Rochas M A, Tufenkji A E, Levillain P, Houin G
Laboratoire de Pharmacocinétique, Université Paul Sabatier, Toulouse, France.
Arzneimittelforschung. 1991 Dec;41(12):1286-8.
Usual methods allowing the measurement of the free concentration of a drug in serum, i.e. equilibrium dialysis, ultrafiltration and ultracentrifugation, are generally based on a physical separation of the bound and free fractions. During this, variations or errors may occur which are probably at the origin of the variability of the previously published results for methotrexate (CAS 59-05-2). In order to verify these results as well as to experience a technique recently applied to rifampicine the first derivative spectroscopic analysis was used to estimate the bound and free fractions of methotrexate in human serum and serum albumin (HSA). Free drug concentrations were measured at 377 nm and the bound form at 372.5 nm. In human serum, bound methotrexate was 54.1% on average for total concentrations ranging from 10(-5) mol/l to 10(-3) mol/l, without any saturation. With HSA, a saturation occurred. Scatchard analysis showed one family of binding sites characterized by 2 binding sites and an affinity constant of 3200 mol/l, in mean, values close to that previously calculated using equilibrium dialysis.
常用的用于测定血清中药物游离浓度的方法，即平衡透析、超滤和超速离心，通常基于结合部分和游离部分的物理分离。在此过程中，可能会出现变化或误差，这可能是先前发表的甲氨蝶呤（CAS 59-05-2）结果变异性的根源。为了验证这些结果以及体验一种最近应用于利福平的技术，采用一阶导数光谱分析法来估计人血清和血清白蛋白（HSA）中甲氨蝶呤的结合部分和游离部分。在377nm处测量游离药物浓度，在372.5nm处测量结合形式。在人血清中，总浓度范围为10（-5）mol/l至10（-3）mol/l时，结合甲氨蝶呤平均为54.1%，没有任何饱和现象。对于HSA，出现了饱和现象。Scatchard分析显示有一类结合位点，其特征为有2个结合位点，平均亲和常数为3200mol/l，该值与先前使用平衡透析计算的值相近。
