Liquid Chromatography-mass Spectrophotometry Research Articles

Enhanced rapamycin production through kinetic and purification studies by mutant strain of Streptomyces hygroscopicus NTG-30-27

Research work was implemented to describe the kinetics of cell growth, substrate utilization and product formation using a mutant strain of Streptomyces hygroscopicus NTG-30-27 in a 3-L bioreactor under optimized condition. Various substrate inhibition mathematical models were applied and it was found that the cell growth and substrate utilization kinetic data fitted well to those models. Andrew’s kinetic model was fitted very well (R2 = 0.998) with our experimental data among different models tested for analysis whereas Luedeking–Piret model suggested that our product is mixed growth associated. The values for maximum specific growth rate (µmax), doubling time (td), saturation constant (KS), inhibition constant (KI) and yield coefficient (YX/S) were found to be 0.03985 h−1, 17.16 h, 2.076 g/l, 0.009 g/l and 0.290 g g−1. Final rapamycin yield with mutant strain was found to be 531.4 mg/l on its 5th day of fermentation which is 6.7-fold higher than the wild type (79.31 mg/l). The effect of aeration on rapamycin production was studied by batch fermentation in a stirred tank reactor (STR) using S. hygroscopicus NTG-30-27. Dynamic behaviour and aeration efficiency of the reactor, as well as rheology pattern of the fermentation broth, were determined by calculating volumetric mass transfer coefficient (KLa) of the process using “Dynamic gassing out method”. KLa was found to be 54.53 h−1 which is quite significant for rapamycin production. Further purification and structural analysis of the extracted sample were carried out by liquid chromatography–mass spectrophotometry (LC–MS) technique in positive ionization mode and molecular mass was found to be 936 D. Finally, 90.62% purified rapamycin was obtained from the study.

Early Neurodevelopmental Exposure to Low Lead Levels Induces Fronto-executive Dysfunctions That Are Recovered by Taurine Co-treatment in the Rat Attention Set-Shift Test: Implications for Taurine as a Psychopharmacotherapy Against Neurotoxicants.

Lead (Pb2+) is a developmental neurotoxicant that causes lifelong cognitive dysfunctions. In particular, Pb2+-induced frontoexecutive dysfunctions emerge later in life when the cortex is fully myelinated, thereby permitting the ability to assess the extent to which Pb2+ has developmentally impacted higher order cognitive and behavioral systems. The present study evaluated the effects of developmental Pb2+-exposure (150ppm lead acetate in the drinking water) in Long Evans Hooded rats through the Attention Set-Shift Test (ASST) between postnatal days (PND) 60-90. Treatment groups were comprised of Control (0ppm), Perinatal (150ppm), and Perinatal+Taurine (150ppm+0.05% Taurine in the drinking water) rats (N=36; n=6 per treatment group for each sex). Frontoexecutive functions were evaluated based on trials-to-criterion (TTC) and errors-to-criterion (ETC) measures for simple and complex discriminations (SD & CD), intradimensional and extradimensional shifts (ID & ED), as well as reversals (Rev) of the CD, I-, and ED stages, respectively. Post-testing, the prelimbic (PrL), infralimbic (IL), orbital ventral frontal (OV), orbital ventro-lateral (OVL), and hippocampal (HP) brain regions were extracted and processed through Liquid Chromatography/Mass Spectrophotometry (LC/MS) for determining the GABA and Taurine ratios relative to Glutamate, Dopamine, Norepinephrine, Epinephrine, and Serotonin. The ASST data revealed that Perinatal rats are negatively impacted by developmental Pb2+-exposures evidenced by increased TTC and ETC to learn the SD, ID, and ID-Rev with unique sex-based differences in frontoexecutive dysfunctions. Moreover, Perinatal+Taurine co-treated rats exhibited a recovery of the frontoexecutive dysfunctions observed in Perinatal rats to levels equivalent to Control rats across both sexes. The LC/MS data revealed altered brain sub-region specific patterns across the PrL, IL, OV, OVL, and HP in response to developmental Pb2+-exposure that produced an altered neurochemical signaling profile in a sex-dependent manner, which may underlie the observed frontoexecutive dysfunctions, cognitive inflexibility, and associated motivation deficits. When taurine co-treatment was administered concurrently for the duration of developmental Pb2+-exposure, the observed frontoexecutive dysfunctions were significantly reduced in both ASST task performance and neurochemical ratios that were comparable to Control levels for both sexes. Altogether, the data suggest that taurine co-treatment may facilitate neuroprotection, mitigate neurotransmitter excitability balancing, and perhaps ameliorate against neurotoxicant exposures in early development as a potential psychopharmacotherapy.

