A Rapid Chemiluminescence Assay for Measurement of Folate in Small Volumes of Breast Milk.

Early life exposure to folate has long lasting effects on development and health. Newborns obtain part of their folate from maternal milk. Studies on health effects of milk folate require rapid, affordable and reliable measurements in large numbers of samples from cohort studies. Recently, a competitive chemiluminescence assay for quantification of folate has become available for automated diagnostic measurement of folate in human serum or plasma. We tested if this method ("FOLA" from Siemens Healthcare) could also be used for human milk. To minimize interference and matrix effects, samples had to be skimmed, diluted seven times with demineralized water, and heated for 5 min at 90 °C. Folate could thus be measured in a linear range between 8.4 and 111.7 nM, with recoveries for the most relevant form, 5-methyltetrahydrofolate (5-MeTHF), of 96%-107%. Results were comparable to those with a recently validated Liquid Chromatography/Mass Spectrometry method (Y = 0.998X - 0.2; R2 = 0.807). The FOLA method was subsequently used for samples from the LIFE Child cohort in Germany, providing first data of breast milk folate in this country (range: 6.2-100.7 nM). This technique could indeed prove useful for large cohorts with multiple samplings.

Vitamin E and vitamin E acetate absorption from self-assembly systems under pancreas insufficiency conditions.

We determined the bioavailability of vitamin E from self-assembly structures in patients with diagnosed chronic pancreas insufficiency. Vitamin E solubilized in dispersed inverted bicontinuous cubic phase and in micellar formulation was delivered directly to the small intestine by tube-feeding. A cross-over study with randomization of 6 subjects and 2 treatments including a combined dose of 18 mg (27 IU) of vitamin E (RRR-[5,7-methyl-((2)H6)]-α-tocopherol) and 27 mg (27 IU) vitamin E acetate (RRR-[5-methyl-(2)H3]-α-tocopheryl acetate) was applied over a time period of 1 h. Plasma samples were collected for 56 h and analyzed by liquid chromatography-mass spectrometry. Appearance of labeled tocopherols originating from the treatment started at 25 h and reached Cmax (0.6-4.6 µM depending on subject) in the 7-9 h window. From the Tmax onwards, both forms of tocopherols diminished slowly to 30-50% of their maxima within 56 h. Strong inter-individual variation was observed in the plasma appearance curves (relative standard deviation varied between 38-45%). No significant discrimination was found between the absorption of free or acetylated forms of deuterated α-tocopherol confirming that application of acetylated α-tocopherol provides the same bioavailability as free α-tocopherol. This observation is valid in both dispersed inverted bicontinuous cubic phase and micellar formulations. Furthermore, since the area-under-the-curve values from cubic phase and from micellar formulations are similar, the cubic phase formulation could represent an alternative delivery system for lipophilic micronutrients in conditions or studies where polysorbate-based micelles cannot be generated.

Do GC/MS methods available for furan and alkylfurans in food provide comparable results? - An interlaboratory study and safety assessment of 2-pentylfuran in food.

A call for data on the occurrence of alkylfurans in food and feed from EFSA triggered the development of new methods to cover next to furan also 2- and 3-methylfuran, 2,5-dimethyl- and 2-ethylfuran as well as 2-pentylfuran. A significant variability was noticed when comparing analysis of 2-pentylfuran and furans in the same matrix performed by different laboratories. To assess the variability an interlaboratory study including eight laboratories was organised. The highest variabilities were observed when analysing cereals, with measurements of 2-pentylfuran indicating concentrations from 8 mg/kg up to more than 1000 mg/kg in the same sample. This study illustrates that the analysis of 2-pentylfuran requires special attention, and that additional method development would be necessary to ensure reliable and reproducible determination of 2-pentylfuran at contamination level. Moreover, a recent evaluation of the EFSA Panel on Food Additives and Flavourings indicates that concerns for genotoxicity, reason why it was grouped with the shorter alkylfurans, are now ruled out. We question the need and justification to include 2-pentylfuran in the analytical method as requested by EFSA, from both the analytical and the safety perspective.

The Longevity Protein Klotho: A Promising Tool to Monitor Lifestyle Improvements.

