Assessing the effects of model Maillard compound intake on iron, copper and zinc retention and tissue delivery in adult rats

Abstract

The behaviour of dietary Maillard reaction compounds (MRP) as metal chelating polymers can alter mineral absorption and/or retention. Our aim in this study was to analyse the long-term effects of the consumption of model MRP from glucose–lysine heated for 90 min at 150 °C (GL) on iron, copper and zinc whole-body retention and tissue delivery. For 88 days, weaning rats were fed a Control diet or one containing 3% GL, until reaching the adult stage. During the experimental period a mineral balance was conducted to investigate the mineral retention. At day 88, the animals were sacrificed, blood was drawn for haemoglobin determination and some organs were removed. Copper and zinc balances were unaffected (Cu: 450 vs. 375 μg; Zn: 6.7 vs. 6.2 mg for Control and GL groups, respectively) and no change was observed in whole-body delivery. Iron retention, too, was unaltered (11.2 mg for Control and GL groups) but due to the tendency toward decreased body weight in the GL group (248 vs. 233 g for the Control and GL groups), whole-body iron concentration was 13% higher in the GL group than in the Control group. Absorbed iron accumulated particularly in the liver (144 vs. 190 μg g⁻¹ for the Control and GL groups), thus reducing haemoglobin levels. The long-term intake of MRP induced iron accumulation in the body but this did not result in enhanced iron functionality, since the haemoglobin concentration declined. Taking into account the findings of our research group's studies of young and adult rats, we now corroborate the hypothesis that the negative effect of GL MRP consumption on iron functionality takes place regardless of the animals’ stage of life.

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Introduction

The processing of foods rich in protein and carbohydrates or fat promotes the development of the Maillard reaction (MR). Various browning compounds, known as Maillard reaction products (MRP), are responsible for producing the characteristic flavours, colours and taste that make cooked foods desirable for consumers.¹ MRP are associated with certain positive biological activities such as antimicrobial,^2,3 antihypertensive⁴ or microbiota modulation.⁵ However, these compounds may also provoke undesirable nutritional effects, some of which are related to protein damage⁶ and mineral availability,⁷ since MRP behave as anionic polymers, forming stable complexes with metal cations such as iron, copper and zinc.⁸

Due to the complex chemical composition of foods, MRP models are commonly investigated in animal studies in order to analyse the biological effects produced, since such studies are straightforward to perform and this approach simplifies the results obtained and their interpretation. One of the most widely studied model systems is glucose–lysine (GL), because lysine is very sensitive to thermal treatment and is highly reactive in the Maillard reaction. Consequently, the availability of lysine also decreases more than that of other amino acids. Some in vitro assays have observed a faster decrease in copper, iron and zinc solubility in the presence of MRP from the GL model system compared with those derived from a glucose–methionine system. This outcome is due to the rapid participation of lysine in the MR compared with others and the ability of MRP to act as metal chelators.⁹

The effects derived from the presence of MRP on iron, copper and zinc availability and antioxidant status have been shown in vitro,^10–12 and in vivo.¹³ Glucose–lysine-heated mixtures have been analysed for their potential iron-chelating activity¹⁴ and it has been observed that the presence of these compounds decreases iron solubility under intestinal conditions.¹⁵ In the case of zinc, after in vitro digestion of casein–glucose–fructose heated mixtures, the percentage of the precipitated mineral was found to be significantly higher than in the raw sample.¹¹

In vivo studies, mainly performed in young rats, are limited and revealed controversial results.⁷ A study conducted to assess the effects of model MRP on copper metabolism in weaning rats for 3 weeks concluded that diets containing GL or glucose–methionine heated mixtures increased the levels of copper absorption and retention, and of accumulated copper in some organs.¹⁰ Biological experiments on iron absorption in young rats fed diets containing MRP derived from amino acid-sugar¹⁴ or from protein-sugar^16,17 model-systems are discordant, but most of them have observed no variations in iron digestibility. Data concerning the effects of MRP consumption on mineral metabolism of adult rats are even scarcer. In this case, a mechanism of adaptation to the diet is more likely to occur, and the initial adverse effects of some dietary toxics may be mitigated. To better understand the long-term impact of MRP consumption on the metabolism of trace minerals and their possible health implications, in this study we evaluate the effects of model GL MRP, when included in a balanced diet on iron, copper and zinc retention and on tissue delivery in adult rats.

