Simultaneous quantitation of folic acid and 5-methyltetrahydrofolic acid in human plasma by HPLC–MS/MS and its application to a pharmacokinetic study

A sensitive method based on high-performance liquid chromatography-tandem mass spectrometry （LC- MS/MS） has been developed for the simultaneous determination of folic acid （FA） and its active meta- bolite, 5-methyltetrahydrofolic acid （5-M-THF）, in human plasma. The analytes were extracted from plasma with methanol solution containing 10 mg/mL of 2-mercaptoethanol and 0.025% （v/v） ammonium hydroxide. FA and 5-M-THF were more stable after the addition of 2-mercaptoethanol and ammonium hydroxide in the sample preparation procedures of this study than they were in the previously published methods. Chromatographic separation was performed on a Hedera ODS-2 column using a gradient elution system of acetonitrile and 1 mM ammonium acetate buffer solution containing 0.6% formic acid as mobile phase. LC-MS/MS was carried out with an ESI ion-source and operated in the multiple reaction monitoring （MRM） mode. The assay was linear over the concentration ranges of 0.249-19.9 ng/mL for FA, and 5.05-50.5 ng/mL for 5-M-THF. The developed LC-MS/MS method offers increased sensitivity for quantification of FA and 5-M-THF in human plasma and was applicable to a pharmacokinetic study of FA and 5-M-THF.

Effects of electroacupuncture on cochlear morphology and expression of aquaporins in rats with AVP-induced endolymphatic hydrops

Objective: The objective of this study was to investigate the effects of EA on EH and the regulation of AQP2 and AQP7 protein expression in rats.Methods: Twenty-four rats were allocated randomly to four groups of blank group, EH group,EH+tolvaptan group and EH + EA group(n = 6 per group). EH rat model was established by intraperitoneally injection of arginine vasopressin(AVP). EA was administered at acupoints "Baihui(百会 GV 20)"and "Tinggong(听宫 SI 19)". Rats in the EH + tolvaptan group and EH+ EA group were treated with tolvaptan and EA, respectively, after EH establishment. Hematoxylin-eosin staining was used to measure the cochlear hydrops degree, and then the ratio of scala media(SM) area to SM + scala vestibuli(SV) area(R value) was calculated. Immunohistochemical method was used to observe AQP2/AQP7 protein expression in the rat cochlear lateral wall after treatment.Results: ①There was no endolymphatic hydrops in the blank group. Reissner' s membrane was extended markedly and bulged into SV in cochleae of the EH group and endolymphatic hydrops was noted. Statistical analysis revealed that R value in the EH group showed a significant increase compared with that in the blank group(0.42 ± 0.02 vs. 0.31 ± 0.05, P=0.000). The distension of Reissner's membrane was less obvious in the EH + tolvaptan group and EH + EA group when compared with the EH group. R value in the EH + tolvaptan group and the EH + EA group was significantly less than that in EH group(0.32±0.04 vs. 0.42 ± 0.02, =0.001;0.35 ± 0.05 vs. 0.42 ± 0.02, P=0.012). The degree of the hydrops in the EH + EA group was not different from that in the EH + tolvaptan group(0.35 ± 0.05 vs. 0.32 ±0.04,P= 1.000). ②The AQP2 protein expression in the rat cochlear lateral wall of EH group was significantly increased when compared with the blank group(12.74 ± 5.18 vs. 5.92 ± 1.52, P = 0.014). The AQP2 protein expression in the rat cochlear lateral wall of EH + tolvaptan group and EH + EA group were all lower than that of the EH group(6.52 ± 2.73 vs. 12.74 ± 5.18. P = 0.029;6.95 ± 3.10 vs. 12.74 ± 5.18, P = 0.047).The AQP2 protein expression in the rat cochlear lateral wall of EH + EA group was not different from that in the EH + tolvaptan group(6.95 ± 3.10 vs. 6.52 ± 2.73, P= 1.000).③The AQP7 protein expression in the rat cochlear lateral wall of EH group was significantly increased when compared with the blank group(30.32 ± 6.39 vs 16.64 ± 3.21, P=0.000). The AQP7 protein expression in the rat cochlear lateral wall of EH + tolvaptan group and EH + EA group were all lower than that of the EH group(18.32 ± 2.45 vs.30.32 ± 6.39, P= 0.001;19.54 ± 4.61 vs. 30.32 ± 6.39, P= 0.003). The AQP7 protein expression in the rat cochlear lateral wall of EH + EA group was not different from that in the EH + tolvaptan group(19.54 ±4.61 vs. 18.32 ± 2.45, P= 1.000).Conclusions: These results indicate that repeated EA stimulation exerted the same effects as tolvaptan application on AQPs levels and subsequent aquaretic effects. And dehydrating effect of EA on the inner ear might be associated with its down-regulation of both AQP2 and AQP7 protein expression, thereby provide a potential molecular mechanism involved in the treatment of Meniere's disease by EA.