Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium-magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3-N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3-N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.

Magnesium oxide (MgO) is extensively used as a dietary magnesium (Mg) source in dairy cow diets. However, dairy operations can benefit from other Mg sources. Thus, we evaluated the replacement of dietary MgO with calcium­magnesium hydroxide (CaMg(OH)2) in diets with and without ruminal buffer and their effects on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. The study used 20 lactating Holstein cows that were blocked in groups of four and randomly assigned to one of the four treatments. The ruminal content, feed, feces, and urine were collected for analysis of the microbiota composition, ruminal fermentation, nitrogen metabolism, and apparent nutrient digestibility. There was no interaction effect of dietary buffer use and Mg source, while buffer improved total tract apparent digestibility of the dry matter and fiber components; CaMg(OH)2 increased the ruminal concentration of butyrate and the abundance of butyrate-producing bacteria. In summary, we conclude that using CaMg(OH)2 can improve ruminal fermentation regardless of buffer use, which indicates that we can take advantage of the mineral formulation in the diet to modulate the ruminal microbiota composition.

Unveiling unstable non-acid incidence in Holstein cows fed with corn silage or sugarcane.

We aimed to evaluate the incidence of unstable non-acid milk (UNAM) in cows fed either sugarcane or corn silage. Second, we aimed to evaluate the effect of daily variation (d 1 to 4) and alcohol grades (72, 78, and 80%) on UNAM incidence. The experiment was conducted as a split-plot crossover design, with 2 periods and 2 roughage types (sugarcane or corn silage). Thirteen multiparous Holstein cows with an average of 281 ± 29 d in milk were randomly distributed into 2 diets. Individual blood (analysis of total proteins, albumin, urea, calcium, phosphorus, magnesium, iron, chloride, glucose, and lactate) and milk samples (analysis of protein, fat, lactose and total solids, somatic cell count, and characterization of the protein profile) were collected during the last 4 d of each period. For UNAM identification, the alcohol test was conducted in milk samples at 4°C; specifically, if the sample presented the formation of clots, this would be noted as positive for UNAM. In addition, the Dornic acidity analysis was performed in the same samples to evaluate the true milk acidity. The use of sugarcane and higher degrees of alcohol were associated with increased UNAM. We observed no daily variation in UNAM. Nevertheless, we found no roughage type effect on the variables most commonly associated with UNAM, such as changes in salts in the casein micelle and, consequently, the zeta potential and the κ-casein (CN) fraction. The Pearson correlation analysis showed that the zeta potential and the concentrations of αS2-CN, blood ionic calcium, lactate, and glucose increased as the incidence of UNAM increased, showing a positive correlation among these variables. In contrast, the concentrations of lactose, phosphorus, and potassium decreased as UNAM increased, presenting a negative correlation. This study brought important discoveries to unveil why cows manifest UNAM. For instance, higher alcohol grades and cows fed with sugarcane had increased the incidence of UNAM. Additionally, animals with a higher incidence of UNAM (sugarcane-fed cows) were related to increased ionic calcium and glucose and changes in milk protein profile, with lower levels of BSA, ß-CN, and α-lactalbumin and greater αS1-CN content, all of which were correlated with UNAM. Nonetheless, this trial also provides evidence for the need for further studies to better understand the physiological mechanisms that directly affect the stability of milk protein.

[Improved quality of stored packed red blood cells by mechanical rinsing]. / Verbesserte Qualität gelagerter Erythrozytenkonzentrate durch maschinelle Autotransfusion.