Materials and methods

Chemicals

The chemicals used were of analytical grade and obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Sigma Chemical, St Louis, MO, USA) unless mentioned otherwise.

Sample preparation

Glucose and lysine-HCl were used to prepare the model system. Equimolar mixtures of glucose–lysine-HCl, 40% moisture and prepared in unbuffered distilled water, were heated in open receptacles in an oven (Model 20000210, Selecta, Barcelona, Spain) at 150 °C for 90 min to obtain the GL sample. This procedure was intended to simulate normal home cooking methods. After heating, the reaction was stopped by cooling in an ice bath and the products were then removed, frozen, lyophilized and stored at 4 °C as described by Delgado-Andrade et al.¹⁵ until required for preparing the diet. The product obtained was highly water soluble since it was produced from lysine-HCl to improve its final solubility.

Preparation of diets

The AIN-93G purified diet for laboratory rodents (Dyets Inc, Bethlehem, PA) was used as the Control diet. The GL sample was added to the AIN-93G diet to reach a final concentration of 3%. This diet was termed GL. The analysis of GL diet revealed no modification of the overall nutrient composition, compared with the Control diet (AIN-93G). The mean ± standard deviation (SD) (n = 4) contents of the main nutrients of the diets were: 81.4 ± 0.8 g per kg moisture, 176.6 ± 3.1 g per kg protein, 78.1 ± 0.9 g per kg fat, 44.1 ± 0.2 mg per kg iron, 60.0 ± 0.1 mg per kg copper and 4.0 ± 0.1 mg per kg zinc. The higher MRP content of the GL diet compared with the Control one was confirmed by analysing their furosine and hydroxymethylfurfural (HMF) contents following the procedures described by Delgado-Andrade et al.¹⁸ The data obtained for furosine were (mean ± SD) 28.8 ± 0.5 and 1787.1 ± 7.3 mg per kg diet for the Control and GL diets respectively. The results for HMF were (mean ± SD) 0.44 ± 0.06 and 5.15 ± 0.08 mg per kg diet for the Control and GL diets, respectively.

Biological assays

Thirty four weanling Wistar rats weighing 40.8 ± 0.3 g (mean ± standard error (SE)), supplied by Charles River Laboratories, Spain, S.A., were used in the study. Twenty four rats were randomly distributed into two groups (12 animals per group), and each group was assigned to one of the dietary treatments. The animals were individually housed in metabolic cages in an environmentally controlled room under standard conditions (temperature: 20–22 °C with a 12 h light–dark cycle and 55–70% humidity). The rats had ad libitum access to their diets and demineralised water (Milli-Q ultrapure water system, Millipore Corps., Bedford, MA, USA). The remaining ten animals were sacrificed by anaesthesia overdose at day 0, and their initial Fe, Cu and Zn body content was analysed. The iron, copper and zinc balances for the entire experimental period, termed “retention”, were calculated from the difference between the final iron, copper and zinc body contents of each animal and the average initial content of the element (2.26 ± 0.08 mg Fe; 107.2 ± 11.0 μg Cu, 1.43 ± 0.03 mg Zn). Fe, Cu and Zn intake was monitored during this period. Six animals from each group were sacrificed by anaesthesia overdose on day 88 to calculate their final Fe, Cu and Zn body content. None of their organs were extracted. The remaining six animals in each group were anaesthetized with sodium pentobarbital (5 mg per 100 g of body weight) (Abbott Laboratories, Granada, Spain) and terminal exsanguination was performed by cannulation of the carotid artery. Haemoglobin levels were determined using the commercial Kit B263 from Linear Chemicals (Barcelona, Spain). The liver, right kidney, spleen, small intestine and right femur were removed, weighed and frozen at −80 °C until iron, copper and zinc analysis.

