Research On The Evaluation And Intervention Techniques Of Nutritional Status Of Biotin Deficiency Rats

BackgroundBiotin has long been recognized for its role as a coenzyme for five carboxylases which take part in the process of neoglycogenesis, amino acid decomposition, fatty acid synthesis and energy metabolism in humans and mammals. Biotin also plays a role in affecting chromatin structure and mediating gene regulation of histone biotinylation in the downstream events of the histone modification. Biotin deficiency has adverse effects on insulin secretion that lead to diabetes and immunological disease and so forth. The intestine of humans and other mammals can synthesize biotin and thus biotin deficiency is rare. The incidence of marginal biotin deficiency and suboptimal levels have been reported with increased frequency in women during pregnancy and in those on long term therapy with anti-convulsant agents and antibiotic recently. In view of the severe outcomes followed, biotin deficiency make a great challenge to the research on estimated average requirement of biotin and evaluation of biotin status. Research about the estimated average requirement of biotin, no matter human trials or animal experiments have not been reported. Therefore, this study try to explore the estimated average requirement of biotin using animal experiments with referring to the standard method of EAR defined in the Chinese Dietary Reference Intakes(2013). In recent years, the researches of indicators for biotin status evaluation have increased, however, inconformity do exist among researchers. With regard to the representativeness, stability and feasibility of the indicators, we finally select 3-hydroxyisovaleryl-carnitine as the "golden standard" for biotin status. Therefore we initiated this study with the following objectives:(1) establishing an UPLC-MS/MS method for determination of 3HIA-carnitine, linear regression relationship between 3HIA-carnitine and serum biotin was used for the validation of the accuracy of 3HIA-carnitine; (2) developing a suitable biotin deficiency rat model;(3) exploring the EAR of biotin by intragastic administration of diferent doses of biotin to biotin deficency rats model with consideration of changes in glucose and lipid indexes as well as the indicators for biotin status; (4) exploring the relationship between diet biotin and intestinal bacteria by intragastic administration of soluble dietary fiber to non-biotin and non-fiber deficiency rats model. Also, intensive study for the effects and its functional mechanism of biotin on glucose and lipid metabolism were omni-directinally studied from the characteristics of blood biochemistry, histomorphology and wide-ranged genome.MethodThis study discussed the nutritional status of biotin from four parts: 1 To establish an UPLC-MS/MS method for determination of 3HIA-carnitine Based on the correlative references,a robust and sensitive method for quantitative analysis of 3HIA-carnitine in blood was developed using ultra performance liquid chromatography tandem mass spectrometry with stable isotope stamp technique.The standard chromatography peaks and optimal resolution for 3HIA-carnitine and its isotope-labeled internal standard were compared under three kinds of mobile phases and two flow velocities. The separation effects of four kinds of solid phase extration columns (WCX、PLS、WAX、MCX)as well as the different concentrations solutions for leach and elution were evaluated and optimized. The methodology validation was used to prove the accuracy,reliable and sensitive of the method. The linear correlation between 3HIA-carnitine and serum biotin determined using the microorganism assay were used to validate the adaptablity of 3HIA-carnitine for assessing the biotin status.2 To establish the biotin deficiency rat model and explore the EAR of biotin2.1 The establishment of biotin deficiency rat model70 healthy SD rats weighting(100±10) grams were randomly assigned to normal group (control) and non-biotin group(NB).The normal group were given the normal feed for maintenance and the NB group were given the non-biotin feed with casein replaced with equal raw egg white. Body weight and diet intake were weekly recorded and changes in hair and feces were monitored as well. At the 0th,8th week and 10th week, blood was drawn from angular arteries of rats to detect the serum biotin and plasma 3HIA-carnitine.10 rats were executed at 10th week to observe the changes in pathomorphism and gene detection.2.2 Study on the dose-effect relationship of biotinThe rats were reallocated into normal group(control), non-biotin control group(NB), low-dose biotin group(NBL), medium-dose biotin group(NBM) and high-dose biotin group(NBH). The normal group were given the normal feed for maintenance, NB group given the non-biotin feed with casein replaced with equal the other groups were given the non-biotin feed with casein replaced with equal cooked egg white. Then the rats in NBL, NBM and NBH group were given 2μg/d、4μg/d、10μg/d biotin through administration of gavage respectively. The rats in normal group and non-biotin group were given the same amount of distilled water.Ended the experiment after 8 weeks(20th of the total time) administration of gavage. The rats were executed for the blood and organs. The serum were separated to detect the glucose, insulin, blood lipid contents; the liver, kidney, spleen and