Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: Collaborative Study, Final Action 2021.07.

BACKGROUND: Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective, and antimicrobial factors to the neonate. OBJECTIVE: To evaluate method reproducibility of AOAC First Action Official Method 2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. METHODS: Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct-assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a four-parameter calibration regression. RESULTS: After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable Horwitz Ratio (HorRatR) values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin cleanup LC-UV), showed negligible difference between results. CONCLUSION: The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing (MLT) study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official MethodSM. HIGHLIGHTS: A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described.

Analysis of Taurine in Infant Formulas and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: First Action 2022.03.

BACKGROUND: Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. OBJECTIVE: To evaluate the analytical performance of a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for compliance with AOAC Standard Method Performance Requirements (SMPR®) for taurine analysis described in SMPR 2014.013. METHOD: Following protein precipitation with Carrez solutions, taurine is extracted and separated by HILIC with detection by triple quadrupole MS using multiple reaction monitoring (MRM). Stable isotope labeled (SIL) taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. RESULTS: The method was shown to meet the requirements specified in the SMPR with a linear range of 0.27-2700 mg/hg RTF (ready-to-feed), a limit of detection of 0.14 mg/hg RTF, acceptable recovery of 97.2-100.1%, and acceptable repeatability of 1.6-6.4% relative standard deviation. Additionally, the method was found to have no statistically significant bias compared with reference values for National Institute of Standards and Technology (NIST) 1849a certified reference material (CRM) (P-value = 0.95) and 1869 CRM (P-value = 0.31), and with results from AOAC 997.05 (P-value = 0.10). CONCLUSIONS: A recent review of the method and validation data by the Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) found that this method met all the criteria for analysis of taurine specified in SMPR 2014.013 and voted to adopt this method as First Action AOAC Official MethodSM2022.03. HIGHLIGHTS: A method for the analysis of taurine in infant formulas and adult nutritionals by HILIC-MS/MS is described. A single-laboratory validation (SLV) study demonstrated the applicability of the method to meet requirements of SMPR 2014.013. In December 2022, the SPIFAN ERP voted to adopt this method as First Action AOAC Official Method 2022.03.

Enhanced Automated Online Immunoaffinity Liquid Chromatography-Fluorescence Method for the Determination of Aflatoxin M1 in Dairy Products.

BACKGROUND: Aflatoxin M1 (AFM1) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B1, which is converted in the cow liver by hydroxylation to AFM1 and is then expressed in milk. AFM1 is regulated in milk and other dairy products because it can cause serious health issues, such as liver and kidney cancers, in humans and is an immunosuppressant. OBJECTIVE: To optimize the chromatographic protocol and to extend the matrix scope to include a wider range of dairy products: whey powder, whey protein concentrate, whey protein isolate, liquid milk, skim milk powder, whole milk powder, adult nutritional products, and yogurt. METHODS: AFM1 is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM1 in the resulting extract is concentrated using an automated RIDA®CREST IMMUNOPREP® online cartridge coupled to quantification by HPLC-fluorescence. RESULTS: The method was shown to be accurate, with acceptable recovery (81.2-97.1%) from spiked samples. Acceptable precision was confirmed, with a relative standard deviation (RSD) for repeatability of 6.6-11.2% and an RSD for intermediate precision of 7.5-16.7%. Method LOD and robustness experiments further demonstrated the suitability of this method for routine compliance testing. Analysis of an international proficiency trial sample generated results that were comparable with the value assigned from alternative independent methods. CONCLUSION: A method with improved chromatography for high-throughput, routine testing of AFM1 in an extended range of dairy products is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit for purpose. HIGHLIGHTS: Single-laboratory validation of an automated online immunoaffinity cleanup fluorescence HPLC method for AFM1 in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM1 with enhanced chromatographic performance. Method applicable to the analysis of AFM1 in an extended range of milk and milk-based products.

