Continuous fluorimetric assay for human aldehyde dehydrogenase and its application to blood analysis

doi:10.1016/0003-2670(95)00476-9

Analytica Chimica Acta

Volume 319, Issues 1–2, 30 January 1996, Pages 209-219

https://doi.org/10.1016/0003-2670(95)00476-9 Get rights and content

Abstract

A highly fluorogenic synthetic substrate for human “low K_m” aldehyde dehydrogenase (ALDH) isozymes, 7-methoxy-1-naphthaldehyde, was identified. The new substrate is characterized by submicromolar or low micromolar Michaelis constants (K_m) for both cytosolic and mitochondrial ALDH from the human liver. The cytosolic ALDH oxidizes the naphthaldehyde substrate with a rate almost equal to that of the respective acetaldehyde reaction, while the mitochondrial isozyme is ca. 25 fold less active. Thanks to a high fluorescence yield of the oxidation product, i.e., the corresponding naphthoate (φ = 0.41), reaction rates as low as 0.1 nM min⁻¹ can be measured reproducibly. The emission spectral characteristics of the naphthoate product differ significantly from that of the aldehyde (λ_max at 395 and 475 nm, respectively), allowing a selective observation of the former, and there is also marked difference between the naphthoate and the corresponding alcohol (λ_max at 355 nm), helping to eliminate possible interferences from alcohol dehydrogenase and/or aldehyde reductase in the fluorimetric assay. As a possible application of the new assay, it is shown that the ALDH activity can easily be detected, by continuous monitoring of the fluorescence increase, in 1000-fold diluted hemolysates, using 7-methoxy-1-naphthaldehyde and NAD⁺ as substrates, and there is no necessity for prior removal of hemoglobin. Normal ALDH level in the blood of healthy volunteers has been measured, and the result, 4.9 U l⁻¹ (n = 27), agrees with the literature data obtained for the acetaldehyde oxidation activity, and exceeds by more than 20-fold the lower detection limit of the method. The present method is free of a background drift, characteristic of all the previously proposed spectral assays for ALDH.

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Citation Excerpt :
The aldehydes were dissolved in 30% aqueous acetonitrile stock solutions (3 mM), and their concentrations determined spectrophotometrically, using molar extinction coefficients of 14,200 for MONAL-62 and 13,500 for DANAL, and corresponding values for carboxylates were 7100 and 11,500, respectively.14 8-Azatheophylline was synthesized according to known procedures.15 NAD+, glutathione (GSH) and dithiothreitol (DTT) and dithioerythritol (DTE) were purchased from SIGMA (St. Louis, USA).
We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme.
Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD⁺. Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate.
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Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids. Both compounds were previously found to be excellent substrates for the ALDH from erythrocytes and for the purified class I (cytosolic) ALDH from human liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class III ALDH present in human saliva. The apparentK_mfor the former compound reacting with saliva ALDH is 0.24 μm, with the reaction rate (V_max) close to that of benzaldehyde oxidation. There is also a fully competitive inhibition of the fluorogenic oxidation of the MONAL-62 by benzaldehyde. Both NAD⁺and NADP⁺can be used as oxidants in this reaction, with comparable rates, a fact previously reported for the human class III aldehyde dehydrogenase. In human liver homogenate (cytosolic + microsomal fraction), the ALDH activity is easily detectable using either MONAL-71 or MONAL-62, with specific activities of approximately 2.5 and 3.2 units per gram of protein, respectively. The low apparentK_mvalues, 0.85 and <0.03 μm, respectively, together with the inhibition profile by propionic aldehyde (ID₅₀in the micromolar range) indicate that both compounds are oxidized primarily by the class I ALDH, further confirmed by low activity (0.4 U/g) with NADP⁺as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the activity measured with MONAL-71, 0.4 U/g, is much lower than that with MONAL-62 (5.1 U/g with NAD⁺and 3.1 U/g with NADP⁺), the latter being virtually insensitive to 1 mmpropionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured selectively with the two fluorogenic substrates described. In all experiments, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mm4-methylpyrazole, a known inhibitor of the alcohol dehydrogenase, did not change the resultant ALDH activities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed diminished class I ALDH activities (0.7 and 0.03 U/g), but no evidence for class III ALDH induction. The above observations are discussed in relation to the mechanism of detoxication of cyclophosphamide.
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Fluorimetry/phosphorimetryContinuous fluorimetric assay for human aldehyde dehydrogenase and its application to blood analysis

Abstract

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FEBS Lett.

Biochem. Pharmacol.

Pharmacol. Biochem. Behav.

Alcohol

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Alcohol

Alcohol

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Protein Sci.

Alcohol and Alcoholism

Fluorimetry/phosphorimetry
Continuous fluorimetric assay for human aldehyde dehydrogenase and its application to blood analysis