BACKGROUND: The transfusion of packed red blood cells (PRBC) is associated with various side effects, including storage damage to PRBCs. The cells change their structure, releasing potassium as well as lactate. Mechanical rinsing, available in many hospitals, is able to remove toxic substances and possibly minimizes the negative side effects of transfusion. OBJECTIVE: The primary aim of our study was to improve the quality of PRBCs before transfusion. The effects of different washing solutions on PRBC quality were analyzed. MATERIAL AND METHODS: This in vitro study compares 30 mechanically washed PRBCs. They were either processed with standard normal saline 0.9% (n = 15, N group) or a hemofiltration solution containing 4 mmol/l potassium (n = 15, HF group) by a mechanical rinsing device (Xtra, LivaNova, Munich, Germany). A subgroup analysis was performed based on the storage duration of the processed PRBCs (7, 14, 37 days). Samples were taken before washing (EKprä), immediately after washing (EKpost) and 10 h later (EKpost10h), after storage in the "wash medium" at room temperature. Concentrations of ATP (probability of survival in transfused erythrocytes), lactate, citrate and electrolytes (potassium, sodium, chloride, calcium) were tested. RESULTS AND CONCLUSION: Mechanical rinsing improves pretransfusion quality of PRBC. Washing with a hemofiltration solution results in a more physiological electrolyte composition. Even 10 h after mechanical rinsing with a hemofiltration solution, the quality of 37-day-old PRBC is comparable to young PRBC that have been stored for 7 days and have not been washed. Washing stored PRBC increases the ATP content, which subsequently leads to an increased probability of survival of red cells after transfusion.

Review: Physiology and nutrition of late gestating and transition sows.

The physiology during late gestation and the transition period to lactation changes dramatically in the sow, especially during the latter period. Understanding the physiological processes and how they change dynamically as the sow approaches farrowing, nest building, giving birth to piglets, and producing colostrum is important because these processes greatly affect sow productivity. Glucose originating from assimilated starch accounts for the majority of dietary energy, and around farrowing, various organs and peripheral tissues compete for plasma glucose, which may become depleted. Indeed, physical activity increases shortly prior to farrowing, leading to glucose use by muscles. Approximately ½ to 1 d later, glucose is also needed for uterine contractions to expel the piglets and for the mammary gland to produce lactose and fat for colostrum. At farrowing, the sow appears to prioritize glucose to the mammary gland above the uterus, whereby insufficient dietary energy may compromise the farrowing process. At this time, energy metabolism in the uterus shifts dramatically from relying mainly on the oxidation of glucogenic energy substrates (primarily glucose) to ketogenic energy supplied from triglycerides. The rapid growth of mammary tissue occurs in the last third of gestation, and it accelerates as the sow approaches farrowing. In the last 1 to 2 wk prepartum, some fat may be produced in the mammary glands and stored to be secreted in either colostrum or transient milk. During the first 6 h after the onset of farrowing, the uptake of glucose and lactate by the mammary glands roughly doubles. Lactate is supplying approximately 15% of the glucogenic carbon taken up by the mammary glands and originates from the strong uterine contractions. Thereafter, the mammary uptake of glucose and lactate declines, which suggests that the amount of colostrum secreted starts to decrease at that time. Optimal nutrition of sows during late gestation and the transition period should focus on mammary development, farrowing performance, and colostrum production. The birth weight of piglets seems to be only slightly responsive to maternal nutrition in gilts; on the other hand, sows will counterbalance insufficient feed or nutrient intake by increasing mobilization of their body reserves. Ensuring sufficient energy to sows around farrowing is crucial and may be achieved via adequate feed supply, at least three daily meals, high dietary fiber content, and extra supplementation of energy.

The transition period is a short period of the reproductive cycle spanning from 7­10 d prepartum to 3­5 d postpartum in sows. Nonetheless, it is highly important for the productivity of sows because it is when the majority of piglet deaths occur. Most piglets die either during the birth process or within the first days postpartum, and mammary development, fetal growth, farrowing process, and colostrum production have profound impacts on piglet survival and growth. Nutrition during this critical period can greatly affect these physiological processes, and the most effective feeding strategy needs to be elaborated. Around farrowing, the sow may suffer from inadequate energy from assimilated starch, and liver glycogen seems not to be able to supply sufficient glucose to meet the demand for nest building, uterine contractions, and colostrum production. The sow seems to prioritize glucose for the mammary gland above the uterus but may suffer from depletion due to nest building. The insufficient energy status of the sow compromises the farrowing process and prolongs the duration, thereby increasing the need for farrowing assistance and stillbirths. Nutritional strategies to alleviate these challenges include adequate feed supply, number of daily meals, dietary fiber content, and extra supplementation of energy to sows around farrowing.

