Early and advanced stages of Maillard reaction in infant formulas: Analysis of available lysine and carboxymethyl-lysine.

Although the literature on the Maillard reaction in infant formulas is extensive, most studies have focused on model systems, and in only a few cases on real food systems. Therefore, the objective of the present study was to determine the status of the Maillard reaction, both the early and advanced phases, in a variety of commercial infant formulas available on the Swedish market. Ten powder and liquid milk-based infant formulas from three manufacturers were selected to determine available lysine and CML contents, the two established indicators of the reaction. The products were also characterized with respect to protein content, carbohydrates composition, water content and water activity. In order to be able to compare the impact of different processing steps applied on powder and liquid formulas, the solid formulas contained similar ingredients as their corresponding liquid ones. Our findings showed that powder and liquid formulas contained similar available lysine concentrations regardless of the manufacturer, showing 27.14-36.57% decrease in the available lysine, compared to the reference skim milk powder in this study. The CML concentrations were in a broad range of 68.77-507.99 mg / kg protein. In the case of one manufacturer, liquid infant formulas had significantly higher CML content, compared to the powder products (p < 0.05). The results from this study are a step taken towards better understanding of the extent of the Maillard reaction in real complex systems of infant formulas.

Review of analytical methods for measurement of oat proteins: The need for standardized methods.

This review summarizes the analytical methods that have been developed for quantification and characterization of oat proteins. These include sampling, sample preparation, extraction, quantification, separation, detection, and characterization of oat proteins. The review also provides a comparison of different methods for the determination of protein fraction of oat and the efficiency thereof. We conclude that there is a need for further validation of existing data or methods and for a standard methodology to quantify oat proteins.

On the Importance of Processing Conditions for the Nutritional Characteristics of Homogenized Composite Meals Intended for Infants.

The nutritional quality of infant food is an important consideration in the effort to prevent a further increase in the rate of childhood obesity. We hypothesized that the canning of composite infant meals would lead to elevated contents of carboxymethyl-lysine (CML) and favor high glycemic and insulinemic responses compared with milder heat treatment conditions. We have compared composite infant pasta Bolognese meals that were either conventionally canned (CANPBol), or prepared by microwave cooking (MWPBol). A meal where the pasta and Bolognese sauce were separate during microwave cooking (MWP_CANBol) was also included. The infant meals were tested at breakfast in healthy adults using white wheat bread (WWB) as reference. A standardized lunch meal was served at 240 min and blood was collected from fasting to 360 min after breakfast. The 2-h glucose response (iAUC) was lower following the test meals than with WWB. The insulin response was lower after the MWP_CANBol (-47%, p = 0.0000) but markedly higher after CANPBol (+40%, p = 0.0019), compared with WWB. A combined measure of the glucose and insulin responses (ISIcomposite) revealed that MWP_CANBol resulted in 94% better insulin sensitivity than CANPBol. Additionally, the separate processing of the meal components in MWP_CANBol resulted in 39% lower CML levels than the CANPBol. It was therefore concluded that intake of commercially canned composite infant meals leads to reduced postprandial insulin sensitivity and increased exposure to oxidative stress promoting agents.

Heat-treated high-fat diet modifies gut microbiota and metabolic markers in apoe-/- mice.

