Medication Exposure in Highly Adherent Psychiatry Patients.

Medication exposure is dependent upon many factors, the single most important being if the patient took the prescribed medication as indicated. To assess medication exposure for psychotropic and other medication classes, we enrolled 115 highly adherent psychiatry patients prescribed five or more medications. In these patients, we measured 21 psychotropic and 38 nonpsychotropic medications comprising a 59 medication multiplex assay panel. Strict enrollment criteria and reconciliation of the electronic health record medication list prior to study initiation produced a patient cohort that was adherent with 91% of their prescribed medications as determined by comparing medications detected empirically in blood to the electronic health record medication list. In addition, 13% of detected medications were not in the electronic health record medication list. We found that only 53% of detected medications were within the literature-derived reference range with 41% below and 6% above the reference range specific to each medication. When psychotropic medications were analyzed near trough-level, only sertraline was found to be within the literature-derived reference range for all patients tested. Concentrations of the remaining medications indicated extensive exposure below the reference range. This is the first study to empirically and comprehensively assess medication exposure obtained in comorbid polypharmacy patients, minimizing the important behavioral factor of adherence in the study of medication exposure. These data indicate that low medication exposure is extensive and must be considered when therapeutic issues arise, including the lack of response to medication therapy.

Assessment of Patient Medication Adherence, Medical Record Accuracy, and Medication Blood Concentrations for Prescription and Over-the-Counter Medications.

Importance: Inaccurate medication records and poor medication adherence result in incomplete knowledge of therapy for patients. Objective: To study accuracy of medical records and patient adherence by measuring blood concentrations of medications. Design, Setting, and Participants: This cross-sectional study validated a serum-based liquid chromatography-tandem mass spectrometry assay to simultaneously quantify 263 medications used for acute and chronic conditions. The assay panel was applied to 3 clinical patient cohorts: residual serum from 1000 randomly selected samples sent for routine clinical chemistry testing between April 8 and October 6, 2015 (residuals cohort), 50 prospectively enrolled patients in a gastroenterology clinic between March 1 and March 15, 2016, who were prescribed more than 5 medications (gastroenterology care cohort), and a convenience cohort of 296 patients with hypertension who sought care in an emergency department (ED care cohort) between July 1, 2012, and April 25, 2013. Integrated data analysis of the cohorts was performed from August 22 to November 29, 2017. Main Outcomes and Measures: Medication serum concentrations, electronic health record medication lists, and predicted drug interactions. Results: Of the 1346 total samples, 1000 came from the residuals cohort (640 women and 360 men; median age, 60 years [interquartile range (IQR), 44-71 years]), 50 from the gastroenterology care cohort (30 women and 20 men; median age, 66 years [IQR, 62-70 years]), and 296 from the ED care cohort (160 women and 136 men; median age, 59 years [IQR, 52-66 years]). Median medication adherence, defined as the subset of detected medications from the prescription record, was 83% (IQR, 50%-100%) in the residuals cohort, 100% (IQR, 84%-100%) in the gastroenterology care cohort, and 78% (IQR, 57%-100%) in the ED care cohort. Patients adherent to 1 medication were more often adherent to other medications. Among patients prescribed 3 medications or more, there were no significant associations between medication adherence and sex or number of prescribed medications, and there was a modest association between adherence and age. By comparing detected vs prescribed medications, we detected a median of 0 (IQR, 0-2) medications per patient that were not listed in the electronic health record in the residuals cohort, 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the gastroenterology care cohort, and 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the ED care cohort. A total of 435 patients (43.5%) in the residuals cohort had no discrepancy between the electronic health record and detected medication lists, 22 patients (44.0%) in the gastroenterology care cohort had no discrepancy between the electronic health record and detected medication lists, and 41 patients (13.9%) in the ED care cohort had no discrepancy between the electronic health record and detected medication lists. Half of adverse drug reaction alerts occurred among medications detected without prescription. Conclusions and Relevance: Comprehensive medication monitoring offers promise to improve adherence, the accuracy of medical records, and the safety for patients with polypharmacy.

Medication adherence, medical record accuracy, and medication exposure in real-world patients using comprehensive medication monitoring.

