Human erythrocyte d-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups D. Gabriel, L. Pivetta, V. Folmer, J.C.M. Soares, G.R. Augusti, C.W. Nogueira, G. Zeni, J.B.T. Rocha * Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 8 June 2004; revised 1 December 2004; accepted 30 March 2005 Abstract The heme pathway enzyme d-aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. This sulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to selenium organic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediated reactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose and ribose concentrations on d-ALA-D activity in vitro. These reducing sugars inhibited d-ALA-D with efficacies in the order fructose Z ribose O glucose. The possible mechanism of glucose inhibition was investigated using lysine, DTT, and t-butylamine. Oxidation of the enzyme’s critical sulfhydryl groups was not involved because DTT had no effect. We concluded that high concentrations of reducing sugars or their autoxidation products inhibit d-ALA-D by a mechanism not related to thiol oxidation. Also, we are not able to demonstrate that the formation of a Schiff base with the critical lysine residue of the enzyme is involved in the inhibition of d-ALA-D by hexoses. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Ebselen; Diabets; Porphobilinogen synthase; Oxidative stress 1. Introduction Diabetes is characterized by hyperglycemia and is treated with insulin or drugs to ameliorate this symptom (Strowig and Raskin, 1992; Pinero-Pilona et al., 2002). The disease leads to debilitating secondary complications that shorten the patient’s life span. Recently, the molecular mechanisms underlying these secondary com- plications have been more thoroughly investigated. Evidence suggests that non-enzymatic glucose-mediated reactions such as autoxidation (Hunt et al., 1988; Wolff and Dean, 1987a,b; Carubelli et al., 1994; Parthiban et al., 1995), protein cross-linking (Namiki et al., 1977; Li et al., 1996; Day et al., 1979; Beswick and Harding, 1985) and AGEs formation (Strowig and Raskin, 1992; Sensi et al., 1995; Soluis et al., 1999; Rahbar et al., 1999; Chevalier et al., 2002; Forbes et al., 2003) are involved. There have been numerous studies on the deleterious effects of hyperglycemia on the properties of physiologically abundant proteins such as hemoglobin (Schwartz, 1995), albumin (Day et al., 1979), and collagen (Fu et al., 1992). However, published data on the effects of * Corresponding author. Fax: C55 552208031. E-mail address: jbtrocha@yahoo.com.br (J.B.T. Rocha). 1065-6995/$ - see front matterÓ 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.03.017 www.elsevier.com/locate/cellbi Cell Biology International 29 (2005) 669e674