Liquid Chromatography-mass Spectrometry-based Method Research Articles

Metabolomics perspectives into the co-exposure effect of polycyclic aromatic hydrocarbons and metals on renal function: A meet-in-the-middle approach

Studies on the dose effects of kidney impairment and metabolomes in co-exposure to polycyclic aromatic hydrocarbons (PAHs) and metals are limited. We aimed to identify overall associations and metabolic perturbations in 130 participants (53 petrochemical workers and 77 controls) exposed to a PAHs-metals mixture in Southern China. The urinary 7 hydroxylated PAHs and 15 metal(loid)s were determined, and serum creatinine, beta-2 microglobulin, and estimated glomerular filtration rate were health outcomes. The liquid chromatography-mass spectrometry-based method was applied to serum metabolomics. Generalized weighted quantile sum (gWQS) regressions were used to estimate the overall dose-response relationships, and pathway analysis, “meet-in-the-middle” approach, and mediation effect analyses were conducted to identify potential metabolites and biological mechanisms linking exposure with nephrotoxic effects. Our results indicated that renal function reduction was associated with a PAHs-metals mixture in a dose-dependent manner, and 1-hydroxynaphthalene and copper were the most predominant contributors among the two families of pollutants. Furthermore, the metabolic disruptions associated with the early onset of kidney impairment induced by the combination of PAHs and metals encompassed pathways such as phenylalanine-tyrosine-tryptophan biosynthesis, phenylalanine metabolism, and alpha-linolenic acid metabolism. In addition, the specifically identified metabolites demonstrated excellent potential as bridging biomarkers connecting the reduction in renal function with the mixture of PAHs and metals. These findings shed light on understanding the overall associations and metabolic mechanism of nephrotoxic effects of co-exposure to PAHs and metals.

Metabolomics: a review of liquid chromatography mass spectrometry-based methods and clinical applications

Abstract Introduction Metabolomics is a rapidly growing field that aims to understand the complex metabolic pathways involved in health and disease. Liquid chromatography mass spectrometry (LC-MS) based untargeted metabolomics has emerged as a powerful tool for investigating the metabolic changes associated with various diseases, and for identifying potential biomarkers for early disease detection and treatment monitoring. This review provides a comprehensive overview of LC-MS based untargeted metabolomics and its clinical applications. Content The advantages and challenges of untargeted metabolomics are discussed, encompassing sample preparation, data processing, sample storage, acquisition mode selection, column strategy, and annotation. The latest advancements in LC-MS technology and data analysis techniques are reviewed, which have facilitated the more accurate and sensitive detection of metabolites in biological samples. The clinical applications of untargeted metabolomics are emphasized, including its utilization in disease diagnosis, treatment monitoring, and personalized medicine. Examples are provided of how biomarkers for various diseases, such as cancer, coronary heart disease, and infectious disease, have been identified through the application of untargeted metabolomics. Summary and outlook Overall, LC-MS based untargeted metabolomics is a powerful tool for investigating the complex metabolic changes associated with various diseases, and has the potential to transform clinical diagnosis, treatment, and personalized medicine.

Abstract 702: Accurate Quantification Of Peptides In The Renin Angiotensin Aldosterone System By Mass Spectrometry

Aim/Background: The Renin Angiotensin Aldosterone System (RAAS) is responsible for the regulation of vascular hemodynamics. When dysregulated, the RAAS contributes to cardiovascular pathologies, such as hypertension, which affects ~40-50% of the US population. Angiotensin (Ang) peptides are the primary effector molecules of the classical RAAS and alternative RAAS pathways. 1 The accurate quantification of these vasoactive peptides using a high throughput method may provide a better understanding of the mechanisms underlying hypertension that standard blood pressure cuff measurements cannot capture. Methods: A liquid chromatography-mass spectrometry-based method was developed to simultaneously measure four (4) angiotensin peptides (Ang (1-7), Ang (1-9), Ang I, and Ang II) in human serum. The peptides are isolated prior to measurement using a two-step precipitation followed by solid phase extraction. Peptides were quantified using stable isotope labeled peptides as internal standards. Results: Calibration curves spanning a concentration range of 1 - 1000 fmol/mL were established for all peptides. Linear regression analysis established R 2 values ≥ 0.98. Estimated method limits of detection (LOD) are at or below 5 fmol/mL for all 4 peptides. For initial assessments, RAAS peptides were quantified in 25 commercial serum samples. Median values (concentration ranges) were as follows: Ang (1-7) 3.7 fmol/mL (LOD - 37.9 fmol/mL), Ang (1-9) 3.8 fmol/mL (LOD - 16.4 fmol/mL), Ang II 82.0 fmol/mL (LOD - 549.6 fmol/mL), Ang I 27.2 fmol/mL (LOD - 324.0 fmol/mL). Ang II always occurred in the highest concentration, except for one sample where Ang I was the most abundant peptide. In most samples, Ang I was about 4 times lower, and Ang (1-9) and Ang (1-7) were about 25 times lower than Ang II. In one sample, Ang I was about 30 time higher, and Ang (1-7) was about 3 times higher than Ang II. Conclusion: This mass spectrometry method enables accurate RAAS peptide quantification in serum samples. Application of this method will lead to the generation of new data that may provide unique, mechanistic insights into hypertension, as well as renal diseases, arteriosclerosis, and other cardiovascular pathologies associated with the RAAS pathway.