Fluorometric assay of iron(II) lactate hydrate and ammonium ferric citrate in food and medicine based on poly(sodium-p-styrenesulfonate)-enhanced Ag nanoclusters.

Poly(sodium-p-styrenesulfonate)-enhanced and D-penicillamine stabilized Ag nanoclusters (PSS-DPA-AgNCs) were prepared using one-step ultraviolet irradiation combined with microwave heating method, and the effects of the AgNCs photo-luminescence performance based on different types of polyelectrolytes and energy suppliers were studied detailedly. The as-prepared AgNCs can be used as a viable fluorescent probe for monitoring indirectly iron(II) lactate hydrate (ILH) and ammonium ferric citrate (AFC), respectively. The fluorescence (FL) quenching of PSS-DPA-AgNCs by Fe3+ (it is obtained from oxidized ILH/ionized AFC) mainly derives from a dynamic quenching process. Excellent linear relationships exist between the FL quenching degree of the AgNCs and the concentrations of ILH/AFC in the range of 0.17-6.00/0.067-3.33 µmol·L-1, and corresponding limit of detection (at 3σ/slope) is 12.4/6.04 nmol·L-1. Moreover, the AgNCs probe was extended to the assays of ILH in tablets, solid beverage or ILH additive and AFC in two kinds of edible salts or syrup with satisfactory results compared with the standard 1, 10-phenanthroline method. In addition, the AgNCs probe reveals a good temperature sensing capability.

[Determination of seven indicative ingredients in oral liquids of Chinese medicine by liquid chromatography-electrospray ionization-ion mobility spectrometry].

A two-dimensional separation and analysis method, based on liquid chromatography-electrospray ionization-ion mobility spectrometry (LC-ESI-IMS), is developed for the determination of seven indicative ingredients (danshensu, glycyrrhizic acid, gastrodin, chlorogenic acid, puerarin, baicalin, and rutin) in oral liquids of Chinese medicine. The sample was first separated on an ACQUITY UPLC BEH C18 column (50 mm×1 mm, 1.7 µm). The post-column effluent was directed to an adjustable flow splitter with a split ratio of 50:1. The low-flow and high-flow outlets were connected to an ion mobility spectrometer and a triple quadrupole mass spectrometer, respectively. The experimental conditions for LC, spray voltage, drift tube temperature, gas pre-heating temperature, and drift gas velocity were systematically optimized. The limits of detection (LODs) and quantitation (LOQs) for the seven analytes were 2-10 µg/mL and 5-25 µg/mL, respectively. The proposed method was applied for the analysis of real oral liquids of Chinese medicine samples. By coupling LC and IMS, two-dimensional separation could be achieved based on hydrophobicity difference and ionic mobility disparity, thus providing more comprehensive measurement information than LC or IMS used alone.

Development and validation of in-line near-infrared spectroscopy based analytical method for commercial production of a botanical drug product.

