Targeted Liquid Chromatography-tandem Mass Spectrometry Research Articles

Proteomic assay for rapid characterisation of Staphylococcus aureus antimicrobial resistance mechanisms directly from blood cultures.

Staphylococcus aureus is one of the most common pathogens causing bloodstream infection. A rapid characterisation of resistance to methicillin and, occasionally, to aminoglycosides for particular indications, is therefore crucial to quickly adapt the treatment and improve the clinical outcomes of septic patients. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples. A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3')-III, ANT(4')-I, and AAC(6')-APH(2''), directly from positive blood cultures and in less than 70min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains. This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3')-III (n = 16/16), and ANT(4')-I (n = 20/20), and 94% for AAC(6')-APH(2'') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4')-Ia gene. The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.

Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Altered neopterin and IDO in kynurenine metabolism based on LC-MS/MS metabolomics study: Novel therapeutic checkpoints for type 2 diabetes mellitus

BackgroundThis study assessed the alternations of kynurenine pathway (KP) and neopterin in type 2 diabetes mellitus (T2DM) and explored possible differential metabolites. MethodsA fresh residual sera panel was collected from 80 healthy control (HC) individuals and 72 T2DM patients. Metabolites/ratios of interest including tryptophan (TRP), kynurenine (KYN), 5-hydroxytryptamine (5HT), kynurenic acid (KA), xanthurenic acid (XA), neopterin (NEO), KA/KYN ratio and KYN/TRP ratio were determined using a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics approach, and the difference between groups was assessed. Supervised orthogonal partial least squares-discriminant analysis and differential metabolite screening with fold change (FC) were performed to identify distinct biomarkers. The diagnostic performance of KP metabolites in T2DM was evaluated. ResultsSignificant decreases of TRP, 5HT, KA, XA, and KA/KYN and increases of KYN/TRP and NEO in T2DM compared to HC group were observed (P < 0.05). The KP metabolites panel significantly changed between T2DM and HC groups (Q2: 0.925, P < 0.005). 5HT (FC: 0.63, P < 0.01) and NEO (FC: 3.27, P < 0.01) were proven to be distinct differential metabolites. A combined testing of fasting plasma glucose and KYN/TRP showed good value in the prediction of T2DM (AUC: 0.904, 95% CI 0.843–0.947). ConclusionsThe targeted LC-MS/MS metabolomics study is a powerful tool for evaluating the status of T2DM. This study facilitated the application of KP metabolomics into future clinical practice. 5HT and NEO are promising biomarkers in T2DM. KYN/TRP was highly associated with the development of T2DM and may serve as a potential treatment target.

Urinary markers of Mycobacterium tuberculosis and dysbiosis in paediatric tuberculous meningitis cases undergoing treatment

BackgroundThe pathogenesis of tuberculous meningitis (TBM) involves infection by Mycobacterium tuberculosis in the meninges and brain. However, recent studies have shown that the immune response and inflammatory processes triggered by TBM can have significant effects on gut microbiota. Disruptions in the gut microbiome have been linked to various systemic consequences, including altered immunity and metabolic dysregulation. Inflammation caused by TBM, antibiotic treatment, and changes in host immunity can all influence the composition of gut microbes. This complex relationship between TBM and the gut microbiome is of great importance in clinical settings. To gain a deeper understanding of the intricate interactions between TBM and the gut microbiome, we report innovative insights into the development of the disease in response to treatment. Ultimately, this could lead to improved outcomes, management strategies and quality of life for individuals affected by TBM.MethodWe used a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach to investigate metabolites associated with gut metabolism in paediatric participants by analysing the urine samples collected from a control group (n = 40), and an experimental group (n = 35) with confirmed TBM, which were subdivided into TBM stage 1 (n = 8), stage 2 (n = 11) and stage 3 (n = 16).FindingsOur metabolomics investigation showed that, of the 78 initially selected compounds of microbiome origin, eight unique urinary metabolites were identified: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine as urinary markers of dysbiosis in TBM.ConclusionThese results – which are supported by previous urinary studies of tuberculosis – highlight the importance of gut metabolism and of identifying corresponding microbial metabolites as novel points for the foundation of improved management of TBM patients.