Development of a fast and robust liquid chromatography-mass spectrometry-based metabolomics analysis method for neonatal dried blood spots

Dried blood spot (DBS) samples have been widely used in many fields including newborn screening, with the advantages in transportation, storage and non-invasiveness. The DBS metabolomics research of neonatal congenital diseases will greatly expand the understanding of the disease. In this study, we developed a liquid chromatography-mass spectrometry-based method for neonatal metabolomics analysis of DBS. The influences of blood volume and chromatographic effects on the filter paper on metabolite levels were studied. The levels of 11.11 % metabolites were different between 75 μL and 35 μL of blood volumes used for DBS preparation. Chromatographic effects on the filter paper occurred in DBS prepared with 75 μL whole blood and 6.67 % metabolites had different MS responses when central disks were compared with outer disks. The DBS storage stability study showed that compared with − 80 °C storage, storing at 4 °C for 1 year had obvious influences on more than half metabolites. Storing at 4 °C and − 20 °C for short term (< 14 days) and − 20 °C for longer term (1 year) had less influences on amino acids, acyl-carnitines and sphingomyelins, but greater influences on partial phospholipids. Method validation showed that this method has a good repeatability, intra-day and inter-day precision and linearity. Finally, this method was applied to investigate metabolic disruptions of congenital hypothyroidism (CH), metabolic changes of CH newborns were mainly involved in amino acid metabolism and lipid metabolism.

Plasma GM2 ganglioside potential biomarker for diagnosis, prognosis and disease monitoring of GM2-Gangliosidosis.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal β-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of β-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Amino Acids from Urine as Possible Biomarkers for Early Detection of Vancomycin Nephrotoxicity

Abstract: Drug-induced nephrotoxicity is an important therapeutic concern, as many endogenous compounds are filtered through the kidneys for excretion into urine. Vancomycin is a drug of last resort used to treat multiple drug-resistant infections, and is primarily used in paediatrics to treat infections caused by gram-positive organisms resistant to beta-lactam antibiotics. Vancomycin is primarily (80–90%) excreted through the kidney. To identify biochemical markers useful for the early diagnosis of nephrotoxicity, amino acid profiling was performed in young Wistar rats treated with vancomycin. A liquid chromatography mass spectrometry-based method was developed for targeted amino acid analysis from urine samples collected after dosing with vancomycin (300 mg/kg). Alterations in amino acids levels were observed in urine immediately after the first dosing, and increased in prominence during the course of treatment. Nephrotoxicity was confirmed using established methods such as histopathological evaluation and clinical chemistry analysis. Of note, a significant change in amino acid levels in urine was observed well before any noticeable increase in traditional markers. This suggests that quantification of amino acids from urine could be a good alternative to blood-based analysis in neonates and children as a strategy for the detection of kidney injury at an earlier stage than any existing methods. Keywords: Amino acid profiling, LC-MS, Method validation, Nephrotoxicity, Targeted analysis, Vancomycin.

Overview of Recent Liquid Chromatography Mass Spectrometry-Based Methods for Natural Toxins Detection in Food Products.