Near-infrared (NIR) spectroscopy is one of the most successful pharmaceutical process analytical tools. For botanical drug products (BDPs), many studies have applied NIR spectroscopy for rapid analysis of botanical raw materials, extracts and formulations. However, the real-time process analysis reported for BDPs was still mainly conducted on lab- or pilot- scale equipment, where some essential conditions of the materials and process parameters can be easily controlled for NIR spectral measurement. Due to the chemical and physical characteristics of the commercial production of BDPs, it is challenging to develop in-line NIR methods with enough robustness for industrial-scale application. This is the first reported case study of the development and validation of the in-line NIR method for the commercial production of BDPs, taking Compound Danshen Dripping Pill (CDDP) as an example. An in-line NIR method was developed for simultaneous measurement of the three critical quality attributes, i.e. the relative density, the moisture content and the content of 3,4-dihydroxyphenyl lactic acid (danshensu, a key active compound), during the extract concentration process. The NIR spectra and sample collection lasted for three years (67 production batches) to cover the variability of raw materials and process conditions. NIR calibration models were established respectively, with determination coefficients (r2) of 0.9905, root mean square errors of prediction (RMSEP) of 0.004 for the relative density in the range of 1.042-1.184; r2 of 0.9870, RMSEP of 1.1% for the moisture content in the range of 50.8-83.0%; r2 of 0.9870, RMSEP of 0.461 mg/g for danshensu in the range of 2.563-8.869 mg/g. Then, all the method validation parameters (accuracy, precision, range, specificity, linearity, robustness, detection and quantitation limits) were discussed according to the characteristics of the commercial production of BDPs. The NIR method development and validation strategy proposed may also be applied in the future for the commercial production of other BDPs.

Effects of clay mineral supplementation on particle-associated and epimural microbiota, and gene expression in the rumen of cows fed high-concentrate diet.

The increased concentrate amounts in cow diets may initiate changes in both particle-associated (PaM) and epimural microbiota (EpM) with the potential for promoting the establishment of pathogens. Clay minerals have shown promising potentials in binding harmful microorganisms and metabolites due to their high adsorption capacity. This study evaluated the effects of a clay-mineral based product (CM) on PaM, EpM, fermentation parameters, and epithelial gene expression in cows fed a high-concentrate diet. Eight rumen-cannulated non-lactating Holstein cows received a concentrate mix supplemented with CM or not (CON) in a change-over design with an initial 100% roughage diet phase (RD, 1 week), followed by intermittent 65%-high-concentrate-diet phases (HC1, HC2; 1 and 2 week duration, respectively), interrupted by 1 week roughage only. Rumen samples for short-chain fatty acids, ammonia, and lactate quantification, as well as PaM, and epithelial biopsies for EpM examination and epithelial gene expression were collected via the cannula once during each feeding phase. Phylogenetic distance analysis of Illumina MiSeq sequencing of the 16S rRNA gene region V345 showed a clear clustering of RD microbiota compared to HC in PaM, showing the impact of the high-concentrate diet on the bacterial community. In the EpM this effect was less pronounced, due to higher variability in RD. In the PaM, a decrease (P < 0.01) of community diversity occurred with the onset of HC feeding, while in the EpM there was an increase in diversity (P < 0.05). In the PaM, CM increased the relative abundance of genus Butyrivibrio (P < 0.01), a commensal bacterium of the rumen, which was, with 6.4%, the second most abundant genus. There, the CM supplementation decreased the genera Lactobacillus, Fusobacterium, and Treponema (P = 0.05), which are potentially either lactate producing or opportunistic pathogens. In the EpM, CM decreased the relative abundance of Succiniclasticum genus (P < 0.01), a possible endotoxin producer, and increased bacteria that are associated with a normobiotic rumen, such as Campylobacter (P = 0.06). Barrier function genes were upregulated in HC2 and nutrient transport genes downregulated in HC1 (P < 0.05); however, there was little effect on pro-inflammatory genes at the epithelium. The CM showed a significant decreasing effect on the cellular metabolism genes HMGCS1 (P = 0.04). Our results suggest that CM supplementation can increase the relative abundance of commensal microbiota and decrease bacteria that could negatively impact the rumen milieu and health during high-concentrate feeding.

Salvia clandestina L.: unexploited source of danshensu.

We report the characterisation of Salvia clandestina L. shoots and roots aqueous extract using HPLC-ESI/MS-TOF. Among the 29 compounds detected, we observed the presence of danshensu (3-(3,4-dihydroxy-phenyl) 2-hydroxy-propinic acid), a powerful antioxidant and a cardio-protective agent. The danshensu content found in the shoots of S. clandestina was considerable (4.96 mg g-1 DW) if compared with previous studies on S. miltiorrhiza Bunge, the main source of this compound. We also determined the total phenolic concentration and we evaluated their antioxidant activity by ABTS, FRAP and Superoxide anion scavenging methods. All the three assays confirmed a greater antioxidant activity for the shoots in comparison to roots. S. clandestina shoots may represent a valuable and natural unexploited source of danshensu and other phenolic compounds, so that it may be useful for future applications in functional foods and pharmaceutical industries.