BACKGROUND: High-fat diet has been known to have adverse effects on metabolic markers, as well as the gut microbiota. However, the effect of heat processing of high-fat diet, which leads to formations of advanced glycation end products (AGEs) has not been clearly distinguished from the effect of unheated fat. This study compared the effect of high-fat diet with heat-treated high-fat diet on adiposity, atherosclerosis and gut microbiota composition in the caecum of apoe (-/-) mice. METHOD: Male apoe (-/-) mice were fed either low-fat (LF) control diet, high-fat (40 E% saturated fat, HF) control diet, or heat-treated high-fat (200 °C for 10 min, HT) diet, for 8 weeks. The plasma samples were used in the analysis of Nε-carboxy-methyl-lysine (CML) and Nε-carboxy-ethyl-lysine (CEL). The heart samples were analysed for atherosclerotic plaques, and the DNA from caecum was extracted and analysed for microbiota composition using 16S rRNA gene sequencing on a Miseq instrument. Additionally, the functions of microbial communities were also predicted based on the bacterial 16S rRNA gene sequence using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). RESULTS: Here we found that HT modifies gut microbiota composition and host adiposity. Prediction of bacterial gene functions based on 16S rRNA gene sequence revealed that HF increased bacterial genera enriched in lipid metabolism genes, while HT did not. Plasma CML and CEL increased 1.7 and 2.5 times, respectively, in mice fed HT as compared to mice fed HF. Despite lower adiposity, mice fed HT maintained atherosclerosis and displayed enlarged spleens. CONCLUSIONS: The results suggested that heat processing of high-fat diet modifies the substrates reaching the lower gut of apoe (-/-) mice, resulting in different effects on gut microbiota composition. AGEs seem to maintain the effect on atherosclerosis, despite lower adiposity, and causing enlarged spleens, which possibly reflect elevated levels of inflammation in the body.

Application of a dye-binding method for the determination of available lysine in skim milk powders.

A dye-binding method using Acid Orange 12 was investigated regarding its suitability for the quantification of available lysine, as a means of monitoring the Maillard reaction in skim milk powders. The method was evaluated by analyzing a wide range of milk powders produced by three different drying methods and stored under various conditions. A pilot-scale freeze-dryer, spray-dryer and drum-dryer were used to produce skim milk powders and the samples were stored at two temperatures (20 °C and 30 °C) and two relative humidities (33% and 52%) under strictly controlled conditions. Moreover to validate the method, two protein isolates; bovine serum albumin and casein were investigated for their available lysine content. The results demonstrate the suitability of this method for measuring the available lysine in skim milk powders with good precision and high reproducibility. The relative standard deviations obtained from the 125 freeze-dried powders were 1.8%, and those from the 100 drum-dried samples were all 1.9%. The highest variation was found for the spray-dried powders, which showed relative standard deviations between 0.9% and 6.7%.

Barley malt increases hindgut and portal butyric acid, modulates gene expression of gut tight junction proteins and Toll-like receptors in rats fed high-fat diets, but high advanced glycation end-products partially attenuate the effects.

Barley malt, a product of controlled germination, has been shown to produce high levels of butyric acid in the cecum and portal serum of rats and may therefore have anti-inflammatory effects. The aim of the study was to investigate how four barley malts, caramelized and colored malts, 50-malt and 350-malt, differing in functional characteristics concerning beta-glucan content and color, affect short-chain fatty acids (SCFA), barrier function and inflammation in the hindgut of rats fed high-fat diets. Male Wistar rats were given malt-supplemented high-fat diets for four weeks. Low and high-fat diets containing microcrystalline cellulose were incorporated as controls. All diets contained 70 g kg(-1) dietary fiber. The malt-fed groups were found to have had induced higher amounts of butyric and propionic acids in the hindgut and portal serum compared with controls, while cecal succinic acid only increased to a small extent. Fat increased the mRNA expression of tight junction proteins and Toll-like receptors (TLR) in the small intestine and distal colon of the rats, as well as the concentration of some amino acids in the portal plasma, but malt seemed to counteract these adverse effects to some extent. However, the high content of advanced glycation end-products (AGE) in caramelized malt tended to prohibit the positive effects on occludin in the small intestine and plasma amino acids seen with the other malt products. In conclusion, malting seems to be an interesting process for producing foods with positive health effects, but part of these effects may be destroyed if the malt contains a high content of AGE.

Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians.

PURPOSE: Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. METHODS: Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. RESULTS: Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p < 0.01, Student's t test). A multivariate analysis demonstrated that there was no association between the fMN and factors such as age, sex, intake of vitamins/minerals, serum folic acid and vitamin B12 levels, physical activity, and body mass index. The mean Hb adduct levels of acrylamide and glycidamide showed no significant differences between vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p < 0.04) between the two groups. CONCLUSIONS: These data suggest that the vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the metabolic rates of acrylamide was indicated. In addition, the observed significant difference in the background fMN in the two groups demonstrated that the MN analysis method has a sensitivity applicable to the biomonitoring of human lifestyle factors.