Rapid Analysis of Taurine in Infant Formula and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

BACKGROUND: Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. OBJECTIVE: A rapid compliance method for the analysis of taurine that is applicable to infant formula and milk-based nutritional products is described. METHOD: Following protein precipitation with Carrez solutions, taurine in the sample extract is separated by hydrophilic interaction liquid chromatography (HILIC) with detection by triple quadrupole mass spectrometry using multiple reaction monitoring (MRM). Stable isotope-labeled taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. RESULTS: The method was shown to be accurate, with acceptable recovery of 99.6% (range = 91.1-106.5%). Results for National Institute of Standards and Technology (NIST)-certified reference materials showed no statistical bias for NIST 1849a (P = 0.96) and NIST 1869 (P = 0.88) when compared with reference values. No bias was found when results were compared with those of an international reference method, AOAC Official MethodSM997.05 (P = 0.18). Repeatability was estimated to be 3.1% RSDr (range: 2.4-4.0%, HorRat: 0.3), and intermediate precision was estimated to be 4.9% RSDiR (range: 2.2-7.7%). CONCLUSIONS: Successful single-laboratory validation demonstrates that this rapid method is suitable for use in high-throughput laboratories as part of routine product compliance release testing of taurine in nutritional products. HIGHLIGHTS: A method for the analysis of taurine in infant formula and adult nutritionals by hydrophilic interaction liquid chromatography-mass spectrometry (LC-MS) is described. The method is suitable for use in high-throughput laboratories for routine product compliance testing of taurine. A single-laboratory validation study demonstrated the method to be accurate, precise, and fit for purpose.

Serotonergic Paracrine Targets in the Intestinal Mucosa.

Serotonin functions as a neurotransmitter in the enteric nervous system. Aside from its neurotransmitter role, serotonin also is a paracrine mediatorial signal in the digestive tract. It is a major paracrine signaling molecule in the integrated physiology of several classes of cells in the intestinal mucosa. Paracrine action can be initiation or suppression of activity in populations of cells that make up divergent phenotypic classes. This underlies phenotypic plasticity in single classes and links single classes to other neighboring phenotypic classes, thereby forming a single and higher-order organization in which different categories of function are integrated to work in harmony as a single homeostatic entity at higher levels of physiological organization. Phenotypic classes of cells that are linked by serotonergic paracrine signaling at upper levels of functional organization in the small intestine are (1) enterochromaffin cells; (2) enteric mast cells; (3) spinal sensory afferents; (4) sympathetic postganglionic neurons; (5) enteric neurons.

Multiple rather than specific autoantibodies were identified in irritable bowel syndrome with HuProt™ proteome microarray.

Immune activation and several autoantibodies might be involved in the pathophysiology of irritable bowel syndrome (IBS). We aimed to identify serum biomarkers for IBS by HuProt™ microarray. IBS patients met Rome III criteria were enrolled. Control groups included healthy controls (HCs) and disease controls (DCs). In stage I, we profiled sera from IBS and control groups with HuProt™ microarrays. Based on significant different proteins in stage I, IBS focused microarrays were constructed and validated in a larger cohort in stage II, then decision tree models were generated to establish a combination of biomarkers. In stage III, 4 purified proteins were verified by ELISA. Finally, we analyzed the correlation of autoantibodies with symptoms. In stage I, we identified 47 significant different proteins including 8 autoantibodies of IgG, 2 of IgA between IBS and HCs; 13 autoantibodies of IgG, 13 of IgA between IBS and DCs. In stage II, we found the positive rates of 14 IgG and IgA autoantibodies in IBS were significantly higher than HCs. Five autoantibodies of IgG and 7 IgA were comprehensively involved in differentiating IBS and HCs with the sensitivity and specificity to diagnose IBS as 40%-46.7% and 79.4%-86.3%. The median optical density value of ELAVL4 (IgG) and PIGP (IgA) were significantly higher in IBS than HCs. Parts of autoantibodies above were related to IBS symptoms. We found a combination of autoantibodies to differentiate IBS with HCs, but no specific autoantibodies could serve as serum biomarkers for IBS.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: First Action 2021.07.

