| Betaine, as a methyl donor, is distributed widely in animals, plants, and microorganisms. It participates in the methionine cycle in organism. Betaine can obviously improve the symptoms due to lacking of methyl group in organism, and can be used to treat hyperhomocysteine syndrome, protect liver and kidney, heart and blood vessels. It also has lots of other beneficial pharmacological effects such as inhibition of unceasingly growing tumor cells and protection of cells against hyperosmotic damage.Study on the pharmacokinetics of betaine can illustrate the rule of the betaine's dynamic transformation in the body, provide important theoretical parameters for the study of pharmacodynamics and theoretical foundations for the development of new drugs and health foods with betaine.Part one Analytical methods of betaine hydrochloride in biological samples by HPLC-ELSDObjective: According to the structural characteristics of betaine, establish a set of HPLC-ELSD analytical methods for serum, fecal and tissues in rats.Method: (1)The HPLC consisted of Hypersil NH2 analytical column(250×4.6mm, 5μm) as solid phase, Acetonitrile - water - triethylamine (85:15:0.08) as mobile phase, flow rate of 1.0ml/min, using the United States-based Alltech ELSD 2000 evaporative light scattering detector, drift tube temperature of 86.0℃, carrier gas flow rate of 2.2L ? min-1. (2)Basic methods of biological samples treatment: taken 100μl samples, add 900μl of methanol for extraction and protein deposition, then filtered by 0.22μm microporous membrane, drying by distillation of 90℃water bath, solid residues dissolved with 150μl mobile phase, 20μl for injection.Results: There was a wide linear range in various biological samples (serum for 0.5052 ~19.8588μg), the r value of regression equation was 0.9977~0.9999, the limit of detection was 0.03μg and the limit of quantification was 0.11μg. The precision(RSD) of injection in three different concentrations (high, medium, low) were 1.74%,1.64% and 0.50%,the precision(RSD) of repeatabilit y for the analysis were 1.76%,3.31% and 6.32%,the accuracy were 98.57%, 104.97% and 83.20%,the absolute recovery were 93.98%, 91.40% and 80.59%,the within-day precision(RSD) were 5.69~7.87% and the between-day precision(RSD) were 3.77~6.40%.Conclusions: The measurement of betaine in biological samples was achieved by HPLC-ELSD method. The method was sensitive,specific and simple enough to determine the concentration of betaine and to obtain pharmacokinetic parameters.Part two The Pharmacokinetic study of betaine hydrochloride in ratsObjective: To study the pharmacokinetic characteristics in rats after ig. and iv. with two doses by the established method of quantitative analysis of betaine and obtain reliable pharmacokinetic parameters.Method: (1) Twenty Wistar rats were divided into 2 groups randomly, each group has 10 rats. One group ig. betaine hydrochloride 450mg·Kg-1 or 112.5 mg·Kg-1, about 0.5ml blood samples were collected from tail vein before administration and at 10min,30 min,1h,1.5h,2h,3h,4h,6h,8h,12h,18h,24h after administration, another group iv. betaine hydrochloride 450mg·Kg-1 or 112.5 mg·Kg-1, about 0.5ml blood samples were collected from tail vein before administration and at 5 min,15 min,30 min,45min,1h,1.5h,2h,3h,4h,6h,8h,12h,24h after administration. Operate by the pretreatment of serum samples, analysis and calculate the betaine content (Xa + X0), deduct endogenous betaine content X0 Correspondingly and record Xa. (2) The data was automatic handled with 3P97 pharmacokinetic software, calculating the main pharmacokinetic parameters of each rat with the best compartment model as the standard, using statistical moment method for calculating AUC and MRT values and calculating the absolute bioavailability.Results: The mean serum concentration-time curves of betaine hydrochloride after ig. with two doses were confirmed to open two-compartment model, however, the mean serum concentration-time curves of betaine hydrochloride after iv. with two doses were confirmed to open three -compartment model. The main pharmacokinetic parameters were:(1)Group ig.450mg·kg-1 : t1/2(ka)=1.14h,t1/2(ke)=4.34h,T(peak)=2.69h,MRT(0～t) =7.06h,CL/F(s)=0.371L·kg-1·h-1 , V/F(c)= 2.257L·kg-1 ;( 2 ) Group ig.112.5mg·kg-1 : t1/2(ka)=0.84h , t1/2(ke)=4.39h , T(peak)=1.80h , CL/F(s)=0.193L·kg-1·h-1 ,V/F(c)=1.229L·kg-1,MRT(0～t) =6.49h;(3)Group iv.450mg·kg-1 : t1/2 pi=0.18h,t1/2 alpha=1.30h,t1/2 beta=13.77h, Vc=0.198L·kg-1·h-1,CL(s)= 0.182L·kg-1·h-1,MRT(0～t) =3.10h;(4)Group iv.112.5mg·kg-1: t1/2 pi=0.25h,t1/2 alpha=1.03h,t1/2 beta=5.48h,Vc=0.326L·kg-1·h-1,CL(s)= 0.181L·kg-1·h-1,MRT(0～t) =3.65hConclusions: The result showed that BTH was absorbed quickly and the time reaching to the peak was about 2h, then eliminated slowly after ig. , the t1/2(ke) was about 4.3h and the MRT(0～t) 7h. The serum concentration of Bet declined quickly after iv. in rats, the t1/2 pi was about 0.2h and the t1/2 alpha 1.2h, it was distributed quickly and eliminated slowly, the t1/2 beta of high dosage group was higher than low dosage group significantly.Part three Study on distribution of betaine hydrochloride in rats'tissuesObjective: To study the distribution characteristics in rats before administration and after ig. betaine hydrochloride 450mg·Kg-1 by the established method of quantitative analysis of betaine.Method: (1) Determination of endogenous betaine in rats'tissues: seven Wistar rats were killed by cervical dislocation, all main tissues were taken out, Operate by the pretreatment of tissue samples, analysis and calculate the betaine content X0. (2) Ten Wistar rats were killed by cervical dislocation after ig. betaine hydrochloride 450mg·Kg-1 2h, all main tissues were taken out, Operate by the pretreatment of tissue samples, analysis and calculate the betaine content (Xa + X0).Results: Endogenous betaine can be detected in heart, lung, liver, spleen, kidney, testicle and intestine, there was a high content in liver, kidney and testicle. Betaine can not be detected in muscle and fat after ig. betaine hydrochloride 450mg·kg-1 2h and there had an unsharpness change in testicle, however, the content of betaine was heightened quickly in brain, heart, lung, spleen and intestine, it also congregated in liver and kidney.Conclusions: The endogenous betaine mainly concentrated in the liver, kidney and testicle, it congregated in liver, kidney and intestine after administration. Part four Study on excretion of betaine hydrochloride in ratsObjective: To illustrate the relationship of betaine excretion and metabolism, consummate the rule of the betaine in the body through the study of betaine excretion in urine and feces.Method: Ten Wistar rats were taken into the metabolic cage for collecting the urine and feces within 24 hours before administration and after ig. betaine hydrochloride 450mg·Kg-1, then operate by the pretreatment of excretion samples, analysis and calculate the betaine content.Results: The total content of betaine from urine excretion after administration within 24 hours was about 10 times of the content before administration, but the total elimination of the volume only to the dosage of 7.79%; There had an unsharpness change in the content of betaine from feces excretion before and after administration.Conclusions: The betaine elimination primarily through metabolism after administration, the ratio of elimination from urine excretion was very small. We found that betaine elimination hardly through feces excretion by comparing the content before and after administration.
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