Chemical activation of piperidine by formaldehyde and formation of lysine-specific Maillard reaction products

Piperidine is considered as a lysine-specific Maillard reaction product that can be formed from free lysine through decarboxylation and deamination reactions or through cyclization of pent-4-en-1-amine, the counterpart of acrylamide from lysine. Due to the importance and reactivity of piperidine its further interaction products in glucose/lysine model system was investigated. A useful strategy based on Py-GC/MS analysis was developed using an isotope labelling technique to identify reaction products incorporating piperidine moieties. Products simultaneously possessing five lysine carbon atoms (C2′–C6′) and the Nε-amino group from lysine in addition to glucose carbon atoms were targeted using specifically labelled precursors such as [¹⁵Nα]Lysine·2HCl, [¹⁵Nε]Lysine·2HCl, [U-¹³C₆]Lysine·2HCl, [¹³C-6]Lysine·2HCl and [U-¹³C₆]Glucose. Detailed labelling studies using specifically ¹³C-enriched sugars have shown that the piperidine can form reactive 1-methylidenepiperidinium ion with formaldehyde, which is able to undergo further aldol addition reactions to form compounds, such as 3-(piperidin-1-yl)propanal and 3-(pyridin-1(4H)-yl)propanal. Furthermore, these studies have also demonstrated that oxidation of piperidine into di- and tetrahydropyridine derivatives can generate reactive eneamine moieties capable of nucleophilic attack at carbonyl groups and formation of pyridine derivatives.     

Amino acid-dependent formation pathways of 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone in the Maillard reaction

The formation pathways of two furanoids, 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone (DMHF) were studied by GC–MS in the Maillard-type model system based on glucose and selected amino acids. The reaction was performed in 0.01 M phosphate buffer by heating a 1:1 mixture of [¹³C6] glucose and [¹²C6] glucose with amino acid. There is only one major formation pathway for DMHF in which the glucose carbon skeleton stayed intact. Formation pathways for 2-acetylfuran were more complicated. They formed either from glucose or from glucose and glycine. In the presence of glycine, the [C-5] unit of glucose combined with formaldehyde from glycine leads to 2-acetylfuran. For other amino acids, either cyclisation of intact glucose or recombination of glucose fragments can lead to 2-acetylfuran formation. These results indicate a competitive trend in controlling Maillard reaction. Therefore, besides changing Miallard reaction impact factors (temperature, time, pH etc.), inhibiting or preventing the competitive reaction cascade may direct desired pathways of Maillard reaction.     

Elucidation of the mechanism of pyrrole formation during thermal degradation of 13C-labeled l-serines

Pyrolysis of [¹³C-1], [¹³C-2] and [¹³C-3]-labelled l-serines generated mono-substituted methyl and ethyl derivatives of pyrroles and pyrazines among other compounds. Analyses of label incorporation into the pyrroles have indicated their formation through aldol condensation of acetaldehyde with different α-aminocarbonyl compounds followed by cyclization and loss of water (Knorr pyrrole synthesis). Comparison of the label incorporation patterns of the α-aminocarbonyls involved in the formation of methyl and ethyl-substituted pyrroles with that of similarly substituted pyrazines, revealed their common origin. In addition, α-aminocarbonyls involved in the formation of 2- and 3-substituted pyrroles had identical label distribution patterns, indicating their formation through the same carbonyl precursors. Furthermore, the major pathway (55%) leading to the formation of the α-aminocarbonyl precursors of methyl-substituted pyrroles involved aldol addition of formaldehyde to glycolaldehyde, whereas the only pathway leading to the formation of the α-aminocarbonyl precursors of ethyl-substituted pyrroles involved the interaction of alanine — formed in situ — with glycolaldehyde.     

