Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. Free radicals are formed disproportionately in diabetes by glucose oxidation, nonenzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus. Changes in oxidative stress biomarkers, including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione levels, vitamins, lipid peroxidation, nitrite concentration, nonenzymatic glycosylated proteins, and hyperglycemia in diabetes, and their consequences, are discussed in this review. In vivo studies of the effects of various conventional and alternative drugs on these biomarkers are surveyed. There is a need to continue to explore the relationship between free radicals, diabetes, and its complications, and to elucidate the mechanisms by which increased oxidative stress accelerates the development of diabetic complications, in an effort to expand treatment options.

Diabetes, oxidative stress, and antioxidants: a review

Benzyl Alcohol is an aromatic alcohol used in a wide variety of cosmetic formulations as a fragrance component, preservative, solvent, and viscosity-decreasing agent. Benzoic Acid is an aromatic acid used in a wide variety of cosmetics as a pH adjuster and preservative. Sodium Benzoate is the sodium salt of Benzoic Acid used as a preservative, also in a wide range of cosmetic product types. Benzyl Alcohol is metabolized to Benzoic Acid, which reacts with glycine and excreted as hippuric acid in the human body. Acceptable daily intakes were established by the World Health Organization at 5 mg/kg for Benzyl Alcohol, Benzoic Acid, and Sodium Benzoate. Benzoic Acid and Sodium Benzoate are generally recognized as safe in foods according to the U.S. Food and Drug Administration. No adverse effects of Benzyl Alcohol were seen in chronic exposure animal studies using rats and mice. Effects of Benzoic Acid and Sodium Benzoate in chronic exposure animal studies were limited to reduced feed intake and reduced growth. Some differences between control and Benzyl Alcohol-treated populations were noted in one reproductive toxicity study using mice, but these were limited to lower maternal body weights and decreased mean litter weights. Another study also noted that fetal weight was decreased compared to controls, but a third study showed no differences between control and Benzyl Alcohol-treated groups. Benzoic Acid was associated with an increased number of resorptions and malformations in hamsters, but there were no reproductive or developmental toxicty findings in studies using mice and rats exposed to Sodium Benzoate, and, likewise, Benzoic Acid was negative in two rat studies. Genotoxicity tests for these ingredients were mostly negative, but there were some assays that were positive. Carcinogenicity studies, however, were negative. Clinical data indicated that these ingredients can produce nonimmunologic contact urticaria and nonimmunologic immediate contact reactions, characterized by the appearance of wheals, erythema, and pruritus. In one study, 5% Benzyl Alcohol elicited a reaction, and in another study, 2% Benzoic Acid did likewise. Benzyl Alcohol, however, was not a sensitizer at 10%, nor was Benzoic Acid a sensitizer at 2%. Recognizing that the nonimmunologic reactions are strictly cutaneous, likely involving a cholinergic mechanism, it was concluded that these ingredients could be used safely at concentrations up to 5%, but that manufacturers should consider the nonimmunologic phenomena when using these ingredients in cosmetic formulations designed for infants and children. Additionally, Benzyl Alcohol was considered safe up to 10% for use in hair dyes. The limited body exposure, the duration of use, and the frequency of use were considered in concluding that the nonimmunologic reactions would not be a concern. Because of the wide variety of product types in which these ingredients may be used, it is likely that inhalation may be a route of exposure. The available safety tests are not considered sufficient to support the safety of these ingredients in formulations where inhalation is a route of exposure. Inhalation toxicity data are needed to complete the safety assessment of these ingredients where inhalation can occur.

Final report on the safety assessment of Benzyl Alcohol, Benzoic Acid, and Sodium Benzoate.

Our aim was to evaluate oxidative stress in diabetes mellitus by the measurement of biophysical parameters (changes) of hemoglobin macromolecule and some biochemical parameters on two groups of diabetic patients, non-insulin dependent diabetes mellitus (NIDDM), and insulin- dependent diabetes mellitus (IDDM). This study has been conducted on 45 NIDDM, 30 IDDM, compared to 20 healthy subjects. Blood glucose, glycosylated hemoglobin (HbA1c), fructosamine, plasma insulin, plasma malondialdehyde (MDA), erythrocyte reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathio ne reductase (GSH-Red) were measured. Also the biophysical parameters, electrical conductivity of hemoglobin, auto-oxidation rate of hemoglobin, and hemoglobin derivatives were measured. Diabetic patients (NIDDM, IDDM) have significant increases in blood glucose, glycosylated (HbA1c), fructosamine with a concomitant significant decrease in Plasma insulin compared to control. Diabetic patients (NIDDM, IDDM) have a significant high level of MDA compared to control. Diabetic patients (NIDDM, IDDM) have a significant increase in SOD activity, and a significant decrease of GSH compared to control. Significant increases in electrical conductivity of Hb, hemoglobin auto-oxidation rate and hemoglobin derivatives in diabetic patient compared to control. IDDM have a higher level of hemoglobin auto- oxidation rate when compared to NIDDM. Diabetic patients undergo an important oxidative stress that is low in NIDDM compared to IDDM, suggesting metabolic differences between the two types of diabetes. Met-Hb is an important indicator for oxidative stress in diabetes.

