Nutrition 26 (2010) 449–458 www.nutritionjrnl.com Special article Erythritol is a sweet antioxidant Gertjan J.M. den Hartog, Ph.D.a,*, Agnes W. Boots, Ph.D.a, Aline Adam-Perrot, Ph.D.b,y, Fred Brouns, Ph.D.c, Inge W.C.M. Verkooijen, M.Sc.d, Antje R. Weseler, Ph.D.a, Guido R.M.M. Haenen, Ph.D.a, and Aalt Bast, Ph.D.a a Department of Pharmacology and Toxicology, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands b Cargill R&D Center Europe, Vilvoorde, Belgium c Department of Human Biology, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands d Clinical Trial Center Maastricht, Maastricht, The Netherlands Manuscript received November 21, 2008; accepted May 13, 2009.Abstract Objective: Hyperglycemia, oxidative stress, and the onset and progression of diabetic complications are strongly linked. Reduction of oxidative stress could be of utmost importance in the long-term treat- ment of diabetic patients. The chronic nature of the disease calls for a mode of antioxidant intake that can be sustained easily, e.g., by the diet. Erythritol, a simple polyol, could be such a compound. It is orally available, well tolerated, and its chemical structure resembles that of mannitol, a well-known hydroxyl radical (HO) scavenger. Methods: We studied the antioxidant properties of erythritol in vitro and subsequently determined its antioxidant activity and its vasoprotective effect in the streptozotocin diabetic rat. Results: Erythritol was shown to be an excellent HO radical scavenger and an inhibitor of 2,20 -azo- bis-2-amidinopropane dihydrochloride–induced hemolysis but inert toward superoxide radicals. High- performance liquid chromatographic and electron spin resonance spectroscopy studies showed that the reaction of erythritol with hydroxyl radicals resulted in the formation of erythrose and erythrulose by abstraction of a carbon-bound hydrogen atom. In the streptozotocin diabetic rat, erythritol displayed an endothelium-protective effect and, in accordance with the in vitro experiments, erythrose was found in the urine of erythritol-consuming rats. Conclusion: Erythritol acts as an antioxidant in vivo and may help protect against hyperglycemia- induced vascular damage. Ó 2010 Elsevier Inc. All rights reserved.Keywords: Erythritol; Diabetes; Oxidative stress; RatIntroduction oxygen radicals (most notably the hydroxyl radical [HO]) and lowers antioxidant defenses . Reducing hyperglycemia by Diabetes mellitus (DM) is a chronic disease with a rapidly replacing sucrose with sweeteners will probably reduce theincreasing prevalence characterized by hyperglycemia. It is as- amount of oxidative stress. The remaining oxidative damagesociated with a high risk of vascular, renal, neuronal, and ocular can be prevented by supplementing with antioxidants. Prevent-damage. Patients with DM often develop diabetic complica- ing hyperglycemia and supplying appropriate levels of antiox-tions including myocardial infarction, stroke, blindness, and idants seems a rational approach to reduce the amount ofgangrene . A large body of evidence indicates that these di- oxidative damage and thus the onset and development of dia-abetic complications are linked to oxidative stress . There is betic complications in patients with DM . Because of theconsiderable evidence that hyperglycemia causes formation of chronic nature of the disease, supplementation with antioxi- dants also has to be long term. Intake of appropriate levels of antioxidants with food or drink would be an acceptable proce- y dure and easy for the patient. Based on its molecular character- Current address: Tate Lyle Ingredients France, Villeneuve d’Ascq,France. istics, erythritol may be a good antioxidant to incorporate in *Corresponding author. Tel.: þ31-43-388-2916; fax: þ31-43-388-4149. food and beverage formulations. However, its antioxidant E-mail address: email@example.com (G. J. M. den Hartog). properties have thus far not been studied in detail. Erythritol0899-9007/10/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2009.05.004