The contribution of coloured Maillard reaction products to the total colour of browned glucose/l-alanine solutions and studies on their formation

 Thermal treatment of aqueous solutions of glucose and l-alanine in the presence of furan-2-carboxaldehyde (mixture I) resulted in the formation of a variety of coloured compounds, amongst which (1R,8aR)- and (1S,8aR)-4-(2-furyl)-7-[(2-furyl)methylidene]-2hydroxy-2H,7H,8aH-pyrano[2,3-b]pyran-3-one (1a/1b) and 3,5-dihydroxy-2-[(E)-(2-furyl)methylidene]methyl-5,6-dihydropyran-4-one(2) were identified as the most intense by application of the colour dilution analysis (CDA). To study how the colorant formation is influenced by the solvent, the Maillard mixture was then heated in a water/methanol mixture (mixture II). Besides 1a/1b and 2, additional coloured compounds were detected in mixture II, amongst which (E)- and (Z)-6-hydroxymethyl-2-methoxy-4-[(2-furyl)methylene]-2H-pyran-3-one (3a/3b), (E)- and (Z)-2-methoxy-4-[(2-furyl)methylene]-2H-pyran-3-one (4a/4b) as well as (1R,8aR)- and (1S,8aR)-4-(2-furyl)-7-[(2-furyl)methylidene]-2-methoxy-2H,7H,8aH-pyrano[2,3-b]pyran-3-one (5a/5b) could be distinguished from the less colour-active by application of CDA. To measure the contribution of these colorants to the overall colour of the browned Maillard mixtures I and II, colour activity values were calculated as the ratio of the concentration to the visual detection threshold of each colorant. By application of this colour activity concept, 3.3% of the total colour of the Maillard mixture II was shown to be caused by the 2H,7H,8aH-pyrano[2,3-b]pyran-3-one chromophore (1a/1b and 5a/5b). Based on a labelling experiment with glucose-6-[¹³C₁], the formation pathway leading to this key chromophore, involving a retro-aldol cleavage of the C(6) carbon from the hexose skeleton, was clarified. Received: 7 May 1998     

Elucidation of the formation of CO2 in culinary dry products

 The formation of CO₂ in tomato powder, chosen as an example of a dry culinary product, was investigated at room temperature and at low values of water activity (a _w). CO₂ formation correlated well with parameters that represent the beginning and progression of the Maillard reaction. In the absence of O₂, CO₂ formation decreased. Pectin and depolymerized pectin did not influence CO₂ formation while galacturonic acid (GalA) had a large effect. Determination of ¹³CO₂ in low-moisture model systems revealed that CO₂ was not formed by decarboxylation of GalA alone. Only a small proportion of [1-¹³C]glycine and GalA was degraded by the Strecker pathway; however, glucose reacted with the labelled amino acid forming ¹³CO₂ which amounted to over 90% of the total CO₂ formed. Therefore, CO₂ could be used as an early indicator for the beginning of the Maillard reaction in dry culinary products. Received: 28 October 1996     

Elucidation of the formation of CO2 in culinary dry products

 The formation of CO₂ in tomato powder, chosen as an example of a dry culinary product, was investigated at room temperature and at low values of water activity (a _w). CO₂ formation correlated well with parameters that represent the beginning and progression of the Maillard reaction. In the absence of O₂, CO₂ formation decreased. Pectin and depolymerized pectin did not influence CO₂ formation while galacturonic acid (GalA) had a large effect. Determination of ¹³CO₂ in low-moisture model systems revealed that CO₂ was not formed by decarboxylation of GalA alone. Only a small proportion of [1-¹³C]glycine and GalA was degraded by the Strecker pathway; however, glucose reacted with the labelled amino acid forming ¹³CO₂ which amounted to over 90% of the total CO₂ formed. Therefore, CO₂ could be used as an early indicator for the beginning of the Maillard reaction in dry culinary products. Received: 28 October 1996     

Mechanism of formation of sulphur aroma compounds from l-ascorbic acid and l-cysteine during the Maillard reaction

