Ion-exchange Liquid Chromatography Research Articles

Platelet amino acid spectrum and gut microbiota, their links in patients with coronary artery disease and atrial fibrillation

Background. The aim of our work was to identify the links between platelet amino acid (AA) spectrum and gut microbiota composition in patients with coronary artery disease (CAD) and atrial fibrillation (AF) and to evaluate them. Materials and methods. Three hundred patients were enrolled in the study. They were divided into 3 groups: I (CAD) — 149 patients with CAD but without arrhythmias, II (CAD + AF) — 124 people with CAD and AF paroxysm, and control group (CG) — 27 individuals without CAD and arrhythmias. Platelet AA level was assessed by method of ion-exchange liquid column chromatography. Gut microbiota composition was studied by 16-S rRNA sequencing. Results. A significant increase in isoleucine (10.73 %), leucine (12.63 %) and a decrease in threonine (23.05 %), serine (5.06 %), glycine (32.21 %) and valine (30.83 %) platelets levels was found in patients with CAD and AF (P < 0.05). In addition, they had a significant increase in Bacteroides spp., Faecalibacterium prausnitzii, Actinobacter spp., Streptococcus spp., Ruminococcus spp. and a decrease in Lactobacillus spp., Bifidobacterium spp., Eubacterium rectale (P < 0.05). Platelet glutamine acid, valine, glycine, asparagine acid, threonine had the highest number of significant correlations with gut microbiota species (P < 0.05). Actinobacter spp., Blautia spp., Streptococcus spp., Akkermansia muciniphila and Roseburia inulinivorans had the highest number of significant correlations with platelet amino acids (P < 0.05). Conclusions. Platelet amino acid spectrum and gut microbiota composition in patients with coronary artery disease and atrial fibrillation are closely linked.

Trace elements and arsenic speciation in Paracentrotus lividus from North-West Mediterranean Sea.

Levels of 14 trace elements (Al, Ti, V, Cr, Mn, Co, Ni, Zn, As, Se, Cd, Sn, Hg, and Pb) and 5 arsenic species (arsenite/As(III), arsenate/As(V), monomethylarsonic acid/MA, dimethylarsinic acid/DMA, and arsenobetaine/AsB) were assessed in the gonads of sea urchin samples (Paracentrotus lividus) from North-West Mediterranean Sea (French coast). The samples were collected from 13 sites characterized by different types and levels of chemical contamination. Trace elements levels were measured by inductively coupled plasma-mass spectrometry (ICP-MS) following microwave (acid) digestion in a closed system, whereas As speciation analysis was carried out by ion-exchange liquid chromatography coupled with ICP-MS after microwave-assisted extraction. High levels of trace elements were found in sea urchins sampled from Corsica whereas the samples from Théoule were found to be the least contaminated. From all the analyzed urchin samples, none showed Cd, Hg, or Pb concentrations above the regulatory levels set by the European Community (EC) No. 1881/2006 for seafood or bivalve mollusks. Regarding arsenic speciation, AsB was confirmed to be the predominant species. Inorganic As (As(III) + As(V)) was mainly constituted by As(III), which was quantified in all samples. Methylated As forms (MA and DMA) represent 9 to 23% of the total As.

Extraction, Isolation, Characterization, and Biological Activity of Sulfated Polysaccharides Present in Ascidian Viscera Microcosmus exasperatus.

Ascidians are marine invertebrates that synthesize sulfated glycosaminoglycans (GAGs) within their viscera. Ascidian GAGs are considered analogues of mammalian GAGs and possess great potential as bioactive compounds, presenting antitumoral and anticoagulant activity. Due to its worldwide occurrence and, therefore, being a suitable organism for large-scale mariculture in many marine environments, our main objectives are to study Microcosmus exasperatus GAGs regarding composition, structure, and biological activity. We also aim to develop efficient protocols for sulfated polysaccharides extraction and purification for large-scale production and clinical applications. GAGs derived from M. exasperatus viscera were extracted by proteolytic digestion, purified by ion-exchange liquid chromatography, and characterized by agarose gel electrophoresis and enzymatic treatments. Anticoagulant activity was evaluated by APTT assays. Antitumoral activity was assessed in an in vitro model of tumor cell culture using MTT, clonogenic, and wound healing assays, respectively. Our results show that M. exasperatus presents three distinct polysaccharides; among them, two were identified: a dermatan sulfate and a fucosylated dermatan sulfate. Antitumoral activity was confirmed for the total polysaccharides (TP). While short-term incubation does not affect tumor cell viability at low concentrations, long-term TP incubation decreases LLC tumor cell growth/proliferation at different concentrations. In addition, TP decreased tumor cell migration at different concentrations. In conclusion, we state that M. exasperatus presents great potential as an alternative GAG source, producing compounds with antitumoral properties at low concentrations that do not possess anticoagulant activity and do not enhance other aspects of malignancy, such as tumor cell migration. Our perspectives are to apply these molecules in future preclinical studies for cancer treatment as antitumoral agents to be combined with current treatments to potentiate therapeutic efficacy.