Aging is not a disease; it is a natural evolution of human physiology. Medical advances have extended our life expectancy, but chronic diseases and geriatric syndrome continue to affect the increasingly aging population. Yet modern medicine perpetuates an approach based on treatment rather than prevention and education. In order to help solve this ever-growing problem, a new discipline has emerged: lifestyle medicine. Nutrition, physical activity, stress management, restorative sleep, social connection, and avoidance of risky substances are the pillars on which lifestyle medicine is founded. The aim of this discipline is to increase healthspan and reduce the duration of morbidity by making changes to our lifestyle. In this review, we propose the use of klotho protein as a novel biomarker for lifestyle medicine in order to quantify and monitor the health status of individuals, as no integrative tool currently exists.

Interlaboratory Study to Evaluate a Testing Protocol for the Safety of Food Packaging Coatings.

According to European regulations, migration from food packaging must be safe. However, currently, there is no consensus on how to evaluate its safety, especially for non-intentionally added substances (NIAS). The intensive and laborious approach, involving identification and then quantification of all migrating substances followed by a toxicological evaluation, is not practical or feasible. In alignment with the International Life Sciences Institute (ILSI) and the European Union (EU) guidelines on packaging materials, efforts are focused on combining data from analytics, bioassays and in silico toxicology approaches for the risk assessment of packaging materials. Advancement of non-targeted screening approaches using both analytical methods and in vitro bioassays is key. A protocol was developed for the chemical and biological screening of migrants from coated metal packaging materials. This protocol includes guidance on sample preparation, migrant simulation, chemical analysis using liquid chromatography (LC-MS) and validated bioassays covering endocrine activity, genotoxicity and metabolism-related targets. An inter-laboratory study was set-up to evaluate the consistency in biological activity and analytical results generated between three independent laboratories applying the developed protocol and guidance. Coated packaging metal panels were used in this case study. In general, the inter-laboratory chemical analysis and bioassay results displayed acceptable consistency between laboratories, but technical differences led to different data interpretations (e.g., cytotoxicity, cell passages, chemical analysis). The study observations with the greatest impact on the quality of the data and ultimately resulting in discrepancies in the results are given and suggestions for improvement of the protocol are made (e.g., sample preparation, chemical analysis approaches). Finally, there was agreement on the need for an aligned protocol to be utilized by qualified laboratories for chemical and biological analyses, following best practices and guidance for packaging safety assessment of intentionally added substances (IAS) and NIAS to avoid inconsistency in data and the final interpretation.

The fat:carbohydrate energy ratio of the weaning diet programs later susceptibility to obesity in male sprague dawley rats.

Dietary fat intake, which is high during suckling, is markedly reduced when food and drinks are introduced into the diet. We investigated whether alterations in the fat:carbohydrate (CHO) content of the weaning diet influenced the later development of adiposity and insulin sensitivity. Three groups of male rats (24/group) were fed from age 16-37 d (phase I) with weaning diets varying in their fat:CHO energy (E) ratios, 10:70 low-fat, high-CHO (LFHC); 30:50 medium-fat, medium-CHO (MFMC), and 60:30 high-fat, high-CHO (HFLC), on an isocaloric basis. Then, all groups consumed ad libitum first a low-fat diet (13% fat E) for 30 wk (phase II) and subsequently a high-fat diet (45% fat E) for another 18 wk (phase III). At the end of phase I, the group fed the HFLC diet demonstrated higher plasma glucose and insulin responses to an oral glucose tolerance test (P < 0.05), but this effect was transient and did not persist into adulthood (phases II and III). By contrast, when challenged with a high-fat diet later in life (age 35.3-53.3 wk), the LFHC group had greater gains in weight (as percent initial weight) and body fat (as absolute and percent body weight) than the other 2 groups that had been weaned with diets higher in fat (P < 0.04 for all). These results provide evidence that metabolic programming by altering the dietary fat:CHO ratio can occur during the weaning period and emphasizes the importance of the fat:CHO ratio of the complementary diet and its relation to the susceptibility to develop adiposity later in life.

Promoting the "3Rs" principle in developmental biology with early and convenient diagnosis of pregnancy in mice.

