The bioavailability of iron picolinate is comparable to iron sulfate when fortified into a complementary fruit yogurt: a stable iron isotope study in young women.

PURPOSE: A technological gap exists for the iron (Fe) fortification of difficult-to-fortify products, such as wet and acid food products containing polyphenols, with stable and bioavailable Fe. Fe picolinate, a novel food ingredient, was found to be stable over time in this type of matrix. The objective of this study was to measure the Fe bioavailability of Fe picolinate in a complementary fruit yogurt. METHODS: The bioavailability of Fe picolinate was determined using stable iron isotopes in a double blind, randomized cross-over design in non-anemic Swiss women (n = 19; 25.1 ± 4.6 years). Fractional Fe absorption was measured from Fe picolinate (2.5 mg 57Fe per serving in two servings given morning and afternoon) and from Fe sulfate (2.5 mg 54Fe per serving in two servings given morning and afternoon) in a fortified dairy complementary food (i.e. yogurt containing fruits). Fe absorption was determined based on erythrocyte incorporation of isotopic labels 14 days after consumption of the last test meal. RESULTS: Geometric mean (95% CI) fractional iron absorption from Fe picolinate and Fe sulfate were not significantly different: 5.2% (3.8-7.2%) and 5.3% (3.8-7.3%) (N.S.), respectively. Relative bioavailability of Fe picolinate versus Fe sulfate was 0.99 (0.85-1.15). CONCLUSION: Therefore, Fe picolinate is a promising compound for the fortification of difficult-to-fortify foods, to help meet Fe requirements of infants, young children and women of childbearing age.

Improvement of AOAC Official Method 984.27 for the determination of nine nutritional elements in food products by Inductively coupled plasma-atomic emission spectroscopy after microwave digestion: single-laboratory validation and ring trial.

A single-laboratory validation (SLV) and a ring trial (RT) were undertaken to determine nine nutritional elements in food products by inductively coupled plasma-atomic emission spectroscopy in order to improve and update AOAC Official Method 984.27. The improvements involved optimized microwave digestion, selected analytical lines, internal standardization, and ion buffering. Simultaneous determination of nine elements (calcium, copper, iron, potassium, magnesium, manganese, sodium, phosphorus, and zinc) was made in food products. Sample digestion was performed through wet digestion of food samples by microwave technology with either closed or open vessel systems. Validation was performed to characterize the method for selectivity, sensitivity, linearity, accuracy, precision, recovery, ruggedness, and uncertainty. The robustness and efficiency of this method was proved through a successful internal RT using experienced food industry laboratories. Performance characteristics are reported for 13 certified and in-house reference materials, populating the AOAC triangle food sectors, which fulfilled AOAC criteria and recommendations for accuracy (trueness, recovery, and z-scores) and precision (repeatability and reproducibility RSD and HorRat values) regarding SLV and RT. This multielemental method is cost-efficient, time-saving, accurate, and fit-for-purpose according to ISO 17025 Norm and AOAC acceptability criteria, and is proposed as an improved version of AOAC Official Method 984.27 for fortified food products, including infant formula.

Analysis of advanced glycation endproducts in dairy products by isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The particular case of carboxymethyllysine.

A fully validated multiple-transition recording isotope dilution liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of N(epsilon)-carboxymethyllysine (CML) and lysine in dairy products is described. Internal standards were [N-1',2'-(13)C(2)]CML and [1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]lysine, and the method was validated by evaluating the selectivity, linearity, precision (repeatability and reproducibility) and trueness, using both powder and liquid products. For liquid dairy products, the repeatability and reproducibility was 2.79% and 11.0%, while 4.85% and 4.92% were determined for powder dairy products, respectively. The trueness of the method ranged from -9.6% to -3.6% for powder and from -0.99% to 6.8% for liquid dairy products. The limit of detection for CML was estimated to be 8 ng CML per mg protein while the limit of quantification was 27 ng CML per mg protein. The method encompasses a proteolytic cleavage mediated by enzymatic digestion to reach a complete release of the amino acids prior to a sample cleanup based on solid phase extraction, and followed by LC-MS/MS analysis of CML and lysine residues. To ensure a suitable performance of the enzymatic digestion, CML measurements were compared to values obtained with an acid hydrolysis-mediated proteolysis. Finally, the method was employed for the analysis of CML in various dairy products. The values compare well to the data available in the literature when similar methods were used, even if some discrepancies were observed upon comparison with the results obtained by other techniques such as enzyme-linked immunosorbent assay and GC-MS.

