Lysophosphatidylcholines modulate immunoregulatory checkpoints in peripheral monocytes and are associated with mortality in people with acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening disease characterised by high-grade inflammation and immunoparesis, which is associated with a high incidence of death from sepsis. Herein, we aimed to describe the metabolic dysregulation in ALF and determine whether systemic immune responses are modulated via the lysophosphatidylcholine (LPC)-autotaxin (ATX)-lysophosphatidylcholinic acid (LPA) pathway. METHODS: Ninety-six individuals with ALF, 104 with cirrhosis, 31 with sepsis and 71 healthy controls (HCs) were recruited. Pathways of interest were identified by multivariate statistical analysis of proton nuclear magnetic resonance spectroscopy and untargeted ultraperformance liquid chromatography-mass spectrometry-based lipidomics. A targeted metabolomics panel was used for validation. Peripheral blood mononuclear cells were cultured with LPA 16:0, 18:0, 18:1, and their immune checkpoint surface expression was assessed by flow cytometry. Transcript-level expression of the LPA receptor (LPAR) in monocytes was investigated and the effect of LPAR antagonism was also examined in vitro. RESULTS: LPC 16:0 was highly discriminant between ALF and HC. There was an increase in ATX and LPA in individuals with ALF compared to HCs and those with sepsis. LPCs 16:0, 18:0 and 18:1 were reduced in individuals with ALF and were associated with a poor prognosis. Treatment of monocytes with LPA 16:0 increased their PD-L1 expression and reduced CD155, CD163, MerTK levels, without affecting immune checkpoints on T and NK/CD56+T cells. LPAR1 and 3 antagonism in culture reversed the effect of LPA on monocyte expression of MerTK and CD163. MerTK and CD163, but not LPAR genes, were differentially expressed and upregulated in monocytes from individuals with ALF compared to controls. CONCLUSION: Reduced LPC levels are biomarkers of poor prognosis in individuals with ALF. The LPC-ATX-LPA axis appears to modulate innate immune response in ALF via LPAR1 and LPAR3. Further investigations are required to identify novel therapeutic agents targeting these receptors. IMPACT AND IMPLICATIONS: We identified a metabolic signature of acute liver failure (ALF) and investigated the immunometabolic role of the lysophosphatidylcholine-autotaxin-lysophosphatidylcholinic acid pathway, with the aim of finding a mechanistic explanation for monocyte behaviour and identifying possible therapeutic targets (to modulate the systemic immune response in ALF). At present, no selective immune-based therapies exist. We were able to modulate the phenotype of monocytes in vitro and aim to extend these findings to murine models of ALF as a next step. Future therapies may be based on metabolic modulation; thus, the role of specific lipids in this pathway require elucidation and the relative merits of autotaxin inhibition, lysophosphatidylcholinic acid receptor blockade or lipid-based therapies need to be determined. Our findings begin to bridge this knowledge gap and the methods used herein could be useful in identifying therapeutic targets as part of an experimental medicine approach.

Dysregulation of the Lysophosphatidylcholine/Autotaxin/Lysophosphatidic Acid Axis in Acute-on-Chronic Liver Failure Is Associated With Mortality and Systemic Inflammation by Lysophosphatidic Acid-Dependent Monocyte Activation.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation, monocyte dysfunction, and susceptibility to infection. Lysophosphatidylcholines (LPCs) are immune-active lipids whose metabolic regulation and effect on monocyte function in ACLF is open for study. APPROACHES & RESULTS: Three hundred forty-two subjects were recruited and characterized for blood lipid, cytokines, phospholipase (PLA), and autotaxin (ATX) concentration. Peripheral blood mononuclear cells and CD14+ monocytes were cultured with LPC, or its autotaxin (ATX)-derived product, lysophosphatidic acid (LPA), with or without lipopolysaccharide stimulation and assessed for surface marker phenotype, cytokines production, ATX and LPA-receptor expression, and phagocytosis. Hepatic ATX expression was determined by immunohistochemistry. Healthy volunteers and patients with sepsis or acute liver failure served as controls. ACLF serum was depleted in LPCs with up-regulated LPA levels. Patients who died had lower LPC levels than survivors (area under the receiver operating characteristic curve, 0.94; P < 0.001). Patients with high-grade ACLF had the lowest LPC concentrations and these rose over the first 3 days of admission. ATX concentrations were higher in patients with AD and ACLF and correlated with Model for End-Stage Liver Disease, Consortium on Chronic Liver Failure-Sequential Organ Failure Assessment, and LPC/LPA concentrations. Reduction in LPC correlated with higher monocyte Mer-tyrosine-kinase (MerTK) and CD163 expression. Plasma ATX concentrations rose dynamically during ACLF evolution, correlating with IL-6 and TNF-α, and were associated with increased hepatocyte ATX expression. ACLF patients had lower human leukocyte antigen-DR isotype and higher CD163/MerTK monocyte expression than controls; both CD163/MerTK expression levels were reduced in ACLF ex vivo following LPA, but not LPC, treatment. LPA induced up-regulation of proinflammatory cytokines by CD14+ cells without increasing phagocytic capacity. CONCLUSIONS: ATX up-regulation in ACLF promotes LPA production from LPC. LPA suppresses MerTK/CD163 expression and increases monocyte proinflammatory cytokine production. This metabolic pathway could be investigated to therapeutically reprogram monocytes in ACLF.

