Metabolomics of Human Semen: A Review of Different Analytical Methods to Unravel Biomarkers for Male Fertility Disorders.

BACKGROUND: Human life without sperm is not possible. Therefore, it is alarming that the fertilizing ability of human spermatozoa is continuously decreasing. The reasons for that are widely unknown, but there is hope that metabolomics-based investigations may be able to contribute to overcoming this problem. This review summarizes the attempts made so far. METHODS: We will discuss liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), infrared (IR) and Raman as well as nuclear magnetic resonance (NMR) spectroscopy. Almost all available studies apply one of these methods. RESULTS: Depending on the methodology used, different compounds can be detected, which is (in combination with sophisticated methods of bioinformatics) helpful to estimate the state of the sperm. Often, but not in all cases, there is a correlation with clinical parameters such as the sperm mobility. CONCLUSIONS: LC-MS detects the highest number of metabolites and can be considered as the method of choice. Unfortunately, the reproducibility of some studies is poor, and, thus, further improvements of the study designs are needed to overcome this problem. Additionally, a stronger focus on the biochemical consequences of the altered metabolite concentrations is also required.

Differences in the sperm metabolomes of smoking and nonsmoking men†.

Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.

[An Update on the Epidemiological Situation of Alveolar Echinococcosis: Recording and Reporting Structures in Austria, Switzerland and Germany]. / Epidemiologisches Update: Aktuelles zur epidemiologischen Situation der alveolären Echinokokkose und deren Erfassungs- und Meldestrukturen in Österreich, der Schweiz und Deutschland.

OBJECTIVE: Alveolar echinococcosis (AE) is a rare disease in Austria, Switzerland and Germany (DACh) caused by an infection with the parasite Echinococcus multilocularis. The aim of the study was to describe differences in the detection and reporting systems of alveolar echinococcosis in Austria, Switzerland and Germany and to describe epidemiological trends. METHODOLOGY: As part of an epidemiological update on 6th September 2019 in Ulm, Germany, experts and representatives discussed differences in the reporting and recording systems as well as the current epidemiological situation. RESULTS: Since 2004, Austria has had an obligation to report suspected cases, diseases and deaths of alveolar echinococcosis by name in accordance with §1 Para. 1 of the Epidemiegesetz 1950 (EpidemieG) and the Ordinance on Notifiable Communicable Diseases. According to §7 Para. 3 of the German Infection Protection Act (IfSG), Germany has also been subject to a reporting obligation since 2001, but not by name. In addition, national registers are available in both countries, which can be used to answer scientific questions. In Switzerland, there is no obligation to report human alveolar echinococcosis since 1997. Efforts are currently being made to implement a national register for alveolar echinococcosis in Switzerland. Despite different reporting and recording systems, a similar epidemiological trend can be observed for DACh. CONCLUSIONS: In Austria, Switzerland and Germany there is a slightly increasing trend of human cases with alveolar echinococcosis. The direct comparability is limited due to different reporting obligations. The structures often do not allow a joint answering of scientific questions concerning diagnostics, treatment and care.

Worldwide literature on epidemiology of human alveolar echinococcosis: a systematic review of research published in the twenty-first century.

PURPOSE: Human alveolar echinococcosis (AE) is a potentially lethal zoonosis caused by the cestode Echinococcus multilocularis. The aim of this systematic review is to establish a comprehensive global AE literature overview taking into account the epidemiologically relevant AE research of the twenty-first century. METHODS: We systematically searched the global literature published from 2001 through 2018 via MEDLINE, EMBASE, the Russian databases eLIBRARY.RU, CyberLeninka, the Chinese databases CNKI, VIP, Journals. RESEARCH: ac.ir (Farsi language-based), Jordan E-Library (Arab language-based) and supplementary Google Scholar, in accordance with the PRISMA guidelines. QGIS software was used for the mapping of the affected countries. RESULTS: We have listed 154 relevant publications in the final literature synopsis in consideration of our quality assessment. Including non-autochthonous cases, human AE was reported in 36 countries within the northern hemisphere from 2001 to 2018. The first publication of AE in Tajikistan, Pakistan, South Korea, Belgium, the Netherlands, Slovakia, Hungary, Lithuania, Latvia, Slovenia and Morocco occurred in this century; further first cases in Taiwan, Thailand, and Denmark were considered to be non-autochthonous by the authors. The highest total case numbers (n ≥ 100 in a single article) were reported in France, Germany, Switzerland, Poland, and Lithuania, including China and Kyrgyzstan with by far the highest prevalence figures. CONCLUSIONS: Our paper emphasises the increasing spread of reported cases and the rise in its numbers in the literature of the twenty-first century, especially in western, northern and eastern Europe, as well as in central Asia. Epidemiological studies on human infections are lacking in many parts of the world.