Kinetics of ventilation-induced changes in diaphragmatic metabolism by bilateral phrenic pacing in a piglet model.

Perioperative necessity of deep sedation is inevitably associated with diaphragmatic inactivation. This study investigated 1) the feasibility of a new phrenic nerve stimulation method allowing early diaphragmatic activation even in deep sedation and, 2) metabolic changes within the diaphragm during mechanical ventilation compared to artificial activity. 12 piglets were separated into 2 groups. One group was mechanically ventilated for 12 hrs (CMV) and in the second group both phrenic nerves were stimulated via pacer wires inserted near the phrenic nerves to mimic spontaneous breathing (STIM). Lactate, pyruvate and glucose levels were measured continuously using microdialysis. Oxygen delivery and blood gases were measured during both conditions. Diaphragmatic stimulation generated sufficient tidal volumes in all STIM animals. Diaphragm lactate release increased in CMV transiently whereas in STIM lactate dropped during this same time point (2.6 vs. 0.9 mmol L-1 after 5:20 hrs; p < 0.001). CMV increased diaphragmatic pyruvate (40 vs. 146 µmol L-1 after 5:20 hrs between CMV and STIM; p < 0.0001), but not the lactate/pyruvate ratio. Diaphragmatic stimulation via regular electrodes is feasible to generate sufficient ventilation, even in deep sedation. Mechanical ventilation alters the metabolic state of the diaphragm, which might be one pathophysiologic origin of ventilator-induced diaphragmatic dysfunction. Occurrence of hypoxia was unlikely.

Qualitative and quantitative analyses of Compound Danshen extract based on 1H NMR method and its application for quality control.

In this study, a new approach using 1H NMR spectroscopy combined with chemometrics method was developed for qualitative and quantitative analyses of extracts of Compound Danshen Dripping Pills (CDDP). For the qualitative analysis, some metabolites presented in Compound Danshen extract (CDE, extraction intermediate of CDDP) were detected, including phenolic acids, saponins, saccharides, organic acids and amino acids, by the proposed 1H NMR method, and metabolites profiles were further analyzed by selected chemometrics algorithms to define the threshold values for product quality evaluation. Moreover, three main phenolic acids (danshensu, salvianolic acid B, and procatechuic aldehyde) in CDE were determined simultaneously, and method validation in terms of linearity, precision, repeatability, accuracy, and stability of the dissolved target compounds in solution was performed. The average recoveries varied between 84.20% and 110.75% while the RSDs were below 6.34% for the three phenolic acids. This 1H NMR method offers an integral view of the extract composition, allows the qualitative and quantitative analysis of CDDP, and has the potential to be a supplementary tool to UPLC/HPLC for quality assessment of Chinese herbal medicines.

Valores de lactato sérico e sua correlação com parâmetros clínicos de cães saudáveis, mensurados durante atendimento ambulatorial veterinário / Serum lactate values and its correlation with clinical parameters evaluated at veterinary ambulatorial exam