All management and experimental procedures carried out in this study were in strict accordance with the current European regulations (86/609 E.E.C.) regarding laboratory animals. The Bioethics Committee for Animal Experimentation at our institution (EEZ-CSIC) approved the study protocol.

Analytical techniques

To determine the mineral retention, the whole cadavers of the six animals sacrificed by anaesthesia overdose from both dietary treatments were weighed, lyophilized and homogenized. Aliquots of them, together with the diets consumed, were separately and completely digested by the addition of concentrated HNO₃, HClO₄ and by heating at high temperatures (210–220 °C) in a sand beaker. On the other hand, at the same time, the liver, spleen, kidney, small intestine and femur of the other six animals per dietary treatment were dry-ashed in a muffle furnace (Selecta, Mod.366, Barcelona, Spain) at 450 °C, and the white ashes obtained were dissolved using HCl/HNO₃/H₂O (1 : 1 : 2). All samples (including aliquots of diets, cadavers and whole organs) were diluted with deionised water to an appropriate volume for iron, copper and zinc measurement. The total Fe, Cu and Zn contents were determined by atomic absorption spectrophotometry (AAS) in a Perkin-Elmer Analyst 700 spectrophotometer (Norwalk, CT, USA). Standard solutions were prepared from stock Tritisol solutions of iron (FeCl₃ in 15% HCl, 1000 mg Fe, Merck), copper (CuCl₂ in H₂O, 1000 mg Cu, Merck) and zinc (ZnCl₂ in 0.06% HCl, 1000 mg Zn, Merck). The Control diet was used as an internal control to assess the precision of the AAS determinations. The inter-assay coefficients of variation were 3.21%, 6.99% and 1.93% for iron, copper and zinc, respectively. Pig kidney standard (certified reference material BCR no. 186, Community Bureau of Reference, Brussels, Belgium) was simultaneously used to quantify the accuracy of three elements: iron: measured value 311 ± 5 μg g⁻¹, certified value 299 ± 10 μg g⁻¹; copper: measured value 32.2 ± 0.5 μg g⁻¹, certified value 31.9 ± 0.4 μg g⁻¹; zinc: measured value 126 ± 4 μg g⁻¹, certified value 128 ± 4 μg g⁻¹ (mean ± SD of ten determinations).

All glassware and polyethylene sample bottles were washed with 10M nitric acid, and Milli-Q water was used throughout.

The parameters calculated for the balance study were mineral retention (final mineral body content − initial mineral body content) and retention rate or R/I (%) (mineral retention/mineral intake × 100).

Statistical analysis

After confirming the normal distribution of the data, Student's unpaired t-test was applied to compare the means that showed a significant variation (P < 0.05). Analyses were performed using Statgraphics Plus, version 5.1, 2001.

Results and discussion

Food intake and body weight

The animals’ body weight at different moments of the trial and their cumulative food intake are shown in Fig. 1. With slight variations, food consumption was stable between diets throughout the whole experimental period. Previous research findings differ concerning the effects of MRP on food intake. While the consumption of a high MRP diet by a healthy population increases the food intake,¹⁹ animal assays have led to contradictory conclusions: in one study, when mice were fed diets containing high or low MRP levels, there was no effect on food consumption;²⁰ however our research group reported a decreased food intake when younger rats were fed the same browning products as those employed in this trial.²¹ Differences between human and animal trials arise because in the human diet the presence of MRP is the major factor giving rise to pleasant flavours and aromas, which are essential to improve food palatability and consumer acceptability and thus to increase consumption.¹ The decreased food intake in animal assays could be related, on the one hand, to excessive amounts of MRP introduced into the diet, compared with human trials, with the aim of investigating the deleterious effects associated with MRP consumption. On the other hand, it could be caused by the satiating effects of a less digestible diet. Larger and more indigestible particles are known to be retained in the stomach for longer periods,²² and this situation could apply to the excessive presence of MRP, as they are less digestible,²³ especially those with a higher molecular weight. In this respect, Kimiagar et al.²⁴ described a slower rate of stomach emptying in rats fed on Maillard browned egg albumin.

