Cow's Milk Antigens Content in Human Milk: A Scoping Review.

The functionality of breast milk in terms of immunity is well-known. Despite this, a significant proportion of breastfed infants exhibit sensitization to different potentially allergenic proteins and clinical reactivity (including anaphylaxis) early in life and before the introduction of complementary feeding for the first time. The potential induction of early oral tolerance to overcome early allergic sensitization through exposure to allergens in breast milk also remains controversial and not yet well-established. The objective of this scoping review is to provide a critical appraisal of knowledge about the content of cow's milk antigens in human milk. The amount of dietary derived milk antigens found in human milk and the analytical methodologies used to detect and quantify these antigens, the allergic status of the mother, the stage of lactation, the time of sampling (before or after ingestion of food), and the impact of human milk allergen on the infant were the outcomes that were assessed. Allergy risk was explored in all reviewed studies and could help to better elucidate its role in the context of allergic disease development. According to the included literature, we can conclude that there are mainly fragments derived from bovine proteins in human milk, and the presence of potentially allergenic molecules is greater in the milk of mothers with an allergic tendency. A clear relationship between maternal diet and allergen content in breast milk could not be firmly concluded though. Also, infants receiving milk from human milk banks, where donor milk is pasteurized for preservation, may be subject to greater risk of allergy development, especially for ß-lactoglobulin.

Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum.

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

Human Milk Oligosaccharides (HMOs) and Infant Microbiota: A Scoping Review.

Human milk oligosaccharides (HMOs) are the third most abundant solid component of breast milk. However, the newborn cannot assimilate them as nutrients. They are recognized prebiotic agents (the first in the newborn diet) that stimulate the growth of beneficial microorganisms, mainly the genus Bifidobacterium, dominant in the gut of breastfed infants. The structures of the oligosaccharides vary mainly according to maternal genetics, but also other maternal factors such as parity and mode of delivery, age, diet, and nutritional status or even geographic location and seasonality cause different breast milk oligosaccharides profiles. Differences in the profiles of HMO have been linked to breast milk microbiota and gut microbial colonization of babies. Here, we provide a review of the scope of reports on associations between HMOs and the infant gut microbiota to assess the impact of HMO composition.

Breast Milk: A Source of Functional Compounds with Potential Application in Nutrition and Therapy.

Breast milk is an unbeatable food that covers all the nutritional requirements of an infant in its different stages of growth up to six months after birth. In addition, breastfeeding benefits both maternal and child health. Increasing knowledge has been acquired regarding the composition of breast milk. Epidemiological studies and epigenetics allow us to understand the possible lifelong effects of breastfeeding. In this review we have compiled some of the components with clear functional activity that are present in human milk and the processes through which they promote infant development and maturation as well as modulate immunity. Milk fat globule membrane, proteins, oligosaccharides, growth factors, milk exosomes, or microorganisms are functional components to use in infant formulas, any other food products, nutritional supplements, nutraceuticals, or even for the development of new clinical therapies. The clinical evaluation of these compounds and their commercial exploitation are limited by the difficulty of isolating and producing them on an adequate scale. In this work we focus on the compounds produced using milk components from other species such as bovine, transgenic cattle capable of expressing components of human breast milk or microbial culture engineering.

Association between Breast Milk Mineral Content and Maternal Adherence to Healthy Dietary Patterns in Spain: A Transversal Study.

The composition of breast milk is influenced by many factors, some of which dependent on the mother and others on the child. Changes in lactation and other factors depending on the mother's physiology and anthropometric characteristics, as well as her nutritional status and diet, are of key importance. Breast milk minerals have been extensively studied with highly uneven results. In this work, a comparison will made with data across the world. To understand the factors that might explain the disparity, several minerals (Na, K, Ca, P, Mg, Fe, Se and I) have been analyzed using ICP-MS in a set of human milk samples (n = 75). The samples had an identical geographical origin (Galicia, in northwestern Spain) but different lactation circumstances, including maternal anthropometric data, lactating time, newborn sex and maternal adherence to healthy dietary patterns (Mediterranean Diet, MD, or Atlantic Diet, AD). The required concentrations of essential elements reported in the literature are similar to those found in these Spanish women. A univariate approach revealed that factors such as lactating time, body mass index (BMI) and newborn sex have a significant influence in breastmilk mineral content. According to multivariate linear regression analysis, minerals in milk are particularly associated with lactating time, but also with newborn sex, maternal BMI, age and diet pattern in some cases. More precisely, these results suggest that the iron and selenium concentrations in the milk of Galician donors may be positively influenced by maternal adherence to AD and MD, respectively.

