In vitro post-ovulatory oocyte ageing in grass carp Ctenopharyngodon idella affects H4K12 acetylation pattern and histone acetyltransferase activity.

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio.

Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.

A fast SALLE GC-MS/MS multi-analyte method for the determination of 75 food packaging substances in food simulants.

A simple and fast method was developed for the simultaneous determination of 75 plastic food contact material (FCM) in liquid food simulants, at levels of a few ng g-1. The method employs an optimised salt-assisted liquid-liquid extraction for all EU-regulated ethanol/H2O food simulants, in the presence of 10% NaCl (simulants A and C) or 5% NaCl (simulant D1), and dichloromethane as the extracting solvent. Gas chromatography with triple-quad MS operating in multiple reaction monitoring acquisition was used, applying isotope dilution with selected deuterated compounds. Adequate sensitivity was demonstrated for all analytes. The results also showed sufficient accuracy for the majority of substances, with recoveries of 70-120% and repeatability (expressed as relative standard deviations, RSDs) smaller than 15%. The method was applied to the analysis of FCM multilayer items after undergoing migration testing according to the specifications of the current EU legislation in force.

Potential of Unconventional Seed Oils and Fats from West African Trees: A Review of Fatty Acid Composition and Perspectives.

Seed oils sourced from West Africa are generally not well-characterized, but likely to have an untapped potential. This review aims to make an overview of fatty acid (FA) composition of unconventional seed oils from semi-arid West African trees and evaluate potential for new and enhanced uses and for improving local livelihoods and biodiversity conservation. A total of 111 studies on FA composition were found, covering 31 species. Only 69 of the studies (62%) were included in the review, as 38% had unreliable or incomplete results. There was a clear link between taxonomic kinship and FA composition. Over 20 potentially interesting and underexploited oils were found, including oils with properties similar to palm oil, olive oil, coconut oil, shea butter, and cotton seed oil. About half of the oils have promising potential for cosmetics. One third of the oils were relatively saturated, indicating properties for structuring food and heat resistance. Most of the species had multiple uses and oil production could be profitable in co-production with other non-timber forest products. Furthermore, establishment of sustainable oil production and domestication of oil trees could promote biodiversity conservation. Enhanced oil production in semi-arid West Africa is promising, but several practical constraints remain to be overcome.

Circulating Levels of Muscle-Related Metabolites Increase in Response to a Daily Moderately High Dose of a Vitamin D3 Supplement in Women with Vitamin D Insufficiency-Secondary Analysis of a Randomized Placebo-Controlled Trial.

Recently, we demonstrated negative effects of vitamin D supplementation on muscle strength and physical performance in women with vitamin D insufficiency. The underlying mechanism behind these findings remains unknown. In a secondary analysis of the randomized placebo-controlled trial designed to investigate cardiovascular and musculoskeletal health, we employed NMR-based metabolomics to assess the effect of a daily supplement of vitamin D3 (70 µg) or an identically administered placebo, during wintertime. We assessed the serum metabolome of 76 postmenopausal, otherwise healthy, women with vitamin D (25(OH)D) insufficiency (25(OH)D < 50 nmol/L), with mean levels of 25(OH)D of 33 ± 9 nmol/L. Compared to the placebo, vitamin D3 treatment significantly increased the levels of 25(OH)D (-5 vs. 59 nmol/L, respectively, p < 0.00001) and 1,25(OH)2D (-10 vs. 59 pmol/L, respectively, p < 0.00001), whereas parathyroid hormone (PTH) levels were reduced (0.3 vs. -0.7 pmol/L, respectively, p < 0.00001). Analysis of the serum metabolome revealed a significant increase of carnitine, choline, and urea and a tendency to increase for trimethylamine-N-oxide (TMAO) and urinary excretion of creatinine, without any effect on renal function. The increase in carnitine, choline, creatinine, and urea negatively correlated with muscle health and physical performance. Combined with previous clinical findings reporting negative effects of vitamin D on muscle strength and physical performance, this secondary analysis suggests a direct detrimental effect on skeletal muscle of moderately high daily doses of vitamin D supplements.

