Podcasts about lc ms ms

Dr. Michelle Peace is an internationally recognized forensic toxicologist and a Full Professor in the Department of Forensic Science at Virginia Commonwealth University. The National Institute of Justice has funded her team to study the proliferation of semi-synthetic THC analogs in the unregulated market and define their relevant biomarkers. Her work impacts public health and public safety policies and initiatives. At CannMed 25 Michelle will present “Why Some People “Green Out”? The Analysis of Unregulated “Hemp-derived” Cannabis Products”. Her team used a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze unregulated hemp products. All the products they analyzed were mislabeled in some way, either misrepresenting the cannabinoids contained therein or their concentrations.  During our conversation we discuss What has caused the rise of semi-synthetic cannabinoids in the market?  How Michelle got involved testing unregulated hemp and cannabis products  Some of the adverse events that have occured from consuming semi-synthetic cannabinoids  How the LS-MS/MS method allows Michelle's team to see compounds other labs can't   How consumers can protect themselves from mislabeled products Whether federal legalization of cannabis would eliminate the semi-synthetic market  Thanks to This Episode's Sponsor: PRICH Biotech PRICH Biotech, Corp. is a vertically integrated company dedicated to the cultivation, manufacture and dispensing of medicinal Cannabis in Puerto Rico.  With over 500,000 square feet of state-of-the-art facilities, Prich uses the highest standards of agricultural and manufacturing practices to guarantee the highest standard of medicinal cannabis. Their mission is to offer a natural and unique experience through medicinal cannabis that raises the patient's well-being and quality of life. Learn more at prichbiotech.com.  Additional Resources Register for CannMed 25 Meet the CannMed 25 Speakers Review the Podcast CannMed Archive

We look at 5 recent publications in the field of toxicology.Ning He, et al.  Contextual bias in forensic toxicology decisions: A follow-up empirical study from China. (2024) Journal of Forensic Sciences, doi: 10.1111/1556-4029.15520.Kesmen, E. et al. Bioinformatics-driven untargeted metabolomic profiling for clinical screening of methamphetamine abuse. (2024) Forensic Toxicology, doi: 10.1007/s11419-024-00703-2.Schuller, M. et al. Electromembrane extraction of drugs of abuse and prescription drugs from micropulverized hair. (2024) Journal of Analytical Toxicology, doi: 10.1093/jat/bkae051.Schackmuth, M. et al. Identification of fentanyl analogs and potential biomarkers in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS). (2024) Journal of Chromatography B, doi: 10.1016/j.jchromb.2024.124303.Trobbiani, S. et al. Increasing the linear dynamic range in LC-MS: is it valid to use a less abundant isotopologue? (2017) Drug Testing and Analysis, doi: 10.1002/dta.2175You can try out NotebookLM yourself at https://notebooklm.google/Contact us at toxpod@tiaft.orgFind out more about TIAFT at www.tiaft.orgThe Toxpod is a production of The International Association of Forensic Toxicologists. The opinions expressed by the hosts are their own and do not necessarily reflect the views of TIAFT or their employers.You can send us a text message using this link!

Xylazine Pharmacokinetics in Patients Testing Positive for Fentanyl and Xylazine  Clinical Chemistry This study of xylazine pharmacokinetics used plasma samples from 28 patients who had urine screens positive for xylazine and fentanyl. The patients were being treated for skin lesions, most commonly, then shortness of breath or opioid overdose. At least two subsequent plasma samples were analyzed for xylazine and xylazine metabolites by LC-MS/MS. The median terminal half-life for xylazine in plasma was 12 hours (range 6-21 hours). Animal studies show xylazine to be extensively metabolized, with little unchanged xylazine eliminated in urine. The two most abundant metabolites were oxo-x and sulfone-x, which did not have a window of detection longer than xylazine. Researchers had no information as to the timing or route of xylazine ingestion or if additional xylazine was consumed during the study period. These factors could affect the accuracy of the results.   Read this issue of the ASAM Weekly Subscribe to the ASAM Weekly Visit ASAM

Jessica Kennedy, MD, Infectious Diseases Fellow at the University of South Florida Division of Infectious Diseases, discusses the scientific basics around many of the primary Infectious Diseases tests ordered by providers. Dr. Kennedy discusses the scientific basis for EIA/ELIZA, Quantiferon, chemiluminant immunoassays, lateral flow assays, RT-PCR, Fungitell, and LC/MS-MS. In explaining these diagnostic assays, Dr. Kennedy helps the provider understand the limitations and advantages of each test and when they are best used in a clinical scenario.

In this episode of The New Frontier podcast, we're joined by Daniel Schulz-Jander, Senior Director of Mass spectrometry Bioanalysis at QPS Netherlands (Groningen). Daniel explains how he uses immune-precipitation and immunoaffinity LC–MS techniques for his work relating to macromolecular pharmaceuticals like gene therapies, as well as their benefits and challenges. Daniel also covers bottom-up, middle-down and top-down approaches and their suitability for his work in clinical support versus research and discovery.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.549867v1?rss=1 Authors: Zubizarreta, L., Jalabert, C., Silva, A. C., Soma, K. K., Quintana, L. Abstract: Steroids play a crucial role in modulating brain and behavior. While traditionally it is considered that the brain is a target of peripheral hormones produced in endocrine glands, it has been discovered that the brain itself produces steroids, known as neurosteroids. Neurosteroids can be produced in brain regions involved in the regulation of social behaviors and can act locally regulating behaviors like reproduction and aggression. Here, for the first time in a teleost fish, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify a panel of 8 steroids in both plasma and brain. We use the weakly electric fish Gymnotus omarorum, a species which shows non-breeding aggression in both sexes, to characterize these hormonal profiles in wild non-breeding adults. We show that: 1) systemic steroids in the non-breeding season are similar in both sexes, although only males have circulating 11-KT, 2) brain steroid levels are sexually dimorphic, as females display higher levels of AE, T and E1, and only males had 11-KT, 3) systemic androgens such as AE and T in the non-breeding season are potential precursors for neuroestrogen synthesis, and 4) estrogens, which play a key role in non-breeding aggression, are detectable in the brain (but not the plasma) in both sexes. These data fall in line with previous reports in G. omarorum which show that non-breeding aggression is dependent on the estrogenic pathway, as has also been shown in bird and mammal models. Overall, our results constitute a fundamental groundwork to understanding the complexity of hormonal modulation, its potential sex differences, the role of neurosteroids and the interplay between central and peripheral hormones in the regulation of behaviors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546157v1?rss=1 Authors: Karnam, S., Maurya, S., Ng, E., Choudhary, A., Thobani, A., Flanagan, J. G., Gronert, K. Abstract: Purpose: Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to its neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. However, regulation of lipoxin formation and cellular targets for their neuroprotective activity in glaucoma remain to be defined. We investigated if ocular hypertension and inflammatory cytokines regulate astrocyte lipoxin pathway and if LXB4 can regulate astrocyte reactivity. Design: Experimental study. Subjects/Participants/Controls: C57BL/6J mice were administered silicon oil into the anterior chamber to induce ocular hypertension (n=40). Age and gender-matched mice served as control subjects (n=40). Methods: RNAscope in-situ hybridization, RNA-seq, and qPCR to analyze gene expression. LC/MS/MS lipidomics to assess functional expression of the lipoxin pathway. Retinal flat mounts and immunohistochemistry (IHC) to assess macroglia reactivity. OCT quantified the retinal layer thickness in vivo, and ERG assessed retinal function. Primary human brain astrocytes were used for in vitro reactivity experiments. Non-human primate optic nerves were used to assess gene and functional expression of the lipoxin pathway. Main Outcome Measures: Intraocular pressure, RGC function, OCT measurements, gene expression, in situ hybridization, lipidomic analysis, and IHC. Results: Gene expression and lipidomic analysis established functional expression of the lipoxin pathway in the mouse retina, optic nerve of mice and primates, as well as human brain astrocytes. Ocular hypertension caused significant dysregulation of this pathway, with an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-lipoxygenase activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity in the mouse retina. Reactive human brain astrocytes also showed a marked increase in 5-LOX. Administration of LXB4 regulated the lipoxin pathway, restored and amplified LXA4 generation, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. Conclusions: The lipoxin pathway is functionally expressed in retina and brain astrocytes, optic nerves of rodents and primates, a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4 neuroprotective action is inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539472v1?rss=1 Authors: Sangster, M., Shahriar, S., Niziolek, Z., Carisi, M. C., Lewandowski, M., Budnik, B., Grishchuk, Y. Abstract: 1 Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1, the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1-/- mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC-MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1-/- mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1-/- tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1-/- brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537143v1?rss=1 Authors: Manolis, D., Hasan, S., Ettelaie, C., Maraveyas, A., O'Brien, D. P., Kessler, B. M., Kramer, H. B., Nikitenko, L. L. Abstract: Background: G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) signalling is implicated in skin-related and cardiovascular diseases, migraine and cancer. However, beyond its agonists and receptor activity-modifying proteins (RAMPs), proteins which bind to CLR and define its properties in primary human cells remain insufficiently understood. Aim: We aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed. Materials and methods: Immunoprecipitation (IP) of core- and terminally-glycosylated CLR from primary in vitro cultured HDLEC was conducted using rabbit polyclonal anti-human CLR serum (with pre-immune serum serving as a control) and confirmed by immunoblotting. Total HDLEC and co-immunoprecipitated CLR proteomes were analysed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative in-situ proximity ligation assay (PLA) using ZEISS LSM 710 confocal microscope and ZEN Blue 3.0 and Image J software was performed to confirm LC-MS/MS findings. All experiments were repeated at least three times (biological replicates). For statistical analysis of PLA data, distribution was analysed using Shapiro-Wilk normality test followed by an unpaired t-test or Mann-Whitney test with a p-value of less than or equal to 0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed using t-test with a permutation-based false discovery rate (FDR)-adjusted p-value of less than or equal to 0.006 interpreted as significant. Results: A total of 4,902 proteins were identified and quantified by LC-MS/MS in primary HDLEC and 46 were co-immunoprecipitated with CLR (p less than 0.006). Direct interaction with the GPCR was confirmed for five of these by PLA (p less than 0.01). Conclusions: This is the first study of its kind to identify novel binding partners of CLR expressed in primary human cells. Our integrative quantitative approach, combining immunoprecipitation of core- and terminally-glycosylated CLR, LC-MS/MS, and PLA, could be applied in a similar fashion to study its interactome in a variety of human cells and tissues, and its contribution to a range of diseases, where the role of this GPCR is implicated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Special guests Dawn Duffield, PhD of KCAS and Barry Jones, PhD of Crinetics join John and Dom in the 66th episode of "The Weekly Bioanalysis" to discuss the ways Hybrid LC-MS/MS has advanced in recent years and how the technology has gone from "new" and somewhat obscure to much more mainstream as a leading option for program success within the pharmaceutical and biopharmaceutical industries."The Weekly Bioanalysis" is a podcast dedicated to discussing Bioanalytical news, tools and services related to the Pharmaceutical, Biopharmaceutical and Biomarker industries. Every month, KCAS will bring you another 60 minutes (or so) of friendly banter between our two finest Senior Scientific Advisors as they chat over coffee and discuss what they've learned about the Bioanalytical world the past couple of weeks. The Weekly Bioanalysis is brought to you by KCAS.KCAS is a progressive growing contract research organization of well over 250 talented and dedicated individuals with growing operations in Kansas City, Doylestown, PA, Milan, Italy, and Lyon, France, where we are committed to serving our clients and improving health worldwide. Our experienced scientists provide stand-alone bioanalytical services to the Pharmaceutical, Biopharmaceutical, Animal Health and Medical Device industries.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.01.530696v1?rss=1 Authors: Becker-Krail, D., Ketchesin, K. D. D., Xue, X., Wilson, R., Lam, T. T., Williams, K., Nairn, A., Tseng, G., Logan, R. W. Abstract: Substance use disorders (SUDs) are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based (LC-MS/MS) quantitative proteomics, along with a data-independent acquisition (DIA) analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveals cocaine and morphine differentially alters diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.530344v1?rss=1 Authors: Arime, Y., Saitoh, Y., Ishikawa, M., Kamiyoshihara, C., Uchida, Y., Fujii, K., Takao, K., Akiyama, K., Ohkawa, N. Abstract: BACKGROUND: One of the critical unmet medical needs in schizophrenia is a remedy for cognitive deficits. However, the neural circuit mechanisms of them remain unresolved. In addition, despite the patients with schizophrenia cannot stop taking antipsychotics due to a high rate of discontinuation-induced relapse, previous studies using animal models of schizophrenia have not considered these clinical situations. METHODS: Here, we employ multi-dimensional approaches, including histological analysis in the prelimbic cortex, LC-MS/MS-based in vivo dopamine D2 receptor occupancy analysis for antipsychotic drugs, in vivo calcium imaging and behavioral analyses of mice using chemogenetic manipulation, to investigate neural mechanisms and potential therapeutic interventions for working memory deficit in a mouse model with chronic phencyclidine (PCP) administration that resembles the schizophrenia symptomatology. RESULTS: Chronic PCP administration led to abnormalities in excitatory and inhibitory synapses, including dendritic spines of pyramidal neurons, vesicular glutamate transporter 1 (VGLUT1) positive terminals, and parvalbumin (PV) positive GABAergic interneurons, in layer 2-3 of the prelimbic cortex. Continuous olanzapine, which achieved a sustained therapeutic window of dopamine D2 receptor occupancy (60-80%) in the striatum, did not affect these synaptic abnormalities and working memory deficit in the PCP-treated mice. We found that the selective prelimbic PV activation, using hM3D(Gq)-DREADD system confirmed by in vivo calcium imaging, restored working memory deficit, even under continuous olanzapine treatment. CONCLUSIONS: Our study raises a possibility that intervention in prefrontal PV neurons leads to an add-on therapy to antipsychotics targeting amelioration of treatment-resistant cognitive deficits in schizophrenia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.30.526247v1?rss=1 Authors: Turlo, A. J., Hammond, D. E., Ramsbottom, K. A., Soul, J., Gillen, A., McDonald, K., Peffers, M. J., Clegg, P. D. Abstract: Variation in Mesenchymal Stromal Cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs is primarily attributed to secretion of various biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome composition remains largely unknown. Here, we examined the influence of donor age, sex and tissue source, on the protein profile of the equine MSC secretome. Initially, we used dynamic metabolic labelling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically-secreted based on the rate of isotope label incorporation into newly synthesised proteins released into the extracellular space. Next, we analysed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS proteomics. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of two proteins (CTHRC1, LOX), which has been validated with western blot and enzymatic activity assay. There was limited evidence of sex-related differences in protein abundance. In conclusion, this study provides evidence that tissue source and donor age contribute to heterogeneity in the protein composition of MSC secretomes which may influence the effects of MSC-based cell therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.515175v1?rss=1 Authors: Obis, E., Sol, J., Andres-Benito, P., Martin-Gari, M., Mota-Martorell, N., Galo-Licona, J. D., Pinol-Ripoll, G., Portero-Otin, M., Ferrer, I., Jove, M., Pamplona, R. Abstract: Aims: Non-targeted lipidomics analysis was conducted in post-mortem human frontal cortex area 8 (GM) and white matter of the frontal lobe centrum semi-ovale (WM) to identify lipidomes in middle-aged individuals with no neurofibrillary tangles and senile plaques, and cases at progressive stages of sporadic Alzheimer's disease (sAD). Methods: Lipidomic analysis using an LC-MS/MS platform was carried out in selected cases with suitable post-mortem lacking co-morbidities and concomitant brain pathologies. Complementary data were obtained using RT-qPCR and immunohistochemistry. Results: The WM shows an adaptive lipid phenotype resistant to lipid peroxidation, characterized by a lower fatty acid unsaturation, peroxidizability index, and higher ether lipid content than the GM. Changes in the lipidomic profile more marked in the WM than in GM occur in AD with disease progression. WM alterations are characterized by a decline in the content of branched fatty acid esters of hydroxy fatty acids (FAHFA), diacylglycerols (DG), triacylglycerols (TG), glycerophospholipids (GP) (especially ether lipids), and sphingolipids (especially sulfatides, ceramides, and glycosphingolipids). The GM acquires a fatty acid profile prone to peroxidation in sAD, while WM reinforces its peroxidation-resistant trait. Transcriptomic data point to additional defects of peroxisomal function. Conclusions: Four functional categories are associated with the different lipid classes affected in sAD: membrane structural composition, bioenergetics, antioxidant protection, and bioactive lipids, with deleterious consequences affecting both neurons and glial cells favoring disease progression. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

We look at 5 recent publications in the field of toxicology.Huen-Yin Tang M, et al.  Ropinirole metabolite mimics a new psychoactive substance (4-HO-MET) in LC-MS/MS. (2022) Forensic Science International, https://doi.org/10.1016/j.forsciint.2021.111151.Shang, Q. et al.  Multi-extraction system with identical supported semi-liquid membrane: Enhanced stability for coextraction of acidic and basic drugs. (2022) Talanta,  https://doi.org/10.1016/j.talanta.2022.123485.Khan, T.  Kamini, a little recognised source of illicit opioid: A case series of 12 patients. (2022) Drug and Alcohol Review,  DOI: 10.1111/dar.13475. Trigg, S.  The alprazolam analogue 4'-chloro deschloroalprazolam identified in seized capsules. (2022) Drug Testing and Analysis, DOI: 10.1002/dta.3325. Brooks-Lim, E.  Fatal ingestion of Taxus baccata: English yew. (2022) Journal of Forensic Sciences,  DOI: 10.1111/1556-4029.14941.Contact us at toxpod@tiaft.orgFind out more about TIAFT at www.tiaft.orgThe Toxpod is a production of The International Association of Forensic Toxicologists. The opinions expressed by the hosts are their own and do not necessarily reflect the views of TIAFT.

We're joined by Mass Spectrometry (Mass Spec) expert, Michelle Dubuke, Ph.D.,  to learn all about mass spec! From learning the practical applications of mass spec to how researchers are using the tool to discover new insight into biological aging and cancer development, Dubuke breaks it all down to showcase how expansive mass spec is.About Michelle Dubuke:Michelle Dubuke is currently the Manager, Field Application Scientist at Seer. Dubuke is a mass spectrometrist and protein biochemist focused on translational Precision Medicine workflows. Her previous experience was in varied areas of mass spectrometry, including small molecule LC-MS/MS, targeted and untargeted proteomics, DESI imaging, and intact protein analysis in an academic core facility. She has an established publication record in protein biochemistry, bottom-up proteomics, and targeted metabolomics. About SLAS:SLAS (Society for Laboratory Automation and Screening) is an international professional society of academic, industry and government life sciences researchers and the developers and providers of laboratory automation technology. The SLAS mission is to bring together researchers in academia, industry and government to advance life sciences discovery and technology via education, knowledge exchange and global community building.  For more information about SLAS, visit www.slas.org.SLAS publishes two peer-reviewed and MEDLINE-indexed scientific journals, SLAS Discovery and SLAS Technology. For more information about SLAS and its journals, visit www.slas.org/publications.Registration is now open for the 2022 AI Data Pipelines for Life Sciences Symposium in Seattle, WA, September 26-27.This two-day symposium will allow participants to explore how AI data pipelines are integrated into the life sciences. Attendees will learn about MLOPS, applications, techniques, and architectures of data and their uses in the life sciences. The SLAS 2022 Bio Entrepreneurship Symposium will allow emerging bio entrepreneurs, start-up companies, academics and those considering bio-entrepreneurship to explore the start-up ecosystem. Register by visiting: https://www.slas.org/events-calendar/slas-2022-bio-entrepreneurship-symposium/attend/register/

A presentation and discussion with Dr. Owusu on his recently published paper in JMSACL entitled Development and validation of a novel LC-MS/MS assay for C-peptide in human serum.

