Iodine intake in healthy Japanese aged from 6 to 70 years residing in the same district

Abstract

Iodine is an essential component of thyroid hormones and a dietary micronutrient for humans, and adequate iodine intake is necessary to maintain thyroid function. A population’s iodine intake and nutritional status are assessed based on urinary iodine excretion. There are few studies on iodine nutritional status for all age groups residing in the same area in Japan. Between 2010 and 2017, a total of 769 healthy subjects aged 6.4–73 years in three sites in Yokohama City, were enrolled in the survey. The urinary iodine concentration (UIC), iodine to creatinine (Cr) ratio (UI/Cr) and estimated 24-h urinary iodine excretion (UIE) in single spot urine samples were measured, and habitual dietary iodine intake was assessed by food frequency questionnaires. The estimated 24-h UIE was calculated using individual predicted 24-h creatinine excretion by the validated equations developed for healthy Japanese children and adults which vary by age, gender and anthropometry. The median UIC for all participants was 219 μg/L, suggesting adequate iodine intake for this population. There was an increasing trend in median UI/Cr and estimated 24-h UIE by age. A significant correlation between UIC and UI/Cr (r = 0.6378), UIC and estimated 24-h UIE (r = 0.6804), and UI/Cr and estimated 24-h UIE (r = 0.5756) were observed. These estimates can be feasible, convenient and alternative methods to 24-h urine collection in order to assess iodine status in some populations such as ethnically or racially homogeneous and well-nourished people. Additional studies are required to validate these findings.

IODINE is an essential component of thyroid hormones and also a dietary micronutrient for humans. Deficient as well as excessive iodine intakes induce thyroid dysfunction [1-3]. Iodine in foods is almost completely absorbed from the intestine and then partially taken up by the thyroid gland, and approximately 90% of dietary iodine is rapidly excreted into the urine [4]. Therefore, urinary iodine excretion is a sensitive indicator of recent iodine intake and also a good predictor of dietary iodine consumption at the population level. Iodine-deficiency disorders (IDD) is a wide spectrum of adverse consequences and the World Health Organization (WHO), the United Nations Children’s Fund (UNICEF) and the Iodine Global Network (IGN), a successor of International Council for the Control of Iodine Deficiency Disorders (ICCIDD) have recommended the criteria to classify a population’s iodine intake and nutritional status based on the median urinary iodine concentration (UIC) values in school-aged children (SAC) [2, 5]. Although UIC is a currently well-validated biomarker for populations, there has been an ongoing debate on the validity of various measurements and methods for assessing iodine status [6-11].

Japan has been regarded as an iodine replete or even iodine excessive country without iodine fortification. We have reported the iodine status of schoolchildren [12, 13] in Hokkaido and Tokyo, and of pregnant and lactating women [14, 15] in Chiba. However, national data on iodine nutrition is still missing at present, and there are few epidemiological studies on iodine nutritional status for all age groups residing in the same area in Japan.

Materials and Methods

Study population

An epidemiological survey was conducted in three facilities located in the same district (Hodogaya-ku, Yokohama City), i.e., the Hodogaya elementary school, the Nishiya middle school and the Savai medical checkup clinic where the healthy individuals regularly have a general health examination, in 2017, 2012 and 2010, respectively. Schoolchildren and adults were asked to provide a single spot urine sample and to complete a food frequency questionnaire (FFQ). There were 660 and 303 students in the Hodogaya elementary school and the Nishiya middle school, respectively. During the three-month period 1,085 apparently healthy adults visited the Savai medical checkup clinic, and subjects without past and present history of thyroid disease were requested tor provide residual samples of their serum and urine for their health checkup and to fill out a questionnaire. To measure serum thyroid stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), thyroid autoantibodies or urinary iodine and creatinine (Cr) concentrations, serum and urine samples were immediately stored at –30°C until analysis. The urine samples were not acidified.

