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Advanced glycation endproducts form during ovalbumin digestion in the presence of fructose: Inhibition by chlorogenic acid
Fitoterapia 120 (2017) 1–5
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Advanced glycation endproducts form during ovalbumin digestion in the presence of fructose: Inhibition by chlorogenic acid
MARK
Yasmin Bains, Alejandro Gugliucci⁎, Russell Caccavello Glycation, Oxidation and Disease Laboratory, Dept. of Research, College of Osteopathic Medicine, Touro University-California, Vallejo, CA, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Advanced glycation Fructose Inflammation Ovalbumin
Background: One mechanism by which fructose could exert deleterious effects is through intestinal formation and absorption of pro-inflammatory advanced glycation endproducts via the Maillard reaction. We employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that advanced glycation endproducts (AGEs) are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions. Methods: OVA was subjected to simulated gastric and intestinal digestion using standard models, in presence of fructose or glucose (0–100 mM). Peptide fractions were analyzed by fluorescence spectroscopy and intensity at Excitation: λ370 nm, Emission: λ 440 nm. Results: AGE adducts formed between fructose and OVA, evidenced by the peptide fractions (< 5 kDa) at times (30 min) and concentration ranges (10 mM) plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction was inhibited by chlorogenic acid at concentrations compatible with those found in the gut. The reaction was also inhibited by aminoguanidine, a specific antiglycation agent. Conclusion: Our study showed fructose-AGE formation on a ubiquitous dietary protein under model physiological conditions. Our study also suggests ways to decrease the damage: enteral fructose-AGE formation may be partially inhibited by co-intake of beverages, fruits and vegetables with concentrations of phenolics high enough to serve as anti-glycation agents.
1. Introduction The association of high fructose diets with metabolic syndrome, diabetes and obesity is undeniable [1–5]. Large epidemiological studies have so demonstrated, notably for soda and fruit juices consumption. Nevertheless, correlation cannot adjudicate causation and some researchers maintain it is just due to extra calories, not to specific mechanistic action. One mechanism by which fructose could exert deleterious effects in metabolism and inflammation is via its potency in the Maillard reaction. Indeed, fructose is 7–10 times more potent a glycating agent than glucose due to the instability of the Heyns product it produces [6–10]. These early glycation products undergo further rearrangement, dehydration, and condensation reactions to produce irreversibly cross-linked derivatives called advanced glycation endproducts (AGEs), some of which are fluorescent. Fructose-specific AGEs, however have not been well characterized. Moreover, except for the liver, fructose concentrations in serum are low (micromolar range). Therefore, the role of endogenous fructose glycation (with the possible exception of the polyol pathway) as a significant pathogenic process is
difficult to substantiate [11]. Until recently, little attention has been paid to the possibility of endogenous formation of AGEs between fructose and proteins, precisely where they are more concentrated – the intestinal lumen [12] According to this hypothesis, fructose derived AGEs form in the intestines when fructose to glucose ratios exceed 1:1, but not after consumption of sucrose, or equal monomers of fructose and glucose [12]. Foods and beverages sweetened with high fructose corn syrup (HFCS) contain elevated levels of fructose [13] [14–15]. The same is true for apple juice [14–15]. Through careful epidemiological studies using NHANES data, over the past 5 years evidence for an independent effect of excess fructose ingestion and a range of inflammatory conditions has been found (asthma, chronic bronchitis and arthritis [12–13,16–19]. The authors suggest these associations may be mediated through in situ enteral formation of fructose AGEs, which, after being absorbed, may contribute to inflammatory diseases via engagement of the proinflammatory receptor for advanced glycation endproducts (RAGE) [12,20–21]. Other food AGEs (preformed in cooking) have been shown to actually be absorbed and be proinflammatory in animal and some human intervention studies
Abbreviations: AGE, advanced glycation endproducts; AG, aminoguanidine; RAGE, receptor for advanced glycation end products ⁎ Corresponding author at: Touro University-California, 1310 Club Drive, 94592 Vallejo, CA, USA. E-mail address: [email protected] (A. Gugliucci). http://dx.doi.org/10.1016/j.fitote.2017.05.003 Received 28 March 2017; Received in revised form 11 May 2017; Accepted 16 May 2017 Available online 17 May 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
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4. Fluorescence spectroscopy
[22–24]. However, several other studies have not shown this effect so this topic remains controversial [25–28]To shed some light on the luminal formation of AGES by fructose, we recently conducted a proof of principle study where we showed that fluorescent AGE adducts form between fructose and amino acids at times and concentration ranges plausibly found in the intestines, whereas no detectable reaction occurs with glucose [29]. The reaction was inhibited by chlorogenic acid and Ilex paraguariensis extracts (containing high concentrations of chlorogenic acid), suggesting that intake of food or herbal teas containing polyphenols may help dampen this process. In order to shed some light on the initial mechanism suggested by the above-mentioned hypothesis, in this follow up study we employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that indeed AGEs are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions.
The interaction between fructose and ovalbumin peptides was studied by fluorescence spectroscopy. AGE fluorescence spectra between 390 and 600 nm (excitation, 370 nm) were recorded at room temperature on a Biotek Synergy. 4.1. SDS-page Samples were dissolved to a protein concentration of 1 mg/ml or 1/ 1 for peptide fractions in 10 mM Tris-HCl buffer, pH 8, containing 2.5% SDS, 5% 2-β-mercaptoethanol, and 10 mM EDTA and heated at 100 °C for 10 min. SDS-PAGE was performed using precast Mini-PROTEAN TGX 4–20% gels (Bio-Rad, USA). Following the electrophoretic gels were stained with silver nitrate. A LMW Calibration Kit for SDS (Amersham Biosciences, USA) was used.