The sulphur aroma compounds produced from a phosphate-buffered solution (pH 8) of l-cysteine and l-, l-[1-¹³C] or l-[4-¹³C] ascorbic acid, heated at 140 ± 2 °C for 2 h, were examined by headspace SPME in combination with GC-MS. MS data indicated that C-1 of l-ascorbic acid was not involved in the formation of sulphur aroma compounds. The sulphur aroma compounds formed by reaction of l-ascorbic acid with l-cysteine mainly contained thiophenes, thiazoles and sulphur-containing alicyclic compounds. Among these compounds, 1-butanethiol, diethyl disulphide, 5-ethyl-2-methylthiazole, cis and trans-3,5-dimethyl-1,2,4-trithiolane, thieno[2,3-b]thiophene, thieno[3,2-b]thiophene, cis and trans-3,5-diethyl-1,2,4-trithiolane, 1,2,5,6-tetrathiocane, 2-ethylthieno[2,3-b]thiophene, 2,4,6-trimethyl-1,3,5-trithiane and cyclic octaatomic sulphur (S₈) were formed solely by l-cysteine degradation, and the rest by reaction of l-ascorbic acid degradation products, such as hydroxybutanedione, butanedione, acetaldehyde, acetol, pyruvaldehyde and formaldehyde with l-cysteine or its degradation products, such as H₂S and NH₃. A new reaction pathway from l-ascorbic acid via its degradation products was proposed.     

Detection of Maillard products of lactose in heated or processed milk by HPLC/DAD

 Two HPLC methods with diode array detection were used to separate Maillard products from milk components. The Maillard products 4-(β-d-galactopyranosyloxy)-2-hydroxy-2-methyl-2H-pyran-3(6H)-one (2), 1-[3-(β-d-galactopyranosyloxy)-2-furanyl]-1-ethanone (4), 4-(β-d-galactopyranosyloxy)-5-(hydroxymethyl)-2-methyl-3(2H)-furanone (5), isomaltol (7), maltol (9), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (6), and 5-hydroxymethyl-2-furfuraldehyde (8) were determined using a water-methanol gradient. Furthermore, 4,5-dihydroxy-2-(β-d-galactopyranosyloxy)-5-methyl-2-cyclopenten-1-one (3) was separated from the milk components by applying the interaction reagent octylamine. Several heated or processed milk samples were analyzed, and formation of the Maillard products was determined and quantified. Thus it was found that 2 and 3 are early products of the Maillard reaction in milk, whereas after prolonged heating 4, 5, and particularly 9 and 6 become more important. Compounds 7 and 8 were not detected, even if the samples were heated under stringent conditions. Received: 25 June 1998     

Detection of Maillard products of lactose in heated or processed milk by HPLC/DAD

 Two HPLC methods with diode array detection were used to separate Maillard products from milk components. The Maillard products 4-(β-d-galactopyranosyloxy)-2-hydroxy-2-methyl-2H-pyran-3(6H)-one (2), 1-[3-(β-d-galactopyranosyloxy)-2-furanyl]-1-ethanone (4), 4-(β-d-galactopyranosyloxy)-5-(hydroxymethyl)-2-methyl-3(2H)-furanone (5), isomaltol (7), maltol (9), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (6), and 5-hydroxymethyl-2-furfuraldehyde (8) were determined using a water-methanol gradient. Furthermore, 4,5-dihydroxy-2-(β-d-galactopyranosyloxy)-5-methyl-2-cyclopenten-1-one (3) was separated from the milk components by applying the interaction reagent octylamine. Several heated or processed milk samples were analyzed, and formation of the Maillard products was determined and quantified. Thus it was found that 2 and 3 are early products of the Maillard reaction in milk, whereas after prolonged heating 4, 5, and particularly 9 and 6 become more important. Compounds 7 and 8 were not detected, even if the samples were heated under stringent conditions.     

Isolation and identification of 3,4-dideoxypentosulose as specific degradation product of oligosaccharides with 1,4-glycosidic linkages

Although it is well known that arginine side chains of proteins become extensively modified in the course of the advanced Maillard reaction, to date only few possible arginine derivatives are known. Recently, N-δ-[5-(3-hydroxypropyl)-4-oxo-imidazolon-2-yl]-l-ornithine (PIO) was identified as a new arginine derivative, formed exclusively from the side-chain of peptide-bound arginine and degradation products of oligosaccharides with 1,4-glycosidic linkages. In the present paper, a previously unknown C5 dicarbonyl compound, namely 3,4-dideoxypentosulose (3,4-DDPS), was isolated by means of semi-preparative RP-HPLC as the corresponding chinoxaline after heating lactose followed by trapping the 1,2-dicarbonyl compound with o-phenylenediamine. Identification was achieved using LC–MS as well as ¹H- and ¹³C-NMR. Synthesis of 3,4-DDPS as the corresponding 2-hydroxy-dihydro-pyran-3-one proved the structure analysis. During heating of N-α-hippuryl arginine (Hip-Arg) with 2-hydroxy-dihydro-pyran-3-one, Hip-PIO was formed as the only arginine derivative. The formation of 3,4-DDPS from oligosaccharides with 1,4-glycosidic linkages follows a new and quantitatively important pathway of carbohydrate degradation in foods.     