Microsampling Devices for Routine Therapeutic Drug Monitoring-Are We There Yet?

BACKGROUND: The use of microsampling for therapeutic drug monitoring (TDM) is increasingly feasible as sensitive methods have become more accessible. There exists an increasing interest in the use of microsampling, and new microsampling devices and techniques can potentially improve patient convenience and care, among other features. This review provides an update on currently validated methods for measuring drugs pertinent to TDM, including data from clinical samples. METHODS: A literature record search was undertaken, including PubMed and Google Scholar. Reports that included the use of microsampling to measure concentrations of drugs associated with TDM were reviewed and included if data from patient samples were reported. The studies are described in brief, including sample preparation and analyte stability, with the most pertinent findings reported. RESULTS: Sensitive analyses and innovative designs and materials have resulted in an increasing number of reported evaluations and validations for measuring drugs using microsamples. Novel designs largely overcome common problems associated with traditional dried blood spot sampling. Although examples of patient self-sampling are rare at present, studies that evaluated feedback found it to be largely positive, revealing the feasibility of microsampling for TDM. CONCLUSIONS: Microsampling is suited to the TDM of numerous drugs in diverse situations, and it will play an increasingly important role. The issues with traditional dried blood spot samples have been largely overcome by employing novel methods to obtain volumetric samples.

Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.

BACKGROUND: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma. METHODS: After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%-96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species. RESULTS: There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at -20 °C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C. CONCLUSIONS: A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4 °C, separated within 2 hours of collection and stored at 4 °C before analysis, with long-term storage at -20 °C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.

Use of a small particle solid-core packing for improved efficiency and rapid measurement of sirolimus and everolimus by LC-MS/MS.

Measurement of whole blood sirolimus and everolimus is required in order to optimize patient treatment following solid organ transplant. Assay by LC-MS/MS is increasingly preferred; however efficient use of the instrument and short turnaround times are crucial. Use of a 1.6 µm solid-core packing HPLC column (Cortecs) gave significant increases in efficiency, sensitivity and throughput compared with an existing method, following simple protein precipitation of small-volume (20 µL) whole blood samples. Sirolimus, everolimus and the stable isotopic internal standard ((13) C2 D4 - everolimus) eluted at around 0.8 min, and total analytical run time was 2.2 min, saving almost 4 min per sample compared with an existing method. Within-assay imprecision (CV) was 3.3-8.5%, and between-assay imprecision was 2.2-10.8%. Retrospective assay of external quality assurance samples and comparison of patient samples assayed in parallel showed only small differences (between +6.8 and -1.9%) in results using the Cortecs column when compared with the existing method. No significant interferences or ion suppression were observed. Copyright © 2015 John Wiley & Sons, Ltd.

The influence of training and experience on memory strategy.