2. Material and Methods 4.2. Statistical analysis All reagents were purchased from Sigma St Louis, MO. OVA digestion was conducted employing classical models [30–32].
Experiments were run 3 times in duplicates. Data are mean +/− SD. All differences found are significant p < 0.05. Significance of differences was assessed with the Student's t-test.
2.1. In vitro gastric digestion 5. Results and discussion
Ovalbumin (OVA, A-5503, 98% purity, Sigma, MO, USA) was dissolved in simulated gastric fluid (SGF, 150 mM NaCl, 20 mM KH2PO4) at pH 1.2, to a final concentration of 25 mg/ml (0.42 mM) and subjected to an in vitro gastric digestion at 37 °C with porcine pepsin (EC 3.4.23.1, 3440 units/mg, P7012, Sigma) at an enzyme/ substrate ratio of 1:20 w/w (172 units/mg) [31]. Digestion was conducted in 50 ml cell culture tubes under rotatory movement at 100 rpm. After 2 h the reaction was stopped by adding 0.1 M NaOH to titrate pH to 7.0. At least triplicate digestions were conducted for each condition.
This study was designed as a follow up of our earlier proof of principle study [29] where we showed that unlike glucose, fructose forms fluorescent advanced glycation endproducts with basic and neutral aminoacids at conditions and times compatible with those in the digestive system lumen. In this follow-up work we have simulated the gastrointestinal proteolysis of OVA using a classic in vitro digestion system in two steps, which mimics the successive passage through the stomach and duodenum. We show that fructose produces AGE-peptides in OVA during simulated digestion, at times and concentrations compatible with physiological conditions. The allergenic potential of OVA [30–32] may be thus enhanced by endogenous formation of AGEs during digestion when consumed with fructose. Fig. 1 shows digestion of OVA (lane 1) during the stomach (lane 2) and intestinal phase (lanes 3 and 5) of simulated digestion. Peptic digestion of OVA (lane 1, time 0) leads to multiple peptides of decreasing molecular weights, as shown in lane 2, which is enhanced by intestinal digestion. The pattern of digestion is in agreement with previous reports [30–31]. The peptide fractions (< 10 kDa, lanes 4 (control) and 6 (fructose) run off the gels for the most part, indicating that they contain peptides smaller than 5 kDa as well as free aminoacids. No significant difference in the digestion pattern was seen between
2.2. In vitro duodenal digestion Duodenal digestions were performed by using, as the starting material, the 120 min gastric digests as described above adjusted to pH 7.0, with the addition of 1 mM CaCl2, 1 mM MgCl2, and a 0.125 M bile salt mixture B3426 (Sigma). After preheating at 37 °C for 15 min, a 10 × solution of pancreatin from porcine pancreas (containing all pancreatic enzymes) in NaHCO3 buffer ph 8. The final composition of the mixture was 0. 4 mM OVA at an enzyme/substrate ratio of 1:25 w/ w, 20 mM HCO3, 1 mM CaCl2, 1 mM MgCl2, ph 7.0. Aliquots were taken at different times and digestion was stopped by changing pH to 2.0. The mixture was chilled in ice and peptides were immediately separated. At least triplicate digestions were conducted for each condition. Digestions were performed in the absence or presence of a range of fructose or glucose concentrations (0–100 mM), with and without the presence of glycation inhibitors. Aminoguanidine (AG, the classic AGE inhibitor, Sigma St Louis, MO) as well as 5-caffeoylquinic acid (cholorogenic acid, Sigma St Louis, MO) were employed as inhibitors.
3. Peptide separation Digested peptides and free aminoacids (< 10 kDa) were separated by ultrafiltration through 0.5 ml Amicon Ultra Centrifugal filters spun at 14,000g for 30 min in a refrigerated centrifuge at 4 °C. Samples (triplicates of ultrafiltrates) were read in a Biotek Synergy H1. Top fluorescence was recorded at Excitation: λ370 nm, Emission: λ 440 nm against blanks, corrected by absorbance at 280 nm and expressed in AU as previously described.
Fig. 1. SDS-PAGE analysis of OVA during in vitro simulated stomach and intestines digestion. Samples were run in 4–20% polyacrylamide gels under reducing conditions. OVA was digested with pepsin for 2 h at 37 °C to simulate gastric digestion, followed by digestion with pancreatic enzymes for 2 h at 37 °C to simulate enteral digestion, as described in Methods. MWM: molecular weight markers. 1: OVA at time 0; 2: OVA after 2 h pepsin; 3: OVA after 2 h pancreatic enzymes; 4: peptide fraction of 4; 5:OVA + 50 mM fructose after 2 h pancreatic enzymes; 6: peptide fraction of 5.
2
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A
Fig. 2. Emission absorption spectrum of AGEs in the peptide fraction of OVA digestion. OVA was digested in the presence of 50 mM fructose or buffer (control) for 2 h of intestinal digestion. The peptide fraction was obtained by ultrafiltration through 10 kDa cut off Amicon filters. Spectrum was obtained after excitation at 370 nm using OVA incubated in the absence of fructose as blank.
