Aldose reductase (AR) is an NADPH-dependent reductase, which acts on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined as the first enzyme in the so-called polyol pathway, in which glucose is transformed into sorbitol by AR and then to fructose by an NAD+-dependent dehydrogenase. An exaggerated flux of glucose through the polyol pathway (as can occur in diabetes) with the subsequent accumulation of sorbitol, was originally proposed as the basic event in the aethiology of secondary diabetic complications. For decades this has meant targeting the enzyme for a specific and strong inhibition. However, the ability of AR to reduce toxic alkenals and alkanals, which are products of oxidative stress, poses the question of whether AR might be better classified as a detoxifying enzyme, thus raising doubts as to the unequivocal advantages of inhibiting the enzyme. This paper provides evidence of the possibility for an effective intervention on AR activity through an intra-site differential inhibition. Examples of a new generation of aldose reductase “differential” inhibitors (ARDIs) are presented, which can preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. Some selected inhibitors are shown to preferentially inhibit enzyme activity on glucose or glyceraldehyde and 3-glutathionyl-4-hydroxy-nonanal, but are less effective in reducing 4-hydroxy-2-nonenal. We question the efficacy of D, L-glyceraldehyde, the substrate commonly used in in vitro inhibition AR studies, as an in vitro reference AR substrate when the aim of the investigation is to impair glucose reduction.

The chaperone behaviour of bovine serum albumin was compared with that of alpha-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with alpha-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than alpha-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family.

Sorbitol dehydrogenase (L-iditol:NAD؉ 2-oxidoreductase, E.C. 1.1.1.14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens ␣-crystallin. An ␣-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55°C. Moreover, an ␣-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55°C for at least 40 min. The protection by ␣-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 M NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the ␣-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by ␣-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of ␣-crystallin against the inactivation of SDH induced at 55°C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of ␣-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with ␣-crystallin. Two different adducts between ␣-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M r of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M r aggregates of ␣-crystallin. Even though it had a reduced efficiency with respect to ␣-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of ␣-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of ␣-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

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Although glucose is one of the most important physio-pathological substrates of aldose reductase, it is not an easy molecule for in vitro investigation into the enzyme. In many cases alternative aldoses have been used for kinetic characterization and inhibition studies. However these molecules do not completely match the structural features of glucose, thus possibly leading to results that are not fully applicable to glucose. We show how aldose reductase is able to act efficiently on L-idose, the C-5 epimer of D-glucose. This is verified using both the bovine lens and the human recombinant enzymes. While the kcat values obtained are essentially identical to those measured for D-glucose, a significant decrease in KM was observed. This can be due to the significantly higher level of the free aldehyde form present in L-idose compared to D-glucose. We believe that L-idose is the best alternative to D-glucose in studies on aldose reductase.

Aldose reductase (AKR1B1), the first enzyme in the polyol pathway, is likely involved in the onset of diabetic complications. Differential inhibition of AKR1B1 has been proposed to counteract the damaging effects linked to the activity of the enzyme while preserving its detoxifying ability. Here, we show that epigallocatechin gallate (EGCG), one of the most representative catechins present in green tea, acts as a differential inhibitor of human recombinant AKR1B1. A kinetic analysis of EGCG, and of its components, gallic acid (GA) and epigallocatechin (EGC) as inhibitors of the reduction of L-idose, 4-hydroxy2,3-nonenal (HNE), and 3-glutathionyl l-4-dihydroxynonanal (GSHNE) revealed for the compounds a different model of inhibition toward the different substrates. While EGCG preferentially inhibited L-idose and GSHNE reduction with respect to HNE, gallic acid, which was still active in inhibiting the reduction of the sugar, was less active in inhibiting HNE and GSHNE reduction. EGC was found to be less efficient as an inhibitor of AKR1B1 and devoid of any differential inhibitory action. A computational study defined different interactive modes for the three substrates on the AKR1B1 active site and suggested a rationale for the observed differential inhibition. A chromatographic fractionation of an alcoholic green tea extract revealed that, besides EGCG and GA, other components may exhibit the differential inhibition of AKR1B1.

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