450 G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458is a simple polyol (1,2,3,4-butanetetrol), present in small quan- adding a 200-mL aliquot to 2 mL of demineralized water. Thetities in melons and peaches, and currently produced in large percentage of hemolysis was calculated from the ratio ofquantities for use as a low-calorie, tooth-friendly bulk sweet- hemolysis in the test sample to 100% hemolysis. The timeener . Extensive toxicologic testing has shown that erythritol needed to obtain 50% hemolysis was determined for eachis well tolerated and has no toxic effects, even after consump- concentration by ﬁtting the data with the three-parameter sig-tion of large quantities [6,7]. In addition, it has no impact on moid model from SigmaPlot 2001 for Windows (SPSS Inc.,blood insulin or glucose levels, which renders it a useful and Chicago, IL, USA).safe food component for patients with DM. A compound thatclosely resembles erythritol is the therapeutically applied pol- Electron paramagnetic resonance spectroscopyyol mannitol, a well-known HO radical scavenger [8,9]. How-ever, in contrast to mannitol, of which about 75% remains The formation of radical intermediates during scavengingunabsorbed, erythritol is rapidly and virtually completely (up of hydroxyl radicals by erythritol was studied with an elec-to 90%) absorbed from the gut , which makes it easy to tron spin resonance spectrometer (EMX, Bruker GmbH,reach appropriate systemic concentrations required to neutral- Karlsruhe, Germany) with 5,50 -dimethyl pyrroline N-oxideize the highly reactive HO radicals. (DMPO) as a spin trap. The spectrometer settings were: The goal of the present study was to evaluate the antioxi- power, 20 mW; center ﬁeld, 3488 G; modulation amplitude,dant properties of erythritol in vitro and subsequently study 0.5 G; time constant, 20.48 ms.its potential protective effect in vivo in the streptozotocin-in- Identiﬁcation of oxidation productsduced diabetic rat. Erythritol (100 mM) was treated with a Fenton reagent (10 mM H2O2, 1 mM Fe[II]SO4, and 1.04 mM ethylenedia-Materials and methods minetetra-acetic acid) for 30 min. Analysis of oxidation prod- ucts was carried out according to Nascimento et al. . InHydroxyl radical scavenging short, samples were treated with 2,4-dinitrophenylhydrazine (2.5 mM) in the presence of perchloric acid to form the 2,4- The rate constants for the reaction with HO radicals of dinitrophenylhydrazones of any present carbonyl compound.erythritol and related polyols were determined according to After 60 min of shaking at room temperature the solution wasHalliwell et al. . Erythritol and other polyols (xylitol, sor- analyzed by high-performance liquid chromatographybitol, and mannitol) were tested in concentrations ranging (column Hypersil (Supelco Inc., Bellefonte, PA, USA)from 0 to 8 mM. 5 mm, eluent 25:75 acetonitrile:0.1% triﬂuoroacetic acid, detection at 365 nm).Superoxide radical scavenging The identity of the reaction products was determined by comparing their retention times with those for pure erythrose The superoxide radical scavenging activity of erythritol was (2,3,4-trihydroxybutanal) and erythrulose (1,3,4-trihydroxy-determined according to Kirkova et al.  using nitroblue tet- 2-butanone) that underwent the same derivation procedure.razolium (NBT; 50 mM) as detector for superoxide radicalsgenerated by xanthine 0.1 mM and xanthine oxidase 10 mU/ Antioxidant activity of erythritol in the streptozotocin-mL. Possible interference by inhibition of xanthine oxidase induced diabetic ratby the test compounds was checked by measuring the rate ofuric acid formation spectrophotometrically at 293 nm. The study protocol was approved by the ethics committee for animals of the Universiteit Maastricht. Twenty rats (Wis-Hemolysis assay tar, 10 male, 10 female, 200–250 g of body weight) were supplied by Charles River Nederland BV (Maastricht, Neth- To determine the antioxidant activity of erythritol, its erlands) and kept under standard conditions. Standard foodeffect on 2,20 -azobis-2-amidinopropane dihydrochloride– (Ssniff Spezialdiaten GmbH, Soest, Germany) and acidiﬁed ¨induced hemolysis was investigated according to Vosters drinking were provided ad libitum. The study’s protocoland Neve . In short, a red blood cell suspension was in- ` was approved by the ethics committee for animals of thecubated with erythritol (0–50 mM) for 5 min at 37 C after Universiteit Maastricht.which 2,20 -azobis-2-amidinopropane dihydrochloride(50 mM) was added. At regular intervals (0–300 min), 200- Induction of DMmL aliquots were taken and diluted in 2 mL of 0.9% NaCl so-lution. The extent of hemolysis was determined by treating Diabetes mellitus was induced according to the method ofthe sample with Hemoglobina TC (potassium hexacyanofer- Hasselbaink et al. . The rats were anaesthetized with hal-rate [III] 2.4 mM, potassium cyanide 3 mM) for hemoglobin othane and subsequently treated with 70 mg/kg of streptozo-determination. The absorbance of the mixture was measured tocin in citrate buffer (100 mM, pH 4.5) by intravenousat 540 nm. One hundred percent hemolysis was obtained by injection in the tail vein. Controls received citrate buffer