B
fructose-incubated samples (lane 5) and controls (lane 3). Ovalbumin was chosen as a model protein for several reasons. First, it is a key nutritional protein in our diets. Second, its digestion has been thoroughly studied. Finally, it is particularly interesting since it is somewhat resistant to peptic digestion and its peptides are allergenic and pose a problem to some children [30–31]. More interestingly, extensive glycation of OVA (as in cooking) has already been substantiated as factor enhancing the antigen uptake and presentation by human dendritic cells in vitro [32]. Therefore, a known pathway exists for OVA peptides to interact with antigen presenting cells in the gut. Would that be enhanced by these peptides containing fructose-derived AGEs formed in situ? If this is true, they would engage RAGE on top of their intrinsic known deleterious action and this would help explain the epidemiological evidence showing that only fructose in excess of glucose is strongly associated with asthma, arthritis and other inflammatory ailments. The OVA concentrations chosen were adequate to mimic a meal of an average protein consumption of 1 g/kg weight per day, in 3 meals. That is 20–25 g protein, diluted in about 1 l of gastric fluid. As shown in Fig. 2, simulated digestion of OVA in the presence of fructose (but not with glucose,) leads to the formation of compounds, present in the molecular weight fraction < 10 kDa that possess the characteristic fluorescence spectrum of AGEs at excitation 370 nm. Glucose incubation did not produce changes in the spectrum over the control (data not shown). Of note, fluorescent compounds were also seen at time 0, indicating the purified OVA already contains AGEs. Fig. 3 shows time (3A) and dose dependent (3B) formation of AGE fluorescence in peptides during simulated digestion. Notably, at 50 mM fructose AGE fluorescence is already apparent after just 30 min duodenal digestion, a time frame well compatible with the digestive process. At 120 min of intestinal digestion AGE formation is already apparent at 10 mM fructose concentrations. We show a time and dose dependent formation of fluorescent AGEs between fructose (but not glucose) and OVA during simulated digestion at concentrations plausibly found in the gastrointestinal lumen (10–50 mmol/l). Indeed, in the typical Western diet over 200 mmoles of fructose in liquid form can be ingested in two glasses of apple juice or high fructose corn syrup sweetened soda, usually together with a load of protein and fat that delays gastric motility [11]. This usually leads to 12–18 g (up to 130 mmoles) of excess fructose that is not co-absorbed with glucose and therefore has an increased luminal residence time [12]. Even if we allowed for an exaggerated 1/5 dilution of these amounts due to fluid changes and poor absorption, these concentrations are compatible with
Fig. 3. Time and concentration dependent formation of AGE-peptides during OVA digestion A) Concentration dependent fructose AGE formation. OVA was incubated in the presence or absence (blanks) of fructose for 2 h of stomach conditions and 2 h of intestinal conditions as described in Methods, for 3 h. B) Time dependent fructose AGE formation. OVA was incubated in the presence or absence (blanks) of 50 mM fructose for 2 h of stomach conditions and the indicated times of intestinal conditions as described in Methods. Data are means +/− SD of triplicates. Top fluorescence (expressed in arbitrary units) was recorded at Excitation: λ350 nm, Emission: λ 440 nm against blanks (OVA incubated under the same conditions in the absence of fructose). Experiments were run 3 times. Data are mean +/− SD. All differences found are significant, p < 0.05.
the ones shown here to allow for formation of AGEs. It is important to point out that extended contacts between fructose and proteins, peptides and amino acids is greatly enhanced by fructose malabsorption [33–34]. This usually follows ingestion of food or beverages that contain a ratio of fructose to glucose > 1 [17–18]. Otherwise, enhanced absorption of fructose by glucose would tend to shorten contact times. For instance, fructose malabsorption due to large intake of high fructose corn syrup (HFCS) and apple juice has been shown [33–36]. Table 1 shows inhibition of AGE formation by aminoguanidine and phenolics. AGE formation was inhibited by aminoguanidine (AG) with an IC 50 of 8 mM. AG inhibited fructose AGE fluorescence formation in a concentration dependent manner which attests to the specificity of the reaction explored. AG is a specific AGE formation inhibitor, very efficacious and widely used in animal models to study the role of glycation in disease [29,37–39]. Due to AG toxicity, we reasoned that better candidates to avoid intraluminal AGE formation by fructose would be known dietary compounds, especially those with poor absorption which get concentrated in the lumen. Chlorogenic acids 3
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Table 1 Inhibition of fructose AGE formation during simulated digestion of ovalbumin. Aminoguanidine (mmol/l)
Inhibition of AGE formation (%)
Chlorogenic acid (mmol/l)
Inhibition of AGE formation (%)
Caffeic acid (mmol/l)
Inhibition of AGE formation (%)
0 0.5 2 10
0 10 +/− 2 30 +/− 3 60 +/− 6
0 0.5 2 10
0 5 +/− 2 20 +/− 3 50 +/− 6
0 0.5 2 10
0 6 +/− 3 18 +/− 4 50 +/− 8
OVA was digested in the presence of 50 mM fructose or buffer (control) for 2 h of intestinal digestion, in the presence of the shown concentrations of inhibitors. The peptide fraction was obtained by ultrafiltration through 10 kDa cut off Amicon filters. Top fluorescence (expressed in arbitrary units) was recorded at Excitation: λ350 nm, Emission: λ 440 nm against blanks (OVA incubated under the same conditions in the absence of fructose). Experiments were run 3 times. Data are mean +/− SD. All differences found are significant, p < 0.05.
