Interrelationships between renal TCA-cycle metabolism and ammoniagenesis: studies with 13C-NMR and GC-MS.
Interrelationships between renal TCA-cycle metabolism and ammoniagenesis: studies with 13C-NMR and GC-MS.
复制标题
肾脏 TCA 循环代谢与氨生成之间的相互关系：13C-NMR 和 GC-MS 的研究。
DOI：
10.1159/000415700
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发表时间：
1988
影响因子：
--
通讯作者：
Segal,S
中科院分区：
文献类型：
--
作者：
Nissim,I;Yudkoff,M;Segal,S
从Charles River Breeding Laboratories获得体重在250和300 g之间的雄性Sprague-Dawley大鼠。如前所述，通过氯化铵给药诱导慢性代谢性酸中毒并制备肾小管[6，17-19]。将小管（25-30 mg湿重/ml）在水浴中在25-ml带塞锥形瓶中的4 ml含有2.5%白蛋白的Krebs-Henseleit缓冲液（pH7.4）（透析的牛级分V）中于37 ℃孵育指定的时间。用95%02：5%C02预处理缓冲液。通过加入2.5 mM [3- 13 C]丙氨酸（99原子%过量; MSD Ltd.）向4 ml细胞悬液中加入400 µl 40%高氯酸终止培养。用KOH中和后，上清液用于测定代谢物以及用GC-MS或NMR测量同位素丰度。
Male Sprague-Dawley rats weighing between 250 and 300 g were obtained from Charles River Breeding Laboratories. Induction of chronic metabolic acidosis by ammonium chloride administration and preparation of tubules were carried out as described pre-viously [6, 17-19]. Tubules (25-30 mg wet weight/ml) were incubated at 37 C for the times indicated in 4 ml of Krebs-Henseleit buffer (pH 7.4) containing 2.5% albumin (dialyzed bovine fraction V) in stoppered 25-ml Erlenmeyer flasks in a water bath. The buffer was pregassed with 95% 02: 5% CO2. Incubations were initiated by addition of 2.5 mM [3-13C] alanine (99 atom% excess; MSD Ltd.) to 4 ml of cell suspension and terminated by addition of 400 µl of 40% perchloric acid to each flask. After neutralization with KOH, the supernatants were utilized for determination of metabolites as well as measurement of isotopic abundance with either GC-MS or NMR.