Environ Monit Assess (2016) 188:226 DOI 10.1007/s10661-016-5221-7

The concentration of iodine in horse serum and its relationship with thyroxin concentration by geological difference Mariko Mochizuki & Noriyuki Hayakawa & Fumiko Minowa & Akihiro Saito & Katsumi Ishioka & Fukiko Ueda & Kimihiro Okubo & Hiroyuki Tazaki

Received: 5 August 2015 / Accepted: 3 March 2016 # Springer International Publishing Switzerland 2016

Abstract In this study, iodine and thyroxin (T4) concentrations in the serum of 69 horses were investigated. Higher iodine concentrations were obtained from the horses housed in Chiba Prefecture. In contrast, T4 concentrations of horses at Shizuoka Prefecture were higher than those of horses at Chiba Prefecture. There was a significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations of horses at Saitama and Shizuoka prefectures. Although a significant correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefecture, the distribution area

of the data was separated from the data of horses housed in Saitama and Shizuoka prefectures. A higher iodine concentration in the environment is expected in the sampling area at Chiba Prefecture. Thus, it was suggested that the concentrations of iodine in the serum of horses are influenced by geological differences. It was thought that equine serum is a useful sample for monitoring.

M. Mochizuki (*) Department of Applied Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo 180-8602, Japan e-mail: [email protected]

K. Ishioka Department of Veterinary Nursing, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo 180-8602, Japan

N. Hayakawa Veterinary Medical Teaching Hospital, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo 180-8602, Japan

Keywords Geological difference . Iodine . Monitoring . Sea algae supplement

F. Ueda Laboratory of Veterinary Public Health, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo 180-8602, Japan

F. Minowa Minowa Horse Clinic, 4-4-5, Higashikoujiya, Oota, Tokyo 144-0033, Japan

K. Okubo Department of Otolaryngology, Graduate School of Medicine, Nippon Medical School, 1-1-5, Senda-Gi, Bunkyo, Tokyo 113-8603, Japan

A. Saito Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555, Japan

H. Tazaki Laboratory of Bimolecular Chemistry, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo 180-8602, Japan

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Introduction

Materials and methods

The understanding of environment status by wildlife monitoring is thought to be important in the public health field of veterinary science. Thus, to determine the state of the environment, the body burden of several wildlife elements has been reported (Mochizuki et al. 2008, 2012a, 2013). However, a lack of epidemiological information such as age makes the analysis of data obtained from wildlife difficult. In contrast, it was thought that epidemiological data of domestic animals are more complete than those of wildlife. Furthermore, while the living area of domestic animals is confined to a specific area, the complete delineation of the living area of wildlife is difficult. Thus, it was thought that studies using domestic animals can produce significant results in the field of monitoring using animals. Iodine is an essential element for humans and animals, and thyroid adenoma caused by iodine deficiency is observed in the interiors of continents, which are regions where there is little opportunity to absorb iodine from the environment. Thus, iodine deficiency is one of the three most prevalent nutritional deficiencies in animals and humans, along with iron and vitamin A deficiencies (Bhutta and Salam 2012), and it is thought to affect over 740 million people worldwide, approximately 13 % of the world’s population (WHO 2009). However, a lack of iodine is not problematic for most Japanese people because iodine is naturally obtained from marine products (Nagai 2012). Instead, the excess ingestion of iodine often becomes problematic in Japan. There are also areas where the level of environmental iodine is high. The Minami-Kanto gas field (a natural aqueous gas resource) located in Chiba Prefecture is such an area in Japan (Kaneko 2005). Recently, we obtained blood samples from horses living in the Minami-Kanto gas field region. In addition, a sea algae supplement was supplied to the horses at an equestrian club, although the living area environment of those horses was thought to contain higher iodine contents. Thus, because a higher body burden of iodine was thought to be expected in the animals living in this area and the horses that were supplied with the algae supplement were thought to be a model of iodine excess, we compared the data of the aforementioned horses with those of horses living in other areas.