free fructose beverages (HFCS sweetened soft drinks, fruit drinks, and apple juice) and the above mentioned outcomes, independent of potential confounders including smoking and exposure to in-home smoke [13,18–19] and has recently been reviewed by us [11]. Limitations are that we did not measure non-fluorescent specific AGEs. However since many AGEs are non-fluorescent, we may be underestimating the formed AGEs. LC-MS/MS studies to characterize these adducts and animal testing of their immunogenicity are warranted to fully establish this mechanism.
are a family of esters formed between certain trans cinnamic acids and (−)-quinic acid and are major phenolics compounds in coffee, yerba mate tea, strawberries, pineapple, apple, sunflower, blueberries [40–43]. We and others have shown a potent effect of these compounds as glycation inhibitors in model systems under intracellular conditions [29,44–45]. Cholorogenic acid (CGA) was as potent fructose-AGE inhibitor as AG, with an IC50 of 10 mM. In this regard, these results build up on our prior report showing AGE formation between fructose and amino acids in short time and physiological conditions and give plausibility to the proposed mechanism to explain the epidemiological association between fructose malabsorption (due to ingestion of beverages with a ratio of fructose to glucose > 1 and up to 2) and inflammatory diseases (asthma, chronic bronchitis and arthritis). It has been proposed that the association is due to RAGE activation by AGE adducts formed in situ in the intestines when fructose, peptides and amino acids co-exists for abnormally long periods [12] Using data from 2801 adults aged 20–55 years from the National Health and Nutrition Examination Survey (NHANES), DeChristopher et al. found that independent of all covariates, regular intake of sodas (≥ 5 times p/wk) vs never/seldom consumption was associated with nearly twice the likelihood of having chronic bronchitis [18], but not with diet soda. In another cross sectional study with NHANES, children who regularly consumed any combination of high excess free fructose beverages, including apple juice, fruit drinks and HFCS sweetened soft drinks were five times more likely to have asthma vs seldom/never consumers, independent of potential confounders. Regular apple juice drinkers were twice as likely vs seldom/never consumers to have asthma. There was no association with orange juice, with a 1:1 fructose to glucose ratio. Further, young adults who regularly consumed any combination of high excess free fructose beverages were three times as likely to have non-wear-andtear, non-age associated arthritis vs seldom/ never consumers [17]. Our results suggest that AGE adducts form between fructose and OVA which can be found in peptide fractions (< 5 kDa) at times and concentration ranges plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction is inhibited by chlorogenic acid at concentrations compatible with those found in the gut and by AG, a specific antiglycation agent. These findings add another layer of support to our previous report that fructose-AGE adducts may be forming in situ (in the intestinal lumen) and contributing to circulating AGE load [29]. This phenomenon may be of pathogenic significance, as US average per capita HFCS consumption levels remain high vs other parts of the world [14,46], and the fructose to glucose ratio in US soft drinks [14–15,46–47] has been found to exceed levels generally recognized as safe. In this regard, this study provides further in vitro evidence for the mechanism proposed by DeChristopher which explains how consumption of high fructose corn syrup, but not sucrose, could result in airway mucus hypersecretion that overwhelms the airways and contributes to asthma, chronic bronchitis, and other co-morbidities including non-wear-and-tear, non-age-associated arthritis, and possibly other pro-inflammatory chronic diseases [13,18–19]. The proposed hypothesis is supported by cross sectional epidemiological studies with nationally representative data including intake frequency of high excess
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Fructositis Triggered Bronchitis, Arthritis, & Auto-Immune Reactivity Merely a Side bar in the Etiology of Metabolic Syndrome II (to be Defined)? – Evidence and a Hypothesis, (2012). L.R. DeChristopher, J. Uribarri, K.L. Tucker, The link between soda intake and asthma: science points to the high-fructose corn syrup, not the preservatives: a commentary, Nutr Diabetes 6 (11) (2016) e234. M.I. Goran, S.J. Ulijaszek, E.E. Ventura, High fructose corn syrup and diabetes prevalence: a global perspective, Glob. Public Health 8 (1) (2013) 55–64. E.E. Ventura, J.N. Davis, M.I. Goran, Sugar content of popular sweetened beverages based on objective laboratory analysis: focus on fructose content, Obesity (Silver Spring) 