Evaluating the extent of protein damage in dairy products: simultaneous determination of early and advanced glycation-induced lysine modifications.

An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine lysine (Lys), N(epsilon)-fructosyllysine (FL), N epsilon-carboxymethyllysine (CML), and pyrraline (Pyr) in dairy products. The presented approach entails protein cleavage via enzymatic digestion to liberate the aforementioned compounds, which were then quantified using a stable isotope dilution assay. LC-MS/MS analysis was performed by positive electrospray ionization recording two transition reactions per analyte in selected reaction monitoring mode. The CML and Lys values obtained with enzymatic digestion were compared to those acquired with acid hydrolysis HCl (6 mol/L), and the two proteolysis methods yielded comparable quantifications. Allowing for the fact that the investigated compounds are formed during different stages of the glycation process, the method is able to reveal the progress of protein glycation in dairy products.

Absence of 2'-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/MS.

The contribution of DNA adduct formation in the carcinogenic action of the mycotoxin ochratoxin A (OTA) has been subject to much debate. Recently, a carbon-bonded ochratoxin A-2'-deoxyguanosine adduct (dGuoOTA) formed by photochemical reaction in vitro has been shown by 32P-postlabeling/TLC to comigrate with a spot detected in DNA isolated from rat and pig kidney following exposure to OTA. Considering the large body of evidence arguing against covalent DNA binding of OTA and the poor resolution and specificity of postlabeling analysis, we developed a stable isotope dilution LC-MS/MS method to analyze dGuoOTA in kidney DNA isolated from rats treated with OTA. dGuoOTA and nitrogen-15-labeled dGuoOTA (15N(5)-dGuoOTA) were prepared by photoirradiation of OTA in the presence of dGuo or nitrogen-15-labeled dGuo. Conditions for DNA hydrolysis were optimized using a synthetic oligonucleotide containing dGuoOTA to ensure complete release of dGuoOTA. The LOD of the method (S/N > 3) was 10 fmol dGuoOTA on-column. However, dGuoOTA was not detected in DNA samples isolated from male F344 rats treated with OTA for up to 90 days at doses known to cause renal tumor formation. Detection limits, calculated for each individual sample based on the absolute LOD and the amount of DNA injected, were as low as 3.5 dGuoOTA/10(9) nucleotides. These data are consistent with previous results showing lack of DNA adduct formation by OTA and demonstrate that dGuoOTA is not formed in biologically relevant amounts under physiological conditions in vivo.

N(epsilon)-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response.

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: enzymatic digestion versus hydrochloric acid-mediated hydrolysis.

A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.

Quantitative determination of four nitrofuran metabolites in meat by isotope dilution liquid chromatography-electrospray ionisation-tandem mass spectrometry.

A confirmatory method based on isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the low-level determination of residues of four nitrofuran veterinary drugs in meat, e.g., furazolidone, furaltadone, nitrofurantoin, and nitrofurazone. The procedure entails an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into the 2-nitrobenzaldehyde (NBA) imine-type derivatives. Liquid-liquid extraction and clean-up on a polymeric solid phase extraction cartridge are then performed before LC-MS/MS analysis by positive electrospray ionisation (ESI) applying multiple reaction monitoring of three transition reactions for each compound. Reliable quantitation is obtained by using one deuterated analogue per analyte (d4-NBA derivative) as internal standard (IS). Validation of the method in chicken meat was conducted following the European Union (EU) criteria for the analysis of veterinary drug residues in foods. The decision limits (CCalpha) were 0.11-0.21 microg/kg, and the detection capabilities (CCbeta) 0.19-0.36 microg/kg, thus below the minimum required performance limit (MRPL) set at 1 microg/kg by the EU. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.

Analysis of matrix-bound nitrofuran residues in worldwide-originated honeys by isotope dilution high-performance liquid chromatography-tandem mass spectrometry.