Determination of bile acids in serum of pigs exposed to polychlorinated biphenyls by liquid chromatography-mass spectrometry

Exposure to polychlorinated biphenyls (PCBs) has been linked to dyslipidemia. Under acute exposure to PCBs, it has been observed that the secretion of bile acids (BAs) can be impacted, limiting (indirectly) lipid absorption in the gut. In this context, two non-targeted metabolomics studies on pig serum have recently suggested that BA concentrations may fluctuate under exposure to current non-dioxin-like (NDL)-PCB levels in food, reflecting the acute effects of such chronic exposure. The objective of this research is to implement a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for BA analysis in order to validate the findings of previous metabolomics studies, in which BA levels in serum samples from pigs exposed to environmental doses of NDL-PCBs were highlighted to be affected. The proposed LC-MS method involves the use of a C18-pentafluorophenyl LC column, which is not usually selected for the separation of BAs, but shows better performance for the separation of isomers than typical C18 columns. This LC-MS method shows excellent analytical performance such as low limits of detection (LODs) (≤1 ng/mL for most BAs) and good linearity (R2 > 0.994), while no matrix effect was observed. A total of 13 BAs have been quantified, while further BA isomers could be detected and semi-quantified. The application of this targeted LC-MS method confirmed previous findings, suggesting that exposure to low doses of NDL-PCBs decreases the concentration of BAs (i.e., glycochenodeoxycholic acid, hyodeoxycholic acid and taurochenodeoxycholic acid) while the effect on the precursors (cholic acid and chenodeoxycholic acid) is less pronounced.

Repurposing dried blood spot device technology to examine bile acid profiles in human dried fecal spot samples.

Dried blood spot (DBS) analysis has existed for >50 years, but application of this technique to fecal analysis remains limited. To address whether dried fecal spots (DFS) could be used to measure fecal bile acids, we collected feces from five subjects for each of the following cohorts: 1) healthy individuals, 2) individuals with diarrhea, and 3) Clostridioides difficile-infected patients. Homogenized fecal extracts were loaded onto quantitative DBS (qDBS) devices, dried overnight, and shipped to the bioanalytical lab at ambient temperature. For comparison, source fecal extracts were shipped on dry ice and stored frozen. After 4 mo, frozen fecal extracts and ambient DFS samples were processed and subjected to targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics with stable isotope-labeled standards. We observed no differences in the bile acid levels measured between the traditional extraction and the qDBS-based DFS methods. This pilot data demonstrates that DFS-based analysis is feasible and warrants further development for fecal compounds and microbiome applications.NEW & NOTEWORTHY Stool analysis in remote settings can be challenging, as the samples must be stored at -80°C and transported on dry ice for downstream processing. Our work indicates that dried fecal spots (DFS) on Capitainer quantitative DBS (qDBS) devices can be stored and shipped at ambient temperature and yields the same bile acid profiles as traditional samples. This approach has broad applications for patient home testing and sample collection in rural communities or resource-limited countries.

Electrochemistry-mass spectrometry bridging the gap between suspect and target screening of valsartan transformation products in wastewater treatment plant effluent

Degradation of xenobiotics in wastewater treatment plants may lead to the formation of transformation products with higher persistence or increased (eco-)toxic potential compared to the parent compounds. Accordingly, the identification of transformation products from wastewater treatment plant effluents has gained increasing attention. Here, we show the potential of electrochemistry hyphenated to liquid chromatography and mass spectrometry for the prediction of oxidative degradation in wastewater treatment plants using the antihypertensive drug valsartan as a model compound. This approach identifies seven electrochemical transformation products of valsartan, which are used to conduct a suspect screening in effluent of the main wastewater treatment plant in the city of Münster in Germany. Apart from the parent compound valsartan, an electrochemically predicted transformation product, the N-dealkylated ETP336, is detected in wastewater treatment plant effluent. Subsequently, a targeted liquid chromatographytandem mass spectrometry method for the detection of valsartan and its electrochemical transformation products is set up. Here, electrochemical oxidation is used to generate reference materials of the transformation products in situ by hyphenating electrochemistry online to a triple quadrupole mass spectrometer. Using this setup, multiple reaction monitoring transitions are set up without the need for laborious and costly synthesis and isolation of reference materials for the transformation products. The targeted method is then applied to extracts from wastewater treatment plant effluent and the presence of ETP336 and valsartan in the samples is verified. The presented workflow can be used to set up targeted analysis methods for previously unknown transformation products even without the need for expensive high-resolution mass spectrometers.

Breeding from 1891 to 2010 did not increase the content of amylase/trypsin-inhibitors in wheat (Triticum aestivum)

The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951–2010). There were also no significant changes in ATI distribution.