G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458 451only. After 7 d, blood glucose was determined with a glucose Thiobarbituric acid-reactive substances (TBARS) were mea-meter (Lifescan BV, Maastricht, Netherlands). sured in plasma according to Lepage et al. . The 10 diabetic rats were divided in two groups: those thatreceived normal drinking water (group D, n ¼ 5) and those Chemicalsthat received erythritol-supplemented water (group DE,n ¼ 5) for 21 d. The 10 non-diabetic rats were also divided Erythritol, xylitol, sorbitol, and mannitol were providedin two groups: normoglycemic rats that received normal by Cargill (Vilvoorde, Belgium). Dinitrophenylhydrazinedrinking water (group N, n ¼ 5) and those that received (DNPH) and high-performance liquid chromatographic elu-erythritol-supplemented water (group NE, n ¼ 5) for 21 d. ents were purchased from Fluka GmbH (Fluka Chemie The amount of solution consumed by each rat was moni- GmbH, Buchs, Switzerland) and recrystallized from acetoni-tored. The concentration of the erythritol solution was read- trile before use. Streptozotocin was purchased from Sigmajusted daily to ensure that each rat had a daily consumption Chemical Co. (St. Louis, MO, USA). All other chemicalsof 1000 mg of erythritol per kilogram of body weight. were of reagent grade.Weight and blood glucose levels of the rats were determinedweekly. Statistical evaluationEx vivo aortic reactivity measurements The data are expressed as mean 6 standard error. Differ- ences between groups were investigated with Student’s t test After sacriﬁcing the rats, with CO2/O2 blood was rapidly and those with a P value 0.05 were considered statisticallycollected by venipuncture. The thoracic parts of the aortas signiﬁcant.were excised. These were immediately placed in Krebs-Ringer buffer (NaCl 118 mM, KCl 4.4 mM, CaCl2 Results2.5 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, glucose10 mM, and NaHCO3 25 mM) after which excess fat and Hydroxyl and superoxide radical scavenging properties ofconnective tissue were removed. Rings approximately erythritol and related polyols4 mm in length were cut and mounted between stainless steelhooks in a 20-mL organ bath ﬁlled with Krebs-Ringer buffer, Figure 1 presents the effects of erythritol, xylitol, sorbitol,kept at 37 C, and bubbled with 95% O2/5% CO2. An isomet- and mannitol on hydroxyl radical–induced deoxyribose deg-ric force transducer (Grass FT03, West Warwick, RI, USA) radation. Erythritol scavenged HO radicals with a rate con-was attached to each strip, after which the resting tension stant of 1.18 3 109 M/s. In comparison, xylitol, sorbitol, andwas constantly readjusted to 1.0 3 g for 60 min. Integrity 1.6of the tissues was checked with phenylephrine (10 mM) and kS(x109 M-1s-1)carbachol (100 mM), followed by a washout period of 1.430 min. The aortic rings were then submaximally contractedwith phenylephrine (10 mM). When a stable contraction was 2.2 1.2established, a carbachol concentration–response curve(10 nM to 100 mM) was recorded. When a stable relaxation 2.0 1.0 4 5 6was obtained after the last addition of carbachol, sodium 1.8 Number of hydroxyl groupsnitroprusside (SNP; 100 mM) was added to the organ bathto induce maximum relaxation. 1.6 1/A532 The pD2 [negative logarithm of the concentration that pro- 1.4duces 50% of the maximal response (EC50)] values and max- 1.2imum effect of carbachol were obtained by ﬁtting the data Mannitol(four-parameter logistic curve, SigmaPlot 2001 for Win- Sorbitol 1.0 Xylitoldows, SPSS Inc., Chicago, IL, USA). Total relaxation (relax- Erythritol 0.8ation by carbachol and SNP) and the SNP fraction (part of thetotal relaxation caused by SNP) were calculated from the 0.6data. 0 2 4 6 8 10 Concentration (mM)Blood antioxidant and oxidative stress parameters Fig. 1. Effect of test compounds on the HO radical-induced degradation of 2-deoxyribose. Values are means of three experiments. SEMs for A532 were Erythrocyte glutathione (GSH) and glutathione disulﬁde approximately 2% of the measured value; error bars have been omitted for clarity. Fitting using linear regression (least squares method) was performed(GSSG) were determined according to Baker et al.  using SigmaPlot 4.01 for Windows (SPSS Inc., Chicago, IL, USA). (Inset)with the modiﬁcations of Vandeputte et al. . The trolox Correlation between the rate constant for HO radical scavenging and theequivalent antioxidant capacity (TEAC) was determined in number of hydroxyl groups present in the tested polyols. A532; absorbancedeproteinized plasma according to Arts et al. . at a wavelength of 532 nm.
