Identification of a rare [G γ(A γδß)0 ] -thalassemia using tandem mass spectrometry.

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (δß-) thalassemia has large deletions in the ß globin gene cluster involving δ- and ß-globin genes, leading to absent or reduced synthesis of both δ- and ß-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of δß-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, ß, and Aγ globin proteins with increased G γ globin protein in the affected individuals. The diagnosis of G γ(A γδß)0 -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Maternal nutritional status and milk volume and composition in India: an observational study.

BACKGROUND: Human milk provides essential nutrition for infants, and its benefits are well established. We lack data on the influence of maternal nutritional status on milk volume and composition in low-middle income countries. OBJECTIVE: We aimed to 1) assess lactation performance (human milk volume, macronutrient composition, and infant energy intake) in Indian females and 2) examine the associations between maternal anthropometry (BMI, percentage body fat) and lactation performance. METHODS: We conducted an observational study among 232 mother-infant dyads, 2 to 4 mo postpartum in Haryana, India. We used deuterium oxide dose-to-mother technique to measure milk volume and maternal percentage body fat and collected human milk samples to determine macronutrient and energy concentrations. Adjusted multiple linear regression models were used to examine the associations between maternal anthropometry and lactation performance. RESULTS: The mean BMI and percentage body fat of mothers were 21.7 ± 3.6 kg/m2 and 29.5 ± 7.7, respectively. Milk volume and macronutrient composition were similar to the reference values (means ± standard deviations: milk volume, 724 ± 184 mL/d; median (25th, 75th percentile); protein, 9.9 (8.3, 11.7) g/L; fat, 41.0 ± 15.2 g/L; energy density, 0.71 ± 0.14 kcal/g; lactose, 65.5 (55.3, 71.3) g/L). Maternal BMI and percentage body fat were not significantly associated with macronutrient composition. Both maternal BMI and percentage body fat were negatively associated with milk volume (-7.0, 95% CI: -12.4, -1.6 mL/d; -3.5, 95% CI: -6.0, -1.1mL/d, respectively) but there were no effects on the total energy intake of infants after adjusting for covariates. CONCLUSION: Most mothers had a normal BMI and milk of similar composition and volume to reference values. Future work in populations with a greater burden of underweight and/or obesity are needed to examine the underlying mechanisms between maternal body composition and milk volume. This trial was registered at The Clinical Trials Registry- India as CTRI/2017/01/007636.

Quantification of aflatoxin and ochratoxin contamination in animal milk using UHPLC-MS/SRM method: a small-scale study.

Mycotoxin contamination in animal milk is an emerging concern around the globe. Here we developed and validated an ultrahigh-performance liquid chromatography and mass spectrometry-selected reaction monitoring (UHPLC/MS-SRM) method to quantify low concentrations of aflatoxins (AFs) and ochratoxins (OTs) in routinely consumed animal milk samples collected from southern India. Stable isotope dilution methodology was applied to quantify AFB1, AFB2, AFG1, AFG2, AFM1, AFM2 and OTA, OTB in n = 38 different milk samples, using 1 mL of milk. Bioanalytical parameters including method accuracy, precision, recovery, regression analysis and stability were assessed. Dynamic ranges for quantification were between 15.6-1000 pg/mL for AFB1, AFB2, AFG1, and OTA; 7.8-500 pg/mL for AFM1, AFM2 and OTB; 78.6-5000 pg/mL for AFG2. Method accuracy ranged between 80-120%, with ± 15% precision. Recoveries for spiked standards were > 88% in water and 75% in milk, with limits of quantification (LOQ) ranging between 31.3 pg/mL for AFB1, AFB2, AFG1 and OTA, 15.6 pg/mL for AFM1, AFM2 and OTB and 156 pg/mL for AFG2. R2 values for regression analyses ranged between 0.9991-0.9999. AFB2 [mean: 38 pg/mL (0.038 µg/kg)] was quantified in goat milk, AFM1 was quantified in cow, goat, pasteurized milk [mean: 331 pg/mL (0.331 µg/kg), 406 pg/mL (0.406 µg/kg), 164 pg/mL (0.164 µg/kg)]. Additionally, 90% of cow, goat and pasteurized milk samples were above European Union (EU) limits of 50 pg/mL (0.05 µg/kg) and 40% of cow and goat milk samples were above the Food Safety Standards Authority of India (FSSAI) limit of 500 pg/mL (0.5 µg/kg). AFM2 was also quantified in cow, goat, and pasteurized milk samples [mean: 249 pg/mL (0.249 µg/kg), 375 pg/mL (0.375 µg/kg), 81 pg/mL (0.081 µg/kg)]. Our dynamic ranges for quantification are lower than other published methods, with need for a smaller volume of milk. This validated method can be applied for routine quantification of mycotoxins in milk. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-021-04986-w).