Video of this interview: https://youtu.be/o15HCjtSkug Chubbyemu video https://youtu.be/ChPeG19qKUo Christopher's recount of his case: https://youtu.be/7BpymtR2vpA Tweet me: https://www.twitter.com/hemereview IG me: https://www.instagram.com/hemereview FB me: https://www.facebook.com/hemereview 2,4-Dinitrophenol (DNP) is a chemical that is not approved for weight loss. It is a classic case of efficacy, but without safety in the setting of weight loss. This setting of weight loss can be prone to chemical misuse, further exacerbating understanding in human systems. Christopher is one of 2 - 3 cases documented in literature to have survived an accident with DNP for weight loss. As of 2021, one organization has permission from the US FDA to investigate DNP in some neurodegenerative disorders. These are rare diseases with limited standards of care, therefore extenuating circumstances to find anything that could potentially improve patient outcomes. References: Role of dantrolene in dinitrophenol (DNP) : A continuing question? Am J Emerg Med. 2019 Jun;37(6):1216.e1-1216.e2. https://pubmed.ncbi.nlm.nih.gov/30948257/ 2,4-dinitrophenol (DNP): a weight loss agent with significant acute toxicity . J Med Toxicol. 2011 Sep;7(3):205-12. https://pubmed.ncbi.nlm.nih.gov/21739343/ Ambient temperature and mortality from unintentional . JAMA. 1998 Jun 10;279(22):1795-800. https://pubmed.ncbi.nlm.nih.gov/9628710/ Beware the yellow slimming pill: fatal 2,4-dinitrophenol. BMJ Case Rep. 2016 Apr 4;2016. https://pubmed.ncbi.nlm.nih.gov/27045052/ Dantrolene is not the answer to 2,4-dinitrophenol poisoning: more heated debate. BMJ Case Rep. 2018 Dec 19;11(1):e225323. https://pubmed.ncbi.nlm.nih.gov/30573533/ The effect of 2,4-dinitrophenol on adipose-tissue metabolism. Biochem J. 1969 Feb;111(4):431-44. https://pubmed.ncbi.nlm.nih.gov/4388239/ The mechanism of uncoupling of oxidative phosphorylation by 2,4-dinitrophenol. J Biol Chem. 1967 Oct 25;242(20):4577-83. https://pubmed.ncbi.nlm.nih.gov/4964808/ MECHANISMS OF DINITROPHENOL TOXICITY. Simon E.W. Biological Reviews. Vol 28. (4). Nov 1953. Identification and Importance of Brown Adipose Tissue in Adult Humans. https://www.nejm.org/doi/full/10.1056/NEJMoa0810780 Mitochondrial uncoupling as a target for drug development for the treatment of obesity. Obes Rev. 2001 Nov;2(4):255-65. https://pubmed.ncbi.nlm.nih.gov/12119996/ LC-MS-MS analysis of 2,4-dinitrophenol and its phase I and II metabolites. J Ana Toxicol. Jan-Feb 2007;31(1):55-61. https://pubmed.ncbi.nlm.nih.gov/17389084/ Uncouplers of oxidative phosphorylation. Environ Health Perspect. 1990 Jul;87:213-8. https://pubmed.ncbi.nlm.nih.gov/2176586/ Texas Woman Charged with Selling Misbranded https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/press-releases/texas-woman-charged-selling-misbranded-drug --- Support this podcast: https://anchor.fm/chubbyemu/support

Patient CE's personal recount of case: https://youtu.be/7BpymtR2vpA Patient CE's Interview: https://youtu.be/o15HCjtSkug Chubbyemu video on this case: https://youtu.be/ChPeG19qKUo Heme Review video version: https://youtu.be/jgDUMNaVqHg 2,4-Dinitrophenol (DNP) is a chemical that is not approved for weight loss in the United States. It is a classic case of efficacy, but without safety in the setting of weight loss. This setting of weight loss can be prone to chemical misuse, further exacerbating understanding in human systems. Christopher is one of 2 - 3 cases documented in literature to have survived an accident with DNP for weight loss. As of 2021, one organization has permission from the US FDA to investigate DNP in some neurodegenerative disorders. These are rare diseases with limited standards of care, therefore extenuating circumstances to find anything that could potentially improve patient outcomes. Tweet me: https://www.twitter.com/hemereview IG me: https://www.instagram.com/hemereview FB me: https://www.facebook.com/hemereview References: Role of dantrolene in dinitrophenol (DNP) : A continuing question? Am J Emerg Med. 2019 Jun;37(6):1216.e1-1216.e2. https://pubmed.ncbi.nlm.nih.gov/30948257/ 2,4-dinitrophenol (DNP): a weight loss agent with significant acute toxicity . J Med Toxicol. 2011 Sep;7(3):205-12. https://pubmed.ncbi.nlm.nih.gov/21739343/ Ambient temperature and mortality from unintentional . JAMA. 1998 Jun 10;279(22):1795-800. https://pubmed.ncbi.nlm.nih.gov/9628710/ Dinitrophenol as a Medicine. Cells. 2019 Mar; 8(3): 280. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468406/ The Interaction of Highly Active Uncouplers With Mitochondria. Biochimica et Biophysica Acta, 639 (1981) 225-242. Beware the yellow slimming pill: fatal 2,4-dinitrophenol. BMJ Case Rep. 2016 Apr 4;2016. https://pubmed.ncbi.nlm.nih.gov/27045052/ Dantrolene is not the answer to 2,4-dinitrophenol poisoning: more heated debate. BMJ Case Rep. 2018 Dec 19;11(1):e225323. https://pubmed.ncbi.nlm.nih.gov/30573533/ The effect of 2,4-dinitrophenol on adipose-tissue metabolism. Biochem J. 1969 Feb;111(4):431-44. https://pubmed.ncbi.nlm.nih.gov/4388239/ The mechanism of uncoupling of oxidative phosphorylation by 2,4-dinitrophenol. J Biol Chem. 1967 Oct 25;242(20):4577-83. https://pubmed.ncbi.nlm.nih.gov/4964808/ MECHANISMS OF DINITROPHENOL TOXICITY. Simon E.W. Biological Reviews. Vol 28. (4). Nov 1953. Identification and Importance of Brown Adipose Tissue in Adult Humans. https://www.nejm.org/doi/full/10.1056/NEJMoa0810780 Mitochondrial uncoupling as a target for drug development for the treatment of obesity. Obes Rev. 2001 Nov;2(4):255-65. https://pubmed.ncbi.nlm.nih.gov/12119996/ LC-MS-MS analysis of 2,4-dinitrophenol and its phase I and II metabolites. J Ana Toxicol. Jan-Feb 2007;31(1):55-61. https://pubmed.ncbi.nlm.nih.gov/17389084/ Uncouplers of oxidative phosphorylation. Environ Health Perspect. 1990 Jul;87:213-8. https://pubmed.ncbi.nlm.nih.gov/2176586/ Texas Woman Charged with Selling Misbranded https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/press-releases/texas-woman-charged-selling-misbranded-drug --- Support this podcast: https://anchor.fm/chubbyemu/support

What do we want? Good chromatography! How do we get it? We don't know! Columns, maybe?Good chromatography is essential to any LC-MS/MS method. Setting it up is often one of the earlier steps in developing a new method. While it can be an iterative process, setting up good chromatographic conditions at the start can set you up for success later on. This podcast will discuss our laboratories efforts to setup chromatographic conditions for the analysis of ethyl-glucuronide and buprenorphine (plus metabolites). We will present what we tried, why it seemed like it a good/bad idea, and along the way talk through some basic chromatography concepts. The goal is for attendees to be more informed when they approach setting up their own chromatographic methods.This session is the second in a 4 part series in which Dr. Hayden will invite attendees to witness in real time his journey bringing mass spec testing to a clinical lab. During these interactive sessions, attendees will be encouraged to help troubleshoot, and offer advice as desired.You can watch the recording of the first session: "Getting going with mass spectrometry: Josh installs a mass spectrometer".Subsequent sessions anticipated include:Part 3. Getting going with mass spectrometry: Josh tries to do sample preparationPart 4. Getting going with mass spectrometry: Josh analyzes peaks

This is the first in a 3-Part Series of "The Weekly Bioanalysis" wherein Dom and John, our hosts (and Bioanalytical specialists at KCAS) answer the question "What is Bioanalysis?""The Weekly Bioanalysis" is a podcast dedicated to discussing Bioanalytical news, tools and services related to the Pharmaceutical, Biopharmaceutical and Biomarker industries. Every week, KCAS will bring you another 30 minutes of friendly banter between our two finest Senior Scientific Advisors as they chat over coffee and brief themselves on what they've learned about the Bioanalytical world the past week.The Weekly Bioanalysis is brought to you by KCAS. KCAS is a progressive growing contract research organization of well over 100 talented and dedicated individuals committed to serving our clients and improving health worldwide. Our experienced scientists provide stand-alone bioanalytical services to the Pharmaceutical, Biopharmaceutical, Animal Health and Medical Device industries.

This is the third in our 3-Part Series of "The Weekly Bioanalysis" wherein the specialists at KCAS answer the question "What is Bioanalysis?" This week, Dom and John host their first-ever guests to the podcast to discuss Hybrid LC-MS/MS vs Ligand Binding Assays."The Weekly Bioanalysis" is a podcast dedicated to discussing Bioanalytical news, tools and services related to the Pharmaceutical, Biopharmaceutical and Biomarker industries. Every week, KCAS will bring you another 30 minutes of friendly banter between our two finest Senior Scientific Advisors as they chat over coffee and brief themselves on what they've learned about the Bioanalytical world the past week.The Weekly Bioanalysis is brought to you by KCAS. KCAS is a progressive growing contract research organization of well over 100 talented and dedicated individuals committed to serving our clients and improving health worldwide. Our experienced scientists provide stand-alone bioanalytical services to the Pharmaceutical, Biopharmaceutical, Animal Health and Medical Device industries.

This week's episode of "The Weekly Bioanalysis" features a discussion about when it is advantageous to combine Hybrid LC-MS/MS vs Ligand Binding Assays and why it so great to have both under one roof. Dom and John go into detail about when and why using both of these approaches can help projects and studies."The Weekly Bioanalysis" is a podcast dedicated to discussing Bioanalytical news, tools and services related to the Pharmaceutical, Biopharmaceutical and Biomarker industries. Every week, KCAS will bring you another 30 minutes of friendly banter between our two finest Senior Scientific Advisors as they chat over coffee and brief themselves on what they've learned about the Bioanalytical world the past week.The Weekly Bioanalysis is brought to you by KCAS. KCAS is a progressive growing contract research organization of well over 100 talented and dedicated individuals committed to serving our clients and improving health worldwide. Our experienced scientists provide stand-alone bioanalytical services to the Pharmaceutical, Biopharmaceutical, Animal Health and Medical Device industries.

Dom and John continue our three-part series on Discovery, Non-GLP, Non-Clinical Drug Development. This episode, we focus on Discovery LC/MS-MS & Universal Assays."The Weekly Bioanalysis" is a podcast dedicated to discussing Bioanalytical news, tools and services related to the Pharmaceutical, Biopharmaceutical and Biomarker industries. Every week, KCAS will bring you another 45-60 minutes of friendly banter between our two finest Senior Scientific Advisors as they chat over coffee and brief themselves on what they've learned about the Bioanalytical world the past week.The Weekly Bioanalysis is brought to you by KCAS. KCAS is a progressive growing contract research organization of well over 100 talented and dedicated individuals committed to serving our clients and improving health worldwide. Our experienced scientists provide stand-alone bioanalytical services to the Pharmaceutical, Biopharmaceutical, Animal Health and Medical Device industries.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.392266v1?rss=1 Authors: Phapale, P., Palmer, A., Gathungu, R. M., Kale, D., Brugger, B., Alexandrov, T. Abstract: Liquid chromatography-mass spectrometry(LC-MS)-based untargeted metabolomics studies require high-quality spectral libraries for reliable metabolite identification. We have constructed EMBL-MCF, an open LC-MS/MS spectral library that currently contains over 1600 fragmentation spectra from 435 authentic standards of endogenous metabolites and lipids. The unique features of the library are presence of chromatographic profiles acquired with different LC-MS methods and coverage of different adduct ions. The library covers many biologically important metabolites with some unique metabolites and lipids as compared to other public libraries. The EMBL-MCF spectral library is created and shared using an in-house developed web-application at https://curatr.mcf.embl.de/. The library is freely available online and also integrated with other mass spectral repositories. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.03.365585v1?rss=1 Authors: Gotti, C., Roux-Dalvai, F., Joly-Beauparlant, C., Leclercq, M., Mangnier, L., Droit, A. Abstract: In the past few years, LC-MS/MS in DIA mode has become a strategy of choice for deep coverage of complex proteomes and accurate quantification of low abundant species. However, there is still no consensus in the literature on the best acquisition parameters and processing tools to use. We present here the largest benchmark of DIA proteomic workflows on Orbitrap instruments ever published. Using a complex proteomic standard, we tested 36 workflows including 4 different acquisition schemes and 6 different software tools (DIA-NN, DIA-Umpire, OpenSWATH, Scaffold-DIA, Skyline and Spectronaut-Pulsar-X) with or without the use of a spectral library. By reporting the protein identifications, linearity, reproducibility, as well as sensitivity and specificity in 28 pairwise comparisons, we propose here a guide book for the users to choose the best DIA strategy for their data. Moreover, our data has been deposited on ProteomeXchange for testing or developing new DIA processing tools. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318055v1?rss=1 Authors: Cantres-Velez, J., Blaize, J., Vierra, D., Boisvert, R., Garzon, J., Piraino, B., Tan, W., Deans, A., Howlett, N. G. Abstract: Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 ( FANCD2 -/- ) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.293092v1?rss=1 Authors: Davies, V., Wandy, J., Weidt, S., van der Hooft, J. J. J., Miller, A., Daly, R., Rogers, S. Abstract: Tandem mass spectrometry (LC-MS/MS) is widely used to identify unknown ions in untargeted metabolomics. Data Dependent Acquisition (DDA) chooses which ions to fragment based upon intensity observed in MS1 survey scans and typically only fragment a small subset of the ions present. Despite this inefficiency, relatively little work has addressed the development of new DDA methods, partly due to the high overhead associated with running the many extracts necessary to optimise approaches in busy MS facilities. In this work, we firstly provide theoretical results that show how much improvement is possible over current DDA strategies. We then describe an in silico framework for fast and cost efficient development of new DDA acquisition strategies using a previously developed Virtual Metabolomics Mass Spectrometer (ViMMS). Additional functionality is added to ViMMS to allow methods to be used both in simulation and on real samples via an instrument application programming interface (API). We demonstrate this framework through the development and optimisation of two new DDA methods which introduce new advanced ion prioritisation strategies. Upon application of the here developed methods to two complex metabolite mixtures, our results show that they are able to fragment more unique ions than standard DDA acquisition strategies. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.04.236356v1?rss=1 Authors: Petrera, A., von Toerne, C., Behler, J., Huth, C., Thorand, B., Hilgendorff, A., Hauck, S. M. Abstract: The plasma proteome is the ultimate target for biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which is however still difficult to comprehensively access. The high structural complexity of the plasma proteome can be addressed by either a system-wide and unbiased tool such as mass spectrometry (LC-MS/MS) or a highly sensitive targeted immunoassay such as the Proximity Extension Assays (PEA). In order to address relevant differences and important shared characteristics, we tested the performance of LC-MS/MS in data-dependent and -independent acquisition modes and PEA Olink to measure circulating plasma proteins in 173 human plasma samples from a Southern German population-based cohort. We demonstrated the measurement of more than 300 proteins with both LC-MS/MS approaches applied, mainly including high abundance plasma proteins. By the use of the PEA technology, we measured 728 plasma proteins, covering a broad dynamic range with high sensitivity down to pg/ml concentrations. In a next step, we quantified 35 overlapping proteins with all three analytical platforms, verifying the reproducibility of data distributions, measurement correlation and gender-based differential expression. Our work highlights the limitations and the advantages of both, targeted and untargeted approaches, and prove their complementary strengths. We demonstrated a significant gain in proteome coverage depth and subsequent biological insight by platforms combination a promising approach for future biomarker and mechanistic studies. Copy rights belong to original authors. Visit the link for more info

Dr. Ted Achacoso is the Global Founding Pioneer of Health Optimization Medicine and Practice (HOMe/HOPe) which detects and corrects imbalances at the level of the metabolome leveraging multi-omics, epigenetics, bioenergetics, gut immune systems, chronobiology, exposomics and evolutionary medicine. He’s also the Founder of BioBalance Institute in Manilla providing proof of concept that Health Optimization Medicine & Practice is an Economically Superior Clinical Practice. He’s Double Board Certified in Nutritional & Anti-Aging Medicine with focus on AI, Medical Informatics and Mathematics of Consciousness, and wrote the first-ever Connectome of C. Elegans into a book. https://homehope.org https://biobalanceinstitute.com https://troscriptions.com SHOW NOTES

