Tredget E E, Forsyth N, Uji-Friedland A, Chambers M, Ghahary A, Scott P G, Hogg A M, Burke J F
Department of Surgery, University of Alberta, Edmonton, Canada.
J Chromatogr. 1993 Jan 29;612(1):7-19. doi: 10.1016/0378-4347(93)80361-7.
The use of gas chromatography-mass spectrometry (GC-MS) and 18O2, a stable isotope which is incorporated into collagen during the post-translational conversion of proline to hydroxyproline, offers the potential advantages of high levels of sensitivity and specificity as compared to other techniques for measuring rates of collagen synthesis and degradation in vitro and vivo. Trifluoracetylation and methanol esterification of hydroxyproline yields two derivatives of hydroxyproline: N,O-trifluoroacetyl methyl 4-hydroxy-L-proline (N,O-TFA-Hyp) and N-trifluoroacetyl methyl 4-hydroxy-L-proline (N-TFA-Hyp). In the past, N-TFA-Hyp, which yields the 16O/18O-containing m/z 182/184 ion pair [M-COOH3]+ when analyzed by electron impact ionization GC-MS, has been proposed for analysis of 18O-enriched collagen. Although N,O-TFA-Hyp can be converted to N-TFA-Hyp by solvolysis, we find that this leads to degradation of the chromatography in GC-MS and demonstrate here that this extra chemical step is unnecessary if the m/z 278/280 ion pair (representing the [M-COOCH3]+. fragment) is measured by selected ion monitoring. By labelling fibroblasts in culture with 18O2, a sample of isotope-enriched collagen was obtained which was used to calibrate the GC-MS over the range 0.5-49% atom percent enrichment (APE). The greater sensitivity of 18O2 versus [15N]proline for labelling newly synthesized collagen was demonstrated by the finding of a ten-fold higher enrichment in the former isotope when administered to cell cultures at the same precursor APE. Thus, the approach described herein permits the determination of total hydroxyproline and APE on the same sample avoiding additional processing steps while maintaining the quality of chromatography and the sensitivity of detection. Measurement of absolute rates of both collagen synthesis and intracellular degradation of newly synthesized collagen in cell cultures is thus possible. Preliminary results comparing collagen metabolism in pairs of fibroblasts from hypertrophic scars and normal skin in post-burn patients are presented.
气相色谱 - 质谱联用（GC - MS）技术与18O2（一种在脯氨酸向羟脯氨酸的翻译后转化过程中掺入胶原蛋白的稳定同位素）的使用，与其他用于测量体外和体内胶原蛋白合成与降解速率的技术相比，具有高灵敏度和高特异性的潜在优势。羟脯氨酸的三氟乙酰化和甲醇酯化产生两种羟脯氨酸衍生物：N，O - 三氟乙酰基 - 4 - 羟基 - L - 脯氨酸甲酯（N，O - TFA - Hyp）和N - 三氟乙酰基 - 4 - 羟基 - L - 脯氨酸甲酯（N - TFA - Hyp）。过去，有人提出通过电子轰击电离GC - MS分析时产生含16O/18O的m/z 182/184离子对[M - COOH3]+的N - TFA - Hyp用于分析富含18O的胶原蛋白。尽管N，O - TFA - Hyp可通过溶剂解转化为N - TFA - Hyp，但我们发现这会导致GC - MS中的色谱降解，并且在此证明，如果通过选择离子监测测量m/z 278/280离子对（代表[M - COOCH3]+片段），则这个额外的化学步骤是不必要的。通过用18O2标记培养中的成纤维细胞，获得了富含同位素的胶原蛋白样品，该样品用于在0.5 - 49%原子百分比富集（APE）范围内校准GC - MS。当以相同的前体APE施用于细胞培养物时，发现前一种同位素的富集程度比[15N]脯氨酸高十倍，这证明了18O2在标记新合成胶原蛋白方面比[15N]脯氨酸具有更高的灵敏度。因此，本文所述方法允许在同一样品上测定总羟脯氨酸和APE，避免了额外的处理步骤，同时保持了色谱质量和检测灵敏度。因此，可以测量细胞培养物中胶原蛋白合成的绝对速率以及新合成胶原蛋白的细胞内降解速率。本文还展示了比较烧伤后患者肥厚性瘢痕和正常皮肤中成纤维细胞对之间胶原蛋白代谢的初步结果。