BACKGROUND: Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of immunoprotective, growth, and antimicrobial factors to the neonate. OBJECTIVE: To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005. METHOD: Following dilution of the sample in buffer, an automated, label-free, real-time optical biosensor immunoassay was used in a direct assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a 4-parameter calibration regression. RESULTS: The analytical range (0-200 mg/hg), method detection limit (0.8 mg/hg), recovery (96.1-109.2%), and repeatability (1.0-5.3%) complied with the requirements given in the lactoferrin SMPR. The method was shown to be specific for native, intact lactoferrin; thermally denatured lactoferrin generated no measurable binding response. CONCLUSION: The method described is suitable for the quantification of intact, undenatured lactoferrin in milk products, infant formulas (bovine milk protein-based, soy protein-based, and amino acid-based), and adult nutritionals and has been demonstrated to meet the performance requirements defined in SMPR 2020.005. HIGHLIGHTS: A single-laboratory validation (SLV) of an automated biosensor immunoassay for the determination of intact, undenatured lactoferrin is described.

Motor behavior of mouse large intestine: A Minireview.

Mice with a recessive gene which reduces the number of ganglion cells of the distal colon and rectum and produces megacolon, imitating Hirschsprung disease, are discussed as a model for integrative control of the large intestinal smooth musculature by the enteric division of the autonomic nervous system (ie, the brain-in-the-gut). Investigative approaches, such as propulsion of artificial pellets in preparations of whole colon in organ baths in vitro and innovative approaches capitalizing on neurogenetic technologies (eg, optogenetics), are considered in view of potential application in the development of novel therapeutic mechanisms to selectively evoke and control gastrointestinal motility patterns, such as the small intestinal digestive motility pattern, interdigestive pattern, and reversed direction of powerful propulsive motility during emesis. This minireview relates to the paper titled: "Motor patterns in the proximal and distal mouse colon which underlie formation and propulsion of feces," appearing in this issue of Neurogastroenterology and Motility.

Analysis of α-Tocopherol Stereoisomers in Fortified Infant Formula by Chiral Chromatography.

BACKGROUND: Direct measurement of the bioavailable α-tocopherol content presents a significant analytical challenge and requires chiral separation of the α-tocopherol stereoisomers. OBJECTIVE: The objective of the study was to validate an analytical method for the analysis of α-tocopherol stereoisomers in infant formulas and dairy products. METHOD: Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the α-tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection. RESULTS: The method was shown to be accurate, with spike recoveries of 91.9-108.8% for RRR-α-tocopherol and 90.1-104.7% for α-tocopherol, with no statistical bias against NIST 1849a certified reference material (P-value = 0.54) and an HPLC-UV analytical method (P-value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR-α-tocopherol and 4.6% RSDr (HorRat = 0.4) for α-tocopherol. CONCLUSIONS: A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of α-tocopherol is described. In a single analytical run, the method can quantify: (i) the total α-tocopherol content; (ii) the nutritionally important RRR-α-tocopherol and/or 2 R, 4'-ambo, 8'-ambo-α-tocopherol contents; (iii) the amount of all-rac-α-tocopherol, all-rac-α-tocopheryl acetate, or all-rac-α-tocopheryl succinate fortified into the product. HIGHLIGHTS: An accurate and precise chiral chromatographic method for the analysis of isomeric forms of α-tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection.

BACKGROUND: Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. OBJECTIVE: A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. METHOD: AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. RESULTS: The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81-112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4-12% RSD and intermediate precisions of 5-13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. CONCLUSIONS: A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. HIGHLIGHTS: An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.

Differential Thermal Isomerization: Its Role in the Analysis of Vitamin D3 in Foods.