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.17.156265v1?rss=1 Authors: Huan, Y., Wei, J., Su, T., Gao, Y. Abstract: Background. Major depressive disorder (MDD) is a prevalent complex psychiatric disorder with a high prevalence rate. Because MDD is a systemic multifactorial disorder involving complex interactions and disturbances of various molecular pathways, there are no effective biomarkers for clinical diagnosis. Urine is not subjected to homeostatic control, allowing it to reflect the sensitive and comprehensive changes that occur in various diseases. In this study, we examined the urine proteome changes in a CUMS mouse model of MDD. Methods. Male C57BL/6 mice were subjected to chronic unpredictable mild stress for 5 weeks. The tail suspension test (TST) and sucrose consumption test (SCT) were then applied to evaluate depression-like behaviors. The urine proteomes on day 0 and day 36 in the CUMS group were profiled by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Results. A total of 45 differential proteins were identified, 24 of which have been associated with the pathogenic mechanisms of MDD, while 10 proteins have been previously suggested as MDD biomarkers. There was an average of two differential proteins that were identified through 1048574 random combination statistical analyses, indicating that at least 95% of the differential proteins were reliable and not the result of random combination. The differential proteins were mainly associated with blood coagulation, inflammatory responses and central nervous system development. Conclusions. Our preliminary results indicated that the urine proteome can reflect changes associated with MDD in the CUMS model, which provides potential clues for the diagnosis of clinical MDD patients. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.15.150052v1?rss=1 Authors: Das, T., Kamle, A., Kumar, A., Chakravarty, S. Abstract: Understanding the molecular basis of sex differences in neural response to acute hypoxic insult has profound implications for the effective prevention and treatment of ischemic stroke. Global hypoxic-ischemic induced neural damage has been studied recently under the well-controlled, non-invasive, reproducible conditions using zebrafish model. Our earlier report on sex difference in global acute hypoxia induced neural damage and recovery in zebrafish prompted us for comprehensive study on the mechanisms underlying the recovery. An omics approach for studying quantitative changes in brain proteome upon hypoxia insult following recovery was undertaken using iTRAQ-based LC-MS/MS approach. The results shed light on altered expression of many regulatory proteins in zebrafish brain upon acute hypoxia following recovery. The sex difference in differentially expressed proteins along with the proteins expressed in uniform direction in both the sexes was studied. Core expression analysis by Ingenuity Pathway analysis (IPA) showed a distinct sex difference in the disease function heatmap. Most of the upstream regulators obtained through IPA were validated at the transcriptional level. Translational upregulation of H3K9me3 in male led us to elucidate the mechanism of recovery by confirming transcriptional targets through ChIP-qPCR. The upregulation of H3K9me3 level in male at 4 hr post-hypoxia appears to affect the early neurogenic markers nestin, klf4 and sox2, which might explain the late recovery in male, compared to female. Acute hypoxia-induced sex-specific comparison of brain proteome led us to reveal many differentially expressed, which can be further studied for the development of novel targets for better therapeutic strategy. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.19.105197v1?rss=1 Authors: Ping, L., Kundinger, S. R., Duong, D. M., Yin, L., Gearing, M., Lah, J. J., Levey, A. I., Seyfried, N. T. Abstract: Alzheimer's disease (AD) is characterized by an early, asymptomatic phase (AsymAD) in which individuals exhibit amyloid-beta (A{beta}) plaque accumulation in the absence of clinically detectable cognitive decline. Here we report an unbiased multiplex quantitative proteomic and phosphoproteomic analysis using tandem mass tag (TMT) isobaric labeling of human post-mortem cortex (n=27) across pathology-free controls, AsymAD and symptomatic AD individuals. With off-line high-pH fractionation and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on an Orbitrap Lumos mass spectrometer, we identified 11,378 protein groups across three TMT 11-plex batches. Immobilized metal affinity chromatography (IMAC) was used to enrich for phosphopeptides from the same TMT-labeled cases and 51,736 phosphopeptides were identified. Of these, 48,992 were quantified by TMT reporter ions representing 33,652 unique phosphosites. Two reference standards in each TMT 11-plex were included to assess intra- and inter-batch variance at the protein and peptide level. This comprehensive human brain proteome and phosphoproteome dataset will serve as a valuable resource for the identification of biochemical, cellular and signaling pathways altered during AD progression. Copy rights belong to original authors. Visit the link for more info

Hosts Tracy Swain and JaVonne Williams are joined by Amy J. Reisinger, President and Chief Executive Officer of SteelFusion Clinical Toxicology Laboratory, LLC. Ms. Reisinger studied Biology/Pre-Medicine at The University of North Carolina in Wilmington, North Carolina and completed the Graduate Cardiovascular Perfusion Program at Duquesne University in Pittsburgh, Pennsylvania. Ms. Reisinger began her professional career as an Extra Corporeal Membrane Oxygenator (ECMO) Specialist and Histologist at Children’s Hospital of Pittsburgh, Pennsylvania, where she provided perfusion to neonates and assisted the medical examiner with autopsies. She became a Research Scientist at GlaxoSmithKline, Research Triangle Park, North Carolina, where she provided regulatory support to the pharmaceutical industry. Ms. Reisinger was responsible for performing the gross and microscopic examinations of animals utilized in toxicology studies as well as evaluating the significance of the anatomic and clinical pathology, as well as organ weight data from such studies. SteelFusion prepares, processes, analyzes, validates, reports and conducts research and testing of oral fluid and urine [e.g. A pilot study of the management of mental health services and treatment for abuse (SAMHSA) and deaths]. Enzyme-linked immunosorbent assay (ELISA)Liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS). Her vision is to become more important in the area of ​​quality assurance and compliance. SteelFusion is a WBENC – Certified Women Owned Business. Amy is also one of the featured authors in the "Women in Business – Leading the Way" Book released in Spring 2020. To learn more about Amy Reisinger and her company’s structure (including forensic medicine, drugs and alcohol, rehabilitation centers, correctional facilities, services for youth, doctors, workers compensation and employment assessment), visit her website https://www.steelfusionlabs.com/, Email: amy@steelfusionlabs.com, Facebook: Facebook.com/SteelFusionLabs & Twitter: @SteelFusionLabs and other social media platforms.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.30.071076v1?rss=1 Authors: Smalley, J. L., Kontou, G., Choi, C., Ren, Q., Albrecht, D., Abiraman, K., Rodriguez Santos, M. A., Bope, C. E., Deeb, T. Z., Davies, P. A., Brandon, N. J., Moss, S. J. Abstract: Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanism neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of Kcc2 from the plasma membrane of murine forebrain. We resolved these using Blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS. Purified Kcc2 migrated as distinct molecular species of 300, 600 and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N and C-termini of Kcc2 was obtained. The 300kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. Intriguingly, lower levels of Kcc1 were also found in this species suggesting the existence of mixed Kcc2/Kcc1 heterodimers. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed association of Kcc2. These included spectrins, ankyrins, and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5 and Lmtk3. Finally, we used LC-MS/MS on highly purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that the Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity. Copy rights belong to original authors. Visit the link for more info

Of the minority of men taking steroids who actually get blood tests, 99% of them get inaccurate assays. Most doctors don't realize the importance of high sensitivity testing when it comes to men in general, and even more so in steroid users. The difference between high sensitivity testing and what you have probably been getting in the past can be the entire reference range, or in some cases, it may even be detecting metabolites or analogs of other hormones. This is not limited to just testosterone either. Measuring Estradiol via Roche ECLIA (Electrochemiluminescence Immunoassay) instead of liquid chromatography with tandem mass spectrometry (LC/MS-MS) has similar limitations, as do other hormone tests with insufficient analytical sensitivity. https://youtu.be/8mw-jZpmrsQ The Most Accurate Total Testosterone Blood Test The gold standard for accurately measuring total testosterone levels is high-performance liquid chromatography with tandem mass spectrometry (LC/MS-MS). Recent studies have shown that the current assays used in total testosterone testing lack the required sensitivity to produce accurate results when working with very low concentrations. This can be especially problematic when trying to accurately measure total testosterone levels in severely testosterone deficient men, normal or testosterone deficient women, and children. In addition, these tests have proven via my own personal tests to detect synthetic anabolic androgenic steroids as testosterone. This issue can be corrected by newer methods based on mass spectrometry. The Most Accurate Free Testosterone Blood Test The gold standard for accurately measuring free testosterone levels is equilibrium dialysis. Equilibrium ultrafiltration is also sufficient. A comparison of results obtained via equilibrium dialysis and equilibrium ultrafiltration found that the two methods correlated closely (r = 0.97) [R]. Direct analog enzyme immunoassay (EIA) is inaccurate and has shown in my own personal tests to detect synthetic anabolic androgenic steroids as testosterone. My Blood Test Results Comparing ECLIA And EIA Vs. LC/MS-MS And Equilibrium Ultrafiltration This is an example that very clearly shows how important high sensitivity testing can be. I went in for a blood test during a Nandrolone-only experiment. I had no Testosterone in my system whatsoever. Expectedly, by assessing my Total Testosterone level via liquid chromatography with tandem mass spectrometry (LC/MS-MS) and my Free Testosterone level via equilibrium ultrafiltration, we can see that my Testosterone levels were crashed. Both the total and the free were lower than a healthy female. This is what you should see in your blood work if you’re on just Nandrolone. The only Testosterone being produced in my body was indirectly via the trace amounts of androgens produced in my adrenal cortex, which is why the value wasn't completely bottomed out at 0. I've mentioned many times the importance of getting high sensitivity testing done for hormone levels and how Nandrolone will register as Testosterone in primitive garbage blood tests. This is another great example of this. In addition to high sensitivity testing, I had the same blood tested using electrochemiluminescence immunoassay (ECLIA) for my Total Testosterone level, and direct analog enzyme immunoassay (EIA) for my Free Testosterone level. These were the test results using the exact same blood sample with the terrible default assays that doctors will use to determine how to treat you, and that labs will give you in the majority of your blood work panels. According to ECLIA and EIA, I had a normal Total Testosterone and Free Testosterone level. This just one example of why getting accurate hormone testing is critical. My Testosterone levels were actually in the gutter, but ECLIA and EIA couldn't even tell the difference between Testosterone and 19-nortestosterone in my blood. Where To Order Accurate Blood Tests Most labs offer total testosterone (LC/MS-MS), and some offer free testosterone (equilibrium ultrafiltration). However, very few self-pay labs offer free testosterone (equilibrium dialysis). Of the ones that do offer it, it is extremely expensive and it does not come in a package with a total testosterone test. If your doctor will write you a requisition for it, great. If not, just get equilibrium ultrafiltration. Personally, I drive to the US just so I can get LC/MS-MS and equilibrium ultrafiltration testing done. This is the exact test I order every time to accurately check my testosterone levels: Testosterone, Free, Equilibrium Ultrafiltration With Total Testosterone, LC/MS-MS