Fig. 1 Weight evolution (g) and cumulative food intake (g) of weaning rats fed the Control (none MRP added) or the GL (MRP added) diets for 88 days. Global food intake during the trial was as follows: 1311.6 ± 35.4 and 1281.8 ± 43.3 for the Control and GL group, respectively. No significant variations were found between groups (P > 0.05).

The above-mentioned slight variations in food intake during the experimental period led to the animals in the GL group having a lower final weight; nevertheless, the difference was not statistically significant (Fig. 1). A similar weight pattern was previously observed by Pastoriza et al.²⁵ after feeding rats a diet including 10% bread crust or its soluble fractions for three months. On the other hand, Sebekova et al.²⁶ observed an increase in body weight when rats consumed a diet containing 25% bread crust but not when the proportion of bread crust was 5%.

Mineral balance and tissue delivery

Data from the iron, copper and zinc balances are shown in Table 1. Regarding the mineral supply to the rats used in our study, it should be stressed that all the experimental diets met the requirements detailed for growing rats, since they were basically the commercial AIN-93G diet²⁷ to which the synthetized MRP were added.

Table 1 Iron, copper and zinc balance in weaning rats fed the Control (none MRP added) or the GL (MRP added) diets for 88 days

Mineral		Control group	GL group
Values are mean ± SE, n = 6. Different letters within a row indicate significant differences between groups (P > 0.05).
Fe	Intake (mg)	56.6 ± 2.6	56.6 ± 2.4
	Body content (mg)	13.5 ± 0.8	13.5 ± 0.6
	Retention (mg)	11.2 ± 0.8	11.2 ± 0.6
	Body concentration (μg per g weight)	54 ± 1^A	61 ± 2^B
Cu	Intake (μg)	8073 ± 367	7488 ± 322
	Body content (μg)	557 ± 30	482 ± 13
	Retention (μg)	450 ± 30	375 ± 13
	Body concentration (μg per g weight)	2.3 ± 0.1	2.2 ± 0.1
Zn	Intake (mg)	52.8 ± 2.4	50.4 ± 2.2
	Body content (mg)	8.1 ± 0.3	7.6 ± 0.4
	Retention (mg)	6.7 ± 0.3	6.2 ± 0.4
	Body concentration (μg per g weight)	33 ± 1	34 ± 2

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Although there were variations in the mineral intake at different moments of the trial (data not shown), the total consumption of each element did not vary significantly between the two experimental diets. The intake of the GL diet induced a non-significant trend toward decreasing the copper and zinc content and retention in the body. In the case of iron, these parameters remained fairly stable during the experimental period. As expected, the retention rates for all three elements (R/I%) remained unchanged (P > 0.05) compared with the Control group (Fe: 20.1 vs. 19.8%; Cu: 5.7 vs. 5.1%; Zn: 12.9 vs. 12.3% for the Control and GL groups, respectively).

Although the animals fed the two experimental diets had the same iron intake, the slight decreases (P > 0.05) in the final body weight of the rats that consumed the GL diet were sufficient to induce a higher iron concentration in their bodies (54 ± 1 vs. 61 ± 2 μg of Fe per g body weight for the Control and GL groups respectively; P < 0.05) (Table 1). This accumulation is striking, since, from a physiologic standpoint, homeostatic regulation effectively inhibits excessive iron uptake in the organism.²⁸ Taking into account that the only difference between the diets was the presence of model MRP in the GL diet; it is suggested that these products might be responsible for the accumulation, resulting from processes bypassing homeostatic control. On the other hand, no changes were observed in the copper or zinc concentrations in the body after consumption of the diet containing the assayed MRP.

