Amount Of Internal Standard Research Articles

Isotope dilution with isotopically labeled biomass: An effective alternative for quantitative metabolomics

BackgroundState-of-the-art quantitative metabolomics relies on isotope dilution using internal standards (IS) derived from fully 13C labeled biomass. By spiking samples and external standards with known amounts of IS, the spike characterization demands are kept to a minimum. In fact, it is sufficient to experimentally assess the isotopic enrichment of the IS. This study develops the yeast derived IS toolbox further, (1) by characterizing the concentration levels of hydrophilic metabolites in a yeast fermentation batch and (2) by exploring the analytical figures of merit of one-point IS versus multipoint external calibration using IS, the established gold-standard for quantitative metabolomics. ResultsIndependent reverse isotope dilution experiments using different chromatographic methods over a period of several months, delivered a list of 83 13C-labeled metabolites with fully characterized concentration and their uncertainty, covering 5 orders of magnitude, from the nanomolar to the low millimolar range. The 13C-labeled yeast-derived IS showed excellent intermediate stability with 92 % of molecules showing inter-method RSDs ≤30 % (75 % of molecules showed RSDs ≤15 %) over a timeframe of five months. One-point internal standardization with the characterized labeled biomass achieved figures of merit equivalent to multipoint calibrations for the majority of metabolites. SignificanceThe proposed calibration workflow rationalizes time and standard expenditure and is particularly beneficial for laboratories dealing with wide-target assays and small analysis batches. The present assessment serves as a seminal study for further developments of the concept towards absolute quantification from archive high-resolution MS data of U13C-biomass-spiked samples and the implementation of quick biomass recalibration with each experiment, promising seamless transition between internal standards derived from different fermentation batches.

A Rapid Method for the determination of Dehydrated alcohol (Ethanol) content in Clobetasol propionate foam by Non-polar Capillary Gas chromatography

Clobeatsol propionate foam is a topical class 1 corticosteroid used to treat itching and inflammatory arthritis on the skin occurred by allergic reactions, psoriasis and eczema. In the pharmaceutical aerosol products, Dehydrated alcohol (Ethanol) is the primary ingredient and its concentration level in the formulation composition plays a significant role in the regulation of aerosol rate, droplet shape and particle size. It can also act as solubilizer for active pharmaceutical ingredient, topical disinfectant and skin permeation enhancer. Hence accurate assay of Ethanol is a crucial quality control component. A simple and rapid gas chromatography method was developed to quantify Ethanol content in Clobetasol propionate foam drug product using fused silica glass tube non-polar capillary column (HP-5, 30 m, 0.53 mm, 1.5 µm). Ethanol in the sample was diluted with methanol after adding suitable amount of internal standard, the isopropyl alcohol solution.The developed method is precise, linear and accurate in the range of 5 mg/mL to 15 mg/mL of nominal concentration of ethanol i.e. 10 mg/mL . The presented method has an advantage of a very quick gas chromatographic separation (less than 16 min) and therefore is highly suitable for in-process and stability analysis of Ethanol content in Clobetasol propionate foam drug product.

Ultra‐performance liquid chromatography‐tandem mass spectrometry method for analysis of tau in human cerebrospinal fluid without the need of immunocapture

AbstractBackgroundReliable tau protein measurement in CSF is required for detection of Alzheimer’s disease pathology when combined with Aβ42/40 measurement. Tau is present at a very low concentration in CSF, has 6 different isoforms and many forms with post‐translational modification including methylation, glycosylation and phosphorylation. Immunoassays are sensitive enough to measure low tau concentration however poor linearity and lack of specificity in regards to tau’s isoforms have been reported. Thus, mass spectrometry is a candidate alternative technology. Additionally, the possibility of avoiding immunocapture would be important for a protein with multiple isoforms.MethodHere we describe our efforts to develop an LC‐MS/MS (triple quadrupole) assay for quantification of tau in CSF. Sample preparation includes selective protein precipitation, neutralization and on‐filter digestion with trypsin without the need of immunocapture. Following trypsinization the peptide mixture is washed/centrifuged through the filters, injected onto the analytical column and eluted with water/ acetonitrile/0.1% formic acid gradient. For quantification we use the tryptic peptide ‐ GAAPPGQK [(156‐163, m/z‐ 363.26/526.28 (quantifier ion)], which is present in all tau isoforms and does not contain a phosphorylation site, and whose quantity is directly proportional to tau concentration. The same peptide labelled with 15N is used as the internal standard (IS) (m/z‐ 368.19/267.14 and 368.19/533.28, quantifier ions).ResultThe method is still under development however several parameters have been established: surrogate matrix (0.5% serum in water), amount of IS added at the beginning of sample preparation (4ng/mL), lower (350pg/mL) and upper (30000pg/mL) limit of measuring interval, analytical recovery of human peptide from the filters (67‐83%), duplicates precision (5.0 3.9%). No background peaks found at the retention time of the analyte/IS from a matrix‐matched double blank sample confirms good assay specificity. Collection of a full set of imprecision/accuracy data using surrogate matrix and human CSF are underway.ConclusionThe assay will be assessed for tau quantification in human CSF by evaluating each parameter of the full validation process. This newly developed and validated assay will be important for direct measurement of tau in CSF samples, and may be applied as a candidate reference measurement procedure for CSF tau in standardization projects on this biomarker.

