Helmut F. Erbersdobler

Metabolic Transit and in vivo Effects of Melanoidins and Precursor Compounds Deriving from the Maillard Reaction

Metabolic transit data on food-borne advanced MRPs (Maillard reaction products) termed melanoidins are yet not completely elucidated and it is still an open question whether isolated melanoidin structures undergo metabolic biotransformation and subsequently cause physiological effects in vivo. Advanced MRPs, acting as premelanoidins, and melanoidins are formed under severe heat treatment of foods and are ingested with the habitual diet at considerable amounts. Metabolic transit data are known for Amadori compounds classified as early MRPs, like, e.g., fructose-lysine. For rats and humans, the percentages of ingested free versus protein-bound fructose-lysine excreted in the urine were found within ranges of 60–80% and 3–10%, respectively. Balance studies on free advanced MRPs are still lacking, but protein-bound low-molecular-weight premelanoidins and high-molecular-weight melanoidins have already been investigated in animal experiments using 14C-tracer isotopes. The amount of ingested radioactivity absorbed and excreted in the urine was found at levels ranging from 16 to 30% and from 1 to 5% for premelanoidins and melanoidins, respectively. These different metabolic transit data of premelanoidins and melanoidins can be explained by the following mechanisms involved: (i) intestinal degradation by digestive and microbial enzymes; (ii) absorption of these compounds or their degradates, and (iii) tissue retention. Structure specific in vivo effects have been identified for protein-bound premelanoidins on intestinal microbial activity, xenobiotic biotransformation enzymes and further glycation reactions. The latter are hypothesized to be involved in the aging process and in the course of different diseases. Further investigations are needed to clarify synergistic in vivo effects of dietary ingested melanoidins and endogenously formed glycation products.

Dietary α-Linolenic Acid, EPA, and DHA Have Differential Effects on LDL Fatty Acid Composition but Similar Effects on Serum Lipid Profiles in Normolipidemic Humans

Our aim was to study the effects of increased dietary intake of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on serum lipids and LDL fatty acid compositions. To this end, a controlled parallel study was conducted in 74 healthy normolipidemic men and women aged 19-43 y. Participants were randomly assigned to 1 of 3 interventions and consumed a total intake of 4.4 g/d ALA (ALA group), 2.2 g/d EPA (EPA group), and 2.3 g/d DHA (DHA group) for 6 wk. Fatty acid ethyl esters were incorporated into margarines, which replaced the participants normal spread. The ALA, EPA, or DHA intake led to a significant enrichment of the LDL with the respective (n-3) fatty acid. In addition, LDL EPA contents in the ALA group increased by 36% (P < 0.05) with no changes in LDL DHA. The EPA intervention led to an additional enrichment with DHA (24%; P < 0.001), whereas the DHA intervention further increased the amount of EPA (249%; P < 0.001). ALA, EPA, or DHA intake did not affect fasting serum concentrations of total and LDL cholesterol, but fasting serum triacylglycerol concentrations significantly decreased in the EPA (-0.14 mmol/L) and DHA (-0.30 mmol/L) interventions and also in the ALA intervention (-0.17 mmol/L). DHA intake significantly increased serum HDL cholesterol, whereas no changes were found with ALA or EPA intake. In conclusion, the present data support the hypothesis that isolated dietary ALA, EPA, and DHA intakes lead to differential enrichment in LDL due to interconversion. Moderate amounts of ALA, EPA, and DHA are effective in improving lipid profiles of normolipidemic humans.

Determination of Nϵ-carboxymethyllysine in milk products by a modified reversed-phase HPLC method

Abstract A modified reversed-phase-HPLC method with o-phthalaldehyde pre-column-derivatisation for determination of N ϵ -carboxymethyllysine in food samples is presented. It is shown, that the method has to be modified if applied to milk products, including specific modifications in sample preparation and chromatographic separation conditions. The increased selectivity of a double endcapped RP 18 phase is necessary for reliable separation of N ϵ -carboxymethyllysine in hydrolysates of complex products like cheese. With a detection limit of 0.5 pMol the method shows high sensitivity and a very good reproducibility ( s =2.81%). In total, several different milk products ( n =50) as well as fresh, processed and ripened cheese samples ( n =50) were analysed. The highest amounts of N ϵ -carboxymethyllysine were found in a whey cheese (1016 mg/kg protein), evaporated milk (1691 mg CML/kg protein), coffee cream (613 mg CML/kg protein) and cocoa milk (413 mg CML/kg protein). N ϵ -carboxymethyllysine could not be detected in UHT milk, fresh, processed and ripened cheese. The results show that N ϵ -carboxymethyllysine can give valuable information on lysine damage in severely heat-treated milk products and in products, with added sugar, pre-damaged constituents or stabilising agents. ©

A case-control study of the effect of infant feeding on celiac disease.

