Development of an ionic‐liquid‐based dispersive liquid–liquid microextraction method for the determination of antichagasic drugs in human breast milk: Optimization by central composite design

Chagas disease constitutes a major public health problem in Latin America. Human breast milk is a biological sample of great importance for the analysis of therapeutic drugs, as unwanted exposure through breast milk could result in pharmacological effects in the nursing infant. Thus, the goal of breast milk drug analysis is to inquire to which extent a neonate may be exposed to a drug during lactation. In this work, we developed an analytical technique to quantify benznidazole and nifurtimox (the two antichagasic drugs currently available for medical treatment) in human breast milk, with a simple sample pretreatment followed by an ionic‐liquid‐based dispersive liquid–liquid microextraction combined with high‐performance liquid chromatography and UV detection. For this technique, the ionic liquid 1‐octyl‐3‐methylimidazolium hexafluorophosphate has been used as the “extraction solvent.” A central composite design was used to find the optimum values for the significant variables affecting the extraction process: volume of ionic liquid, volume of dispersant solvent, ionic strength, and pH. At the optimum working conditions, the average recoveries were 77.5 and 89.7%, the limits of detection were 0.06 and 0.09 μg/mL and the interday reproducibilities were 6.25 and 5.77% for benznidazole and nifurtimox, respectively. The proposed methodology can be considered sensitive, simple, robust, accurate, and green.     

Detection of milk oligosaccharides in plasma of infants

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.     

蛋白质组学在人乳蛋白质研究中的应用

人乳是新生儿最理想的天然食物，蛋白质是人乳中最主要的营养成分之一。随着蛋白质组学技术的发展，利用蛋白质组学的方法研究人乳蛋白质也取得了一些研究成果。本文综述了近年来蛋白质组学技术在人乳蛋白质研究中的应用，分别从人乳蛋白质的组成研究、动态变化、人乳与其他来源乳汁的蛋白质差异比较、人乳磷酸化蛋白和糖基化蛋白研究、人乳内源肽的研究及人乳蛋白与疾病等几个方面进行了阐述。蛋白质组学技术使人乳蛋白质的研究进入了微量营养研究的时代，人乳蛋白质组学的研究成果将为母婴健康提供更好的保障。     

Simultaneous Determination of Cadmium,Mercury, Lead,Arsenic, Copper,and Zinc in Human Breast Milk by ICP‐MS/Microwave Digestion

Abstract

An analytical method using microwave digestion and inductively coupled argon plasma-mass spectrometry (ICP‐MS) analysis was developed for the measurement of Cd, Hg, Pb, As, Cu, and Zn in human breast milk. We applied external calibration, internal calibration, and standard addition for reference material and pooled milk analysis. Method performances were defined in terms of detection limits, accuracy, and precision. Accuracy varied between 93% and 105% and precision between 3% and 8%. External calibration and background interferences were checked through a calibrator addition procedure. Our method has shown high accuracy, precision, and sensitivity, as well as linearity within a wide range of values. Our methodology, developed by treatment of reference material and pooled milk samples, was applied for determination of Cu, Zn, As, Cd, Hg, and Pb in 120 human breast milk samples.     

Comparative two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of human milk to identify dysregulated proteins in breast cancer

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC‐associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one‐dimensional polyacrylamide gel electrophoresis (1D‐PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC‐MS/MS) analysis. The upregulated proteins in BC versus control are alpha‐amylase, gelsolin isoform a precursor, alpha‐2‐glycoprotein 1 zinc isoform CRA_b partial, apoptosis‐inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.     

