Decosterd L A, Karagiannis A, Roulet J M, Bélaz N, Appenzeller M, Buclin T, Vogel P, Biollaz J
Département de Médecine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
J Chromatogr B Biomed Sci Appl. 1997 Dec 5;703(1-2):25-36. doi: 10.1016/s0378-4347(97)00396-4.
PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.
在临床研究中，对氨基马尿酸（PAH，N-(4-氨基苯甲酰基)甘氨酸）清除率的测量已用于肾血浆流量测定长达50年。血浆或尿液中PAH的定量通常在重氮化反应后通过比色法进行，但测量必须针对空白血浆中观察到的非特异性残留反应进行校正。我们开发了一种高效液相色谱法（HPLC），使用梯度洗脱离子对反相色谱，分别在273和265nm处进行紫外检测，以特异性测定PAH及其代谢物NAc-PAH。分离在室温下于ChromCart（125mm×4mm内径）Nucleosil 100-5μm C18AB柱上进行，使用甲醇-缓冲液pH 3.9 1:99→15:85的梯度洗脱，持续15分钟。pH 3.9的缓冲水溶液由375ml柠檬酸钠-柠檬酸溶液（每升含21.01g柠檬酸和8.0g NaOH）、2.7ml 85%的H3PO4、1.0g庚烷磺酸钠混合而成，并用超纯水补足至1000ml。尽管在12名健康受试者中，NAc-PAH占未改变排泄的PAH的10.2±2.7%，但N-乙酰转移酶活性似乎对PAH清除率没有显著影响。使用从健康志愿者收集的尿液和血浆样本，比较了HPLC和比色法的性能。两种技术获得的结果之间存在良好的相关性（血浆和尿液的r分别为0.94和0.97）。然而，比色法测得的尿液中PAH浓度高于HPLC法，而血浆中PAH浓度低于HPLC法。因此，比色法测得的肾清除率（ClR）和全身清除率（Cls）系统性地更高（分别为35.1%和17.8%）。根据HPLC数据（n = 143）计算的肾脏排泄的PAH分数ClR/ClS，正如预期的那样，始终<1（平均值 = 0.73±0.11），而比色法给出的平均提取率为0.87±0.13，这意味着一些不符合生理的值（>1）。总之，HPLC不仅能够同时定量PAH和NAc-PAH，还可能提供更准确和精确的PAH清除率测量。