Natural toxins include a wide range of toxic metabolites also occurring in food and products, thus representing a risk for consumer health. In the last few decades, several robust and sensitive analytical methods able to determine their occurrence in food have been developed. Liquid chromatography mass spectrometry is the most powerful tool for the simultaneous detection of these toxins due to its advantages in terms of sensitivity and selectivity. A comprehensive review on the most relevant papers on methods based on liquid chromatography mass spectrometry for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides and furocoumarins in food is reported herein. Specifically, a literature search from 2011 to 2021 was carried out, selecting a total of 96 papers. Different approaches to sample preparation, chromatographic separation and detection mode are discussed. Particular attention is given to the analytical performance characteristics obtained in the validation process and the relevant application to real samples.

Fatty Acid Substitutions Modulate the Cytotoxicity of Puwainaphycins/Minutissamides Isolated from the Baltic Sea Cyanobacterium Nodularia harveyana UHCC-0300.

Puwainaphycins (PUW) and minutissamides (MIN) are structurally homologous cyclic lipopeptides that exhibit high structural variability and possess antifungal and cytotoxic activities. While only a minor variation can be found in the amino acid composition of the peptide cycle, the fatty acid (FA) moiety varies largely. The effect of FA functionalization on the bioactivity of PUW/MIN chemical variants is poorly understood. A rapid and selective liquid chromatography–mass spectrometry-based method led us to identify 13 PUW/MIN (1–13) chemical variants from the benthic cyanobacterium Nodularia harveyana strain UHCC-0300 from the Baltic Sea. Five new variants identified were designated as PUW H (1), PUW I (2), PUW J (4), PUW K (10), and PUW L (13) and varied slightly in the peptidic core composition, but a larger variation was observed in the oxo-, chloro-, and hydroxy-substitutions on the FA moiety. To address the effect of FA substitution on the cytotoxic effect, the major variants (3 and 5–11) together with four other PUW/MIN variants (14–17) previously isolated were included in the study. The data obtained showed that hydroxylation of the FA moiety abolishes the cytotoxicity or significantly reduces it when compared with the oxo-substituted C18-FA (compounds 5–8). The oxo-substitution had only a minor effect on the cytotoxicity of the compound when compared to variants bearing no substitution. The activity of PUW/MIN variants with chlorinated FA moieties varied depending on the position of the chlorine atom on the FA chain. This study also shows that variation in the amino acids distant from the FA moiety (position 4–8 of the peptide cycle) does not play an important role in determining the cytotoxicity of the compound. These findings confirmed that the lipophilicity of FA is essential to maintain the cytotoxicity of PUW/MIN lipopeptides. Further, a 63 kb puwainaphycin biosynthetic gene cluster from a draft genome of the N. harveyana strain UHCC-0300 was identified. This pathway encoded two specific lipoinitiation mechanisms as well as enzymes needed for the modification of the FA moiety. Examination on biosynthetic gene clusters and the structural variability of the produced PUW/MIN suggested different mechanisms of fatty-acyl-AMP ligase cooperation with accessory enzymes leading to a new set of PUW/MIN variants bearing differently substituted FA.

Current Challenges and Recent Developments in Mass Spectrometry-Based Metabolomics.

High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry-based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.

Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry.

Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein-specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography-mass spectrometry-based method for direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC-OBE-nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.

Quantitative analysis of 3’- and 6’-sialyllactose in human milk samples by HPLC-MS/MS: A validated method for the comparison of two consecutive lactation periods in the same woman

Human milk oligosaccharides (HMOs) are complex carbohydrates that exist naturally in mother’s milk. Due to their myriad beneficial biological effects, HMOs are in the focus of current researches. Some of these complex carbohydrates are already commercially available utilizing their health-promoting benefits not just in infant formulas but in dietary supplements. Among the acidic oligosaccharides 3′- and 6′-sialyllactoses (SLs) are the most abundant in human milk. Extending the health benefits of these trisaccharides, 3′-SL and 6′-SL are expected to be active ingredients in infant- and follow-on formula and novel foods, therefore reliable and easy-to-perform analytical methods are urgently needed for their characterization. Herein we report a graphitized carbon liquid chromatography-mass spectrometry-based method for the separation and quantitation of sialyllactoses in human milk. Compounds were separated as their alditol forms and were quantified using maltotriose as internal standard. The developed bioanalytical method was validated in terms of selectivity, accuracy, precision, recovery, carry-over, matrix effects and stability. Two consecutive lactation periods of the same individual were monitored for the first time and a slight decrease in SL concentrations were found during the first month of the second lactation.

Metabolomics of Children and Adolescents Exposed to Industrial Carcinogenic Pollutants.

