| Iodine is an essential trace element in human, plays an important role in body growth and metabolism. Iodine deficiency is a public health problem for populations in many countries. Practices show that universal salt iodization (USI) is an effective solution to this problem. Nowadays, both potassium iodate and potassium iodide are the major salt iodization additives. However, potassium iodate has strong oxidizing property and iodate should be reduced to iodide before effectively used by the body. In recent years, some scholars query the safety of iodized salt with potassium iodate, since that potassium iodate might cause oxidative damage of organism. However, until now the safety of potassium iodate is not completely documented. This study develop a new method to analyze iodate and iodide in the tissue homogenate using an high performance liquid chromatography-inductively coupled plasma mass to spectrometry (HPLC-ICP-MS) assay, and apply the method to demonstrate the iodate reduction process in tissues and plasma of rats in vitro for the first time. The study also detect total antioxidant capacity and reduced nicotinamide adenine dinucleotide phosphate (NADPH) to explore the effects of different doses of potassium iodate on the anti-oxidative state of the samples. This study may lay the scientific foundation of revealing potassium iodate reduction process in vivo and provide reference for the future safety evaluation of potassium iodate.ObjectivesTo develop a new HPLC-ICP-MS method for analyzing iodate and iodide in biological tissue homogenates; to study iodate reduction process in liver, kidney, thyroid gland and plasma of rats using the new established method; to explore the effects of potassium iodate on total antioxidant capacity and NADPH concentration of tissue homogenates. This can provide scientific reference for the study of iodate reduction process in vivo.MethodsTwelve healthy SPF Wistar rats in 12 weeks old, sex in half, were fed with normal feedstuff. After rats were sacrificed, liver, kidney, thyroid and blood samples were obtained, and plasma were separated.1. Adjust HPLC-ICP-MS instrument parameters, column mobile phase composition, flow rate, etc. to optimize its working condition for a better species separation. Explore the detail methods in sample pre-treatment, such as the choices for the purification column, the medium solution to keep iodate stable, ratios of tissue homogenate vs. iodate solution and so on. Then, explore the effects of different potassium iodate concentration and incubation time on tissue homogenate. Based on the earlier results, establish the experimental system, and use potassium iodate and iodide mix standard solution to evaluate the accuracy and precision.2. Add the same dose of potassium iodate in the homogenate of liver, kidney, thyroid and plasma respectively; incubate for different time (0,7.5,15,30,60 min). Then determine the iodate reduction situations using HPLC-ICP-MS.3. Measure the total antioxidant capacity of tissue homogenates and plasma by ABTS method. Then add different doses of potassium iodate (0,0.25,0.5,1,2 ?g) to observe the changes of total antioxidant capacity.4. Add different doses of potassium iodate (0,0.25,0.5,1,2 ?g) in tissue homogenates, measure NADPH concentration by fluorimetric NADPH assay (red fluorescence).Results1. The HPLC-ICP-MS method was provided with favorable linear relation (correlation coefficients were greater than 0.9990), good separation of iodine species, high sensitivity, and high recovery rate. The average recovery rate of iodate were 97.0%-105.3%, iodide were 95.5%-102.8%. The relative standard deviation were<3.0%, showed a good precision The detection limit concentration of iodate was 0.17 ?g/L, and the iodide ion was 0.12 ?g/L.2. The difference between different incubate time group about degree of iodate reduction in all the sample time had statistically significant (P<0.05). Compared with non-incubation group, the degree of iodate reduction increased significantly. Tissue samples showed the same trend, which was, rapid increased first and then leveled off During incubation, the degree of iodate reduction in liver tissue increased from 15% to 99%, kidney from 16% to 84%, thyroid from 4% to 31%. However, iodate reduction degree in plasma gradually increased from zero to 13% with the incubation time prolonged. The reduction degree had differences between males and females. The liver tissue and plasma of male group had larger degree of reduction than the female group, there were statistically significant differences at each time point (P<0.05). Kidney male group were statistically significant different from female group only at 60 min incubation group (P<0.05). Between the thyroid, no gender differences in the degree of reduction were statistically significant (P>0.05), but the proportion of exogenous peaks in female group were higher than male group at each time point (P<0.05).3. The total antioxidant capacity baselines of different tissue were statistically significant (P<0.05). And gender differences were statistically significant (P<0.05).The total antioxidant capacity of female were liver> kidney> thyroid tissue, while male were thyroid> liver> kidney tissue. After reacting with different doses of potassium iodate, total antioxidant capacity in all tissue type decreased, the overall differences were statistically significant (P< 0.05). The consumptions of potassium iodate were different, female group of liver and kidney decreased more than male group, whereas in thyroid gland the male group declined more. The results for plasma were the females> males (P<0.05), but potassium iodate only decreased the results of male group (P<0.05), had no effects on female group (P>0.05).4. NADPH concentrations between tissues were statistically significant (P<0.05). And gender differences were statistically significant in all the tissue (P<0.05). Female were liver> kidney?thyroid tissue, while male were liver> kidney> thyroid. With dose of potassium iodate increased, only liver homogenates NADPH decreased statistically significantly (P< 0.05).Conclusions1. This study has established a new HPLC-ICP-MS method for iodate and iodide separation in the rat tissue homogenate. And successfully apply it in the iodine speciation analysis of tissue and plasma of rats.2. The liver, kidney, thyroid and blood plasma of rats can be reduced iodate to iodide in vitro. Reducing capacity differences between tissue and plasma lead to different reaction trends when incubated with potassium iodate. Tissue reducing capacity were liver> kidney> thyroid. There are some differences in gender, too. For liver, kidney, plasma, male group have a stronger reducing power, while in thyroid the female is higher.3. Potassium iodate can decrease total antioxidant capacity of rats tissue, the downward trend are liver> kidney> thyroid. For liver, kidney, plasma, the female group have more total antioxidants than male. However, the overall reduction activity show the male group> female group; on the contrary, thyroid female group have higher activity. Different doses of potassium iodate have no effect on the total antioxidant capacity of plasma in female rats.4. Potassium iodate can decrease the NADPH concentration of rat liver significantly, while have no significant changes in kidney and thyroid. The process of iodate reduction in tissue may also depend on other reducing substances in addition to NADPH. |
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