Food contaminant acrylamide increases expression of Cox-2 and nitric oxide synthase in breast epithelial cells.

Acrylamide has been discovered in foods cooked at high temperature. A potentially harmful effect of this dietary component has been suggested by data indicating its association with increased breast cancer. This study investigated the potential effects of acrylamide in nontumorigenic breast cells by assessing expression levels of inducible nitric oxide synthase (iNOS) and cycloogenase-2 (Cox-2) and NOS activity, which are known to be early molecular changes in disease formation. Treatment of cells with acrylamide increased levels of iNOS (both expression and activity) and Cox-2. Its potent metabolite, glycidamide, also induced both iNOS and Cox-2, with induction of iNOS occurring at a lower concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), another food-borne carcinogen, was found to induce Cox-2 expression. Combining acrylamide with PhIP did not result in a further increase. These studies suggest that further research is needed to determine the role of carcinogens formed from cooking foods in inducing early molecular changes associated with breast cancer.

Acrylamide formed at physiological temperature as a result of asparagine oxidation.

Acrylamide is a probable human carcinogen that is neurotoxic to both humans and animals. It is known to be formed during cooking of foods at temperatures higher than 120 degrees C. The present study demonstrates that acrylamide can also be formed at physiological conditions (37 degrees C, pH 7.4) when asparagine is incubated in the presence of hydrogen peroxide (H(2)O(2)). The formation of acrylamide under these conditions is dependent on the incubation time and the concentration of H(2)O(2). Thus, the results raise the question of the possible endogenous formation of acrylamide in pathological conditions that are associated with long-term oxidative stress. Further studies are therefore warranted to clarify the possible endogenous formation of acrylamide and its significance in chronic conditions that are known to be associated with oxidative stress.

Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.

Acrylamide: a dietary carcinogen formed in vivo?

Acrylamide, a chemical formed during heating of human foods, reacts with N-terminal valine in hemoglobin (Hb) and forms stable reaction products (adducts). These adducts to N-terminal valine in Hb have been used to estimate daily intake of acrylamide. Daily intake of acrylamide estimated from Hb adduct levels was higher than daily intake estimated from dietary questionnaires, possibly indicating other sources of exposures. Therefore, in this study the possible endogenous formation of acrylamide was investigated by treating mice with FeSO 4, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-hydrochloric acid (MPTP), or methamphetamine (METH). Acrylamide Hb adducts were determined, and a significant increase ( p < 0.05) in acrylamide Hb adduct levels was observed 24 h following treatment with FeSO 4 and 72 h following treatment with MPTP or METH. The results of this study show that acrylamide Hb adduct levels are increased in mice treated with compounds known to induce free radicals, thus suggesting the endogenous production of acrylamide.

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection.

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.

Relationships between biomarkers of exposure and toxicokinetics in Fischer 344 rats and B6C3F1 mice administered single doses of acrylamide and glycidamide and multiple doses of acrylamide.

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells and carcinogenic in rodents. AA is also formed in many commonly consumed starchy foods during cooking. Our previous toxicokinetic investigations of AA and its important genotoxic metabolite, glycidamide (GA), in rodents showed that AA is highly bioavailable from oral routes of administration, is widely distributed to tissues and that the dietary route, in particular, favors metabolism to GA. Measurements of DNA adducts in many tissues supported the hypothesis that AA is carcinogenic in rodent bioassays through metabolism to GA. The current investigation describes the development and validation of methodology for measuring hemoglobin (Hb) adducts with AA and GA in the same rodents previously used for toxicokinetic and DNA adduct measurements. The goal was to investigate possible relationships between these circulating biomarkers of exposure and serum toxicokinetic parameters for AA and GA and tissue GA-DNA adducts in rodents from both single and repeated dosing with AA. Significant correlations were observed between GA-Hb and liver GA-DNA adducts for either single or multiple dosing regimens with AA. Using available GA-Hb adduct data, empirical and allometric relationships permitted estimation of liver DNA adducts in humans in the range of 0.06-0.3 adducts/10(8) nucleotides. This approach may prove useful in extrapolating human cancer risks from findings in rodent bioassays.

Factors that influence the acrylamide content of heated foods.

