荧光高效液相色谱法测定三种失眠模型大鼠脑组织氨基酸类神经递质的含量

目的测定睡眠剥夺大鼠脑组织氨基酸类神经递质的含量。方法复制药物诱导失眠动物模型、平台水环境诱导失眠动物模型、刺激诱导失眠动物模型,以Agilent 1100荧光检测器高效液相系统为检测工具,Agilent ZORBAX SB-Aq(250 mm×4.6 mm,5μm)为色谱柱,柱温25℃,激发波长λex=357 nm,发射波长λem=455 nm,甲醇-50 mmoL/L醋酸钠缓冲液(pH=6.5)为流动相,采取梯度洗脱,测定正常组及模型组大鼠脑组织中谷氨酸(Glu)、甘氨酸(Gly)、γ-氨基丁酸(γ-GABA)、牛磺酸(Tau)的含量。结果谷氨酸、甘氨酸、γ-氨基丁酸、牛磺酸分别在10.06～0.0503、10.13～0.0506、10.05～0.0502、10.03～0.0501μg/mL范围内,其浓度与峰面积呈良好的线性关系(r分别为0.99995、0.99995、0.99985、0.99990)。测得药物诱导失眠大鼠脑组织中Glu、Gly、Tau、γ-GABA的含量为(0.2042±0.0145)、(0.0086±0.0005)、(0.0919±0.0024)、(0.0421±0.0011)μg;平台水环境诱导失眠大鼠脑组织中Glu、Gly、Tau、γ-GABA的含量为(0.2144±0.0159)、(0.0085±0.0004)、(0.0966±0.0035)、(0.0433±0.0012)μg;刺激诱导失眠大鼠脑组织中Glu、Gly、Tau、γ-GABA的含量为(0.1818±0.0043)、(0.0084±0.0005)、(0.0824±0.0033)、(0.0414±0.0018)μg;正常大鼠脑组织中Glu、Gly、Tau、γ-GABA的含量为(0.1744±0.0038)、(0.0085±0.0004)、(0.0791±0.0022)、(0.0406±0.0012)μg。结论本实验建立的方法能满足同时测定大鼠脑组织中谷氨酸、甘氨酸、γ-氨基丁酸、牛磺酸的含量测定的需要,Glu、Tau、γ-GABA与失眠可能存在一定的量效关系,三种失眠动物模型均能较好的反映出脑内氨基酸类神经递质的变化。

Measurement of amino acid neurotransmitters in brain tissues of three insomnia rat models

Objective To determine the contents of amino acid neurotransmitters in the brain tissues of insomnia rat models. Methods Thirty-two healthy male Sprague-Dawley rats were randomly divided into normal control group and three groups of insomnia rat models induced by intra-cerebral injection of p-chlorophenylalanine （PCPA） , platform in water environment, and restraint stimulation, respectively, 8 rats in each group. The contents of glutamate （ Glu）, glycine （Gly） , γ-aminobutyric （γ-GABA） and taurine （Tau） in brain tissues of all the rats were measured by a HPLC system, using a chromatographic column Agilent ZORBAX SB-Aq （250 mm × 4.6 ram, 5 μm）, temperature of 25% , excitation wavelength 357 nm, emission wavelength 455 nm, and methanol-sodium acetate buffer （50 mmol/L, pH 6. 5） as mobile phase and gradient elution. Results Good linear standard curves of Glu, Gly, γ-GABA and Tau were obtained in the range of 10. 06 to 0. 0503, 10. 13 to 0. 0506, 10.05 to 0. 0502, and 10.03 to 0. 0501 μg/mL, with a correlation coefficient of O. 99995, 0. 99995, 0. 99985 and O. 99990, respectively. Contents of Glu, Gly, γ-GABA and Tau in the PCPA- induced insomnie group were 0. 2042 ±0.0145, 0. 0086 ± 0. 0005, 0. 0919 ±0. 0024, and 0. 0421 ± 0. 0011μg, respectively. Contents of Glu, Gly, γ-GABA and Tau in the platform in water environment-induced insomnie group were 0. 2144 ±0159, 0. 0085±0. 0004, 0. 0966 ±0. 0035, and 0. 0433 ±0. 0012 μg, respectively. Contents of Glu, Gly, γ-GABA and Tau in the restraint stimulation-induced insomnic group were 0. 1818±0. 0043, 0. 0084 ± 0. 0005, 0. 0824 ± 0. 0033, and 0.0414 ±0. 0018 μg, respectively. While the contents of Glu, Gly, γ-GABA and Tau in the control group were 0. 1744±0.0038, 0.0085 ±0.0004, 0.0791 ±0.0022, and 0.0406±0.0012 μg, respectively. Conclusions This method can satisfy the demands of simultaneous determination of Glu, Gly, Tau and γ-GABA concentrations in rat brain tis- sues. There may be certain amount-effect relationship between the Glu, Tau, γ-GABA contents and insomnia. All the three types of insomnia animal models can well reflect the changes of amino acid neurotransmitter contents in the brain tissues.