This paper investigates whether, and if so how much, prior training and experience overwrite the influence of the constraints of the task environment on strategy deployment. This evidence is relevant to the theory of soft constraints that focuses on the role of constraints in the task environment (Gray, Simms, Fu, & Schoelles, Psychological Review, 113: 461-482, 2006). The theory explains how an increase in the cost of accessing information induces a more memory-based strategy involving more encoding and planning. Experiments 1 and 3 adopt a traditional training and transfer design using the Blocks World Task in which participants were exposed to training trials involving a 2.5-s delay in accessing goal-state information before encountering transfer trials in which there was no access delay. The effect of prior training was assessed by the degree of memory-based strategy adopted in the transfer trials. Training with an access delay had a substantial carry-over effect and increased the subsequent degree of memory-based strategy adopted in the transfer environment. However, such effects do not necessarily occur if goal-state access cost in training is less costly than in transfer trials (Experiment 2). Experiment 4 used a fine-grained intra-trial design to examine the effect of experiencing access cost on one, two, or three occasions within the same trial and found that such experience on two consecutive occasions was sufficient to induce a more memory-based strategy. This paper establishes some effects of training that are relevant to the soft constraints theory and also discusses practical implications.

A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.

BACKGROUND: Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. METHODS: Whole-blood samples (20 µL) were treated with ammonium bicarbonate (20 µL), zinc sulfate (20 µL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 µL). After centrifugation, 20 µL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). RESULTS: Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 µg/L for sirolimus, 1-41 µg/L for everolimus), and a lower limit of quantitation of 1 µg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. CONCLUSIONS: A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice.

With the notable exception of humans, uric acid is degraded to (S)-allantoin in a biochemical pathway catalyzed by urate oxidase, 5-hydroxyisourate (HIU) hydrolase, and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase in most vertebrate species. A point mutation in the gene encoding mouse HIU hydrolase, Urah, that perturbed uric acid metabolism within the liver was discovered during a mutagenesis screen in mice. The predicted substitution of cysteine for tyrosine in a conserved helical region of the mutant-encoded HIU hydrolase resulted in undetectable protein expression. Mice homozygous for this mutation developed elevated platelet counts secondary to excess thrombopoietin production and hepatomegaly. The majority of homozygous mutant mice also developed hepatocellular carcinoma, and tumor development was accelerated by exposure to radiation. The development of hepatomegaly and liver tumors in mice lacking Urah suggests that uric acid metabolites may be toxic and that urate oxidase activity without HIU hydrolase function may affect liver growth and transformation. The absence of HIU hydrolase in humans predicts slowed metabolism of HIU after clinical administration of exogenous urate oxidase in conditions of uric acid-related pathology. The data suggest that prolonged urate oxidase therapy should be combined with careful assessment of toxicity associated with extrahepatic production of uric acid metabolites.

An NZW-derived interval on chromosome 7 moderates sialadenitis, but not insulitis in congenic nonobese diabetic mice.

Autoimmune lymphocytic infiltration of the salivary glands, termed sialadenitis, is a pathologic feature of Sjögren's syndrome (SjS) that is also prominent in nonobese diabetic (NOD) mice. Genetic factors regulate sialadenitis, and a previous (NOD x NZW)F2 study detected linkage to murine chromosome (Chr) 7. The locus, subsequently annotated as Ssial3, maps to the distal end of Chr7 and overlaps a region associated with type 1 diabetes susceptibility in NOD mice. To examine whether Ssial3 could contribute to both diseases, or was specific for SjS, we generated a congenic mouse strain that harbored an NZW-derived Chr7 interval on the NOD genetic background. This congenic strain exhibited reduced sialadenitis compared with NOD mice and confirmed Ssial3. This reduction, however, did not ameliorate saliva abnormalities associated with SjS-like disease in NOD mice, nor were congenic mice protected against insulitis (lymphocytic infiltration of the pancreatic islets) or diabetes onset. Thus, the Ssial3 locus appears to have a tissue-specific effect for which the NZW allele is unable to prevent other autoimmune traits in the NOD mouse. Anomalous increases for antinuclear Ab production and frequency of marginal-zone B cells were also identified in congenic mice, indicating that the NZW-derived Chr7 interval has a complex effect on the NOD immune system.

Bibliometric analysis of the literature relating to silicone hydrogel and daily disposable contact lenses.