Risk of dietary and breastmilk exposure to mycotoxins among lactating women and infants 2-4 months in northern India.

Mycotoxins are carcinogenic secondary metabolites of fungi that have been linked to infant growth faltering. In this study, we quantified co-occurring mycotoxins in breast milk and food samples from Haryana, India, and characterized determinants of exposure. Deterministic risk assessment was conducted for mothers and infants. We examined levels of eight mycotoxins (Aflatoxin B1 , B2 , G1 , G2 , M1 , M2 ; Ochratoxin A, B) in 100 breast milk samples (infants 2-4 months) using ultra-high-performance liquid chromatography tandem mass spectrometry. Aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ) and deoxynivalenol (DON) were detected in several food items (n = 298) using enzyme-linked immunosorbent assays. We report novel data on the presence of mycotoxins in breast milk samples from India. Whereas breast milk concentrations (AFM1 median: 13.7; range: 3.9-1200 ng/L) remain low, AFM1 was detected above regulatory limits in 27% of animal milk samples. Additionally, 41% of infants were above provisional maximum tolerable daily intake (PMTDI) limits for AFM1 due to consumption of breast milk (mean: 3.04, range: 0.26-80.7 ng kg-1 bw day-1 ). Maternal consumption of breads (p < 0.05) was associated with breast milk AFM1 exposure. AFB1 (µg/kg) was detected in dried red chilies (15.7; 0-302.3), flour (3.13; 0-214.9), groundnuts (0; 0-249.1), maize (56.0; 0-836.7), pearl millet (1.85; 0-160.2), rice (0; 0-195.6), wheat (1.9; 0-196.0) and sorghum (0; 0-63.5). FB1 (mg/kg) was detected in maize (0; 0-61.4), pearl millet (0; 0-35.4) and sorghum (0.95; 0-33.2). DON was not detected in food samples. Mothers in our study exceeded PMTDI recommendations for AFB1 due to consumption of rice and flour (mean: 75.81; range: 35.2-318.2 ng kg-1 bw day-1 ). Our findings show the presence of Aflatoxin B1 and M1 at various levels of the food chain and in breast milk, with estimated intakes exceeding PMTDI recommendations. Aflatoxins are known carcinogens and have also been linked to stunting in children. Their presence across the food system and in breast milk is concerning, thus warranting further research to replicate and expand on our findings and to understand implications for maternal and child health.

Applications of quantitative metabolomics to revolutionize early diagnosis of inborn errors of metabolism in India.