Effect of heat treatment on the antioxidant activities of camel milk alpha, beta and total caseins

This study aimed to evaluate the effect of various heating temperatures on the antioxidant activities of camel milk caseins. The samples were processed with three different heat treatments: Pasteurization at low and high temperatures and boiling. Fresh camel milk (unheated) was used as a control. Camel milk caseins were separated by fast ion exchange liquid chromatography (FPLC) and identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS page). The antioxidant activities of caseins were measu- red by three different in vitro methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) radical scavenging activity and ferric reducing power assay (FRAP). The antioxidant activity evaluated by the DPPH assay decreased significantly (p<0.05) with the increase in heat treatment of caseins. However, there was no significant difference in ABTS radical scavenging activity and Ferric Reducing Antioxidant Power assay (FRAP) of heat-treated camel caseins compared to unheated onesStill, a decrease was observed in those activities by the increase of temperature in the different casein concentrations. Besides, whatever the concentration tested and the methods applied, the antioxidant activity of beta-casein (β-CN) was more pronounced than the alpha-casein (α-CN). Therefore, camel milk casein could be used as a natural source of antioxidants which may have a potential application in the food and nutraceutical industries. Throughout the different heat treatments applied, pasteurization at low temperature could be the most suitable alternative to preserve the antioxidant properties of camel milk.

The Influence of Traditional and Immobilized Yeast on the Amino-Acid Content of Sparkling Wine

This article focuses on the effect of yeast strains, vintage, and must sugar content on the amino-acid content of sparkling wines produced by the traditional method. In the experiment, the amino-acid concentrations before and after secondary fermentation, depending on the type of yeast used (basic wine without secondary fermentation, wine fermented with immobilized yeast, and wine fermented with classical Champagne yeast) and the sugar content of the must (170, 190, and 210 g per liter), and the vintage (2010 and 2011), were evaluated. Concentrations of 20 free amino acids in 18 wine variants were analyzed by ion-exchange liquid chromatography with postcolony ninhydrin derivatization and photometric detection. Results of the study show an increase in all the amino acids represented, except ornithine, after secondary fermentation. The average content of each amino acid in the basic wine, wine fermented with immobilized yeast, and wine fermented with classical Champagne yeast was higher in the variant where classical yeast was used. In this variant, the concentrations of alanine, glutamic acid, lysine, arginine, phenylalanine, valine, and glycine were almost twice as high as in the other variants. A higher proportion of most amino acids was observed in the year 2011; only for amino acids lysine, leucine, phenylalanine, tyrosine, ornithine, histidine, and methionine was a higher concentration observed in the year 2010. A higher concentration of released amino acids was also observed in wine produced from must with a higher sugar content (21° NM).

The content of amino acids in the grain of different varieties of winter peas and products of its processing