Simultaneous Determination of L- and D-Amino Acids in Proteins: A Sensitive Method Using Hydrolysis in Deuterated Acid and Liquid Chromatography-Tandem Mass Spectrometry Analysis.

Determination of the L- and D-amino acid composition in proteins is important for monitoring process-induced racemization, and thereby protein quality loss, in food and feed. Such analysis has so far been challenging due to the need for sample hydrolysis, which generates racemization, thereby leading to an overestimation of D-amino acids. Here, validation of an LC-MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed, was performed in combination with deuterated HCl hydrolysis. This approach eliminated a racemization-induced bias in the L- and D-amino acid ratios. The LC-MS/MS method was applied for the analysis of 18 free amino acids, with a quantification limit of either 12.5 or 62 ng/mL, except for D-phenylalanine, for which quantification was impaired by background interference from the derivatization agent. For hydrolyzed samples, the composition of 10 L- and D-amino acids pairs could be determined in protein. The average relative standard deviation was 5.5% and 6.1%, depending on the type of hydrolysis tubes. The method was applied on a green protein isolate (lucerne), which contained an average of 0.3% D-amino acids. In conclusion, this method allows for an unbiased analysis of L- and D-amino acid ratios in complex protein samples, such as food and feed.

Mechanism behind the degradation of aqueous norbixin upon storage in light and dark environment.

Buffered aqueous solutions of norbixin were stored in light and dark, and analyzed using mass spectrometry. Compounds with both higher and lower masses than norbixin were detected, suggesting the formation of oxidation products and oxidative cleavage products of norbixin. The norbixin oxidation products included compounds containing several oxidations. The amounts of oxidation products of norbixin increased during storage in both light and dark, but in light, the development accelerated. Scavengers of superoxide radical anion (superoxide dismutase), hydrogen peroxide (catalase), hydroxyl radicals (mannitol) and singlet oxygen (sodium azide) and carbon-centered radicals (DMPO) were tested to determine if any of the reactive species were involved in the degradation of norbixin. Of these, only DMPO decreased the bleaching of norbixin indicating the involvement of carbon-centered radicals. Multiple oxidations of norbixin might be a result of a radical chain reaction involving peroxyl and carbon-centered radicals even though not detectable with electron spin resonance.

Incorporation of bixin in aqueous media: Self-formulation with sorbitol ester of norbixin.

We have previously reported how the natural food colorant, bixin, was enzymatically modified by appending sorbitol to the bixin scaffold. The resulted product, sorbitol ester of norbixin (SEN) was expected to be more hydrophilic. The present study aimed to investigate the physical behaviour of SEN in aqueous media. The property of SEN was studied together with non-reacted bixin as separation of the two compounds was unsuccessful. The SEN molecules behaved as a bolaamphiphile in aqueous media, underwent self-association and develop a hydrophilic aggregate. SEN-aggregates could uptake the non-reacted bixin molecules inside its hydrophobic moiety and dispersed it in aqueous media. Aggregation of SEN molecules with incorporated bixin resulted in a hypsochromic shift of the absorption spectra indicting H-aggregation. Dynamic light scattering showed the formation of aggregates with an average hydrodynamic radius 38 ±â€¯2 nm. The dispersibility of the aggregates was affected by pH and the ionic strength of the media.

Correction: Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion.

Correction for 'Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion' by Jeske H. J. Hageman et al., Food Funct., 2019, 10, 2102-2113.

Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion.