Daily dietary iodine intake assessed by FFQ

The average dietary iodine intake (DII) was estimated in middle school children and adult subjects including the elementary school children’s caregivers using a semi-quantitative FFQ developed and validated by the authors [15] and given in μg iodine per day. It contained 50 different iodine-rich food items classified in 10 categories with a specified serving size. For each food item, participants indicated their average frequency of consumption in the previous month by checking 1 of 10 frequency categories ranging from “almost never” to “3 times/day”. The selected frequency category for each food item was converted to a daily intake. The DII from each food was calculated on the basis of frequency of consumption and iodine content of the specific food in the Japanese Food Composition Table 2010 [16] or other database when insufficient information was available.

Analytical methods

The serum TSH and FT4 concentrations were measured by electrochemiluminescence immunoassay using ECLusys TSH and FT₄ (Roche Diagnostics K.K., Tokyo, Japan). The reference range for TSH and FT4 defined by the manufacturer were 0.5–5.0 μIU/mL and 0.9–1.7 ng/dL, respectively. The antithyroid peroxidase antibodies (TPOAb), antithyroglobulin antibodies (TgAb) and the thyroid stimulating hormone receptor antibody (TRAb) were measured in serum by RIA using TPOAb Cosmic II (500), TgAb Cosmic II and TRAb Cosmic Ⅲ (RSR Limited, Cardiff, UK), respectively. The TPOAb, TgAb and TRAb values above the manufacturer’s reference limit (less than 16 IU/mL, 28 IU/mL and 2 IU/L, respectively) were considered positive.

The iodine concentration in the urine samples obtained from children in the Nishiya middle school and the Savai Clinic subjects was measured at Hitachi Chemical Co., Ltd., Kawasaki, Japan using the modified microplate method based on the ammonium persulfate digestion on microplate (APDM) method with spectrophotometric detection of the Sandell-Kolthoff reaction [17]. The analytical sensitivity of this assay was 1.39 μg/dL and the intra-assay and inter-assay coefficients of variation were 4.8–5.9% and less than 10%, respectively. The UIC in the samples from the Hodogaya elementary school was determined using inductive coupled mass spectrometry (ICP-MS) at Kotobiken Medical Laboratories, Inc., Tokyo, Japan. The sensitivity of this assay was 3 μg/L and the intra-assay and inter-assay coefficients of variation were 1.5–6.9% and 1.8–6.3%, respectively. ICP-MS is generally considered as the gold standard for urinary iodine analysis, and the UIC values measured by the APDM method were highly correlative with those by ICP-MS (r = 0.9753). The urinary creatinine concentration was measured by colorimetric enzymatic assay. All the urine samples were assayed in duplicate.

To ensure the quality of urinary iodine analyses both iodine laboratories, i.e. Hitachi Chemical Co., Ltd. and Kotobiken Medical Laboratories, Inc. have participated in the Ensuring the Quality of Urinary Iodine Procedures (EQUIP) program in the Centers for Disease Control and Prevention (CDC), USA [18].

UIC was expressed as a concentration in μg iodine per 1,000 mL of urine (μg/L) or relative to creatinine excretion (UI/Cr, μg/gCr). Since the 24-h UIE is regarded as representative of 24-h iodine intake, the estimated 24-h UIE was calculated based on measurement of iodine and Cr concentrations in spot urine samples and using anthropometry-based 24-h Cr reference values for healthy, well-nourished children and adults as suggested previously [19, 20]. The equation used for Japanese is as follows: For adults, estimated Cr excretion (mg/24 h) = height (cm) × 7.39 + weight (kg) × 15.12 + age (year) × –12.63 for males; height (cm) × 5.09 + weight (kg) × 8.58 + age (year) × –4.72 for females [21]. For children aged between 2 and 18 years, estimated Cr excretion (mg/24 h) = height (cm) × 0.94 + weight (kg) × 35.01 – 221 for boys; height (cm) × 2.74 + weight (kg) × 19.57 – 330 for girls [22]. The body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. For assessment of iodine nutrition in populations four methods are recommended, i.e., UIC, the goiter rate, serum TSH and Tg. UIC is a sensitive indicator of recent iodine intake [7]. Adequate iodine intake corresponds to the median UIC values in the range 100–299 μg/L, and UIC ≥300 μg/L indicates excessive iodine intake although the term ‘excessive’ means in excess of the amount required to prevent and control IDD [2]. For conversion to S.I. units: 1μg/dL = 78.799 nmol/L.