Chiral and water‐soluble poly(2‐oxazoline)s

We describe the synthesis and characterization of the first water‐soluble and chiral poly(2,4‐disubstituted‐2‐oxazoline)s. While poly(2,4‐dimethyl‐2‐oxazoline)s are water soluble up to 100 °C, aqueous solutions of poly(2‐ethyl‐4‐methly‐2‐oxazoline) exhibit a lower critical solution temperature. This is discussed in context with its constitutional isomers poly(2‐oxazoline)s and poly(2‐oxazine)s. Circular dichroism spectroscopy revealed strong Cotton effects, which are also responsive to temperature in aqueous solution. It is therefore hypothesized that structures, comparable to polyproline helices, are formed in aqueous solution. In contrast to polyproline, poly(2,4‐disubstituted‐2‐oxazoline)s are highly water soluble and therefore represent very interesting pseudo‐polypeptides that may be useful to develop responsive biomimetic biomaterials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Comparative study of the instrumental couplings of high performance liquid chromatography with microwave-assisted digestion hydride generation atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry for chiral speciation of selenomethionine in breast and formula milk

A comparison of chiral separation and analysis of selenomethionine in breast and formula milk, using high performance liquid chromatography (HPLC) on a glycopeptide teicoplanin-based chiral stationary phase (Chirobiotic T), coupled to atomic fluorescence spectrometry (AFS) and inductively coupled plasma (ICP) MS detectors has been performed. The coupling HPLC-microwave-assisted digestion hydride generation requires on-line post-column analytes treatment, and a severe sample clean-up for fat and proteins elimination using centrifugation and ultrafiltration. Underivatized -selenomethionine enantiomers were completely resolved in 10 min using unbuffered water mobile phase at 1 ml min⁻¹ flow. Good selectivity and sensitivities (detection limits 3.1 and 3.5 ng ml⁻¹ as Se for - and -selenomethionine, respectively) were obtained, and method robustness and simplicity, together to the low cost of AFS detector, makes it suitable for infant milk routine analysis. HPLC–ICP-MS coupling exhibits very low detection limits (0.9 ng ml⁻¹, as Se) for each -selenomethionine enantiomers, but the method suffers from matrix influence, that produces a poor S/N ratio and low reliability.

The methods were applied to breast and formula milk samples with recoveries of 80% of the total selenium presence, which is attributable to the existence of other unknown species. -Selenomethionine was the only isomer present in breast milk, but a 30% of -selenomethionine was also detected in formula milk.     

Synthesis,Characterization, Novel Interaction of DNA,Antioxidant and Antimicrobial Studies of New Water Soluble Metallophthalocyanines Posture Eight Hydroxyphenyl Moiety via 1,3,4‐oxadiazole Bridge

The new peripheral 2(3),9(10),16(17),23(24)‐tetra‐5‐[4,4′‐diphenol]‐phenyl‐[1,3,4]‐oxadiazole substituted metallophthalocyanine (MPc) complexes has been well designed and executed. Due to high conjugation and excellent solubility in water makes them potential use in DNA binding and cleavage studies. Fourier transform infrared spectroscopy, nuclear magnetic resonance, electron spin ionization mass spectra data, and elemental analysis confirmed the well‐defined saddle‐like distorted structures for these substituted MPc complexes. The successful synthesis of these novel water soluble MPc moieties were employed as an effective DNA binding with calf thymus DNA was monitored using ultraviolet?visible spectral titrations and cleavage pBR322 DNA conceded in the absence of reductant by agarose gel electrophoresis method. The results indicate that all these water soluble complexes significantly show excellent binding and modest cleavage sensitivity activity. It is noteworthy that 6 and 7 exhibit potential antimicrobial and appreciable antioxidant activity with other water soluble phthalocyanines.     