The 3Rs stand for Replacement of animals in experiments, Reduction in the number of experimental animals, and Refinement of experiments to minimize animal pain and stress. We propose to address Reduction and Refinement in the use of mice as experimental models in developmental research. This study focuses on the maternal percentage of weight increase at gestational day 8 (%WI(GD8)) to diagnose pregnancy early in BALB/c mice. We documented sensitivity, specificity, false positive and negative rates and probability of pregnancy associated with %WI(GD8). This predictive model of pregnancy allows for significant reduction in the number of mice to be sacrificed in developmental research. Reported observations and literature suggest that this model is independent of litter size and should be applicable to other mice strains. This procedure allows mice pregnancy detection before midgestation and proposes an ethically sound approach to experimental animal use by optimizing the number of mice used and refining animal manipulation.

Evaluation of a strategy for difficult embryo transfers from a prospective series of 2,046 transfers.

OBJECTIVE: To evaluate an embryo transfer strategy for difficult transfers (DiTs). DESIGN: Prospective, nonrandomized, observational, cohort study. SETTING: A hospital fertility center in France. PATIENTS: Data were collected on all embryo transfers conducted using the strategy between February 2014 and February 2020. INTERVENTIONS: Anatomical characteristics that could cause DiT were identified by transvaginal ultrasound and the catheter was adapted accordingly. Transfer was guided by transvaginal ultrasound. After passage through the cervix, a rest period was introduced to allow any contractions to stop before embryo deposition in the uterus. MAIN OUTCOME MEASURES: The primary criterion was the percentage of pregnancies per transfer (P/T) after an easy transfer (EaT) or a DiT. The secondary criteria included the anatomical causes of DiT and the patients' levels of discomfort. RESULTS: Of 2,046 transfers, 257 (12%) were DiTs: minor difficulties (n = 152; 7.4%), major difficulties (n = 96; 4.7%), very significant difficulties (n = 7; 0.3%), or impossible (n = 2; 0.1%). The most common causes of DiTs were endocervical crypts (54%), tortuous cervical canal (36%), and marked uterine anteversions (30%). Several causes were often responsible for DiTs. There was no statistically significant difference in the P/T between the EaTs (n = 1,789, 41%) and all degrees of DiT (n = 257, 37%). In addition, there was no statistically significant difference between the level of patient-reported discomfort in the EaT and DiT groups. CONCLUSIONS: This study demonstrated that an adapted embryo transfer strategy, monitored by transvaginal ultrasound, led to similar pregnancy rates regardless of whether the transfer was easy or difficult.

Comparison of two models of intrauterine growth restriction for early catch-up growth and later development of glucose intolerance and obesity in rats.

Two models of intrauterine growth restriction, maternal food restriction (FR), and dexamethasone (DEX) exposure were compared for early postnatal catch-up growth and later development of glucose intolerance and obesity in Sprague-Dawley rats. Mated dams were randomly divided into three groups at 10 days gestational age. Group FR was food restricted (50% of nongestating rats) during the last 11 days of gestation; Group DEX received DEX injections during the last week of gestation, and Group CON, the control group, had no intervention. Birth weight, catch-up growth, body weight, and food intake were measured in male offspring for 22 wk. Body composition, blood glucose, and plasma insulin in response to a glucose load were assessed at 8, 16, and 22 wk. Pups from both FR and DEX dams had similarly lower birth weights than CON (22% and 25%, P < 0.0001), but catch-up growth, which occurred during the suckling period, was much more rapid in FR than DEX offspring (6 vs. 25 days, 95% CI). Postweaning, there were no significant differences between groups in food intake, body weight, body fat, and plasma insulin, but baseline plasma glucose at 22 wk and 2-h glucose area-under-the-curve at 8 and 22 wk were greater only in FR vs. CON offspring (P < 0.05), thereby contrasting with the lack of significant differences between DEX and CON. These results suggest that prenatal food restriction is a more sensitive model than DEX exposure for studies aimed at investigating the link between low birth weight, early postnatal catch-up growth, and later development of glucose intolerance.

Nondairy creamer, but not milk, delays the appearance of coffee phenolic acid equivalents in human plasma.