Technological and microbiological factors affecting the polyphenolic profile of Montenegrin red wines

The main objective of this study was to determine the impact of three commercial yeasts (BDX, ICVD21 and BM4x4) and lactic acid bacteria (VP41 and ALPHA) on the phenolic profile of Montenegrin red wines. Wines made of two autochthonous grape varieties (Vranac and Kratosija) and international grape variety (Cabernet Sauvignon) were examined. The research was performed during the 2012 and 2013 vintage and twenty-one phenolic compounds, including stilbenes, hydroxycinnamic and hydroxybenzoic acids, and flavonols have been identified in the wine after alcoholic and malolactic fermentation, using the liquid chromatography-mass spectrophotometry method. The influence of different commercial yeasts on all analysed phenolic compounds was statistically significant (p ??0.05) for all examined wines, and there were also statistically significant differences between varietal wines. After alcoholic and malolactic fermentation, the highest average content of p-coumaric acid was determined in Vranac wines. Additional analysis of stilbenes after alcoholic fermentation of varietal wines of the 2013 vintage revealed that the total content of stilbenes was the highest in Vranac wines, where a significant increase of their concentration was observed in wines inoculated with commercial yeast. A similar trend was observed in all studied wine varieties.

Plasminogen activator inhibitor-1 and tenascin-C secreted by equine mesenchymal stromal cells stimulate dermal fibroblast migration in vitro and contribute to wound healing in vivo

BackgroundImpaired cutaneous wound healing is common in humans, and treatments are often ineffective. Based on the significant emotional and economic burden of impaired wound healing, innovative therapies are needed. The potential of mesenchymal stromal cell (MSC)–secreted factors to treat cutaneous wounds is an active area of research that is in need of refinement before effective clinical trials can be initiated. The aims of the present study were to (i) study which MSC-secreted factors stimulate dermal fibroblast (DF) migration in vitro and (ii) evaluate the potential of these factors to promote wound healing in vivo. MethodsTo this end, MSCs were isolated from the peripheral blood of healthy horses, a physiologically relevant large animal model appropriate for translational wound-healing studies. Conditioned medium (CM) from cultured equine MSCs was analyzed using liquid chromatography-mass spectrophotometry (LC-MS/MS) to identify secreted proteins of interest. Double-stranded RNA-mediated interference (RNAi) was used to silence the genes encoding selected proteins, and the effects of CM from these transfected MSCs on migration of cultured equine DF cells in vitro and full-thickness wounds in mice were evaluated. ResultsWe found that MSC-derived plasminogen activator inhibitor-1 (PAI-1) and tenascin-C significantly increased DF migration in vitro and improved wound healing in vivo by decreasing time to wound closure. DiscussionThese results suggest that in a complex wound environment, MSC-secreted factors PAI-1 and tenascin-C contribute to the positive effect of therapeutically applied MSC CM on wound healing.

Are Processed Bilberry Products a Good Source of Phenolics?

Although we confirmed the common belief among consumers that the consumption of fresh berries is more beneficial than consuming processed products, this study showed that various bilberry-derived products can also be valuable source of phenolic compounds all year round.