Behaviour of protein-stabilised emulsions under various physiological conditions

Emulsion forms a major part of many processed food formulations. During the past few decades, the physico-chemical properties of oil-in-water emulsions under various food processing conditions have been extensively studied. However, over the recent years, interest has turned to understanding the behaviour of emulsions during consumption, i.e. physiological processing. In general, on ingestion, an emulsion is exposed to a relatively narrow range of physical (e.g. shear and temperature) and biochemical (e.g. dilution, pH, pepsin, pancreatin, mucins and bile salts) environments as it passes through the mouth into the stomach and then the intestines. There is currently limited knowledge of the physico-chemical and structural changes, which an emulsion may undergo when it passes through the physiologically active regime. A better understanding of the gastro-intestinal processing of emulsions would allow manipulation of physico-chemical and interfacial properties to modulate lipid ingestion, improve bioavailability of lipid soluble nutrients and reduce absorption of saturated fats, cholesterol and trans fats.Food emulsions are commonly stabilised by proteins, as they are not only excellent emulsifiers but also provide nutritional benefits to the product. The effects of digestion conditions on interfacial protein structures are complicated because of potential breakdown of these structures by proteolytic enzymes of the gastrointestinal tract. Studies dealing directly with the behaviour of protein-based emulsions under digestion conditions are very limited. This paper provides an overview of the behaviour of oil-in-water emulsions stabilised with globular proteins, namely lactoferrin and β-lactoglobulin. Recent advances in understanding the interactions between interfacial proteins on oil droplets and various physiological materials (e.g. enzymes and bile salts) in in vitro digestion systems are considered. Major emphasis is placed on the recent work carried out in our laboratory at Massey University on the behaviour of milk protein based emulsions (lactoferrin or β-lactoglobulin) during their passage through the gastro-intestinal tract.     

One pot synthesis of 6,12‐dihydro‐1,3,7,9‐tetramethyl‐5H, 11H‐dipyrido[1,2‐a:1′,2′‐d]pyrazine‐2,8‐dione by hilbert‐johnson reaction of 2‐chloromethyl‐3,5‐dimethyl‐4‐methoxypyridine

By heating 2‐chloromethyl‐3,‐5‐dimethyl‐4‐methoxypyridine (1) either neat or in solution methoxy group cleavage was achieved, followed by dimerisation to poorly soluble 6,12‐dihydro‐1,3,7,9‐tetramethyl‐5H,11H‐dipyrido[1,2‐a:1′,2′‐d]pyrazine‐2,8‐dione (3) in almost quantitative yield with methyl chloride evolution. To our knowledge this is the first example of such Hilbert‐Johnson preparation of dipyridopyrazine‐diones. Recrystallization of 3 from the hydrochloric acid yielded 6,12‐dihydro‐2,8‐dihydroxy‐1,3,7,9‐tetramethyl‐dipyrido[1,2‐a:1′,2′‐d]pyrazinediylium dichloride (4) , neutralization of which gave back the pyrazine‐2,8‐dione 3. The molecular structures of both compounds 3 and 4 have been unambiguously confirmed by single crystal X‐ray structure analysis.     

Selenium and vitamin E concentrations in human milk and formula milk from Hungary

The metabolic roles of vitamin E and selenium are closely related, and to a very great extent, each can compensate for the deficiency of the other. The aim of the study was to determine and compare the Se and vitamin E (α- and γ-tocopherol) contents of breast milk and commercially available infant formulas in Hungary. The Se content was measured by instrumental neutron activation analysis (INAA), while the α-, and γ-tocopherol concentrations were determined by high performance liquid chromatography (HPLC). The mean Se concentration was 17.4±2.8 μg/L in transitional and 13.8±2.3 μg/L in mature milk. It was found that, all of the starter (ST), the follow-on (FO) and the specialized formulas (SF) had lower Se content than breast milk. Transitional breast milk resulted in a higher Se intake (14 μg/day) than mature milk (11 μg/day). The daily Se intakes in Hungarian infants were within the Recommended Dietary Allowance (RDA) range. The natural vitamin E contents of human milk were similar during the early and late lactation. Mature breast milk had 3.30±1.13 mg/L α-TE concentration and this was significantly higher than that of in ST (1.98±1.57), and FO (1.77±0.78), or in SF ready to feed preparations (1.03±0.74). The present study suggests that the formulas for the optimal development of young infants, should contain concentrations of these antioxidants on a level which is comparable to that of the human milk.     