Purpose. Determine amino acid content, biological and nutritional value of grain of different winter pea varieties and products of its processing. Methods. The amino acid share was determined by ion exchange liquid chromatography. Mathematical processing of the obtained data was performed by variance analysis of a one-factor field experiment. Amino acid and integral scores were determined by calculation. Results. Winter pea grain of ‘NS Moroz’, ‘Baltrap’ and ‘Enduro’ varieties had high amino acid content. In the composition of essential amino acids, lysine and leucine share was the highest – 1.53–1.77%, and methionine share was the lowest – 0.25–0.28% depending on winter pea variety. The main amino acid of winter pea grain was glutamic acid, the share of which was 3.25–3.30% of the share of all amino acids. The share of aspartic acid was 2.30–2.37% depending on the variety. In the composition of essential amino acids, cystine share was the lowest – 0.31–0.37% depending on the variety. The share of essential amino acids in winter pea was 40–41% of their total. The share of tryptophan decreased by 30–43%, isoleucine – by 20–24%, methionine – by 8–29%, phenylalanine, lysine, leucine – by 8–12%, valine share – by 5–9%, threonine one – by 1–5%. In the composition of essential amino acids, cystine share decreased most – by 58–73%, histidine – by 36–43%, glycine – by 42–45% compared to grain. Alanine share decreased by 19–25%, glutamic acid – by 12–15%, tyrosine – by 8–18%, aspartic acid – by 9–12%. The share of serine and arginine decreased least of all – by 3–8%. However, the amount of essential amino acids decreased by only 15–17% depending on the varietal cha­racteristics. It was found that the amino acid score of winter pea grain and products of its processing was non-deficient. Calculations showed that 100 g of winter pea grain satisfied the biological need of adults with isoleucine by 55–58%, valine – 51–42%, tryptophan – 41–50%, lysine – 37–40%, threonine – 39–40%, leucine – 36–38%. The highest daily requirement was provided by 100 g of grain processing products with arginine and glutamic acid – by 20–26%, depending on winter pea variety. For the rest of the essential amino acids, this figure was 6–16% for flour and 2–15% for cereals due to a decrease in the amino acid content in these products. Conclusions. The share of amino acids, biological and nutritional value of grain of different winter pea varieties and products of its processing, such as cereals and flour were determined. In the amino acid composition, the share of essential amino acids predominated, but 100 g of grain and products of its processing most meet the need for essential amino acids. The share of amino acids is reduced in the products of winter pea processing, especially in cereals. The amino acid score of winter pea grain of ‘NS Moroz’, ‘Baltrap’ and ‘Enduro’ varieties and products of its processing was balanced.

Variability levels of selected amino acids among mandarins produced in Uruguay

Nutraceutical properties of mandarins are of great interest to promote their consumption. The occurrence of free amino acids in foods is relevant to assess the nutritional value of it. To learn more about the amino acids� occurrence and variability between species, a targeted metabolomics study in �Ellendale�, �Willowleaf� and �Page� varieties was performed through ion exchange liquid chromatography coupled to tandem mass spectrometry. The studied amino acids were asparagine, glutamic acid, glutamine, histidine, methionine, phenylalanine, proline, threonine and tyrosine. The difference between two consecutive seasons was evaluated, as well as the influence of fruit maturity of �Page� collected in two periods of 2015. The analytical methodology was validated. The concentration of the compounds through principal component analysis, separated well apart the three cultivars in both harvests, showing a particular profile for each of them. When comparing mature and immature cultivar �Page�, the amino acids with higher levels in mature samples were histidine, asparagine, glutamine and glutamic acid. The profiles were different due to genetic diversity, and the climatic conditions. These results add value to citric production.

THE ROLE OF BETA-BLOCKERS IN VETIANGINAL THERAPY IN THE CORRECTION OF VIOLATIONS OF THE AMINO ACID SPECTRUM IN PATIENTS WITH UNSTABLE ANGINA WITH CONCOMITANT DIABETES MELLITUS

The purpose of the study: to study the effect of bisoprolol and nebivolol on the spectrum of essential blood plasma AK in patients with concomitant diabetes mellitus (DM) type 2 in order to optimize drug treatment of this pathology in the future.Material and methods. The study involved 70 patients with NA with concomitant DM type 2, which was divided into two groups: group I (37 patients) as part of basic antianginal therapy received bisoprolol, group II (33 patients) received nebivolol. The control group (CG) consisted of 18 healthy individuals. All patients were tested for irreplaceable blood plasma AA by ion exchange liquid chromatography on an automatic analyzer AK T-339 produced by «Microtechna» (Czech Republic, Prague) at the Institute of Biochemistry named after OV Palladin NAMS of Ukraine. The following essential AAs were identified: arginine, valine, histidine, isoleucine, leucine, lysine, methionine, threonine, phenylalanine. Examination of patients was performed at the beginning of treatment and after 20 days. Results. It was determined that in patients with NA with concomitant DM type 2 under the influence of antianginal therapy in both groups significantly decreased the total amount of irreplaceable AA compared with control group, indicating an increase in intracellular metabolism as a compensatory response in coronary circulation. It should be noted that in patients of group II, in contrast to patients of group I, under the influence of treatment the level of histidine remained normal. The level of threonine significantly decreased compared to CG and the rate of patients in group I, the level of valine significantly increased compared to pre-treatment, the level of isoleucine significantly decreased compared with CG and with the rate of patients of group I. Importantly, ammonia levels returned to normal under the influence of basic therapy, which included nebivolol.Conclusion. The dynamics of changes in the levels of individual irreplaceable AA blood plasma, due to the mechanisms of their metabolic transformations in patients with NA with concomitant DM type 2 indicates greater effectiveness of nebivolol and the feasibility of its prescription іn complex of a basic antianginal therapy.