BACKGROUND: For nutritional purposes, the measurement of vitamin D3 (defined as the sum of vitamin D3 and previtamin D3) is required to obtain an accurate and reliable estimate of its content in foods. An often neglected aspect in the development of methods for the analysis of vitamin D3 is accounting for any potential analytical bias in the results associated with differential thermal isomerization between previtamin D and vitamin D. CONCLUSIONS: For LC-UV methods using a vitamin D2 internal standard, cold saponification, or direct lipid extraction techniques should be avoided, unless chromatographic separation of vitamin D2, vitamin D3, and their previtamin forms is achieved so that UV absorbance corrections can be made. For both LC-UV and LC-MS methods using calciferol internal standards, the simplest solution to avoid analytical bias due to the presence of previtamin D is to utilize heating conditions (typically during saponification) such that previtamin D and vitamin D in the sample and the internal standard reach an equivalent equilibrium state prior to instrumental analysis. Only under such circumstances is the integration of previtamin D unnecessary to obtain accurate results for vitamin D3. HIGHLIGHTS: A detailed discussion of the quantitation of vitamin D3 in food with concise recommendations for avoiding measurement bias as a consequence of differential thermal isomerization.

Determination of Sorbic Acid in Cheese by High Performance Liquid Chromatography.

BACKGROUND: Sorbic acid (E, E-2, 4-hexadienoic acid) is added as a preservative to cheese because of its fungistatic and antimicrobial activity. OBJECTIVE: A facile method for the analysis of sorbic acid that is applicable to sliced processed cheese and grated cheese products. METHOD: A cheese sample and dry-ice mixture was blended and sorbic acid was extracted with methanol and analyzed by HPLC-ultraviolet with external standardization. A large sample size was used to overcome sample inhomogeneity due to imprecise sorbic acid addition techniques during production and sorbic acid migration through the fat over time. RESULTS: The method was shown to be accurate for both processed cheese and grated Cheddar cheese, with acceptable spike recovery (93.7, 103.7%, respectively), and no bias (α = 0.05) against an international reference method (p = 0.59, p = 0.13, respectively) was found. Acceptable precision was confirmed for both processed cheese slices and grated Cheddar cheese, with repeatability of 5.3% and 4.3% relative standard deviation, respectively, and intermediate precision Horwitz ratio values of 1.3 and 1.7 for processed cheese slices and grated Cheddar cheese, respectively. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. CONCLUSIONS: A method for high-throughput, routine testing of sorbic acid is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose.

Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry.

BACKGROUND: Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. OBJECTIVE: A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. METHOD: Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography‒tandem mass spectrometry (LC-MS/MS), with quantitation accomplished by internal standard technique. RESULTS: The method was shown to be accurate, with acceptable recovery (thiamine, 99.3-101.1%; pantothenic acid, 99.2-108.6%). A certified reference material (NIST 1849a), showed no statistical bias (α = 0.05) for thiamine (P = 0.64); although a statistically significant bias (P < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC-MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (RSDr) (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. CONCLUSIONS: This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.

Serotonergic Integration In the Intestinal Mucosa.

Mucosal serotonin (5-HT) is a key paracrine signaling molecule in the integrated physiology of enterochromaffin cells, enteric mast cells, spinal afferent nerves and the enteric nervous system (ENS). Enterochromaffin cells release 5-HT as a paracrine signal to enteric mast cells, spinal afferents and neurons in the ENS. Enteric mast cells release multiple mediators of paracrine signaling, among which are histamine and the serine proteases, chymase and tryptase, as well as serotonin. Some of these mediators diffuse to receptors on afferent nociceptive and mechanosensitive terminals and sensitize the terminals in a manner that may underlie abdominal pain and distension induced pain in the irritable bowel syndrome. Substance P and calcitonin gene-related peptide (CGRP), released by spinal afferent innervation, degranulate enteric mast cells. Substance P and CGRP are significant factors in mucosal inflammation evoked by bacteria in the colonic microbiome. Binding of immunoglobulin antibodies to FcεRI receptors, on enteric mast cells, degranulate the mast cells and release paracrine mediators that overlay integrative microcircuitry in the ENS. An overlay of histamine "calls up" from the ENS library of programed gut behaviors, a defensive program consisting of a sequence of copious mucosal secretions, increased blood flow and powerful orthograde propulsion organized to move threats out of the colonic lumen. Symptoms of acute watery diarrhea, cramping abdominal pain and incontinence are associated with "running" of the defense program. Intestinal behavioral programs stored in the ENS library are described as working like digital "apps".

[Unique characteristics of "the second brain" - The enteric nervous system].