Statistics

Since the UIC, UI/Cr, UIE and DII were distributed asymmetrically and skewed [23], their logarithmically transformed values were used to normalize the distribution. The results were presented as mean, SD, median, and interquartile range (IQR, 25^th and 75^th percentiles). Differences between paired data or groups were examined using one-way ANOVA with Tukey’s multiple comparison test, the Kruskal-Wallis test and Dunn’s multiple comparison test. Differences between two unmatched groups for normally and non-normally distributed data were tested using the unpaired t test and Mann-Whitney test, respectively. Simple linear regression analysis was used to test for correlations among UIC, UI/Cr, estimated 24-h UIE and DII. A p-value less than 0.05 was considered significant. Data processing and statistical analysis were performed using GraphPad Prism 8.0 from GraphPad Software Inc. San Diego, CA, U.S.A.

Ethical approval

This study was conducted in accordance with the Declaration of Helsinki. The study protocol was reviewed and approved by the local ethical committee of Savai Clinic on May 1, 2010 (No. 2010-001), and by the ethics review board of Japanese Red Cross Hokkaido College of Nursing on January 15 2014 (No. 170). The clinical trial/study was not registered. Informed written consent was obtained from the schoolchildren’s caregivers and all the checkup clinic participants at their initial visit.

Results

Overall, 769 participants aged 6.4–73 years were included in this survey and 608 subjects provided their urine samples. The number of subjects in the school survey was 129 of 660 students in the Hodogaya elementary school and 139 of 303 students in the Nishiya middle school. In the Savai medical checkup clinic 340 of 1,085 adults agreed to participate in the survey (Table 1). In total, DII by FFQ was obtained from 568 subjects, i.e., 91 students from the middle school, 161 caregivers from the elementary school and 316 adults from the checkup clinic. All participants were without past and present history of thyroid disorders.

Table 1 Characteristics of participants

		Elementary school	Middle school	Check-up Clinic
Number of Subjects	Total	129	139	340
	Male/female	67/62	74/65	193/147
Age, years	Total	9.9 ± 1.7	13.5 ± 0.9	47.6 ± 12.2
	Range	6.4–12.8	12.3–15.3	16.0–73.0
	Male/female	9.7 ± 1.7/10.1 ± 1.7	13.6 ± 0.9/13.4 ± 0.9	48.2 ± 12.6/47.3 ± 11.6
Height, cm	Total	136.4 ± 12.1	156.2 ± 7.3	164.5 ± 8.3
	Male/female	136.3 ± 11.9/136.5 ± 12.5	158.4 ± 7.5/153.6 ± 6.1*	169.7 ± 5.7/157.5 ± 5.8*
Weight, kg	Total	32.4 ± 8.9	45.2 ± 8.0	61.5 ± 1.2
	Male/female	33.2 ± 9.4/31.6 ± 8.3	46.5 ± 7.6/43.5 ± 8.3*	68.7 ± 10.2/54.4 ± 8.4*
BMI, kg/cm2	Total	17.1 ± 2.7	18.5 ± 2.6	23.0 ± 3.5
	Male/female	17.5 ± 3.1/16.6 ± 2.1	18.5 ± 2.1/18.4 ± 3.2	23.9 ± 3.3/21.9 ± 3.4*

Values are mean ± SD except for number of subjects. *: Fisher’s exact test

Characteristics of the participants

In the elementary school there were no differences of age, height, weight or BMI between males and females while in the middle school the males had higher (p = 0.001) and heavier (p = 0.0325) values than the females. In the adult subjects more males were included and their height, body weight and BMI values were higher than those of the females (p < 0.0001) (Table 1).

