Lee W N, Bassilian S, Guo Z, Schoeller D, Edmond J, Bergner E A, Byerley L O
Research and Education Institute, Harbor University of California, Los Angeles Medical Center, Torrance 90502.
Am J Physiol. 1994 Mar;266(3 Pt 1):E372-83. doi: 10.1152/ajpendo.1994.266.3.E372.
Fractional biosynthesis of palmitate, stearate, and cholesterol was determined with deuterated water (2H2O) using mass isotopomer analysis in Hep G2 and MCA sarcoma cells in culture. The method employed differs from previous ones in that the number of deuterium atoms from 2H2O incorporated into newly synthesized molecules was determined and not assumed. After correction for background natural abundances, the isotopomer distribution due to deuterium incorporation in fatty acids and cholesterol was shown to follow a simple binomial distribution depending on the deuterium enrichment in water (p) and the maximum number of deuterium atoms incorporated per molecule (N). Under a wide range of 2H2O enrichments, N could be determined to be 17 for palmitate, 20 for stearate, and 20 for cholesterol by regression analysis or from a series of consecutive mass isotopomer ratios. The fraction derived from de novo synthesis was given by the ratio of the observed to the theoretical deuterium enrichment, which is the product (N x p). The new synthesized fraction of palmitate and stearate by Hep G2 cells for the length of the experiment was found to be 77 and 65%, respectively. These values were confirmed by experiments with [U-13C]glucose as the precursor. In MCA sarcoma cells grown in lipid-poor medium, the average values for fractional synthesis of palmitate, stearate, and cholesterol were 70, 35, and 70%, respectively. This approach should be generally applicable to the simultaneous determined of fractional synthesis of a number of compounds with either deuterium or 13C tracers. Its application is only limited by the accuracy of mass spectrometric analysis.
在培养的Hep G2细胞和MCA肉瘤细胞中，使用重水（2H2O）通过质量同位素异构体分析来测定棕榈酸、硬脂酸和胆固醇的部分生物合成。所采用的方法与之前的方法不同之处在于，测定了从2H2O掺入新合成分子中的氘原子数量，而不是假设其数量。在校正背景自然丰度后，脂肪酸和胆固醇中由于氘掺入导致的同位素异构体分布显示遵循简单的二项分布，这取决于水中的氘富集度（p）和每个分子掺入的最大氘原子数（N）。在广泛的2H2O富集范围内，通过回归分析或一系列连续的质量同位素异构体比率，可以确定棕榈酸的N为17，硬脂酸的N为20，胆固醇的N为20。从头合成衍生的部分由观察到的氘富集与理论氘富集的比率给出，理论氘富集是乘积（N×p）。在实验期间，Hep G2细胞新合成的棕榈酸和硬脂酸部分分别为77%和65%。这些值通过以[U-13C]葡萄糖为前体的实验得到了证实。在贫脂培养基中生长的MCA肉瘤细胞中，棕榈酸、硬脂酸和胆固醇部分合成的平均值分别为70%、35%和70%。这种方法通常应适用于使用氘或13C示踪剂同时测定多种化合物的部分合成。其应用仅受质谱分析准确性的限制。