452 G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458mannitol displayed rate constants of 1.42, 1.56, and 200 1901.62 3 109 M/s, respectively. An interesting correlation 180 100 170was observed between the number of hydroxyl groups in t50(min) 160the compound under investigation and its rate constant for 150the reaction with HO radicals (Fig. 1, inset). 80 140 130 Figure 2 shows the effect of erythritol on superoxide rad- 120 Haemolysis (%) 0 10 20 30 40 50ical–induced reduction of NBT. Erythritol at concentrations [Erythritol] (mM) 60of up to 2 mM did not lower the NBT reduction rate. Cate-chol, which was used as a reference compound, completelysuppressed NBT reduction at 30 mM. 40 0 mM erythritol 2.5 mM erythritol 20 5 mM erythritolHemolysis assay 10 mM erythritol 20 mM erythritol 50 mM erythritol Figure 3 shows the effect of erythritol (0–50 mM) on 2,20 - 0azobis-2-amidinopropane dihydrochloride–induced hemolysis. 0 50 100 150 200 250Erythritol caused a concentration-dependent increase in lag time Time (min)(the time it takes before hemolysis starts). The lag time was Fig. 3. Effect of erythritol (0–50 mM) on 2,20 -azobis-2-amidinopropane di-quantiﬁed by determining the time needed to obtain 50% hemo- hydrochloride–induced hemolysis. Data points represent the average of threelysis. The relation between the increase in lag time and the eryth- separate experiments. Curves were ﬁtted using SigmaPlot (three-parameterritol concentration is displayed in the inset of Figure 3. sigmoidal). Erythritol concentration-dependently shifted the curve to the right. (Inset) Relation between erythritol concentration and t50. t50, time needed to obtain 50% hemolysis.Electron spin resonance Oxidation products Scavenging of HO radicals by sugars probably proceeds According to the mechanism proposed in Figure 4, expo-by abstraction of a hydrogen atom, as shown in Figure 4. This sure of erythritol to HO radicals results in the formation ofmechanism was veriﬁed by spin trapping and analysis using a carbonyl group containing the products erythrose or eryth-an electron spin resonance spectrometer. rulose. Figure 6 shows chromatograms of erythritol solution Exposure of erythritol to HO radicals in the presence of before (Fig. 6B) and after (Fig. 6A) exposure to HO radicalsDMPO yielded a 1:1:1:1:1:1 spectrum (Fig. 5) with hyperﬁne followed by treatment with DNPH. Exposure of erythritol tosplitting AN ¼ 15.8 G, AH ¼ 22.3 G. This is consistent with HO radicals resulted in the formation of an at least 5-generation of an adduct of a carbon-centered radical with carbonyl group containing DNPH reactive oxidation prod-DMPO (DMPO-R) . Also visible in this spectrum is a sig- ucts. The two most prominent products had chromatographicnal from a DMPO-OH adduct (1:2:2:1) with hyperﬁne split- properties identical to those of the hydrazones of erythroseting AN ¼ 15.0 G, AH ¼ 14.8 G which is the result of [retention time (RT) 3.99 min] and erythrulose (doublescavenging of HO radicals by DMPO (AN and AH are split- peak at RT 5.09 min and RT 5.41 min).ting constants due to nitrogen or hydrogen nuclei). 0.16 Erythritol in the streptozotocin-induced diabetic rat General characteristics NBT reduction (ΔA560.min-1) 0.12 Blood glucose levels were increased 7 d after treatment with streptozotocin (25.0 6 1.4 mM) compared with control rats (6.7 6 0.2 mM). Mannitol 0.08 Sorbitol Xylitol Blood antioxidant and oxidative stress parameters Erythritol Catechol Antioxidant levels and status were determined at the end 0.04 of the study in the animals by measuring erythrocyte GSH and GSSG concentrations and the TEAC value. As presented in Table 1, no differences among the different experimental 0.00 groups were observed in GSH and GSSG levels. In diabetic 0 500 1000 1500 2000 2500 rats, the erythritol-supplemented rats (DE group) tended to μ Concentration (μM) have slightly higher GSH (172 versus 162 mM) and slightlyFig. 2. Effect of test compounds on the rate of NBT reduction in a system lower GSSG (15 versus 14 mM) levels than normoglycemicconsisting of xanthine and xanthine oxidase. Values are means 6 SEMs of rats that drank water (D group). The same effect of erythritolthree experiments. NBT, nitroblue tetrazolium. supplementation was observed in the normoglycemic rats.