Exploration of Blood Metabolite Signatures of Colorectal Cancer and Polyposis through Integrated Statistical and Network Analysis

Colorectal cancer (CRC), one of the most prevalent and deadly cancers worldwide, generally evolves from adenomatous polyps. The understanding of the molecular mechanisms underlying this pathological evolution is crucial for diagnostic and prognostic purposes. Integrative systems biology approaches offer an optimal point of view to analyze CRC and patients with polyposis. The present study analyzed the association networks constructed from a publicly available array of 113 serum metabolites measured on a cohort of 234 subjects from three groups (66 CRC patients, 76 patients with polyposis, and 92 healthy controls), which concentrations were obtained via targeted liquid chromatography-tandem mass spectrometry. In terms of architecture, topology, and connectivity, the metabolite-metabolite association network of CRC patients appears to be completely different with respect to patients with polyposis and healthy controls. The most relevant nodes in the CRC network are those related to energy metabolism. Interestingly, phenylalanine, tyrosine, and tryptophan metabolism are found to be involved in both CRC and polyposis. Our results demonstrate that the characterization of metabolite-metabolite association networks is a promising and powerful tool to investigate molecular aspects of CRC.

Association of food insecurity on gut microbiome and metabolome profiles in a diverse college-based sample

Voluntary caloric restriction (e.g., eating disorders) often results in alterations in the gut microbiota composition and function. However, these findings may not translate to food insecurity, where an individual experiences inconsistent access to healthy food options. In this study we compared the fecal microbiome and metabolome of racially and ethnically diverse first year college students (n = 60) experiencing different levels of food access. Students were dichotomized into food secure (FS) and food insecure (FI) groups using a validated, 2-question screener assessing food security status over the previous 30 days. Fecal samples were collected up to 5 days post survey-completion. Gut microbiome and metabolome were established using 16S rRNA amplicon sequencing, targeted liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry. FI students experienced significantly greater microbial diversity with increased abundance of Enterobacteriaceae and Eisenbergiella, while FS students had greater abundance of Megasphaera and Holdemanella. Metabolites related to energy transfer and gut–brain-axis communication (picolinic acid, phosphocreatine, 2-pyrrolidinone) were elevated in FI students (q < 0.05). These findings suggest that food insecurity is associated with differential gut microbial and metabolite composition for which the future implications are unknown. Further work is needed to elucidate the longitudinal metabolic effects of food insecurity and how gut microbes influence metabolic outcomes.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.

Association between Higher Circulating Leucine-Rich α-2 Glycoprotein 1 Concentrations and Specific Plasma Ceramides in Postmenopausal Women with Type 2 Diabetes.

Background: Although ceramides are involved in the pathophysiology of cardiovascular disease and other inflammation-associated disorders, there is a paucity of data on the association between plasma ceramides and inflammatory biomarkers in type 2 diabetes mellitus (T2DM). Therefore, we explored whether there was an association between plasma leucine-rich α-2 glycoprotein 1 (LRG1) concentrations (i.e., a novel proinflammatory signaling molecule) and specific plasma ceramides in postmenopausal women with T2DM. Methods: We measured six previously identified plasma ceramides, which have been associated with increased cardiovascular risk [plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)], amongst 99 Caucasian postmenopausal women with non-insulin-treated T2DM (mean age 72 ± 8 years, mean hemoglobin A1c 6.9 ± 0.7%), who consecutively attended our diabetes outpatient service during a 3-month period. Plasma ceramide and LRG1 concentrations were measured with a targeted liquid chromatography-tandem mass spectrometry assay and a Milliplex® MAP human cardiovascular disease magnetic bead kit, respectively. Results: In linear regression analyses, higher plasma LRG1 levels (1st tertile vs. 2nd and 3rd tertiles combined) were associated with higher levels of plasma Cer(d18:1/16:0) (standardized β coefficient: 0.289, p = 0.004), Cer(d18:1/18:0) (standardized β coefficient: 0.307, p = 0.002), Cer(d18:1/20:0) (standardized β coefficient: 0.261, p = 0.009) or Cer(d18:1/24:1) (standardized β coefficient: 0.343, p < 0.001). These associations remained significant even after adjusting for age, body mass index, systolic blood pressure, total cholesterol level, hemoglobin A1c, insulin resistance and statin use. Conclusions: The results of our pilot exploratory study suggest that higher plasma LRG1 concentration was associated with higher levels of specific high-risk plasma ceramide molecules in elderly postmenopausal women with metabolically well-controlled T2DM, even after adjusting for known cardiovascular risk factors and other potential confounding variables.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect β-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of β-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the β-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6′)-Ib, ANT(2)-I, and APH(3′)-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6′)-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Plasma ceramides independently predict all-cause mortality in men aged 85.