Chlorogenic acids (CGA) are antioxidants found in coffee. They are becoming of interest for their health-promoting effects, but bioavailability in humans is not well understood. We hypothesized that adding whole milk or sugar and nondairy creamer to instant coffee might modulate the bioavailability of coffee phenolics. Nine healthy participants were asked to randomly drink, in a crossover design, instant coffee (Coffee); instant coffee and 10% whole milk (Milk); or instant coffee, sugar, and nondairy creamer already premixed (Sugar/NDC). All 3 treatments provided the same amount of total CGA (332 mg). Blood was collected for 12 h after ingestion and plasma samples treated using a liquid-liquid extraction method that included a full enzymatic cleavage to hydrolyze all CGA and conjugates into phenolic acid equivalents. Hence, we focused our liquid chromatography-Electrospray ionization-tandem MS detection and quantification on caffeic acid (CA), ferulic acid (FA), and isoferulic acid (iFA) equivalents. Compared with a regular black instant coffee, the addition of milk did not significantly alter the area under the curve (AUC), maximum plasma concentration (C(max)), or the time needed to reach C(max) (T(max)). The C(max) of CA and iFA were significantly lower and the T(max) of FA and iFA significantly longer for the Sugar/NDC group than for the Coffee group. However, the AUC did not significantly differ. As a conclusion, adding whole milk did not alter the overall bioavailability of coffee phenolic acids, whereas sugar and nondairy creamer affected the T(max) and C(max) but not the appearance of coffee phenolics in plasma.

Plasma appearance and correlation between coffee and green tea metabolites in human subjects.

Coffee and green tea are two of the most widely consumed hot beverages in the world. Their respective bioavailability has been studied separately, but absorption of their respective bioactive phenolics has not been compared. In a randomised cross-over design, nine healthy subjects drank instant coffee and green tea. Blood samples were collected over 12 h and at 24 h to assess return to baseline. After green tea consumption, (-)-epigallocatechin (EGC) was the major catechin, appearing rapidly in the plasma; (-)-EGC gallate (EGCg) and (-)-epicatechin (EC) were also present, but (-)-EC gallate and C were not detected. Dihydroferulic acid and dihydrocaffeic acid were the major metabolites that appeared after coffee consumption with a long time needed to reach maximum plasma concentration, suggesting metabolism and absorption in the colon. Other phenolic acid equivalents (caffeic acid (CA), ferulic acid (FA) and isoferulic acid (iFA)) were detected earlier, and they peaked at lower concentrations. Summations of the plasma area under the curves (AUC) for the measured metabolites showed 1.7-fold more coffee-derived phenolic acids than green tea-derived catechins (P = 0.0014). Furthermore, we found a significant correlation between coffee metabolites based on AUC. Inter-individual differences were observed, but individuals with a high level of CA also showed a correspondingly high level of FA. However, no such correlation was observed between the tea catechins and coffee phenolic acids. Correlation between AUC and maximum plasma concentration was also significant for CA, FA and iFA and for EGCg. This implies that the mechanisms of absorption for these two classes of compounds are different, and that a high absorber of phenolic acids is not necessarily a high absorber of catechins.

Rosemary (Rosmarinus officinalis L.) leaf extract limits weight gain and liver steatosis in mice fed a high-fat diet.

The objective of this study was to investigate the effects of rosemary (Rosmarinus officinalis L.) leaf extract (RE) on the prevention of weight gain and associated metabolic disorders in mice fed a high-fat diet. For this purpose, RE was administered for 50 days at 20 or 200 mg/kg body weight (BW) to mice fed a high-fat diet. Body weight was monitored during the study and body composition was measured before and at the end of the intervention. Glucose tolerance, assessed by an intraperitoneal glucose tolerance test (IPGTT), and hepatic and faecal lipid contents were determined at the end of the study. Treatment with 200 mg/kg BW of RE induced a significant reduction of weight and fat mass gain (-64% and -57%, respectively) associated with an increase of faecal lipid excretion. This effect appears to be related to the inhibition of pancreatic lipase activity induced by RE, as demonstrated IN VITRO. While glucose tolerance and fasting glycaemia were not affected by RE treatment, hepatic triglyceride levels were decreased by 39% in RE-treated mice. Administration of the lower dose of RE (20 mg/kg BW) was ineffective on all the parameters measured. In conclusion, our results demonstrate that consumption of 200 mg/kg BW of RE can limit weight gain induced by a high-fat diet and protect against obesity-related liver steatosis.

