beef jerky makes the protein-bound thyroxine difficult to extract completely. Therefore, the method of measuring the organic iodine content in dried beef was subsequently adopted to determine the levels of thyroxine. The iodine level in dried beef primarily comes from the blood and comprises both organic and inorganic iodine, and 90% of organic iodine is thyroid hormone [19–22]. Therefore, the total iodine and inorganic iodine levels were measured in dried beef, and their differential values were considered as the levels of organic iodine. There are two types of thyroid hormones, T3 and T4, with the latter being more prevalent. Therefore, the T4 levels are used to indicate the levels of thyroid hormone in dried beef and can be calculated based on the organic iodine (Io) content, according to the following formula (1):
T4 (µg/kg) = Io/ (4 × 127) × 777 × 90% (1)
Where:
4: one T4 molecule contains 4 iodine atoms;
127: iodine relative atomic weight;
777: T4 relative molecular weight;
90%: 90% of organic iodine is thyroid hormone in the tissues.
To extract the inorganic iodine from dried beef, a 0.4 g sample was placed in a 5 mL centrifuge tube, to which 3 mL of deionized water and two stainless steel or ceramic grinding beads with a diameter of 2 mm were added. Next, a pipe cover was installed, and the apparatus was placed into a vertical vibrating ball grinder and ground for 120 s at a frequency of 65 Hz. The centrifuge tube and its contents were then centrifuged for 5 min at 4,000 rpm. The supernatant was removed, the process was repeated three times, and the supernatants were pooled at the end. Deionized water was added to the supernatant to reach a final volume of 10 mL. Next, 1.0 mL of 20% (w/w) trichloroacetic acid solution was added to the supernatants for precipitation of the protein, and the samples were placed into a constant-temperature water bath at 37°C for 30 min, followed by centrifugation at 3,500 rpm for 10 min. The thyroid hormone combined with protein and the dissociative thyroid hormone (heavier than water and insoluble in water) settled at the bottom of the tube, separate from the inorganic iodine. Then, 4.0 mL of the supernatant was carefully extracted and filtered with a 5-µm syringe aqueous-phase filter membrane to measure the inorganic iodine content.
The extracted inorganic iodine was measured using ICP-MS [23]. The recovery rate was used to evaluate the effects of the extraction of inorganic iodine from the samples. A total of 1 mL of an iodine standard solution was added to 0.4 g of dried beef paste to reach an iodine content of 50 µg/L in the supernatants in the end. The recovery was calculated using the following formula:
Recovery % = (inorganic iodine after standard iodine was added – inorganic iodine in the sample)/(standard iodine added) × 100%
The recovery and intra-assay variation coefficients (cv%) of the method for extracting and measuring the inorganic iodine level in dried beef are shown in Table 3. The average recovery was 93.8 ± 2.3% with a range of 89.7–96.8%, and the intra-assay variation coefficients ranged from 2.4–2.9%.
Table 3
|
Population
|
Area
(km2)
|
Altitude
(m)
|
Annual mean temperature
(℃)
|
Annual mean precipitation (mm)
|
Naqu
|
90,000
|
16,195
|
4,450
|
-2.2
|
400
|
Chanang
|
83,000
|
2,163
|
3,620
|
8.2
|
420
|
The recovery of the method for measuring the inorganic iodine level in dried beef |
The total iodine levels in dried beef was measured using AsIII–Ce4+ catalytic spectrophotometry and dry ash sampling, and the detection limit of this method was 6 µg/kg for a 0.5 g sample [24]. The reference material 8418 (wheat gluten, NIST of USA) with iodine content of 60±13 µg/kg was used to control the quality of the test. The ICP-MS method is not suitable for the determination of total iodine in dried beef because of the high inorganic salt content of samples after ashing.
Data Input And Statistical Analysis
The collected data were sorted and summarized. Excel software was used to establish a database and the statistical analyses were performed using SPSS20.0. The D test (R. B. D’ Agostino) was used as a normality test to determine the type of distribution of the quantitative data. For skewed distribution data, the median values were adopted to describe the centralized trend and indicate the mean levels. The interquartile range was used to determine the degree of dispersion. The Wilcoxon signed-rank test was adopted to determine the significance of the differences between two groups of samples, and a value of p < 0.05 was accepted as statistically significant. For the normal distribution data, the mean values and standard deviations were adopted to describe the mean levels and the degree of dispersion. The statistical t-test was adopted for significance assessment, and a value of p < 0.05 was accepted as statistically significant.