Aims: The aim of this study was to investigate the association between the duration of breast-feeding and the age at the first gluten introduction into the infant diet and the incidence and age at onset of celiac disease. Methods: In a case-control study, 143 children with celiac disease and 137 randomly recruited gender- and age-matched control children were administered a standardized questionnaire. Multivariate-adjusted odds ratios (OR) as estimates of the relative risk and corresponding 95% confidence intervals (95% CI) were calculated. Results: The risk of developing celiac disease decreased significantly by 63% for children breast-fed for more than 2 months (OR 0.37, 95% CI 0.21–0.64) as compared with children breast-fed for 2 months or less. The age at first gluten introduction had no significant influence on the incidence of celiac disease (OR 0.72, 95% CI 0.29–1.79 comparing first gluten introduction into infant diet >3 months vs. ≤3 months). Conclusions: A significant protective effect on the incidence of celiac disease was suggested by the duration of breast-feeding (partial breast-feeding as well as exclusive breast-feeding). The data did not support an influence of the age at first dietary gluten exposure on the incidence of celiac disease. However, the age at first gluten exposure appeared to affect the age at onset of symptoms.

Metabolic transit of Amadori products

In several studies, the absorption and urinary excretion of free and protein bound Amadori products were measured in rats and humans. Both, in vitro tests with everted intestinal sac preparations and in vivo experiments, showed that there is no active intestinal transport of these compounds but an absorption by diffusion. Trials with tissue slices have shown that there was an uptake into the cells of the liver, kidneys and muscles. Metabolism of Amadori products, if it exists in animals, tends to be very low. Micoorganisms in the large intestines decompose the Amadori products almost completely. The profile of urinary excretion of Amadori products after the ingestion of test meals showed a rapid elimination of the absorbed part, while the fecal output, although low because of the hind gut fermentation, persisted up to 3 days. Only 1-3% of the ingested amounts of protein bound Amadori products were recovered in the urine, which suggests a low absorption rate.

RAGE-mediated MAPK activation by food-derived AGE and non-AGE products

Investigating the cellular effects of food compounds formed by heat treatment during processing, we recently demonstrated the expression of the receptor for advanced glycation endproducts (RAGE) and the p44/42 MAP kinase activation by casein-N(epsilon )-(carboxymethyl)lysine (casein-CML), a food-derived AGE, in the intestinal cell line Caco-2. In this work, we report a Caco-2 p44/42 MAP kinase activation by bread crust and coffee extract. After identification, quantification, and synthesis of two key compounds formed in association with the process-induced heat impact applied to bread dough and coffee beans, those compounds, namely the AGE pronyl-glycine and the non-AGE N-methylpyridinium, were also demonstrated for the first time to activate the p44/42 MAP kinase through binding to RAGE in Caco-2 cells. Blocking of RAGE by an antagonistic antibody and expression of C-terminally truncated RAGE resulted in a reduced Caco-2- and HEK-293-MAP kinase activation. These findings unequivocally point to a RAGE-mediated activating effect of chemically defined food-derived, thermally generated products, both, AGEs and non-AGEs, on cellular signal transduction pathways involved in inflammatory response and cellular proliferation.

Determination of Nε -carboxymethyllysine by a reversed-phase high-performance liquid chromatography method

Abstract To determine Ne -carboxymethyllysine (CML) in foods a RP-HPLC method after derivatisation with o -phthaldialdehyde was developed. To prevent an overestimation of the CML values by the formation of CML from Amadori products during hydrolysis a borohydride reduction precedes the hydrolysis. A comparison of the determination with and without reduction shows that during hydrolysis 2–12 times more CML than originally present can be formed. With the analytical conditions described in this paper it is possible to obtain measurable amounts of this trace substance in spite of the much higher values for other amino acids. The CML contents in selected processed food items varied between 11 mg in a preparation from mixed cereals for infants to 408 mg/kg protein in a processed malt product. CML is suitable as indicator of heat damage in processed or stored foods, being more stable than the Amadori compounds determined, e.g. in form of furosine.