Comparison of the fatty acid profile of Spanish infant formulas and Galician women breast milk

The importance of dietary lipids during childhood is evident, as they are necessary for correct growth and development of the newborn. When breastfeeding is not possible, infant formulas are designed to mimic human milk as much as possible to fulfill infant’s requirements. However, the composition of these dairy products is relatively constant, while human milk is not a uniform bio-fluid and changes according to the requirements of the baby. In this study, breast milk samples were donated by 24 Spanish mothers in different lactation stages and different infant formulas were purchased in supermarkets and pharmacies. Gas chromatography coupled to flame ionization detection was used for the fatty acid determination. Compared to breast milk, first-stage formulas are apparently very similar in composition; however, no major differences were observed in the fatty acid profiles between formulas of different lactation stages. The Galician women breast milk has a fatty acid profile rich in oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. When comparing human milk with formulas, it becomes evident that the manufacturers tend to enrich the formulas with essential fatty acids (especially with α-linolenic acid), but arachidonic and docosahexaenoic acid levels are lower than in breast milk. Additionally, the obtained results demonstrated that after 1 year of lactation, human milk is still a good source of energy, essential fatty acids, and long-chain polyunsaturated fatty acids for the baby

Comparison of the fatty acid profile of Spanish infant formulas and Galician women breast milk.

The importance of dietary lipids during childhood is evident, as they are necessary for correct growth and development of the newborn. When breastfeeding is not possible, infant formulas are designed to mimic human milk as much as possible to fulfill infant's requirements. However, the composition of these dairy products is relatively constant, while human milk is not a uniform bio-fluid and changes according to the requirements of the baby. In this study, breast milk samples were donated by 24 Spanish mothers in different lactation stages and different infant formulas were purchased in supermarkets and pharmacies. Gas chromatography coupled to flame ionization detection was used for the fatty acid determination. Compared to breast milk, first-stage formulas are apparently very similar in composition; however, no major differences were observed in the fatty acid profiles between formulas of different lactation stages. The Galician women breast milk has a fatty acid profile rich in oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. When comparing human milk with formulas, it becomes evident that the manufacturers tend to enrich the formulas with essential fatty acids (especially with α-linolenic acid), but arachidonic and docosahexaenoic acid levels are lower than in breast milk. Additionally, the obtained results demonstrated that after 1 year of lactation, human milk is still a good source of energy, essential fatty acids, and long-chain polyunsaturated fatty acids for the baby.

A facile method for the fabrication of magnetic molecularly imprinted stir-bars: A practical example with aflatoxins in baby foods.

A fast and facile method for the fabrication of magnetic molecularly imprinted stir-bars (MMIP-SB) has been developed, using a combination of imprinting technology and magnetite. Magnetite was prepared in the laboratory from the raw and embedded into molecularly imprinted polymers through a process of bulk polymerization. This novel design was applied to the analysis of aflatoxins, one of the most important groups of mycotoxins in terms of occurrence and toxicity. In the context of food safety, molecularly imprinted polymers are a promising tool to achieve selective and accessible methods of extraction for different residues and contaminants. Considering the toxicity of aflatoxins, a dummy template was preferred for the synthesis of the imprinted polymers. A rapid and affordable extraction method for isolating five different aflatoxins that may be present in food was developed. The MMIP-SB was used as a conventional stir-bar and combined with high performance liquid chromatography and mass spectrometry for the determination of aflatoxin M1 in milk powder (infant formulas) and aflatoxins B1, B2, G1 and G2 in cereal-based baby foods. The results showed an average recovery of 60%, 43, 40, 44 and 39%, respectively, and RSD below 10%. These in-house prepared stir-bars featured good stirring and extraction performance, and recognition abilities, offering a good alternative to more complicated.