Determination of Dipeptidyl Peptidase-4 Inhibitors by Spectrophotometric and Chromatographic Methods

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of oral anti-diabetic agents used in the treatment of type 2 diabetes mellitus and work to enhance the effect of incretin hormones. There has been significant research on wide range of analytical techniques that could be useful in the estimation of DPP-4 inhibitors in formulations and biological matrices. Analytical methods such as ultraviolet (UV) spectrophotometry, mass spectroscopy, NMR spectroscopy, HPLC, high pressure thin layer chromatography, ultra performance liquid chromatography, liquid chromatography‒mass spectrophotometry and capillary zone electrophoresis have been reported for the estimation of DPP-4 inhibitors in single and/or in combination with other drugs. This comprehensive review covers most of the spectrophotometric and chromatographic methods described for the determination of sitagliptin, alogliptin, vildagliptin, linagliptin, saxagliptin and teneligliptin in pure forms, in different pharmaceutical dosage forms and biological fluids. From the review it can be deduced that a large number of chromatographic methods have been developed, and HPLC-UV methods have been commonly used in the detection and evaluation of DPP-4 inhibitors.

Green synthesis of zinc oxide nanoparticles using aqueous extract of Garcinia mangostana fruit pericarp and their photocatalytic activity

We reported a facile, green and eco-friendly approach for the synthesis of zinc oxide nanoparticles (ZnO NPs) using aqueous extract of Garcinia mangostana (G. mangostana) fruit pericarp as reducing agent as well as capping agent. Biosynthesized ZnO NPs were characterized by various analytical tools using X-ray diffraction, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy and UV–Vis spectroscopy. The results showed that ZnO NPs synthesized by aqueous extract of G. mangostana fruit pericarp with high purity, mostly spherical in shape with an average size of 21 nm. The photocatalytic activity of biosynthesized ZnO NPs was evaluated by carrying out the degradation of malachite green dye under solar irradiation. The extent of MG dye degradation was monitored spectrophotometrically by measuring absorbance at its characteristics \(\lambda _{\max }\) value of 615 nm. Degradation products were detected using liquid chromatography–mass spectrophotometry technique. The biosynthesized ZnO NPs showed an excellent photocatalyst performance due to the small size and high purity.

Chlorpromazine Impacts on the Length and Width ofSarcophaga haemorrhoidalis(Diptera: Sarcophagidae) Larvae: Potential Forensic Implications

Abstract The effect of chlorpromazine (CPZ) on the larval length and width of Sarcophaga haemorrhoidalis (Fallen) (Diptera: Sarcophagidae) was investigated under laboratory conditions for possible implications for forensic investigations. Fresh beef was treated with one of four concentrations of CPZ (0, 0.5, 1.0, and 1.5 μg per g of beef) to simulate postmortem concentrations in CPZ-dosed corpses. Ten replicates of 50 S. haemorrhoidalis eggs each were placed on the treated beef and maintained at 27°C. The length and width of 2 d old and 4 d old larvae were measured for all treatments and replicates, and the presence of CPZ in the treated meats was qualitatively verified using liquid chromatography–mass spectrophotometry coupled with electrospray ionization. Pearson's product-moment correlation coefficient revealed significant correlations of CPZ concentration with both larval length and width at the two larval ages (P < 0.001). However, the mean length and width of larvae fed on CPZ-treated beef did not d...

Solid state fermentation of fig ( Ficus carica L.) by-products using fungi to obtain phenolic compounds with antioxidant activity and qualitative evaluation of phenolics obtained

Abstract An optimized method of solid-state fermentation (SSF) is reported for the valorization of fig (Ficus carica L.) by-products. Four fungal strains were screened for their capacity to enhance the release of total phenolic compounds (TPCs). Temperature, pH, moisture, and mineral composition were evaluated and optimized using a Box–Hunter–Hunter design and then central composite design was performed. The optimized SSF conditions were as follows: A. niger HT4 for 36 h at 40 °C; moisture, 60%; pH: 5.0; mineral composition (g/L): NaNO3: 0.57; KH2PO4: 3.04; MgSO4·7H2O: 1.52; and KCl: 5.37. Under the optimal SSF conditions, the experimental values (10.19 ± 0.04 mg of GAE/g of dm) matched with those predicted (10.37 mg of GAE/g of dm) within a 95% confidence level. A total of eight phenolic compounds were identified by High Performance Liquid Chromatography-Mass Spectrophotometry (HPCL-MS). The optimized SSF bioprocess increased the TPC release and antioxidant activity values up to 5.48 and 98.54 times, respectively, showing its feasibility as a reliable alternative for the use of fig by-products.