BACKGROUND: Poor adherence to medication regimens and medical record inconsistencies result in incomplete knowledge of medication therapy in polypharmacy patients. By quantitatively identifying medications in the blood of patients and reconciling detected medications with the medical record, we have defined the severity of this knowledge gap and created a path toward optimizing medication therapy. METHODS AND FINDINGS: We validated a liquid chromatography-tandem mass spectrometry assay to detect and/or quantify 38 medications across a broad range of chronic diseases to obtain a comprehensive survey of patient adherence, medical record accuracy, and exposure variability in two patient populations. In a retrospectively tested 821-patient cohort representing U.S. adults, we found that 46% of medications assessed were detected in patients as prescribed in the medical record. Of the remaining medications, 23% were detected, but not listed in the medical record while 30% were prescribed to patients, but not detected in blood. To determine how often each detected medication fell within literature-derived reference ranges when taken as prescribed, we prospectively enrolled a cohort of 151 treatment-regimen adherent patients. In this cohort, we found that 53% of medications that were taken as prescribed, as determined using patient self-reporting, were not within the blood reference range. Of the medications not in range, 83% were below and 17% above the lower and upper range limits, respectively. Only 32% of out-of-range medications could be attributed to short oral half-lives, leaving extensive exposure variability to result from patient behavior, undefined drug interactions, genetics, and other characteristics that can affect medication exposure. CONCLUSIONS: This is the first study to assess compliance, medical record accuracy, and exposure as determinants of real-world treatment and response. Variation in medication detection and exposure is greater than previously demonstrated, illustrating the scope of current therapy issues and opening avenues that warrant further investigation to optimize medication therapy.

Managing Psychotropic Medications in Complex, Real-World Patients Using Comprehensive Therapeutic Drug Monitoring.

There are multiple treatment options for depression, anxiety, psychosis, and other psychiatric disorders, and psychiatry patients are often comorbid with complex, polypharmacy treatment regimens. Unlike cardiovascular disease and diabetes, there are no readily available biomarkers to gauge treatment success with psychotropic medications, often resulting in subjective determination of medication therapy effectiveness. The physiochemical properties of psychiatric medications in general lend themselves to quantitative measurement in blood, offering an avenue to optimize treatment for each patient. Herein, we describe a novel application that employs comprehensive therapeutic drug monitoring of both psychiatric and nonpsychiatric medications to holistically personalize therapy for complex psychiatry patients.

Lipid composition of viral envelope of three strains of influenza virus - not all viruses are created equal.

While differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31- H3N2, PR8- H1N1, and VN1203- H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, while in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species specific biomarkers of viral pathogenicity.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Peroxisomes are required for lipid metabolism and muscle function in Drosophila melanogaster.

Peroxisomes are ubiquitous organelles that perform lipid and reactive oxygen species metabolism. Defects in peroxisome biogenesis cause peroxisome biogenesis disorders (PBDs). The most severe PBD, Zellweger syndrome, is characterized in part by neuronal dysfunction, craniofacial malformations, and low muscle tone (hypotonia). These devastating diseases lack effective therapies and the development of animal models may reveal new drug targets. We have generated Drosophila mutants with impaired peroxisome biogenesis by disrupting the early peroxin gene pex3, which participates in budding of pre-peroxisomes from the ER and peroxisomal membrane protein localization. pex3 deletion mutants lack detectible peroxisomes and die before or during pupariation. At earlier stages of development, larvae lacking Pex3 display reduced size and impaired lipid metabolism. Selective loss of peroxisomes in muscles impairs muscle function and results in flightless animals. Although, hypotonia in PBD patients is thought to be a secondary effect of neuronal dysfunction, our results suggest that peroxisome loss directly affects muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in Drosophila physiology, specifically in muscle cells may reveal novel aspects of PBD etiology.

The serine hydrolase ABHD6 Is a critical regulator of the metabolic syndrome.

The serine hydrolase α/ß hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Diacylglycerol kinase delta promotes lipogenesis.

We have studied the relationship between diacylglycerol kinase delta (DGKδ) and lipogenesis. There is a marked increase in the expression of DGKδ during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGKδ, there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGKδ promotes lipogenesis. Lipid synthesis is decreased in DGKδ knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids, which is higher in the wild type cells than in the DGKδ knockouts. In comparison, there was no change in lipid synthesis in DGKε knockout mouse embryo fibroblasts. We also demonstrate that the DGKδ knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGKδ markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.

Sum of the parts: mass spectrometry-based metabolomics.