Variability Assessment of 90 Salivary Proteins in Intraday and Interday Samples from Healthy Donors by Multiple Reaction Monitoring-Mass Spectrometry.

Saliva is an attractive sample source for the biomarker-based testing of several diseases, especially oral cancer. Here, we sought to apply multiplexed LC-MRM-MS to precisely quantify 90 disease-related proteins and assess their intra- and interindividual variability in saliva samples from healthy donors. We developed two multiplexed LC-MRM-MS assays for 122 surrogate peptides representing a set of disease-related proteins. Saliva samples were collected from 10 healthy volunteers at three different time points (Day 1 morning and afternoon, and Day 2 morning). Each sample was spiked with a constant amount of a 15 N-labeled protein and analyzed by MRM-MS in triplicate. Quantitative results from LC-MRM-MS were calculated by single-point quantification with reference to a known amount of internal standard (heavy peptide). The CVs for assay reproducibility and technical variation were 13 and 11%, respectively. The average concentrations of the 99 successfully quantified proteins ranged from 0.28±0.58ng mL-1 for profilin-2 (PFN2) to 8.55±8.96 μg mL-1 for calprotectin (S100A8). For the 90 proteins detectable in >50% of samples, the average CVs for intraday, interday, intraindividual, and interindividual samples were 38%, 43%, 45%, and 69%, respectively. The fluctuations of most target proteins in individual subjects were found to be within±twofold. Our study elucidated the intra- and interindividual variability of 90 disease-related proteins in saliva samples from healthy donors. The findings may facilitate the further development of salivary biomarkers for oral and systemic diseases.

Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling

Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1(-/-) mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-β-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

A simple and specific quantitative method for determination of dictamnine in Dictamni Cortex by 1H NMR spectroscopy

Dictamni Cortex (Baixianpi) is a commonly used medicinal herb in traditional Chinese medicines (TCMs), possessing various bioactivities. Quinoline alkaloids are regarded as one of the main active components in this herb. In the present study, a more convenient, specific and accurate analytical method using 1H NMR has been developed for the quantitative determination of dictamnine, one of the main active quinoline alkaloids in Dictamni Cortex. In a less crowded region δ 8.0–8.5, the signal of H-5 of dictamnine was well separated from other signals in chloroform-d1. The quantity of dictamnine was calculated by means of the integral value of H-5 of dictamnine to that of the known amount of internal standard, pyrazine. This assay was precise and reproducible, with RSDs less than 3%. The recovery results of dictamnine were observed between 92.10 and 108.10% and the limit of detection (LOD) and limit of quantitation (LOQ) were 3.3 μg ml−1 and 13.2 μg ml−1, respectively. This newly developed method was proven to be highly accurate, precise and robust, for the determination of dictamnine in Dictamni Cortex. The results obtained from 1H NMR method were also compared with those from conventional HPLC method, and no remarkable difference was found between these two methods.

Determination of 2‐ethylhexyl nitrate in diesel fuel

The aim of this study was to develop and validate fast and easily applicable GC/MS assay for the quantification of the substance that increases cetane number in diesel fuel (2-ethylhexylnitrate, 2-EHN). These requirements were fulfilled best by a headspace GC-MS assay with negative chemical ionization with methane (HS-GC/MS). Chromatographic separation is achieved using a DB5-MS capillary column after the addition of known amount of internal standard (o-nitrotoluene). The limit of detection was 0.009% v/v for 2-EHN and the limit of quantification was 0.03% v/v. The HS-GC/MS method was applied for the quantification of cetane improver in spiked diesel fuel and real diesel fuel. The method is linear over the studied range (0.03-0.3%, v/v), with satisfactory intra- and inter-assay precision, and the relative standard deviations are lower than 10%. Good accuracy is achieved with bias <10% at all levels tested.

Multiplex panel of metabolomic markers for early detection of prostate cancer.