Correction of imbalance of essentialaminoacids of blood plasma in Patients with stableangina

Introduction. Ischemic heart disease is the leading nosology calunit in the structure of cardiovascular diseases interms of disability and mortality among the population of Ukraine. The purpose. To improve the treatment of patients with stable angina by studying the effect of L-arginine on the balance of essential amino acids in blood plasma. Material and methods. It was examined 85 patients with stable angina. They were divided into two groups: group Ipatients received antianginal basic therapy, group II patients received basic antianginal therapy and L-arginine. The amino acid spectrum of patients' blood plasma was studied by ion-exchange liquid column chromatography, using an automatic amino acid analyzer T-339 Microtechna (Czech Republic, Prague). Results and discussion. In patients with stable angina who received basic therapy and L-arginine, in contrast to patients who received only basic therapy, plasma levels of arginine became normalized, which probably contributes to the synthesis of NO. The level of valine, leucine and isoleucine, which provide the synthesis of acyl-CoA and succinyl-CoA, became also normalized.Conclusion. Administration of L-arginine to patients with stable angina together with antianginal therapy helps to correct plasma amino acid imbalances, which is likely to effectively affect the course of the disease and prognosis.

The effect of L-arginine on the balance of essential amino acids in plasma of the patients with stable angina

Background. Despite the significant achievements of clinical medicine in the prevention, diagnosis and treatment of coronary heart disease, the levels of morbidity, disability and mortality among the population of Ukraine from this pathology remain consistently high. The purpose. To improve the treatment of patients with stable angina by studying the effect of L-arginine on the balance of essential amino acids in blood plasma. Material and methods. It was examined 67 patients with stable angina. They were divided into two groups: group Ipatients received antianginal basic therapy, group II patients received basic antianginal therapy and L-arginine. The amino acid spectrum of patients' blood plasma was studied by ion-exchange liquid column chromatography, using an automatic amino acid analyzer T-339 Microtechna (Czech Republic, Prague). Results and discussion. In patients with stable angina who received basic therapy and L-arginine, in contrast to patients who received only basic therapy, plasma levels of arginine became normalized, which probably contributes to the synthesis of NO. The level of valine, leucine and isoleucine, which provide the synthesis of acyl-CoA and succinyl-CoA, became also normalized. Conclusion. Administration of L-arginine to patients with stable angina together with antianginal therapy helps to correct plasma amino acid imbalances, which is likely to effectively affect the course of the disease and prognosis.

The study of the cardioprotective action of the thick extract of parsnip herb

Topicality. Modern drugs used to treat diseases of the cardiovascular system generally have a number of side effects. In this regard, the problem of developing and studying safe and effective drugs for the use in cardiology becomes relevant for scientists. Pharmacological drugs with the metabolic and cytoprotective action of plant origin are increasingly used for the treatment of cardiovascular diseases; however, their range is quite limited. The latest information on the use of herbal medicines based on the promising types of the medicinal plant raw material of the domestic flora, in particular parsnip (Pastinaca sativa L.) herb, determines the urgency of creating new potential cardioprotectors of plant origin. Aim. To study amino acids and phenolic compounds in the thick extract of parsnip herb, as well as the cardioprotective activity of this extract. Materials and methods. The thick extract of parsnip herb (TEPH) was chosen as the study object. The study of the amino acid composition of TEPH was performed by ion exchange liquid chromatography on a T 339 automatic analyzer. Phenolic compounds were studied by high performance liquid chromatography (HPLC). Identification of components was performed by retention time and compliance of UV spectra with standard substances, the quantitative content was determined by calculating the area of peaks on the chromatogram. The study of the effectiveness of the use of the plant substance of TEPH for the correction of disorders in the body of rats under conditions of heart disease with high doses of adrenaline was performed at the premises of the Central Research Laboratory of the NUPh. The experiment was performed on 25 nonlinear white male rats weighing 180-220 g. The drug “Tricardin” LLC “Ternopharm” (Ukraine) was used as a reference drug. Results and discussion. As a result of the study of the amino acid composition of TEPH, 18 amino acids were identified; their total content was almost 10 % in the extract. Rutin and chlorogenic acid in the amount of 3.37 mg/100 g and 0.41 mg/100 g, respectively, were identified in the extract by HPLC. The results of the study of the effect of the thick extract of parsnip on the integrated indicators of the state of white rats on the model of acute adrenaline myocarditis indicate that when treating animals with TEPH in the doses of 100 mg/kg and 200 mg/kg the heart mass coefficient increased by 1.25 and 1.2 times compared to the group of intact animals, indicating a decrease in the manifestations of exudation compared to animals of the control pathology. Conclusions. Amino acids and phenolic compounds in TEPH have been studied. As a result, the presence of 18 amino acids has been found; their content in the extract is almost 10 %. The extract contains 3.37 mg/100 g of rutin and 0.41 mg/100 g of chlorogenic acid. The study conducted has confirmed the cardioprotective properties of TEPH, indicating the relevance of further comprehensive physicochemical, pharmacological and preclinical pharmacological studies of this herbal medicine and emphasizing the prospects for its further study, implementation in production and use in the treatment of cardiovascular diseases.