Studies on metabolomes of carcinogenic pollutants among children and adolescents are limited. We aim to identify metabolic perturbations in 107 children and adolescents (aged 9-15) exposed to multiple carcinogens in a polluted area surrounding the largest petrochemical complex in Taiwan. We measured urinary concentrations of eight carcinogen exposure biomarkers (heavy metals and polycyclic aromatic hydrocarbons (PAHs) represented by 1-hydroxypyrene), and urinary oxidative stress biomarkers and serum acylcarnitines as biomarkers of early health effects. Serum metabolomics was analyzed using a liquid chromatography mass spectrometry-based method. Pathway analysis and "meet-in-the-middle" approach were applied to identify potential metabolites and biological mechanisms linking carcinogens exposure with early health effects. We found 10 potential metabolites possibly linking increased exposure to IARC group 1 carcinogens (As, Cd, Cr, Ni) and group 2 carcinogens (V, Hg, PAHs) with elevated oxidative stress and deregulated serum acylcarnitines, including inosine monophosphate and adenosine monophosphate (purine metabolism), malic acid and oxoglutaric acid (citrate cycle), carnitine (fatty acid metabolism), and pyroglutamic acid (glutathione metabolism). Purine metabolism was identified as the possible mechanism affected by children and adolescents' exposure to carcinogens. These findings contribute to understanding the health effects of childhood and adolescence exposure to multiple industrial carcinogens during critical periods of development.

Assessment of NAD+metabolism in human cell cultures, erythrocytes, cerebrospinal fluid and primate skeletal muscle

The reduction-oxidation state of NAD+/NADH is critical for cellular health with NAD+ and its metabolites playing critical roles in aging and pathologies. Given the inherent autooxidation of reduced dinucleotides (i.e. NADH/NADPH), and the well-established differential stability, the accurate measurement of NAD+ and its metabolites is technically challenging. Moreover, sample processing, normalization and measurement strategies can profoundly alter results. Here we developed a rapid and sensitive liquid chromatography mass spectrometry-based method to quantify the NAD+ metabolome with careful consideration of these intrinsic chemical instabilities. Utilizing this method we assess NAD+ metabolite stabilities and determine the presence and concentrations of NAD+ metabolites in clinically relevant human samples including cerebrospinal fluid, erythrocytes, and primate skeletal muscle.

Profiling and Quantification of Regioisomeric Caffeoyl Glucoses in Berry Fruits.

On the basis of a recently developed tandem mass spectrometry-based hierarchical scheme for the identification of regioisomeric caffeoyl glucoses, selected berry fruits were profiled for their caffeoyl glucose ester content. Fresh edible berries profiled, including strawberries, raspberries, blueberries, blackberries, red currant, black currant, lingonberries, gooseberries, and juices of elderberries, goji berries, chokeberries, cranberries, açai berries, sea buckthorn berries, Montmorency sour cherries, and pomegranates, were investigated. 1-Caffeoyl glucose was found to be the predominant isomer in the majority of samples, with further profiling revealing the presence of additional hydroxycinnamoyl glucose esters and O-glycosides with p-coumaroyl, feruloyl, and sinapoyl substituents. A quantitative liquid chromatography-mass spectrometry-based method was developed and validated, and all caffeoyl glucose isomers were quantified for the first time in edible berries.

Elucidation of the Recognition Sequence of Sortase B from Bacillus anthracis by Using a Newly Developed Liquid Chromatography-Mass Spectrometry-Based Method.

Sortases are enzymes that are responsible for the attachment of secreted proteins to the cell wall of Gram-positive bacteria. Hereby, the sortases recognize short, five-residue amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is widely used in protein engineering for modification of target proteins. The majority of protocols rely on the high activity of the well-characterized Staphylococcus aureus SrtA and variants thereof, while sortases from other classes are not used for this purpose. This can be attributed to the lower activity of other sortases and to the limited sequence specificity data available for the different sortases. We set out to determine the sequence specificity of Bacillus anthracis SrtB. To this end, we developed a new method for sequence specificity determination of sortases or other bond-forming enzymes that recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed via liquid chromatography and mass spectrometry. With this, peptide sequences that are recognized by the sortase and function as substrates can be determined and quantified. The method, developed with the highly active evolved SrtA from S. aureus, allowed for the first time unbiased in-depth analysis of the sequence specificity for SrtB from B. anthracis, which is 104-fold less active than SrtA from S. aureus.