[Implementation of ultrasound education in the medical degree curriculum - the Ulm model]. / Implementierung der Ultraschallausbildung in das Kerncurriculum des Medizinstudiums ­ das Ulmer Modell.

Ultrasound is the most commonly used diagnostic method in everyday clinical practice aside from conventional radiography. For prospective physicians, there is a gap between the increased requirements in everyday clinical practice and the skills acquired thus far, especially when dealing with sonography. In this work, we would like to present the structure of our sonography course and its implementation in the Ulm core curriculum of medical studies. The sonography course is based on two pillars. The Sonography Seminar (Pillar 1) has a lecturer who regularly conveys interdisciplinary theoretical content, fundamentals and possible fields of application for sonography during 13 appointments of 45 minutes. The internship (Pillar 2) consists of four appointments of two hours each. The groups of four students are supervised by a student tutor. In addition to the technical basics and an introduction to the device customer, the organ systems liver, gallbladder and biliary tract, kidneys and adrenal glands, spleen, pancreas, abdominal vessels and lymph nodes are mediated. The focus is on the realistic exercises with high-end ultrasound equipment used in small groups with a final practical test. The structure and procedure of the course was evaluated as being very positive by the students.

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF-7 Cells.

The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17ß-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes in vitro. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd2+). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd2+. Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound's estrogenic potency. Co-exposure to Cd2+ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd2+-specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the complementary use of metabolomics and cellular and molecular assays.

Attenuation of Postoperative Acute Liver Failure by Mesenchymal Stem Cell Treatment Due to Metabolic Implications.

OBJECTIVE: To prevent posthepatectomy acute liver failure after extended resection by treatment with mesenchymal stem cells (MSCs). BACKGROUND: Liver tumors often require extended liver resection, overburdening metabolic and regenerative capacities of the remnant organ. Resulting dysfunction and failure may be improved by the proregenerative characteristics of MSCs. METHODS: Extended liver resection was performed in (DPPIV)-deficient F344-Fischer rats. Wild-type animals served as donors of peritoneal adipose-derived MSCs. These were predifferentiated in vitro into hepatocytic cells and delivered to the liver by splenic application. Liver-related blood parameters (international normalized ratio, bilirubin, aspartate aminotransferase, alanine aminotransferase) and liver histology (hematoxylin-eosin, Sudan III) were determined to monitor liver function. Metabolic changes were assessed by metabolomic analyses in the remnant liver and the serum. Liver damage and regeneration were quantified by determination of the apoptotic and proliferation rates. RESULTS: MSCs supported survival after partial hepatectomy. They decreased liver-related blood parameters indicative for the improvement of liver function. The extensive lipid accumulation in hepatocytes illustrating the metabolic overload after resection was attenuated. Treatment with MSCs normalized imbalance of amino acids, acylcarnitines, sphingolipids, and glycerophospholipids in the liver and blood. Furthermore, MSCs decreased the apoptotic rate and increased the proliferation rate. The experimental time period (48 hours) was too short to allow for integration of MSCs into the host liver. Thus, the mode of action was probably indirect. CONCLUSIONS: MSCs ameliorated hepatic dysfunction and improved liver regeneration after extended resection by paracrine mechanisms. They may represent a new therapeutic option to treat posthepatectomy acute liver failure.

Optimization of metabolomics of defined in vitro gut microbial ecosystems.

The metabolic functionality of a microbial community is a key to the understanding of its inherent ecological processes and the interaction with the host. However, the study of the human gut microbiota is hindered by the complexity of this ecosystem. One way to resolve this issue is to derive defined communities that may be cultured ex vivo in bioreactor systems and used to approximate the native ecosystem. Doing so has the advantage of experimental reproducibility and ease of sampling, and furthermore, in-depth analysis of metabolic processes becomes highly accessible. Here, we review the use of bioreactor systems for ex vivo modelling of the human gut microbiota with respect to analysis of the metabolic output of the microbial ecosystem, and discuss the possibility of mechanistic insights using these combined techniques. We summarize the different platforms currently used for metabolomics and suitable for analysis of gut microbiota samples from a bioreactor system. With the help of representative datasets obtained from a series of bioreactor runs, we compare the outputs of both NMR and mass spectrometry based approaches in terms of their coverage, sensitivity and quantification. We also discuss the use of untargeted and targeted analyses in mass spectroscopy and how these techniques can be combined for optimal biological interpretation. Potential solutions for linking metabolomic and phylogenetic datasets with regards to active, key species within the ecosystem will be presented.