Nitazoxanide: A Review of Analytical Methods.

Nitazoxanide is an antiprotozoal and anthelmintic agent, which is mostly used in the treatment and prevention of wide variety of protozoa, helminthes and gram negative organisms. This article studies published analytical techniques that are reported so far for the determination of nitazoxanide in bulk, pharmaceutical formulation and biological samples. They include various techniques like spectrophotometry, electrochemical methods, capillary electrophoresis, high performance liquid chromatography, high performance thin layer chromatography, and liquid chromatography-mass spectrophotometry.

Discovery of Antimalarial Drugs from Streptomycetes Metabolites Using a Metabolomic Approach.

Natural products continue to play an important role as a source of biologically active substances for the development of new drug. Streptomyces, Gram-positive bacteria which are widely distributed in nature, are one of the most popular sources of natural antibiotics. Recently, by using a bioassay-guided fractionation, an antimalarial compound, Gancidin-W, has been discovered from these bacteria. However, this classical method in identifying potentially novel bioactive compounds from the natural products requires considerable effort and is a time-consuming process. Metabolomics is an emerging “omics” technology in systems biology study which integrated in process of discovering drug from natural products. Metabolomics approach in finding novel therapeutics agent for malaria offers dereplication step in screening phase to shorten the process. The highly sensitive instruments, such as Liquid Chromatography-Mass Spectrophotometry (LC-MS), Gas Chromatography-Mass Spectrophotometry (GC-MS), and Nuclear Magnetic Resonance (1H-NMR) spectroscopy, provide a wide range of information in the identification of potentially bioactive compounds. The current paper reviews concepts of metabolomics and its application in drug discovery of malaria treatment as well as assessing the antimalarial activity from natural products. Metabolomics approach in malaria drug discovery is still new and needs to be initiated, especially for drug research in Malaysia.

Molecular and Proteomic Analysis of Levofloxacin and Metronidazole Resistant Helicobacter pylori.

Antibiotic resistance in bacteria incurs fitness cost, but compensatory mechanisms may ameliorate the cost and sustain the resistance even under antibiotics-free conditions. The aim of this study was to determine compensatory mechanisms of antibiotic resistance in H. pylori. Five strains of levofloxacin-sensitive H. pylori were induced in vitro to develop resistance. In addition, four pairs of metronidazole-sensitive and -resistant H. pylori strains were isolated from patients carrying dual H. pylori populations that consist of both sensitive and resistant phenotypes. Growth rate, virulence and biofilm-forming ability of the sensitive and resistant strains were compared to determine effects of compensatory response. Proteome profiles of paired sensitive and resistant strains were analyzed by liquid chromatography/mass spectrophotometry (LC/MS). Although there were no significant differences in growth rate between sensitive and resistant pairs, bacterial virulence (in terms of abilities to induce apoptosis and form biofilm) differs from pair to pair. These findings demonstrate the complex and strain-specific phenotypic changes in compensation for antibiotics resistance. Compensation for in vitro induced levofloxacin resistance involving mutations of gyrA and gyrB was functionally random. Furthermore, higher protein translation and non-functional protein degradation capabilities in naturally-occuring dual population metronidazole sensitive-resistant strains may be a possible alternative mechanism underlying resistance to metronidazole without mutations in rdxA and frxA. This may explain the lack of mutations in target genes in ~10% of metronidazole resistant strains.