A mensuração do lactato sérico é utilizada na rotina médica como marcador prognóstico de pacientes em estado de emergência. Sua interpretação não deve ser feita de forma isolada, mas conjunta aos demais parâmetros clínicos, pois seus valores podem sofrer interferência do estresse metabólico ou ambiental, contenção física e/ ou manipulação dos pacientes. Assim, buscou-se mensurar os valores do lactato sérico e parâmetros clínicos de cães saudáveis, bem como as suas correlações, durante o atendimento clínico ambulatorial veterinário. Para isso, foram avaliados 80 cães, machos ou fêmeas, com idade de um a oito anos, atendidos para revacinação anual polivalente. Foram considerados cães saudáveis os que não apresentaram intercorrências clínicas nos últimos 60 dias e alterações nos exames físicos e nos valores de hemograma e glicemia sérica. Foram mensurados inicialmente o peso corporal, a frequência cardíaca (FC) e respiratória (FR), tempo de preenchimento capilar (TPC), coloração de mucosas, temperatura retal (TR), periférica (TP) e a diferença entre TR e a TP, o Delta T°C. Por último, realizaram-se os exames de hemograma e glicemia sérica, juntamente com a mensuração do lactato sérico, utilizando para isso um lactímetro portátil, por meio da amostra sanguínea obtida da veia cefálica. Além disso, havendo a correlação dos valores do lactato séricos com o peso corporal os cães foram divididos conforme o cálculo do 33° e 66° percentil. Os cães avaliados evidenciaram valores médios de 18,3±12,1 kg de peso corporal e 3,0±1,9 anos de idade; FC de 126,6±29,1bpm, FR de 66±24mpm, TR de 38,9±0,4°C, TP de 31,5±1,0°C, Delta TºC de 7,3±1,0°C e lactato sérico de 3,2±0,4mmol/L; com este último, evidenciando intervalo de confiança a 95% de 3,1-3,3mmol/L e correlação significativa (p<0,05) dos seus valores com o peso corporal (r=0,6) e a frequência cardíaca (r=0,4). Os valores do lactato sérico obtidos foram comparados entre os grupos de cães conforme o peso corporal, evidenciando diferenças significativas (p<0,05) entre eles. Dessa forma, pode se concluir que os valores do lactato sérico em cães hígidos sob atendimento ambulatorial é de 3,2mmol/L, com o intervalo de confiança de 3,1-3,3mmol/L, ressaltando a influência que a FC e o peso corporal podem exercer nos seus valores.(AU)

The measurement of serum lactate is used in the medical routine as a prognosis marker of emergency patients. Its interpretation should not be done disconnectedly from the other clinical parameters once metabolical or environmental stress as well as restraint and/or manipulation of patients can interfere. Thus we tried to measure the levels of serum lactate and clinical parameters of healthy dogs, as their correlation during veterinarian outpatient clinical care. For that we evaluated 80 dogs, males and females, with age ranging from one to eight years, met for polyvalent annual revaccination. We considered to be healthy those dogs that had no clinical events in the last 60 days or alteration in physical exams, blood exam values and serum glycemia. We initially measured body weight, heart rate (HR) and respiratory (RR), capillary refill time, mucosa's coloring, rectal temperature (RT), peripheral temperature (PT) and the difference between RT and PT, Delta T°C. Finally we did the blood exam and the serum glycemia, as well as the serum lactate measurement. For that we used a portable lactimeter, using the blood sample taken from the cephalic vein. Furthermore, when there was correlation between the serum lactate values and the body weight, we divided the dogs according to the calculation of 33 and 66 percentile. Evaluated dogs showed average values of 18.3±12.1 kg of body weight and 3.0±1.9 of age; with HR of 126.6±29.1bpm, RR of 66±24mpm, RT of 38.9±0.4°C, PT of 31.5±1,0°C, Delta T°C of 7.3±1.0°C and serum lactate of 3.2±0.4mmol/L; with the latter showing range of 3.1-3.3mmol/L with 95% of reliability and significant correlation (p<0.05) between its values and the body weight (r=0.6) and the heart rate (r=0.4). The serum lactate values obtained were compared between the dogs' groups according to their body weight, showing distinguished differences between them. Thereby we concluded that the serum lactate values in dogs under outpatient care is 3.2mmol/L, with a trust gap of 3.1-3.3mmol/L, highlighting the influence that HR and body weight can have on its values.(AU)

Combined chromatography and mass spectrometry for the molecular characterization of food emulsifiers.