Quantitation of estragole by stable isotope dilution assays

Various calibration strategies for the quantitation of the phenylpropane estragole by gas chromatography-mass spectrometry were developed and compared. For application in stable isotope dilution assays, two deuterium labelled estragole isotopologues were synthesized. Of these, [3′,3′-²H₂]estragole was prepared by Wittig reaction of 4-methoxy-phenylacetaldehyde with [²H₃]methyl-triphenyl-phosphonium bromide, whereas [1″,1″,1″-²H₃]estragole was obtained by demethylation of estragole and deuteromethylation of the resulting 4-allylphenole.Besides estragole isotopologues, 1,2,4-trimethoxybenzene and 4-propylanisole were also tested as internal standards (I.S.) for the determination of estragole in fennel tea.[1″,1″,1″-²H₃]Estragole, 1,2,4-trimethoxybenzene, and 4-propylanisole revealed linear calibration functions and, therefore, were suitable for estragole quantitation. In contrast to this, [3′,3′-²H₂]estragole could only be applied as I.S. if it was added to the extracts in stoichiometric deficiency compared to unlabelled estragole. Moreover, due to its different chemical and physical properties, 1,2,4-trimethoxybenzene showed a recovery as low as 77%, whereas the other I.S. revealed recovery rates close to 100%. Considering the “real” values of estragole in fennel tea, the choice of the I.S. obviously is less important than the way of preparing the tea. In contrast to the common method for tea preparation, squeezing of the teabags increased the estragole content significantly by 50%.     

Reactive intermediates and carbohydrate fragmentation in Maillard chemistry

Much research is devoted to the elucidation of mechanisms in the Maillard reaction. Model studies with reactive intermediates and ¹³C-labelled precursors have contributed significantly to our understanding of flavour formation in the Maillard reaction. Several examples are discussed here: The formation of methyl pyrazines and 2-acetyl-1-pyrroline, the role of ARP's and deoxyglycosones and the formation of carbohydrate fragments from reducing sugars, 3-deoxy-glucosone and ARP's. It is concluded that carbohydrate fragments, and also flavour substances derived therof, are formed from the starting reducing sugars (or the corresponding imines), deoxyglycosones, and possibly ARP's. A general scheme for flavour formation in the Maillard reaction is proposed.     

Evaluation of Maillard Reaction Variables and Their Effect on Heterocyclic Amine Formation in Chemical Model Systems

Heterocyclic amines (HCAs), highly mutagenic and potentially carcinogenic by‐products, form during Maillard browning reactions, specifically in muscle‐rich foods. Chemical model systems allow examination of in vitro formation of HCAs while eliminating complex matrices of meat. Limited research has evaluated the effects of Maillard reaction parameters on HCA formation. Therefore, 4 essential Maillard variables (precursors molar concentrations, water amount, sugar type, and sugar amounts) were evaluated to optimize a model system for the study of 4 HCAs: 2‐amino‐3‐methylimidazo‐[4,5‐f]quinoline, 2‐amino‐3‐methylimidazo[4,5‐f]quinoxaline, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, and 2‐amino‐3,4,8‐trimethyl‐imidazo[4,5‐f]quinoxaline. Model systems were dissolved in diethylene glycol, heated at 175 °C for 40 min, and separated using reversed‐phase liquid chromatography. To define the model system, precursor amounts (threonine and creatinine) were adjusted in molar increments (0.2/0.2, 0.4/0.4, 0.6/0.6, and 0.8/0.8 mmol) and water amounts by percentage (0%, 5%, 10%, and 15%). Sugars (lactose, glucose, galactose, and fructose) were evaluated in several molar amounts proportional to threonine and creatinine (quarter, half, equi, and double). The precursor levels and amounts of sugar were significantly different (P < 0.05) in regards to total HCA formation, with 0.6/0.6/1.2 mmol producing higher levels. Water concentration and sugar type also had a significant effect (P < 0.05), with 5% water and lactose producing higher total HCA amounts. A model system containing threonine (0.6 mmol), creatinine (0.6 mmol), and glucose (1.2 mmol), with 15% water was determined to be the optimal model system with glucose and 15% water being a better representation of meat systems.     