Our finding that acrylamide is formed during heating of food initiated a range of studies on the formation of acrylamide. The present paper summarizes our follow-up studies on the characterization of parameters that influence the formation and degradation of acrylamide in heated foods. The system designed and used for studies of the influence of added factors was primarily homogenized potato heated in an oven. The net content of acrylamide after heating was examined with regard to the following parameters: heating temperature, duration of heating, pH and concentrations of various components. Higher temperature (200 degrees C) combined with prolonged heating led to reduced levels of acrylamide, due to elimination/degradation processes. At certain concentrations, the presence of asparagine or monosaccharides (in particular fructose, glucose and glyceraldehyde) was found to increase the net content of acrylamide. Addition of other free amino acids or a protein-rich food component strongly reduced the acrylamide content, probably by promoting competing reactions and/or covalently binding of formed acrylamide. The pH-dependence of acrylamide formation exhibited a maximum around pH 8; lower pH enhanced elimination and decelerated formation of acrylamide. In contrast, the effects of additions of antioxidants or peroxides on acrylamide content were not significant. The acrylamide content of heated foods is the net result of complex reactions leading to both the formation and elimination/degradation of this molecule.

Investigations of factors that influence the acrylamide content of heated foodstuffs.

The acrylamide content of heated foodstuffs should be considered to be the net result of complex reactions leading to the formation and elimination/degradation of this compound. The present study, involving primarily homogenized potato heated in an oven, was designed to characterize parameters that influence these reactions, including the heating temperature, duration of heating, pH, and concentrations of various components. Higher temperature (200 degrees C) combined with prolonged heating times produced reduced levels of acrylamide, due to elimination/degradation processes. At certain concentrations the presence of asparagine or monosaccharides (in particular, fructose and also glucose and glyceraldehyde) was found to increase the net content of acrylamide. Addition of other free amino acids or a protein-rich food component strongly reduced the acrylamide content, probably by promoting competing reactions and/or covalently binding acrylamide formed. The dependence on pH of the acrylamide content exhibited a maximum around pH 8; in particular, lower pH was shown to enhance elimination and decelerate formation of acrylamide. In contrast, the effects of additions of antioxidants or peroxides on acrylamide content were small or nonexistent.

Analysis of acrylamide, a carcinogen formed in heated foodstuffs.

Reaction products (adducts) of acrylamide with N termini of hemoglobin (Hb) are regularly observed in persons without known exposure. The average Hb adduct level measured in Swedish adults is preliminarily estimated to correspond to a daily intake approaching 100 microg of acrylamide. Because this uptake rate could be associated with a considerable cancer risk, it was considered important to identify its origin. It was hypothesized that acrylamide was formed at elevated temperatures in cooking, which was indicated in earlier studies of rats fed fried animal feed. This paper reports the analysis of acrylamide formed during heating of different human foodstuffs. Acrylamide levels in foodstuffs were analyzed by an improved gas chromatographic-mass spectrometric (GC-MS) method after bromination of acrylamide and by a new method for measurement of the underivatized acrylamide by liquid chromatography-mass spectrometry (LC-MS), using the MS/MS mode. For both methods the reproducibility, given as coefficient of variation, was approximately 5%, and the recovery close to 100%. For the GC-MS method the achieved detection level of acrylamide was 5 microg/kg and for the LC-MS/MS method, 10 microg/kg. The analytic values obtained with the LC-MS/MS method were 0.99 (0.95-1.04; 95% confidence interval) of the GC-MS values. The LC-MS/MS method is simpler and preferable for most routine analyses. Taken together, the various analytic data should be considered as proof of the identity of acrylamide. Studies with laboratory-heated foods revealed a temperature dependence of acrylamide formation. Moderate levels of acrylamide (5-50 microg/kg) were measured in heated protein-rich foods and higher contents (150-4000 microg/kg) in carbohydrate-rich foods, such as potato, beetroot, and also certain heated commercial potato products and crispbread. Acrylamide could not be detected in unheated control or boiled foods (<5 microg/kg). Consumption habits indicate that the acrylamide levels in the studied heated foods could lead to a daily intake of a few tens of micrograms.