Serum samples Blood was collected from 69 horses housed at five equestrian clubs in Japan. The sample collection area is shown in Fig. 1. The sampling period was between December 2011 and October 2013. Blood was not drawn immediately after grazing, exercising, feeding, or grooming. The horse feed consisted of hay, hay cube, and various pellets. Only the horses housed in an equestrian club (Chiba 1) were provided feed supplemented with seaweed. The diagnosis of enlargement of the thyroid gland in horses in Chiba 1 (female/gelding: n = 5/9; average age, 16.50 ± 1.43 years), with the exception of one horse (gelding, 25 years old), was confirmed by palpations performed by a veterinarian. The enlargement of the thyroid gland was not observed in the horses housed in Saitama Prefecture (n = 6/6; average age, 15.92 ± 1.84 years), Shizuoka Prefecture (n = 9/16; average age, 14.40 ± 1.42 years), Chiba 2 (n = 11/0, male; n = 1; age unknown), and Chiba 3 (n = 3/2; age unknown). Approximately 20 mL of blood from each horse was withdrawn using a syringe with an 18-gauge syringe needle (Nipro, Japan), which was then immediately transferred into vacuum blood collection tubes (Venoject® II, Terumo Corporation, Japan). The blood samples of Shizuoka in the vacuum blood collection tube were transported to the Shizuoka Institute of Science and Technology. Other samples were transported to the Nippon Veterinary and Animal Science University. On the same day, the serum was transferred to Eppendorf tubes after centrifugation (3000 rpm for 15 min). Eppendorf tubes of serum were placed in a deep freezer (Panasonic Healthcare Co., Ltd., Japan) at −30 °C just before analysis. The present study was conducted following the ethical policies of the Nippon Veterinary and Life Science University, Japan, for experimental animals. Although the same serum samples were analyzed in another study (Takahasi 2013), the data were not presented by each prefecture in the study by Takahashi. Analytical methods The serum T4 concentrations were determined by a chemiluminescent enzyme immunoassay using an IMMULITE 1000 system (Siemens Healthcare GmbH, Germany) in the veterinary teaching hospital of the Nippon Veterinary and Life Science University and the outside inspection institute (Monolis Inc., Japan). The

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Fig. 1 The sample collection area in the present study

Saitama (n=12) Shizuoka (n=25)

Chiba 3 (n=5)

Chiba 1 (n=15)

Chiba 2 (n=12)

concentration of serum iodine was determined using an inductively coupled plasma mass spectrometer (ICP-MS, Agilent Technologies) in the Japan Food Research Laboratories (Tokyo, Japan). A 0.1-mL aliquot of the serum sample was placed in a tube, and 1 % nitric acid (Kanto Kagaku, Tokyo, Japan) was added to reach a final volume of 10 mL. A semi-quantitative analysis was employed. The following operating condition of ICPMS was selected: radio frequency power, 1600 W; carrier gas flow rate, 0.7 L/min; plasma gas flow, 15 L/min; sampling depth, 8.0 mm; acquisition mode, spectrumpeak hopping; and integration time, 0.1 s/point. The mass-to-charge ratio of iodine was 127. The analytical accuracy of 20 elements, including iodine, was also investigated in our previous study (Takahasi 2013). The recovery rate of iodine was 92.5 ± 5.8 %.

Statistical analysis The data were analyzed using Lotus 2001 and Excel 2010 software. The concentrations of T4 and iodine in the serum are represented as the mean values ± SEM. The significance of differences in the concentrations of T4 and iodine was analyzed using the Dunett multiple comparison test (SPSS Statistic 19, IBM Japan, Tokyo, Japan). The statistical significance of a correlation (Spearman’s rank-correlation) was tested using SPSS Statistics 19 (IBM Japan, Tokyo, Japan) software. The equal probability ellipse was analyzed using JMP (SAS Institute Japan, Tokyo, Japan).