Rapid determination of β-lactam antimicrobial resistance in bacteria by a liquid chromatography-mass spectrometry-based method.

Conventional antimicrobial susceptibility tests (ASTs) are very time consuming and insufficiently precise to promptly select a proper antimicrobial treatment. This difficulty disrupts the management of infections and exacerbates the development of antimicrobial resistance. Generally, antimicrobial resistance involves the chemical modification of an antimicrobial compound to an inactive form by an enzyme released by bacteria. This modification causes a structural change and is followed by a characteristic mass shift of the antimicrobials. Using this mechanism, we developed a new liquid chromatography-mass spectrometry method to rapidly determine the degree of resistance of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium), Escherichia coli, and Staphylococcus aureus to amoxicillin, ampicillin, and penicillin G, respectively. This method was successfully applied to 20 bacterial isolates from Korean slaughterhouses and farms. There were 18-Da mass shifts in resistant strains compared with susceptible strains of Salmonella Typhimurium, E. coli, and S. aureus, and the intensities of the hydrolyzed penicillin mass spectra were much higher in resistant strains than those in susceptible strains, which together indicate the reliability of this method. A comparison of the mass spectrometry-derived results with that from conventional ASTs revealed an identical classification of the tested bacteria according to sensitivity and resistance. Notably, this assay method requires only 2h for determining the susceptibility status of a strain. This newly developed method is able to determine the extent of antimicrobial resistance qualitatively and quantitatively within a very short time and could be used to replace conventional AST methods. Graphical abstract Rapid determination of β-lactam antimicrobial resistance in bacteria by LC-MS/MS.

Roles of sphincter of ODDI laxity in bile duct microenvironment in patients with cholangiolithiasis: From the perspective of the microbiome and metabolome

Background Bile duct microenvironment plays several key roles in cholangiolithiasis occurrence. Sphincter of Oddi laxity (SOL) is associated with cholangiolithiasis, probably due to enhanced reflux of intestinal contents that changes the microenvironment. However, the microenvironment has not been investigated comprehensively. Study Design Patients with cholangiolithiasis were consecutively recruited and their bile was collected intraoperatively for high-throughput experiments. Pyrosequencing of 16S ribosomal RNA gene was performed to characterize the microbiota in the bile. A liquid chromatography mass spectrometry-based method was used to profile bile composition. Clinical manifestation, microbiome, and bile composition were compared between patients with and without SOL. Results Eighteen patients with SOL and 27 patients without SOL were finally included. Patients with SOL showed more severe inflammation. Bacteria in the bile duct were overwhelmingly aerobes and facultative anaerobes. Proteobacteria and Firmicutes were the most widespread phylotypes, especially Enterobacteriaceae . Compared with those without SOL, patients with SOL possessed more varied microbiota. In the SOL group, pathobionts, such as Bilophila and Shewanella algae had richer communities, and harmless bacteria were reduced. Metabolomics analysis showed the differences in bile composition between groups were mainly distributed in lipids and bile acids. Particularly, the increased abundance of Bilophila involved in taurine metabolism was associated with reduced contents of taurine derivatives in the bile of patients with SOL. Conclusions A bile duct microenvironment with more severe bacterial infection and stronger lithogenicity was found in patients with SOL. The findings suggest a possible mechanism of cholangiolithiasis and provide the basis for future strategies for prevention of cholangiolithiasis recurrence.

Curiouser and Curiouser: The Evolving Story of the Mechanisms Involved in Puberty.