There's been a growing amount of hype around the "Deca only cycle". While it is most commonly referred to as the Deca only cycle, it is actually based on the compound Nandrolone being used on its own. The decanoate ester being abbreviated as "Deca" has just become synonymous nowadays in the bodybuilding community with Nandrolone itself. Seeing the potential merits of Nandrolone as a makeshift hormone replacement therapy alternative to Testosterone, I stopped using Testosterone and instead started using Nandrolone on its own with exogenous Estradiol for 3 months and paid over $1000 for an elaborate blood panel to assess how it affected my health markers. https://youtu.be/kLScNddgkks How Nandrolone Could Potentially Be A Superior HRT Alternative To Testosterone The primitive thought process is that Nandrolone used in conjunction with Testosterone will lead to horrible side effects, but Nandrolone used on its own will just result in all of the benefits of steroids with a near absence of the androgenic or estrogenic side effects associated with Testosterone use. In reality, it's a lot more nuanced than that. The reason why I found this experiment worth pursuing is the lack of androgenicity of Nandrolone in the body. Nandrolone 5α-reduces in tissues that express 5α-reductase to the much less androgenic metabolite Dihydronandrolone (DHN). Nandrolone is basically the only anabolic steroid that is going to maintain 100% anabolic activity of the Nandrolone in muscle tissue where you want it, but also be converted into a much less androgenic metabolite with a lower binding affinity in certain areas of the body where you wouldn't want Nandrolone to bind. The two areas of concern for most individuals being hair follicles and skin. By converting to DHN in these areas, Nandrolone (and by extension DHN) causes less hair loss and acne than Testosterone (and by extension DHT). In addition, some men are genetically predisposed to high levels of aromatization and estrogen receptor expression and can't even use TRT doses of Testosterone without experiencing estrogenic side effects. Nandrolone is not a potent substrate for aromatase, and mainly converts to a weaker estrogen called Estrone (Estradiol is about 10-fold more potent than Estrone). Nandrolone is also mildly estrogenic on its own via its ability to act as an estrogen receptor alpha (ERα) agonist [R]. Overall, Nandrolone is much less androgenic and estrogenic than Testosterone, and may provide symptom relief in those seeking a viable hormone replacement therapy alternative. In this context, Nandrolone may also have great potential as an efficacious alternative to Testosterone as an anabolic agent for some individuals who are prone to androgenic and/or estrogenic side effects. The Neurotoxicity And Cardiotoxicity Of Nandrolone Based on the limited data available, Nandrolone has shown to be more deleterious to cardiovascular and neurological health than testosterone. https://www.youtube.com/watch?v=Gv_v0mJy6Bg By extrapolating the data, we start to get a clearer picture as to why this likely is. Nandrolone is mildly estrogenic on its own, and it does not aromatize nearly enough to create as much Estradiol as Testosterone does. Comparing the effect of testosterone with that of 19-nortestosterone (Nandrolone) and Stanozolol (Winstrol) on neurotoxicity we can clearly see that Estrogen is what protects neurons in the brain, not Testosterone itself. In this study, a physiologic dosage of Testosterone was neuroprotective [R]. Testosterone only amplified neurotoxicity at supraphysiological dosages. The neuroprotective effect of a physiologic dosage of Testosterone was completely eliminated when the aromatase inhibitor Anastrozole (Arimidex) was co-administered, suggesting that the intrinsic toxicity of Testosterone as an androgen is only counterbalanced by its aromatization into 17β-estradiol. As opposed to testosterone, Nandrolone does not appear to aromatize sufficiently into estrogen. As you would expect, Nandrolone was neurotoxic at every single dose evaluated regardless of Arimidex being co-administered or not. If Nandrolone was inherently able to provide enough estrogen receptor alpha (ERα) activation to balance out its androgenicity without even requiring aromatization (it acts as an estrogen on its own to some extent), we would see a neuroprotective effect at equivalent dosages to a physiologic concentration of Testosterone when no AI is used, but that does not appear to be the case either. The anti-androgen flutamide attenuated the neurotoxicity of all three androgens, thus further reinforcing that physiologic dosages of androgens without a sufficient amount of opposing estrogens, or supraphysiological dosages of androgens may facilitate neuronal death. I suspect that the same applies for the inherent cardiotoxicity of Nandrolone as well. Just because you can get your Estradiol levels up to 15 pg/mL with a gram of Deca only, that ratio of androgens to estrogen in the body is way off of what would otherwise be optimal for health based on what I've seen. This is reinforced by the fact that Flutamide (an anti-androgen) was able to attenuate the neurotoxicity of Nandrolone. By preventing Nandrolone from binding to androgen receptors, it is no longer able to transcribe its effects in tissues. Hair loss and acne are one thing, cardiotoxicity and neurotoxicity are another thing and should ultimately take precedence obviously. However, just because Nandrolone monotherapy cannot produce a sufficient ratio of androgens to estrogens, that doesn't mean that there isn't a potential loophole. That loophole is exogenous Estradiol administration. Exogenous Estradiol Use With Nandrolone Only Cycles As we've seen, Estrogen produced via aromatase is what provides neuroprotection from the androgenicity of Testosterone, not the Testosterone itself. We also know that Nandrolone is not able to produce enough estrogenic activity in the body to facilitate this same level of neuroprotection. I theorize that Nandrolone in conjunction with exogenous Estradiol to replace this otherwise missing component could attenuate a significant amount of the deleterious impact Nandrolone has on the heart and brain. In addition, by providing a sufficient amount of exogenous estrogen, libido, muscle growth, fat loss, and several other aspects of health and performance should be more optimized. It isn't a coincidence that cardiovascular disease rates skyrocket once women hit menopause and stop producing Estrogen properly. The same negative effects will apply in men with low Estrogen levels. The lack of sufficient Estrogen is often addressed in Deca only cycles by adding an adjunct anabolic steroid that aromatizes into Estrogen or Estrogen analogs. Obviously for those seeking to minimize androgenic side effects, the ideal way to go about achieving sufficient Estrogen receptor activation is probably not going to be by adding more steroids to their protocol. This is where exogenous Estradiol comes into play, and I believe the majority of Deca only cycles would be more sustainable from a health perspective, and successful in a bodybuilding context as well with its inclusion. I have yet to see one person on a Deca only cycle achieve a sufficient Estradiol level relative to their Nandrolone dosage via a sensitive assay Estradiol blood test. The following blood test result was submitted by an individual on over 1000 mg per week of Deca only. Over a gram of androgens relative to a 19.2 pg/mL Estradiol level is far from ideal in my opinion. I had a good conversation with Vigorous Steve as well about his Deca only cycle experience. He told me that his Estradiol was 12 pg/mL on 1000 mg of Deca per week after 4 weeks, and he ended up adding 25 mg DHEA per day just to bring it up to 25 pg/mL. When it comes to Nandrolone use on its own, most would benefit from more Estrogen in my opinion. My Weekly Nandrolone And Estradiol Dosage For "HRT" Most guys doing Deca only cycles are evaluating Nandrolone at dosages of 600 mg or higher per week for short blasts. My experiment was based on its potential as an alternative long term HRT option for those prone to androgenic side effects. Or alternatively, its potential as a compound to swap to periodically throughout the year from TRT to reverse some of the androgenic side effects of Testosterone and DHT while still maintaining the same amount of muscle mass. Every blood test I've seen of Deca only cycle users was on high doses of Nandrolone without a sufficient amount of Estrogen. I wanted to see how Nandrolone on its own at a "therapeutic dose" would affect my blood work if I had a sufficient amount of Estrogen provided through exogenous Estradiol. Long-term, the only way Nandrolone monotherapy could be even relatively safe in a cardiovascular context would be with exogenous Estradiol supplementation from what I've seen. And even then, I'm sure it has major drawbacks that will likely accumulate over the years. With that being said, it is still something I wanted to explore nonetheless, as it is one of the few compounds that can actually support supraphysiological muscle growth with a relatively minimal impact on androgenic alopecia. Oral micronized Estradiol tablets have quite a few drawbacks. A few of the most notable drawbacks are that oral Estrogen pills can be somewhat liver toxic, they spike SHBG through the roof, and they result in the production of clotting factors in the blood that do not develop with forms of administration that skip the first pass. The two most viable methods of administration that skip the first pass are transdermal topical application, or injection. I chose to topically apply transdermal Estradiol gel (Estrogel) for this experiment. I used 100 mg of Nandrolone phenylpropionate (NPP) per week split into daily injections using an insulin pin rotating between my glutes and ventroglutes. I also applied 2.5 grams of transdermal Estrogel (delivering 1.5 mg Estradiol) to my inner thighs every day for over 3 months straight. Blood Pressure Changes On Nandrolone One of the first things I noticed was that it was a struggle to keep my blood pressure in check on NPP, even at the mild dose I was using. What that was caused by exactly, I'm not sure. I assumed it was Aldosterone prior to this blood work. When I'm on Testosterone, even when I was using TRT as high as 200 mg per week, I could keep my blood pressure at 110/70 with ease. Even if I ate terribly, I could still hold 115/75 without even trying on Testosterone. Within the first week of switching to NPP it became way harder to control my systolic blood pressure. My diastolic blood pressure was fine for the entire 3 months, but my systolic blood pressure would consistently be around 125-128. That is not normal for me, and is borderline stage 1 hypertension. The fact that I even had to try to lower my blood pressure showed to me that Nandrolone is a lot harder to manage in this regard. This is consistent with almost every single person I know who has blasted high doses of Deca. They all had significant issues with blood pressure. Most of the guys who thought they had normal blood pressure were actually stage 1 hypertensive and didn't even realize that their results were indicative of cardiovascular stress. My 125-128 systolic occurred without being in a calorie surplus, without any weight changes, and on what I would consider a very low dose of NPP. The exact same diet, weight, lifestyle, etc. would have me at 110/70 on TRT. Muscle Growth And Strength On Nandrolone I maintained my muscle and do not feel that there was a substantial difference between the anabolic potency of Nandrolone compared to Testosterone. At the very least, the anabolic activity of Nandrolone is comparable to Testosterone, but the androgenic activity is far less than that of Testosterone. In certain contexts for certain individuals, Nandrolone will be the desirable alternative because of this. Reduced Libido On Nandrolone - Deca Dick? My libido was extremely subdued on NPP. That's one of the most obvious things I noticed during my experiment. I had a libido and would still want to have sex, but my libido was much lower than it is on regular TRT. On TRT I can barely go one day without sex before it starts to consume my mind.  On Nandrolone only, I can easily go a couple days barely even thinking about it. However, when it came time to get the job done, I could still get the job done and stay hard the entire time without any issues in erection quality. It was a bit harder to reach orgasm though. On top of the lack of androgenicity causing a reduction in libido, Nandrolone also has progestogenic activity and binds to the Progesterone receptor. Excessive Progesterone is notorious for killing libido and causing erectile dysfunction, and it seems that Nandrolone has similar effects in many individuals via this pathway in conjunction with its 5α-reduction into DHN. My drive was also lower, and I felt less aggressive overall. In many individuals Testosterone and DHT levels will strongly influence libido, drive, aggression, motivation and productivity. Personally, even if I have that support via DHT or DHT derivatives, the increased motivation and drive is actually more counterproductive in a work productivity context because my libido gets way too high. Even when I had high testosterone levels and 0 DHT in my body I still had sex on my mind far more than I would like. When that happens, I can barely get anything done, and then I end up depleting myself of energy for the day through excessive sex. The subdued and normalized libido on Nandrolone is welcomed for me because of this. I don't think this is necessarily just because I'm a good responder to Nandrolone, I think it has more so to do with the fact that I was using exogenous Estrogen during this experiment with the Nandrolone. Despite androgens driving aggression and drive, libido and erection quality is largely dictated by adequate Estrogen levels. With all that being said, DHT (with sufficient Estrogen via Testosterone aromatization) is blatantly better for sexual support than Nandrolone, and testosterone itself, even if you completely inhibited 5α-reductase and nuked DHT, still provides better libido and erection quality than Nandrolone does at equivalent "therapeutic" doses for the majority of people. My Blood Work Results On A Deca Only Cycle For HRT I don't like taking shots in the dark when it comes to something that I see potential in. There is a lot of theory thrown back and forth in the community on Deca only cycles, and I needed to see for myself how Nandrolone in conjunction with exogenous Estradiol would impact my personal blood work. I wanted to check markers of oxidative stress, inflammation, kidney function, Aldosterone, Prolactin, hormone levels via sensitive assay testing, and an array of other health markers that are often debated about but very infrequently actually tested for to reinforce statements made. Expectedly, high dose Deca only cycle blasts will almost always result in low HDL levels, subpar Estradiol levels, and an array of other out of range values that are less common and are more individual dependent. To date I have yet to see someone get their blood work checked with exogenous Estradiol being used in conjunction with Nandrolone at a "therapeutic" dose. This is what I wanted to evaluate. Complete Blood Count with Differential/Platelets I was actually expecting far worse from my blood test results. At a "therapeutic" dose, it doesn't seem like my hematology was negatively affected at all. Comprehensive Metabolic Panel In my metabolic panel, nothing was really off to the point that would cause concern. My BUN being high is likely just the result of being muscular and having a high protein diet. Lipid Panel Going into the lipid panel, we can see the number one most common blood test result among steroid users. My HDL is low. LDL is also borderline high, but not overly concerning when I can see that my Triglycerides are pretty low. The reason why my HDL was too low was that my Estrogen levels were too low. Again, this just reinforces the fact that Nandrolone does not sufficiently aromatize into Estrogen. I will get into my Estrogen level and why it was still too low even with Estrogel administration later once we get to that part of the blood test results, but my HDL could have been in range if my Estrogen level was in check. If I didn't use the Estrogel my HDL likely would have been in the single digits. I know I can get my HDL into the reference range if my estradiol levels were doubled, which I have the leeway to do. Iron And Total Iron Binding Capacity Getting into Iron and TIBC we can see that everything looks pretty normal here. Total Testosterone And Free Testosterone Expectedly, by assessing my Total Testosterone level via liquid chromatography with tandem mass spectrometry (LC/MS-MS) and my Free Testosterone level via equilibrium ultrafiltration, we can see that my Testosterone levels were crashed. Both the total and the free were lower than a healthy female. This is what you should see in your blood work if you’re on just Nandrolone. The only Testosterone being produced in my body was indirectly via the trace amounts of androgens produced in my adrenal cortex, which is why the value wasn't completely bottomed out at 0. I've mentioned many times the importance of getting high sensitivity testing done for hormone levels and how Nandrolone will register as Testosterone in primitive garbage blood tests. This is another great example of this. In addition to high sensitivity testing, I had the same blood tested using electrochemiluminescence immunoassay (ECLIA) for my Total Testosterone level, and direct analog enzyme immunoassay (EIA) for my Free Testosterone level. These were the test results using the exact same blood sample with the terrible default assays that doctors will use to determine how to treat you, and that labs will give you in the majority of your blood work panels. According to ECLIA and EIA, I have a normal Total Testosterone and Free Testosterone level. Hilarious. This just one example of why getting accurate hormone testing is critical. My Testosterone levels are actually in the gutter, but the stupid primitive tests that doctors and labs give out as defaults for people is so f*cking stupid that it can't even tell the difference between Testosterone and 19-nortestosterone in my blood. Renin Activity and Aldosterone My renin activity and Aldosterone appeared to be normal. This is one of the main things I wanted to check because there's a lot of speculation around the effect Nandrolone is going to have on Aldosterone levels. When it comes to the Deca only cycle, there's something going on that throws off homeostatic mechanisms that regulate blood pressure that does not appear to be Estrogen related or Aldosterone related. At least based on my blood work, my Aldosterone was definitely not at a level that could imply any kind of negative effect on blood pressure. My Aldosterone level was low if anything. Granted, some markers in the serum can be relatively worthless when compared to actual tissue concentrations, but at least based on my blood work, Aldosterone does not appear to be the culprit. The first thing many jump to when explaining blood pressure regulation is the effect Nandrolone supposedly has on spiking Aldosterone through the roof, but it just doesn't appear to be the case in my experience as you can see yourself here. Vitamin B12 and Folate My B12 and Folate levels were normal. Pregnenolone Pregnenolone appeared to be normal and within the reference range for men which is notable, as many assume that Nandrolone will shut down the production of precursor steroids. That does not appear to be the case either. I assumed precursor hormone levels like Pregnenolone would be less affected than many seem to think as most circulating Pregnenolone is derived from the adrenal cortex. Dihydrotestosterone (DHT) Expectedly, my DHT was very low. This is because I have almost no Testosterone being produced to 5α-reduce into DHT. If my Testosterone is low, my DHT will be low as well. DHT Backdoor Pathway Contrary to popular belief, there is a backdoor pathway via Pregnenolone that can create DHT as well, which contributes to the chunk of DHT I have in my blood. Hemoglobin A1c Hemoglobin A1c appeared to be normal at 5.1%. Thyroxine (T4) My Free T4 was 1.28 ng/dL, which is acceptable. DHEA-Sulfate My DHEA was in range and actually on the high end of normal. Being on exogenous Nandrolone or Testosterone does not shut down DHEA production. Cortisol Cortisol was "normal" apparently, although it looks a bit high to me. I believe this result was mostly sleep hygiene related rather than entirely Nandrolone related. Thyroid Stimulating Hormone (TSH) My TSH is too high. I've never had a TSH this high before. I have had a TSH in the 2's before, this isn't the first time, but never this high. However, based on my resting heart rate and my morning waking temperature and my mid-day temperature, my metabolic rate seems to be the same as it usually is on TRT, and I have had no standout hypothyroidism symptoms. Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) Expectedly, LH and FSH were undetectable. Prolactin My Prolactin was on the low end of normal. This was another interesting health marker to see on Nandrolone, as many will often jump to assuming that Nandrolone spikes Prolactin levels through the roof. That does not appear to be the case though. Prostate-Apecific Antigen (PSA) My PSA level was normal, and did not change from my previous blood work on Testosterone for TRT. D-Dimer My D-Dimer was normal. A friend of mine had a very high D-Dimer level on a Deca only cycle and he wanted me to check mine to see if there was a pattern. It looks like the elevated D-Dimer was case-specific for him and was probably caused by something completely unrelated, as my D-Dimer is normal. C-Reactive Protein C-Reactive Protein is one of the primary markers we have for assessing inflammation in the body. A C-Reactive Protein level of 0.34 mg/L is not overly concerning, although I would like to see it below 0.3 mg/L. I had undetectable C-Reactive Protein levels in the past on TRT, and on Nandrolone it jumped up to 0.34, which is notable. Estradiol, Sensitive My Estradiol (E2) level determined via LC/MS-MS was only 15.4 pg/mL, despite administering 2.5 grams of Estrogel per day. This was disappointing, as I would have liked to see my E2 at least around 30 pg/mL based on the amount of Estrogel I was applying daily. Evidently, my inner thigh was not absorbing the Estrogel very well. This is one of the recommended areas of application, but my results were not even close to in line with the average blood levels found in the Estrogel pharmacokinetics studies. With daily administration of 2.5 g or 5 g Estrogel (corresponding to 1.5 mg or 3 mg estradiol, respectively), mean serum estradiol concentrations of approximately 80 pg/ml (294 pmol/L) and 150 pg/ml (551 pmol/L), respectively, are maintained. Administration of Estrogel also results in increased serum estrone concentrations, producing a physiological estradiol/estrone ratio of approximately one. Therefore, serum concentrations of both estradiol and estrone and the serum estradiol/estrone ratio provided by ESTROGEL® are consistent with physiological levels observed during the follicular phase of the normal menstrual cycle. My inner thigh isn't very hairy at all as I manscape fairly regularly, so I expected at least a 40 pg/mL E2 based on the amount of Estrogel I was applying. I was overly generous with my dose based on the off chance that I would encounter an absorption issue, and my E2 was still way below where I expected it to be. Based on the pharmacokinetics outlined by Merck, 80 pg/mL is the average E2 level for someone applying 2.5 grams of Estrogel per day. There's no way I could have predicted that I would have an absorption issue so problematic that it would result in five times lower absorption than the average. If my E2 was closer to 30 pg/mL, I expect that my HDL would have been pushed into the reference range, and all Estradiol driven physiologic functions likely would have been more optimized. To me, this just reinforced further that Nandrolone is a subpar source of Estradiol as I was using a high dose of transdermal E2 and still could barely reach a satisfactory E2 level. To increase my E2 levels for similar future experiments I will either have to find a better application area, add some DMSO to my Estrogel to increase absorption, or consider Estradiol injections instead. Homocysteine My Homocysteine level was higher than I would like. Normally my Homocysteine is closer to 8-8.5 umol/L. Earlier in the year when I did a shorter Nandrolone experiment for a month using 200 mg per week (double the dose I used for this experiment) I had a Homocysteine level around 8.5, so I doubt this spike was Nandrolone related. This is one of the main markers I always have my eye on because I am homozygous for the C667T polymorphism. Gamma-Glutamyl Transferase (GGT) My GGT looked good. I was worried that this would be cranked through the roof as it is a marker of oxidative stress. Magnesium My magnesium level looked okay. Copper and Zinc My zinc level looked okay. My copper level may be a bit low, which I am now addressing by eating an ounce of beef liver every day. Progesterone My Progesterone was normal, which is notable as it is another precursor hormone that many assume drops to zero when exogenous anabolic steroids are present in the body. Insulin My insulin level was good. Estrone Expectedly, my Estrone was a bit high. This can be a major problem with exogenous Estradiol and Nandrolone unfortunately. Estrone Level Increase From Exogenous Estradiol The ratio of Estrone-to-Estradiol is skewed with massive elevations in Estrone with oral Estrogen administration. Fortunately, this unhealthy ratio can be avoided for the most part with transdermal Estradiol administration. High levels of serum Estrone sulfate (E1S) were found after long-term oral estrogen treatment of commonly prescribed dosages, whereas there was a small increase in E1S levels after transdermal Estradiol (E2) therapy. The mean maximum E1S levels were more than 20-fold higher with oral estradiol (E2) when compared with the 0.05 mg/day transdermal estradiol patch. This is consistent with the 20-fold higher dose of E2 when compared with the transdermal dose [R]. Estrone Level Increase From Nandrolone Nandrolone also significantly elevates concentrations of Estrone in plasma [R]. During a pilot study evaluating the possible beneficial effect of Nandrolone Decanoate (ND) on bone metabolism in patients with rheumatoid arthritis there was a significant increase in the serum levels of Estrone [R]. Despite the fact that Estrone can convert to Estradiol, we can clearly see that the amount this actually happens in the body is minimal based on the consistently skewed ratios of androgens to Estradiol in the blood test results of Deca only users (or Nandrolone in general). Ferritin My Ferritin level is too low. This is likely the result of phlebotomizing too frequently in 2019. Estriol My Estriol level was undetectable, which was expected. Triiodothyronine (T3) My Free T3 level was 2.7 pg/mL. It's not low enough for me to be overly concerned, however, it is suboptimal and should be in the low 3's at least. This is something I will need to address moving forward. With that being said, I like to look at my resting heart rate as well as my body temperature for a more accurate assessment of my metabolism, and both are where I want them to be. My waking temperature has consistently been 98 degrees Fahrenheit, and my midday temperature has consistently been 98.6 degrees Fahrenheit. Sex Hormone-Binding Globulin Expectedly, my sex hormone-binding globulin (SHBG) was low. While this isn't as relevant for a Nandrolone only user as Nandrolone has a very low affinity for SHBG, this is a value I would still like to see in the reference range, especially if I was on TRT. If I had the Estradiol level I was shooting for, I'm confident that my SHBG would have been in the reference range. My Overall Experience On Nandrolone And Exogenous Estrogen For HRT I was expecting to see a bunch of red flags in my blood work, but nothing really stood out as a major concern to me except for the spike in systolic blood pressure, and the high Estrone level. A before and after echocardiogram and calcium scoring would have been nice to see, but unfortunately I can only afford to do so much in these experiments, and the blood work was expensive enough as is. I felt good throughout the entire experiment, I maintained my physique, my libido and penis were functional, and my blood work looked pretty good considering that each issue was something more so related to my Estradiol administration than the Nandrolone itself. Estrone being out of range is a concern, as I would need to use even more exogenous Estradiol to achieve what I would consider a more therapeutic E2 level, which would likely push my Estrone up even higher. The difficulty in controlling the blood pressure spike is also a huge concern and could be a deal breaker. If I gave this experiment more time, it is entirely possible that certain things would have become problematic that appeared to be fine during my three month assessment, like my libido or sense of well-being. It is also possible that despite maintaining a healthy Estrogen level, the same neurological and cardiovascular issues we see in a significant amount of the Nandrolone data could still accumulate over time. In addition, healthy looking serum levels of Estradiol may not necessarily reflect adequate localized Estrogen receptor activation in each tissue. With Testosterone, there is a regulated amount of aromatization occurring in each tissue to satisfy however much Estrogen receptor (ER) activation we need. In the context of Deca only cycles, or Nandrolone monotherapy, there's nothing else I can refer to other than serum levels, my libido, sense of well-being, other cardiovascular health markers, etc. In other words, just because you feel good and your Estrogen levels look good on paper, that doesn't mean that an exogenously administered source of Estrogen is providing the same therapeutic ER activation in all tissues like it would if it were regulated via aromatase. With that being said, you could also argue the opposite as adequate receptor activation via exogenous hormone therapy is essentially all HRT boils down to to begin with in the context of any hormone. More than 95% of our endogenous Testosterone is produced in the testes. Testosterone is supplied to target tissues in the blood, just like most other hormones in the body. If you inject exogenous Testosterone, it then goes into the blood and is supplied to target tissues. If you inject anything it goes into the blood and then is carried to the areas that it is needed. Estrogen replacement has been deemed satisfactory for fulfilling the same functions as endogenously produced Estrogen in women for years, and synthetic Estrogen analogs are handed out like candy to millions of young girls (including teenagers). Is it healthy? Estrogen analogs like Ethinyl Estradiol probably aren't ideal for regulating Estrogen dependent functions, and they definitely aren't ideal for developing women who haven't fully matured. However, there is tons of data to support the fact that exogenous Estradiol is well-tolerated, has a strong safety profile, and can still fulfill physiologic functions sufficiently. In an ideal world, this would be a regulated process in the body in each tissue (aromatization). My experiments do not necessarily reflect what I believe are best practice with these hormones, which should be noted. This was an experiment, and not something that I would recommend someone else do. Using an exogenous progestogen with estrogel certainly isn't what I would consider an optimal HRT protocol, or what is indicative of an ideal means of providing androgenic and estrogenic support in tissues. With that being said, I don't see a better way to go about utilizing Nandrolone on its own for HRT. Should it even be considered as an HRT alternative though? That's the question, and I believe it is largely going to be individual dependent, with a significant amount of users having poor outcomes in one aspect or another. I do believe there are a minority of individuals who are very prone to androgenic and/or estrogenic side effects from exogenous Testosterone use that may benefit from exploring Nandrolone though, and it should not be discarded as a potentially viable alternative simply because it is not the primary bioidentical hormone that men produce.

Today, I’m bringing you an interview with my good friend Andrea Jang.  Andrea is a fertility acupuncturist in the San Francisco bay area.  Her clinic was the first clinic other than my own, to embrace the Practically Fertile Methodology.  We’ve been working closely together for two years. What Andrea has added to her practice recently is functional medicine. According to Andrea, Conventional lab ranges are inaccurate, bell curves are used, and only limited tests are ordered. Unlike conventional lab tests, functional lab values are more sensitive to reveal problems.  Andrea wants to offer the Comprehensive Thyroid Evaluation for only $147 (Regularly $350). And this test will be Available to the first 10 people who contact her.   Offer includes: Complete thyroid panel including 9 key thyroid tests plus a one on one Functional Medicine consultation to review the test results. T3 Uptake  T3, Free (FT3)  T3, Reverse, LC/MS/MS   T3, Total  T4 (Thyroxine), Total  T4, Free (FT4)  Thyroid Peroxidase Antibodies  Thyroglobulin Antibodies  TSH (Thyroid Stimulating Hormone)  Most tests run only 1-2 of these markers and miss the key underlying imbalances. This offer also includes a 25% discount on a supplement purchase through Fullscript and a Thyroid Health Education card.  (Lab testing not available for residents in NY, NJ, RI. ) To register, text “Thyroid” to 415.496.JANG (5264) or go to jangacupuncture.com and click the email icon to get in touch. 

