Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone.
Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone.
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从猫牙槽骨中同时提取和测定环核苷酸、前列腺素和 DNA。
DOI：
10.1016/0003-2697(81)90075-0
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发表时间：
1981
影响因子：
2.9
通讯作者：
Z. Davidovitch
中科院分区：
文献类型：
--
作者：
J. Shanfeld;J. Jones;Z. Davidovitch
本文介绍了从少量矿化骨中同时提取cAMP、cGMP、PGE_2、PGE_2、α和DNA的方法，并对这些物质进行了随后的分析。不同的溶剂对脂肪酸的提取效率进行了测试，发现水或40%的乙醇可以提取90%以上的标记前列腺素。为了避免酶降解，这些物质是在−5°C下提取的，需要一种在提取过程中不会冻结的溶剂。冷冻的猫牙槽骨标本在40%乙醇中匀浆，加入5 mM EDTA以抑制磷酸二酯酶。提取小等分的匀浆，用于DNA的荧光光度分析。离心后的上清液用石油醚提取，去掉中性脂，然后用乙酸乙酯分配。乙酸乙酯层用氮气浸泡，与测定缓冲液重组，并测定前列腺素E_2和前列腺素F_2α。部分水相组分用于cAMP结合分析，其余部分用柱层析洗脱cGMP进行放射分析。以每微克脱氧核糖核酸为单位，猫牙槽骨中cAMP、cGMP、PGE_2、α的含量分别为0.346±0.049、0.026±0.001、5.52±1.46pg、1.00±0.29pg。在样品等分稀释或向cAMP、cGMP或PGE2中加入标准后计算的值与各自的值没有显著差异(P<；0.05)。在每个检测系统的灵敏度范围内，从同一样本中测定牙槽骨中cAMP、cGMP、PGE_2和PGF_2α是可行的。
A method for the simultaneous extraction of cAMP, cGMP, PGE2, PGF2α, and DNA from a small sample of mineralized bone and the subsequent assay of these substances is described. Various solvents were tested for efficiency of extraction for the fatty acids, and water or 40% ethanol was found to extract more than 90% of labeled prostaglandin. In order to avoid enzymatic degradation, the substances were extracted at −5°C requiring a solvent which would not freeze during extraction. Frozen alveolar cat bone samples were homogenized in 40% ethanol in the presence of 5 mm EDTA to inhibit phosphodiesterase. Small aliquots of the homogenate were withdrawn for the spectrofluorophotometric assay of DNA. After centrifugation, the supernatant was extracted first with petroleum ether, in order to take out neutral lipids, followed by ethyl acetate partition. The ethyl acetate layer was dired with N2gas, reconstituted with assay buffer, and assayed for PGE2and PGF2α. A portion of the aqueous fraction was used for cAMP binding assay, while the rest was column chromatographed to elute the cGMP for radioassay. On the basis of per microgram of DNA, values for each of the following in cat alveolar bone were: 0.346 ± 0.049 pmol for cAMP, 0.026 ± 0.001 pmol for cGMP, 5.52 ± 1.46 pg for PGE2, and 1.00 ± 0.29 pg for PGF2α. Values calculated after the dilution of the sample aliquots or addition of standards to cAMP, cGMP, or PGE2showed no significant difference (P < 0.05) to their respective values. Within the limits of the sensitivity for each of the assay systems, it is feasible to measure cAMP, cGMP, PGE2, and PGF2αin alveolar bone from the same sample.