Oxidative stress in diabetes mellitus

Hippurate, the glycine conjugate of benzoic acid, is a normal constituent of the endogenous urinary metabolite profile and has long been associated with the microbial degradation of certain dietary components, hepatic function and toluene exposure, and is also commonly used as a measure of renal clearance Here we discuss the potential relevance of hippurate excretion with regard to normal endogenous metabolism and trends in excretion relating to gender, age, and the intestinal microbiota Additionally, the significance of hippurate excretion with respect to disease states including obesity, diabetes, gastrointestinal diseases, impaired renal function, psychological disorders and autism, as well as toxicity and parasitic infection, are considered

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Hippurate: the natural history of a mammalian-microbial cometabolite.

Beneficial effects of Murraya koenigii leaves on antioxidant defense system and ultra structural changes of pancreatic β-cells in experimental diabetes in rats

Glycine conjugation of benzoic acid is catalyzed by the mitochondrial enzymes benzoyl-coenzyme A(CoA) synthetase and benzoyl-CoA: glycine N-acyltransferase and requires ATP, CoA, and glycine as cosubstrates. Lipoic acid (LA), an important endogenous and also therapeutic compound, depletes hepatic CoA; therefore, it may interfere with glycine conjugation. To test this hypothesis, LA (0.5-1.5 mmol/kg ip) was given to anesthetized, glycine-loaded rats 1 hr before administration of benzoic acid (1 mmol/kg iv). LA inhibited glycine conjugation of benzoic acid in a dose-dependent manner as indicated by: 1) reduced clearance of benzoic acid from blood; 2) delayed appearance of benzoylglycine in blood; and 3) decreased excretion of benzoylglycine in urine. LA also decreased urinary excretion of injected benzoylglycine, indicating that reduced excretion of this metabolite after benzoic acid injection is caused by diminished formation and impaired renal transport of benzoylglycine. Urine formation was decreased by LA in a dose-dependent fashion, and acute renal failure was evident in rats receiving the highest dose. LA depleted hepatic CoA, carnitine, and glutathione, but not ATP, whereas it increased the hepatic concentration of glycine. In isolated and solubilized rat liver mitochondria, LA inhibited both benzoyl-CoA synthetase (IC50 approximately 1.5 mM) and benzoyl-CoA:glycine N-acyltransferase (IC50 approximately 0.3 mM). Thus, depletion of CoA and inhibition of the pertinent enzymes seem responsible for impairment of glycine conjugation of benzoic acid by LA. LA may also impair renal tubular cell function, compromising the tubular secretion of benzoylglycine and causing acute renal failure.

Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine.

In the course of glycine conjugation, benzoic acid is successively converted into benzoyl-CoA and benzoylglycine by mitochondrial enzymes (i.e. benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively), utilizing ATP, CoA, and glycine. Large doses of benzoate deplete CoA from the liver, suggesting that the supply of CoA may limit the capacity for glycine conjugation. Because fibrates are known to increase hepatic CoA synthesis, we examined whether treatment with fenofibrate or bezafibrate enhanced the capacity of rats to conjugate benzoic acid with glycine. Dietary administration of fenofibrate or bezafibrate (2.5 mmol/kg of feed, for 10 days) increased hepatic CoA levels 8-10-fold, while not affecting hepatic ATP levels; only fenofibrate elevated, albeit moderately, the concentration of glycine in liver. Hepatic mitochondria isolated from fibrate-fed rats, compared with those from controls, exhibited unchanged benzoyl-CoA synthetase activity but higher benzoyl-CoA hydrolase and lower benzoyl-CoA/glycine N-acyltransferase activities. Feeding with either fibrate increased liver mass by 50-60%. Control and fibrate-fed rats were administered benzoate at different doses, one to produce a large demand for CoA (i.e. 2 mmol/kg, iv) and two others to produce smaller demands for CoA (i.e. 1 mmol/kg or 2 mmol/kg plus glycine, iv). Fenofibrate-fed rats, and to a lesser extent bezafibrate-fed animals, exhibited increased glycine conjugation capacity, as indicated by faster disappearance of benzoate from the blood and appearance of benzoylglycine in the blood and urine, compared with controls; however, fibrates were not more effective in rats receiving the benzoate dose that produced the greatest demand for CoA. In contrast, benzoylglycine formation from benzoate (0.1-1 mM) was not enhanced in liver slices from fibrate-fed rats; moreover, it was lower than control levels in slices from bezafibrate-fed animals. Bezafibrate, but not fenofibrate, given to rats in a single dose (0.5 mmol/kg, ip) decreased the elimination and glycine conjugation of benzoate, indicating that bezafibrate is a direct inhibitor of glycine conjugation. In summary, fibrates influence glycine conjugation in a complex manner. Some fibrate-induced alterations (i.e. increased benzoyl-CoA hydrolase and decreased glycine transferase activities and direct inhibition by bezafibrate) can potentially hinder conjugation of benzoate with glycine, thus precluding conclusions regarding whether increased CoA availability enhances glycine conjugation. Fibrate-induced hepatomegaly appears to significantly contribute to the increased glycine conjugation capacity of rats treated with fenofibrate or bezafibrate.