BACKGROUND: Bovine milk fat is increasingly used in infant formula (IF). The triacylglycerol (TAG) structure of bovine milk fat might be beneficial for digestion and absorption. We investigated the release of fatty acids (FAs) of IF containing different fat blends and compared this to human milk. METHODS: Fresh human milk was sampled and two IFs were produced; one containing 100% vegetable fat (IF1) and one with 67% bovine milk fat and 33% vegetable fat (IF2). Using a static in vitro infant digestion model, consisting of a gastric and duodenal phase, the time dependent release of individual free fatty acids (FFA) was studied and analysed using GC-MS, and residual TAG levels were determined by GC-FID. RESULTS: Human milk and the IFs showed comparable total FA release. In the gastric phase, 4-11% of lipolysis occurred, and mainly short (SCFA)- and medium chain fatty acids (MCFA) were released. In the duodenal phase, lipolysis proceeded with release of C4:0 but was marked by a fast release of long-chain fatty acids (LCFA). The digestion of the IFs resulted in different FFA profiles during and at the end of digestion. IF2 gave more release of C4:0-C11:0, which reflects the FA composition of bovine milk. CONCLUSION: The addition of bovine milk fat to IF resulted in a total FA release comparable to an IF with only vegetable fat and human milk. However, it did lead to a different time-dependent release of individual FAs, which might result in differences in absorption and other health effects in vivo.

Pre-meal and postprandial lipaemia in subjects with the metabolic syndrome: effects of timing and protein quality (randomised crossover trial).

Non-fasting TAG - postprandial lipaemia (PPL) - are to a higher degree associated with cardiovascular risk compared with fasting TAG. Dietary protein, especially whey proteins (WP), may lower PPL. We hypothesised that a WP pre-meal (17·6 g protein) consumed 15 v. 30 min before a fat-rich meal reduces the PPL response in subjects with the metabolic syndrome (MetS) and that a WP pre-meal has more potent effects than casein and gluten pre-meals. A total of sixteen subjects with the MetS completed an acute, randomised, crossover trial. WP pre-meals were consumed 15 and 30 min, and casein and gluten 15 min before a fat-rich meal. Blood samples were drawn 360 min postprandially to determine metabolite and hormone responses, S-paracetamol (for assessment of gastric emptying) and amino acids. Insulin and glucagon responses were affected by both timing and protein type (for all P <0·01), with significantly higher concentrations for WP given at -15 min than WP at -30 min and higher responses compared with gluten for the first 30 min after pre-meal consumption (for all P <0·05). The PPL responses changed neither by timing nor by protein type. Glucose-dependent insulinotropic peptide but not glucagon-like peptide 1 responses differed between the three protein types. S-paracetamol concentration was higher for WP (-30 min) than for WP (-15 min) 15 min after the main meal (P = 0·028), and higher for casein and gluten than for WP at time point 30 min (for all P <0·05). In conclusion, the PPL response was not changed by ingestion of a 17·6 g protein pre-meal, whereas both timing and protein quality affected hormone secretion (insulin and glucagon).

Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol.

Bixin is one of the most used yellow-orange food colorants in the food industry. The polyene chain of bixin makes it highly hydrophobic and less suitable for water-based food formulations. Lipase-catalyzed reactions of bixin with sorbitol were studied to synthesize a new derivative of bixin with potential hydrophilic properties. Interestingly, we show that the lipase-catalyzed reaction of bixin leads to a transesterification reaction and formation of a transesterified product, sorbitol ester of norbixin (SEN). The reaction efficiency was optimized with various immobilized lipases at different water activity levels in the organic solvent, 2-methyl-2-butanol. Among the examined lipases, immobilized Candida antarctica lipase B (Novozyme 435) provided the highest reaction yield at a water activity close to zero. Tetrahydrofuran (THF) was used as co-solvent to improve bixin solubility. The optimization of the reaction conditions with 20% THF lead to a total reaction yield of 50% of SEN.

Consumption of Whey in Combination with Dairy Medium-Chain Fatty Acids (MCFAs) may Reduce Lipid Storage due to Urinary Loss of Tricarboxylic Acid Cycle Intermediates and Increased Rates of MCFAs Oxidation.