A sensitive and selective isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) method is presented for the simultaneous analysis of the metabolites of four nitrofuran veterinary drugs, that is, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone, in honey samples. The method entails a combined hydrolysis of protein-bound drug metabolites and derivatization of the resulting metabolites with 2-nitrobenzaldehyde (NBA) during an overnight incubation, followed by a liquid-liquid extraction and a cleanup on a polymeric solid-phase extraction cartridge. Mass spectral acquisition is carried out in the positive ion mode by applying multiple reaction monitoring (MRM) of three diagnostic transition reactions for each analyte under survey. A reliable quantification is obtained by the use of one deuterated analogue per analyte (NBA-d(4) derivative). The method has been validated in honey according to the European Union criteria for the analysis of veterinary drug residues in food. Expressed in underivatized nitrofuran metabolite concentrations, the decision limits (CCalpha) ranged within 0.07-0.46 microg/kg, and the detection capabilities (CCbeta) were within 0.12-0.56 microg/kg. The method has been successfully applied in a survey of honeys of various geographical origins, showing that furazolidone is the main nitrofuran antibiotic administered to treat bacterial diseases of bees.

Preparation of stable isotope-labeled 2-nitrobenzaldehyde derivatives of four metabolites of nitrofuran antibiotics and their comprehensive characterization by UV, MS, and NMR techniques.

A convenient method is presented for the preparation of the carbon-13-labeled 2-nitrobenzaldehyde derivatives of the nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 1-aminohydantoin (AH), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), with the purpose of using them as internal standards for the quantification of trace levels of nitrofuran residues by liquid chromatography-tandem mass spectrometry in foods of animal origin. The synthesis encompasses the nitration of [1,2,3,4,5,6-(13)C(6)]toluene prior to chromyl compound-mediated oxidation of the methyl group into the corresponding aldehyde. The four metabolites of nitrofuran antibiotics were derivatized independently with the resulting ring-labeled 2-nitrobenzaldehyde (NBA) to obtain the target compounds. Both the isotopically enriched and native substances were used to perform a comprehensive fragmentation study by electrospray ionization (ESI) collision-induced dissociation (CID) mass spectrometry (MS). Full characterization of the nitrofuran derivatives was accomplished with ultraviolet (UV) and exhaustive nuclear magnetic resonance (NMR) analysis. A major advantage of the described procedure is that it can be extended to the preparation of other carbon-13-labeled derivatives of metabolites of nitrofuran antibiotics.

The effects of coffee on enzymes involved in metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rats.

The effects of coffee on the metabolism and genotoxicity of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were investigated. Coffee diminished the bacterial mutagenicity of PhIP in the Ames reversion assay through inhibition of cytochrome P450 1A2 (CYP1A2), a key enzyme involved in the metabolic activation of PhIP. When given as part of the diet (0, 1 or 5% w/w) to male Fischer-344 rats for 2 weeks, coffee affected the expression of hepatic enzymes involved in PhIP metabolism. Coffee increased the expression of CYP1A2 by 16-fold in the 5% coffee-treated group, and approximately half of this inductive effect was attributed to caffeine. Coffee also increased the expression of enzymes involved in the detoxication of PhIP. A 2-fold increase in expression of glutathione S-transferase alpha was observed, UDP-glucuronosyl transferase (UGTs) activities of p-nitrophenol increased 2-fold, while N(2)-and N3-glucuronidation of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP) increased by 1.3-fold in the 5% coffee-treated over the control group. The amount of PhIP (0.75 mg/kg, 24 h) eliminated in urine as the N(2)-and N3-glucuronide conjugates of HONH-PhIP increased by 1.8- and 2.5-fold, respectively, in the 5% coffee-treated group over control rats, suggesting either increased rates of N-oxidation of PhIP or N-glucuronidation of HONH-PhIP. Despite the strong induction of CYP1A2, there was no increase in PhIP-DNA adduct formation in colon and pancreas while liver adducts decreased by 50% over control animals. These data suggest that the effect of coffee on inhibition of PhIP N-oxidation and ensuing DNA damage is more important in vivo than its effect on induction of PhIP N-hydroxylation.