Two-dimensional electrophoretic analysis of human milk proteins

Identification of human milk proteins and formulation of a two-dimensional map is a first step in a project which intends to survey human milk proteins by two-dimensional electrophoresis. Thirty-four proteins have been identified using the Iso-Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species-specific, and, therefore, breast feeding confers a decided advantage for the infant. Using antisera for identification has revealed relationships between molecules which have not been previously noted. The antibody recognizes a common epitope between the IgA alpha chain and lactoferrin, and between the IgD d chain and beta casein. Milk protein concentrations vary longitudinally, diurnally, and individually. Identification of the proteins contributes meaning to the varying patterns. Knowledge of human milk proteins will help to elucidate human nutrition and health needs.     

Synthesis and characterization of new alternating,amphiphilic, comblike copolymers of poly(ethylene oxide) macromonomer and N‐phenylmaleimide

A surface‐active p‐vinyl benzyloxy‐ω‐hydroxy‐poly(ethylene oxide) macromonomer containing 22 pendant structural units of ethylene oxide (St–PEO₂₂) was synthesized with an initiation method. Because of its solubility in a large variety of solvents, the free‐radical copolymerization with electron‐acceptor N‐phenylmaleimide (NPMI) was performed at 60 °C in benzene and tetrahydrofuran (THF) as isotropic media and in a water–THF mixture or water as a heterogeneous medium. Oil‐soluble 2,2′‐azobisisobutyronitrile and water‐soluble 4,4′‐azobis(4‐cyanovaleric acid) were used as the initiators at fixed concentrations. Two different St–PEO₂₂/NPMI comonomer ratios (1/1 and 3/7) at a fixed total comonomer concentration in the polymerization system were used. The structures, compositions, and microstructure peculiarities of the obtained alternating, amphiphilic, comblike copolymers were determined by NMR analysis. For the copolymers synthesized in hydrophilic media, differential scanning calorimetry showed, near the endothermic peak attributed to the melting of the poly(ethylene oxide) side chains, the presence of a second peak due to the partially ordered phase that could exist between the crystalline state and the isotropic melt. Also, the thermal stability of the obtained copolymers was studied with thermogravimetric analysis. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 479–492, 2005     

A Facile and Cost‐effective Electroanalytical Strategy for the Quantification of Deoxyguanosine and Deoxyadenosine in Oligonucleotides Using Screen‐printed Graphite Electrodes

The development of electroanalytical methods for the detection and quantification of nucleotides in DNA offers vital implications in assessing the degree of oxidation or epigenetic modification in DNA. Unfortunately, the electrochemical response of oligonucleotides is strongly influenced by the size, composition and nucleic base sequence. In this article, an optimized analytical procedure for the enzymatically breakdown of the oligonucleotides to their corresponding nucleotides for the evaluation of the electrochemical response through the use of square wave voltammetry (SWV) is presented. Enzymatic digestion of oligonucleotides has been optimized in terms of buffer composition, digestion time, strategy for stopping the enzymatic reaction and filtration requirement for enzyme removal, and then compared to an established protocol. Under the optimized protocol SWV response of a number of untreated and enzymatically digested six‐mer oligonucleotides, namely 5′‐GGGGGG‐3′, 5′‐AAAAAA‐3′, 5′‐CGCGCG‐3′ and 5′‐AAACGC‐3′ have been analysed, providing a higher sensitivity for the determination of guanosine and adenosine monophosphate species under digestion conditions with a more facile and cost effective procedure. The novel strategy for the enzymatically treated oligonucleotides in combination with the SWV response provides a proof of principle for feasible applications in the diagnosis of methylated guanosine in DNA as a potential biomarker due to its relation with cancer.     