Our research team has previous research experience in the field of trace minerals–MRP interaction, but always with young rats; nevertheless, deepening the insights into the effects on mineral metabolism in adult rats is the specific novelty of the present study. In the case of iron metabolism we have previously reported that iron retention increases when weaning rats are fed for three weeks a diet containing the same GL model MRP as was used in the present trial, in which the animals also achieved a higher body concentration of iron.¹⁵ Other researchers have described the stability of the iron balance after the inclusion of single Maillard products in the diet in studies of rats²⁹ or when the protein source was casein heated with glucose.¹⁴ In the few studies that have been conducted with human subjects, controversial results have been reported: in one study the inclusion of toasted cereals in the diet had no effect on the iron balance,³⁰ but in another, the consumption of a MRP-rich diet reduced iron bioavailability in male adolescents.¹²

Regarding copper, an earlier study by our team established that its apparent retention, as well as its bioavailability in weaning rats, increased after three weeks’ consumption of a balanced diet including MRP derived from different glucose–amino acid model systems.¹⁰ However, a human trial with a group of male adolescents consuming a rich or poor-MRP diet concluded that a high intake of browning products induces lower copper retention and bioavailability.³¹

With respect to the zinc balance, we have previously reported that Zn retention remains stable when the above-mentioned experimental design is applied to young rats fed the same GL diet.²¹ Other studies have established that the administration of oral or parenteral doses of MRP may affect zinc metabolism, suggesting that browning products could chelate zinc and increase its faecal excretion and/or urinary elimination, thereby affecting its retention.^30,32 Hyperzincuria has been reported,⁷ but other studies have failed to corroborate this, either in animals or in humans.^30,33 A recent study in humans demonstrated that a high MRP intake as part of a balanced diet did not affect zinc bioavailability or its biomarkers.³¹

For our study, the distribution of iron, copper and zinc in body tissues after the consumption of the experimental diets is shown in Table 2. In line with the higher concentration of iron in the body of the animals, given the assayed MRP, there was as an evident increase in the iron content and concentration in the hepatic tissue (P < 0.05) (Table 2), which is indicative of a state of ferric repletion. As it is well-known, iron absorption is essentially regulated by its requirements, and these may increase in response to hematopoietic needs. Iron from the liver is required by the bone marrow in order to enable the synthesis of the heme group and to produce red cells, so that it is transported in the bloodstream by transferrin.²⁸ In parallel with decreasing levels of haemoglobin, hematopoietic needs could increase, thus triggering the mobilisation of the iron stock from the liver. However, the incoming mineral was not functional, since it is accumulated in the liver but cannot be mobilised to prevent the decreased level of haemoglobin in the blood (Table 2). In this respect, some authors have suggested that different organs, including the liver, are the preferential target for MRP in the organism.³⁴ Recently the accumulation of a specific Maillard reaction compound, carboxymethyl-lysine, in the hearts and tail tendons of the animals from the present study was observed.³⁵ MRP have been described as metal chelators,¹³ and they could act as a vehicle toward the final destination of iron in some viscera, perhaps because they have a greater affinity with iron than transferrin, thus leaving hematopoietic needs unresolved. A similar situation has been documented in previous studies by our research group.¹⁵

Table 2 Iron, copper and zinc content and concentration in different organs of weaning rats fed the Control (no MRP added) or the GL (MRP added) diets for 88 days

