The accumulation of advanced glycation end products (AGEs) is associated with accelerated aging [1
] and may be involved in the development of degenerative disease––such as diabetes, cardiovascular disease and Alzheimer’s disease––by promoting insulin resistance, inflammation and oxidative stress [2
AGEs can be endogenously generated and also enter the body when AGEs are consumed from food [8
]. Approximately 10–30% of consumed AGEs are absorbed in the intestine. About 1/3 of consumed AGEs are excreted via urine or feces and 2/3 accumulate in the body [9
]. AGE formation occurs through nonenzymatic reactions among free carbonyl groups of reducing sugars and reactive aldehydes derived from the free amino groups (lysine or arginine) in proteins, lipids and nucleic acids from rearrangement of the Schiff base and Amadori products, known as the Maillard reaction [8
]. AGEs are categorized into fluorescent cross-linking AGEs (pentosidine and crossline), non-fluorescent cross-linking AGEs (imidazolium dilysine cross-links, alkyl formyl glycosyl pyrrole (AFGP) cross-links and arginine-lysine imidazole (ALI) cross-links), and non-cross-linking AGEs (pyrraline, carboxymethyllysine (CML) and carboxyethyllysine (CEL)) [11
]. CML, an indicator of Maillard reaction product, is a major AGE compound [11
]. CML and CEL are lysine modificationwhereas methylglyoxal hydroimidazolone (MG-H1—mainly generated through highly reactive α-dicarbonyl, e.g., methylglyoxal) is an arginine adduct [12
Higher levels of AGEs are produced in thermally processed foods under dry conditions, such as by grilling, broiling, frying and roasting, compared with foods processed slowly at lower temperatures or in water [13
]. Foods contain high levels of AGEs when they are exposed to short-time processing with dry heat (e.g. baking, grilling, barbecuing, broiling, searing, frying, toasting and roasting), high temperature and increased pH compared with longer-time processing with lower temperature and water (e.g. boiling and steaming) [14
The formation of AGEs can potentially be reduced when red meat is consumed with abundant unprocessed plant foods (spices, herbs, fruits and vegetables) [15
]. It is uncertain whether dietary AGEs can play a role in the etiologies of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) or whether only endogenously generated AGEs contribute to these diseases. However, high red meat consumption (especially processed meat) as part of a low-antioxidant, low-fiber, highly available starch standard western diet can increase dietary AGEs [21
]. The effect of dietary AGEs on disease risk markers can differ according to a person’s health status (healthy, with or without T2DM or with CVD) [2
A meta-analysis of 17 randomized controlled trials (RCTs), comparing low-AGE diets with high-AGE diets indicated that low-AGE diets could reduce risk markers of cardiometabolic disease, such as insulin, LDL cholesterol, CRP and adhesion molecules, and inflammatory markers [22
]. A second, smaller meta-analysis found similar effects and noted a high-AGE diet increased circulating AGEs in most, but not all, studies (as assessed using ELISA) [2
As we previously reported [24
], a diet high in red and processed meat and refined grains (HMD) significantly attenuated the insulin sensitivity index (ISI) and significantly increased insulin and glucose levels only in relatively insulin-resistant subjects (fasting insulin > 56 pmol/L, n
= 25), but this was not the case in insulin-sensitive subjects (insulin < 56 pmol/L, n
= 24) compared with a diet high in whole grain, nuts, dairy and legumes (HWD). No differences in hs-CRP, IL-6, fluorescent AGEs or plasma CML (measured by ELISA) were observed between the HMD and HWD [25
To date, no interventions have examined the effect of different dietary patterns on plasma concentrations of advanced glycation end products measured by LC-MS/MS. Only one study has shown increased urinary AGEs measured by LC-MS/MS with a high-AGE diet versus a low-AGE diet [26
]. We have already measured CML by immunoassay and fluorescent AGEs in our previous publication [25
]. However, the method of liquid chromatography–tandem mass spectrometry (LC-MS/MS) may be a more reliable method to study AGEs [27
This present study aimed to investigate the association between dietary patterns and protein-bound AGEs (CML, CEL and MG-H1) using LC-MS/MS, which is a far more specific and sensitive method as the immunoassay may suffer from steric hindrance and interference from anti-AGE antibodies [27
]. It also aimed to examine the association between plasma advanced glycation end products (CML, CEL and MG-H1) and the changes in ISI and other biomarkers found in previously published studies [24
We hypothesized that subjects without T2DM on the HMD would have higher plasma concentrations of advanced glycation end products compared with those on the HWD.
