Blood ALDH1 and GST activity in diabetes type 2 and its correlation with glycated hemoglobin.

There is increasing evidence that oxidative stress (OS) plays a major role in the pathogenesis of diabetes mellitus (DM) and the development of its complications. As one of the consequences of OS is increased lipid peroxidation (LP), the aim of our studies was to check, how the activity of 2 enzymes involved in the detoxification of aldehydes formed during LP, glutathione S-transferase (GST) and aldehyde dehydrogenase 1 (ALDH 1) is changed in patients suffering from DM.GST and ALDH1A1 activities were determined in whole blood samples of DM type 2 patients (n=64) and healthy controls (n=60) using spectrophotometer (for GST activity) and fluorometer (for ALDH1 activity) and they were found to be significantly increased in diabetics when compared with healthy control (p<0.05). Intriguingly, grouping the DM patients on the basis of the glucose level and HbA1c revealed unusually low ALDH activity in the group of patients (n=16) with a relatively high level of these 2 parameters.The increase of ALDH1A1 and GST activity in DM seems to be associated with the severity of the disease and might be a compensatory effect against oxidative stress. Surprisingly low ALDH activity in DM patients with relatively high glucose and HbA1c levels can be a factor predisposing to the development of diabetic complications.

Can lower aldehyde dehydrogenase activity in saliva be a risk factor for oral cavity cancer?

OBJECTIVES: Salivary ALDH3A1 protects the oral cavity from aromatic and medium-chain aliphatic aldehydes originating from food and air pollution and generated during oxidative stress. Due to their reactivity, aldehydes may exhibit an irritating effect as well as cytotoxic, genotoxic, mutagenic and even carcinogenic effects. The aim of this study was to verify whether lower ALDH3A1 activity is a risk factor for oral cavity cancer. SUBJECTS AND METHODS: Fasting saliva samples were collected one day before and about one week after surgery from patients with oral cancer (OCC) (n = 59), other tumours (cysts, neoplasms) (n = 108), gnathic defects and fractures (controls after the surgery) (n = 63), and from healthy volunteers (n = 116). Enzyme activity was measured using a fluorometric method. RESULTS: Total ALDH3A1 activity [U g(-1) ] in patients with OCC was statistically lower than in patients with keratocystic odontogenic tumour (KCOT) (P = 0.00697), odontogenic cysts (OC) (P < 0.00001), neoplasms (P = 0.03343) and the healthy volunteers up to and over 40 years old (P < 0.00001; P = 0.00019). The activity in the saliva of OCC after surgery was lower than in the healthy volunteers (P < 0.00001) and in the groups with fractures (P = 0.00303) and gnathic defects (P = 0.00538). CONCLUSION: Low salivary ALDH activity may be a risk factor for oral cancer development.

Aromatic aldehydes as fluorogenic indicators for human aldehyde dehydrogenases and oxidases: substrate and isozyme specificity.

Substrate properties of a number of potentially fluorogenic aromatic aldehydes of naphthalenes, phenanthrenes and anthracenes and of some coumarin aldehydes towards various forms of the human and rat aldehyde oxidase and dehydrogenase were examined using absorption and emission spectroscopy. It was demonstrated that recombinant human class 1 aldehyde dehydrogenase (ALDH-1) readily oxidizes naphthalene (except for those ortho-substituted), phenanthrene and coumarin aldehydes, whereas the class 3 enzyme (ALDH-3) from human saliva is active only towards 2-naphthaldehyde derivatives. The observed reaction rates in both cases are comparable to those of the best known substrates, and the Km values are typically in the sub-micromolar range. Aldehyde oxidases (AlOx), which are present in mammalian liver, reveal much broader substrate specificity, oxidizing nearly all the compounds examined, including those of the anthracene series, with maximum activity in the micromolar range of substrate concentration. In rat liver, nearly all AlOx activity was located in the cytosolic fraction.

Aldehyde dehydrogenase isoenzymes in tumours--assay with possible prognostic value for oxazaphosphorine chemotherapy.

A novel fluorimetric assay, allowing independent measurement of the activities of two principal cytosolic forms of human aldehyde dehydrogenase, ALDH-1 and ALDH-3 (known as a tumour-associated ALDH) was applied to estimate the activities of these isoenzymes in human liver and thyroid tumours. The assay is based on two artificial substrates, 6-methoxy-2-naphthaldehyde (MONAL-62) and 7-methoxy-1-naphthaldehyde (MONAL-71), exhibiting excellent substrate properties toward various forms of human ALDH (see Wierzchowski et al., 1997, Anal. Biochem. 245, 69-78). We have found significant differences in ALDH activities between malignant and non-malignant tissue fragments, particularly in cancerous livers. Out of 16 tumours examined, only 4 exhibited ALDH-1 activities comparable to that found in the tumour-free tissue (0.5-2.5 U/g), while in the remaining 12 this activity was at least 10-fold lower. The ALDH-3 activity was detectable in about 40% of both tumour and tumour-free liver samples (maximum value 1.5 U/g). Comparison of 13 pathological thyroid fragments revealed ALDH activities in the range of 0.02 to 0.35 U/g, with two malignant samples showing activities of 0.27 and 0.18 U/g. Both substrate specificity and kinetic behaviour of the thyroid ALDH (Km values for the fluorogenic naphthaldehydes as well as propanal inhibition profile) were similar to those of the purified ALDH-1. In 5 thyroid samples traces of ALDH-3 activity was detected, using MONAL-62 and NADP+ as substrates (maximum value 0.04 U/g). Possible prognostic value of the foregoing measurements for cyclophosphamide chemotherapy is discussed.

Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes.

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 apparent Km for the former compound reacting with salvia ALDH is 0.24 microM, with the reaction rate (Vmax) 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 apparent Km values, 0.85 and < 0.03 microM, respectively, together with the inhibition profile by propionic aldehyde (ID50 in 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 mM propionic 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 mM 4-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.

Selective assay of the cytosolic forms of the aldehyde dehydrogenase in rat, with possible significance for the investigations of cyclophosphamide cytotoxicity.

Continuous fluorimetric assay for aldehyde dehydrogenase activity in rat tissue, based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde, is described. The new assay is ca. 2 orders of magnitude more sensitive than the standard procedures and it is not subjected to interferences by other dehydrogenases, although occasionally the aldehyde dehydrogenase activity may be obscured by aldehyde oxidase. The new fluorimetric assay is highly selective for the cytosolic forms of the both rat and human aldehyde dehydrogenase, which are primarily responsible for cyclophosphamide inactivation in vivo. These forms can now be detected in crude homogenates of several rat organs without necessity of subcelluar fractionation.