Qualitative Indicators of Experimental Brucellosis Antigen Preparations Designed for Cellular Tests in vitro

In order to develop the most diagnostically informative methods for carrying out antigen-stimulated cellular tests in vitro a careful selection of stimulating agent (antigen) is required, possessing an adequate activating potential and providing specificity of the reaction.Objective of the study was to identify the qualitative indicators of experimental batches of brucellosis antigen preparations designed for cellular tests in vitro.Materials and methods. Initially we produced antigen complexes of brucellosis microbe on the basis of the vaccine strains of three epidemically significant Brucella species (B. abortus, B. melitensis, B. suis). Quantitative determination of WsAg and PPBC proteins of experimental preparation series was performed applying capillary electrophoresis. Qualitative composition was assessed through ion exchange liquid chromatography with refractometric detection.Results and discussion. We have specified physical-chemical features, investigated chromatographic profiles and composition of protein fractions, as well as tried the produced experimental batches of brucellosis antigen preparations. After analyzing the defined protein and polysaccharide composition of the obtained WsAg samples, one can conclude that WsAg preparation cannot be used for cellular tests as the probability of non-specific lymphocyte reaction manifestation in vitro was experimentally proven. By contrast, complex brucellosis antigen preparation PPBC has an expressed specific activity and specificity under in vitro conditions and the prospects to be used when developing methodological approaches for laboratory diagnosis of brucellosis and assessment of de facto immunity rate in risk contingents after vaccination. The obtained parameters will allow for proper quality provision when manufacturing the developed experimental PPBC preparation designed for cellular tests in vitro, taking into account modern validation and standardization regulations.

Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

Oligonucleotide therapeutics have contributed remarkably to healthcare, being well suited for the treatment of intractable diseases that are difficult to approach using conventional drug modalities. However, as common techniques of oligonucleotide analysis rely on reversed-phase or ion-exchange liquid chromatography and thus employ toxic organic solvents and/or ion-pairing reagents, better alternatives are highly sought after. Poly(N-isopropylacrylamide) (PNIPAAm) is widely used in temperature-responsive chromatography (TRC), which relies on column temperature variation to control the physical properties of the stationary phase and, unlike conventional reversed-phase liquid chromatography, avoids the use of toxic organic solvents and complicated gradient methods. Herein, PNIPAAm copolymer hydrogel-modified silica beads were used for the simultaneous analysis of multiple synthetic oligonucleotides by TRC to recognize differences in the length of single nucleotides, single bases, and the number of phosphorothioated sites. Temperature-responsive elution was observed in all cases. Each separation of all combinations of multiple oligonucleotides was better at higher temperatures above the lower critical solution temperature and was performed without the use of organic solvents and gradient methods. In the case of multiply phosphorothioated oligonucleotides, good separation was achieved using an aqueous solvent and isocratic elution in the absence of ion-pairing reagents. Thus, the developed procedure was concluded to be well suited for oligonucleotide analysis.Graphical abstract

Toxicity, Biodegradability and Detection Methods of Glyphosate; the Most Used Herbicide: A Systematic Review