Results and discussion The normal concentration of iodine in human serum was reported to range from 50 to 100 μg/L (WHO 2009), whereas the mean iodine concentration in the present study was less than 41 μg/L. Although horses have been used in interesting studies such as those of selenium (Streeter et al. 2012), copper (Van Weeren et al. 2003), and zinc (Stark et al. 2001), there have been no reports on the iodine concentration in equine serum. In a study using cattle (Rey-Crespo et al. 2014), most results appeared to be similar to those the results for humans (WHO 2009). In our study using fattening cattle (Takahasi 2013), the mean iodine concentration (n = 38; 103 ± 6.5 μg/L, n = 78; 101 ± 2.8 μg/L) obtained using the same analytical method was similar to that in the above studies (WHO 2009; Rey-Crespo et al. 2014). On the other hand, a higher (>0.2 mg/L) serum iodine concentration was obtained in dairy cattle (Launer and Richte 2005). In our previous study using the same method as the present study, we similarly obtained a higher iodine level for dairy cattle (n = 27, 146 ± 1.1 μg/L) (Takahasi 2013). Thus, it was thought that the iodine concentration in equine serum was low, and this was not an error in the analysis. A high iodine concentration (7.26 mg/kg) (JRA 2004) was reported in protein supplement feeds such as fish meal. Fish meal is a significant source of animal protein for cattle in Japan (Sibui 2012). Furthermore, it was suggested that many disinfectants contain iodine as an ingredient and cause an increase in the iodine concentration in milk

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Iodine concentration (μg/L)

40

35 30

a

b

25

b

20

c

c

15 10 5

ND 0

Chiba1

Chiba2

Chiba3

Saitama Shizuoka

Fig. 2 The mean iodine concentration (μg/L) of horses in each equestrian club. Different letters indicate significant differences (c < b < a, P < 0.05)

＊＊ 2.5 T4 concentration (μg/dL)

(WHO 2009). The higher iodine concentration in the serum of the dairy cattle was thought to be due to the effects of feed and disinfectants. In contrast, riding horses are thought to be fed normally using feed with low iodine content. The iodine content was reported to be between 0.11 and 0.3 mg/kg in roughage and concentrated feed (JRA 2004). Thus, the iodine concentration in the serum of horses was thought to be lower than that in the serum of other animals such as humans and cattle. The mean iodine concentrations of three equestrian clubs at Chiba Prefecture were significant higher (P < 0.01) than those of equestrian clubs at Saitama and Shizuoka prefectures. Yanai et al. (2013) suggested that the mean iodine concentration in the soil of the Kanto area (17.8 mg/kg), including Chiba Prefecture, is higher than that in the soil of the Cyubu area (4.97 mg/kg), including Shizuoka Prefecture. The Minami-Kanto gas field is also located in Chiba Prefecture. Thus, the tendency of low iodine concentrations at Shizuoka Prefecture was thought to reflect the environmental situation. Furthermore, the highest mean iodine concentration (29.33 μg/L) was obtained from horses housed at an equestrian club, Chiba 1, and their mean iodine concentration was significantly higher (P < 0.05) than that of other clubs (Fig. 2). The horses at this equestrian club were provided feed including sea algae known to contain a high concentration of iodine, although the higher iodine concentration in soil was expected in this area. On the other hand, the mean T4 concentrations ranged from 0.19 to 2.11 μg/dL (Fig. 3). In the report of race horses, the mean T4 level and SDs were 2.15 ± 0.75 (yearlings, n = 39) and 2.71 ± 0.72

2 1.5 1

0.5 ND 0 Chiba1

Chiba2

Chiba3

Saitama Shizuoka

Fig. 3 The mean thyroxin concentration (μg/dL) of horses in each equestrian club. **P < 0.01

(2 year-old, n = 39) μg/dL, respectively (JRA 1995). The data by the Japan Racing Association (JRA 1995) were higher than those of our study. However, Malinowski et al. (1996) suggested that the T4 concentration in horse plasma decreases with age, declining from 233 ng/mL at birth to 49 ng/mL at 14 days. Malinowski et al. (1996) also reported that the T4 level in older age groups varies from 36 (6 months, n = 6) to 9 ng/mL (16– 22 years, n = 6). Thus, it was thought that the T4 concentrations of our study are within the normal range of T4 levels. The T4 concentration in the serum of horses in an equestrian club at Shizuoka Prefecture indicated a higher tendency. The significant difference was only obtained from Shizuoka Prefecture and Chiba 1 and 2. The difference between the results of horses in Chiba and those of horses in other areas became clearer when the relationship between iodine and T4 was investigated. There was a significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations in the serum of horses at Saitama and Shizuoka prefectures. A significant correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefecture. Although a similar correlation was observed for the serum of horses housed in the equestrian club, Chiba 1 (r = 0.919, P < 0.001), the distribution area of the horses housed in Chiba 1 was completely different, as shown in Fig. 4. This tendency was similar to the results of our previous reports (Mochizuki et al. 2008). In the investigation of cadmium (Cd) contamination in animals, there was a significant positive correlation between the Cd contents of the