Measurement of plasma adrenocorticotropic hormone, or ACTH, is key in the differential diagnosis of hypothalamic pituitary adrenal disorders. Two-site sandwich immunoassay dominate clinical testing of ACTH in North America.  However, discordant results among different assays have been repeatedly reported.  To help resolve this discrepancy, a multicenter effort developed a liquid chromatography tandem mass spectrometry assay for the intended measurand, that is the biologically active intact ACTH.  A report on that initiative appears in the November 2019 issue of Clinical Chemistry.  We are pleased to have the senior author of that paper , Dr. Mari DeMarcoas our guest in this podcast. 

I recently lowered my weekly TRT dose down to 100 mg Testosterone Propionate split into every day subcutaneous administrations with an insulin pin. Micro-dosing every day results in the most stable blood serum concentrations possible, less aromatization, and more free testosterone.…

Glomerular filtration rate, or GFR, is generally accepted as the best overall index of kidney function, and a decrease in GFR has important implications for prognosis in patient management.  GFR is most commonly estimated by calculation, using the serum concentration of an endogenous filtration marker such as creatinine and demographic variables such as age, sex, and race.  In the March 2019 issue of Clinical Chemistry, a paper investigated the possibility of developing a more accurate estimate of GFR, using a panel of metabolites measured by quantitative liquid chromatography, tandem mass spectrometry without creatinine, cystatin C, or demographic variables.

  Join Dan (@drusyniak) &Howard (@heshiegreshie) as they speak with Dr. Ed Boyer about therapies for opioid use disorder, including kratom, ibogaine, and loperamide. Great stuff, deep science and a dispelling of some myths while recognizing some less than savvy science out there. . . Looking forward to part 2, where we find that the dulcet tones of Dan and Howard are the right frequency to treat your blues. Delicious Links Many of the therapeutics both prescribed and otherwise in the Pharmacological Treatment of Opioid Use Disorder. Mitragynia speciosa korth - self-treatment of opioid withdrawal using kratom. Not only adults are affected by these agents. Neonatal abstinence syndrome due to maternal kratom use. Where can you get these products and agents? Look at the impact of internet pharmacy regulation on opioid analgesics availability. But be careful where you buy. Suspected adulteration of commercial kratom products with 7-hydroxymitragynine. Get intracellular with mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Some more information as described in beta-arrestin 2 knockout mice. How strong do you like your tea? Quantification of Morphine, Codeine, and Thebaine in Home‐Brewed Poppy Seed Tea by LC‐MS/MS. While out guest certainly doesn't believe in the magic of ibogaine, there are many out there that still do. Here's one example of what to look out for . . . in a patient with prolonged multiple cardiac arrhythmias after ingestion of internet purchased ibogaine. Some interesting stuff on addiction research and the use of ibogaine, a “vast, uncontrolled experiment.” Keep your eyes peeled for more cases of tianeptine misuse as reported in poison control center experience with tianeptine: an unregulated pharmaceutical product with potential for abuse. And if you haven't already read it (and why not?) here is a great post about the toxicity of loperamide used for opioid use disorder - The Worst of Both Worlds. Special Thanks Thank you for your continued support. As always, we are looking for feedback - comments, questions, suggestions, recipes, etc. Let us know. Reach us at @toxandhound. We want to hear from you! Thank you to our house band Pretty Simple Duo (@prettysimpleduo), our announcer Josh Shelov (@shelovj), and Reverend Matt Winston of Witness Protection Products. Kraken image by Bryan Alexander. Music "Pamgaea" by Kevin MacLeod. License: CC BY Interested in #FOAMtox? Like this podcast? Take a gander at The Tox and The Hound. It's like a podcast, but for your eyes. Listen on iTunes or Spotify! Earholes happy? Rate and review! Show the love!  

Live Right Now - Episode 009 – Soy: Perfect Food or Evil Imposter? What’s square, white, jiggles, weighs about eight ounces, can make you gag, and often can clear a room in ten seconds? The answer is, tofu, or soy bean curd. This often-maligned product has been known to strike dread in the hearts of the bravest culinary souls, paralyzing them in fear at its mere mention. (Not wholly unlike the feeling you might get when receiving notification of an IRS audit.) Flash back to the flower children of the mid and late 1960s, when a rumor wafted through the grease-filled air that the Golden Arches folks used a sinister form of fibrous soybeans as filler in their burgers. “Ai-ee! Hack! P-tooey! We’ve been poisoned!” In retrospect, what we should have protested instead was the saturated-fat-laden bovine tallow used to deep-fry those golden, salt-covered French fries. (Just as an FYI, though, McDonalds and Taco Bell have indeed been using soy products as filler for decades. Nevertheless, don’t expect to see the International House of Bean Curd popping up soon.) So how many centuries have people been eating tofu? Tradition has it that tofu was invented by Liu An (179–122 B.C.), a prince of the Han Dynasty, supposedly while searching for a substance to help him achieve immortality. But way before then, in 2838 B.C., Chinese Emperor Cheng Nung developed soy cultivation. Soybeans did not, however, grace American soil until Samuel Bowen brought it to the continent and Henry Yonge planted the first soy crop on his farm in Thunderbolt, Georgia, in 1765. Did Henry know when he sowed the seeds of soy he would be saving us from the sorrow of serious sickness and senility? Somebody let a snake loose? New findings are out about tofu and soy products, however, and as I painfully sift through the mountains of information on the subject, I have to ask myself, “Is it actually—gasp!—bad for us?” After decades of aggressive research and marketing and touting the wiggly curd as a miracle cure-all for many of humanity’s maladies, I wonder, alas, is the honeymoon over?  Is mass tofu-phobia justified? Can tofu really make your brain shrivel and encourage dementia and breast cancer? What’s with that? For a substance that has been providing nourishment for humans for so many years, this Rodney Dangerfield of food is getting no respect. But what I’m placing in your to-go bag is whether we should be alarmed about these new studies regarding the safety of eating tofu, or is this junk science? Is it safe to continue making tofu a foundation of our diet? Ignorance may be bliss, but information is a powerful tool, so let’s look objectively at both sides of the issue, and, as my Mom would have said, “Don’t throw the baby out with the bath water.” We need to encourage more funding for further studies and season our own judgment with a generous helping of knowledge. I'd like to emphasize that in every study I looked at showing beneficial effects, the study was either sponsored by the soy industry, or the authors had some kind of financial ties to the soy industry. Follow the money, as they say. When asked about the validity of tofu-phobia, the Indiana Soybean Board responded passionately with this reassurance: “Wendell, I think the important thing is that overreacting and taking things out of context is the biggest problem...Asians have been eating soy foods for centuries and undoubtedly there is no evidence that they have less cognitive function.” Yes, but they were not GMO! And, contrary to what you may have heard, Asians do not consume large amounts of soy. They use small amounts as a condiment (about two teaspoons daily), but not as a primary protein source. And the type of soy they consume is traditionally fermented soy. Soybean crops are also heavily sprayed with chemical herbicides, such as glyphosate (Round-up), which researchers have found to be carcinogenic. The herbicide has been the subject of controversy for years once it became known it causes serious health problems, including endocrine disruption, allergies, asthma, autism-spectrum disorders, gastrointestinal disorders, rhinitis, obesity, leukemia, lymphoma, and other forms of cancer. One of the primary reasons it would be wise for you to avoid soy is more than 90 percent of soybeans grown in the United States are genetically modified. Since the introduction of genetically engineered foods in 1996, we’ve had an upsurge in low birth weight babies, infertility, and other problems in the U.S., and animal studies have shown devastating effects from genetically engineered soy including allergies, sterility, birth defects, and offspring death rates up to five times higher than normal. Soybean crops are also heavily sprayed with chemical herbicides, such glyphosate which a French team of researchers have found to be carcinogenic. Glyphosate, the world’s most widely (vilified) used herbicide linked to Monsanto’s Roundup Ready genetically engineered crops, has been found at alarming levels in a wide range of best-selling foods across the U.S., Food Democracy Now! and The Detox Project announced Monday. The results published in this report are from the first independent glyphosate residue testing of popular American food products performed using liquid chromatography tandem mass spectrometry (LC-MS/MS), the regulatory recognized “gold standard testing methods at an FDA registered laboratory. These newest findings also come as the Environmental Protection Agency (EPA) postponed hearing which were due to explore glyphosate’s link to cancer in humans. Last year, 17 leading global cancer experts from the World Health Organization’s International Agency for Research on Cancer (IARC) sparked a firestorm when they classified glyphosate as a class 2A “probable human carcinogen. On the heels of the growing controversy surrounding glyphosate’s safety, this unique testing project  that started in 2015, has so far found alarming levels of glyphosate in General Mills’ Cheerios and Honey Nut Cheerios, Kellogg’s Corn Flakes, Raisin Bran and Frosted Flakes and PepsiCo’s Doritos Cool Ranch, Ritz Crackers and Stacy’s Simply Naked Pita Chips, as well as many more  famous products at levels that present significant risks according to the latest independent peer-reviewed science on glyphosate. Detoxproject.org Soybeans — even organically grown soybeans — naturally contain “antinutrients” such as saponins, soyatoxin, phytates, trypsin inhibitors, goitrogens and phytoestrogens. Traditional fermentation destroys these antinutrients, which allows your body to enjoy soy’s nutritional benefits. However, most Westerners do not consume fermented soy, but rather unfermented soy, mostly in the form of soymilk, tofu, TVP, and soy infant formula. Mercola.com Fermented Soy Probiotics versus Unfermented Many types of fermented foods are very good for our gastrointestinal tracts, helping to keep points A through Z flowing and in good working order, which is imperative for optimum health. Soybeans are among those foods that are best whether fresh or fermented. Fermented non-GMO organic soy products such as tempeh and miso are much easier for our Earth Suits to digest than processed silken tofu products. Tempeh, a fermented soybean product that comes in cakes, is made from whole soybeans and has a nutty, smoky flavor and is similar to mushrooms in texture. At our home we us it to cook sloppy joes, barbecue, Cajun “steaks,” Caesar salad protein, spaghetti sauce, taco filling, and chili. The grandkids love it, and sneaky chef that I am, I don’t tell them how good it is for them! Four ounces of cooked tempeh contains 17 grams of protein, a mere 204 calories, 15 grams of carbohydrates, and 8 grams of (good) fat. Plus, it’s full of calcium, iron, zinc, and fiber. It’s so much better for you than the same size portion of steak, and doesn’t contain artery-clogging saturated fats, antibiotics, and growth hormones so commonly found in factory farmed beef. A plethora of reasons to make soy the center of our diets abounds. In 2001 in San Diego, California, at the Fourth International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, a mutually agreed-upon conclusion was reached: Non-GMO, organic Soy may possibly have a positive effect on cognitive function. Two preliminary research studies presented at the symposium showed that soy actually improved several aspects of cognitive function, especially verbal memory. Hopefully, this good news will alleviate any concerns you’ve had about the soy-and-dementia issue. The Soy Board reminds people to keep things in perspective. The negative effects were found only in an epidemiological study; however, animal studies suggest just the opposite—soy has beneficial effects on cognitive function.” Hmm? This is somewhat contrary with what Dr. Lon Wright of the Pacific Health Research Institute presents. He has conducted a study of 3,734 middle-aged Japanese-American men that indicates that eating tofu more than twice a week may be linked to dementia. White’s theory is that the phytoestrogens in tofu interfere with the brain’s estrogen receptors and keep the brain from properly using estrogen. His article appeared in an edition of the Journal of the American College of Nutrition. But listen to this:  He says, “I would be violating a cardinal rule if I said my data says you shouldn’t eat tofu [or other soy foods].” Ah-hah! White emphasizes this data can’t be turned into sweeping conclusions, and the findings must be considered preliminary. And according to Beverly Creamer, staff writer for a Honolulu advertiser newspaper, “It’s the first time scientists have labeled a dietary risk factor for the disease that affects 2 percent of the nation’s sixty-five-year-olds and up to 16 percent of eighty-year-olds.” Finally, White’s study was based on processed tofu, which is not fermented, and which could be considered another endorsement for the fermented forms of the bean or edamame. Here’s more spice for the health stew: University of Minnesota scientist Mindy Kurzer, Ph.D., who does extensive research on the humble bean, assures us that there are no data connecting soy and cancer. “There is a theoretical risk that processed soy might promote breast cancer in some way,” Kurzer added, “but it’s purely theoretical at this point.” Forgo the ubiquitous protein bars made with protein isolate. Side effects of soy protein isolate: In animal studies, soy isolate has been linked to allergies, thyroid problems, and even brain damage. Soy has been labeled one of the top seven allergens for people to avoid, as soy isolate is found in a lot of processed foods, including bread and baked goods, soups and sauces, and breakfast cereals and protein bars. There have also been several studies on soy protein and age-related dementia, although many of those studies have been inconclusive. Wellnesstoday.com Perhaps the problem is our American lifestyle. Otherwise-healthy Asians who come to live in America ultimately succumb to the same health maladies as native-borns. Is it the pineal gland-trashing fluoridated water, the pesticides, food coloring, preservatives, fungicides on our produce, or our overly polluted environment? Or is it a disconnection from earth. Consider the negative findings. Until then, open your mind as well as your mouth to the healthy virtues of unprocessed, non-GMO, organic soy products, but don’t go overboard and follow the American mantra, “More is better.” Most of the time, less is more. Sweet and Spicy Peanut Noodles with Avocado and Kale                                                                     (Recipe from: Eat Right Now with Chef Wendell Fowler: The Divinity of Food-2017 Lulu Press)   1 package tempeh (can substitute firm Tofu if preferred) - cut into cubes-protein 3 tbs. coconut oil 2 tbsp. wheat-free soy (Tamari) 2 tbs. maple syrup or brown rice syrup 1 # brown rice noodles or rice noodles 2 avocados 4 cups chopped kale 1 cup fresh or frozen peas Green onion, chopped Ground flax or chia seed   Sauce: 2/4 cups peanut butter 3 tbsp. fresh grated ginger 2 garlic cloves, minced 3 tbsp. wheat-free soy (Tamari) Hot pepper flakes to taste Juice of two fresh limes 2 tbsp. toasted sesame oil   To Prepare: Cook pasta per package instructions, drain and reserve. Cut tempeh into 1/2 inch cubes In a large sauté pan, heat the coconut oil over medium heat and add the tempeh cubes. Sauté gently till the edges begin to brown. Add the soy and syrup and cook 4- 5 minutes longer. Set aside to cool. Keep warm however. Cook noodles to package instructions To make dressing, whisk together the peanut butter, ginger, syrup, soy, lime and sesame oil to a mixing bowl.  Too thick?  Add water. To assemble the dish, fill four bowls with noodles and top with kale, peas, and avocado quarters. Pour about 1/4-1/3 cup of dressing of each and garnish with sesame seeds, chia / flax, avocado wedges and green onion.   Live Right Now theme music is “future soundtrack II” by Adam Henry Garcia from the Free Music Archive licensed under CC BY-NC-SA 4.0  

A high throughput LC-MS/MS method for the determination of phosphatidylethanol (PEth) in clinical samples using a simple automated extraction procedure was published in the May 2018 issue of The Journal of Applied Laboratory Medicine.  This work describes an improved automated approach to analysis of the alcohol marker phosphatidylethanol that uses liquid chromatography mass spectrometry instead of conventional HPLC.

Judith Stone

mTOR-Inhibitoren (Synonym: Rapamycin) sind Wirkstoffe, die sowohl in der Immunsuppression als auch in der antiproliferativen Therapie systemisch und lokal Verwendung finden. In Vorversuchen unserer Arbeitsgruppe mit Zellkulturen zeigte sich schnell ein großes Adsorptionspotential von Rapamycin – beziehungsweise von dessen Derivat Everolimus – an die Oberflächen von Zellkulturflaschen. Diese Adsorption hängt im Wesentlichen von zwei Faktoren ab: dem Proteingehalt des Mediums und der spezifischen Oberfläche der Zellkulturflaschen. Um dies zu zeigen, wurden verschiedene gebräuchliche Zellkulturflaschen (Nunclon, Ultra-low-attachment, Weichglas, unbehandelte Polystyren-Oberflächen und Polystyren Oberflächen beschichtet mit Collagen 1 beziehungsweise Poly-D-Lysin) sowie Duranglas-Petrischalen ausgewählt. Die Oberflächen der Zellkulturflaschen wurden eine Stunde mit Medium mit einer definierten Menge an Everolimus bedeckt, gespült und wiederum eine Stunde mit DMSO bedeckt. DMSO löst die Substanz wieder von der Oberfläche ab. Die Everolimuskonzentrationen im Medium nach einer Stunde und in der DMSO-Lösung wurden mittels LC-MS/MS bestimmt. Es zeigte sich signifikante Adsorption von Everolimus in absteigender Reihenfolge: Ultra-low-attachment > Unbehandeltes Polystyren > Collagen 1 > Nunclon > Poly-D Lysin > Weichglas > Duranglas (bei 10% FCS in Medium) und Ultra-low-attachment > Unbehandelt > Collagen 1 > Weichglas > Poly-D-Lysin > Duranglas (bei 30% FCS in Medium). Im Folgeversuch wurden vier der Zellkulturflaschen ausgewählt (Nunclon, Unbehandelt, Collagen 1, Duranglas-Petrischalen) und untersucht, ob die reine Adsorption von Everolimus an die Oberfläche ohne Everolimus im Medium negative Effekte auf das Zellwachstum hat. Dies konnte bei drei Zelllinien (293T, VSMC, HUVEC) mittels Zellzählung demonstriert werden. Bei allen drei Zelllinien wurden p-p70s6K- Western Blots durchgeführt. Die p-p70s6K ist ein downstream gelegenes Phosphorylierungsprodukt von mTOR, welches wiederum von Rapamycin/ Everolimus gehemmt wird. Teilweise zeigte sich hier eine absteigende Phosphorylierung. Bei HUVEC- Zellen wurde zusätzlich die Expression von VEGF und p-p70s6K mittels ELISA untersucht. VEGF ist ein Faktor, der Wachstumssignale spezifisch an Gefäß- Endothelzellen vermittelt. Hier konnte entgegen der Erwartungen sogar eine Zunahme der Expression mit steigender Everolimuskonzentration gemessen werden. Neuere Studien legen jedoch nahe, dass VEGF nicht ausschließlich über TOR aktiviert wird. Bei p-p70s6K zeigte sich die erwartete Abnahme der Expression. Die Versuche weisen auf eine signifikante Beeinflussung des Zellwachstums durch Everolimusadsorption an Oberflächen hin. Inwiefern sich Adsorption bei Zellversuchen mit Everolimus in Lösung auswirkt, ist noch unklar. Eine Minderung der Everolimuswirkung wäre denkbar. Um die Oberflächenadsorption bei Versuchen mit Everolimus möglichst gering zu halten, empfiehlt unsere Arbeitsgruppe anhand der Versuchsergebnisse die Kultivierung auf wenig absorbierenden Oberflächen wie Duranglas beziehungsweise die Erhöhung der FCS-Konzentration in Lösung, soweit von den Zellen toleriert.

