Low J E, Borch R F, Sladek N E
Cancer Res. 1982 Mar;42(3):830-7.
The rates at which 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer composition, buffer concentration, and pH. Conversion of 4-hydroperoxycyclophosphamide to 4-hydroxycyclophosphamide in 0.5 M Tris buffer, pH 7.4, 37 degrees, was first-order (k = 0.016 min-1), but subsequent conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein under these conditions was negligible. Phosphoramide mustard and acrolein were readily generated from 4-hydroperoxycyclophosphamide or 4-hydroxycyclophosphamide when either of these agents was placed in phosphate buffer. Conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was first-order with respect to 4-hydroxycyclophosphamide (k = 0.126 min-1 in 0.5 M phosphate buffer, pH 8, 37 degrees) as well as first-order with respect to phosphate serving as a catalyst. The rate-determining step in the reaction was pH dependent only insofar as the hydrogen ion concentration governed the relative amounts of monobasic and dibasic phosphate present. Pseudo-first-order rate constants were 0.045 M-1 min-1 for monobasic phosphate and 0.256 M-1 min-1 for dibasic phosphate. The role of phosphate in this reaction was as that of a bifunctional catalyst. The reaction was not subject to specific or general, acid or base, catalysis. Other bifunctional catalysts such as glucose-6-phosphate and bicarbonate also catalyzed the reaction, albeit less efficiently. Aldophosphamide apparently exists only transiently; its presence could not be established by 31P nuclear magnetic resonance spectroscopy. We conclude that, in the reaction sequence 4-hydroxycyclophosphamide leads to aldophosphamide leads to phosphoramide mustard + acrolein, the conversion of 4-hydroxycyclophosphamide to aldophosphamide is rate limiting and is subject to bifunctional catalysis; this reaction can proceed efficiently only in the presence of a bifunctional catalyst. Assuming that the oncotoxic specificity of cyclophosphamide resides with 4-hydroxycyclophosphamide and that its cytotoxic effect at therapeutic doses is largely mediated by phosphoramide mustard released within cells, these observations offer the possibility that the intracellular concentration of bifunctional catalysts, whether in the form of inorganic phosphates, organic phosphates, enzymes, or other species, serve as important determinants with regard to the oncotoxic potential and specificity of cyclophosphamide. Similarly, the concentration of bifunctional catalysis in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via acrolein, hemorrhagic cystitis.
测定了4-氢过氧环磷酰胺和4-羟基环磷酰胺转化为磷酰胺芥和丙烯醛的速率，该速率是缓冲液组成、缓冲液浓度和pH值的函数。在pH 7.4、37℃的0.5M Tris缓冲液中，4-氢过氧环磷酰胺转化为4-羟基环磷酰胺是一级反应（k = 0.016 min⁻¹），但在这些条件下，随后4-羟基环磷酰胺转化为磷酰胺芥和丙烯醛的反应可忽略不计。当将这两种试剂中的任何一种置于磷酸盐缓冲液中时，磷酰胺芥和丙烯醛很容易从4-氢过氧环磷酰胺或4-羟基环磷酰胺中生成。4-羟基环磷酰胺转化为磷酰胺芥和丙烯醛对于4-羟基环磷酰胺是一级反应（在pH 8、37℃的0.5M磷酸盐缓冲液中k = 0.126 min⁻¹），对于作为催化剂的磷酸盐也是一级反应。反应中的限速步骤仅取决于pH值，因为氢离子浓度决定了存在的磷酸一氢盐和磷酸二氢盐的相对量。磷酸一氢盐的准一级速率常数为0.045 M⁻¹ min⁻¹，磷酸二氢盐的准一级速率常数为0.256 M⁻¹ min⁻¹。磷酸盐在该反应中的作用是双功能催化剂的作用。该反应不受特殊或一般的酸或碱催化。其他双功能催化剂，如6-磷酸葡萄糖和碳酸氢盐，也催化该反应，尽管效率较低。醛磷酰胺显然仅短暂存在；通过³¹P核磁共振光谱无法确定其存在。我们得出结论，在4-羟基环磷酰胺→醛磷酰胺→磷酰胺芥 + 丙烯醛的反应序列中，4-羟基环磷酰胺转化为醛磷酰胺是限速步骤，并且受到双功能催化；该反应仅在双功能催化剂存在下才能有效进行。假设环磷酰胺的肿瘤毒性特异性在于4-羟基环磷酰胺及其在治疗剂量下的细胞毒性作用很大程度上由细胞内释放的磷酰胺芥介导，这些观察结果提供了一种可能性，即双功能催化剂的细胞内浓度，无论是以无机磷酸盐、有机磷酸盐、酶或其他物质的形式，都是环磷酰胺肿瘤毒性潜力和特异性的重要决定因素。同样，尿液中双功能催化的浓度以及尿液的pH值对于环磷酰胺通过丙烯醛诱导出血性膀胱炎的可能性可能很重要。