Whey Versus Casein as a Protein Source during the Weaning Period: Impact on Growth and Later Adiposity and Glucose Homeostasis in a Rat Model of Intrauterine Growth Restriction.

The impact of early life protein source (whey vs. casein) on short- and long-term glucose homeostasis and adiposity is unknown and was investigated in this study. At the end of the suckling period, non-IUGR (intrauterine growth restriction) and IUGR pups were separated from dams and were randomized into four groups. From age 21-49 days, non-IUGR and IUGR pups were fed ad-libitum chow or a semi-synthetic diet (20% from protein; casein or whey) and from age 50-199 days, all groups were fed ad-libitum chow. Food intake, body composition, glucose, and insulin homeostasis were assessed. Among the chow groups, IUGR had slower growth and higher fasting glucose at age 42 days, as well as higher fasting and AUC glucose at age 192 days relative to non-IUGR. The whey IUGR group had a slower growth rate and higher fasting glycemia in early life (age 21-49 days) and higher HOMA-IR later in life (age 120-122 and 190-192 days) relative to casein IUGR. This study shows the potential advantage of casein relative to whey during weaning on short term energy intake, growth, and glucose homeostasis in an IUGR model and reveals, for the first time, its long term impact on insulin sensitivity, which may have implications for later metabolic health, particularly in small-for-gestational-age populations at risk of type 2 diabetes.

Mineral oil hydrocarbons in foods: is the data reliable?

The contamination of foods with mineral oil hydrocarbons (MOH) is a serious concern, requiring in most cases tedious mitigation measures that span across the whole food supply chain. A major issue today is the significant variability of the results generated by laboratories. This study was therefore designed to achieve a deeper insight into the analytical procedures used by commercial laboratories, identifying possible gaps and suggesting improvements that will enhance the reliability of the MOH data, an important prerequisite for risk assessment. In total six different food matrices, i.e. infant formula (IF), cocoa butter, cocoa powder, biscuits, fruit-based baby food containing biscuit and roast and ground coffee were subjected to comparative inter-laboratory studies, as well as one vegetable oil analysed within the frame of a professionally conducted proficiency test. The results indicate that on some matrices with possibly low amounts of MOH contamination, the current methodologies cannot reliably conclude whether or not a food sample is indeed contaminated with mineral oils (<10 mg/kg food). Urgently needed are: (i) an aligned and fully validated sample preparation strategy tested on a range of different food matrices; (ii) a confirmation of positive flame ionisation detection (FID) results by confirmatory methods such as mass spectrometry - in line with the CEN Standard and the Joint Research Centre (JRC) Guidance Document, (iii) a more detailed root-cause analysis in the reports of laboratories through the use of mineral oil markers, and (iv) a fully validated official method for the concerned foods with a limit of application <10 mg/kg food.

Safety of supplementing infant formula with long-chain polyunsaturated fatty acids and Bifidobacterium lactis in term infants: a randomised controlled trial.

Probiotics and long-chain PUFA (LC-PUFA) may be beneficial supplements for infants who are not breast-fed. The aim of the present study is to evaluate the safety of an infant formula containing the LC-PUFA DHA and arachidonic acid (AA) and the probiotic Bifidobacterium lactis by comparing the growth rate of infants fed the supplemented and unsupplemented formulas. One hundred and forty-two healthy, term infants were enrolled in a single-centre, randomised, double-blind, controlled, parallel-group trial, and allocated to receive either standard or probiotic and LC-PUFA-containing experimental formulas. The infants were fed with their assigned formulas for 7 months. The primary outcome (weight gain) and the secondary outcomes (length, head circumference and formula tolerance) were measured throughout the study. LC-PUFA status was assessed at 4 months of age and immune response to childhood vaccines was measured at 7 months of age. There was no significant difference in growth between the two groups. The 90 % CI for the difference in mean weight gain was - 0.08, 3.1 g in the intention-to-treat population and 0.1-3.8 g in the per protocol population, which lay within the predefined boundaries of equivalence, - 3.9-3.9. There were no significant differences in mean length and head circumference. DHA and AA concentrations were higher in infants in the experimental formula group compared with the control formula group. No influence of the supplements on the response to vaccines was observed. Growth characteristics of term infants fed the starter formula containing a probiotic and LC-PUFA were similar to standard formula-fed infants.