The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk.

Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality.

Pathway and time-resolved benzo[a]pyrene toxicity on Hepa1c1c7 cells at toxic and subtoxic exposure.

Benzo[a]pyrene (B[a]P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 µM) or a subtoxic (50 nM) concentration of B[a]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B[a]P, which was followed by quantitative analysis of the concentration of intracellular B[a]P and seven representative degradation products. Exposure with 5 µM B[a]P led to a maximal intracellular concentration of 1604 pmol/5 × 10(4) cells, leveling at 55 pmol/5 × 10(4) cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B[a]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 µM), and 150 (only 50 nM) proteins were found to be regulated in response to B[a]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B[a]P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B[a]P specific effects.

High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry.

The constitutive activity of the ghrelin receptor is of high physiological and pathophysiological relevance. In-depth structure-activity relationship studies revealed a palmitoylated ghrelin receptor ligand that displays an in vitro binding affinity in the low nanomolar range. Activity studies revealed inverse agonistic as well as antagonistic properties and in vitro metabolic analysis indicated a high stability in blood serum and liver homogenate. For metabolic testing in vivo, a combined approach of stable isotopic labeling and mass spectrometry-based analysis was established. Therefore, a heavy isotopic version of the peptide containing a (13)C-labeled palmitic acid was synthesized and a 1:1 ratio of a (12)C/(13)C-peptide mixture was injected into rats. Biological samples were analyzed by multiple reaction monitoring allowing simultaneous peptide detection and quantification. Measurements revealed a suitable bioavailability over 24h in rat serum and subsequent high-resolution mass spectrometry investigations showed only negligible degradation and slow body clearance. Hence, this method combination allowed the identification and evaluation of a highly potent and metabolically stable ghrelin receptor ligand in vivo.

Effects of ceftiofur treatment on the susceptibility of commensal porcine E.coli--comparison between treated and untreated animals housed in the same stable.

BACKGROUND: Healthy farm animals have been found to act as a reservoir of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli). Therefore, the objective of the study was to determine the input of antimicrobial active ceftiofur metabolites in the stable via faeces and urine after intramuscular administration of the drug to pigs and the elucidation of the Escherichia coli ESBL resistance pattern of treated and untreated pigs housed in the same barn during therapy. METHODS: For determination of the minimal inhibitory concentration (MIC) the method of microdilutionaccording to the recommended procedure of the Clinical and Laboratory Standards Institute was used. Inaddition to that, a qualitative determination was performed by agar dilution. Unsusceptible E. coli speciesselected via agar dilution with cefotaxime were confirmed by MALDI-TOF and ESBL encoding genes wereidentified by PCR. The amounts of ceftiofur measured as desfuroylceftiofur (DFC) in the different probes (plasma, urine, faeces and dust) were analysed by UPLC-MS/MS. RESULTS: In a first experiment two groups of pigs (6 animals per group) were housed in the same barn in two separated boxes. One group (group B) were treated with ceftiofur according to the licence (3 mg/kg administered intramuscularly (i.m.) on three consecutive days, day 1-3). During a second treatment period (day 29-31) an increased rate of ESBL resistant E. coli was detectable in these treated pigs and in the air of the stable. Moreover, the second group of animals (group A) formerly untreated but housed for the whole period in the same stable as the treated animals revealed increased resistance rates during their first treatment (day 45-47) with ceftiofur. In order to investigate the environmental input of ceftiofur during therapy and to simulate oral uptake of ceftiofur residues from the air of the stable a second set of experiments were performed. Pigs (6 animals) were treated with an interval of 2 weeks for 3 days with different doses of ceftiofur (3 mg/kg, 1 mg/kg and 0.3 mg/kg i.m.) as well as with 3 mg/kg per os) and the renal and biliary excretion of ceftiofur as its active metabolite were measured in comparison to the plasma levels. In addition to that, probes of the sedimentation dust and the air of the stable were analysed for drug residues. CONCLUSION: The present study shows that treatment of several animals in a stable with ceftiofur influences the resistance pattern of intestinal Escherichia coli of the treated as well as untreated animals housed in the same stable. During therapy with the drug which was administered by injection according to the licence we detected nameable amounts of ceftiofur and its active metabolites in the dust and air of the stable.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Identification of lipidomic biomarkers for coexposure to subtoxic doses of benzo[a]pyrene and cadmium: the toxicological cascade biomarker approach.