High Burden of Vitamin D Deficiency in Pregnancy and its Association with Low Birth Weight in a Prospective Cohort in South India

ObjectiveThis study was conducted to determine the prevalence of vitamin D deficiency in a prospective cohort of pregnant women and its association with birth outcomes in Bangalore, India.MethodsPregnant women (n=419) were recruited in their first trimester to a perinatal cohort study between 2008 and 2014 and followed up until delivery. Vitamin D status was assessed through determination of serum 25‐hydroxyvitamin D (25(OH)D) using tandem liquid chromatography mass spectrophotometry (LCMS/MS, CV 1.6%) in maternal samples at enrollment and in cord blood at delivery. Binomial and linear regression models were used to evaluate the associations between maternal and infant vitamin D status and with birth outcomes, including infant birth weight and preterm birth.ResultsThe mean serum 25‐hydroxyvitamin D concentration in both maternal and cord blood was 37 ± 19 nmol/L with nearly 82% of mothers having vitamin D deficiency (25(OH)D &lt;50 nmol/L). The mean birth weight of children born in this study was 2915 ± 431 grams with 13% of the children being classified as having low birth weight (&lt;2,500 grams). Children born to women with serum vitamin D concentrations in the lowest quintile had significantly lower birth weight, compared to children born to women with vitamin D concentrations in the highest four quintiles (mean difference: 148 grams; 95% Confidence Interval: 45, 252 grams; p‐value &lt;0.01).ConclusionsVitamin D deficiency is present in four out of five pregnant women in our cohort in an urban tropical setting and is associated with adverse birth outcomes such as lower birth weight. Studies with longer follow‐up are urgently needed to determine impact of poor vitamin D status on functional outcomes during childhood, and if vitamin D supplementation should be a part of standard antenatal care.Support or Funding InformationDivision of Nutritional Sciences, Cornell University

Direct conversion of theophylline to 3-methylxanthine by metabolically engineered E. coli.

BackgroundMethylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monomethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonasputida, ndmA, ndmB, and ndmD, provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3-methylxanthine by a metabolically engineered strain of E. coli.ResultsHere we report the construction of E. coli strains with ndmA and ndmD, capable of producing 3-methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3-methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3-methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-methylxanthine were also produced, attributed to slight activity of NdmA at the N3-position of theophylline. The 1- and 3-methylxanthine products were separated by preparative chromatography with less than 5 % loss during purification and were identical to commercially available standards. Purity of the isolated 3-methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chromatography-mass spectrophotometry or nuclear magnetic resonance.ConclusionsWe were able to biologically produce and separate 100 mg of highly pure 3-methylxanthine from theophylline (1,3-dimethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD. This microbial conversion represents a first step to develop a new biological platform for the production of methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0395-1) contains supplementary material, which is available to authorized users.

227. Cancer Cell Susceptibility To Treatment With a Retroviral Replicating Vector Encoding Cytosine Deaminase and 5-FC Is Likely To Be Common

We are currently investigating the clinical utility of a Retroviral Replicating Vector (RRV), Toca 511 (vocimagene amiretrorepvec) which encodes an optimized yeast cytosine deaminase (yCD2) transgene, in recurrent high grade glioma (NCT01470794, NCT01156584 & NCT01985256). Toca 511, which is based on amphotropic murine gamma retroviruses, selectively infects tumors without immediate cell killing. Expression of virally-delivered yCD2 converts orally administered 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in infected tumor cells. There are several variables that could affect how well this combination therapy works in different tumors. By integrating a combination of RNA-seq, Liquid Chromatography/Mass Spectrophotometry (LC/MS), and PCR analysis with biochemical and virological assays, key criteria have been identified that help predict how Tocagen's Toca 511 RRV gene therapy will perform in several different tumor types. Efficient production and secretion of 5-FU by Toca511 infected tumor cells are expected to kill other uninfected cells in the immediate tumor microenvironment (the so-called “bystander” effect) and may, thereby, also influence the extent of induction of anti-tumor immune responses in animal models and patients. A panel of 9 established human tumors derived from 3 different tissue types (brain, colon and breast), each with unique histology, growth characteristics and morphology, was used to evaluate parameters central to effective antitumor activity in different indications. Parameters measured included: the rate of cell division; the rate of infection; the levels of CD gene integration and expression; intracellular and extracellular concentrations of 5-FC, 5-FU and downstream metabolites; gene expression levels of viral susceptibility genes and genes for pyrimidine metabolism. The data showed that: 1) all the tumor lines tested infected quite readily; 2) the rate of infection (varied 7-fold) was not dependent on the rate of cell division; 3) the expression of CD mRNA and protein were highly correlative with conversion of 5-FC to 5-FU; 4) all tumors were readily able to import 5-FC; 5) while LC/MS analysis established all tumors were readily able to import 5-FC, RNA-seq analysis correlated intracellular 5-FC levels with expression of SLC29A1, which codes for the main 5-FC transporter. Further, in vitro data from infected cell extracts, showed that the production of 5-FU could be augmented by increasing the 5-FC concentrations up to 5mg/mL. This suggests that infected tumor cells in vivo can kill uninfected nearby tumor cells better with higher doses of 5-FC. In summary, these studies identified multiple factors that influence Toca 511 gene therapy in the 9 tumor cell lines in culture. Data from this initial integrative approach further supports the conclusion that Toca 511 therapy is likely to be effective in killing multipletumortypes.