Effects of Fibrates on the Glycine Conjugation of Benzoic Acid in Rats

Conjugation with glycine, a reaction important in the elimination of carboxylic acids (e.g. benzoic and salicylic acids), takes place in hepatic mitochondria and uses ATP, coenzyme A, and glycine. Although normal ATP supply does not limit glycine conjugation in vivo (Gregus, Z., et al., Drug Metab. Dispos. 20, 234, 1992), ATP deficiency may impair it. This hypothesis was tested by examining the effect of ATP depletors (oligomycin, fructose, and ethionine) on glycine conjugation and elimination of benzoic acid in rats. Pretreatment with the mitochondrial ATP synthesis inhibitor oligomycin (0.5-2 mg/kg, intraportally) decreased glycine conjugation of benzoic acid markedly and in a dose-dependent manner, as indicated by the delayed elimination of benzoate and delayed appearance of benzoylglycine in blood. Oligomycin also dramatically diminished urinary excretion of benzoylglycine, because it inhibited not only formation of benzoylglycine from benzoate, but also the renal transport of benzoylglycine. Treatment with fructose (a consumer of both cytosolic and mitochondrial ATP) or ethionine (a consumer of cytosolic ATP) depleted hepatic ATP from approximately 2.5 micromol/g to levels comparable with those observed after administration of 1 mg/kg oligomycin (approximately 1.2 micromol/g). Despite this, elimination of benzoate and formation of benzoylglycine were decreased less by fructose than by oligomycin and only negligibly by ethionine. ATP depletors did not influence hepatic glycine levels, and only oligomycin lowered coenzyme A levels in liver. However, the oligomycin-induced decline of hepatic coenzyme A levels was delayed, contrary to impairment of glycine conjugation, which was almost immediate. In summary, impairment of benzoylglycine formation by ATP depletors apparently correlates with their capacity to diminish ATP levels in hepatic mitochondria (i.e. at the site of glycine conjugation). These observations suggest that limited availability of mitochondrial (but not cytosolic) ATP reduces glycine conjugation capacity. Therefore, mitochondrium toxic agents and pathological mitochondrial injuries acting in liver may compromise glycine conjugation by decreasing ATP supply.

Does hepatic ATP depletion impair glycine conjugation in vivo

1. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (0.1-0.5 mmol/kg i.p.) delayed the disappearance of injected benzoate from blood and diminished the urinary excretion of the formed benzoylglycine, but elevated the blood levels of benzoylglycine in rat, suggesting that these herbicides interfere with both the formation and the renal transport of benzoylglycine. 2. Inhibition of the renal excretion of benzoylglycine by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in rat injected with benzoylglycine. 3. Inhibition of benzoylglycine formation from benzoic acid by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in renal pedicles-ligated rats injected with benzoate. 4. Neither 2,4-D nor 2,4,5-T influenced the hepatic concentrations of ATP, coenzyme A (CoA) or glycine; therefore, it is unlikely that they inhibit glycine conjugation of benzoic acid by diminishing the availability of co-substrates. 5. Although the chlorophenoxyacetic acids did not appear to be a substrate for the mitochondrial acyl-CoA synthetases, both 2,4-D and 2,4,5-T diminished the activity of benzoyl-CoA synthetase (but not that of benzoyl-CoA:glycine N-acyltransferase) in solubilized hepatic mitochondria. These findings suggest that 2,4-D and 2,4,5-T impair benzoylglycine formation in rat by inhibiting benzoyl-CoA synthetase.

Éva Halászi

Papers

Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine.

Effects of Fibrates on the Glycine Conjugation of Benzoic Acid in Rats

Does hepatic ATP depletion impair glycine conjugation in vivo

Effect of chlorophenoxyacetic acid herbicides on glycine conjugation of benzoic acid.