Cortisol: the emergency responder hormone that when chronically elevated, makes you fat, stupid and unsexy. Nobody wants an excess of cortisol, but like all things in physiology there’s a sweet spot; low cortisol doesn’t feel right either. Which you have? Symptoms of high cortisol Symptoms of low cortisol Fatigue Extreme fatigue Muscle weakness Muscle or joint pains Depression, anxiety and irritability Depression, irritability Loss of emotional control Salt craving Cognitive difficulties Abdominal pain New or worsened high blood pressure Low blood pressure, even fainting Glucose intolerance that may lead to diabetes Low blood sugar (hypoglycaemia) Headache Nausea, diarrhea or vomiting Bone loss, leading to fractures over time Weight loss and decreased appetite Source: Mayo Clinic Both sets of symptoms are very similar, in fact, all of these symptoms could be attributable to half the diseases known to man. The only way to know for sure what’s going on with your cortisol is to do the test. I tested over 500 endurance athletes And all but a handful had low free cortisol. Free cortisol is the active fraction of the hormone, but it only accounts for 1% of the total cortisol production and so forms just part of the puzzle. I can’t give you any more information than this because free cortisol is all a saliva test measures. Luckily the testing has gotten more sophisticated Mark Newman is an analytical chemist and the founder of Precision Analytical, a lab using GC and LC-tandem mass spectrometry to measure not just free cortisol, but also the metabolites tetrahydrocortisol and tetrahydrocortisone. Their DUTCH test is a game changer. The extra information in this test enables me to be a better detective. Now I can differentiate between problems with cortisol production and cortisol clearance. The two have very different implications. Not just cortisol The DUTCH also measures no less than eight androgens, eight oestrogens and two metabolites of progesterone. The results can be overwhelming at first, but as you begin to appreciate the pathways that these hormones take, the picture becomes clear. Testosterone Testosterone can metabolise down one of two pathways: the more androgenic alpha pathway that leads to the hormone DHT, thinning scalp hair and prostate problems for older men or a potentially more desirable and less androgenic beta pathway. The good news is that once you understand your metabolism, there are nutritional supplements that you can take to inhibit the alpha pathway. Oestrogen Saliva isn’t a very accurate way to measure oestrogen. Not only is urine more precise, but it also enables us to test the metabolites that tell us all about both phase 1 and phase 2 metabolism. Oestradiol is the most abundant oestrogen, and its friend oestrone breaks down in one of two different directions. This can produce two very different, and not always desirable outcomes. The only way to know what’s happening to you is to do the test Establishing a baseline During this interview, Mark makes an important point. Let’s say you feel good right now. Wouldn’t it be nice to have the complete picture of your hormone metabolism for future reference? Or perhaps you suspect a problem. Either way, you can order a DUTCH though me and then together we figure out a plan to optimise your hormones. Don’t be that guy guessing using the list of symptoms you found on the Mayo Clinic website. Here’s the outline of this interview with Mark Newman: 0:00:50    Mark Newman is an analytical chemist with a master's degree in forensic science. 0:01:07    Mark started with urine testing then moved onto saliva testing. 0:01:41    DUTCH stands for Dried Urine Test for Comprehensive Hormones. 0:02:11    The DUTCH combines the best of saliva and urine. 0:02:51    Educational videos. 0:03:32    The DUTCH is the culmination of Mark's life work. 0:05:01    My first saliva test showed high cortisol at night, and low cortisol during the day. 0:05:51    We've run over 546 saliva tests now. 0:06:12    All the results look the same. 0:06:31    The question is WHY low cortisol? 0:07:01    Mark has managed data from one million saliva results. 0:07:23    Mark started with the idea that cortisol makes you fat, yet salivary cortisol isn’t higher in fat people. 0:07:40    The free cortisol is just one piece of the puzzle. 0:08:07    With urine testing, you also get to look at the metabolites. 0:08:24    Free cortisol is only about 1% of the total. 0:08:53    40% of patient that have low free cortisol do have elevated production (metabolites). 0:09:45    What drives cortisol production also drives clearance. 0:10:00    In obesity there is high production AND clearance, overall free cortisol is often low. 0:10:33    In hypothyroidism cortisol clearance is sluggish. 0:11:06    The DUTCH is the only way to get all three dimensions. 0:11:51    I get the impression all the tests will look dated in 10 years time. 0:12:32    Mark is skeptical when I say "everybody's low". 0:13:01    Reference ranges can be very problematic. 0:13:38    You cannot easily compare results from different tests. 0:14:13    Not everybody has low cortisol. 0:14:55    Morning serum cortisol is better than nothing. 0:15:08    Saliva is better still and DUTCH better yet. 0:15:27    The term adrenal fatigue needs some work. 0:15:44    Adrenal glands are not like ovaries (they don’t tend to give out with age). 0:16:16    The problem with cortisol is probably often in the brain, not the adrenal gland. 0:17:01    The misinformation has been driven by oversimplification. 0:18:03    How are the reference ranges for the DUTCH defined. 0:18:20    You start with a bunch of healthy people. 0:18:35    The range is then two standard deviations either side of the mean. 0:18:53    This is useful for finding pathology. 0:19:17    Reference ranges require more thought than simple maths. 0:20:00    Some reference ranges are set up so that everyone is low. 0:20:23    The analytes are unique and so are the reference ranges. 0:21:07    People are critical of blood tests, both blood is very well established and standardised. 0:21:35    Saliva testing has some shoddy standards for some tests. 0:22:06    Intranasal ACTH experiment. 0:22:30    If the lab is not curious enough to do that type of experiment you have to wonder. 0:23:16    Everyone should get test to establish a baseline. 0:23:30    Hormonal symptoms overlap a lot. 0:23:51    Then when people read or hear about symptoms, they say gee that's me. 0:24:18    Lots of things can drive cortisol. 0:24:59    Hormones are worth exploring around menopause. 0:25:28    Everybody is tired. 0:26:17    Mark's sister-in-law is healthy in spite of low hormones, should anything happen in the future, having the baseline would be important. 0:27:20    DHEA comparison. 0:27:34    Mark is a skeptic when it comes to labs, he always suspects they could be wrong, even his own…that’s why having multiple markers (as in DUTCH) for hormones can be helpful. 0:28:19    Etiocholanolone and Androsterone. 0:28:43    Inflammation blocks sulfation DHEA à DHEAS. 0:29:24    The DHEA-S can misrepresent what's really going on. 0:29:55    The DUTCH represents 14 hours worth of DHEA production. 0:30:31    The DUTCH paints the big picture of hormones. 0:31:01    Iron overload in master's athletes. 0:31:24    The DUTCH helps me be a better detective. 0:31:36    Inflammation makes prostaglandins that drive aromatase. 0:32:19    Narrowing down a list possibilities to a list of probabilities. 0:32:43    DHEA and women. 0:33:03    DHT is three times as potent as testosterone. 0:33:18    Insulin can push alpha metabolism. 0:33:36    The beta pathway is less androgen. 0:33:42    Saw palmetto, nettles, pygeum, EGCG, progesterone, zinc, finasteride all block the alpha pathway. 0:33:48    DHT can cause acne, thinning scalp hair. 0:34:37    The DUTCH has 4 alpha pathways measured. 0:34:59    PCOS is a leading cause of infertility. 0:35:36    Could we take supplements without doing the tests? 0:35:59    Mark thinks that's a bad idea in some cases. 0:36:50    Pragmatism can work. 0:37:05    Thinning scalp hair can have a number of causes. 0:37:29    If you can afford the test, do it. 0:37:42    Some of the supplements are also expensive. 0:37:51    DIM for oestrogen metabolism. 0:38:03    Pushes oestrogen down the 2-OH pathway. 0:39:11    DIM as a goitrogen. 0:39:39    I3C metabolises to DIM. 0:40:52    Mark's testing matrix. 0:41:18    People take progesterone at night because it helps with sleep. 0:41:27    Oral progesterone is gone four hours later. 0:42:09    Saliva and serum are not good ways to measure oral progesterone supplementation. 0:43:13    Mark has spent 10 years putting the testing matrix together. 0:43:43    What testing oestrogen can tell you about your methylation status. 0:43:47    Oestradiol is the main oestrogen. 0:44:01    Saliva testing for oestrogen is almost useless. 0:44:20    In serum and urine there's a 10 fold difference in oestrogen between pre and postmenopausal women. 0:44:23    In saliva, it's a 2 fold difference at best. 0:44:44    This is one of the main reasons why Mark switched to urine. 0:45:11    DIM acts on phase one metabolism. 0:45:23    Phase two includes methylation. 0:45:34    Genetic defects affect phase two. 0:45:51    Methylation is an important thing to do well. 0:45:56    Mark has two very messed up COMT genes. 0:46:04    Mark's MTHFR genes are fine. 0:46:17    On the DUTCH test, Mark is a poor methylator. 0:47:40    The DUTCH is cheap to ship (filter paper is light). 0:48:07    The test is still expensive, unfortunately. 0:48:33    Precision Analytical uses GC-MS/MS and LC-MS/MS. 0:49:14    Providers should go to dutchtest.com. 0:49:26    Five tests at half price. 0:50:12    Mark will help doctors interpret the results. 0:50:28    Testing (lab testing, not sample collection) is a four day process. 0:50:55    Organic acids tests are run in batches. 0:51:10    Mark developed organic acids testing for BioTech Lab  (US BioTek). 0:51:49    You can order a DUTCH though me and then I can connect with you on the phone or Skype to explain what the results mean

There are now numerous epidemiological studies that have demonstrated that low plasma concentrations of vitamin D are associated with not only bone related disorders, but with a wide variety of adverse health outcomes.

Die equine rezidivierende Uveitis (ERU) ist eine sehr häufig auftretende autoimmune Augenerkrankung bei Pferden, welche meist mit dem Verlust der Sehfähigkeit der betroffenen Augen einhergeht. Da die ERU das einzig spontane Tiermodell für die humane autoimmune Uveitis darstellt, ist die Erforschung der zugrundeliegenden Pathomechanismen der ERU nicht nur veterinärmedizinisch, sondern auch für die Humanmedizin von großer Bedeutung. Charakteristisch für die ERU sind der Zusammenbruch der Blut-Retina-Schranke (BRS) und die Infiltration von autoaggressiven T-Lymphozyten in das innere Auge mit anschließender Zerstörung retinaler Strukturen. Beim Pferd wird die BRS, aufgrund der weitestgehend avaskulären Retina, hauptsächlich von der äußeren Komponente der BRS gebildet, dem retinalen Pigmentepithel (RPE). Im physiologischen Zustand stellt das RPE durch feste Zell-Zellverbindungen sowohl eine stabile mechanische, als auch durch seine Fähigkeit, mit Mediatoren des Immunsystems kommunizieren und interagieren zu können, eine effektive immunologische Barriere dar. Die im Verlauf der ERU stattfindenden pathophysiologischen Mechanismen, welche für den Zusammenbruch dieser Barriere verantwortlich sind, konnten bislang nicht ausreichend geklärt werden. Vor allem Änderungen im Expressionsmuster des Zelloberflächenproteoms könnten hierbei aufgrund der ständigen Interaktion und Kommunikation der RPE-Zellen mit ihrer Umgebung eine entscheidende Rolle spielen. Deshalb war es das Ziel dieser Arbeit, differentiell regulierte Zelloberflächen-proteine zwischen gesunden und uveitischen RPE-Zellen zu detektieren, welche maßgeblich an der Pathogenese der ERU beteiligt sein könnten. Um so nah wie möglich die am RPE in vivo stattfindenden physiologischen und pathophysiologischen Prozesse widerspiegeln zu können, wurden RPE-Zelloberflächenproteine von gesunden und an ERU erkrankten Pferden in dieser Studie mittels einer neuartigen in situ Biotinylierungsmethode angereichert und anschließend massenspektrometrisch analysiert. Dabei konnten insgesamt 148 Proteine identifiziert werden, von denen 81,8 % Plasmamembranproteine waren, was deutlich für den Erfolg der neuartigen Anreicherungsmethode sprach. Unter den 148 insgesamt identifizierten Proteinen befanden sich 27 differentiell regulierte Proteine, wovon in uveitischem RPE drei hoch- und 24 herunterreguliert waren. Neben den für RPE-Zellen klassischen Proteinen wie RPE65, Rhodopsin und S-Arrestin konnten auch mehrere Proteine detektiert werden, die unseres Wissens zuvor noch nicht in RPE-Zellen beschrieben wurden, wie der Glukosetransporter 4, Synaptotagmin 1 und Peripherin 2. Funktionell besonders interessant fanden wir die vier Proteine Synaptotagmin 1, Basigin, Collectrin und Perpherin 2, welche alle mit einer verminderten Expression in uveitischem RPE zu finden waren. Interessanterweise ergab sich aus einer Pathway-Analyse für alle vier Proteine eine Beteiligung an „Visual Functions“ und „Immunological Diseases“. Mittels weiterführender Analysen wie der Durchflusszytometrie, der Immunhistologie und der Quantifizierung der Protein-Fluoreszenzintensitäten ist es gelungen die bereits massenspektrometrisch identifizierte verminderte Expression von Synaptotagmin 1, Basigin, Collectrin und Perpherin 2 zu verifizieren und die Proteine näher zu charakterisieren. Die in dieser Arbeit präsentierte neuartige in situ Biotinylierungsmethode zur Anreicherung von Oberflächenproteinen, welche anschließend mittels LC-MS/MS identifiziert wurden, erwies sich als sehr effektive und innovative Methode, um Oberflächenproteine so nah wie möglich in ihrem physiologischen und pathophysiologischen in vivo Vorkommen zu untersuchen. Daher liefert der in dieser Arbeit generierte Datensatz der differentiell regulierten Proteine zwischen gesunden und uveitischen RPE-Zellen eine solide Grundlage für weitere funktionelle Analysen zur Aufklärung der Pathogenese der ERU.

Frontotemporal dementia is the second most common neurodegenerative disease in people younger than 65 years. Patients suffer from behavioral changes, language deficits and speech impairment. Unfortunately, there is no effective treatment available at the moment. Cytoplasmic inclusions of the DNA/RNA-binding protein TDP-43 are the pathological hallmark in the majority of FTLD cases, which are accordingly classified as FTLD-TDP. Mutations in GRN, the gene coding for the trophic factor progranulin, are responsible for the majority of familiar FTLD-TDP cases. The first genome-wide association study performed for FTLD-TDP led to the identification of risk variants in the so far uncharacterized gene TMEM106B. Initial cell culture studies revealed intracellular localization of TMEM106B protein in lysosomes but its neuronal function remained elusive. Based on these initial findings, I investigated the physiological function of TMEM106B in primary rat neurons during this thesis. I demonstrated that endogenous TMEM106B is localized to late endosomes and lysosomes in primary neurons, too. Notably, knockdown of the protein does neither impair general neuronal viability nor the protein level of FTLD associated proteins, such as GRN or TDP-43. However, shRNA-mediated knockdown of TMEM106B led to a pronounced withering of the dendritic arbor in developing and mature neurons. Moreover, the strong impairment of dendrite outgrowth and maintenance was accompanied by morphological changes and loss of dendritic spines. To gain mechanistic insight into the loss-of-function phenotypes, I searched for coimmunoprecipitating proteins by LC-MS/MS. I specifically identified the microtubule-binding protein MAP6 as interaction partner and was able to validate binding. Strikingly, overexpression of MAP6 in primary neurons phenocopied the TMEM106B knockdown effect on dendrites and loss of MAP6 restored dendritic branching in TMEM106B knockdown neurons, indicating functional interaction of the two proteins. The link between a lysosomal and a microtubule-binding protein made me study the microtubule dependent transport of dendritic lysosomes. Remarkably, live cell imaging studies revealed enhanced movement of dendritic lysosomes towards the soma in neurons devoid of TMEM106B. Again, MAP6 overexpression phenocopied and MAP6 knockdown rescued this effect, strengthening the functional link. The MAP6-independent rescue of dendrite outgrowth by enhancing anterograde lysosomal movement provided additional evidence that dendritic arborization is directly controlled by lysosomal trafficking. From these findings I suggest the following model: TMEM106B and MAP6 together act as a molecular brake for the retrograde transport of dendritic lysosomes. Knockdown of TMEM106B and (the presumably dominant negative) overexpression of MAP6 release this brake and enhance the retrograde movement of lysosomes. Subsequently, the higher protein turnover and the net loss of membranes in distal dendrites may cause the defect in dendrite outgrowth. The findings of this study suggest that lysosomal misrouting in TMEM106B risk allele carrier might further aggravate lysosomal dysfunction seen in patients harboring GRN mutations and thereby contribute to disease progression. Taken together, I discovered the first neuronal function for the FTLD-TDP risk factor TMEM106B: This lysosomal protein acts together with its novel, microtubule-associated binding partner MAP6 as molecular brake for the dendritic transport of lysosomes and thereby controls dendrite growth and maintenance.