Enteric nervous system (ENS) is composed of intestinal submucosal and myenteric plexuses. ENS may independently regulate intestinal digestive and absorptive function, and it is also known as "the second brain" or gut brain. ENS has significant specificity relative to central nervous system (CNS) in properties and functional activities of neurons and neural circuits. ENS is connected with CNS through the feedback pathway (brain-gut-axis) of sympathetic and parasympathetic nerves and peripheral primary sensory afferent nerves to form the bidirectional brain-gut-axis, which may affect emotion, appetite and behavioral states of individuals. Gastrointestinal functional disorder (GIFD) induced by ENS dysfunction may not only cause abnormal gastrointestinal function but also has been implicated in cognitive and mood disorders, such as irritable bowel syndrome (IBS). GIFD would influence deeply the quality of life in patients. Nevertheless, in the worldwide, ENS has so far received much less attention as compared with CNS. The depth of research and scale of investment in ENS studies have been much lower than those in CNS studies. The situation in China is even more evident. From ENS research history, an outstanding problem is to ignore largely the unique properties of ENS and apply mechanically the hypotheses formed in CNS studies to ENS researches. In this review, the structure and function of ENS are briefly introduced, and the importance of extraordinary characteristics of ENS is illustrated by the problems encountered in our studies.

Establishing and Sustaining a Culture of Evidence-Based Practice: An Evaluation of Barriers and Facilitators to Implementing the Best Practice Spotlight Organization Program in the Australian Healthcare Context.

BACKGROUND: Nurses and midwives are central to the implementation and delivery of quality care through evidence-based practice (EBP). However, implementation of EBP in nursing and midwifery is under-researched with few examples of systematic and sustained change. The Registered Nurses Association of Ontario's Best-Practice Spotlight Organization (BPSO) Program was adopted in South Australia as a framework to systematically implement EBP in two diverse and complex healthcare settings. METHODS: The study was a post-implementation, mixed-method evaluation conducted at two healthcare settings in Adelaide, South Australia utilizing qualitative and quantitative data. Proctor's implementation evaluation framework guided the evaluation design. Information sources included; interviews, focus groups, questionnaires, and document review. RESULTS: Clinical and executive staff (n = 109 participants) from a broad range of stakeholder groups participated in the interviews, focus groups, and returned questionnaires. A number of facilitators directly affecting program implementation were identified; these pertained to embedding continuity into the program's implementation and delivery, a robust governance structure, and executive sponsorship. Barriers to implementation were also identified. These barriers pertained to organizational or workforce challenges; staff turnover and movement (e.g., secondment), insufficient staff to allow people to attend training, and a lack of organizational commitment to the program, especially at an executive level. As a result of successful implementation, it was observed that over three years, the BPSO program positively influenced the uptake and implementation of EBP by clinicians and the organizations into which they were introduced. CONCLUSIONS: The BPSO model can be translocated to new healthcare systems and has the potential to act as a mechanism for establishing and sustaining EBP change. This study was the first to apply an implementation evaluation framework to the BPSO program, which allowed for structured analysis of facilitating or impeding factors that affected implementation success. The findings have important implications for other health systems looking to translocate the same or similar EBP programs, as well as contributing to the growing body of implementation evaluation literature.

Sera with anti-enteric neuronal antibodies from patients with irritable bowel syndrome promote apoptosis in myenteric neurons of guinea pigs and human SH-Sy5Y cells.