G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458 453 O2 H O H C OH H C OH H OH + O2 -HO2 H OH H OH H OH H OH H OH CH2OH CH2OH CH2OH CH2OH H OH + HO Erythrose H OH CH2OH CH2OH CH2OH Erythritol HOH2C C OH + O2 O2 C OH O -HO2 H OH H OH H OH CH2OH CH2OH CH2OH Erythrulose Fig. 4. Proposed mechanism for the scavenging of HO radicals by erythritol. HO radicals attack erythritol by abstracting carbon-bound hydrogen atoms, gen-erating two different primary erythrityl radicals. In the presence of oxygen, these carbon-centered radicals are converted into the corresponding peroxyl radicals,which after expulsion of a protonated superoxide radical (HO2), yield erythrose or erythrulose.The TEAC did not differ among experimental groups. How- presents the carbachol concentration–response curves fromever, a trend was observed that the diabetic rats (D and DE rats in the D and DE groups. Total relaxation in the aortic seg-groups) displayed somewhat lower TEAC values than their ments from group DE was greater, although not signiﬁcantly.normoglycemic control counterparts (N and NE groups). In The pD2 value for carbachol in the aortic rings from groupaddition to the antioxidant parameters, we assessed oxidative DE was higher and approached the value found in the aorticdamage in the rats by measuring TBARS levels in plasma. rings of groups N and NE (Table 2).Similar TBARS levels were found in all four experimentalgroups (Table 1). DiscussionUrine production and analysis Urine from all four groups was collected for 24 h. Samples Erythritol is intended for use as a bulk sweetener in, e.g.,were treated with DNPH and the amount of erythrose hydra- confectionery, chewing gum, beverages, and bakery prod-zones was determined by high-performance liquid chroma- ucts. Because it does not affect glucose and insulin levels,tography. The total amount of erythrose excreted (urinary it is safe for diabetics. Its structural properties resemble thoseerythrose concentration 3 urine production in 24 h) was of mannitol, a well-known antioxidant. Because oxidativehigher in the DE group (262 6 37 nmol) compared with damage has been implicated in the pathogenesis and develop-the NE group (28 6 6 nmol, P 0.05; Fig. 7). ment of diabetic complications, the antioxidant activity of erythritol was investigated in vitro and in vivo.Endothelial function The hydroxyl radical scavenging activity of erythritol and Endothelial function was determined by measuring the re- other polyols such as xylitol, sorbitol, and mannitol weresponse of isolated aortic rings to phenylephrine, carbachol, evaluated in the deoxyribose assay (Fig.1). The rate constantand SNP. As presented in Table 2, phenylephrine 100 mM for the reaction was found to be close to that of mannitol. Incaused an identical contraction in the aortic rings from all fact, a good correlation between the number of hydroxylfour groups. Figure 8 shows the carbachol concentration– groups in a polyol and its rate of HO radical scavengingresponse curves recorded with rings from diabetic rats (D was found (Fig. 1, inset). Erythritol is therefore an appealinggroup) and normoglycemic rats (N and NE groups). The car- HO radical scavenger that combines a high scavenging ratebachol concentration–response curve from aortic rings from constant with good bioavailability. Erythritol did not affectdiabetic rats differed markedly from the curves obtained superoxide-induced reduction of NBT at concentrations ofwith rings from the normoglycemic rats. The pD2 value for up to 2 mM. This did not come as a surprise, because eryth-carbachol was lower in the aortic rings obtained from the di- ritol does not possess a hydroxyl group attached to anabetic animals (Table 2). The curve was shifted to the right aromatic moiety, which has been shown to be a major struc-and total relaxation was lowered (Fig. 8, Table 2). Figure 9 tural requirement for superoxide scavengers .