Food emulsifiers are widely used to stabilise water-fat emulsions such as mayonnaise and dressings. They are prepared by oligomerisation of a poly-alcohol (as e.g. glycerol or citric acid) followed by a reaction with fatty acids. In order to gain insight in the chemical composition of different emulsifiers, a range of chromatographic methods including gas chromatography, size exclusion chromatography, normal phase- and reversed phase liquid chromatography either or not in combination with mass spectrometry was deployed. The different methods turned out to be highly complementary. By combining the information from different methods the polar head group and the fatty acid part of the emulsifier can be characterised in detail. Mass spectrometry is indispensable for establishing the number of polar molecules in the head group as well as for establishing the correct combinations of fatty acids in one molecule. Ten commercial emulsifiers were described at the level of number and type of polar groups and fatty acids present.

Fingerprint analysis, multi-component quantitation, and antioxidant activity for the quality evaluation of Salvia miltiorrhiza var. alba by high-performance liquid chromatography and chemometrics.

Salvia miltiorrhiza Bge. var. alba C.Y. Wu and H.W. Li has wide prospects in clinical practice. A useful comprehensive method was developed for the quality evaluation of S. miltiorrhiza var. alba by three quantitative parameters: high-performance liquid chromatography fingerprint, ten-component contents, and antioxidant activity. The established method was validated for linearity, precision, repeatability, stability, and recovery. Principal components analysis and hierarchical clustering analysis were both used to evaluate the quality of the samples from different origins. The results showed that there were category discrepancies in quality of S. miltiorrhiza var. alba samples according to the three quantitative parameters. Multivariate linear regression was adopted to explore the relationship between components and antioxidant activity. Three constituents, namely, danshensu, rosmarinic acid, and salvianolic acid B, significantly correlated with antioxidant activity, and were successfully elucidated by the optimized multivariate linear regression model. The combined use of high-performance liquid chromatography fingerprint analysis, simultaneous multicomponent quantitative analysis, and antioxidant activity for the quality evaluation of S. miltiorrhiza var. alba is a reliable, comprehensive, and promising approach, which might provide a valuable reference for other herbal products in general to improve their quality control.

Comparative pharmacokinetic and tissue distribution profiles of four major bioactive components in normal and hepatic fibrosis rats after oral administration of Fuzheng Huayu recipe.

Fuzheng Huayu recipe (FZHY) is a herbal product for the treatment of liver fibrosis approved by the Chinese State Food and Drug Administration (SFDA), but its pharmacokinetics and tissue distribution had not been investigated. In this study, the liver fibrotic model was induced with intraperitoneal injection of dimethylnitrosamine (DMN), and FZHY was given orally to the model and normal rats. The plasma pharmacokinetics and tissue distribution profiles of four major bioactive components from FZHY were analyzed in the normal and fibrotic rat groups using an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Results revealed that the bioavailabilities of danshensu (DSS), salvianolic acid B (SAB) and rosmarinic acid (ROS) in liver fibrotic rats increased 1.49, 3.31 and 2.37-fold, respectively, compared to normal rats. There was no obvious difference in the pharmacokinetics of amygdalin (AMY) between the normal and fibrotic rats. The tissue distribution of DSS, SAB, and AMY trended to be mostly in the kidney and lung. The distribution of DSS, SAB, and AMY in liver tissue of the model rats was significantly decreased compared to the normal rats. Significant differences in the pharmacokinetics and tissue distribution profiles of DSS, ROS, SAB and AMY were observed in rats with hepatic fibrosis after oral administration of FZHY. These results provide a meaningful basis for developing a clinical dosage regimen in the treatment of hepatic fibrosis by FZHY.

Growth of Desulfovibrio vulgaris when respiring U(VI) and characterization of biogenic uraninite.