Proteomics analysis of human breast milk to assess breast cancer risk

Detection of breast cancer (BC) in young women is challenging because mammography, the most common tool for detecting BC, is not effective on the dense breast tissue characteristic of young women. In addition to the limited means for detecting their BC, young women face a transient increased risk of pregnancy‐associated BC. As a consequence, reproductively active women could benefit significantly from a tool that provides them with accurate risk assessment and early detection of BC. One potential method for detection of BC is biochemical monitoring of proteins and other molecules in bodily fluids such as serum, nipple aspirate, ductal lavage, tear, urine, saliva and breast milk. Of all these fluids, only breast milk provides access to a large volume of breast tissue, in the form of exfoliated epithelial cells, and to the local breast environment, in the form of molecules in the milk. Thus, analysis of breast milk is a non‐invasive method with significant potential for assessing BC risk. Here we analyzed human breast milk by mass spectrometry (MS)‐based proteomics to build a biomarker signature for early detection of BC. Ten milk samples from eight women provided five paired‐groups (cancer versus control) for analysis of dysregulatedproteins: two within woman comparisons (milk from a diseased breast versus a healthy breast of the same woman) and three across women comparisons (milk from a woman with cancer versus a woman without cancer). Despite a wide range in the time between milk donation and cancer diagnosis (cancer diagnosis occurred from 1 month before to 24 months after milk donation), the levels of some proteins differed significantly between cancer and control in several of the five comparison groups. These pilot data are supportive of the idea that molecular analysis of breast milk will identify proteins informative for early detection and accurate assessment of BC risk, and warrant further research. Data are available via ProteomeXchange with identifier PXD007066.     

Water‐Soluble Fluorescent Probes for Selective Recognition of Lysine and Its Application in an Object Carry‐and‐Release System

A water‐soluble fluorescent probe PEG‐TPA‐5′ was synthesized, which shows an excellent selectivity to detect Lys in aqueous phase. An object carry‐and‐release system is established by applying PEG‐TPA‐5′ as carrier and Lys as chemical stimulating source.     

Real time evolution of liquid crystalline nanostructure during the digestion of formulation lipids using synchrotron small-angle X-ray scattering

The role of the digestion of lipids in facilitating absorption of poorly water-soluble compounds, such as vitamins, is not only an important nutritional issue but is increasingly being recognized as an important determinant in the effectiveness of lipid-based drug formulations. It has been known for some time that lipids often form complex liquid crystalline structures during digestion and that this may impact drug solubilization and absorption. However, until recently we have been unable to detect and characterize those structures in real time and have been limited in establishing the interplay between composition, digestion, and nanostructure. Here, we establish the use of an in vitro lipid digestion model used in conjunction with synchrotron small-angle X-ray scattering by first confirming its validity using known, nondigestible liquid crystalline systems, and then extend the model to study the real time evolution of nanostructure during the digestion of common formulation lipids. The formation of liquid crystalline structures from unstructured liquid formulations is discovered, and the kinetics of formation and dependence on composition is investigated.     

Synthesis,characterization, and comparison of properties of new fluorinated poly(arylene ether)s containing phthalimidine moiety in the main chain

Four new poly(arylene ether)s have been prepared by the reaction of N‐phenyl‐3,3‐bis(4‐hydroxyphenyl)phthalimidine (PA) with four different perfluoroalkylated monomers namely 1,3‐bis(4′‐fluoro‐3′‐trifluoromethyl benzyl) benzene, 4,4′‐bis(4′‐fluoro‐3′‐trifluoromethyl benzyl) biphenyl, 2,6‐bis(4′‐fluoro‐3′‐trifluoromethyl benzyl) pyridine, and 2,5‐bis(4′‐fluoro‐3′‐trifluoromethyl benzyl) thiophene. The poly(arylene ether)s were characterized by different spectroscopic, thermal, mechanical, and electrical techniques. The poly(arylene ether) containing quadriphenyl unit in the main chain showed very high glass transition temperature of 291°C and outstanding thermal stability upto 556°C for 10% weight loss under a 4:1 nitrogen:oxygen mixture. The polymers were soluble in a wide range of organic solvents. Transparent thin films of these polymers exhibited tensile strengths upto 75 MPa and elongation at break upto 32%. The films of these polymers showed low water absorption of 0.26%. Copyright © 2009 John Wiley & Sons, Ltd.