Sometimes, like a giant jigsaw puzzle, concepts in endocrinology take time to develop, to form a coherent picture of how endocrine systems are integrated to support physiological responses. Such, it can be argued, has been the case with respect to our understanding of the mechanisms underlying puberty. For more than 50 years, it has been known that mammals experience a period of agonadal restraint during development, in which reproductive function is held in check (1). As puberty approaches, this restraint is switched off and pulsatile GnRH secretion is activated. What caused the juvenile period to end remained a matter of conjecture, although a shift in steroid feedback was suggested by observations showing that immature rats had enhanced sensitivity to the negative feedback effects of estradiol (2), whereas the onset of ovarian cyclicity could be advanced by prepubertal injections of estradiol (3). In the past 5 decades, several other important pieces of the puzzle have been filled in. The brain was itself found to be a site of gonadal steroid synthesis (4, 5). Data emerged to indicate that neurosteroid synthesis might contribute to reproductive function (6). Dramatic advances were made in understanding the neurotransmitter and neuropeptide circuitry responsible for regulating the onset of reproductive function, in particular the kisspeptin system (7, 8). However, what actually triggered the change in feedback control of gonadotrophin secretion at puberty remained obscure. A shift from a predominantly inhibitoryhypothalamic environment toone inwhichexcitatory neurotransmitters activated the GnRH neurons was believed to occur (9, 10), but what caused this shift remained unknown. In the current issue of Endocrinology, Kenealy et al (11) develop the novel idea that in female primates, the control of GnRH secretion before and during puberty may actually operate in a relatively similar hypothalamic steroidal milieu: only the source of the estradiol is different. To determine hypothalamic steroid concentrations, perfusates were collected in vivo from the stalk-median eminence (S-ME) region of 32 female rhesus monkeys at different stages of prepubertal and pubertal development. The samples were assayed for estradiol, estrone, testosterone, androstenedione, and progesterone, using a sensitive and specific liquid chromatography-mass spectrometry-based method. Concomitantly, GnRH in the perfusates, as well as LH and steroid levels in circulating serum samples from the same animals, were also assayed. As expected, GnRH and LH levels followed a similar developmental pattern, levels at midpuberty being higher than either prepuberty or early puberty. Circulating estradiol levels also were higher in midpuberty. The steroid measurements in the S-ME perfusates, however, exhibited a strikingly different pattern. Although androstenedione, testosterone, and progesterone did not change significantly in the perfusates across the different stages of puberty, estrone and estradiol were found to be high in the S-ME perfusates from prepubertal animals, declining in midpuberty and early puberty, respectively. Because serum estradiol levels increased over the same period, the S-ME to serum ratio fell dramatically in the midpubertal animals, more than 5-fold in the case of estradiol and more than 60-fold in the case of estrone. These results suggest that although the prepubertal period is indeed characterized by a period of GnRH inhibition that does not depend on ovarian hormone secretion, it is not in fact steroid independent. Rather, it may be mediated at least in part by estradiol produced in situ,

Roles of Sphincter of Oddi Laxity in Bile Duct Microenvironment in Patients with Cholangiolithiasis: From the Perspective of the Microbiome and Metabolome

Bile duct microenvironment plays several key roles in cholangiolithiasis occurrence. Sphincter of Oddi laxity (SOL) is associated with cholangiolithiasis, probably due to enhanced reflux of intestinal contents that changes the microenvironment. However, the microenvironment has not been investigated comprehensively. Patients with cholangiolithiasis were consecutively recruited and their bile was collected intraoperatively for high-throughput experiments. Pyrosequencing of 16S ribosomal RNA gene was performed to characterize the microbiota in the bile. A liquid chromatography mass spectrometry-based method was used to profile bile composition. Clinical manifestation, microbiome, and bile composition were compared between patients with and without SOL. Eighteen patients with SOL and 27 patients without SOL were finally included. Patients with SOL showed more severe inflammation. Bacteria in the bile duct were overwhelmingly aerobes and facultative anaerobes. Proteobacteria and Firmicutes were the most widespread phylotypes, especially Enterobacteriaceae. Compared with those without SOL, patients with SOL possessed more varied microbiota. In the SOL group, pathobionts, such as Bilophila and Shewanella algae had richer communities, and harmless bacteria were reduced. Metabolomics analysis showed the differences in bile composition between groups were mainly distributed in lipids and bile acids. Particularly, the increased abundance of Bilophila involved in taurine metabolism was associated with reduced contents of taurine derivatives in the bile of patients with SOL. A bile duct microenvironment with more severe bacterial infection and stronger lithogenicity was found in patients with SOL. The findings suggest a possible mechanism of cholangiolithiasis and provide the basis for future strategies for prevention of cholangiolithiasis recurrence.

Metabolomics and Natural-Products Strategies to Study Chemical Ecology in Nematodes.

This review provides an overview of two complementary approaches to identify biologically active compounds for studies in chemical ecology. The first is activity-guided fractionation and the second is metabolomics, particularly focusing on a new liquid chromatography–mass spectrometry-based method called isotopic ratio outlier analysis. To illustrate examples using these approaches, we review recent experiments using Caenorhabditis elegans and related free-living nematodes.