pancreas were weighted and fixed in the 4% formaldehyde solutions for pathomorphism observation. Another piece of liver and muscle samples were saved in the RNA preserving fluid at 4 centigrade for the rat wide-range genome expression profile chip analysis. The contents in caecum were obtained under sterile conditions and saved at -20 centigrade for the 16S rDNA gene sequencing of intestinal bacteria.3 The effects of soluble dietary fiber on biotin status50 healthy SD rats were induced to biotin deficiency with intervention of non-biotin and non-fiber feed for 12 weeks. At 10th week,10 rats were killed to do the pathomorphism observation and wide-range genome expression profile chip analysis. Then allocating the rest rats into non-biotin and non-fiber group(NBD), biotin intervention group(NBDB), resistance dextrin intervention group(NBDF) and biotin+resistance dextrin intervention group(NBDBF).Another 10 normal rats and 10 non-biotin rats model were selected to complete the research plan. The normal group were given the normal feed for maintenance and the NB group were given the non-biotin feed with casein replaced with equal raw egg white, the other groups given the non-biotin as well as non-fiber feed with casein replaced with equal raw egg white.Then the rats in NBDB, NBDF and NBDBF group were given 4μg/d biotin, lg/d resistance dextrin and 4μg/d biotin+lg/d resistance dextrin through gavage administration respectively. The rats in other groups were given the same amount of distilled water. After intervention for 8 weeks, the operations were the same as that described in part 2.4 Observation on fetal growth of fertilized female of biotin deficiency (preliminary experiment)This study was a preliminary experiment.10 healthy female rats and 6 healthy and sextual maturity male rats were put into metabolism cages by one to one for mating. After the female rats pregnant then randomly allocated the female rat into normal group (control), biotin deficiency control group (MC). The control group was given normal feed, the MC given model a feed.4 biotin deficient females rats were mated according to the method above, then forming the biotin deficiency group(Model).At the 20th day of pregnant executed the rats according to the standard SOP operational criterion for reproductive and developmental toxicities.Results1 To establish an UPLC-MS/MS method for determination of 3HIA-carnitine 25 μl 200μg/L isotope-labeled internal standard of 3HIA-carnitine and 100μl plasma were diluted with 1% formic acid water solution to 2ml. The compound was purified and extracted using MCX solid-phase extraction column(activated using 3ml methyl alcohol and 3ml deionized water).The leaching, elution and redissolve processes were completed by using 2ml 20% methyl alcohol water solution,3ml 5% ammonia water-methyl alcohol solution and 1ml 5% methyl alcohol-water solution. The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (2.1 mm×50mm,1.7μm), with a mobile phase composed of 0.1% formic acid in water-methanol with gradient elution:0-2min:5%～10%B; 2.0～6.0min: 10%～100%B; 6.0～7.0min:100%～5% B. Analyte quantification was performed in the positive electrospray ionization mode and multiple monitoring. The quantification and qualification were performed using the internal standard of 3-hydroxyisovaleryl carnitine. The correlation coefficient was 0.9995 within the linear range of 0.1-20.0μg/L in the plasma. Moreover, in three standard levels, rate of recovery ranged from 99.60% to 107.00%.The RSD of six parallel determination for the 3HIA-carnitine was 4.80%. The detection and quantification limits for were 0.04μg/L and 0.1 μg/L respectively.The linear relationship between serum biotin and plasma 3HIA-carnitine was negative, the correlation equation was:Y=139.67-0.10976*x; the correlation coefficient was 0.79 being statistically significant (P=0.0001).2 To establish the biotin deficiency rat model and explore the EAR of biotin2.1 The establishment of biotin deficiency rat modelIn the non-biotin group rats hair began to be rough and lose, activities reduced, weight gain and diet intake decreased in 6th week. The fur conditions became severe after feeding model feed for 8 weeks. The serum biotin detected in 2nd day of NB group was lower than the control group(P<0.05) while the plasma 3HIA-carnitine increased slightly(P>0.05). At 8th week, serum biotin significantly declined and plasma 3HIA-carntine increased markedly in the non-biotin group(P<0.05). After different doses of biotin intervention for 8 weeks, the rats diet intakes and weight gain increased as well as the hair and behavior been the same as the normal group. Compared with the non-biotin group, the contents of serum biotin and plasma 3HIA-carnitine recovered to normal level of control group. Also, there has been dose-reaction relationship between the different dose level of biotin for intervention.2.2 Study on the dose-effect relationship of biotinGlucose and insulin contents in the non-biotin group were lower than that in the control group(P<0.05); the changes of LDL in non-biotin increased (P<0.05). The blood glucose rose remarkably (P<0.05).The indicators representing the function of liver and kidney such as increased AST and BUN or decreased ALT remained abnormal despite the intervention of biotin(P<0.05). The results showed that low-dose biotin(2μg/d) was high enough to maintain the normal glucose level, considering