Key meat flavour compounds formation mechanism in a glutathione-xylose Maillard reaction

The formation mechanism of meat flavours formed from a glutathione-xylose Maillard reaction was studied using a group of model reactions with [¹³C₅] xylose/xylose (1:1), heated at 132 °C for 90 min. Volatiles, especially the aroma-active compounds, and non-volatiles were analysed respectively with GC-O-MS and LC-TOF-MS. Analysis of the mass spectra showed that furfural, 2-furfurylthiol and thiophene were ¹³C₅-labelled and hence stem from xylose, whereas 2-methyl-3-furanthiol, 2-methyl-thiophene and 2-pentyl-thiophene, which showed similar formation patterns with only unlabelled compounds, may be from thiamine and/or cysteine carbons. In the process of Maillard reactions, GSH can be capable of cleaving in the position of glutamyl and cysteinyl, and then form 5-oxoproline or pyroglutamic acid (PCA), cysteine, and Cys-Gly dipeptide, which can form cyclic (Cys-Gly).     

Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows

The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B₁₂, given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B_12. To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-¹³C]glucose, [¹³C]NaHCO₃ and 3[1-¹³C,²H₃] methionine. Milk and plasma concentrations of folic acid and vitamin B₁₂ increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 ± 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism.     

Antioxidant formation by γ-irradiation of glucose–amino acid model systems

The efficacy of γ-irradiation for the formation of Maillard reaction products (MRPs) from glucose and lysine/glycine and the antioxidant potential of MRPs thus formed were examined. Formation of MRPs was observed by monitoring absorbance at 284 nm and 420 nm. Upon irradiation, there was a dose-dependent increase in absorbance (r² = 0.99) at both the wavelengths. Irradiation of glucose/lysine solution resulted in higher absorbance at 284 nm than did that of glucose/glycine solution. Similarly, increase in absorbance at 420 nm was observed upon irradiation in both the systems. No significant absorbance was observed with unirradiated solution of glucose and lysine/glycine. These findings thus clearly revealed the formation of intermediate products and brown complexes (of Maillard reaction) upon irradiation of glucose/amino acid solution. A fluorescence was also observed in irradiated glucose/amino acid solution, whereas, none was seen in non-irradiated solution. These observations further confirmed the formation of MRPs, as fluorescent compounds are known to be precursors of brown pigments formed during the Maillard reaction. These MRPs exhibited excellent antioxidant activity, as measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene bleaching assays. MRPs, formed at a 40 kGy dose, scavenged up to 62% of DPPH radical and 82% of β-carotene was protected from bleaching. Reducing power of MRPs, estimated using the ferricyanide, method was also increased as compared to non-irradiated solutions. Further, these MRPs were able to scavenge hydroxyl radical and superoxide anion radical to the extents of 33% and 58%, respectively. These MRPs could chelate iron to an extent of 32% under in vitro conditions. Thus, these studies clearly demonstrated that radiation technology could be employed for obtaining novel antioxidants from sugar and amino acid combinations.     

Tea Polyphenols as Inhibitors of Furan Formed in the Maillard Model System and Canned Coffee Model

In this article, the effects of sugars and amino acids on furan formation via the Maillard reaction in low‐moisture model systems were investigated. Glucose and alanine are important furan precursors, and the effects of the heating temperature, heating time, and molar ratio of glucose to alanine on furan formation were studied in glucose/alanine model system by response surface methodology. The heating temperature greatly affected furan formation. The maximum furan concentration was obtained with a glucose‐to‐alanine molar ratio of 0.83:1.00, by heating at 151 °C for 41 min. Tea polyphenols effectively inhibited furan formation in the glucose/alanine model and a canned coffee model. A high inhibition rate of 42.4% ± 1.5% was obtained in the canned coffee model during sterilization procedure with addition of 84 mg (the mass fraction is 12.1%) of tea polyphenols (99%). However, the content of aromatic components in the canned coffee model was significantly reduced at the same time. This study provides evidence for a good furan inhibitor that can be used in food processing.     