There were no adult subjects with overt thyroid dysfunction. Based on serum TSH and FT4 values, the prevalence of subclinical hypothyroidism was 3.4% (9/268 subjects) and serum TgAb and/or TPOAb were positive in 2 of these 9 subjects. Subclinical hyperthyroidism was 3.0% (8/268 subjects) and serum TRAb was negative in all 8 subjects.

UIC, UI/Cr, estimated 24-h UIE and DII values in three sites

The median UIC values ranged from 213 to 242 μg/L and there were no significant differences among the three age groups. The median UI/Cr value in the elementary school was higher than that in the middle school (184 vs. 128 μg/gCr). The median estimated 24-h UIE value in the checkup clinic was higher than those in the elementary and middle schools (218.2 vs. 159.0 and 161.4 μg/day). The median DII in the checkup clinic was higher than that in the middle school (388.8 vs. 265.9 μg/day) (Table 2).

Table 2 UIC, UI/Cr, estimated 24-h UIE and DII in three sites

		Elementary School	Middle School	Check-up Clinic
	n	121	117	334
UIC, μg/L	Total	216 (131, 428)	242 (134, 378)	213 (125, 422)
	Range	37–11,753	37–4,330	25–16,800
	Male/female	216/221	256/229	212/222
UI/Cr, μg/gCr	Total	1841) (113, 438)	1282) (82, 294)	164 (96, 378)
	Range	43–5,950	43–4,790	17–19,853
	Male/female	195/183	149/110	160/182
Estimated 24-h UIE, μg/day	Total	159.03) (91.0, 323.7)	161.44) (97.4, 386.6)	218.25) (118.5, 509.6)
	Range	21.4–9,336	43.5–5,542	25.0–16,687
	Male/female	191.0/120.0*	226.2/106.9**	265.7/179.9***
DII, μg/day	Total	Not Available	265.96) (137.9, 590.0)	388.87) (175.1, 778.8)
	Range		8.6–3,086	0–8,305
	Male/female		280.4/248.8	371.9/430.7

Values are median (IQR) and range. UIC, urinary iodine concentration; UI/Cr, urinary iodine/creatinine; UIE, urinary iodine excretion; DII, dietary iodine intake

1) vs. 2) : p = 0.0043; 3) vs. 5) : p = 0.0067; 4) vs. 5): p = 0.0485 (ANOVA, Kruskal-Wallis test), 6) vs. 7) : p = 0.0131; *: p = 0.0049, **: p = 0.0001, ***: p = 0.0007 (unpaired t test, Mann-Whitney test)

Differences of UIC, UI/Cr, estimated 24-h UIE and DII values by gender

There were no significant differences of median UIC, UI/Cr and DII values between males and females. The median estimated 24-h UIE of males was higher than that of females in all age groups (Table 2).

Age-related changes of UIC, UI/Cr, estimated 24-h UIE, Cr concentration and DII

The median UIC varied between 186 and 259 μg/L and showed no significant change with age, while the median UI/Cr increased from 96 to 243 μg/gCr (Fig. 1). The mean urinary Cr concentration decreased with age from 520 to 115 mg/dL. The estimated 24-h UIE value in subjects more than 60 y was higher than that in children less than 12 y (284.2 vs. 148.0 or 154.0 μg/day). There was no significant difference in median DII except between the subjects more than 60 y and those who were 13–18 y (393.8 vs. 228 μg/day) (Fig. 2) (Supplementary Table 1).