the changes of biotin status indicators, we concluded that 2μg/d was the EAR of biotin for rat. Also the injure caused by biotin deficiency was irreversible.The rats wide-range genome expression profile analysis showed that there has been great differences in the pathways of fatty acid degradation、PPAR signaling pathway、fatty acid metabolism、arachidonic acid metabolism and biosynthesis of unsaturated fatty acids between control and biotin deficiency rats.3 The effects of soluble dietary fiber on biotin statusThe symptoms and hair removal on lower jaw and limbs of rats in NBD group was more severer than NB group, the diet intake and weight gain decreased as well. Compared with the NB group, there was no statistical difference in the serum biotin of NBD group (P>0.05) while plasma 3HIA-carnitine increased significantly (P<0.05). There has been no statistic significance between the glucose and lipid indexes between NB and NBD groups.With the intervention of resistance dextrin for 8 weeks, the hair lost phenomenon of rats in NBDF group became more severe with decrease of diet intake levels and weight gain. However, the rats in NBDB and NBDBF groups constantly grow new hairs at 4th week and the outlook became the same as rats control group at 6th week. When compared with the NBD group, the serum biotin continued to decline and plasma 3HIA-canitine increase in the NBDF group.The glucose was slightly higher and insulin slightly lower than that in NBD group(P>0.05). However, the biotin and 3HIA-carnitine in NBDB and NBDBF groups showed significantly change in the contents to recover to the normal level as the control group. At the same time, the interaction between biotin and resistance dextrin was analyzed, the results showed to be negative.The results showed that fiber deficiency might aggravate the symptoms of deficient; the effects of dietary fiber on intestinal biotin was unapparent after intervention of resistance dextrin.4 Observation on fetal growth of fertilized female of biotin deficiency (preliminary experiment)The ratio of pessaries for the female rats in three groups were 100%, but the rate of conception were 100% for the normal group and biotin deficiency control group while 50% for biotin deficiency group. Compared with the MC group, the weight gain of pregnancy rats in model was significantly lower (P<0.05). The ratio for absorptive fetus in MC group was 10.64% while 100% for the model group(P<0.05). The rate of fetal rats bone hypoplasia and spinal cord cavity in MC group were significantly higher than from those normal fetal (P<0.05).The serum prolactin(PRL), progesterone(P) and luteinizing hormone(LH) in MC group were lower than that in model rats, the estradiol(E2) higher(P<0.05). When compared with the control group, the PRL, E2 declined while the P, LH and follicle-stimulating hormone(FSH) levels in MC group showed an obvious tendency to increase(P>0.05).Serum biotin was lowest and 3HIA-carnitine highest in the model pregnancy rats, significantly different from the control and MC group(P<0.05). While the plasma 3HIA-carnitine in MC group was slightly higher than that in the control group. The results shows that biotin deficiency occured before pregnant might caused absorptive fetus or even sterile for the maternal; biotin deficiency happened during pregnancy might increase the fetal malformations rates.Conclusion1. This study established a sensitive and precise method to determine the plasma 3HIA-carnitine using the UPLC-MS/MS technology. The results were stability by using isotope-labeled 3HIA-carnitine and qualified and quantified by using mass spectrum technology. At the same time, linear correlation analysis was done between 3HIA-carnitine and biotin assayed by microorganism assay. The results showed 3HIA-carnitine could precisely reflect the biotin status.2. Through feeding the rats model feed for 10 weeks, compositing the serum biotin, plasma 3HIA-carnitine and glucose as well as the lipid indexes, all the outcomes proved the successful induction of biotin deficiency rats model.3.After intervention of non-biotin feed for 2 days and 8 weeks, the decline range of serum biotin of female rats and increase range of 3HIA-carntine was smaller than that of male rats. The results showed there might be a certain protective mechanism for female rats to avoid the risk brought by biotin deficiency.4.The rats wide-range genome expression profile analysis showed that biotin can significantly affect the synthesis and metabolism of fatty acid genes expression. This study will validate the results through pathways of fatty acid degradation, PPAR signaling pathway, fatty acid metabolism, arachidonic acid metabolism and biosynthesis of unsaturated fatty acids.5. Referring to the standard method of EAR defined in the Chinese Dietary Reference Intakes(2013), combining the results from animal experiments,we initially deduced the EAR of biotin for humans was 20μg/d.6. Remove the fiber from the non-biotin feed could aggravate the conditions of biotin deficiency. Administration of soluble dietary fiber independently might have no function on improvement of biotin status. The functions of resistance dextrin on intestinal was not seen in this study. Whether other kinds of dietary fiber has the similar affects need more exploration.7.The tentative experiment showed that biotin deficiency might lead to fetal absorption or even infertility.