L-Rhamnose: progenitor of 2,5-dimethyl-4-hydroxy-3 [2H]-furanone formation by Pichia capsulata?

 2,5-Dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, 1), an important aroma constituent, was detected at concentrations of up to 2 mg/l after 4 days of growth of Pichia capsulata on casein peptone culture medium containing L-(+)-rhamnose (2). Blank samples without yeast contained no 1 after the incubation period. In parallel experiments five samples of 2 exhibiting different [¹³C] abundance were given to P. capsulata. The volatile compounds formed were isolated and analysed for their [¹³C]/[¹²C] ratios by on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). A positive correlation between the isotopes in 1 and 2 was observed; thus, 2 served as the carbon source for 1. However, 1 was formed from a postulated intermediate of 2 generated during thermal sterilization, as 1 was neither detected after sterile filtration of the culture medium nor after separate thermal sterilization of the casein peptone and 2. This observation was confirmed by an experiment investigating the time course of the formation of 1. Received: 4 June 1996     

Mechanism of pyrazole formation in [13C-2] labeled glycine model systems: N–N bond formation during Maillard reaction

Studies with ¹³C-2 labeled glycine in model systems containing 3-hydroxy-2-butanone or glyceraldehyde have indicated that the β-dicarbonyl compounds, the immediate precursors of pyrazoles, are produced in Maillard model systems through two pathways. One pathway involves dehydration of α,β-dihydroxy carbonyl compounds with elimination of the α-hydroxyl group and the other through aldol condensation of an α-hydroxy carbonyl compound with simple aldehydes to produce α,β-dihydroxy carbonyl moiety that can undergo the above-mentioned dehydration to produce β-dicarbonyl structures. The conversion of β-dicarbonyls into pyrazoles can be achieved through 1,3-diimine formation by reaction with either two ammonia molecules or with a primary amine and an ammonia. After imine–enamine isomerizations the resulting dienamine can be oxidized to form pyrazole rings similar to the oxidation of two thiol moieties into disulfide linkages. The α-dicarbonyl species can serve as hydrogen acceptors since in their absence no pyrazole formation was detected. Glycine/3-hydroxy-2-butanone system generated 3,4,5-trimethyl-pyrazole (associated with the aroma of tequila) and 1,3,4,5-tetramethyl-pyrazole whereas, glycine/glyceraldehyde generated 1,5-dimethyl and 1,3,5-trimethyl-pyrazoles.     

The production of intrinsically labeled milk and meat protein is feasible and provides functional tools for human nutrition research

Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce intrinsically l-[1-¹³C]phenylalanine-labeled milk and meat protein that would permit in vivo assessment of postprandial protein digestion and absorption kinetics in humans. One lactating dairy cow was continuously infused with 420 μmol of l-[1-¹³C]phenylalanine/min for 96 h, with plasma and milk being collected before, during, and after isotope infusion. Twenty-four hours after infusion, the cow was slaughtered to produce intrinsically labeled meat. Levels of l-[1-¹³C]phenylalanine enrichment as high as 40 mole percent excess (MPE) in milk and 1.5 MPE in meat protein were achieved. In a subsequent human proof-of-principle experiment, 2 healthy young males (25 ± 1 yr; 66.2 ± 5.2 kg) each ingested 135 g of l-[1-¹³C]phenylalanine intrinsically labeled minced beef, after which plasma samples were collected at regular time intervals. Plasma l-[1-¹³C]phenylalanine enrichments increased during the first 90 min following beef ingestion, reaching peak plasma enrichment levels of 0.61 ± 0.04 MPE. Whole-body net protein balance, assessed by continuous infusion of l-[ring-²H₅]phenylalanine and l-[ring-²H₂]tyrosine, was higher in the postprandial period compared with basal values (6.4 ± 0.1 vs. −4.5 ± 0.1 μmol/kg per h). In conclusion, the production of intrinsically l-[1-¹³C]phenylalanine-labeled milk and meat protein is feasible and provides functional tools to investigate in vivo protein digestion and absorption kinetics in humans.