In this weight-stable 4-week randomized crossover design, a high-red-and-processed-meat diet and a low-fiber, medium glycemic index/glycemic load diet (HMD) led to a significant increase in plasma CEL concentrations compared with the intake of a diet high in dairy, whole grains, nuts and legumes and low in GI/glycemic load (HWD) in individuals without T2DM. However, HMD did not affect the plasma concentrations of CML and MG-H1 compared with HWD. Moreover, we found that plasma concentrations of CML, CEL and MG-H1 on either the HMD or HWD did not correlate with the insulin sensitivity index, fasting glucose, fasting insulin, lipids or inflammatory markers.
Western-type diets containing high AGEs are partially absorbed in the digestive tract leading to elevated total serum AGE levels with possible negative consequences on health [33
]. CML is produced by both glycoxidation and lipid oxidation [34
], but, in this study, we did not observe higher CML concentrations after HMD than after HWD.
In an observational study of healthy subjects, dietary AGE intake was associated with elevated blood concentrations of CML and MG as assessed by immunoassays independent of age or energy intake [35
]. In contrast, the consumption of AGE-rich foods was not associated with blood CML concentrations in a second observational study of healthy subjects [36
In an acute intervention of crossover design, 19 healthy overweight subjects consumed two test meals of high AGE and low AGE content. Concentrations of CML in plasma and urine as measured by LC-MS/MS were not significantly increased after high-AGE meals [37
In the Cohort on Diabetes and Atherosclerosis Maastricht study (CODAM study) of 450 subjects with increased risk for T2DM and cardiovascular disease, consumption of dietary AGEs (CML, CEL and MG-H1)––as estimated with a dietary AGE database and a food frequency questionnaire––was positively associated with increased free plasma and urinary AGE concentrations, as assessed by UPLC-MS/MS. However, no significant association between dietary AGEs and protein-bound plasma AGEs was observed [38
]. This differs from our finding of an increase in plasma protein-bound CEL from the high-red-and-processed-meat (HMD) diet—this may reflect the very high meat intake not normally seen in a free-living population.
In a double-blind randomized crossover study of 20 healthy overweight subjects, a high-AGE diet for 2 weeks did not alter serum concentrations of protein-bound CML, CEL and MG-H1 as measured by UPLC-MS/MS, compared with a low-AGE diet. However, this study showed significant lower urinary MG-H1 and CEL concentrations after a low-AGE diet. The two diets were matched for energy and macronutrient proportions from protein, fat, and carbohydrate [26
]. Thus, most studies examining CML and MG-H1 did not see an increase in protein-bound AGE after a high-AGE diet, which is in line with our findings here.
In this present study, we found significantly higher plasma CEL concentrations after HMD than after HWD, but only in the insulin-resistant group. Our finding is supported by the observation that CEL is more likely to be contained in meat high in lipids, such as processed meat [39
]. Interestingly, Kellow et al. 2017 [40
] conducted a cross-sectional study to investigate if long-term habitual dietary patterns influence AGE accumulation in skin collagen. They found a positive association between meat consumption and skin AGE levels (r = 0.22; p
< 0.05), and a negative association between cereal consumption and skin AGE levels (r = −0.21; p
< 0.05) in 251 healthy overweight subjects (mean BMI 26 ± 6 kg/m2
) aged 18–80 years. There were significant associations between skin AGE levels and age, anthropometric parameters, such as BMI (r = 0.23; p
< 0.05), body weight (r = 0.24; p
< 0.05) and waist circumference (r = 0.28; p
Courten et al 2016 [26
] showed an effect of a high- and low-AGE diet on insulin sensitivity. The volunteers consumed higher amounts of CEL and CML on the high-AGE diet, so dietary CEL may contribute as much or more than CML to the effect on insulin sensitivity.
In our previous study, we found no differences in fluorescent AGEs or CML between HMD and HWD as assessed by immunoassays [25
]. Consistent with this, protein-bound CML concentrations measured by LC-MS/MS in this study also did not differ between two diets.
A strength of this study is that we measured plasma protein-bound CML, CEL and MG-H1 using LC-MS/MS, which is an accurate, highly sensitive method [27
]. Most intervention studies measured CML concentrations by immunological assays.
This study has several limitations. We did not measure free plasma AGE, which may provide more insight into the relationship between dietary AGE consumption and plasma AGE levels. We did not measure CML, CEL or MG-H1 concentrations in urine or fecal samples.
In conclusion, consumption of high red and processed meat and a low-fiber, medium-glycemic index/glycemic load diet in young individuals without T2DM significantly increased plasma CEL concentrations, but not plasma CML, and MG-H1 concentrations, compared with consumption of a diet high in dairy, whole grains, nuts and legumes and low in GI/glycemic load. Further investigations are needed to clarify the associations between dietary AGE consumption and AGE concentrations in blood (free and protein-bound), urine and feces, which will enable us to identify the role of dietary AGEs in human health. Future work should study the impact of dietary quality/patterns and cooking methods on AGEs, as well as food groups and macronutrients.