Metabolomics is a rapidly growing field of research used in the identification and quantification of the small molecule metabolites within an organism, thereby providing insights into cell metabolism and bioenergetics as well as processes important in clinical medicine, such as disposition of pharmaceutical compounds. It offers comprehensive information about thousands of low-molecular mass compounds (<1500 Da) that represent a wide range of pathways and intermediary metabolism. Because of its vast expansion in the past two decades, mass spectrometry has become an indispensable tool in "omic" analyses. The use of different ionization techniques such as the more traditional electrospray and matrix-assisted laser desorption, as well as recently popular desorption electrospray ionization, has allowed the analysis of a wide range of biomolecules (e.g., peptides, proteins, lipids, and sugars), and their imaging and analysis in the original sample environment in a workup free fashion. An overview of the current state of the methodology is given, as well as examples of application.

Cobalt carbonyl complexes as probes for alkyne-tagged lipids.

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a "tag" that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV(349nm). The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity "pull-down" strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.

Long term myriocin treatment increases MRP1 transport activity.

We investigated the effect of myriocin treatment, which extensively depletes sphingolipids from cells, on multidrug resistance-related protein 1 (MRP1) efflux activity in MRP1 expressing cells and isolated plasma membrane vesicles. Our data reveal that both short term (3 days) and long term (7 days) treatment effectively reduce the cellular sphingolipid content to the same level. Intriguingly, a two-fold increase in MRP1-mediated efflux activity was observed following long term treatment, while short term treatment had no impact. Very similar data were obtained with plasma membrane vesicles isolated from myriocin-treated cells. Exploiting the cell-free vesicle system, Michaelis-Menten analysis revealed that the intrinsic MRP1 activity remained unaltered; however, the fraction of active transporter molecules increased. We demonstrate that the latter effect is due to an enhanced recruitment of MRP1 into lipid raft fractions, thereby promoting MRP1 activity.

Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action.

Mutations of comparative gene identification 58 (CGI-58) in humans cause Chanarin-Dorfman syndrome, a rare autosomal recessive disease in which excess triacylglycerol (TAG) accumulates in multiple tissues. CGI-58 recently has been ascribed two distinct biochemical activities, including coactivation of adipose triglyceride lipase and acylation of lysophosphatidic acid (LPA). It is noteworthy that both the substrate (LPA) and the product (phosphatidic acid) of the LPA acyltransferase reaction are well-known signaling lipids. Therefore, we hypothesized that CGI-58 is involved in generating lipid mediators that regulate TAG metabolism and insulin sensitivity. Here, we show that CGI-58 is required for the generation of signaling lipids in response to inflammatory stimuli and that lipid second messengers generated by CGI-58 play a critical role in maintaining the balance between inflammation and insulin action. Furthermore, we show that CGI-58 is necessary for maximal TH1 cytokine signaling in the liver. This novel role for CGI-58 in cytokine signaling may explain why diminished CGI-58 expression causes severe hepatic lipid accumulation yet paradoxically improves hepatic insulin action. Collectively, these findings establish that CGI-58 provides a novel source of signaling lipids. These findings contribute insight into the basic mechanisms linking TH1 cytokine signaling to nutrient metabolism.

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress.

Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.

Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation.

As technology expands what it is possible to accurately measure, so too the challenges faced by modern mass spectrometry applications expand. A high level of accuracy in lipid quantitation across thousands of chemical species simultaneously is demanded. While relative changes in lipid amounts with varying conditions may provide initial insights or point to novel targets, there are many questions that require determination of lipid analyte absolute quantitation. Glycerophospholipids present a significant challenge in this regard, given the headgroup diversity, large number of possible acyl chain combinations, and vast range of ionization efficiency of species. Lipidomic output is being used more often not just for profiling of the masses of species, but also for highly-targeted flux-based measurements which put additional burdens on the quantitation pipeline. These first two challenges bring into sharp focus the need for a robust lipidomics workflow including deisotoping, differentiation from background noise, use of multiple internal standards per lipid class, and the use of a scriptable environment in order to create maximum user flexibility and maintain metadata on the parameters of the data analysis as it occurs. As lipidomics technology develops and delivers more output on a larger number of analytes, so must the sophistication of statistical post-processing also continue to advance. High-dimensional data analysis methods involving clustering, lipid pathway analysis, and false discovery rate limitation are becoming standard practices in a maturing field.

A mouse macrophage lipidome.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Lipidomics reveals a remarkable diversity of lipids in human plasma.

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2.

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.