Lee Chul, Qiao Xian, Goeger Douglas E, Anderson Karl E
Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA.
Clin Chim Acta. 2004 Sep;347(1-2):183-8. doi: 10.1016/j.cccn.2004.04.015.
Measurement of 5-aminolevulinic acid (ALA) in serum is potentially useful in acute porphyrias, lead poisoning and hereditary tyrosinemia. Because levels of ALA in serum are about 100 times less than in urine, a highly sensitive method is required. We describe a simple and sensitive fluorometric method that does not require HPLC.
ALA is separated from serum using a cation-exchange resin (DOWEX 50WX8-400), followed by addition of acetylacetone and formaldehyde to produce a fluorescent ALA derivative by the Hantzsch reaction. The fluorescence was measured at an excitation wavelength of 370 nm, and the emission peak was near 463 nm. Results were compared with measurements of ALA by a published HPLC method on the same samples.
The fluorometric method was linear for ALA concentrations up to 500 microg/l with a detection limit of about 8.7 microg/l. Within-run variations (N=8) at 25 and 100 microg/l ALA were 3.7% and 3.1%, respectively, and day-to-day variations (N=10) for the same levels were 7.2% and 6.1%, respectively. The regression equation for this method in reference to the HPLC method (Y=0.99X+10.34 for N=34, r=0.98, S(y/x)=36.1) had a slope of near unity and an insignificant y-intercept, although values less than 50 microg/l were generally slightly higher by the fluorometric method than by HPLC. The reference range for serum ALA was 0-79.4 microg/l (35.2+/-22.1, mean+/-S.D., N=42), with a distribution skewed to the left because levels in eight subjects were below the detection limit.
This simple, fluorometric method for serum ALA correlated well with the results of the HPLC method, and may be suitable for assessing biochemical expression of acute porphyrias and response to treatment especially when HPLC is not available.
血清中5-氨基乙酰丙酸（ALA）的检测在急性卟啉病、铅中毒和遗传性酪氨酸血症中可能具有重要意义。由于血清中ALA的水平比尿液中低约100倍，因此需要一种高灵敏度的检测方法。我们描述了一种无需高效液相色谱（HPLC）的简单且灵敏的荧光检测方法。
使用阳离子交换树脂（DOWEX 50WX8 - 400）从血清中分离出ALA，随后加入乙酰丙酮和甲醛，通过汉茨希反应生成荧光性的ALA衍生物。在激发波长370nm处测量荧光，发射峰接近463nm。将结果与对相同样本采用已发表的HPLC方法检测ALA的结果进行比较。
荧光检测方法对于ALA浓度高达500μg/L呈线性关系，检测限约为8.7μg/L。在25μg/L和100μg/L的ALA水平下，批内变异（N = 8）分别为3.7%和3.1%，相同水平的日间变异（N = 10）分别为7.2%和6.1%。该方法相对于HPLC方法的回归方程（N = 34时，Y = 0.99X + 10.34，r = 0.98，S(y/x)=36.1）斜率接近1，y轴截距不显著，尽管荧光检测方法检测低于50μg/L的值通常略高于HPLC方法。血清ALA的参考范围为0 - 79.4μg/L（35.2±22.1，均值±标准差，N = 42），分布向左偏斜，因为有8名受试者的水平低于检测限。
这种用于血清ALA的简单荧光检测方法与HPLC方法的结果相关性良好，尤其在无法使用HPLC时，可能适用于评估急性卟啉病的生化表现及治疗反应。
