Determination of cholesterol in rat hippocampus by

Determination of cholesterol in rat hippocampus by high performance liquid chromatography

key words: high performance liquid chromatography L; cholesterol; Rat sea 1 must deal with horses in time; Ultraviolet detector

Abstract: cholesterol is not only crucial in the occurrence and development of cardiovascular diseases such as atherosclerosis, but also plays a key role in the development and functional activities of the central nervous system [1] A relatively simple high performance liquid chromatography (HPLC) method was used to determine the cholesterol content in rat hippocampus at different developmental stages

1 the technical requirements of materials for products include raw material requirements, sensory requirements, physical and chemical indicators and additive requirements. Materials and methods

1.1 materials

the experimental animal center of Hubei Academy of Medical Sciences provides 10 healthy male Wistar suckling rats (within 24 hours of birth), young rats (28-30 days, body weight 40-60 g) and adult rats (90-100 days, body weight 200-250 g) Acetonitrile, isopropanol, absolute ethanol, etc. are chromatographic pure reagents, and standard cholesterol (HPLC grade) is the product of sigma company of the United States Varian Prostar 210 high performance liquid chromatograph (manufactured by American Varian company), equipped with Prostar 210 high pressure pump, Varian Prostar 500 column temperature box and manual sampler

1.2 methods

1.2.1 preparation of tissue samples: the rats were decapitated rapidly, the hippocampal was weighed, and 10 ml/g was added with absolute ethanol to grind it into a homogenate on the ice table. It was centrifuged twice at 20600 g and 4 ℃ for 20 minutes each time, and the supernatant was diluted with 0.45 μ M microporous filter membrane, take the filtrate and pack it separately, and store it at -70 ℃ for testing

1.2.2 prepare standard solution and determine cholesterol standard cholesterol stock solution (1 g/l): accurately weigh 66.18 mg of cholesterol and dissolve it in 25 ml of absolute ethanol Standard cholesterol working solution: take the standard cholesterol stock solution and dilute it with absolute ethanol to make its concentration be 0.1323, 0.26472, 0.6618, 1.3236, 2.6472 g/l respectively. Agilent reverse phase chromatographic column (Zorbax XdB C18 4.6*250 mm 5 μ m. Agilent), column temperature 38 ℃, acetonitrile isopropanol (4:1, v/v) as the elution flow, relative to the standard stock solution and cholesterol in the sample for separation and elution Flow rate: 0.8 ml/min; Detection wavelength: 208 nm; Injection volume: 20 μ 50. Varian Prostar 325 UV detector and LC workstation V6.2 chromatographic data processing system of American Varian company detect and process chromatographic data to obtain cholesterol chromatogram Calculation result: x=10 × zero point nine nine six five × A × B ÷ cx: content of cholesterol in the sample (mg/g) A: Peak area of cholesterol in the sample 10: Sample dilution multiple (ml/g) B: Content of standard cholesterol (g/l) C: Peak area of standard cholesterol 0.9965: purity of cholesterol standard

statistical processing: the data are expressed in X ± s, and the comparison between variables is tested by F

2 results

2.1 standard working curve under the selected chromatographic conditions, the retention time of cholesterol is 13~15 min. repeat the loading (3 times) with standard cholesterol working solution with cholesterol concentrations of 0.1323, 0.26472, 0.6618, 1.3236 and 2.6472 g/l respectively, and the loading amount is 20 μ 50. Taking the peak area as the ordinate y and the cholesterol concentration as the abscissa x (g/l), the linear regression equation is: y=837148.3+x, the correlation coefficient r=0.99993, P 0.01 The minimum detection limit is 10 mg/l.

2.2 recovery. Accurately weigh 0.1 g (accurate to 0.0002 g) of the sample with a known content (12.8 mg/g), and accurately add 1 ml of cholesterol standard solution with a concentration of 1.3236 g/L After preparing tissue samples according to the above method, add anhydrous ethanol to make five parallel samples to determine the cholesterol content The average recovery is 99.94%

2.3 precision take the cholesterol standard solution with a concentration of 1.3236 g/l and inject it for 5 consecutive times. The measurement results show that the coefficient of variation is 1.2%

2.4 cholesterol content in samples cholesterol chromatograms of hippocampal samples from suckling, young and adult rats Cholesterol was eluted at the same retention time, and its contents were 2.92 ± 0.03,11.20 ± 1.41,12.91 ± 1.25 (mg/g) respectively. After F test and comparison between two groups, the difference was extremely significant (P 0.01)

3 discussion

the lowest detection limit of cholesterol obtained by UV detector is 10 mg/l, and the recovery and precision are high Therefore, this method is sensitive, accurate and reliable, and is of great value to clinical and scientific research

mauch et al. [1] determined that glia factor is a lipoprotein formed by the complex of cholesterol and apolipoprotein E There are few reports on the effects of cholesterol on the development and maturation of rat hippocampus [2,3] We speculate that during the development of rats, the content of cholesterol in hippocampus increases gradually, and cholesterol may play an important role in the development and maturation of rat hippocampus [4]

[References]

[1] mauch DH, negler K, Schumaker s, et al. CNS synaptoge "at the same time, ness promoted by glialderived cholesterol [J] Science, 2001; 294: .

[2] Shen Junxian, Jin Weilin, bow Morphological observation of rat hippocampal neurons cultured in low density in vitro [J] Journal of the Fourth Military Medical University, 2003; 24(6)： .

[3] Lei Gesheng, Wan Yehong, Wang Yuying, et al The electrophysiological characteristics of hippocampal CA1 neurons in rats were studied by infrared visible brain patch clamp technique [J] Journal of the Fourth Military Medical University, 2004; 25(13):.

[4] Hayashi h, campeno have developed static tension and compression, static change, high-frequency tension and compression fatigue, low T Rb, Vance De, et al. glial lipoproteins stimulate axogrowth of central nervous system neurons in completed cultures [J] J Biol Chem, 2004; 14(279):.