BACKGROUND: Sera anti-enteric neuronal antibodies (AENA), neuronal inflammation, and degeneration in myenteric plexus in patients with irritable bowel syndrome (IBS) were reported. Effects of sera AENA in patients with IBS are unclear. METHODS: Patients with IBS met Rome III criteria were enrolled. Controls included healthy subjects (HS) and patients with slow transit functional constipation, inflammatory bowel disease, chronic intestinal pseudo-obstruction, and autoimmune diseases. Indirect immunofluorescence was used to detect AENA. Anti-enteric neuronal antibodies intensities were termed as "1" = weak fluorescence (mild positive); "2" = moderate fluorescence (moderate positive); "3" = very high fluorescence (intensive positive). Intensities of ≥1 were defined as positive and ≥2 were defined as obvious positive. Cultured myenteric neurons of small intestine from guinea pigs and human SH-Sy5Y cells were incubated with fetal bovine serum (FBS), HS sera, or IBS sera with or without AENA. Indirect immunofluorescence with anti-PGP9.5/DAPI/anti-active caspase-3 or TUNEL, Western blot, and flow cytometry were used to detect apoptosis. KEY RESULTS: Overall, 293 patients with IBS were enrolled (41.7 ± 11.5 years). AENA-positive and obvious positive rates in IBS were higher than HS (76.8% vs 33%; 43.7% vs 7%; all P < 0.001). Myenteric neurons incubated with AENA moderate or intensive positive IBS sera showed higher rates of anti-active caspase-3 and TUNEL-positive cells than HS or FBS (20% ± 7.3% and 35% ± 13.3% vs 4.3% ± 1.5% and 0.9% ± 0.4%, respectively; 6.2% ± 2.0% and 10.2% ± 4.6% vs 1.3% ± 1.9% and 0.5%±0.5%, respectively; all P < 0.05). Human SH-Sy5Y cells incubated with AENA moderate or intensive positive IBS sera showed increased cleaved caspase-3 and Bax expression and decreased Bcl-2 expression. Flow cytometry showed apoptosis rates of these two groups were higher than that of AENA mild positive, negative, HS, and FBS (7.6%±0.8% and 10.7%±1.3% vs 5.0%±0.8%, 3.8%±0.3%, 3.4%±0.2% and, 2.8%±0.2%, P < 0.05). CONCLUSIONS AND INFERENCES: The AENA obvious positive rate in patients with IBS was higher than HS, and sera with higher levels of AENA promoted neuronal apoptosis. AENA-mediated neuropathy might exist in a subset of patients with IBS.

A Rapid Method for the Determination of Biotin and Folic Acid in Liquid Milk, Milk Powders, Infant Formula, and Milk-Based Nutritional Products by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Biotin and folate are B-group vitamins that play a critical role in numerous metabolic reactions, and they are supplemented to infant and adult nutritional formulas as free biotin and folic acid. OBJECTIVE: We describe a rapid method for the analysis of biotin and folic acid that is applicable to liquid milk, milk powders, infant formula, and milk-based nutritional products. METHODS: Samples are autoclaved, centrifuged, filtered, and analyzed by HPLC-MS/MS, with quantitation accomplished by the internal standard technique. RESULTS: The method was shown to be accurate, with acceptable spike recovery (biotin: 96.5-108.2%; folic acid: 92.6-104.4%), and no bias (α = 0.05) against either a certified reference material (biotin: P = 0.70; folic acid: P = 0.23) or established analytical method (biotin: P = 0.10; folic acid: P = 0.48) was found. Acceptable precision was confirmed with repeatability relative standard deviation (RSDr) and Horwitz ratio (HorRat) values (biotin: RSDr = 0.5-5.6%, HorRatr = 0.1-0.6; folic acid: RSDr = 2.0-3.1%, HorRatr = 0.3-0.5). Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. CONCLUSIONS: This rapid method is intended for use in high-throughput laboratories as part of the routine product compliance release testing of biotin and folic acid in the manufacturing of infant formulas and adult nutritional products.

Enteric Neurobiology: Discoveries and Directions.

Discovery and documentation of noncholinergic-nonadrenergic neurotransmission in the enteric nervous system started a revolution in mechanisms of neural control of the digestive tract that continues into a twenty-first century era of translational gastroenterology, which is now firmly embedded in the term, neurogastroenterology. This chapter, on Enteric Neurobiology: Discoveries and Directions, tracks the step-by-step advances in enteric neuronal electrophysiology and synaptic behavior and progresses to the higher order functions of central pattern generators, hard wired synaptic circuits and libraries of neural programs in the brain-in-the-gut that underlie the several different patterns of motility and secretory behaviors that occur in the specialized, serially-connected compartments extending from the esophagus to the anus.