Analysis of Naturally Occurring Steroid Hormones in Infant Formulas by HPLC-MS/MS and Contribution to Dietary Intake.

Milk is a natural fluid and as such contains small amounts of naturally occurring steroids. Human milk is recommended as the optimal source of nutrients for infants and young children, and it has been associated to several short- and long-term benefits. For this reason, its composition is used as a reference for designing infant formulas. However, the available information on the hormonal levels of these dairy products is scarce, and it is usually limited to estradiol and estrone. In the present study, six natural sex hormones (pregnenolone, progesterone, estrone, dehydroepiandrosterone, testosterone and androstenedione) have been extracted from sixteen milk-based infant formulas and analyzed with liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The purpose of this research was to quantify natural steroid hormones in various infant formulas, to provide food and nutrition practitioners with information to estimate intakes in children. In addition, data found in the literature was used for comparison. The findings suggest that there are certain similarities between bovine milk and dairy products for infants. Furthermore, the detected levels were in general lower than those observed in human milk and/or colostrum. The reported results represent a valuable addition to the current knowledge on natural hormone content of infant foods.

Evaluation of the discriminative potential of a novel biomarker for estradiol treatments in bovine animals.

The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry (GC-C/IRMS). A serum dipeptide composed of pyroglutamic acid and phenylalanine was identified as a potential biomarker of estradiol treatments in adult cows. To evaluate its potential to pinpoint suspicious samples, samples from prepubertal females under different estrogenic treatments have been analyzed. The results confirmed the up-regulation of the dipeptide in adult bovines. The 2-week-old females exhibited short-lasting responses only in a few animals. The 6-month-old female showed a delayed but clear increase on the biomarker level. The composition of the anabolic preparations, the dose, and/or the administration route are possible additional reasons for the reduced response in young animals. A comparison to previous results reported by various researchers is included.

Structure elucidation and HPLC-MS/MS determination of a potential biomarker for estradiol administration in cattle.

Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 96/22/EC. However, this kind of substances is sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semisynthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e., estradiol-17ß or progesterone) still remains a challenge for European authorities. These target compounds are indeed always present in the animal matrix, and the establishment of reference thresholds appears very difficult because of the extreme variability existing among animals. In 2011, a metabolomics study was performed on serum samples obtained from cows treated with estradiol-17ß (or its ester estradiol benzoate) and from control animals using a high-performance liquid chromatography (HPLC)-LTQ-Orbitrap system. After appropriate data processing and multivariate statistical analysis (orthogonal partial least squares discriminant analysis), it was possible to highlight one potential biomarker candidate of estradiol treatments in bovine animals. Now, this biomarker has been structurally elucidated as a dipeptide, and its usefulness has been tested through a targeted HPLC-MS/MS method. Its presence in the previous samples has been confirmed and also in additional samples from estradiol-treated animals.

Rapid method for quantification of nine sulfonamides in bovine milk using HPLC/MS/MS and without using SPE.

Sulfonamides are antimicrobial agents widely employed in animal production and their residues in food could be an important risk to human health. In the dairy industry, large quantities of milk are monitored daily for the presence of sulfonamides. A simple and low-cost extraction protocol followed by a liquid chromatographic-tandem mass spectrometry method was developed for the simultaneous detection of nine sulfonamides in whole milk. The method was validated at the maximum residue limits established by European legislation. The limits of quantification obtained for most sulfonamides were between 12.5 and 25 µg kg(-1), detection capabilities ranged from 116 to 145 µg kg(-1), and recoveries, at 100 µg kg(-1), were greater than 89±12.5%. The method was employed to analyse 100 raw whole bovine milk samples collected from dairy farms in the northwest region of Spain. All of the samples were found to be compliant, but two were positive; one for sulfadiazine and the other for sulfamethoxipyridazine.

Metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry to screen for the illegal use of estradiol and progesterone in cattle.

Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 93/22/EC, however, this kind of substances are sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semi-synthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e. estradiol-17ß or progesterone) still remains a challenging task for European authorities. As a result of their origin, these target compounds are indeed always present in the analytical matrix, and because the concentration levels of natural steroids are extremely variable from one animal to another, the establishment of reference thresholds appears very difficult. During this preliminary study, metabolomic data was acquired on a high performance liquid chromatography system coupled to high resolution mass spectrometer (HPLC-LTQ-Orbitrap). Serum samples were collected from dairy cows treated or not with sex steroid hormones commonly employed in animal husbandry: estradiol-17ß (or its ester estradiol benzoate) and progesterone. After appropriate data processing and multivariate statistical analyses (OPLS-DA), it was possible to highlight significant metabolic modifications in serum consecutively to the administration of estradiol and/or progesterone. Those differences were used to build predictive models able to suspect illegal administration of these hormones in cattle. Potential biomarker candidates of estradiol and/or progesterone were pointed out, that remains to be structurally elucidated.

Disturbance in sex-steroid serum profiles of cattle in response to exogenous estradiol: a screening approach to detect forbidden treatments.

Estradiol benzoate (EB) has been one of the most widely used estrogenic agents in animal husbandry, as a way of exogenously introducing the natural hormone estradiol-17ß into the animal organism. Estradiol was previously employed to induce anabolic effects or reproductive improvements in cattle. However, the employment of EB in European countries has been permanently forbidden by Directive 2008/97/EC to guarantee consumers' health. Despite this prohibition, the control of estradiol-17ß and its esters continues to be a difficult task for residue-monitoring plans in European Communities because official analyses of natural thresholds for hormones in cattle have not yet been established, leading to a lack of confirmation for any exogenous administration of natural hormones. Several researchers have worked on excretion profiles of metabolites, variation in specific hormonal ratios and metabolomic fingerprints after hormonal treatments. This research focuses on the possible existence of disturbances in the serum profile of animals treated with EB in terms of steroid sex hormones (androgens, oestrogens and progestogens), by investigating the serum levels of several of these hormones. The serum samples were collected from three groups of cows: one treated with an intramuscular injection of EB, one treated with a combination of intravaginal EB and progesterone and a control (non-treated) group. The samples have been analysed by a validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, and 17 natural hormones were identified and quantified. Subsequently, data from the serum profiles were submitted for statistic and multivariate analysis, and it was possible to observe a manifest variation between animal groups. The obtained results can help in the development of a viable screening tool for monitoring purposes in cattle.

A new approach for identifying patients with ovarian epithelial neoplasms based on high-resolution mass spectrometry.

We investigated the serum profiles of patients with ovarian neoplasm using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) to obtain serum "fingerprints" for use in identifying patients with these neoplasms. We used HPLC-HRMS to analyze serum samples from patients with ovarian neoplasms and control subjects. Serum samples from 145 patients were analyzed, including 85 with ovarian epithelial neoplasms. We also compared the results of this serum-fingerprinting approach with the results of the CA-125 test and imaging. Fingerprinting successfully permitted the separation of control patients and patients with ovarian neoplasms. The sensitivity and specificity of the test were between 96% and 100%. When the results of this test were concordant with the results of the CA-125 test, 99% of serum samples were correctly classified as being from a patient with an ovarian neoplasm or with no ovarian neoplasm. We found that a metabolite of molecular weight 472 is the main metabolite in the separation of patients with ovarian neoplasms from control subjects. HPLC-HRMS serum profiling could become a screening test for ovarian neoplasms.

Determination of naturally occurring progestogens in bovine milk as their oxime derivatives using high performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

BACKGROUND: Hormones and hormone-like substances which are present in the environment have been repeatedly accused of being the cause of most endocrine disruption. However, the possible role of endogenous hormones in food of animal origin deserves to be discussed as well. The relation between steroid hormones and several human health problems has been previously reported, such as prostate and breast cancer, perturbation of human reproduction and endocrine disruption on humans and wildlife. This research is particularly concerned with cow's milk, which contains a considerable amount of sex hormones. RESULTS: A liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous detection and quantification of four naturally occurring steroid hormones in commercial bovine milk (pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OHP5), 17-hydroxyprogesterone (P4)). Oxime derivatives of steroids were analyzed in positive ionization and multiple reaction monitoring mode. Methodology has been validated according to Decision 2002/657/EC criteria. CONCLUSION: This method has been successfully used in real samples. It is fast and easy-handling and provides a useful tool for the assessment of progestogens in bovine milk.