13 Background: Prostate cancer (PCa) is the second leading cause of cancer deaths among men in US. Prostate specific antigen (PSA) the clinical standard for early detection of PCa is not specific for the disease necessitating development of additional markers. Metabolites are stable, measurable in biological fluids and function as physiological indicators of the individual. Earlier we described the metabolomic alteration in PCa and nominated sarcosine as a tissue marker for PCa progression. Further, elevated sarcosine levels were seen in cancer urine. The current study describes a multiplex panel of metabolomic markers in urine that include sarcosine, for early detection of PCa. Methods: A sensitive liquid chromatography- mass spectrometry based assay was used to quantify relative levels of tissue specific metabolites in urine of PCa patients. The urine specimens examined in this study were collected at University of Michigan from post DRE and pre-biopsy from clinically challenging patients with PSA between 4-10 ng/mL where biopsy was either positive or negative for tumor. The urine supernatants were recovered post-centrifugation, adjusted to have an osmolarity between 350-400 mOsm/kg, spiked with defined amount of internal standards and evaluated by multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer. The MRM data was examined using random-forest (RF)-to generate a panel of discriminatory compounds that were validated in an independent test set. Results: A panel of 11 metabolites (glutamic acid, sarcosine, spermidine, histidine, methionine, adenosine, inosine, thymine, hydroxyphenyl lactic acid, acetyl valine and lauric acid) when employed in a RF-based 2-stage training model (n=63) delineated PCa and benign with a cross-validated (out-of-bag validation based on 20000 decision trees) accuracy of 76 %. When applied to an independent set of test samples (n=98) from the same cohort the accuracy, sensitivity, specificity, positive predictive value and negative predictive values were 72 %, 77.5 %, 62.5 %, 67.8 % and 73.2 % respectively. Conclusions: The study describes a first-generation multiplex panel of metabolic markers for early detection of PCa in urine supernatants. No significant financial relationships to disclose.

Direct Regulation of Myocardial Triglyceride Metabolism by the Cardiomyocyte Circadian Clock

Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.

The predominance of polyunsaturated fatty acids in the butterfly Morpho peleides before and after metamorphosis

We hypothesized that the polyunsaturated fatty acids of the butterfly were probably derived from the diet and that there might be a great loss of body fat during metamorphosis. To substantiate these hypotheses, we analyzed the fatty acid composition and content of the diet, the larva, and the butterfly Morpho peleides. Both the diet and the tissues of the larva and butterfly had a high concentration of polyunsaturated fatty acids. In the diet, linolenic acid accounted for 19% and linoleic acid for 8% of total fatty acids. In the larva, almost 60% of the total fatty acids were polyunsaturated: linolenic acid predominated at 42% of total fatty acids, and linoleic acid was at 17%. In the butterfly, linolenic acid represented 36% and linoleic acid represented 11% of total fatty acids. The larva had a much higher total fatty acid content than the butterfly (20.2 vs. 6.9 mg). Our data indicate that the transformation from larva to butterfly during metamorphosis drastically decreased the total fatty acid content. There was bioenhancement of polyunsaturated fatty acids from the diet to the larva and butterfly. This polyunsaturation of membranes may have functional importance in providing membrane fluidity useful in flight.

Comment on Wong and Medrano’s “Real-Time PCR for mRNA Quantification” BioTechniques 39 :75-85 (July 2005)

quantitative PCR was introduced in the beginning of the 1990s, several groups used internal synthetic standards in the reactions to account for variances in reaction efficiencies, but these are not mentioned, suggesting that they have fallen out of fashion. These standards were typically identical to the target but lacked a sequence in the middle to be able to distinguish the standard from the target. Most importantly, they had the same primer sequences as the target, indicating simple multiplexing of the standard and the target. These internal standards can be used to absolutely quantify mRNA targets (2) and could solve many of the problems identified in the article by Wong and Medrano. It is easy to imagine such a standard in real-time PCR where the two PCR products (standard and target) could be separated by probes emitting different colors. Synthetic RNA internal standards have three advantages. First, by spiking the RNA sample with known amounts of standards, efficiencies in the reverse transcription step is accounted for. Since the standard is converted into cDNA, differences between tubes in the efficiency of the PCR are accounted for as well. Second, there is no need to worry about which reference gene to use for normalization. Third, absolute quantification could easily be obtained by relating to a known amount of internal standard (2) instead of relating to a relative internal standard, as in the case of a housekeeping gene and an external standard curve. It is not difficult to understand why such synthetic RNA standards are not used today since it takes some effort to produce and handle them without degradation, but it is difficult to understand why they are not considered as a solution or a tool for mRNA quantification using real-time PCR.

Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 +/- 5 vs. 24 +/- 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR-/-) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2--> LDLR-/-; n = 19) or wild-type C57BL/6 littermates (C57 BL/6-->LDLR-/-; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2-->LDLR-/- and C57BL/6-->LDLR-/- mice. Aortic root atherosclerosis was increased by 57% in sPLA2-->LDLR-/- mice compared with C57BL/6-->LDLR-/- controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2alpha-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2-->LDLR-/- compared with C57BL/6-->LDLR-/- mice, indicating significantly increased in vivo oxidative stress in sPLA2--> LDLR-/-. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism.

ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.

Quantitative analysis of retinol and retinol palmitate in vitamin tablets using [formula omitted]-nuclear magnetic resonance spectroscopy

1 H -NMR spectrometry was applied to the quantitative analysis of Vitamin A in four different types of vitamin tablets without any chromatographic purification or saponification. The experiment was performed analysing the H-15 resonance, which appears at δ 4.32 for retinol and δ 4.69 for retinol palmitate, well separated from other resonances in the 1 H -NMR spectrum. Compounds were quantified using the relative ratio of the integral of the H-15 signal to that of a known amount of internal standard (200 μg/ml), anthracene. In order to evaluate the feasibility of avoiding the saponification of retinol palmitate in the preparation of samples, several solvents such as dimethylsulfoxide, n-hexane, methanol, water, and 0.1 M of HCl were tested as possible extraction solvents. Among these, dimethylsulfoxide showed the best yield of retinol palmitate. This method, using dimethylsulfoxide extraction and 1 H -NMR, allows rapid and simple quantitation of retinol palmitate in tablets avoiding tedious saponification.

Quantitative analysis of ephedrine analogues from ephedra species using 1H-NMR.

Four ephedrine analogues such as ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine were determined by (1)H-NMR from Ephedra species. In the region of delta 5.0-4.0, the signals of H-1 attached to the same carbon with a hydroxyl, were well separated from each other in CDCl(3). The amount of each alkaloid was calculated by the relative ratio of the intensity of H-1 signal to the known amount of internal standard, 200 microg of anthracene. This method allows rapid determination of the quantity of four ephedrine alkaloids from Ephedra species. The amount of these alkaloids was in the range of 1.0-2.0% of dry weight depending on the plant materials.

Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using (1)H-NMR.

1H-NMR spectrometry was applied to the quantitative analysis of the bilobalide, ginkgolides A, B, and C in Ginkgo biloba leaves and six kinds of commercial Ginkgo products without any chromatographic purification. The experiment was performed by the analysis of each singlet H-12, which were well separated in the range of delta 6.0-7.0 in the (1)H-NMR spectrum. However, the H-12 protons of bilobalide and ginkgolides may have overlapped with H-6 or H-8 protons of the Ginkgo flavonoids. Therefore, the optimum (1)H-NMR solvent for the analysis of the compound was selected through the evaluation of solvent effects on the resolution of these signals from the compounds. Acetone-d(6)-benzene-d(6) (50 : 50) was found to be the best one among the solvents evaluated. The quantity of the compounds was calculated by the relative ratio of the intensity of each compound to the known amount of internal standard (25 microgram), phloroglucinol. This method allows rapid and simple quantitation of underivatized bilobalide and ginkgolides in 5 min without any pre-purification steps.

Interleukin (IL)-1beta regulation of IL-1beta and IL-1 receptor antagonist expression in cultured human endometrial stromal cells.

The interleukin (IL)-1 system is a major regulator of local cellular interactions during embryonic implantation. Because IL-1beta and IL receptor antagonist (IL-1ra) are both expressed in human endometrium, we hypothesized that an appropriate ratio of IL-1beta to IL-1ra might favor the process of embryo implantation. Therefore, we investigated IL-1 regulation of the quantitative ratio of IL-1beta/IL-1ra messenger RNA (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR, as well as intracellular protein expression after stromal cell solubilization. Confluent stromal cell cultures were stimulated with human IL-1beta (0-1000 IU/mL) for 24 h. After 24 h, total RNA was extracted, reverse transcribed, and coamplified by PCR with a defined amount of internal standard. The quantitative ratio was determined by the density of target to the internal standard. After culture with IL-1beta for 24 and 48 h, stromal cells were solubilized, and the intracellular protein levels of IL-1beta and IL-1ra were measured by enzyme-linked immunosorbent assay. The IL-1beta and IL-1ra mRNA were both up-regulated, and IL-1R tI mRNA was down-regulated, by IL-1beta in a dose-dependent manner. The quantitative ratio of IL-1beta to IL-1ra mRNA was constant with the presence of increasing concentrations of IL-1beta (1-1000 IU/mL). IL-1beta and IL-1ra protein was not detected in conditioned media of cultures before addition of IL-1beta. IL-1beta and IL-1ra protein levels increased with increasing amounts of IL-1beta after solubilization of stromal cells. The IL-1beta was detectable after 12 h of culture, in comparison with IL-1ra, which was detectable after 24 h of IL-1beta stimulation. These results suggest that IL-1 may play a crucial role in embryo-maternal interaction by regulating stromal cell expression of IL-1beta and IL-1ra, resulting in an appropriate ratio during the process of embryonic implantation.