Isoniazid: A Review of Analytical Methods

Isoniazid is an anti –tubercular drug, which is mostly used in the treatment and prevention of tuberculosis. It acts by inhibiting the synthesis of mycolic acid, required for mycobacterial cell wall synthesis in mycobacterium(M) tuberculosis. This article examines published analytical techniques that are reported so far for the determination of isoniazid in bulk, pharmaceutical formulation and biological samples. They include various techniques like spectrophotometry, electrochemical methods, capillary electrophoresis, high performance liquid chromatography, high performance thin layer chromatography, capillary gas chromatography and liquid chromatography-mass spectrophotometry.

Growth inhibitory response and ultrastructural modification of oral-associated candidal reference strains (ATCC) by Piper betle L. extract

Candida species have been associated with the emergence of strains resistant to selected antifungal agents. Plant products have been used traditionally as alternative medicine to ease mucosal fungal infections. This study aimed to investigate the effects of Piper betle extract on the growth profile and the ultrastructure of commonly isolated oral candidal cells. The major component of P. betle was identified using liquid chromatography-mass spectrophotometry (LC-MS/MS). Seven ATCC control strains of Candida species were cultured in yeast peptone dextrose broth under four different growth environments: (i) in the absence of P. betle extract; and in the presence of P. betle extract at respective concentrations of (ii) 1 mg⋅mL−1; (iii) 3 mg⋅mL−1; and (iv) 6 mg⋅mL−1. The growth inhibitory responses of the candidal cells were determined based on changes in the specific growth rates (µ). Scanning electron microscopy (SEM) was used to observe any ultrastructural alterations in the candida colonies. LC-MS/MS was performed to validate the presence of bioactive compounds in the extract. Following treatment, it was observed that the µ-values of the treated cells were significantly different than those of the untreated cells (P<0.05), indicating the fungistatic properties of the P. betle extract. The candidal population was also reduced from an average of 13.44×106 to 1.78×106 viable cell counts (CFU)⋅mL−1. SEM examination exhibited physical damage and considerable morphological alterations of the treated cells. The compound profile from LC-MS/MS indicated the presence of hydroxybenzoic acid, chavibetol and hydroxychavicol in P. betle extract. The effects of P. betle on candida cells could potentiate its antifungal activity.

RESIDUES DEPLETION STUDY AND WITHDRAWAL PERIOD DETERMINATION OF SULPHADIAZINE AND TRIMETHOPRIM PREMIX IN PIGS

The emergence and spread of antibiotic resistant hu man pathogens have been directly linked to the use of antibiotics in livestock production. The purpose of this study was to detect and quantify the concentr ation of the residues of Sulphadiazine (SDZ) and Trimethoprim (TMP) in edible tissues of pigs and to determine the withdrawal period after oral administration of OPTI PRIME ® premix 40%, containing 66.7 g TMP and 333.3 g SDZ per kg to healthy pigs. The depletion profile o f SDZ and TMP was studied in healthy pigs, after or al administration of 1.5 kg OPTIPRIME ® per ton of feeding stuff, for 5 consecutive days. A total of 22 pigs at age 65±2 days and from 27.1-33.0 kg were used. The experimental animals were divided into 4 groups (5 pigs per group), while 2 pigs acted as control animals. All medicated pigs were sacrificed 1, 4, 7 and 11 days after the last administration and muscle, fat, liver and kidney ti ssues were collected and analyzed using a validated liquid chromatography-mass spectrophotometry method. On the 1st day post medication (pm), SDZ was found in muscle and fat at higher concentrations than TMP, whereas higher concentrations of TMP were found in the liver, while both substances were found in high concentrations in kidney samples. On 4th day pm SDZ and TMP could not be quantified or detected in any tissue. On 7th day pm, both substances were found in quantifiable concentrations in 1 out of 4 kidney samples, while on 11th day pm, all observations were below the Limit of Quantification (LOQ) of the method. The results sho w that both substances deplete rapidly in all tissu es. A withdrawal period of 5 days is justified for the co mmercial product OPTIPRIME ® 40% premix in pigs.