Dietary Eicosapentaenoic Acid and Docosahexaenoic Acid Are More Effective than Alpha-Linolenic Acid in Improving Insulin Sensitivity in Rats

In the present study, we investigated whether long-term administration of high dose of α-linolenic acid (ALA) is able to mimic the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) or a mixture of both with respect to insulin sensitivity in male Wistar rats. Furthermore, we intended to test whether these n–3 polyunsaturated fatty acids reveal differential effects on glucose and insulin levels. As a result, plasma glucose and insulin levels were lowered by 35 and 38%, respectively, in the EPA and DHA group compared to the ALA group. Insulin sensitivity was substantially improved, as indicated by a 60% decreased HOMA index after an 8-week EPA and DHA administration, as compared to the effect observed for feeding ALA. However, insulin sensitivity did not differ between animals of the EPA and the DHA group. These results demonstrate that ALA intake at the expense of EPA and DHA in a diet high in n–3 fatty acids does not represent an alternative to raising oily fish consumption with regard to insulin sensitivity. Furthermore, a differential effect of the members of the n–3 family was shown for ALA compared to EPA and DHA, but EPA and DHA revealed comparable effects on insulin sensitivity.

Dietary bread crust advanced glycation end products bind to the receptor for AGEs in HEK-293 kidney cells but are rapidly excreted after oral administration to healthy and subtotally nephrectomized rats.

Abstract: In renal HEK‐293 cells, the dietary Maillard reaction compounds casein‐linked Nε‐carboxymethyllysine (CML), CML, bread crust (BC), and pronyl‐glycine (a key compound formed in association with the process‐induced heat impact applied to bread dough) all showed activation of p38‐MAP kinase. Expression of the C‐terminus truncated receptor for advanced glycation end products (RAGE) resulted in a reduction of HEK‐293‐MAP kinase activation. As these findings suggested a RAGE‐mediated activating effect of CML, BC, and pronyl‐glycine on kidney cellular signal transduction pathways, an in vivo study was performed. Male Wistar rats were subjected to a sham operation (CTRL, n= 20) or to 5/6 nephrectomy (NX, n= 20). Both groups were randomized into two subgroups and fed 20 g of a diet containing either 25% by weight BC or wheat starch (WS). GC‐MS analyses of CML, carboxyethyllysine (CEL), and pentosidine revealed increased levels of CML and CEL in the liver but decreased levels of CML in the kidneys of CTRL and NX rats fed the BC diet compared to those on the WS diet. However, urinary levels of CML were also elevated in the CTRL and NX rats on the BC diet, pointing to enhanced excretion of AGEs after BC administration. Although renal insufficiency in the NX rats was reflected by proteinuria, the renal handling of CML and, presumably, other AGEs was not impaired.

Renal effects of oral maillard reaction product load in the form of bread crusts in healthy and subtotally nephrectomized rats

Abstract: The biological consequences of chronic consumption of Maillard reaction products (MRPs) on renal function in health and renal disease are still incompletely understood. We investigated the metabolic and renal effects of a diet with varying MRP content in healthy and subtotally nephrectomized rats. Male Wistar rats were subjected to sham operation (control, C, n= 12), or to 5/6 nephrectomy (5/6NX, n= 12). Both groups were randomized into subgroups and pair‐fed with either a MRP‐poor or ‐rich diet for six weeks. The diet was prepared by replacing 5% or 25% of wheat starch by bread crust (BC). In spite of pair‐feeding, the rats on the 25% BC diet gained more body weight (C: 183 ± 6 g; C + 5% BC: 197 ± 7 g; C + 25% BC: 229 ± 6 g [P < 0.05]; 5/6NX: 165 ± 10 g; 5/6NX + 5% BC: 202 ± 3 g; 5/6NX + 25% BC: 209 ± 8 g [P < 0.05]) and had a higher organ weight (heart, liver, lung, kidney/remnant kidney). Bread crust‐enriched diet induced proteinuria (C: 15 ± 5 mg/24 h; C + 5% BC: 19 ± 4; C + 25% BC: 26 ± 3 [P < 0.05]; 5/6NX: 30 ± 7 mg/24 h; 5/6NX + 5% BC: 47 ± 9; 5/6NX + 25% BC: 87 ± 19 [P < 0.01]) and a rise in urinary transforming growth factor β1 excretion (C: 0.4 ± 0.1 ng/24 h; C + 5% BC: 0.6 ± 0.1; C + 25% BC: 1.2 ± 0.3; 5/6NX: 0.5 ± 0.1 ng/24 h; 5/6NX + 5% BC: 0.9 ± 0.1; 5/6NX + 25% BC: 1.6 ± 0.2 [P < 0.01]). Plasma creatinine or creatinine clearance were not affected significantly. In conclusion, our data suggests that long‐term consumption of a diet rich in MRPs may lead to damage of the kidneys.