Introduction: Glyphosate is known as the most used world's herbicides and contradictions exist over its classification as a probably carcinogenic for the human. This study aimed to review the newest evidences in toxicity, biodegradability and detection methods of glyphosate.&#x0D; Materials and Methods: To conduct this systematic review, databases such as Scopus, Web of Science, PubMed, and Google Scholar were searched to extract studies on the non-target toxicity, biodegradability and detection methods of glyphosate from 2000 to 2018. The applied key words included glyphosate, herbicide, biodegradation, and bio decomposition. The number of articles retrieved and reviewed was 84 and 23, respectively.&#x0D; Results: Glyphosate could cause endocrine disrupting effects, dermal irritation, embryo toxicity, electrolyte abnormalities, apoptosis, cardiovascular collapse, teratogenicity, and mutagenic effects. High-performance liquid chromatography, UV-visible spectroscopy, gas chromatography/ mass spectrometry, and ion-exchange liquid chromatography were techniques used for detecting glyphosate in soil and water. The biodegradation of glyphosate was performed by various bacteria and fungi microorganisms.&#x0D; Conclusions: Given the high consumption and low rates of biodegradation of glyphosate, more attention should be paid to its toxicity potential in the human's environment and health

Short-Term Effects of Branched-Chain Amino Acids–Enriched Dialysis Fluid on Branched-Chain Amino Acids Plasma Level and Mass Balance: A Randomized Cross-Over Study

The hemodialysis (HD) session per se is a catabolic event contributing to protein-energy wasting via several mechanisms including nutrient losses. Amino acids (AAs) losses in the dialysate are estimated from 6 to 10g per treatment. The HD patient plasma AA concentration is usually lower than in normal subjects. This is even more marked in patients with long dialysis vintage or malnutrition. The aim of the study was to evaluate the effect on mass balance of a branched-chain AA (BCAA)-enriched (valine, isoleucine, leucine) dialysis fluid in a group of 6 stable HD anuric patients, fasting since 12hours. The specific choice of BCAA relied on their key role on protein and muscle anabolism and their usual decreased plasma concentration in HD patients. Each patient was prescribed in a cross-over design and random order, either receiving a standard high-flux HD or an HD treatment using a BCAA-enriched acid concentrate designed to achieve a physiological plasma concentration of BCAAs. HD prescription remained unchanged during the 2 phases of study. Dialysate electrolytes prescription was kept constant for each individual patient, as well as dialysate glucose concentration (5.5 mmol/L). Pre- and post-dialysis BCAAs concentrations were measured by Ion-Exchange Liquid Chromatography. Postdialysis concentrations were corrected for hemoconcentration, and net mass transfer was calculated. Six stable prevalent end-stage kidney disease patients were studied. They consisted of 5 men and 1 woman, aged 69.9years, with body mass index of 25.2kg/m2. Treatment schedule consisted of treatment time 4hours, high-flux polysulfone membrane (1.8m2), blood flow 350mL/minute, and dialysate/blood flow ratio at 1.5. The average BCAAs concentration in dialysate was targeted to physiological levels and assessed in 6 different samples, respectively for plasma valine, isoleucine, and leucine at 271, 78, and 145 μmol/L. With standard dialysate, plasma valine decreased from 204.5 to 130.8 (P=.0014). Plasma isoleucine and leucine changes were not significant, respectively from 65.7 to 59.3 μmol/L and 110.3 to 113.4 μmol/L. When using the BCAA-enriched dialysis fluid, plasma valine increased from 197.2 to 269.2 μmol/L (P=.0001), plasma isoleucine and leucine respectively from 63.2 to 84.7 (P=.0022) and from 107.2 to 161.6 μmoles/L (P=.0002). Dialysis dose estimated from KT/V did not differ between the sessions. The mass transfer with BCAA-enriched dialysate was +115, +16, and+83 μmol per session for leucine, isoleucine, and valine, respectively. In conclusion, the addition of BCAAs at physiological concentration in the dialysis fluid contributes to restore physiological plasma concentrations for valine, isoleucine, and leucine at the end of a dialysis session. As BCAAs are essential to muscle balance, this could help to limit losses of BCAAs, restore physiological BCAAs concentrations, and decrease muscle catabolism observed during the HD treatment. Further outcome-based studies are needed to confirm this hypothesis on a larger scale and longer treatment time.

