Michorowska Sylwia, Wiśniewska Agnieszka, Wolinowska Renata, Wroczyński Piotr, Giebułtowicz Joanna
Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland.
Department of Laboratory Medicine, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland.
Diagnostics (Basel). 2024 Dec 3;14(23):2721. doi: 10.3390/diagnostics14232721.
Aldehyde dehydrogenase class 1 (ALDH1) is an enzyme that is ubiquitously distributed in adult tissues and may serve as a prognostic marker in various cancer types. In blood, 99% of ALDH1 is found in erythrocytes; although, it was also demonstrated that leukocytes and platelets exhibit ALDH activity. No ALDH activity was detected in plasma, even when employing the highly sensitive fluorometric method with 7-methoxy-1-naphthaldehyde as a substrate. However, some reports have been released describing stable and measurable ALDH1 activity in the serum of healthy subjects using 6-methoxy-2-naphthaldehyde as a substrate and a Shimadzu RF-5301 spectrofluorometer.
Our study aimed to verify whether ALDH1 activity can be measured in plasma or serum ( = 80) using 6-methoxy-2-naphthaldehyde as a substrate and a highly sensitive Hitachi F7000 spectrofluorometer, which offers a higher signal-to-noise ratio compared to the Shimadzu RF-5301. Additionally, HPLC with fluorometric detection was used to validate the results ( = 25) and analyze the influence of hemolysis ( = 5) and liver cell damage ( = 15) on ALDH1 activity in serum.
Measurable ALDH activity in serum/plasma was very rarely detected using a spectrofluorometer (2 cases out of 80). However, background drift in assays without coenzyme addition was observed, and it may be easily mistaken for ALDH or oxidase activity. Therefore, the spectrofluorometer drift observed in blank assays and modified by a matrix, e.g., enhanced in protein-rich samples, should be considered in ALDH1 activity assays.
The spectrofluorometric method has limited applicability for determining ALDH activity in plasma and serum. HPLC can measure ALDH1 activity in plasma or serum; however, factors like hemolysis and elevated liver enzymes significantly affect activity and must be considered in diagnostic interpretations. To enhance research quality on ALDH1 as a biomarker for diseases, including cancers, we recommend using control samples, reference materials, and purifying commercially available aldehyde substrates to improve method sensitivity.
1类醛脱氢酶（ALDH1）是一种在成人组织中广泛分布的酶，可能作为多种癌症类型的预后标志物。在血液中，99%的ALDH1存在于红细胞中；不过，也有研究表明白细胞和血小板具有ALDH活性。即使采用以7-甲氧基-1-萘甲醛为底物的高灵敏度荧光法，血浆中也未检测到ALDH活性。然而，有一些报告称，使用6-甲氧基-2-萘甲醛作为底物和岛津RF-5301荧光分光光度计，在健康受试者的血清中检测到了稳定且可测量的ALDH1活性。
我们的研究旨在验证是否可以使用6-甲氧基-2-萘甲醛作为底物以及高灵敏度的日立F7000荧光分光光度计（与岛津RF-5301相比具有更高的信噪比）在血浆或血清中（n = 80）测量ALDH1活性。此外，采用荧光检测的高效液相色谱法来验证结果（n = 25），并分析溶血（n = 5）和肝细胞损伤（n = 15）对血清中ALDH1活性的影响。
使用荧光分光光度计在血清/血浆中极难检测到可测量的ALDH活性（80例中仅2例）。然而，在不添加辅酶的检测中观察到了背景漂移，这可能很容易被误认为是ALDH或氧化酶活性。因此，在ALDH1活性检测中应考虑在空白检测中观察到并经基质修正（例如在富含蛋白质的样品中增强）的荧光分光光度计漂移。
荧光分光光度法在测定血浆和血清中的ALDH活性方面适用性有限。高效液相色谱法可以测量血浆或血清中的ALDH1活性；然而，溶血和肝酶升高之类的因素会显著影响活性，在诊断解读中必须予以考虑。为提高将ALDH1作为包括癌症在内的疾病生物标志物的研究质量，我们建议使用对照样品、参考物质，并对市售醛底物进行纯化以提高方法灵敏度。