454 G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458 O PH DN X X O X X X e os X e hrul os ryt O y thr E O Er A B 0 2 4 6 8 10 Retention time (min) Fig. 6. Chromatograms A (erythritol plus Fenton reagent treated with DNPH) and B (blank, erythritol treated with DNPH). Oxidation products ob- served in chromatogram A are erythrose (3.99 min), unknown 1 (4.85 min), erythrulose, double peak (5.09 and 5.41 min), unknown 2 (6.73 min), un- known 3 (8.11 min), and unknown 4 (9.31 min). The large peak visible in 10 G both chromatograms at time 8.56 min is unreacted DNPH. DNPH, dinitro- phenylhydrazine.Fig. 5. Electron spin resonance spectrum of the mixture containing H2O2(2 mM), 5,50 -dimethyl pyrroline N-oxide (50 mM), erythritol (500 mM),and FeSO4 (1 mM) 75 s after mixing the components of the incubation. X, The antioxidant properties of erythritol in the in vitro ex-peaks belonging to the carbon-centered radical adduct; O, peaks belongingto the 5,50 -dimethyl pyrroline N-oxide OH adduct. periments prompted us to test its antioxidant activity in dia- betic rats. Yokozawa et al.  studied the effect of erythritol on glucose metabolism and oxidative stress in Because of their susceptibility to peroxidation, red blood diabetic rats. Erythritol (100, 200, or 400 mg/kg of bodycells were used as a model to investigate the ability of eryth- weight per day) showed a beneﬁcial effect through loweringritol to prevent oxidative damage in biological membranes. glucose levels of serum (by 15% at the highest dose) and he-Erythritol inhibited radical-induced hemolysis in a concentra- patic and renal tissues. Our in vivo study focused on the HOtion-dependent manner, which indicates that erythritol is also radical scavenging activity of erythritol and the possible pre-able to exert its antioxidant activity in a cellular system. vention of diabetic complications. HO radicals are ex- Schuchmann and von Sonntag  thoroughly investi- tremely reactive and because of that almost instantaneouslygated the reaction between glucose and HO radicals. If theirﬁndings are applicable to the reaction of erythritol with HO Table 1radicals, erythrose and erythrulose would be formed as Antioxidant and oxidative stress parameters in blood*shown in Figure 4. This scheme is supported by the ﬁndings Experimental GSH GSSG TEAC TBARSof Cherqaoui et al.  who detected these compounds after group (mmol/L) (mmol/L) (mmol/L) (mmol/L)electrolysis of an aqueous erythritol solution with platinum N 139 6 12 11.9 6 0.7 589 6 34 3.5 6 0.2electrodes. Our study provides strong evidence that exposi- NE 153 6 13 10.7 6 1.1 638 6 34 3.8 6 0.2tion of erythritol to HO radicals indeed results in the forma- D 162 6 7 14.8 6 1.8 561 6 53 3.7 6 0.2tion of erythrose and erythrulose, among two or three other, DE 172 6 16 14.2 6 0.6 517 6 34 3.9 6 0.5as yet unidentiﬁed, minor oxidation products. The mecha- D, diabetic rats that received normal drinking water; DE, diabetic rats thatnism by which erythrose and erythrulose are formed out of received erythritol-supplemented water; GSH, glutathione; GSSG, glutathi-erythritol appears to be analogous to the ﬁndings of one disulﬁde; N, normoglycemic rats that received normal drinking water; NE, normoglycemic rats that received erythritol-supplemented water;Schuchmann and von Sonntag, as shown by our spin trapping TBARS, thiobarbituric acid-reactive substances; TEAC, trolox equivalentexperiment in which we conﬁrmed the formation of a carbon- antioxidant capacitycentered radical after abstraction of a carbon-bound hydrogen * Values are means 6 SEMs, n ¼ 5 for groups N and NE and n ¼ 4 foratom by a HO radical. groups D and DE.