The capacity of Desulfovibrio vulgaris to reduce U(VI) was studied previously with nongrowth conditions involving a high biomass concentration; thus, bacterial growth through respiration of U(VI) was not proven. In this study, we conducted a series of batch tests on U(VI) reduction by D. vulgaris at a low initial biomass (10 to 20 mg/L of protein) that could reveal biomass growth. D. vulgaris grew with U(VI) respiration alone, as well as with simultaneous sulfate reduction. Patterns of growth kinetics and solids production were affected by sulfate and Fe(2+). Biogenic sulfide nonenzymatically reduced 76% of the U(VI) and greatly enhanced the overall reduction rate in the absence of Fe(2+) but was rapidly scavenged by Fe(2+) to form FeS in the presence of Fe(2+). Biogenic U solids were uraninite (UO2) nanocrystallites associated with 20 mg/g biomass as protein. The crystallite thickness of UO2 was 4 to 5 nm without Fe(2+) but was <1.4 nm in the presence of Fe(2+), indicating poor crystallization inhibited by adsorbed Fe(2+) and other amorphous Fe solids, such as FeS or FeCO3. This work fills critical gaps in understanding the metabolic utilization of U by microorganisms and formation of UO2 solids in bioremediation sites.

Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae.

Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC(50) of 9.07 µM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations.

Conversion of salvianolic acid B into salvianolic acid A in tissues of Radix Salviae Miltiorrhizae using high temperature, high pressure and high humidity.

Salvianolic acid A (Sal A), an important constituent of Radix Salviae Miltiorrhizae (RSM), is effective for the treatment of myocardial infarction (MI) and coronary heart disease due to its potential in the improvement of acute myocardial ischemia. However, its content is very low in RSM. So it is obvious to find a rich source of Sal A or to improve its content by conversion of other related components into Sal A modifying reaction conditions. In this research we focused on the conversion of Sal B into Sal A in aqueous solutions of RSM by using different reaction conditions including pH, temperature, pressure and humidity. During the reactions, the contents of Sal A, Sal B and danshensu in the RSM were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS). The results indicated that the conversion of Sal B into Sal A in RSM tissues under the conditions of a high temperature, high pressure and high humidity was efficient and thereby, was readily utilized to prepare rich Sal A materials in practice.

A simple and reliable method for distinguishing danshen in salvia: simultaneous quantification of six active compositions by HPLC.

A simple and reliable method for distinguishing Danshen is important to evaluate the quality and clinical efficiency of these species. An HPLC method was developed for the determination of protocatechuic aldehyde, salvianolic acid A, salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA in 23 samples of Salvia. The analytes were separated on an Agilent XDB C18 reversed-phase column coupled with a Phenomenex C18 guard column using a gradient elution of acetonitile-0.1% aqueous phosphoric acid as the mobile phase at a flow rate 0.8 mL/min and UV detection at 280 nm. The method allowing the simultaneous quantification of six major active compositions was optimized and validated for linearity, precision, accuracy and limits of detection (LOD) and quantification. The LOD ranged from 0.019 to 0.850 µg/mL (R(2) ≥ 0.9998). Accuracy, precision and reproducibility were all within the required limits. The average recovery between 96.49 and 102.16% and the relative standard deviations were <3.01%. Based on the six compositions content and clustering result, this research results suggest that these six major active compositions could be distinguishing markers for Danshen and non-Danshen.

[Simultaneous determination of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in water extract of mixed salviae miltiorrhizae radix et rhizoma and carthami flos by HPLC].

OBJECTIVE: To develop an HPLC method to determine the contents of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in the water extract of mixed Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos simultaneously. METHOD: The separation were carried out at 30 degrees C on a ZORBAX Eclipse Plus C18 column (4.6 mm x 100 mm, 1.8 microm) with formic acid-500 mmol x L(-1) ammonium formate-water solution (0.5:10:90) as mobile phase A and acetonitrile-formic acid solution (100: 0.5) as mobile phase B in gradient mode at a flow rate of 0.5 mL x min(-1). Detection wavelengths were 280 nm for danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, and 380 nm for hydroxysafflor yellow A. RESULT: The 5 components were separated well with a good linearity (R2 > 0.999 3) in the range of the test concentration. The average recoveries of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, and salvianolic acid B were 99.1%, 102%, 102%, 98.5% and 101%, respectively. CONCLUSION: This method is simple, accurate, and repeatable.