19 (4) (2011) 868–874. L.R. DeChristopher, J. Uribarri, K.L. Tucker, Intakes of apple juice, fruit drinks and soda are associated with prevalent asthma in US children aged 2–9 years, Public Health Nutr. 19 (1) (2016) 123–130. L.R. DeChristopher, J. Uribarri, K.L. Tucker, Intake of high-fructose corn syrup sweetened soft drinks, fruit drinks and apple juice is associated with prevalent arthritis in US adults, aged 20–30 years, Nutr Diabetes 6 (2016) e199. L.R. DeChristopher, J. Uribarri, K.L. Tucker, Intake of high fructose corn syrup sweetened soft drinks is associated with prevalent chronic bronchitis in U.S. Adults, ages 20–55 y, Nutr. J. 14 (2015) 107. L.R. DeChristopher, Excess free fructose and childhood asthma, Eur. J. Clin. Nutr. 69 (12) (2015) 1371. A. Shekhtman, R. Ramasamy, A.M. Schmidt, Glycation & the RAGE axis: targeting signal transduction through DIAPH1, Expert Rev. Proteomics (2016) 1–10. A.M. Schmidt, O. Hori, R. Cao, S.D. Yan, J. Brett, J.L. Wautier, S. Ogawa, K. Kuwabara, M. Matsumoto, D. Stern, RAGE: a novel cellular receptor for advanced glycation end products, Diabetes 45 (Suppl. 3) (1996) S77–S80. J. Uribarri, M.D. del Castillo, M.P. de la Maza, R. Filip, A. Gugliucci, C. LuevanoContreras, M.H. Macias-Cervantes, D.H. Markowicz Bastos, A. Medrano, T. Menini, M. Portero-Otin, A. Rojas, G.R. Sampaio, K. Wrobel, K. Wrobel, M.E. Garay-Sevilla, Dietary advanced glycation end products and their role in health and disease, Adv. Nutr. 6 (4) (2015) 461–473. J. Uribarri, A. Stirban, D. Sander, W. Cai, M. Negrean, C.E. Buenting, T. Koschinsky, H. Vlassara, Single oral challenge by advanced glycation end products acutely impairs endothelial function in diabetic and nondiabetic subjects, Diabetes Care 30 (10) (2007) 2579–2582. T. Koschinsky, C.J. He, T. Mitsuhashi, R. Bucala, C. Liu, C. Buenting, K. Heitmann, H. Vlassara, Orally absorbed reactive glycation products (glycotoxins): an environmental risk factor in diabetic nephropathy, Proc. Natl. Acad. Sci. U. S. A. 94 (12) (1997) 6474–6479. R.D. Semba, S.K. Gebauer, D.J. Baer, K. Sun, R. Turner, H.A. Silber, S. Talegawkar, L. Ferrucci, J.A. Novotny, Dietary intake of advanced glycation end products did not affect endothelial function and inflammation in healthy adults in a randomized controlled trial, J. Nutr. 144 (7) (2014) 1037–1042. N.J. Kellow, G.S. Savige, Dietary advanced glycation end-product restriction for the attenuation of insulin resistance, oxidative stress and endothelial dysfunction: a systematic review, Eur. J. Clin. Nutr. 67 (3) (2013) 239–248. R.D. Semba, A. Ang, S. Talegawkar, C. Crasto, M. Dalal, P. Jardack, M.G. Traber, L. Ferrucci, L. Arab, Dietary intake associated with serum versus urinary carboxymethyl-lysine, a major advanced glycation end product, in adults: the energetics study, Eur. J. Clin. Nutr. 66 (1) (2012) 3–9. T. Henle, Dietary advanced glycation end products–a risk to human health? A call for an interdisciplinary debate, Mol. Nutr. Food Res. 51 (9) (2007) 1075–1078.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Advanced glycation endproducts form during ovalbumin digestion in the presence of fructose: Inhibition by chlorogenic acid
MARK
Yasmin Bains, Alejandro Gugliucci⁎, Russell Caccavello Glycation, Oxidation and Disease Laboratory, Dept. of Research, College of Osteopathic Medicine, Touro University-California, Vallejo, CA, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Advanced glycation Fructose Inflammation Ovalbumin
Background: One mechanism by which fructose could exert deleterious effects is through intestinal formation and absorption of pro-inflammatory advanced glycation endproducts via the Maillard reaction. We employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that advanced glycation endproducts (AGEs) are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions. Methods: OVA was subjected to simulated gastric and intestinal digestion using standard models, in presence of fructose or glucose (0–100 mM). Peptide fractions were analyzed by fluorescence spectroscopy and intensity at Excitation: λ370 nm, Emission: λ 440 nm. Results: AGE adducts formed between fructose and OVA, evidenced by the peptide fractions (< 5 kDa) at times (30 min) and concentration ranges (10 mM) plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction was inhibited by chlorogenic acid at concentrations compatible with those found in the gut. The reaction was also inhibited by aminoguanidine, a specific antiglycation agent. Conclusion: Our study showed fructose-AGE formation on a ubiquitous dietary protein under model physiological conditions. Our study also suggests ways to decrease the damage: enteral fructose-AGE formation may be partially inhibited by co-intake of beverages, fruits and vegetables with concentrations of phenolics high enough to serve as anti-glycation agents.