Iodine concentration of serum (μg/L)

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45 40 35 30 25

20 15 10 5

0 ND

0 ND

1

2

3

4

5

T4 concentration of serum (μg/dL)

Fig. 4 The relationship between the thyroxin and iodine concentrations in horse serum. Filled squares: horses in Chiba 1, red triangle: horses in Chiba 2, blue squares: horses in Chiba 3, black triangle: horses in Saitama Prefecture, circle: horses in Shizuoka Prefecture. Black solid line: equal probability ellipse by horses housed in Saitama and Shizuoka prefectures, red solid line: 95 % equal probability ellipse by horses housed in equestrian club, Chiba 1

kidney and those of the liver of wildlife living in a nonpolluted area (n = 101) (Mochizuki et al. 2008; Ueda et al. 2009). Thus, this correlation was used as an index to understand the situation of the target animals. In fact, the data of Itai-itai disease patients and the animals administered Cd were located far from this index of Cd (Ueda et al. 2009; Mochizuki et al. 2012b). A similar tendency was also obtained in the comparison of the element distribution in the urine of healthy cats and that of cats with urolithiasis (Mochizuki et al. 2011). Thus, it was thought that the different distribution of data reflects the status of animals. At present, we do not possess the analytical skills or equipment to determine the iodine concentration in environmental samples such as the soil of the environments where the horses were fed. However, the Minami-Kanto gas field (a natural aqueous gas resource) is located at Chiba Prefecture where equestrian clubs in the present study are located. This gas field was formed by the accumulation of sea algae and organic matter and has a high concentration of iodine, reaching 100 ppm (Kaneko 2005). Thus, the horses in equestrian club, Chiba Prefecture, are situated in a region in which the level of environmental iodine is high. Although, the occurrence of health hazards has not been reported in animals, including humans, living in this area, the excess ingestion of food and supplements, including iodine, is thought to be the

destabilizing factor for thyroid function. In fact, the enlargements of the thyroid gland have been observed in the horses in Chiba 1 fed feed containing sea algae. In the present study, the data of horses living in the area where higher iodine contents in the environment are expected were collected and were compared with the data of horses living in other areas. Because we observed a difference between the horses in Chiba and horses living in other areas, it was thought that the equine serum is a useful sample for monitoring the environment.

Conclusion From the investigation of T4 and iodine concentration in serum obtained from horses in Japan, higher tendency of iodine concentrations were obtained from the horses housed in Chiba Prefecture. In contrast, T4 concentrations of horses at Shizuoka Prefecture indicated higher tendency than those of horses at Chiba Prefecture. In Chiba Prefecture, there are Minami-Kanto gas field where the level of iodine in environment is high. Thus, higher iodine concentration of horses housed in Chiba was thought to reflect the environmental situation. Further, since the horses housed at an equestrian club Chiba 1 were provided feed including sea alga including a high concentration of iodine, the mean iodine concentration (29.33 μg/L) obtained from horses housed at an equestrian club Chiba 1 was significantly higher (P < 0.05) than that of other clubs. The particularity of horses in Chiba 1 became clearer when the relationship between iodine and T4 was analyzed. A significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations of horses at Saitama and Shizuoka prefectures was obtained in the present study. Although a similar correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefecture, the distribution area of the data of Chiba Prefecture was separated from the data of horses housed in Saitama and Shizuoka prefectures. As mentioned above, the concentrations of iodine in the serum of horses are thought to be influenced by geological differences. Further, the supplement should be supplied in consideration of the geological differences.

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Acknowledgments The authors express their thanks to Mr. Takanori Ban at the Green Hill Riding Club, Ms. Tomoko Yoneya at the I’ll Horse Riding Club, Ms. Keiko Hasegawa at Minimini club, and Mr. Yasuhiro Nakajima, Ms. Kumi Sato, and Ms. Yuko Yokoi at the Palomino Pony Club for their help in sample collection. This study was supported by Grant-in Aid number 23580430 from the Ministry of Education, Science, Sports, Culture and Technology in Japan. Compliance with ethical standards The present study was conducted following the ethical policies of the Nippon Veterinary and Life Science University, Japan, for experimental animals.

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