Fig. 1

Age related changes of UIC and UI/Cr

Median, IQR and range. Whiskers denote ranges. UIC, urinary iodine concentration; UI/Cr, urinary iodine/creatinine

Fig. 2

Age related changes of estimated 24-h UIE and DII

The DII of subjects 12 years or older was assessed. Median, IQR and range. Whiskers denote ranges. UIE, urinary iodine excretion; DII, dietary iodine intake

Correlation among UIC, UI/Cr, estimated 24-h UIE and DII

The UIC value was significantly correlated with UI/Cr (r = 0.6378) and estimated 24-h UIE (r = 0.6804). The UI/Cr value was also significantly correlated with estimated 24-h UIE (r = 0.5756). The DII value was not correlated with UIC (r = 0.0544), UI/Cr (r = 0.0633) or estimated 24-h UIE (r = 0.0233) (Fig. 3).

Fig. 3

Correlation between UIC, UI/Cr, estimated 24-h UIE and DII

UIC, urinary iodine concentration; UI/Cr, urinary iodine/creatinine; UIE, urinary iodine excretion; DII, dietary iodine intake. There was a positive correlation between UIC, UI/Cr and estimated 24-h UIE. The regression line is in red.

Discussion

To assess the iodine status of healthy Japanese from 6 to 70 years old except pregnant and lactating women residing in the same district we conducted an epidemiological survey using single spot urine samples, as recommended by the WHO [5]. The median UIC for all participants combined was 219 μg/L suggesting adequate iodine intake. However, the median UIC in SAC was 216 μg/L and lower than the value of 282 μg/L in our 2002 survey in Tokyo [12] and the value of 265 μg/L in the subnational survey of Japan by us [24]. According to IGN’s Global Scorecard 2021 on iodine nutrition in the general population, there were 135 countries with adequate iodine intake (UIC:100–299 μg/L) and the median UIC was between 200 and 299 μg/L in 46 countries. Japan was ranked 10th among these 46 countries [25].

There are several reports on change in urinary iodine excretion with age [26-28]. Using 24-h urine samples Campanozzi et al. measured 24-h UIE in 1,270 healthy Italian children and adolescents between 6 and 18 y, and there was a significant increasing trend in median 24-h UIE with age [26]. Katagiri et al. measured UIE by 24-h urine collection in 713 adults between 20 and 69 y residing in 20 areas throughout Japan. The 24-h UIE and UI/Cr in the older group (50–69 y) were significantly higher than in the younger group (20–29 y) [27]. Konno et al. measured UIC and UI/Cr in spot urine samples from 4,138 adults between 20 and 80’s residing in Sapporo, Japan. The UI/Cr values of both men and women increased with age while the UIC value remained unchanged. At the same time, the urinary Cr concentration decreased in both males and females suggesting that the change of UI/Cr was possibly due to a concomitant decrease in the Cr concentration by an age-related reduction in muscle mass [28]. In our subjects UI/Cr tended to increase with age and higher estimated 24-h UIE adjusted by predicted Cr excretion was observed in elderly adults as well as in male subjects, while there were no significant differences of median UIC, UI/Cr and DII between males and females. This gender-related difference of UIE was partially inconsistent with the previous reports [26, 29]; however, in a large epidemiological study of 3,350 university students (18–22 years, M/F ratio, 1: 0.73) residing in Kobe, Japan, there was no difference in the median UIC values in spot urine samples by gender [30]. The gender difference of iodine excretion is still in controversy. In the present study, the DII estimated by FFQ was higher than the UIC, UI/Cr and estimated UIE and not well correlated with them presumably due to the difference of evaluation methods since FFQs assess past habitual iodine intake while urinary iodine excretion reflects current iodine intake.

The most precise method of estimating iodine intake is measurements of urinary iodine in 24-h collections [8, 31], and indeed there is a highly significant correlation between 24-h UIE and daily iodine intake [32]. Therefore, 24-h UIE is often considered as the reference standard for a population’s iodine intake and used for validating other methods [8]. However, in field studies it is burdensome to collect 24-h urine samples and currently no cutoffs of 24-h UIE values exist to interpret a population’s iodine status [20].