1. Introduction The association of high fructose diets with metabolic syndrome, diabetes and obesity is undeniable [1–5]. Large epidemiological studies have so demonstrated, notably for soda and fruit juices consumption. Nevertheless, correlation cannot adjudicate causation and some researchers maintain it is just due to extra calories, not to specific mechanistic action. One mechanism by which fructose could exert deleterious effects in metabolism and inflammation is via its potency in the Maillard reaction. Indeed, fructose is 7–10 times more potent a glycating agent than glucose due to the instability of the Heyns product it produces [6–10]. These early glycation products undergo further rearrangement, dehydration, and condensation reactions to produce irreversibly cross-linked derivatives called advanced glycation endproducts (AGEs), some of which are fluorescent. Fructose-specific AGEs, however have not been well characterized. Moreover, except for the liver, fructose concentrations in serum are low (micromolar range). Therefore, the role of endogenous fructose glycation (with the possible exception of the polyol pathway) as a significant pathogenic process is
difficult to substantiate [11]. Until recently, little attention has been paid to the possibility of endogenous formation of AGEs between fructose and proteins, precisely where they are more concentrated – the intestinal lumen [12] According to this hypothesis, fructose derived AGEs form in the intestines when fructose to glucose ratios exceed 1:1, but not after consumption of sucrose, or equal monomers of fructose and glucose [12]. Foods and beverages sweetened with high fructose corn syrup (HFCS) contain elevated levels of fructose [13] [14–15]. The same is true for apple juice [14–15]. Through careful epidemiological studies using NHANES data, over the past 5 years evidence for an independent effect of excess fructose ingestion and a range of inflammatory conditions has been found (asthma, chronic bronchitis and arthritis [12–13,16–19]. The authors suggest these associations may be mediated through in situ enteral formation of fructose AGEs, which, after being absorbed, may contribute to inflammatory diseases via engagement of the proinflammatory receptor for advanced glycation endproducts (RAGE) [12,20–21]. Other food AGEs (preformed in cooking) have been shown to actually be absorbed and be proinflammatory in animal and some human intervention studies
Abbreviations: AGE, advanced glycation endproducts; AG, aminoguanidine; RAGE, receptor for advanced glycation end products ⁎ Corresponding author at: Touro University-California, 1310 Club Drive, 94592 Vallejo, CA, USA. E-mail address: [email protected] (A. Gugliucci). http://dx.doi.org/10.1016/j.fitote.2017.05.003 Received 28 March 2017; Received in revised form 11 May 2017; Accepted 16 May 2017 Available online 17 May 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
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4. Fluorescence spectroscopy
[22–24]. However, several other studies have not shown this effect so this topic remains controversial [25–28]To shed some light on the luminal formation of AGES by fructose, we recently conducted a proof of principle study where we showed that fluorescent AGE adducts form between fructose and amino acids at times and concentration ranges plausibly found in the intestines, whereas no detectable reaction occurs with glucose [29]. The reaction was inhibited by chlorogenic acid and Ilex paraguariensis extracts (containing high concentrations of chlorogenic acid), suggesting that intake of food or herbal teas containing polyphenols may help dampen this process. In order to shed some light on the initial mechanism suggested by the above-mentioned hypothesis, in this follow up study we employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that indeed AGEs are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions.
The interaction between fructose and ovalbumin peptides was studied by fluorescence spectroscopy. AGE fluorescence spectra between 390 and 600 nm (excitation, 370 nm) were recorded at room temperature on a Biotek Synergy. 4.1. SDS-page Samples were dissolved to a protein concentration of 1 mg/ml or 1/ 1 for peptide fractions in 10 mM Tris-HCl buffer, pH 8, containing 2.5% SDS, 5% 2-β-mercaptoethanol, and 10 mM EDTA and heated at 100 °C for 10 min. SDS-PAGE was performed using precast Mini-PROTEAN TGX 4–20% gels (Bio-Rad, USA). Following the electrophoretic gels were stained with silver nitrate. A LMW Calibration Kit for SDS (Amersham Biosciences, USA) was used.
2. Material and Methods 4.2. Statistical analysis All reagents were purchased from Sigma St Louis, MO. OVA digestion was conducted employing classical models [30–32].
Experiments were run 3 times in duplicates. Data are mean +/− SD. All differences found are significant p < 0.05. Significance of differences was assessed with the Student's t-test.
2.1. In vitro gastric digestion 5. Results and discussion
Ovalbumin (OVA, A-5503, 98% purity, Sigma, MO, USA) was dissolved in simulated gastric fluid (SGF, 150 mM NaCl, 20 mM KH2PO4) at pH 1.2, to a final concentration of 25 mg/ml (0.42 mM) and subjected to an in vitro gastric digestion at 37 °C with porcine pepsin (EC 3.4.23.1, 3440 units/mg, P7012, Sigma) at an enzyme/ substrate ratio of 1:20 w/w (172 units/mg) [31]. Digestion was conducted in 50 ml cell culture tubes under rotatory movement at 100 rpm. After 2 h the reaction was stopped by adding 0.1 M NaOH to titrate pH to 7.0. At least triplicate digestions were conducted for each condition.
This study was designed as a follow up of our earlier proof of principle study [29] where we showed that unlike glucose, fructose forms fluorescent advanced glycation endproducts with basic and neutral aminoacids at conditions and times compatible with those in the digestive system lumen. In this follow-up work we have simulated the gastrointestinal proteolysis of OVA using a classic in vitro digestion system in two steps, which mimics the successive passage through the stomach and duodenum. We show that fructose produces AGE-peptides in OVA during simulated digestion, at times and concentrations compatible with physiological conditions. The allergenic potential of OVA [30–32] may be thus enhanced by endogenous formation of AGEs during digestion when consumed with fructose. Fig. 1 shows digestion of OVA (lane 1) during the stomach (lane 2) and intestinal phase (lanes 3 and 5) of simulated digestion. Peptic digestion of OVA (lane 1, time 0) leads to multiple peptides of decreasing molecular weights, as shown in lane 2, which is enhanced by intestinal digestion. The pattern of digestion is in agreement with previous reports [30–31]. The peptide fractions (< 10 kDa, lanes 4 (control) and 6 (fructose) run off the gels for the most part, indicating that they contain peptides smaller than 5 kDa as well as free aminoacids. No significant difference in the digestion pattern was seen between
2.2. In vitro duodenal digestion Duodenal digestions were performed by using, as the starting material, the 120 min gastric digests as described above adjusted to pH 7.0, with the addition of 1 mM CaCl2, 1 mM MgCl2, and a 0.125 M bile salt mixture B3426 (Sigma). After preheating at 37 °C for 15 min, a 10 × solution of pancreatin from porcine pancreas (containing all pancreatic enzymes) in NaHCO3 buffer ph 8. The final composition of the mixture was 0. 4 mM OVA at an enzyme/substrate ratio of 1:25 w/ w, 20 mM HCO3, 1 mM CaCl2, 1 mM MgCl2, ph 7.0. Aliquots were taken at different times and digestion was stopped by changing pH to 2.0. The mixture was chilled in ice and peptides were immediately separated. At least triplicate digestions were conducted for each condition. Digestions were performed in the absence or presence of a range of fructose or glucose concentrations (0–100 mM), with and without the presence of glycation inhibitors. Aminoguanidine (AG, the classic AGE inhibitor, Sigma St Louis, MO) as well as 5-caffeoylquinic acid (cholorogenic acid, Sigma St Louis, MO) were employed as inhibitors.