Plasma methylglyoxal and glyoxal are elevated and related to early membrane alteration in young, complication-free patients with Type 1 diabetes

The reactive aldehydes methylglyoxal and glyoxal, arise from enzymatic and non-enzymatic degradation of glucose, lipid and protein catabolism, and lipid peroxidation. In Type 1 diabetes mellitus (T1DM) where hyperglycemia, oxidative stress, and lipid peroxidation are common, these aldehydes may be elevated. These aldehydes form advanced glycation end products (AGEs) with proteins that are implicated in diabetic complications. We measured plasma methylglyoxal and glyoxal in young, complication-free T1DM patients and assessed activity of the ubiquitous membrane enzyme, Na+/K+ ATPase. A total of 56 patients with TIDM (DM group), 6-22 years, and 18 non-diabetics (ND group), 6-21 years, were enrolled. Mean plasma A1C (%) was higher in the DM group (8.5+/-1.3) as compared to the ND group (5.0+/-0.3). Using a novel liquid chromatography-mass spectrophotometry method, we found that mean plasma methylglyoxal (nmol/l) and glyoxal levels (nmol/l), respectively, were higher in the DM group (841.7+/-237.7, 1051.8+/-515.2) versus the ND group (439.2+/-90.1, 328.2+/-207.5). Erythrocyte membrane Na+/K+ ATPase activity (nmol NADH oxidized/min/mg protein) was elevated in the DM group (4.47+/-0.98) compared to the ND group (2.16+/-0.59). A1C correlated with plasma methylglyoxal and glyoxal, and both aldehydes correlated with each other. A high correlation of A1C with Na+/K+ ATPase activity, and a regression analysis showing A1C as a good predictor of activity of this enzyme, point to a role for glucose in membrane alteration. In complication-free patients, increased plasma methylglyoxal, plasma glyoxal, and erythrocyte Na+/K+ ATPase activity may foretell future diabetic complications, and emphasize a need for aggressive management.

The value of plant collections in ethnopharmacology: a case study of an 85-year-old black cohosh ( Actaea racemosa L.) sample

Ethnopharmacological collections of plants used in traditional medical systems are a valuable but often underappreciated resource for scientific investigation. These collections contain many samples of plants currently employed in herbal and pharmaceutical medicine, and questions on stability and storage life can be examined using these historic collections as vouchers. A sample of black cohosh ( Actaea racemosa L.), collected in 1919 by the physician and plant explorer Henry Hurd Rusby, was recently identified in the collections of The New York Botanical Garden and analyzed for its triterpene glycosidic and phenolic constituents qualitatively and quantitatively by high-performance liquid chromatography–photodiode array detector (HPLC–PDA) and liquid chromatography–mass spectrophotometry (LC–MS). A comparison of the triterpene glycosidic and phenolic constituents of the 85-year-old plant sample with those of a modern collection of Actaea racemosa showed the similarity of the two samples, confirming the stability of the older sample, despite its curation over the years under a variety of conditions. Quantitative analyses indicated that both plant samples have similar amounts of the four major triterpene glycosides, but the total amount of the six major phenolic constituents measured in the 85-year-old plant material is lower than the amount measured in the modern plant material. Methanol extracts of the two plant materials were tested for their antioxidant activity, and both extracts showed similar antioxidant activity.