Inborn errors of metabolism (IEMs) are a group of disorders caused by disruption of metabolic pathways, which leads to accumulation, decreased circulating levels, or increased excretion of metabolites as a consequence of the underlying genetic defects. These heterogeneous groups of disorders cause significant neonatal and infant mortality across the whole world and it is of utmost concern for developing countries like India owing to lack of awareness and standard preventive strategies like newborn screening (NBS). Though the predictive cumulative incidence of IEMs is said to be ∼1:800 newborns, data pertaining to the true prevalence of individual IEMs is not available in the context of Indian population. There is a need for a large population-based study to get a clear picture of the prevalence of different IEMs. One of the best ways to screen for IEMs is by applying advanced liquid chromatography-mass spectrometry (LC-MS) technology using a quantitative metabolomics approaches such as selected or multiple reaction monitoring (SRM or MRM). Recent developments in LC-MS/MRM based quantification of marker metabolites in newborns have opened a novel opportunity to screen multiple disorders simultaneously from a minuscule volume of biological fluids. In this review article, we have highlighted how LC-MS/MRM based metabolomics approach with its high sensitivity and diagnostic capability can make an impact on the nation's public health through NBS programs.

Simple click chemistry-based derivatization to quantify endogenous formaldehyde in milk using ultra-high-performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode.

RATIONALE: Formaldehyde (FA) exposure via environmental pollution or through the food chain poses a serious threat to human health, especially in developing countries like India. Although the addition of FA to food is proscribed, it is often illegally added to foods such as milk to increase the shelf-life. There are challenges in differentiating the endogenous FA content in milk from externally added FA. METHOD: We have developed a simple method using ultra-high-performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode (UHPLC/MS/SRM) for the absolute quantification of endogenous FA in milk. The steps include fat removal, protein precipitation using acid, and spiking with labelled FA (FA*), followed by simple click chemistry-based derivatization using Girard P reagent (GP) and final analysis. RESULTS: A standard curve with FA* was constructed and used for the calculation of endogenous FA in milk. The optimal conditions for the derivatization reaction using 500 µL of milk were: GP, 50 µg; temperature, 37°C; time, 60 min; and 0.1% HCl. The validation parameters such as accuracy (95.84 to 99.73%), precision (2.84 to 8.02%) and spiked recovery (>95%) are within the FDA guidelines. This method is highly sensitive [limit of detection (LOD) of 1 ng/mL] with a dynamic range of 3.12 to 200 ng/mL. The endogenous FA level in pasteurized cow milk is 70 ng/mL (n = 60). The FA content in raw milk samples from cow, goat and buffalo (each n = 10) varied from 134 to 255 ng/mL. CONCLUSIONS: This method is precise and sufficiently sensitive to quantify endogenous FA in milk samples using a minimal sample volume. As it involves simple sample preparation steps, it can be used routinely to quantify endogenous FA.

Quantification of B-vitamins from different fresh milk samples using ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring methods.

Absolute quantification of B-vitamins in milk is becoming imperative to correlate its impact on child/human health. In today's world of changing food habits and environmental pollution, there is concern if milk is what we think it to be. In the present work, ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring (UPHPLC-MS/SRM) methods have been developed and validated for quantitative estimation of 21 different B-vitamins [B1-3, B2-3, B3-2, B5-1, B6-5, B8-1, B9-2 vitamins, total choline (betaine, choline and acetylcholine)] from a drop (50 µL) and B12 from 500 µL of milk. These two methods qualify all essential bio-analytical parameters (recovery >80%, accuracy <±15%, variation <±10%) and are highly reproducible. By using these developed methods, B-vitamins in different fresh milk samples from cow, goat, buffalo and pasteurized cow milk (each 10 and total n = 40) were analysed. Total choline is the highest (6.5-10.5 µg/mL) and vitamin B8 is the lowest (16.1-32.9 ng/mL) in all milk samples. Inverse correlation between vitamin B2 and B5 was observed in milk samples. The ratio of vitamin B5/B2 was checked in all milk samples, it is higher in cow (2.64), equal in goat (1.04) and lower in buffalo (0.42) milk. Total B-vitamin content in cow milk is higher (10.5 µg/mL) compared to other three (goat-7.2, buffalo-6.5, pasteurized-8.8 µg/mL). Vitamin B12 is higher in cow milk (3.6 ng/mL) compared to other two fresh milk samples. Different isomers for vitamin B6 were noticed in the fresh milk samples. The complete profile of water soluble vitamins and the ratio of two abundant B vitamins (B5/B2) in milk will be useful to check the nutritional quality and to differentiate the kind of animal milk.

Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

Quantification of Neurotransmitters from Intact and Regenerating Planarians Using UHPLC-MS/SRM Method.

Freshwater planarian species S. mediterranea is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. It is one of the best model systems available to address the basic biological mechanisms in the regeneration processes. Absolute quantification of metabolites from planarians is imperative to understand their role in the regeneration processes. Here we describe a stable isotope dilution ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay for a sensitive and quantitative assessment of neurotransmitters (NTs) in planaria. We used this method for the simultaneous quantification of 16 NTs from both intact and regenerating planarians.

A method for comparative metabolomics in urine using high resolution mass spectrometry.

Developing a workflow for metabolite profiling from biological fluids using mass spectrometry is imperative to extract accurate information. In this study, urine samples from smokers (n=10) and nonsmokers (n=10) were analyzed using an ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system. For the analysis, two different chromatographic methods [Reversed phase chromatography (RPC) and Hydrophilic interaction liquid chromatography (HILIC)], in two ionization modes (positive and negative) were used. Spiked reserpine (positive ion mode) or taurocholate (negative ion mode) were used for data extraction and normalization. Quality controls (QCs), prepared by pooling urine samples from both smokers and non-smokers (each n=10), were used to assess the reproducibility of the method. The final data output from SIEVE 2.2 after applying a cut-off for QC coefficient of variation (CV) <20% and p-value <0.05 showed 165, 83, 177 and 100 unique components in RP positive/negative, HILIC positive/negative modes, respectively. Statistical analysis showed clustering of the two groups and the QCs, while the variable importance in projection (VIP) scores for the top fifteen metabolites in each of the four modes indicated the metabolites most responsible for the differences. Application of the developed workflow for comparative metabolomic analysis of urine in different diseased models will be of great use in the field of clinical metabolomics.

Serum apolipoprotein A-1 quantification by LC-MS with a SILAC internal standard reveals reduced levels in smokers.

BACKGROUND: Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC-MS can provide misleading results. RESULTS: Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [(13)C6(15)N2]-lysine and [(13)C9(15)N1]-tyrosine in human cells. A stable isotope dilution LC-MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers. CONCLUSION: The concentration of ApoA-1 in nonsmokers was 169.4 mg/dl with an 18.4% reduction to 138.2 mg/dl in smokers. The validated assay will have clinical utility for assessing effects of smoking cessation and therapeutic or dietary interventions in high-risk populations.

Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree.

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC-600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.

A quantitative metabolomics peek into planarian regeneration.

The fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate a whole animal from a small piece of tissue. It is one of the best model systems to address the basic mechanisms essential for regeneration. Here, we are interested in studying the roles of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated the differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. The levels of thiols such as cysteine and GSH are 651 and 1107 ng mg(-1) in planarians. Among the nucleotides, the level of cGMP is the lowest (0.03 ng mg(-1)) and the level of AMP is the highest (187 ng mg(-1)) in both of the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, the thiol (cysteine and GSH) levels are well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in intact planarians, the level of cGMP is also very low in regenerating planarians. Surprisingly, the levels of amines and thiols in head regenerating blastemas are ∼3 times higher compared to those for tail regenerating blastemas. Thus our results strongly indicate the potential roles of amines, thiols and nucleotides in planarian regeneration.

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography-tandem mass spectrometry.

An ultrasensitive stable isotope dilution liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5-2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography-mass spectrometry (GC-MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC-MS and LC-MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.

Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method.

RATIONALE: Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed. METHODS: A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%. RESULTS: The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration. CONCLUSIONS: For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.

Nicotine exposure and metabolizer phenotypes from analysis of urinary nicotine and its 15 metabolites by LC-MS.