G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458 455 * 0,8 300 250 0,6 Erythrose excretion /24hr Relaxation (g) 200 0,4 (nmoles) 150 * 0,2 100 0,0 50 0 -0,2 N NE D DE -8 -7 -6 -5 -4 -3 Experimental group log [carbachol]Fig. 7. Urinary erythrose excretion per 24 h by the four experimental groups. Fig. 8. Carbachol concentration–response curves recorded with aortic rings* P 0.05 compared with NE group. D, diabetic rats that received normal from normoglycemic rats (ﬁlled circles), normoglycemic rats that had con-drinking water; DE, diabetic rats that received erythritol-supplemented wa- sumed erythritol (1000 mg $ kgÀ1 $ dÀ1, open circles), and diabetic rats (tri-ter; N, normoglycemic rats that received normal drinking water; NE, normo- angles). Consumption of erythritol did not have an effect on theglycemic rats that received erythritol-supplemented water. concentration–response curve. The curve recorded with the diabetic rat aortic rings is shifted to the right with a lower maximum effect compared with curves from the normoglycemic rats.react with any biomolecule in their vicinity. It is thereforecrucial that plasma concentrations in the millimolar rangeare reached. To obtain these levels rats were supplemented urine of diabetic rats that were supplemented with erythritolwith erythritol at the highest well-studied dose, i.e., was investigated. Interestingly, erythrose was indeed found1000 mg $ kgÀ1 $ dÀ1 for a period of 3 wk after diabetes in the urine of diabetic rats, indicating that HO radicals arewas induced. HO radical scavenging by erythritol in the di- produced systemically during hyperglycemia and that theseabetic rat was investigated by studying the presence of eryth- have been scavenged by erythritol. However, it should berose and/or erythrulose in the urine of diabetic rats. Under realized that the kinetic consequences of the extremely highnormal circumstances, erythritol is excreted unchanged in reactivity of the HO radical make it questionable that it isthe urine . In the hyperglycemic state, high levels of sys- only scavenged by erythritol. Furthermore, it cannot be ruledtemic oxidative stress are known to be present [3,25]. If out that the erythrose also originates from alcohol dehydroge-HO radicals are among the reactive species formed and suf- nases, which might also explain the presence of erythrose inﬁcient erythritol is present, it would be expected that the ox- the normoglycemic rats.idative metabolites erythrose and erythrulose would be The effect of erythritol on endothelial function was studiedformed. Their hydrophilicity and structural similarity to er- in rings prepared from the thoracic aorta. Contraction was eli-ythritol make it likely that these compounds are excreted cited by stimulating with phenylephrine an a-adrenergic re-by the urine. Therefore, the presence of erythrose and eryth- ceptor agonist. Subsequently, the endothelium-dependentrulose, two of the oxidative metabolites of erythritol, in the relaxation was studied by constructing concentration–Table 2Effect of erythritol on reactivity in aortas from normoglycemic and diabetic rats* Normoglycemic Hyperglycemic Control Erythritol Control ErythritolPE (g) 0.66 6 0.09 0.70 6 0.06 0.62 6 0.02 0.60 6 0.07CAR (g) 0.63 6 0.07 0.60 6 0.07 0.48 6 0.03 0.61 6 0.07CAR pD2 6.12 6 0.05 5.98 6 0.02 5.35 6 0.07 5.65 6 0.05SNP (g) 0.13 6 0.03 0.21 6 0.06 0.30y 6 0.07 0.14 6 0.03TR (g) 0.76 6 0.09 0.81 6 0.09 0.78 6 0.04 0.75 6 0.09SNP fraction (%) 17.4 6 3.1 24.7 6 5.5 36.8 6 7.0y 17.8 6 3.1z CAR, relaxation induced by carbachol 100 mM; pD2, negative logarithm of the concentration that produces 50% of the maximal response (E50); PE, contractioninduced by phenylephrine 10 mM; SNP fraction, contribution of sodium nitroprusside 100 mM to total relaxation; SNP, relaxation induced by sodium nitroprusside100 mM after stable response to carbachol 100 mM was obtained; TR, total relaxation induced by carbachol 100 mM followed by sodium nitroprusside 100 mM * Values are means 6 SEMs, n ¼ 5 for normoglycemic rats that received normal drinking water and normoglycemic rats that received erythritol-supplementedwater and n ¼ 4 for diabetic rats that received normal drinking water and diabetic rats that received erythritol-supplemented water. y P 0.05 compared with normoglycemic rats that received normal drinking water. z P 0.05 compared with diabetic rats that received normal drinking water.