3. Peptide separation Digested peptides and free aminoacids (< 10 kDa) were separated by ultrafiltration through 0.5 ml Amicon Ultra Centrifugal filters spun at 14,000g for 30 min in a refrigerated centrifuge at 4 °C. Samples (triplicates of ultrafiltrates) were read in a Biotek Synergy H1. Top fluorescence was recorded at Excitation: λ370 nm, Emission: λ 440 nm against blanks, corrected by absorbance at 280 nm and expressed in AU as previously described.
Fig. 1. SDS-PAGE analysis of OVA during in vitro simulated stomach and intestines digestion. Samples were run in 4–20% polyacrylamide gels under reducing conditions. OVA was digested with pepsin for 2 h at 37 °C to simulate gastric digestion, followed by digestion with pancreatic enzymes for 2 h at 37 °C to simulate enteral digestion, as described in Methods. MWM: molecular weight markers. 1: OVA at time 0; 2: OVA after 2 h pepsin; 3: OVA after 2 h pancreatic enzymes; 4: peptide fraction of 4; 5:OVA + 50 mM fructose after 2 h pancreatic enzymes; 6: peptide fraction of 5.
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A
Fig. 2. Emission absorption spectrum of AGEs in the peptide fraction of OVA digestion. OVA was digested in the presence of 50 mM fructose or buffer (control) for 2 h of intestinal digestion. The peptide fraction was obtained by ultrafiltration through 10 kDa cut off Amicon filters. Spectrum was obtained after excitation at 370 nm using OVA incubated in the absence of fructose as blank.
B
fructose-incubated samples (lane 5) and controls (lane 3). Ovalbumin was chosen as a model protein for several reasons. First, it is a key nutritional protein in our diets. Second, its digestion has been thoroughly studied. Finally, it is particularly interesting since it is somewhat resistant to peptic digestion and its peptides are allergenic and pose a problem to some children [30–31]. More interestingly, extensive glycation of OVA (as in cooking) has already been substantiated as factor enhancing the antigen uptake and presentation by human dendritic cells in vitro [32]. Therefore, a known pathway exists for OVA peptides to interact with antigen presenting cells in the gut. Would that be enhanced by these peptides containing fructose-derived AGEs formed in situ? If this is true, they would engage RAGE on top of their intrinsic known deleterious action and this would help explain the epidemiological evidence showing that only fructose in excess of glucose is strongly associated with asthma, arthritis and other inflammatory ailments. The OVA concentrations chosen were adequate to mimic a meal of an average protein consumption of 1 g/kg weight per day, in 3 meals. That is 20–25 g protein, diluted in about 1 l of gastric fluid. As shown in Fig. 2, simulated digestion of OVA in the presence of fructose (but not with glucose,) leads to the formation of compounds, present in the molecular weight fraction < 10 kDa that possess the characteristic fluorescence spectrum of AGEs at excitation 370 nm. Glucose incubation did not produce changes in the spectrum over the control (data not shown). Of note, fluorescent compounds were also seen at time 0, indicating the purified OVA already contains AGEs. Fig. 3 shows time (3A) and dose dependent (3B) formation of AGE fluorescence in peptides during simulated digestion. Notably, at 50 mM fructose AGE fluorescence is already apparent after just 30 min duodenal digestion, a time frame well compatible with the digestive process. At 120 min of intestinal digestion AGE formation is already apparent at 10 mM fructose concentrations. We show a time and dose dependent formation of fluorescent AGEs between fructose (but not glucose) and OVA during simulated digestion at concentrations plausibly found in the gastrointestinal lumen (10–50 mmol/l). Indeed, in the typical Western diet over 200 mmoles of fructose in liquid form can be ingested in two glasses of apple juice or high fructose corn syrup sweetened soda, usually together with a load of protein and fat that delays gastric motility [11]. This usually leads to 12–18 g (up to 130 mmoles) of excess fructose that is not co-absorbed with glucose and therefore has an increased luminal residence time [12]. Even if we allowed for an exaggerated 1/5 dilution of these amounts due to fluid changes and poor absorption, these concentrations are compatible with
Fig. 3. Time and concentration dependent formation of AGE-peptides during OVA digestion A) Concentration dependent fructose AGE formation. OVA was incubated in the presence or absence (blanks) of fructose for 2 h of stomach conditions and 2 h of intestinal conditions as described in Methods, for 3 h. B) Time dependent fructose AGE formation. OVA was incubated in the presence or absence (blanks) of 50 mM fructose for 2 h of stomach conditions and the indicated times of intestinal conditions as described in Methods. Data are means +/− SD of triplicates. Top fluorescence (expressed in arbitrary units) was recorded at Excitation: λ350 nm, Emission: λ 440 nm against blanks (OVA incubated under the same conditions in the absence of fructose). Experiments were run 3 times. Data are mean +/− SD. All differences found are significant, p < 0.05.