The median UIC in spot urine specimens from a representative sample of the target group is generally used to assess a population’s iodine status. Since urinary iodine excretion can be influenced by the subject’s inter-and intra-individual variation in their hydration status especially during growth and iodine intake, there is a considerable variability of UIC or 24-h UIE [33] in spot urine samples [34, 35]. Moreover, extensive diurnal [36-38] and seasonal fluctuations of UIC [36, 37, 39-41] or UI/Cr [34, 42] have been reported with conflicting results, while a circadian rhythm in iodine excretion was not observed in some studies [43-46].

UI/Cr also has been alternatively applied to epidemiological surveys of iodine intake; however, the amount of daily Cr excretion varies by age, gender, racial/ethnic background, skeletal muscle mass and protein intake [32, 47]. In addition, the urinary Cr concentration may fluctuate according to the storage condition of urine samples (changes in temperature and pH) [32]. The use of UI/Cr to assess iodine intake in a population remains controversial [32, 48-50].

Another alternative method for 24-h urine collections is to calculate the daily iodine excretions using spot urine samples adjusted by predicted 24-h Cr excretion. Urinary Cr measurements have been used to calculate average 24-h excretion rates of certain analytes determined in spot urine samples. For this purpose appropriate Cr reference values are necessary [51]. This is because Cr excretion declines with age, and reference ranges for certain analytes expressed in terms of an analytes/creatinine ratio are strongly age-dependent [32]. By applying this methodological approach, estimated 24-h UIE can be calculated with prediction equations using data from the sample’s age, gender, ethnicity and anthropometric measures [20, 52] or using published estimates from a different population [19, 23, 43, 53-55]. Estimated 24-h UIE determined by using the former method is a good estimate of actual 24-h UIE [20].

In the present study we estimated 24-h UIE (UI/Cr × predicted 24-h Cr excretion) in spot urine samples using individual predicted 24-h Cr excretion by the validated equations developed for healthy Japanese children and adults which vary by age, gender and anthropometry [21, 22]. There was a highly significant correlation between UIC and UI/Cr, UIC and estimated 24-h UIE, and UI/Cr and estimated 24-h UIE. Although it has been generally recognized that UIC, UI/Cr, and estimated 24-h UIE from spot urine samples could not be used interchangeably, there are several reports on a significant relationship between Cr-adjusted 24-h iodine excretion from single spot urine samples and observed 24-h UIE by 24-h urine collection; however, the results are conflicting. In summary, the following can be reported: 1. The UIC in spot urine samples was well correlated with 24-h UIC [56] or observed 24-h UIE [57] by 24-h urine collection. 2. The UI/Cr in spot urine samples was well correlated with observed UIC [27, 52] or 24-h UIE [57] by 24-h urine collection, 3. The estimated 24-h UIE from spot urine samples was well correlated with observed UIC [53] or 24-h UIE [19, 20, 23, 28, 43] by 24-h urine collection. Although we have not collected 24-h urine sample to assess population’s iodine status, the estimated 24-h UIE from spot urine samples may reflect a reasonable iodine status in an ethnically homogeneous and well-nourished population.

Study limitations and strengths

This study has several limitations. Children less than 6 years were not included in this study group. We used single spot urine samples and the estimated 24-h UIE adjusted by individual predicted 24-h Cr secretion can be influenced by fluctuations in Cr secretion. The major strength is that all the participants were healthy Japanese with a wide age range living in the same district, and more than 600 subjects were included since a study size of 500 individuals is needed to determine the iodine level of a population with a precision of 5% [58, 59].