Wed, 19 Mar 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16825/ https://edoc.ub.uni-muenchen.de/16825/1/Hellmuth_Christian.pdf Hellmuth, Christian ddc:610, ddc:600, Medizinische Fakultät

Klinische Studien zeigen, dass die Zusammensetzung von zirkulierenden, freien AS im Blut, ein Marker für monogene und multigenetische Krankheiten ist. Die Analyse von hohen Probandenzahlen in klinischen Studien wird oftmals durch aufwendige und lange Probenaufarbeitungsschritte begrenzt. Im Rahmen der Metabolomics Plattform, die im Dr. von Haunerschen Kinderspitals etabliert wurde, wurde eine Hochdurchsatzmethode entwickelt, die eine selektive, sensitive, präzise und robuste Quantifizierung von 22 AS aus kleinen Probenvolumina ermöglicht. Im Laufe der Zeit konnten noch weitere Aminosäuren zur Methodik hinzugefügt werden. Dabei können innerhalb von 36 Stunden 96 Proben analysiert werden. Mit Hilfe eines deuterierten, interenen Standards in methanolischer Lösung werden Proteine aus nur 10 μL Probenvolumen gefällt. Zur Quantifizierung der AS wird anschliessend der eingedampfte Überstand derivatisiert und in Kombination mit einem Ionen-Paar- Reagenzes chromatographiert. Der Methodenaufarbeitung liegt eine umfassende und ausführliche Validierung zugrunde, die einer Interday Precision von 3.1 -10.8 % für alle Analyten erzielt. Zusätzlich unterzieht sich unsere Methode jedes Jahr an einem Ringversuch, der eine exakte Bestimmung aller Analyten gewährleistet. Im Zusammenhang mit der Programmierung des Stoffwechsels durch die Ernährung im Säuglingsalter wurde die neu entwickelte AS-Methode zur Quantifizierung von 726 Serum Proben in einer randomizierten klinischen Studie eingesetzt. Dabei wurde der Bezug zwischen Proteinzufuhr (Formelnahrung mit hohem Eiweißanteil bzw. niedriegem Eiweißanteil) und AS-Profil bei 6 Monate alten Säuglingen analysiert. Eine signifikante Veränderung der Plasmakonzentrationen zeigte sich in der Gruppe der formelernährten Kindern mit hohem Proteinanteil für folgende AS: BCAA, Gly, Lys, Met, Phe, Pro, Thr, Trp und Tyr. Essentielle AS werden über die Nahrung aufgenommen und vermutlich über das L-Transportsystem in die Zirkulation freigesetzt. Im Vergleich dazu werden nicht essentielle AS im Darm katabolisiert und mit Hilfe der de novo Synthese in gleichbleibenden Konzentrationen reguliert, sodass kein signifikanter Unterschied in beiden Gruppen beobachtet wurde. Durch eine proteinreiche Nahrung können AS vermehrt an der Aktivierung von Wachstumshormonen (mTOR, IGF-1) teilhaben, was sich im vermehrten Zellwachstum und –proliferation manifestiert. Nichts desto trotz kann die vermehrte Wachstumshormonaktivierung Krankheiten wie Insulinresistenz, Diabetes oder auch Übergewicht hervorrufen. Aufgrund des erhöhten Krankheitsrisikos ist einerseits von einer proteinreichen Ernährung im frühen Säuglingsalter abzuraten, andererseits kann das Stillen bzw. eiweißänhnliche Brustmilchzusammensetzung gesundheitsunterstützend sein. Auch im Rahmen eines Supplementierungsprojektes bei Kühen, hat sich die neu entwickelte AS-Methode bewährt. Dazu wurden jeweils 10 Kühe kurz vor und nach der Geburt mit CLA (CLA-Gruppe) oder mit Linolsäure (Kontroll-Gruppe) supplementiert. Ziel der Studie war es, den Zusammenhang zwischen CLA Supplementierung und AS- Profil im Blut zu analysieren. Dazu wurden zu insgesamt 8 Zeitpunkten vor und nach der Geburt, Blutproben entnommen. Es kristallisierten sich 3 verschiedene Zeitverläufe heraus, wobei die meisten AS-Konzentrationen nach der Geburt abfallen und langsam wieder auf ihre Ausgangswerte ansteigen. AS wie Glu sinken vor der Geburt stark ab, was dafür sprechen könnte, dass der Fetus mit ausreichend Glu versorgt wird und nach der Geburt vermehrt in die Milch transportiert wird. Gly, HPro, MHis und Ser steigen bis zur Geburt an und fallen dann wieder ab wobei Mhis durch den Muskelproteinabbau keinen Konzentrationsanstieg in den ersten 12 Laktationswochen erfährt. Die Supplementierung mit CLA zeigte keinen signifikanten Unterschied beider Gruppen auf das AS-Profil und zeigte somit keine Auswirkungen auf die AS-Synthese.

Signaling networks control and regulate outcomes in cells and organisms in both normal physiology and pathophysiological states. Signaling is traditionally represented and studied as a series of stepwise enzymatic events constituting a cascade. However, it is increasingly apparent that such representations limit understanding of signal transduction since these linear cascades function in an interconnected network that includes extensive cross talk among receptors and pathways. Mass spectrometry (MS)-based proteomics is a useful tool that allows a system-wide investigation of signaling events at the levels of post-translational modifications (PTMs), protein-protein interactions and changes in protein expression on a large scale. This technology now allows accurate quantification of thousands of proteins and their modifications in response to any perturbation. This thesis work is dedicated to the optimization and employment of quantitative mass spectrometry to cellular signaling and an application to segregate two lymphoma subtypes at the levels of protein expression and phosphorylation, employing state of the art liquid chromatography (LC)-MS/MS technologies coupled with improved sample preparation techniques and data analysis algorithms. In the first project I investigated the feasibility of a new, high accuracy fragmentation method called higher energy collisional dissociation (HCD) for the analysis of phospho-peptides. Using this method we were able to measure the phospho-proteome of a single cell line in 24h of measurement time which was a great improvement to previous capabilities. This fragmentation method that was originally thought to be slower and less sensitive than the standard method of low resolution collision induced dissociation (CID) fragmentation. However, our work proves this not to be the case and we showed that HCD outperformed the existing low resolution strategy [1]. In the second project I employed this HCD fragmentation technique on the LTQ-Orbitrap Velos for addressing the clinical question of segregating two subtypes of diffuse B-cell lymphoma (DLBCL). These subtypes are histologically indistinguishable but had been segregated on the basis of a gene expression signature. I employed the recently developed ‘super-SILAC’ approach with a ‘super-SILAC mix’ of multiple labeled cell lines. This heavy reference mix was spiked into several cell lines derived from the two DLBCL subtypes and analyzed LC-MS, resulting in successful segregation based on a distinct proteomic signature [2]. The third project deals with the in-depth analysis of the phospho-proteome of a human cancer cell line on a quadrupole-Orbitrap mass spectrometer using a label-free quantification approach. Our analysis uncovered about 50,000 distinct phosphorylated peptides in a single cell type across a number of cellular conditions allowing assessment of global properties of this large dataset. Strikingly, we found that at least three-quarters of the proteome can be phosphorylated which is much higher than current estimates. We also analyzed phosphotyrosine events using enrichment with anti-phospho-tyrosine antibodies to identify more than 1,500 site specific phosphorylation events. Unexpectedly tyrosine phosphorylated proteins were enriched among higher abundance proteins. The observed difference in phospho-protein abundance correlated with the substrate Km values of tyrosine kinases. For the first time we calculated site specific occupancies using label- free quantification and observed widespread full phosphorylation site occupancy during mitosis. In the final and main project, I applied proteomics and phospho-proteomics to the study of signal transduction in response to transforming growth factor-beta (TGF-β), a multifunctional cytokine. TGF-β signaling regulates many biological outcomes including cell growth, differentiation, morphogenesis, tissue homeostasis and regeneration. The cellular responses to this multifunctional ligand are diverse and can even be opposed to each other, depending on the cell type and the conditions. To shed light on the reasons for the different outcomes, we analyzed the early phospho-proteome and ensuing proteome alterations in response to TGF-β treatment in a keratinocyte cell line. The early SILAC based phospho-proteome analysis uncovered over 20,000 phosphorylation events across five time points (0 to 20 min) of TGF-β treatment. Building on our recent advances in instrumentation, sample preparation, and data analysis algorithms we measured a deep TGF-β responsive proteome at six late time points (6h to 48h) with corresponding controls in only eight days of measurement time. Our label-free approach identified about 8,000 proteins and quantified more than 6,000 of them. This deep proteome covered well established pathways involved in TGF-β signaling, allowing global evaluation at the level of individual pathway members. Combining the TGF-β responsive proteome with an in-silico upstream regulator analysis, we correctly retrieved several known and predicted novel transcription factors driving TGF-β induced cytostasis, de-differentiation and epithelial to mesenchymal transition (EMT). The combined analysis of transcription factor regulation with early phosphorylation changes and proteome changes enabled visualization of the intricate interplay of key transcription factors, kinases and various pathways driving cytostatis, EMT and other processes induced by TGF-β. In summary, my thesis developed a highly efficient phospho-proteomic workflow, which was applied to the measurement of a very deep phospho-proteome of a single cancer cell line allowing analysis of its global features. The main achievement was the first in-depth and combined study of the phospho-proteome and resulting proteome changes following a defined signaling event, in this case leading to a time-resolved view of TGF- β signaling events relevant in cancer.

Background: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. Methods: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. Results: In a bi-center validation total CVs of

The thesis describes the configuration, optimization and evaluation of a novel instrumental platform for fully automated SPE-LC-MS/MS analysis of small molecules, such as drugs, in whole blood. The immunosuppressant Cyclosporine A was chosen as a model analyte, as this drug is predominantly bound to erythrocytes. First, anticoagulated blood is converted into so-called Cell-Disintegrated Blood (CDB) by heat-shock or cryogenic treatment. CDB represents a homogenous blood sample and consists of subcellular particles which do not sediment on standing and do not clog capillaries, sieves or HPLC column packings. For in-line treatment of anticoagulated whole blood, i.e. generation of CDB, a sample mixing unit, two special liquid handling units and two home-made sample processing modules were embedded into a XYZ-autosampler. The module for heat-shock treatment consists of a stainless-steel capillary jacketed with a heating sleeve. Under optimal conditions for sampling and in-line processing of 20 µL of whole blood, it takes 13 seconds at 75 °C to generate CDB. The latter is stored in a holding loop before further treatment. For cryogenic treatment of a blood sample, a stainless-steel processing needle with a large inner diameter was installed in one of the liquid handling units. The autosampler was programmed to introduce the processing needle containing the blood sample (40 µL) into a stand-pipe, which is located in a thermo-flask filled with liquid nitrogen. The processing needle therefore contacts liquid nitrogen and the blood sample is snap-frozen. Optimal conditions were found to be 10 seconds for snap-freezing at -196 °C and 60 seconds for thawing at room temperature. A CDB sample obtained either by heat-shock or cryogenic treatment is further processed by being pumped via a switching-valve through an in-line filter to retain cell nuclei and “cell debris”. It was found that a depth filter packed with spherical hydrophilic silica is optimal. This filter allows at least 200 analysis cycles before it has to be replaced. Next, the CDB sample is pumped on-line via another switching-valve through a SPE column (50 x 0.5 mm ID) at a high flow rate. Due to the special packing material and the very small inner diameter, a high linear flow velocity is achieved and turbulent flow is generated. By this, high-molecular matrix components such as proteins are eluted in the void volume to waste. The low-molecular weight target analyte Cyclosporine A and the Internal Standard Cyclosporine D are retained and extracted by reversed phase partitioning chromatography (RPC). After fractionation of CDB on the SPE column, the analyte and the IS are transferred to a series-connected analytical column and separated from residual matrix components by RPC. Finally, the analyte is detected by a tandem mass spectrometer applying electrospray ionization (ESI) and multiple reaction monitoring (MRM). The optimized method has a total analysis time of less than 11 minutes. The analytical procedure and the instrumental platform were validated for heat-shock treated blood samples with respect to linearity, range (10 - 1000 ng/mL), lower limit of quantitation (10 ng/mL), intra-day and inter-day accuracy and precision, as well as matrix-independent and matrix-dependent recovery (around 100 %). It was shown that the electrospray induced ionization is suppressed by approximately 25 %. These matrix effects, however, can be totally compensated for by the addition of an Internal Standard, i.e. Cyclosporine D. A comparison with a semi-automated SPE-LC-MS/MS method, established in the Institute, revealed a very good agreement. This was shown by Passing and Bablok plots. The robustness of the fully automated SPE-LC-MS/MS analysis platform was monitored during 500 consecutive analysis cycles with heat-shock treated blood samples. The relative standard deviation for the signal response was 15.6 % for Cyclosporine A and 15.2 % for Cyclosporine D. The back pressure of the total system rose only by 52 bar. These findings show that, despite its instrumental and chromatographic complexity, the described analysis platform fulfills the prerequisites to be used in routine clinical-chemical analysis.

Isotope dilution LC-MS/MS methods used in the clinical laboratory typically involve multi-point external calibration in each analytical series. Our aim was to test the hypothesis that determination of target analyte concentrations directly derived from the relation of the target analyte peak area to the peak area of a corresponding stable isotope labelled internal standard compound [direct isotope dilution analysis (DIDA)] may be not inferior to conventional external calibration with respect to accuracy and reproducibility.

Vertebrate embryos are derived from a transitory pool of pluripotent embryonic cells. By the process of induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While at gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue histones isolated from four different developmental stages of Xenopus laevis were analysed by mass spectrometry. Initally, a variety of different protocols for histone extraction from Xenopus laevis embryos and stable cell lines was tested and evaluated. Since non of the available methods worked sufficiently, a new reliable and effective protocol for nuclei preparation and histone extraction was established. Using mass spectrometry, core histone modifications were unambiguously determined. The techniques for identification and quantification of histone modifications by tandem mass spectrometry were improved as well. In total, an average sequence coverage of 68% of modification sites for the four core histones was achived by tryptic digestion after covalent modification of lysine residues with propionic anhydride. Using both LC-MS/MS and MALDI-TOF mass spectrometry, a total number of 2 modifications of H2A and 3 modifications H2B, 39 modifications of H3 and 20 modifications of H4 were identified and quantified. During this developmental period, an increase in the unmodified states, and a shift from histone modifications associated with transcriptionally active to transcriptionally repressive histone marks, was observed. Furthermore, these naturally occurring histone modifications were compared to the histone modifications of murine ES cells, detecting large differences in the methylation patterns of lysines 27 and 36 of histone H3 between pluripotent cells from Xenopus blastulae and murine ES cells. By combining all detected modification transitions, their patterns could be clustered according to their embryonic origin, defining specific histone modification profiles for each developmental stage. These specific histone modification profiles indicated a stepwise maturation of the embryonic epigenome, which may be cause to the progressing restriction of cellular potency during development. This thesis has revealed major quantitative shifts for several histone modifications known to be involved in gene regulation and furthermore enabled the definition of stage specific histone modification profiles accompanying and potentially regulating the transition from pluripotent to determined cell states using an antibody-independent method.

Background: Extensive sets of data are required to investigate the potential use of a therapeutic drug monitoring with individualization of dosage of the antimycotic compound caspofungin. The goal was to develop an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for this aim. Methods: Following protein precipitation, on-line solid phase extraction was performed for sample preparation. As the internal standard compound the veterinary drug tylosin was used. A standard validation protocol was applied. Results: Good reproducibility and accuracy of the method were observed. On-line solid phase extraction resulted in a convenient work-flow and good robustness of the method. Conclusions: This improved LC-MS/MS method was found reliable and convenient. It can be suggested for further work on the clinical pharmacology of caspofungin in the setting of clinical research laboratories.

Background/Aims: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. Methods: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. Results: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 +/- 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffe colorimetric assay) proved high agreement (R-2 = 0.9102). Conclusions: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The `dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost-and time-efficient. Copyright (C) 2012 S. Karger AG, Basel

The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD). Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (-70%) and cGMP (-55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99). We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.

Patients with phenylketonuria (PKU) have to follow a lifelong phenylalanine restricted diet. This type of diet markedly reduces the intake of saturated and unsaturated fatty acids especially long chain polyunsaturated fatty acids (LC-PUFA). Long-chain saturated fatty acids are substrates of mitochondrial fatty acid oxidation for acetyl-CoA production. LC-PUFA are discussed to affect inflammatory and haemostaseological processes in health and disease. The influence of the long term PKU diet on fatty acid metabolism with a special focus on platelet eicosanoid metabolism has been investigated in the study presented here. 12 children with PKU under good metabolic control and 8 healthy controls were included. Activated fatty acids (acylcarnitines C6-C18) in dried blood and the cholesterol metabolism in serum were analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Fatty acid composition of plasma glycerophospholipids was determined by gas chromatography. LC-PUFA metabolites were analyzed in supernatants by LC-MS/MS before and after platelet activation and aggregation using a standardized protocol. Patients with PKU had significantly lower free carnitine and lower activated fatty acids in dried blood compared to controls. Phytosterols as marker of cholesterol (re-) absorption were not influenced by the dietary fatty acid restriction. Fatty acid composition in glycerophospholipids was comparable to that of healthy controls. However, patients with PKU showed significantly increased concentrations of y-linolenic acid (C18:3n-6) a precursor of arachidonic acid. In the PKU patients significantly higher platelet counts were observed. After activation with collagen platelet aggregation and thromboxane B(2) and thromboxane B(3) release did not differ from that of healthy controls. Long-term dietary fatty acid restriction influenced the intermediates of mitochondrial beta-oxidation. No functional influence on unsaturated fatty acid metabolism and platelet aggregation in patients with PKU was detected.

Das Ziel der vorliegenden Arbeit war es, molekulare Vorgänge während der B-Zellentwicklung in der Bursa Fabricii des Haushuhns mittels proteomischer Analysen zu charakterisieren. Hierfür wurden zunächst repräsentative Zeitpunkte der bursalen B-Zellentwicklung für die Probengewinnung definiert. Daran schlossen sich qualitative Proteomanalysen der Bursa Fabricii zu den gewählten Entwicklungszeitpunkten Embryonaltag 10 (ET10), Embryonaltag 18 (ET18), Tag 2 und Tag 28 nach dem Schlupf an. Diese erfolgten durch Vorfraktionierung der Proben mittels 1D-SDS-PAGE und nano-HPLC gefolgt von Tandem-MS-Analysen. Hierbei konnten für die bursalen Proteome zu jedem Zeitpunkt zwischen 1152 und 1392 Proteine identifiziert werden (FDR < 1 %). Überschneidungen der einzelnen Zeitpunkte in 537 allgemeinen Struktur- und Stoffwechsel-Proteinen berücksichtigt, wurden insgesamt 2214 verschiedene Proteine identifiziert. Eine zusätzliche qualitative Analyse aufgereinigter bursaler B-Zellen führte zur Identifizierung von 758 Proteinen. Durch genontologische Analysen konnte festgestellt werden, dass für das Zellwachstum verantwortliche Proteine va. zu den frühen Zeitpunkten zu finden waren, während Proteine, welche eine Rolle für das Immunsystem spielen, eher zu späteren Entwicklungszeitpunkten in Erscheinung traten. Dies spiegelt die Entwicklung der Bursa von der unreifen, embryonalen Wachstums- und Differenzierungsprozessen unterliegenden Bursaanlage, zum fertig ausdifferenzierten, primär-lymphatischen Organ auf molekularer Ebene wider. Konform zu den hohen Proliferationsraten der B-Zellen während der Bursaentwicklung fanden sich in den genontologischen Analysen der B-Zellen besonders hohe Anteile an Proteinen, welche für Zellteilung verantwortlich sind. Proteine, welche in Zusammenhang mit Zellmigration stehen, wurden vor allem in der B-Zell-Probe sowie an ET10 gefunden, was als Hinweis auf eine Beteiligung dieser Proteine an der Einwanderung der B-Zellen in die Bursaanlage betrachtet werden kann. Die anschließende quantitative Proteomanalyse wurde zu denselben Entwicklungszeitpunkten an je sechs biologischen Replikaten mittels 2D-DIGE durchgeführt. In der statistischen Auswertung der quantitativen Veränderungen zeigten sich 402 hochsignifikante Unterschiede zwischen den bursalen Proteomen der verschiedenen Entwicklungszeitpunkte, wobei die sehr große Übereinstimmung der Analyseergebnisse innerhalb der biologischen Replikate die gute Reproduzierbarkeit der Experimente belegte. Die deutlichsten Veränderungen zeigten sich zwischen ET10 und allen weiteren Zeitpunkten, wohingegen innerhalb der übrigen Stadien eine geringere Anzahl signifikanter Unterschiede nachgewiesen wurde. Für die 402 differentiell exprimierten Proteine konnten verschiedene charakteristische Protein-expressionsverläufe nachgewiesen werden, welche Rückschlüsse auf die biologischen Hintergründe ermöglichten. Durch massenspektrometrische Analysen der Proteine mittels MALDI-TOF/TOF und LC-MS/MS gelang es, 203 der 242 zur Identifikation vorgesehenen Spots zu identifizieren. Im Rahmen einer bioinformatischen Auswertung des erhaltenen Datensatzes erbrachten sowohl die genontologische Analysen als auch Pathway-Analysen wesentliche Anhaltspunkte für die Auswahl besonders interessanter und vielversprechender Proteine für weiterführende funktionelle Analysen. Unter den identifizierten, differentiell exprimierten Proteinen fanden sich auffällig viele Vertreter des Aktin-Zytoskelett-Pathways, welcher für die mechanische Stabilisierung von Zellen und deren aktive Bewegungen verantwortlich ist. Dabei fielen in dieser Arbeit sowohl Vinculin als auch Gelsolin durch ihre charakteristischen Expressionsmuster auf. Vinculin zeigte zu Beginn der embryonalen Entwicklung erhöhte Abundanzwerte, welche nach ET18 steil abfielen. Als fokales Adhäsionsprotein stellt es ein Schlüsselprotein in der Regulation der Übertragung von kontraktilen Kräften dar, welche die Voraussetzung für die Migration von Zellen sind. Gelsolin, ein wichtiges Apoptose-Effektorprotein, welches auch in Verbindung mit Zellmotilität gebracht wird, zeigte erhöhte Expressionslevel an ET18, welche über die nachfolgenden Entwicklungszeitpunkte abfielen. Gelsolin konnte in drei verschiedenen Ladungs-Isoformen detektiert werden. Für die Fragestellung dieser Arbeit interessant erschien weiterhin eine Gruppe differentiell exprimierter Proteine des Retinolsäure-Metabolismus. Im Einzelnen wurden die Retinaldehydrogenase 2 (ALDH1A2), das „retinol-binding protein 5“ (RBP5), das „fatty acid-binding protein 7“ (FABP7), und Transthyretin (TTR) mit ähnlichen Proteinexpressions-profilen detektiert, welche ihr Expressionsmaximum jeweils an ET10 aufwiesen. Das könnte ein Hinweis sein, dass die embryonale Entwicklung der Bursa Fabricii von ähnlichen Faktoren gesteuert ist wie die embryonale Ausbildung der sekundär-lymphatischen Organe beim Säuger, bei der Retinolsäure-abhängige Proteine eine entscheidende Rolle spielen. Über die Thematik dieser Arbeit hinausgehend, stellt der umfangreiche proteomische Datensatz dieser Arbeit eine wertvolle Ressource dar, die sowohl für weitere Fragestellungen bezüglich der Bursa Fabricii des Huhns, als auch für die Vervollständigung der Annotation des Hühnergenoms genutzt werden können.

