A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures.
A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures.
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DOI：
10.1016/j.jchromb.2012.02.005
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发表时间：
2012-03-15
影响因子：
3
通讯作者：
Rosenberger, Thad A.
中科院分区：
文献类型：
--
作者：
Bhatt, Dhaval P.;Chen, Xuesong;Geiger, Jonathan D.;Rosenberger, Thad A.
关键词：
在单层原代细胞培养中，基线AMP和ADP水平名义上在中低皮摩尔范围内，因此很难用传统的高效液相色谱方法测量，这些方法通常需要汇集样品或需要使用放射性示踪剂或酶偶联分析的间接检测方法。为了解决这个问题，我们开发了一种高灵敏度和选择性的离子对高效液相色谱荧光检测方法来定量原代培养星形胶质细胞中腺嘌呤核苷酸和腺苷能荷。为此，我们优化了荧光衍生化条件和高效液相色谱参数，实现了对所有腺嘌呤核苷酸的基线分离和定量。核苷酸与氯乙醛在pH 4.5和60℃下孵育60分钟，可转化为相应的1，N6-亚乙基衍生物。在此条件下，1，N6-亚乙基腺苷的衍生化产率可达94.1%。离子配对试剂磷酸四丁基铵的最佳浓度为0.8 mM，以实现腺嘌呤核苷酸的可重复分离。核苷酸标准品的线性范围分别为：腺苷0.16-10.4pmo1，AMP 0.16-20.6pmo1，ADP 0.15-19.2pmo1，ATP 0.15-19.5pmo1。所有腺嘌呤核苷酸的检测下限和定量下限分别约为0.08和0.16pmol。该方法的日内和日间变异分别小于5.1%和3.4%。该方法成功地测定了原代培养星形胶质细胞的所有腺嘌呤核苷酸和腺苷能荷为0.92±0.02。
In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N6-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60° C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N6-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8 mM. Calibration curves of nucleotide standards were linear within the range of 0.16–10.4 pmol for adenosine, 0.16–20.6 pmol for AMP, 0.15–19.2 pmol for ADP, and 0.15–19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92 ± 0.02 in primary astrocyte cell cultures.
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DOI：
10.1083/jcb.85.3.890
发表时间：
1980-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
McCarthy KD;de Vellis J
通讯作者：
de Vellis J
影响因子：
2.9
作者：
RAMOSSALAZAR, A;BAINES, AD
通讯作者：
BAINES, AD
影响因子：
6.2
作者：
Tang, Kim San;Suh, Sang Won;Anderson, Christopher M.
通讯作者：
Anderson, Christopher M.
影响因子：
--
作者：
Weinstein, D E
通讯作者：
Weinstein, D E
影响因子：
2.9
作者：
HAMMER, DF;UNVERFERTH, DV;ALTSCHULD, RA
通讯作者：
ALTSCHULD, RA