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2.6. Biochemical analysis

2.6.1. Determination of plasma methionine, homocysteine,

cysteine and glutathione

Total Hcy, cysteine (Cys) and GSH concentrations were

measured by High Pressure Liquid Chromatography

(HPLC) technique with fluorimetric determination after derivation

of thiols with ABD-F according to Durand et al.

[22]. Total methionine concentration was measured with

electrochemical detection. Briefly, 200 l of plasma and 20

l of 2.5 mM-N-acetylcysteine (used as internal standard

for the determination of Hcy, Cys and GSH) were treated

with 20 l of 10% tri-n-butylphosphine (in dimethylformamide)

for 30 min at 4°C in order to release thiols from

plasma proteins and reduce them. Proteins were precipitated

with 200 l of 0.6 M-cold perchloric acid. After 10 min. of

centrifugation at 4000 g, the supernatant was strained with

0.2 M PTFE filter (Interchim, Montluc﹐on, France) and

immediately measured. Fluorescence detection of Hcy, Cys and GSH 20 l

of 1.55 M-sodium hydroxide, 250 l of 0.125 M-borate

buffer (pH 8) and 30 l of 4.6 mM-ABD-F (7-fluoro-2,1,3benzoxadiazole-

4-sulfonamide) solution (in borate buffer)

were added to 100 lof filtrated supernatant. After derivation

at 50°C for 20 min, the samples were rapidly cooled

and 40 l of 1 M-HCl were added.

Samples (20 l) were analyzed by HPLC (ESA 580

Kontron instruments 400, Strasbourg, France) equipped

with a fluorescence detector (Bio-Tek, SFM 25, Kontron)

set at ex . 385 nm and em . 515 nm. Eluent phase was

composed of a 0.1 M-KH2PO4 buffer (pH 3.25) containing

10% acetonitrile, and the rate was fixed at 1.2 mL/min.

Separation was carried out on a 250 . 4.6 mm, 5 m

diameter Nucleosil C18 analytical column (Macherel-Nagel)

maintained at 35°C. Hcy, Cys and GSH concentrations

were determined by the area quotient of N-acetylcysteine

and Hcy, Cys and GSH peaks respectively, after standard

calibration of thiols.

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  • 1 0 年前

    2.6. 生化分析

    2.6.1. 判定血漿甲硫氨酸,同胱胺酸,半胱胺酸與谷胱甘肽

    根據Durand等人的方法,以有ABD-F的硫醇基衍生後,用高壓液相色譜法(HPLC)與螢光測定總Hcy,半胱胺酸(Cys)與GSH濃度 [22]。總甲硫氨酸濃度以電化學偵測法測量。簡單說來,用20公升,10%三正丁[基]膦(在二甲基甲醯胺裡)三十分鐘,4C,來處理200公升血漿與20公升2.5m的M-N-acetylcysteine(用來當作判定Hcy,Cys與GSH的內部標準) ,以釋出並減少血漿蛋白中的硫醇基。 以200公升,0.6M之冷的過氯酸沉澱蛋白質。以4000g離心十分鐘,用0.2 M四氟乙烯過濾器(Interchim, Montlucon, 法國)過濾漂浮物,然後立刻測量。 螢光測定Hcy,Cys與GSH

    將20公升1.55M氫氧化鈉,250公升0.125M硼酸緩衝液(pH值為8)及30公升of 4.6 mM-ABD-F (7-fluoro-2,1,3benzoxadiazole-4-sulfonamide)溶液(在硼酸緩衝液中),加入100公升過濾後的漂浮物。在50C,20分鐘後,快速冷卻樣本,加入40公升1M氯化氫。樣本(20公升)以配有螢光偵測器(Bio-Tek, SFM 25, Kontron) 的HPLC (ESA 580 Kontron instruments 400, Strasbourg, France)分析,設定在ex . 385 nm and em . 515 nm。淋洗相由0.1 M磷酸鉀緩衝液 (pH值為3.25)含10% 乙腈,速率固定於1.2 mL/min。分離由250 . 4.6 mm,5公尺直徑Nucleosil C18 分析管柱(Macherel-Nagel) 維持在35C。在標準校準硫醇基後, Hcy,Cys與GSH濃度由N-乙酰半胱氨酸的面積商數及Hcy,Cys與GSH各自的高峰決定。