A Novel Animal Model to Investigate the Pathophysiology of Atherosclerosis Through Regulation of Cholesterol Homeostasis and the Highly Atherogenic LDL‐L5

The electronegative low‐density lipoprotein (LDL) L5 is elevated in hypercholesterolemic plasma and can induce apoptosis in coronary endothelial cells. The atherogenic potential of L5 renders it an important therapeutic target for cardiovascular disease but the exact mechanisms how plasma L5 concentrations can be regulated by cholesterol intake remains unclear. In this study, we developed an animal model to investigate the inducibility of plasma L5 by cholesterol and test the LDL‐lowering effect of ezetimibe, an inhibitor of intestinal cholesterol absorption, in male Golden Syrian hamsters. The hamsters were fed diets containing high fat and 1% cholesterol (HFC diet) for 2 weeks and LDLs were purified from hamster plasma by ultracentrifugation followed by ion exchange liquid chromatography. The eluting patterns of LDL in hamsters revealed five subfractions, named L1 to L5, which were similar to those obtained from hypercholesterolemic human plasma. HFC‐fed hamsters had highly elevated plasma L5, expressed as L5/LDL ratio, of 48.8±13.4% (n=6) compared with the hamsters fed with normal chow diet (18.9±2.7%; n=4). When the cholesterol absorption inhibitor ezetimibe was added to the HFC diets at the dose of 1mg/day/kg body weight, the plasma L5 levels were significantly reduced compared to non‐ezetimibe group (32.1±11.3% vs. 48.8±13.4%; n=6). Hamsters fed with normal chow diet also demonstrated a modest reduction of L5 with ezetimibe (14.1±11.3% vs. 18.9±2.7%; n=4). The time interval required to reduce plasma L5 by ezetimibe was only two to four weeks, consistent with the cholesterol‐lowering effect of ezetimibe in human. Therefore, we have established a novel hamster‐L5 model to investigate the pathophysiology of atherosclerosis in vivo. This animal model can also serve as a screening tool to identify LDL‐ and L5‐lowering therapy for cardiovascular disease.Support or Funding InformationThis work was supported by grants NSC101‐2320‐B‐002‐026 from the National Science Council and MOST‐106‐2314‐B‐002 ‐156 ‐MY2 (Dr. Po‐Yuan Chang) from the Ministry of Science and Technology, Taipei, Taiwan.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

51 Effects of modulating early luteal phase progestin concentration on endometrial function in early pregnant mares

Progesterone prepares the endometrium for pregnancy. This requires down-regulation of progesterone receptors in the endometrial epithelium as a prerequisite for the expression of pregnancy-associated proteins. We investigated effects of modulated peripheral progestin concentration in early luteal phase mares on endometrial function on Day 14 of pregnancy. Genitally healthy oestrous mares (n=8; age 4 to 14 years) were inseminated until ovulation and treated with either altrenogest (0.044mg kg−1 once daily orally) on Days 5 to 10 after ovulation (ALT), cloprostenol (125mg once daily intramuscularly) on Days 0 to 3 after ovulation (CLO) or left untreated (CON). The ALT and CLO treatments were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles at random order. On Day 14 after ovulation, endometrial fluid was collected with a cotton roll (Salivette, Sarstedt, Germany) inserted into the uterus and an endometrial biopsy for immunohistological examination was collected. In endometrial fluid, free amino acid concentrations were analysed by ion exchange liquid chromatography with an amino acid analyser (Institut Kuhlmann, Analytik-Zentrum Ludwigshafen, Germany). Cell nuclei staining positive for the progesterone receptor were determined in the luminar and glandular epithelium as well as in the stroma. Statistical analysis was performed by non-parametric Friedman test with subsequent Wilcoxon test. Values are given as mean±standard error of mean. Pregnancy rate was 0.6±0.1 (13 cycles/8 pregnancies), 1.0±0 (8 cycles/8 pregnancies), and 0.7±0.1 (11 cycles/8 pregnancies) in CON, ALT, and CLO cycles, respectively (P=0.062). Conceptus size between Days 10 and 14 did not differ among treatments. The percentage of luminal epithelial cells staining positive for progesterone receptor differed among treatments (CON 72.8±4.1, ALT 70.7±4.7, and CLO 84.1±1.9%; P&amp;lt;0.05) and was higher in CLO than in ALT and CON cycles (P&amp;lt;0.05). Free amino acids glutamic acid and glycine were most abundant in endometrial fluid, but their concentrations did not differ among treatments. The concentrations of the amino acids isoleucine (CON 0.17±0.03, CLO 0.14±0.02, and ALT 0.23±0.04 µmol) and lysine (CON 0.27±0.08, CLO 0.18±0.05, and ALT 0.44±0.13 µmol) were influenced by treatment (P&amp;lt;0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed down-regulation of progesterone receptors in the endometrial epithelium on Day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions.