Artifactual nitration controlled measurement of protein-bound 3-nitro-L-tyrosine in biological fluids and tissues by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

A sensitive and selective method is presented to accurately determine the level of protein-bound 3-nitro-L-tyrosine (NTyr) in rat plasma and kidney samples. This assay is based on isotope dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation entails protein precipitation, acid hydrolysis with 6 N HCl, and solid-phase extraction (using reverse and aminopropyl phase cartridges) prior to the determinative step. For kidney samples, NTyr is converted into its butyl ester to improve sensitivity. The potential formation of artifactual NTyr during the acid hydrolysis step was carefully followed and determined by supplementation of the samples with (13)C-labeled L-tyrosine (Tyr) prior to protein digestion. Hence, the concomitant measurement of formation of (13)C-enriched NTyr enabled the accurate determination of artifactual NTyr. This approach was employed to measure the basal level of protein-bound NTyr in rat plasma and kidney samples, revealing levels in the range of 4-18 micromol/mol of Tyr and 50-68 micromol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereas in the case of plasma the contribution of the artifactual response ranged from 16 to 40%. This method allows the analysis of protein-bound NTyr with a full control of the artifactual nitration of tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr in proteins may allow insight into the role of nitric oxide-derived oxidants under various pathological conditions.

Simultaneous determination of 3-nitrotyrosine and tyrosine in plasma proteins of rats and assessment of artifactual tyrosine nitration.

A sensitive and specific isotope dilution liquid chromatography-electrospray tandem mass spectrometry method was developed for the determination of the 3-nitrotyrosine residue levels in rat plasma proteins. The assay is based on the cleavage of proteins with concentrated hydrochloric acid to release both 3-nitrotyrosine and tyrosine. To control the potential artifactual nitration of tyrosine residues during the proteolysis, samples are spiked with (13)C(9)-labeled tyrosine and the level of (13)C(9)-labeled 3-nitrotyrosine is measured. The clean-up process entails hydrolysate fortification with 2,5,6-d(3)-3-nitrotyrosine, followed by solid-phase extraction on octadecylsilyl (to isolate tyrosine) and aminopropylsilyl (to isolate 3-nitrotyrosine) cartridges. Tyrosine and 3-nitrotyrosine fractions are mixed in an appropriate ratio prior to the analysis. The method was applied to animals exposed to ferric nitrilotriacetate to induce oxidative stress.

Analysis and quantification of DNA adducts of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in liver of rats by liquid chromatography/electrospray tandem mass spectrometry.

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to measure DNA adducts of the carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) with a microbore C-18 reversed-phase column. Quantification of the isomeric adducts N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-N(2)-MeIQx) was achieved using synthetic, isotopically labeled internal standards. The reaction of the N-acetoxy ester of 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) with calf thymus DNA (ct DNA) resulted in formation of these adducts in a ratio of 5:1 (dG-C8-MeIQx:dG-N(2)-MeIQx). The detection limit by LC/ESI-MS/MS in the selected reaction monitoring (SRM) mode ([MH(+) --> MH - 116](+)) (loss of deoxyribose) approached 500 fg (1 fmol) of adduct standard, and 1 adduct per 10(8) DNA bases using 100 microg of DNA following solid-phase extraction. The SRM analysis of rat liver DNA 24 h after an oral dose of MeIQx (10 and 0.5 mg/kg) revealed the presence of isomeric dG-MeIQx adducts at levels of 3.07 +/- 0.84 and 0.45 +/- 0.27 adducts per 10(7) bases, respectively. LC/ESI-MS/MS product ion spectra were acquired on both adducts from the elevated dose of MeIQx for unambiguous adduct identification. The contribution of dG-N(2)-MeIQx to the total adducts in vivo was significantly more important than that observed in vitro. dG-C8-MeIQx was the principal adduct formed at the 10 mg/kg dose, (dG-C8-MeIQx:dG-N(2)-MeIQx (3:2)); however, dG-N(2)-MeIQx was the major lesion detected at the 0.5 mg/kg dose (dG-C8-MeIQx:dG-N(2)-MeIQx 1:10). The striking differences between the relative amounts of dG-C8-MeIQx and dG-N(2)-MeIQx formed in vivo as a function of dose suggest that reactive esters of HONH-MeIQx other than N-acetoxy-MeIQx may be formed in vivo and react preferentially with the N(2) atom of guanine, or that dG-C8-MeIQx is removed at a significantly more rapid rate than dG-N(2)-MeIQx. The dG-N(2)-MeIQx adduct, previously thought to be a minor adduct, is likely to be an important contributor to the genotoxic damage of MeIQx.