456 G. J. M. den Hartog et al. / Nutrition 26 (2010) 449–458 0,8 to induce maximum relaxation. The smooth muscle that lies underneath the endothelium, however, appears to be intact 0,6 as evidenced by the unaltered contractile response to phenyl- ephrine and the total relaxant response to the carbachol–SNPRelaxation (g) 0,4 combination. In the diabetic rats that were consuming erythritol, the endothelium appeared intact, and the response to carbachol was almost identical to that observed in the nor- 0,2 moglycemic rats. Reﬂecting this uncompromised response to carbachol is the smaller SNP contribution to the total relaxa- 0,0 tion of aortic segments in the erythritol-supplemented diabetic rats. Our results are in good agreement with those obtained by -0,2 Dhein et al.  who studied the effect of vitamin E supple- -8 -7 -6 -5 -4 -3 mentation in the streptozotocin-induced diabetic rat. After 7 log [carbachol] mo endothelium-dependent relaxation was found to be de-Fig. 9. Carbachol concentration–response curves recorded with aortic rings creased by 50% in diabetic rats on a medium or high vitaminfrom diabetic rats (closed circles) and diabetic rats that had consumed eryth- E diet, whereas in vitamin E–deprived rats, a complete loss ofritol (1000 mg $ kgÀ1 $ dÀ1, open circles). Consumption of erythritol caused endothelium-dependent relaxation was found . Froma leftward shift of the concentration–response curve and an increased maxi- these results and the results of our study it can be concludedmum effect. that dietary antioxidants can play an important role in prevent- ing vascular damage associated with diabetes. It is neverthe-response curves with the muscarinic receptor agonist carba- less intriguing that two different compounds, vitamin E andchol. In rat aorta the relaxant response to carbachol is medi- erythritol, which differ greatly in terms of lipophilicity andated by nitric oxide (NO) produced in the endothelium. NO structural characteristics such as the presence of an aromaticdiffuses to the vascular smooth muscle cells, where it activates function, demonstrate a similar vasoprotective effect incyclic guanosine monophosphate formation by soluble gua- vivo. The lipid-soluble vitamin E owes its antioxidant activitynylate cyclase, which eventually causes relaxation. Maximum to trapping lipid peroxyl radicals in membranes . Thisrelaxation was induced by the addition of SNP, a compound contrasts sharply with the highly hydrophilic erythritol, whichthat releases NO and thus bypasses endothelial NO produc- will almost exclusively act in the aqueous compartments. Fur-tion. Our in vitro determination of the endothelium-dependent ther mechanistic research into the vasoprotective effect of er-relaxation of aortic rings showed that the carbachol response ythritol is therefore indicated. In addition to its potentialin aortic segments from diabetic rats was markedly attenuated vasoprotective action, we investigated the effect of erythritolafter 3 wk of hyperglycemia. The pD2 value and the maximum supplementation on antioxidant levels and markers of oxida-effect were lowered compared with those in normoglycemic tive stress in blood. Surprisingly, the hyperglycemia to whichrats. Total relaxation of the diabetic rings was not less com- the diabetic rats were subjected did not cause detectablepared with the other groups but a larger part had to be pro- changes in GSH, GSSG, and total antioxidant capacity. Thesevoked by SNP, indicating that the relaxant capacity of ﬁndings contrast with the larger part of data on antioxidantvascular smooth muscle was not affected in the diabetic levels and oxidative stress markers in diabetic patients andrats. The attenuated response to carbachol in diabetic rats laboratory animals [1,2,30,32]. This discrepancy may resultcombined with the unaltered total relaxation provides strong from the relatively short period (21 d) that our diabetic ratsevidence that NO production is hampered in diabetic animals. were hyperglycemic. Their antioxidant reserves may haveIt is known that hyperglycemia is associated with attenuation been sufﬁcient to prevent systemic oxidative damage foror loss of endothelium-dependent relaxation  and that re- that period. The observed endothelial dysfunction may beactive oxygen species mediate this loss . This reduced en- the result of locally generated HO radicals, which ﬁts per-dothelium-dependent vasodilator capacity of the coronary fectly with the observation of Pennathur et al.  who foundand peripheral circulation to acetylcholine or other M3 recep- that glycoxidation reactions in the arterial microenvironmenttor agonists is commonly referred to as endothelial dysfunc- form the ﬁrst step toward heart disease in diabetics. Thus, nottion . Endothelial dysfunction is thought to play an the generalized oxidative stress but rather the very local over-important role in vascular diabetic complications or diabetic production of HO radicals could be responsible for the ef-vasculopathy  and appears to be a stepping stone for ath- fects we observed. Pennathur et al.  also demonstratederosclerosis . In diabetic rats (group D) we found an atten- that a HO radical-like species is implicated in diabetic vas-uated response to the vasorelaxant compound carbachol. The culopathy by comparing oxidation products of amino acidstotal relaxation and the pD2 value were lower in aortic rings oxidized in vitro with those obtained from human arteries.from group D, whereas the total relaxant response (carbachol The protective effects of erythritol need not be restricted toplus the NO-donating compound SNP) was unchanged. This DM. Its unique free radical scavenging properties could beindicates that the endothelium of the diabetic rats was dam- beneﬁcial in other chronic disorders in which oxygen radicalsaged and therefore not capable of generating sufﬁcient NO are responsible for tissue damage. Efﬁcient scavengers of
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