Stability study of veterinary drugs in standard solutions for LC-MS/MS screening in food.

A study on stability of veterinary drugs in standard solutions stored at -80°C and at -20°C was conducted over 1 year. Data were acquired on 152 individual stock standard solutions and also on 15 family mixes and 2 working standard solutions. All solutions were prepared, stored and compared 1 year later against freshly prepared ones by LC-MS/MS. A statistical analysis was performed to set the acceptability criteria, taking into account the variability of standard preparations. In individual stock standard solutions stored at -80°C (12 months) and -20°C (9 months), stability was demonstrated for 141 and 140 out of 152 compounds, i.e. for 92% and 93% of compounds, respectively. Drugs were even more stable when solubilised in either diluted family mixes or working standard solutions, with more than 99% and 94% of compounds found unaltered when stored at -80°C and at -20°C, respectively. In mixes, beta-lactams from the cephalosporin (cefadroxil and cephalexin) and penicillin (amoxicillin and ampicillin) families were found to be the least stable compounds when stored at -20°C (6 months), necessitating storage at -80°C to achieve a 1-year shelf life. The study also evidenced solubility issues for two sulfonamides (sulfadiazine and sulfamerazine) in methanol-based solutions. An independent stability study conducted by a second laboratory confirmed the 1-year stability of 3 family mixes-quinolones, sulfonamides and tetracyclines.

Validation of test portion pooling for Salmonella spp. detection in foods.

Pathogen monitoring programs play a crucial role in the verification of the effectiveness of implemented hygiene control measures. Sampling and testing procedures included in pathogen monitoring involve the analysis of multiple test portions where all samples must be negative for the presence of pathogens for a certain test portion size. Many food safety programs require increased testing due to the risks that a pathogen may be present. Analyzing more than one test portion could prove to be expensive and labor intensive. When more than one test portion for a specified food item is to be tested, the test portions could be combined to form a pooled test portion to reduce laboratory workload, costs of reagents and further confirmatory steps, but only when evidence is available that pooling does not affect on the number of false negative results for different matrices. This study has been performed to demonstrate the equivalence of test portion pooling for Salmonella detection with five different methods using cultural, ELISA and Real Time PCR technologies. Twenty-three (23) different food items including confectionary products, meal components, infant formula, pet food and powdered beverages were validated. Other complementary parameters like impact of minimum and maximum incubation time for pre-enrichment, temperature profile, pH and Salmonella concentration after the pre-enrichment and background flora have also been considered in the study. The results showed that pooling test portions up to 375g for Salmonella detection is valid for the methods that were tested. Relative level of detection (RLOD50) values for 22 of the food items tested were acceptable (i.e. lower than 2.5) when comparing the reference sample size (25g) against the alternative pooled sample size (375g), provided the enrichment broth was pre-warmed and maximum incubation time is respected.

Rationale and design of the TRANSFACT project phase I: a study to assess the effect of the two different dietary sources of trans fatty acids on cardiovascular risk factors in humans.

BACKGROUND: Detrimental effects of consumption of industrial trans fatty acids (TFA) from partially hydrogenated vegetable oils (PHVO) on cardiovascular disease (CVD) risk factors are well documented. However, very little information is available on the effect of natural sources of TFA coming from milk fat, dairy products and ruminant meat. In fact, due to the naturally low level of TFA in milk fat, it is almost impossible to conduct a clinical trial with a limited number of subjects (<200). METHODOLOGY: To compare the effects of industrial and natural dietary sources of TFA, two specific test fats have been designed and produced. A substantial amount of milk fat (130 kg) enriched in TFA has been produced by modification of the cow's diet and selection of cows with the highest TFA content. The level obtained was approximately 4- to 7-fold higher than typically present in milk fat (approximately 20 instead of 3-6 g/100 g of total fatty acids). The control fat is composed of PHVO balanced in saturated fatty acids (lauric, myristic and palmitic). Both experimental fats contain about 20-22% of monounsaturated TFA and the volunteers' daily experimental fat intake (54 g), will represent about 12.0 g/day of TFA or 5.4% of the daily energy (based on 2000 kcal/day). These two test fats have been incorporated into food items and will be provided to 46 healthy subjects under a randomised, double blind, controlled, cross-over design. The primary outcome is high-density lipoprotein cholesterol (HDL-C), which is an independent risk factor for CVD. Other parameters such as low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), and HDL-C level and subclasses will be also to be evaluated. CONCLUSION: We have shown that it is technically feasible to perform a clinical trial on the comparative effects of natural and industrial sources of TFA isomers on CVD risk factors. Results are expected by mid-2006.