Backgroundassessing cardiovascular and mortality risk with conventional biomarkers is challenging in oldest-old due to multimorbidity and polypharmacy. Ceramides are bioactive lipids shown to predict mortality in late middle-aged cohorts.Objectiveto assess whether plasma ceramides have independent prognostic value for mortality among oldest-old (85+).Designlongitudinal cohort study (Helsinki Businessmen Study, HBS) with a 3.5-year follow-up.Setting and subjectssurvivors of HBS (125 men born in 1919–1934) visited the clinic for laboratory and clinical examination.Methodsfunctional status including physical (short physical performance battery) and Montreal Cognitive Assessment (MoCA) cognitive performance was assessed and laboratory examinations included a large set of biomarkers. Plasma ceramide concentration (Cer(d18:1/16:0)) was measured using a targeted liquid chromatography–tandem mass spectrometry assay. Mortality was retrieved from national registers.Resultsmedian age was 88 years, two-thirds had multimorbidity and 59% were on statin treatment. During the follow-up, 22 (18%) men died. In a model adjusted for variables associated with mortality in the whole cohort at P < 0.20 (log glucose, SPPB, MoCA and statin use), Cer(d18:1/16:0) as a continuous trait was associated with increased mortality: hazard ratio (HR) per 1 SD 1.64 (95% confidence interval [CI] 1.23–2.18). Compared with the bottom tertile of Cer(d18:1/16:0), HR of mortality was 5.44-fold (95% CI 1.17–25.3) in the top tertile.Conclusionsthese data raise the hypothesis that plasma ceramide concentrations and especially Cer(d18:1/1:60) may offer a clinically useful biomarker to evaluate prognosis in very old age. Such biomarkers are needed for geriatrics, where multimorbidity and pharmacotherapies, such as statins are prevalent hampering assessment of prognosis using conventional methods.

Microbiome and metabolome profiles of high screen time in a cohort of healthy college students

As screens are increasingly integrated into every facet of modern life, there is growing concern over the potential effects of high screen time. Previous studies have largely utilized self-report data on mood and behavioral aspects of screen time, and no molecular theory has yet been developed. In this study, we explored the fecal microbiome and metabolome of a diverse group of 60 college students, classified by high (≥ 75 min/day) or low (0–75 min/day) self-reported screen time using 16S rRNA amplicon sequencing, targeted liquid chromatography-tandem mass spectrometry, and targeted detection of short-chain fatty acids using gas chromatography-mass spectrometry. Several key taxa and metabolites were significantly altered between groups and found to be highly co-occurrent. Results of pathway and enzyme enrichment analyses were synthesized to articulate an integrated hypothesis indicating widespread mitochondrial dysfunction and aberrant amino acid metabolism. High screen time was also predicted to be significantly associated with type I diabetes, obesity, chronic fatigue syndrome, and various manifestations of inflammatory bowel. This is the first-ever study to report the effects of high screen time at the molecular level, and these results provide a data-driven hypothesis for future experimental research.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 β-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC β-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase. BlaCMY–2–like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like β-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

Association between KLF6 rs3750861 polymorphism and plasma ceramide concentrations in post-menopausal women with type 2 diabetes

Background and aimBased on the emerging role of Kruppel-like factor 6 (KLF6) in lipid metabolism, we examined whether there is a relationship between the KLF6 rs3750861 genetic variant and plasma ceramide levels in people with type 2 diabetes mellitus (T2DM). Methods and resultWe measured six previously identified plasma ceramides, which have been associated with increased cardiovascular risk [Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] amongst 101 Caucasian post-menopausal women with T2DM, who consecutively attended our diabetes outpatient service during a 3-month period. Plasma ceramides were measured by targeted liquid chromatography-tandem mass spectrometry assay. Genotyping of the KLF6 rs3750861 polymorphism was performed by TaqMan-Based RT-PCR system.Overall, 87 (86.1%) patients had KLF6 rs3750861 C/C genotype and 14 (13.9%) had C/T or T/T genotypes. After adjustment for age, diabetes-related variables, use of lipid-lowering drugs and other potential confounders, patients with C/T or T/T genotypes had higher plasma Cer(d18:1/18:0) (0.159 ± 0.05 vs. 0.120 ± 0.04 μmol/L, p = 0.012), Cer(d18:1/20:0) (0.129 ± 0.04 vs. 0.098 ± 0.03 μmol/L, p = 0.008), and Cer(d18:1/24:1) (1.236 ± 0.38 vs. 0.978 ± 0.36 μmol/L, p = 0.032) compared with those with C/C genotype. ConclusionsThe C/T or T/T genotypes of rs3750861 in the KLF6 gene were closely associated with higher levels of specific plasma ceramides in post-menopausal women with T2DM.