SCOPE: The aim of the paper is to investigate whether changes in the metabolome could explain observed changes in body composition in overweight adults after consumption of butter with high level of medium-chain fatty acids (MCFAs) in combination with casein or whey. METHODS AND RESULTS: With GC-TOF and LC-Q/MS, metabolites in plasma and urine from a 12-week randomized double-blinded human intervention including 52-abdominally overweight adults were analyzed. The participants consumed 63 g per day of milk fat (high or low in MCFAs) and 60 g per day of protein (whey or casein). Urinary loss of the tricarboxylic acid cycle metabolites and a concomitantly increase of glycerol in blood were observed in the whey + high-MCFAs group, indicating potential lower anabolic processes, such as lipogenesis, by draining substrates. High intake of MCFAs resulted in elevated level of urinary adipic (independently of protein type) and plasma sebacic acid (with whey), indicating a potential increase in oxidation of MCFAs, which might lead to energy loss. CONCLUSION: The type of protein showed highest effect on the overall metabolic profiles, but ω-oxidation of MCFAs in the liver seemed to be the main reason for the observed reduction in body fat mass after consumption of high MCFAs, independent of type of protein.

Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs.

Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants.Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM.Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL · kg-1 · d-1) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL · kg-1 · d-1) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated.Results: A high bacterial load in the UP (6×105 colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidant markers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal health was also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs fed UVC-treated milk than in pigs fed UP and those fed HP-treated milk in both cecum contents (20% and 10%) and distal intestinal mucosa (24% and 20%) (all P < 0.05).Conclusions: UVC is better than HP treatment in preserving bioactive factors in DM. UVC-treated milk may induce better weight gain, intestinal health, and resistance against bacterial infections as shown in preterm pigs as a model for DM-fed preterm infants.

Angiotensin I-converting enzyme-inhibitory peptides from bovine collagen: insights into inhibitory mechanism and transepithelial transport.

The inhibitory mechanism and transepithelial transport of angiotensin I-converting enzyme (ACE)-inhibitory peptides (VGPV and GPRGF) derived from Alcalase®- and papain-hydrolyzed bovine collagen were investigated. The inhibitory mechanism of VGPV and GPRGF was experimentally determined to be non-competitive and the results were supported by molecular docking data. In silico and in vitro gastrointestinal digestion indicated that VGPV remained resistant to digestive enzymes, while GPRGF was degraded into smaller ACE-inhibitory peptides (GPR and GF). VGPV and GPRGF were transported across monolayers of human intestinal epithelial Caco-2 cells through paracellular pathway and retained their ACE-inhibitory activities. The present study suggests that VGPV and GPRGF may possibly be absorbed and exert antihypertensive effects in vivo.

Dietary modulation of the gut microbiota--a randomised controlled trial in obese postmenopausal women.

The gut microbiota has been implicated in obesity and its progression towards metabolic disease. Dietary interventions that target the gut microbiota have been suggested to improve metabolic health. The aim of the present study was to investigate the effect of interventions with Lactobacillus paracasei F19 or flaxseed mucilage on the gut microbiota and metabolic risk markers in obesity. A total of fifty-eight obese postmenopausal women were randomised to a single-blinded, parallel-group intervention of 6-week duration, with a daily intake of either L. paracasei F19 (9.4 × 1010 colony-forming units), flaxseed mucilage (10 g) or placebo. Quantitative metagenomic analysis of faecal DNA was performed to identify the changes in the gut microbiota. Diet-induced changes in metabolic markers were explored using adjusted linear regression models. The intake of flaxseed mucilage over 6 weeks led to a reduction in serum C-peptide and insulin release during an oral glucose tolerance test (P< 0.05) and improved insulin sensitivity measured by Matsuda index (P< 0.05). Comparison of gut microbiota composition at baseline and after 6 weeks of intervention with flaxseed mucilage showed alterations in abundance of thirty-three metagenomic species (P< 0.01), including decreased relative abundance of eight Faecalibacterium species. These changes in the microbiota could not explain the effect of flaxseed mucilage on insulin sensitivity. The intake of L. paracasei F19 did not modulate metabolic markers compared with placebo. In conclusion, flaxseed mucilage improves insulin sensitivity and alters the gut microbiota; however, the improvement in insulin sensitivity was not mediated by the observed changes in relative abundance of bacterial species.