Early luteal phase progestin concentration influences endometrial function in pregnant mares

In the horse, it is still unclear if and to what extent low progestin concentration contributes to early conceptus loss. In the present study, we have investigated if reduced or elevated progestin concentration in the early luteal phase influences endometrial function and conceptus development. We hypothesized that reduced progestin concentration via delayed downregulation of endometrial progesterone receptors (PR) influences endometrial function in healthy fertile mares while progestin substitution does not. Genitally healthy estrous mares (n = 8; age 4–14 years) were inseminated and treated with either altrenogest (0.044 mg/kg once daily orally) on days 5–10 after ovulation (ALT), cloprostenol (125 μg once daily intramuscularly) on days 0–3 after ovulation (CLO) or left untreated (CON). ALT and CLO treatment were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles. On day 14 after ovulation, endometrial fluid was collected with a cotton roll inserted into the uterus and an endometrial biopsy for immunohistological demonstration of progesterone (PR) receptor distribution was collected. In endometrial fluid, free amino acid concentrations were analyzed by ion exchange liquid chromatography with an amino acid analyzer. Cell nuclei staining positive for the PR were determined in the luminal and glandular epithelium as well as in the stroma. Pregnancy rate tended to differ among treatments. The percentage of luminal epithelial cells staining positive for PR differed among treatments (p < 0.05) and was higher in CLO (84.1 ± 1.9%) than in ALT (70.7 ± 4.7%) and CON cycles (72.8 ± 4.1%). Concentrations of the amino acids isoleucine (CON 0.17 ± 0.03, CLO 0.14 ± 0.02, ALT 0.23 ± 0.04 μmol) and lysine (CON 0.27 ± 0.08, CLO 0.18 ± 0.05, ALT 0.44 ± 0.13 μmol) were influenced by treatment (p < 0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed downregulation of progesterone receptors in the endometrial epithelium on day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions. Enhanced progestin concentration had less clear effects in healthy fertile mares.

Analytical and Sample Preparation Protocol for Therapeutic Drug Monitoring of 12 Thiopurine Metabolites Related to Clinical Treatment of Inflammatory Bowel Disease.

Thiopurines (TP) represent an important therapeutic tool for the treatment of inflammatory bowel diseases (IBD) in the current situation of rising incidence and health care costs. The results of multiple clinical studies aimed at finding correlations between levels of TP metabolites and response of IBD patients to the treatment are, however, often controversial due to variability in analytical and sample preparation procedures among these studies. In this work, therefore, an updated analytical and sample preparation procedure for therapeutic drug monitoring (TDM) of TP metabolites in blood samples obtained from patients with IBD was proposed to establish a unified protocol. An advanced analytical method based on ion-exchange liquid chromatography hyphenated with tandem mass spectrometry (IEC-ESI-MS/MS) was used for the determination of the profiles of 12 individual TP metabolites in the particular steps of sample preparation procedure including blood collection, red blood cells (RBC) isolation, lysis, and storage. Favorable performance parameters of the IEC-ESI-MS/MS method (LLOQs 1–10 nmol/L, accuracy 95–105%, intra-day and inter-day precision < 10%, selectivity demonstrated via no sample matrix interferences) and acceptable stability (peak area fluctuations < 15%) of clinical samples under the proposed sample preparation conditions {(i) EDTA anticoagulant tube for the blood collection; (ii) 4 °C and 4 h between the sample collection and RBC isolation; (iii) phosphate-buffered saline for RBC washing and re-suspendation; (iv) −20 °C for RBC lysis and short-term storage; (v) 50 mmol/L phosphate buffer, pH 7.4, 10 mmol/L DTT as a stabilizing medium for TPN in RBC lysates} demonstrated the suitability of such protocol for a well-defined and reliable routine use in studies on thiopurines TDM.

Hydrophilic Interaction Liquid Chromatography for LC-MS

The retention mechanisms in hydrophilic interaction liquid chromatography (HILIC) are quantitatively described in in silico analysis. Among a variety of polar bonded-phase silica gels, hexylamine-bonded silica gel is stable and can be used in both aqueous HILIC and ion-exchange liquid chromatography with high reproducibility. Hexynyl and phenylhexyl bonded silica gels are also used in HILIC with high reproducibility. The retention mechanisms in aqueous hydrophilic and non-aqueous hydrophilic (normal-phase) liquid chromatography are similar. Both hydrogenbonding and weak electrostatic interaction contributes to retention. Furthermore, strong electrostatic interaction is dominant in ion-exchange liquid chromatography. A suitable liquid chromatographic system for liquid chromatography (LC)-mass spectrophotometer (MS) is proposed.