Die equine rezidivierende Uveitis (ERU) ist eine häufige (10%), spontane, immun-mediierte, wiederkehrende Entzündung des inneren Pferdeauges, die letztendlich zur Erblindung des betroffenen Auges führt. Neben ihrer Bedeutung für die Veterinärmedizin stellt die ERU das einzige spontane Tiermodell für die autoimmun-mediierte Uveitis des Menschen dar, so dass Fortschritte in der Erforschung der ERU auch einen Beitrag für ein besseres Verständnis dieser Erkrankung beim Menschen leisten. Die zugrunde liegenden molekularen Mechanismen am Zielorgan, die zur Entstehung der ERU und den rezidivierenden Entzündungsschüben führen, sind bis heute nicht geklärt. Durch seinen unmittelbaren Kontakt zur Retina ermöglicht der Glaskörper einen indirekten Einblick auf die in der Retina ablaufenden pathophysiologischen Prozesse und stellt ein wertvolles Probenmaterial bei der Erforschung vitreoretinaler Erkrankungen wie der ERU dar. Ziel dieser Arbeit war es, Pathogenese assoziierte Glaskörperproteine und deren funktionelle Interaktionsnetzwerke in der ERU zu identifizieren und zu analysieren. Insgesamt wurden in dieser Arbeit 119 Glaskörperproteine mittels LC-MS/MS identifiziert. Hiervon konnte durch eine Label-free Quantifizierung für 79 Proteine eine differenzielle Expression nachgewiesen werden. 17 Proteine zeigten eine signifikant erhöhte Expression in den Glaskörperproben von an ERU erkrankten Pferden, wohingegen 62 Proteine in ihrer Expression vermindert waren. Durch die mit der Software STRING (Search Tool for Retrieval of Interacting Genes/Proteins) durchgeführte Protein-Interaktionsnetzwerk-Analyse konnten vier Cluster von Proteinen identifiziert werden, die bei der ERU verändert exprimiert werden. Neben einer Gruppe von Plasmaproteinen, einer aus Zelladhäsions-Proteinen bestehenden Gruppe und Proteinen des Wnt-Signalweges wurde erstmalig eine funktionell mit den Matrix-Metalloproteinasen MMP-2 und MMP-9 assoziierte Gruppe identifiziert, für deren Vertreter ebenfalls eine differenzielle Expression in der Pferderetina nachgewiesen wurde. Der Wnt-Inhibitor SFRP-2 war in den Glaskörpern von an ERU erkrankten Pferden vermindert nachweisbar und zeigte eine Assoziation mit der Expression seines Interaktors Wnt3a in der gesunden Pferderetina. Mit A2M konnte durch SF-TAP Aufreinigung ein bislang unbekannter Interaktor für SFRP-2 identifiziert und in Bezug zur ERU weiter charakterisiert werden. Für das Protein Osteopontin konnte eine signifikant erhöhte Expression in den Glaskörperproben und Retinae der Kontrollpferde nachgewiesen werden. Rückblickend erwies sich die Protein-Netzwerkanalyse der durch LC-MS/MS identifizierten, differenziell exprimierten Proteine als geeignete Methode, um Pathogenese assoziierte Glaskörperproteine und deren funktionelle Interaktionsnetzwerke in der ERU zu identifizieren und zu analysieren. Weitere Experimente sind nun notwendig, um die funktionelle Bedeutung der neu identifizierten Proteine und der mit ihnen assoziierten Signalwege in der ERU zu evaluieren.

Background: Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. Method: A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time

Background: The goal of this study was to develop and to validate an improved isotope-dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of methylmalonic acid (MMA) in urine. Methods: A previously described sample preparation protocol requires two solvent extraction steps, including evaporation. The first extraction is to extract the analyte from the sample, and second occurs following derivatization of the extract. In the method described here, the second evaporation step was substituted by on-line solid phase extraction employing column-switching and a permanent co-polymer based extraction cartridge. A standard validation protocol was applied to investigate the performance of the method. Results: The method was found to be linear in the clinically relevant range of concentrations (6-100 mu mol/L). Total coefficients of variation were below 10% and inaccuracy was

Mon, 6 Jul 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10379/ https://edoc.ub.uni-muenchen.de/10379/1/Milojkovic_Jelena.pdf Milojkovic, Jelena

Background: Posaconazole is now widely used for prophylaxis of invasive fungal infections in immunocompromised patients. The pharmacokinetic properties of the drug argue for therapeutic monitoring, but so far described analytical methods have shortcomings with respect to application in a routine setting. The aim of our work was to develop an analytical method suitable for routine use. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. For sample preparation, protein precipitation followed by on-line solid phase extraction was used. SCH 56984, a posaconazole related compound provided by the manufacturer of posaconazole, was used as internal standard. Results: The method was characterized by short hands-on time and an analytical run time of four minutes. Validation data demonstrated acceptable analytical performance. Conclusions: LC-MS/MS with on-line solid phase extraction for sample preparation allows the implementation of a convenient and reliable method for pharmacokinetic monitoring of posaconazole.

Background: Daphniids, commonly known as waterfleas, serve as important model systems for ecology, evolution and the environmental sciences. The sequencing and annotation of the Daphnia pulex genome both open future avenues of research on this model organism. As proteomics is not only essential to our understanding of cell function, and is also a powerful validation tool for predicted genes in genome annotation projects, a first proteomic dataset is presented in this article. Results: A comprehensive set of 701,274 peptide tandem-mass-spectra, derived from Daphnia pulex, was generated, which lead to the identification of 531 proteins. To measure the impact of the Daphnia pulex filtered models database for mass spectrometry based Daphnia protein identification, this result was compared with results obtained with the Swiss-Prot and the Drosophila melanogaster database. To further validate the utility of the Daphnia pulex database for research on other Daphnia species, additional 407,778 peptide tandem-mass-spectra, obtained from Daphnia longicephala, were generated and evaluated, leading to the identification of 317 proteins. Conclusion: Peptides identified in our approach provide the first experimental evidence for the translation of a broad variety of predicted coding regions within the Daphnia genome. Furthermore it could be demonstrated that identification of Daphnia longicephala proteins using the Daphnia pulex protein database is feasible but shows a slightly reduced identification rate. Data provided in this article clearly demonstrates that the Daphnia genome database is the key for mass spectrometry based high throughput proteomics in Daphnia.

S100A8 in pancreatic cancer-associated monocytes is associated with the Smad4 status of pancreatic cancer cells, Outer membrane proteome of Actinobacillus pleuropneumoniae: LC-MS/MS analyses validate in silico predictions, Report: Second International Workshop "2D-DIGE Applications in Proteomics", Protein prenylation in an insect cell-free protein synthesis system, Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation, "goProteomics" website.

S100A8 in pancreatic cancer-associated monocytes is associated with the Smad4 status of pancreatic cancer cells, Outer membrane proteome of Actinobacillus pleuropneumoniae: LC-MS/MS analyses validate in silico predictions, Report: Second International Workshop "2D-DIGE Applications in Proteomics", Protein prenylation in an insect cell-free protein synthesis system, Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation, "goProteomics" website.

Wed, 10 Jan 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/6354/ https://edoc.ub.uni-muenchen.de/6354/1/Georgi_Katrin.pdf Georgi, Katrin

Background: The endocannabinoid 2-arachidonoyl glycerol (2-AG) undergoes spontaneous isomerization to biologically inactive 1-AG. This effect has not been adequately addressed in previous studies that reported 2-AG concentrations in biological samples. Methods: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used for 1-AG and 2-AG analyses. Results: Identical collision-induced disintegration spectra were found for 1-AG and 2-AG. For specific detection of both compounds, which share a common mass transition, baseline chromatographic separation is mandatory, even when applying MS/MS technology with its generally high detection specificity. When using standard chromatographic conditions with the very short run times typically used in LC-MS/ MS methods, co-elution of 2-AG with 1-AG, which is present in human serum, causes false 2-AG results. Conclusions: Our data highlight that the analytical specificity of MS/MS can be limited by interference from isobaric isomers with identical disintegration patterns. The specificity of this technology must be carefully evaluated for each individual application.

Background: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Methods: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. Results: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 mu g/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. Conclusions: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.

Background: Endogenous ligands of cannabinoid receptors ( endocannabinoids), in particular anandamide ( arachidonylethanolamide), have been recognized as being of crucial importance in a variety of physiological functions. Plasma concentrations of anandamide have been measured in a number of investigations; however, discrepant data on "normal'' anandamide plasma concentrations were reported. Since this might be caused by pre-analytical variables, we investigated the impact of different sample handling conditions on measured plasma anandamide concentrations. Methods: Blood samples were taken from healthy volunteers in EDTA- or heparin-containing tubes; whole blood samples were kept at +4 degrees C, room temperature, or 37 degrees C, respectively, for up to 120 min before obtaining plasma by centrifugation. Plasma anandamide concentrations were measured by an isotope-dilution liquid chromatography tandem mass spectrometry ( LC-MS/MS) method. Results: A marked time- and temperature-dependent increase in plasma anandamide concentrations ex vivo was observed in both EDTA- and heparin-containing tubes. Mean anandamide concentrations approximately doubled when EDTA samples were kept at 4 degrees C for 60 min before centrifugation {[}immediately centrifuged, 1.3 mg/L ( SD 0.3 mg/L); 2.8 mg/L ( SD 0.5 mg/L) after storage for 60 min; n=12). After storage of heparinized whole-blood samples for 120 min at 37 degrees C, a mean plasma anandamide concentration of 11.9 mg/L ( SD 1.8 mg/L) was found. In cell-free plasma, no increase in anandamide concentrations was found. Conclusion: Anandamide is released from blood cells ex vivo at a very high rate; therefore, strictly standardized pre-analytical protocols have to be applied for plasma anandamide determination.

Background: Measurement of late-night salivary cortisol concentrations is increasingly used as a screening test in suspected Cushing's syndrome. Cortisol concentrations are typically extremely low in late-night samples and discordant assay-specific reference ranges have been reported. Therefore, the aim of our study was to assess the analytical performance of the first automated cortisol immunoassay specified for salivary measurements and to establish late-night sampling reference-range data for this test. Methods: Salivary cortisol was measured using the Roche Cobas Cortisol assay (Roche Diagnostics). Five salivary pools in different concentration ranges were used to assess the inter-assay imprecision of this test in a two-centre evaluation protocol including two reagent lots. Linearity was tested by serial dilution. Salivary samples were obtained at 23:00 h from 100 apparently healthy volunteers using a commercially available salivary sampling device (Salivette, Sarstedt). A subset of 20 samples was used for method comparison with isotope dilution liquid chromatography-tandem mass spectrometry. Results: Inter-assay coefficients of variation (n=20) between 11.6% and 40.4% were found for mean cortisol concentrations between 12.9 and 2.6 nmol/L, with an estimated functional sensitivity of approximately 5.0 nmol/L. The test also gave linear results in the lowest concentration range between 1.0 and 8.3 nmol/L. Mean late-night salivary cortisol of 5.0 nmol/L was found for healthy individuals; the absolute range was 1.4-16.7 nmol/L, and the 95th percentile was 8.9 nmol/L. Substantially lower concentrations were found with isotope dilution LC-MS/MS compared to immunoassay results (mean concentrations 1.8 and 4.4 nmol/L, respectively). Conclusions: The automated assay investigated was found to offer acceptable analytical performance in the very low concentration range required for late-night salivary cortisol, despite a very short turnaround time. Using this assay, late-night salivary cortisol concentrations below 8.9 nmol/L are typically found in healthy volunteers.

Im Rahmen dieser Arbeit wurden verschiedene Proteome von H. salinarum untersucht, die nach zellulären Kompartimenten unterschieden wurden in (1) das Flagellarmotor-Proteom (2) das Cytosolproteom und (3) das Membranproteom. Die Untersuchung des Flagellarmotors erfolgte hauptsächlich auf struktureller Basis mittels Elektronenmikroskopie. Es konnte eine Struktur mit zwei übereinanderliegenden Ringen isoliert werden, die beide an eine Flagelle gebunden sind. Aus weiteren Aufnahmen und Größenkorrelationen wurde ein Modell zum Flagellarmotor entworfen, welches eine Rotation beider Ringe beinhaltet. An diese Doppelringstruktur sind mehrere Flagellen über einen Hook gebunden, was dieses Modell damit vom bakteriellen Flagellarmotor unterscheidet. Bei der Untersuchung des Cytosolproteoms konnten insgesamt 840 Proteine mittels MALDI-MS-Fingerprint identifiziert werden, was einer Identifizierungsrate von 38% des löslichen Proteoms entspricht (Identifizierungsrate aller löslichen Proteine größer 20 kD: 61%). Es wurde eine massenkompatible Silberfärbung optimiert und ein semi-manuelles Verfahren zur in-Gel Spaltung entwickelt, mit dem 800 Proteine/Tag enzymatisch gespalten werden können. Für die anschließende Identifizierung wurde ein Standardprotokoll für die Proben- und Matrixpräparation entwickelt, welches sich im Vergleich zu anderen automatischen Präparationen als zuverlässiger und sensitiver gezeigt hat. Im Verlauf dieser Arbeit wurde von der Bioinformatikgruppe (Dr. F. Pfeiffer) das web-basierte HALOLEX-System entwickelt, welches als Ziel die vollständige Erfassung aller Fakten zu H. salinarum hat. Die generierten Daten (Gele, Proteinidentifizierungen, MALDI-Peaks, MS/MS-Peaks) werden innerhalb dieser Oberfläche zugänglich gemacht und erlauben detaillierte Nachanalysen. Für das Membranproteom wurde gezeigt, dass der etablierte Zellaufschluss mittels Niedrigsalz-Dialyse zu einer erheblichen Kontamination mit löslichen Proteinen führt. Ein Aufschluss unter Hochsalzbedingungen mit anschließender Dichtegradienten-Zentrifugation reinigt die Membran, jedoch dissoziiert die Zellmembran bei anschließender Niedrigsalz-Behandlung in hohem Maße in nicht pelletierbare Fragmente. Eine optimierte Membranaufarbeitung unter ständigen Hochsalzbedingungen, Dichtegradienten-Zentrifugation, Delipidierung und Solubilisierung in einer Detergenzienmischung (Triton X-100/ASB-14) führte bei anschließender zweidimensionaler Trennung (IEF/SDS) zur Identifizierung von fast ausschließlich peripheren Membranproteinen. Mit einer fluoreszenzmarkierten Membranfraktion konnte gezeigt werden, dass der Verlust von integralen Membranproteinen auf einer nahezu quantitativen Präzipitation der Membranproteine an derem pI beruht. Eine pI-unabhängige Strategie wurde mittels BAC/SDS etabliert, die speziell bei H. salinarum zu einer guten Proteinauftrennung führt. Die Identifizierung eines 13 TM-Antiporters (0,22 TM/kD, GRAVY +0,74) als dominantestes Protein dieser Membranfraktion zeigt die Anwendbarkeit dieses Systems. In einem Vergleich von Membranfraktionen aus aerob und phototroph gewachsenen Kulturen konnten so Unterschiede des Expressionsniveaus von Membranproteinen nachgewiesen werden. Aus theoretischen Berechnungen der vorhergesagten Membranproteine zeigte sich weiterhin, dass bei tryptischer Spaltung nicht ausreichend Peptid-Fragmente generiert werden, um mittels MALDI-Fingerprint-Analyse identifiziert zu werden. Diese (H. salinarum spezifische) Problematik kann mittels MS/MS umgangen werden. Bei der Kombination aus 1-D Gel und LC/MS/MS konnten schließlich 114 integrale Membranproteine identifiziert werden, was 20% des integralen Membranproteoms entspricht.

We have developed a liquid chromatography/mass spectrometry (LC-MS/MS) assay to determine acrylamide in various body fluids. The assay also allows the reliable quantitation of acrylamide in food. In a total of 11 healthy male and female subjects, we were able to show that acrylamide from food given to humans is in fact absorbed from the gut. The half-lives determined in two male subjects were 2.2 and 7 h. Acrylamide was found in human breast milk and penetrated the human placenta (n = 3). The variability of acrylamide concentrations found in this investigation is most likely caused by variable intersubject bioavailability and metabolism. This may be an important indication that the assessment of the risk from acrylamide for the individual may be very difficult without knowing the concentrations of acrylamide in the body. This should be considered in the design of any risk assessment study or post hoc analysis of earlier studies. At this time, we suggest that pregnant women and breast-feeding mothers avoid acrylamide-containing food. Copyright (C) 2002 S. Karger AG, Basel.