One-Carbon Metabolism in Alzheimer's Disease and Parkinson's Disease Brain Tissue.

Disruptions in one-carbon metabolism and elevated homocysteine have been previously implicated in the development of dementia associated with Alzheimer’s disease (AD) and Parkinson’s disease (PD). Moreover, a PD diagnosis itself carries substantial risk for the development of dementia. This is the first study that explores alterations in one-carbon metabolism in AD and PD directly in the human brain frontal cortex, the primary center of cognition. Applying targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS), we analyzed post-mortem samples obtained from 136 subjects (35 AD, 65 PD, 36 controls). We found changes in one-carbon metabolites that indicate inefficient activation of cystathionine β-synthase (CBS) in AD and PD subjects with dementia, the latter seemingly accompanied by a restricted re-methylation flow. Levodopa–carbidopa is known to reduce available vitamin B6, which would explain the hindered CBS activity. We present evidence of temporary non-protein-bound homocysteine accumulation upon levodopa intake in the brain of PD subjects with dementia but not in non-demented PD subjects. Importantly, this homocysteine elevation is not related to levodopa dosage, disease progression, or histopathological markers but exclusively to the dementia status. We hypothesize that this levodopa-induced effect is a direct cause of dementia in PD in susceptible subjects with reduced re-methylation capacity. Furthermore, we show that betaine best correlates with cognitive score even among PD subjects alone and discuss nutritional recommendations to improve one-carbon metabolism function.

Analysis of per- and polyfluoroalkyl substances in Houston Ship Channel and Galveston Bay following a large-scale industrial fire using ion-mobility-spectrometry-mass spectrometry

Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants of concern because of their ubiquitous presence in surface and ground water; analytical methods that can be used for rapid comprehensive exposure assessment and fingerprinting of PFAS are needed. Following the fires at the Intercontinental Terminals Company (ITC) in Deer Park, TX in 2019, large quantities of PFAS-containing firefighting foams were deployed. The release of these substances into the Houston Ship Channel/Galveston Bay (HSC/GB) prompted concerns over the extent and level of PFAS contamination. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based study of temporal and spatial patterns of PFAS associated with this incident revealed presence of 7 species; their levels gradually decreased over a 6-month period. Because the targeted LC-MS/MS analysis was focused on about 30 PFAS molecules, it may have missed other PFAS compounds present in firefighting foams. Therefore, we utilized untargeted LC-ion mobility spectrometry-mass spectrometry (LC-IMS-MS)-based analytical approach for a more comprehensive characterization of PFAS in these water samples. We analyzed 31 samples from 9 sites in the HSC/GB that were collected over 5 months after the incident. Our data showed that additional 19 PFAS were detected in surface water of HSC/GB, most of them decreased gradually after the incident. PFAS features detected by LC-MS/MS correlated well in abundance with LC-IMS-MS data; however, LC-IMS-MS identified a number of additional PFAS, many known to be components of firefighting foams. These findings therefore illustrate that untargeted LC-IMS-MS improved our understanding of PFAS presence in complex environmental samples.

Evaluation of potential metabolomic-based biomarkers of protein, carbohydrate and fat intakes using a controlled feeding study.

Objective biomarkers of dietary exposure are needed to establish reliable diet-disease associations. Unfortunately, robust biomarkers of macronutrient intakes are scarce. We aimed to assess the utility of serum, 24-h urine and spot urine high-dimensional metabolites for the development of biomarkers of daily intake of total energy, protein, carbohydrate and fat, and the percent of energy from these macronutrients (%E). A 2-week controlled feeding study mimicking the participants' habitual diets was conducted among 153 postmenopausal women from the Women's Health Initiative (WHI). Fasting serum metabolomic profiles were analyzed using a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for aqueous metabolites and a direct-injection-based quantitative lipidomics platform. Urinary metabolites were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy at 800MHz and by untargeted gas chromatography-mass spectrometry (GC-MS). Variable selection was performed to build prediction models for each dietary variable. The highest cross-validated multiple correlation coefficients (CV-R2) for protein intake (%E) and carbohydrate intake (%E) using metabolites only were 36.3 and 37.1%, respectively. With the addition of established dietary biomarkers (doubly labeled water for energy and urinary nitrogen for protein), the CV-R2 reached 55.5% for energy (kcal/d), 52.0 and 45.0% for protein (g/d, %E), 55.9 and 37.0% for carbohydrate (g/d, %E). Selected panels of serum and urine metabolites, without the inclusion of doubly labeled water and urinary nitrogen biomarkers, give a reliable and robust prediction of daily intake of energy from protein and carbohydrate.