Determination of Labeled Fatty Acids Content in Milk Products, Infant Formula, and Adult/Pediatric Nutritional Formula by Capillary Gas Chromatography: Collaborative Study, Final Action 2012.13.

A collaborative study was conducted on AOAC First Action Method 2012.13 "Determination of Labeled Fatty Acids Content in Milk Products and Infant Formula by Capillary Gas Chromatography," which is based on an initial International Organization for Standardization (ISO)-International Dairy Federation (IDF) New Work Item that has been moved forward to ISO 16958:2015|IDF 231:2015 in November 2015. It was decided to merge the two activities after the agreement signed between ISO and AOAC in June 2012 to develop common standards and to avoid duplicate work. The collaborative study was performed after having provided highly satisfactory single-laboratory validation results [Golay, P.A., & Dong, Y. (2015) J. AOAC Int. 98, 1679-1696] that exceeded the performance criteria defined in AOAC Standard Method Performance Requirement (SMPR(®)) 2012.011 (September 29, 2012) on 12 products selected by the AOAC Stakeholder Panel on Infant Formula (SPIFAN). After a qualification period of 1 month, 18 laboratories participated in the fatty acids analysis of 12 different samples in duplicate. Six samples were selected to meet AOAC SPIFAN requirements (i.e., infant formula and adult nutritionals in powder and liquid formats), and the other Six samples were selected to meet ISO-IDF requirements (i.e., dairy products such as milk powder, liquid milk, cream, butter, infant formula with milk, and cheese). The fatty acids were analyzed directly in all samples without preliminary fat extraction, except in one sample (cheese). Powdered samples were analyzed after dissolution (i.e., reconstitution) in water, whereas liquid samples (or extracted fat) were analyzed directly. After addition of the internal standards solution [C11:0 fatty acid methyl ester (FAME) and C13:0 triacylglycerols (TAG)] to the samples, fatty acids attached to lipids were transformed into FAMEs by direct transesterification using methanolic sodium methoxide. FAMEs were separated using highly polar capillary GLC and were identified by comparison with the retention times of pure analytical standards. Quantification of fatty acids was done relative to C11:0 FAME as internal standard and to instrument response factors (determined separately using calibration standards mixture). The performance of the method (i.e., transesterification) was monitored in all samples using the second internal standard, C13:0 TAG. RSDR values were summarized separately for labeled fatty acids in SPIFAN materials and ISO-IDF materials due to different expression of results. This method was applied to representative dairy, infant formula, and adult/pediatric nutritional products and demonstrated global acceptable reproducibility precision for all fatty acids analyzed (i.e., 46 individuals and/or groups) for these categories of products.

Rapid identification of stress-related fingerprint from whole bacterial cells of Bifidobacterium lactis using matrix assisted laser desorption/ionization mass spectrometry.

Whole cells of Bifidobacterium lactis were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Characteristic and reproducible mass spectra were obtained in the mass range from 6 to 19 kDa. After several days of bacterial cell storage at 4 degrees C (D0, D2, and D6), only minor signal differences were observed. Under identical and reproducible conditions, fourteen relevant diagnostic ions were identified. Moreover, control- and stress-related fingerprints were rapidly obtained using MALDI-TOFMS by comparison of protein patterns obtained from non-stressed (control) versus stressed cells (addition of bile salts during growth). After quantitative validation of the MALDI-MS data by a statistical approach, two and eight signals were assigned as control- and stress-specific ions, respectively. This work provides the evidence that MALDI-TOFMS can be used for the identification of stress-related fingerprint of B. lactis bacterial cells and could have a high potential for the assessment of the physiological status of the cells.