UNLABELLED: Smokers who inhale less deeply are exposed to lower amounts of the toxic substances present in tobacco smoke. In order to more rigorously assess tobacco smoke exposure, it is necessary to have an accurate method for quantifying nicotine and all of its known metabolites. METHODS: A stable-isotope dilution LC-MRM/MS assay has been developed for quantification of urinary nicotine and the 15 possible metabolites that could arise from known metabolic pathways. Nicotine, cotinine, trans-3´-hydroxy-cotinine, nicotine-N-oxide, cotinine-N-oxide, nornicotine, norcotinine and 4-hydroxy-4-(3-pyridyl)butanoic acid were quantified by direct analysis. The corresponding glucuronide metabolites were quantified after urine hydrolysis with ß-glucuronidase. RESULTS: Nicotine and all 15 nicotine metabolites were quantified by LC-MRM/MS in most urine samples from 61 tobacco smokers. Urinary nicotine and metabolite concentrations ranged from 7.9 to 337.8 µM (mean 75.5 ± 67.8 µM). Three nicotine metabolizer phenotypes were established as reduced metabolizers (ratio < 8), normal metabolizers (ratio 8-30), and extensive metabolizers (ratio > 30). 4-hydroxy-4-(3-pyridyl)butanoic acid, which has not been quantified previously, was an abundant metabolite in all three phenotypes. CONCLUSION: Using this assay it will now be possible to determine whether there are relationships between nicotine exposure and/or metabolizer phenotype with exposure to toxic substances that are present in tobacco smoke and/or to biological response biomarkers to tobacco smoking. This will help in identifying individuals at high risk for developing smoking-related diseases as well as those amenable to smoking cessation programs.

Liquid chromatography/mass spectrometry of pre-ionized Girard P derivatives for quantifying estrone and its metabolites in serum from postmenopausal women.

An ultrasensitive stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry (LC/SRM/MS) assay has been developed for serum estrone, 16α-hydroxyestrone, 4-methoxyestrone, and 2- methoxyestrone. The enhanced sensitivity was obtained by the use of Girard P (GP) pre-ionized derivatives coupled with microflow LC. The limit of detection for each estrogen using 0.5 mL of serum was 0.156 pg/mL and linear standard curves were obtained up to 20 pg/mL. Serum samples from 20 postmenopausal women (10 lifetime non-smokers and 10 current smokers) were analyzed using this new assay. Mean serum concentrations of estrone and 2-methoxyestrone were 14.06 pg/mL (±1.56 pg/mL) and 3.30 pg/mL (±1.00 pg/mL), respectively, for the 20 subjects enrolled in the study. The mean estrone concentration determined by our ultrasensitive and highly specific assay was significantly lower than that reported for the control groups in most previous breast cancer studies of postmenopausal women. In addition (and contrary to many reports) serum 16α-hydroxyestrone was not detected in any of the subjects, and 4-methoxyestrone was detected in only one of the subjects. Furthermore, there were no significant differences in the mean serum concentrations of estrone and 2-methoxyestrone or the ratio of serum 2- methoxyestrone to estrone between the non-smoking and smoking groups. Interestingly, the one subject with measurable serum 4-methoxyestrone (2.3 pg/mL) had the lowest estrone and 2-methoxyestrone concentrations. Using this assay it will now be possible to obtain definitive information on the levels of serum estrone, 4-methoxyestrone, and 2-methoxyestrone in studies of cancer risk using small serum volumes available from previous epidemiology studies.

Differential secreted proteome approach in murine model for candidate biomarker discovery in colon cancer.

The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apc(min) mice were compared with control mice. These findings were then confirmed by Western blot analysis.

Cyclooxygenase-2-mediated metabolism of arachidonic acid to 15-oxo-eicosatetraenoic acid by rat intestinal epithelial cells.

Rat intestinal epithelial cells that permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 microM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E 2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE 2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 microM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-( S)-HETE concentrations were approximately twice that of PGE 2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE 2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE 2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min(-1), respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.