The search for model bioassay systems indicating activation of different toxicological signaling pathways is one of the paramount goals of modern toxicology. Especially coexposure scenarios need to be investigated with respect to synergistic and interdependent effects for the activation of toxicological signaling pathways. The present study introduces an experimental in vitro model system for nontoxic and low-dose coexposures of human mammary carcinoma MCF-7 cells against polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP) and heavy metals such as cadmium. For the first time, a multivariate model that identifies 18 metabolic biomarkers has been shown to be sufficient to separate BP-treated cells from coexposed or control cells. A "toxicological pathway color code model" is introduced to visualize the results. Different biomarker subsets can be associated with specific HER2 signaling steps. A tiered cascade biomarker approach is proposed that could be used to identify profiles associated with tumorigenic potency of environmental toxicants in coexposure scenarios, including possible synergistic or additive effects.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM).

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Metabolomic biomarkers correlating with hepatic lipidosis in dairy cows.

BACKGROUND: Hepatic lipidosis or fatty liver disease is a major metabolic disorder of high-producing dairy cows that compromises animal performance and, hence, causes heavy economic losses worldwide. This syndrome, occurring during the critical transition from gestation to early lactation, leads to an impaired health status, decreased milk yield, reduced fertility and shortened lifetime. Because the prevailing clinical chemistry parameters indicate advanced liver damage independently of the underlying disease, currently, hepatic lipidosis can only be ascertained by liver biopsy. We hypothesized that the condition of fatty liver disease may be accompanied by an altered profile of endogenous metabolites in the blood of affected animals. RESULTS: To identify potential small-molecule biomarkers as a novel diagnostic alternative, the serum samples of diseased dairy cows were subjected to a targeted metabolomics screen by triple quadrupole mass spectrometry. A subsequent multivariate test involving principal component and linear discriminant analyses yielded 29 metabolites (amino acids, phosphatidylcholines and sphingomyelines) that, in conjunction, were able to distinguish between dairy cows with no hepatic lipidosis and those displaying different stages of the disorder. CONCLUSIONS: This proof-of-concept study indicates that metabolomic profiles, including both amino acids and lipids, distinguish hepatic lipidosis from other peripartal disorders and, hence, provide a promising new tool for the diagnosis of hepatic lipidosis. By generating insights into the molecular pathogenesis of hepatic lipidosis, metabolomics studies may also facilitate the prevention of this syndrome.

Does the Quantification of Δ9-Tetrahydrocannabinolic Acid A in Serum/Plasma Provide Any Additional Information About Consumption Pattern from Drivers Under the Influence of Cannabis?

Introduction: Δ9-tetrahydrocannabinolic acid A (THCA-A) is one of the main ingredients of cannabis plants and is converted to the psychoactive substance Δ9-tetrahydrocannabinol (THC) by decarboxylation during heating above ∼90°C. During the consumption of cannabis, a varying proportion of THCA-A is absorbed into the body. Therefore, the quantification of THCA-A in serum/plasma might provide additional information on consumption behavior in driving under the influence of cannabis cases. Materials and Methods: In this study, an already established gas-chromatography mass-spectrometry (GC-MS) method for the quantification of THC, 11-OH-THC, and THC-COOH in serum and plasma samples was extended to include THCA-A. This validated method was then applied to 1228 routinely achieved serum/plasma samples from drivers suspected of cannabis consumption in Western Saxony. Two different grouping systems for chronic/occasional consumption, one system for acute/subacute consumption, Huestis formulas, and the cannabis influence factor (CIF) were used for evaluation. Results: Method validation showed appropriate results for forensic toxicological routine analysis. Limit of detection and lower limit of quantification (LLOQ) for THCA-A were 0.3 and 1.0 ng/mL, respectively. Reproducibility was <11% and accuracy ranged between 104% and 107%. THCA-A was stable in native samples at least for 2 weeks at room temperature or 4°C as well as 1 month at -20°C. Freeze-thaw stability for three cycles and processed sample stability over 3 days was proven. A total of 865 cases with a THC concentration above the German analytical cutoff of 1 ng/mL as well as the analytical LLOQs of 0.9 and 2.5 ng/mL for 11-OH-THC and THC-COOH, respectively, were included in further statistical analysis. In 407 (47.1%) of these samples, THCA-A was quantifiable. Different statistical analyses indicated a correlation between THCA-A and THC concentrations in cases of chronic and acute consumption. In addition, an increase of chronic and acute cases with increasing THCA-A concentrations was observed. However, no correlation between THCA-A and CIF was found. Discussion: These data show that THCA-A might be an additional indicative marker to provide information about consumption frequency and acuteness. Additional studies with known consumption frequencies and times are required to verify these findings.