Conclusion

Iodine intake was adequate in this study population aged between 6 and 70 years. The UIC, UI/Cr and estimated 24-h UIE values from a single spot urine sample were highly correlated with each other suggesting that these estimates can be alternative, feasible and convenient methods to assess iodine status in some populations such as ethnically or racially homogeneous and well-nourished people. Additional studies in other populations with a different range of iodine intake are required to validate these findings.

Acknowledgments

The authors wish to thank the participating children, their caregivers, school teachers in Yokohama Municipal Hodogaya Elementary School and Nishiya High School as well as the Yokohama City Board of Education, who cooperated in the survey. We are grateful for the assistance of Ms. Sheryn Mason in the preparation of the manuscript.

YF, YI, YS and MI had the initial idea and coordinated the project over several years. YF wrote the manuscript. YF and NT did the calculations and the statistical evaluations. All the contributors helped in planning and conducting the experiments, evaluating the results and critically reading the manuscript.

Disclosure

No competing financial interests exist.

Supplementary Table 1 Age related changes in UIC, UI/Cr, estimated 24-h UIE, urinary creatinine concentration and DII

	Age	6–	9–	12–	13–18	20–39	40–49	50–59	60–
	n	45	53	76	66	91	98	72	71
UIC, μg/L	Median (IQR)	223 (126, 415)	186 (121, 371)	230 (125, 452)	259 (151, 429)	186 (118, 296)	218 (125, 452)	210 (114, 432)	241 (138, 554)
	Range	37–3,736	48–3,910	51–11,790	37–4,330	45–5,740	25–4,000	30–12,100	35–16,800
UI/Cr, μg/gCr	Median (IQR)	961) (63, 149)	1112) (81, 154)	1433) (92, 224)	1224) (81, 320)	126.15) (79, 234)	142.56) (85, 392)	1807) (111, 465)	2438) (132, 766)
	Range	23–217.1	35–218	46–1,610	43–4,790	23.9–4,102	17–2,740	33–4,716	40.9–19,870
Estimated 24-h UIE, μg/day	Median (IQR)	154.0 (80.9, 277.2)	148.09) (96.5, 300.9)	196.8 (97.3, 412.5)	161.4 (104.3, 351.4)	195.0 (111.7, 311.4)	183.5 (105.6, 448.8)	242.0 (128.7, 572.3)	284.210) (155.8, 764)
	Range	30.1–2,271	21.4–2,510	43.5–9,336	46.8–5,542	25.0–3,876	29.1–4,177	51.3–6,381	35.8–16,687
Urinary Cr concentration, mg/dL	Mean	520.211)	371.412)	287.713)	189.514)	169.415)	163.816)	123.617)	115.418)
	SD	724.2	644.3	696.8	76.96	93.56	94.91	75.85	56.56
	SEM	108	88.5	79.92	9.473	9.807	9.539	8.878	6.713
DII, μg/day	n	Not available		39	58	132	192	83	64
	Median (IQR)			298.4 (170.3, 789.4)	228.419) (108, 500.9)	305 (164.1, 554.5)	410.1 (215.5, 769)	446.2 (214.6, 724.1)	393.820) (239.3, 1,171)
	Range			23.2–3,086	8.6–3,042	15.8–9,769	0–9,691	13.7–5,881	44.6–7,512

1) vs. 6): p = 0.009; 1) vs.7), and 2) vs. 7) and 8) : p < 0.0001; 1) vs. 8) : p = 0.0006; 3) vs. 8): p = 0.0061; 4) vs. 8): p = 0.0025; 5) vs. 8): p = 0.0001; 6) vs. 8): p = 0.0181, 9) vs. 10): p = 0.0162, 11) vs. 12): p = 0.0003; 11) vs. 13): p = 0.03; 11) vs. 14): p = 0.0003; 11) vs. 15), 16), 17) and 18): p < 0.0001; 12) vs. 16): p = 0.0341; 12) vs. 17): p = 0.092; 12) vs. 18): p = 0.0065, 19) vs. 20): p = 0.0163

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