Development and validation of LC-MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations.

A rapid and sensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the qualitative and quantitative assay of cyproheptadine (CP) in pharmaceutical samples. Diphenylpyraline hydrochloride (DPP) was used as an internal standard (IS). Two multiple reaction-monitoring (MRM) transitions for each analyte were observed: 288.1/96.1 and 288.1/191.2 for CP and 282.1/167.2 and 282.1/116.3 for DPP. The retention time of the drug was 7.29 min. The analytical method was successfully validated for linearity (1-100 ng/ml), intra-day precision, inter-day precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.86 and 0.98 ng/ml, respectively. The proposed method was applied to analyse the cyproheptadine content from seven different syrup formulations.

Application of dummy molecularly imprinted solid-phase extraction in the analysis of cyproheptadine in bovine urine.

Due to the difficulty of monitoring trace levels of cyproheptadine (CYP) residues in complicated biological matrices, specific adsorption materials for the preconcentration and clean-up of CYP are indispensable. In this work, CYP was extracted from urine using dummy molecularly imprinted SPE (DMISPE) to avoid leakage of the imprinting molecules during the desorption phase. For synthesis of DMISPE, azatadine (AZA) was employed as the dummy template, methacrylic acid (MAA) as the monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, and dichloromethane as the porogen solvent. An LC-MS/MS method was used to analyze CYP. Two MRM (multiple reaction monitoring) transitions for each analyte were monitored using diphenylpyraline hydrochloride (DPP.HCl), which was used as an internal standard. The advantages of DMISPE include obtaining less complex chromatograms and reducing ion suppression in ESI. The process efficiencies for DMISPE and SPE were 80% and 12%, respectively. In addition, the demonstrated reusability of the DMISPE cartridges is an advantage compared with single-use SPE cartridges or immunoaffinity materials.

Development and validation of an LC-MS/MS confirmatory method for residue analysis of cyproheptadine in urine of food-producing animals.

The possible off-label and illegal use of cyproheptadine (CYP) as an appetite stimulant for food-producing animals creates the need for methods capable of detecting it. A high-performance liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed to identify CYP in bovine urine, according to Commission Decision 2002/657/EC. Two multiple reaction monitoring (MRM) transitions for each analyte were monitored: 288.1/96.1 and 288.1/191.2 for CYP and 282.1/167.2 and 282.1/116.3 for diphenylpyraline hydrochloride (DPP), which was used as an internal standard. The solid phase extraction technique without a liquid-liquid step gives good results in urine samples from treated animals. The analytical method was successfully validated for linearity (0.15-10 ng/mL), with intraday precision of 9.4%, interday precision of 20.4%, and accuracy of 96.7%. The decision limit (CCalpha) and detection capability (CCbeta) were 0.48 and 0.82 ng/mL, respectively.

Development of a rapid and confirmatory procedure to detect 17beta-estradiol 3-benzoate treatments in bovine hair.

A high-performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for efficient and confirmatory surveillance of illegal use of estradiol benzoate, even when this substance is used in reproductive control. After cryogenic grinding, estradiol benzoate was extracted from hair with acetonitrile for 24 h on a rocking table. The validation of the method was based on Commission Decision 2002/657/EC using the deuterated analogue of estradiol benzoate as internal standard. Decision limit (0.81 ng/g), detection capability (1.38 ng/g), repeatability CV% (13.7), within in laboratory reproducibility CV% (15.6%), and trueness (99.3%) were calculated. Using the proposed methodology the presence of estradiol benzoate in samples obtained from animals treated to synchronize their estrous cycles can be confirmed.