Tissue	Control group	GL group
Values are means ± SE, n = 6. Different letters within a row indicate significant differences between groups (P < 0.05).
Liver
Fe (μg)	882 ± 44^A	1147 ± 66^B
Fe (μg g^−1)	144 ± 9^A	190 ± 9^B
Cu (μg)	51 ± 4	47 ± 3
Cu (μg g^−1)	8.3 ± 0.4	8.0 ± 0.5
Zn (μg)	193 ± 6	190 ± 9
Zn (μg g^−1)	31 ± 1	31 ± 2
Kidney
Fe (μg)	82 ± 3	78 ± 7
Fe (μg g^−1)	107 ± 3	110 ± 5
Cu (μg)	4.6 ± 0.4	4.5 ± 0.5
Cu (μg g^−1)	6.0 ± 0.4	6.4 ± 0.4
Zn (μg)	18 ± 1	18 ± 1
Zn (μg g^−1)	24 ± 1	25 ± 1
Spleen
Fe (μg)	343 ± 22^A	264 ± 28^B
Fe (μg g^−1)	685 ± 40^A	554 ± 26^B
Cu (μg)	0.9 ± 0.1	0.8 ± 0.1
Cu (μg g^−1)	1.8 ± 0.1	1.7 ± 0.1
Zn (μg)	11 ± 1	10 ± 1
Zn (μg g^−1)	22 ± 1	22 ± 1
Small intestine
Fe (μg)	92 ± 4	95 ± 6
Fe (μg g^−1)	20 ± 0.4	19 ± 0.5
Cu (μg)	13 ± 1	12 ± 1
Cu (μg g^−1)	2.8 ± 0.2	2.4 ± 0.1
Zn (μg)	128 ± 8	137 ± 6
Zn (μg g^−1)	27 ± 1	27 ± 1
Femur
Fe (μg)	39 ± 2	38 ± 2
Fe (μg g^−1)	70 ± 1	73 ± 2
Cu (μg)	2.8 ± 0.1	2.7 ± 0.1
Cu (μg g^−1)	4.9 ± 0.3	5.1 ± 0.1
Zn (μg)	167 ± 9	157 ± 4
Zn (μg g^−1)	301 ± 10	299 ± 6
Haemoglobin
(mg dL^−1)	15.7 ± 0.7^A	13.7 ± 0.2^B

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Conversely to what was observed in the liver, iron deposits and concentration in the spleen decreased in the GL group. The spleen is the organ where erythrocytes are destroyed when they get older (100–120 days) and it also acts as a reservoir for red cells.²⁸ It can be hypothesised that decreased iron storage in this tissue would indicate a lower stock of red cells, as the production of heme groups in the bone marrow falls, due to insufficient input of iron from the liver. On the other hand, the renal, femoral and intestinal tissues presented no change in the concentration of iron.

In our study, the copper and zinc balances remained stable (Table 1). There was no apparent effect on the tissue distribution of either of these minerals after consumption of the diet to which the model MRP was added (Table 2). In contrast to these results, other studies^10,36 have reported increased copper bioavailability in animals that consumed MRP diets, this change being induced by a significant accumulation of this element in organs such as liver and kidney. In the case of zinc, little information has been reported about the effects of MRP consumption on zinc deposit in the organs. Our own previous work in this field suggests that zinc concentration remains unchanged in the livers of rats fed a diet containing heated casein–glucose–fructose³⁷ but increases in the kidney after the intake of diets including MRP from the methionine–glucose model system.²¹

As a final observation, it should be noted that in earlier investigations we documented the negative effect on bone mineralization of the GL diet in young rats (40 ± 2 days old).^38,39 However, this effect disappeared when the feeding time was extended to 88 days, probably due to adaptive mechanisms that blocked the toxic action of MRP on the mineral constituents of bone.⁴⁰ In relation to iron, as observed in the present work, the actions recorded in previous research with young rats¹⁵ are reproduced in the adult animals in the present trial. In consequence, there does not appear to exist an adaptive mechanism counteracting the deleterious effects of these model MRP on iron metabolism and/or distribution.

In summary, in our study the long-term intake of glucose–lysine MRP did not alter the iron, copper or zinc balances. However, due to a slight decrease in the final weight of the animals, a certain hyperconcentration of iron was observed in the organism. This circumstance provoked an increased deposit of iron in the liver. However, the levels of functional iron did not rise, because haemoglobin values decreased. These data, together with our previous experience in trials with young animals, corroborate the view that the negative effect of GL MRP on iron functionality occurs regardless of the animals’ stage of life. These findings highlight the interactions that take place between dietary iron and GL MRP, which are among those most often consumed, and hence the importance of taking into account the effects of their consumption on iron bioavailability, so that its functionality can be maintained.

Acknowledgements

This work was supported by a project and a “Ramón y Cajal” postdoctoral contract of the Spanish Ministry of Economy and Competitiveness and the postdoctoral scholarship of S. Pastoriza from the Instituto Danone (Spain).

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