the ones shown here to allow for formation of AGEs. It is important to point out that extended contacts between fructose and proteins, peptides and amino acids is greatly enhanced by fructose malabsorption [33–34]. This usually follows ingestion of food or beverages that contain a ratio of fructose to glucose > 1 [17–18]. Otherwise, enhanced absorption of fructose by glucose would tend to shorten contact times. For instance, fructose malabsorption due to large intake of high fructose corn syrup (HFCS) and apple juice has been shown [33–36]. Table 1 shows inhibition of AGE formation by aminoguanidine and phenolics. AGE formation was inhibited by aminoguanidine (AG) with an IC 50 of 8 mM. AG inhibited fructose AGE fluorescence formation in a concentration dependent manner which attests to the specificity of the reaction explored. AG is a specific AGE formation inhibitor, very efficacious and widely used in animal models to study the role of glycation in disease [29,37–39]. Due to AG toxicity, we reasoned that better candidates to avoid intraluminal AGE formation by fructose would be known dietary compounds, especially those with poor absorption which get concentrated in the lumen. Chlorogenic acids 3
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Table 1 Inhibition of fructose AGE formation during simulated digestion of ovalbumin. Aminoguanidine (mmol/l)
Inhibition of AGE formation (%)
Chlorogenic acid (mmol/l)
Inhibition of AGE formation (%)
Caffeic acid (mmol/l)
Inhibition of AGE formation (%)
0 0.5 2 10
0 10 +/− 2 30 +/− 3 60 +/− 6
0 0.5 2 10
0 5 +/− 2 20 +/− 3 50 +/− 6
0 0.5 2 10
0 6 +/− 3 18 +/− 4 50 +/− 8
OVA was digested in the presence of 50 mM fructose or buffer (control) for 2 h of intestinal digestion, in the presence of the shown concentrations of inhibitors. The peptide fraction was obtained by ultrafiltration through 10 kDa cut off Amicon filters. Top fluorescence (expressed in arbitrary units) was recorded at Excitation: λ350 nm, Emission: λ 440 nm against blanks (OVA incubated under the same conditions in the absence of fructose). Experiments were run 3 times. Data are mean +/− SD. All differences found are significant, p < 0.05.
free fructose beverages (HFCS sweetened soft drinks, fruit drinks, and apple juice) and the above mentioned outcomes, independent of potential confounders including smoking and exposure to in-home smoke [13,18–19] and has recently been reviewed by us [11]. Limitations are that we did not measure non-fluorescent specific AGEs. However since many AGEs are non-fluorescent, we may be underestimating the formed AGEs. LC-MS/MS studies to characterize these adducts and animal testing of their immunogenicity are warranted to fully establish this mechanism.
are a family of esters formed between certain trans cinnamic acids and (−)-quinic acid and are major phenolics compounds in coffee, yerba mate tea, strawberries, pineapple, apple, sunflower, blueberries [40–43]. We and others have shown a potent effect of these compounds as glycation inhibitors in model systems under intracellular conditions [29,44–45]. Cholorogenic acid (CGA) was as potent fructose-AGE inhibitor as AG, with an IC50 of 10 mM. In this regard, these results build up on our prior report showing AGE formation between fructose and amino acids in short time and physiological conditions and give plausibility to the proposed mechanism to explain the epidemiological association between fructose malabsorption (due to ingestion of beverages with a ratio of fructose to glucose > 1 and up to 2) and inflammatory diseases (asthma, chronic bronchitis and arthritis). It has been proposed that the association is due to RAGE activation by AGE adducts formed in situ in the intestines when fructose, peptides and amino acids co-exists for abnormally long periods [12] Using data from 2801 adults aged 20–55 years from the National Health and Nutrition Examination Survey (NHANES), DeChristopher et al. found that independent of all covariates, regular intake of sodas (≥ 5 times p/wk) vs never/seldom consumption was associated with nearly twice the likelihood of having chronic bronchitis [18], but not with diet soda. In another cross sectional study with NHANES, children who regularly consumed any combination of high excess free fructose beverages, including apple juice, fruit drinks and HFCS sweetened soft drinks were five times more likely to have asthma vs seldom/never consumers, independent of potential confounders. Regular apple juice drinkers were twice as likely vs seldom/never consumers to have asthma. There was no association with orange juice, with a 1:1 fructose to glucose ratio. Further, young adults who regularly consumed any combination of high excess free fructose beverages were three times as likely to have non-wear-andtear, non-age associated arthritis vs seldom/ never consumers [17]. Our results suggest that AGE adducts form between fructose and OVA which can be found in peptide fractions (< 5 kDa) at times and concentration ranges plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction is inhibited by chlorogenic acid at concentrations compatible with those found in the gut and by AG, a specific antiglycation agent. These findings add another layer of support to our previous report that fructose-AGE adducts may be forming in situ (in the intestinal lumen) and contributing to circulating AGE load [29]. This phenomenon may be of pathogenic significance, as US average per capita HFCS consumption levels remain high vs other parts of the world [14,46], and the fructose to glucose ratio in US soft drinks [14–15,46–47] has been found to exceed levels generally recognized as safe. In this regard, this study provides further in vitro evidence for the mechanism proposed by DeChristopher which explains how consumption of high fructose corn syrup, but not sucrose, could result in airway mucus hypersecretion that overwhelms the airways and contributes to asthma, chronic bronchitis, and other co-morbidities including non-wear-and-tear, non-age-associated arthritis, and possibly other pro-inflammatory chronic diseases [13,18–19]. The proposed hypothesis is supported by cross sectional epidemiological studies with nationally representative data including intake frequency of high excess
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