Assessment of the action spectrum for photooxidation in full fat bovine milk.

The action spectrum for photooxidation in full fat bovine milk was measured. Samples of milk with air or argon in headspace were exposed to narrow wavelength bands of light in the range 400-700 nm. Photooxidation in terms of off-flavors was measured by a sensory panel, volatile compounds by headspace solid phase micro extraction (SPME-GC-MS), and photobleaching of photosensitizers in milk (riboflavin, protoporphyrin IX and a chlorophyllic compound) by front face fluorescence spectroscopy. The action spectrum deviated significantly from the absorption spectrum of milk. Significant oxidation was induced by wavelengths around 400 nm and 500-650 nm in milk with air in headspace. Argon in headspace gave significant oxidation also at 700 nm. It is suggested that protoporphyrin IX and chlorophyll are responsible for oxidation induced by wavelengths >500 nm, and that also riboflavin is contributing from 400 to 500 nm.

A novel class of fungal lipoxygenases.

Lipoxygenases (LOXs) are well-studied enzymes in plants and mammals. However, fungal LOXs are less studied. In this study, we have compared fungal LOX protein sequences to all known characterized LOXs. For this, a script was written using Shell commands to extract sequences from the NCBI database and to align the sequences obtained using Multiple Sequence Comparison by Log-Expectation. We constructed a phylogenetic tree with the use of Quicktree to visualize the relation of fungal LOXs towards other LOXs. These sequences were analyzed with respect to the signal sequence, C-terminal amino acid, the stereochemistry of the formed oxylipin, and the metal ion cofactor usage. This study shows fungal LOXs are divided into two groups, the Ile- and the Val-groups. The Ile-group has a conserved WRYAK sequence that appears to be characteristic for fungal LOXs and has as a C-terminal amino acid Ile. The Val-group has a highly conserved WL-L/F-AK sequence that is also found in LOXs of plant and animal origin. We found that fungal LOXs with this conserved sequence have a Val at the C-terminus in contrast to other LOXs of fungal origin. Also, these LOXs have signal sequences implying these LOXs will be expressed extracellularly. Our results show that in this group, in addition to the Gaeumannomyces graminis and the Magnaporthe salvinii LOXs, the Aspergillus fumigatus LOX uses manganese as a cofactor.

Time-saving design of experiment protocol for optimization of LC-MS data processing in metabolomic approaches.

We describe a time-saving protocol for the processing of LC-MS-based metabolomics data by optimizing parameter settings in XCMS and threshold settings for removing noisy and low-intensity peaks using design of experiment (DoE) approaches including Plackett-Burman design (PBD) for screening and central composite design (CCD) for optimization. A reliability index, which is based on evaluation of the linear response to a dilution series, was used as a parameter for the assessment of data quality. After identifying the significant parameters in the XCMS software by PBD, CCD was applied to determine their values by maximizing the reliability and group indexes. Optimal settings by DoE resulted in improvements of 19.4% and 54.7% in the reliability index for a standard mixture and human urine, respectively, as compared with the default setting, and a total of 38 h was required to complete the optimization. Moreover, threshold settings were optimized by using CCD for further improvement. The approach combining optimal parameter setting and the threshold method improved the reliability index about 9.5 times for a standards mixture and 14.5 times for human urine data, which required a total of 41 h. Validation results also showed improvements in the reliability index of about 5-7 times even for urine samples from different subjects. It is concluded that the proposed methodology can be used as a time-saving approach for improving the processing of LC-MS-based metabolomics data.