PURPOSE: Publication metrics are derived for the fields of silicone hydrogel (SH) and daily disposable (DD) contact lenses. METHODS: A search of the Scopus database for papers in the fields of SH and DD contact lenses found 979 SH and 291 DD papers. Subject-specific h-indices for SH lenses (hSH-index) and DD lenses (hDD-index) were derived, in relation to five categories - authors, institutions, countries and journals - to serve as measures of impact. A short list of the most impactful entities was generated for each of the above five categories in the SH and DD fields. RESULTS: A paper entitled "Soft contact lens polymers: An evolution" by Nicholson and Vogt was the most highly cited article (495 citations) in both SH and DD fields. The most impactful entities for the SH and DD fields were: authors - Lyndon Jones (hSH = 33) and Philip Morgan (hDD = 15); institutions - the University of Waterloo (hSH = 37) and the University of New South Wales (hDD = 15); countries - the United States (hSH = 45) and the United Kingdom (hDD = 24); and journals - Optometry and Vision Science (hSH = 33) and Contact Lens and Anterior Eye (hDD = 17). Overall, the SH field (hSH = 64) is far more impactful than the DD field (hDD = 34). CONCLUSIONS: Impactful papers, authors, institutions, countries and journals in the SH and DD fields are identified. Optometry is revealed as the leading profession in relation to SH and DD publications.

Immunometabolic analysis shows a distinct cyto-metabotype in Covid-19 compared to sepsis from other causes.

Background: In Covid-19, profound systemic inflammatory responses are accompanied by both metabolic risk factors for severity and, separately, metabolic mechanisms have been shown to underly disease progression. It is unknown whether this reflects similar situations in sepsis or is a unique characteristic of Covid-19. Aims: Define the immunometabolic signature of Covid-19. Methods: 65 patients with Covid-19,19 patients with sepsis and 14 healthy controls were recruited and sampled for plasma, serum and peripheral blood mononuclear cells (PBMCs) through 10 days of critical illness. Metabotyping was performed using the Biocrates p180 kit and multiplex cytokine profiling undertaken. PBMCs underwent phenotyping by flow cytometry. Immune and metabolic readouts were integrated and underwent pathway analysis. Results: Phopsphatidylcholines (PC) are reduced in Covid-19 but greater than in sepsis. Compared to controls, tryptophan is reduced in Covid-19 and inversely correlated with the severity of the disease and IFN-É£ concentrations, conversely the kyneurine and kyneurine/tryptophan ratio increased in the most severe cases. These metabolic changes were consistent through 2 pandemic waves in our centre. PD-L1 expression in CD8+ T cells, Tregs and CD14+ monocytes was increased in Covid-19 compared to controls. Conclusions: In our cohort, Covid-19 is associated with monocytopenia, increased CD14+ and Treg PD-L1 expression correlating with IFN-É£ plasma concentration and disease severity (SOFA score). The latter is also associated with metabolic derangements of Tryptophan, LPC 16:0 and PCs. Lipid metabolism, in particular phosphatidylcholines and lysophosphatidylcolines, seems strictly linked to immune response in Covid-19. Our results support the hypothesis that IFN-É£ -PD-L1 axis might be involved in the cytokine release syndrome typical of severe Covid-19 and the phenomenon persisted through multiple pandemic waves despite use of immunomodulation.

Measurement of posaconazole, itraconazole, and hydroxyitraconazole in plasma/serum by high-performance liquid chromatography with fluorescence detection.

BACKGROUND: Itraconazole and posaconazole are used in the prevention and treatment of invasive fungal infections. However, the oral bioavailability of both compounds varies widely, and dose-serum concentration relationships are poorly defined for these analytes. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of therapeutic drug monitoring. METHODS: Calibrators (n = 7) and internal quality control solutions (n = 3) were prepared in pooled human serum. Sample (100 µL), internal standard solution (25 µL), Tris solution (2 mol/L; pH 10.6), and extraction solvent (methyl tert-butyl ether, 600 µL) were vortex mixed and centrifuged. The solvent layer was removed and evaporated to dryness and the residue reconstituted in water:methanol (1 + 3, 50 µL). A portion (5 µL) of the reconstituted extract was analyzed using a 3-µm Gemini C6 phenyl column with fluorescence detection (excitation 260 nm, emission 350 nm). The method was used to measure itraconazole and hydroxyitraconazole, or posaconazole, in serum samples taken 1-2 hours before the next dose, from patients forming part of a study into management and diagnostic strategies for invasive aspergillosis. RESULTS: Response was linear over the calibration ranges. Accuracy and imprecision were 92-111.4% and 3.2-13.4% (relative standard deviation), respectively. No interferences were noted. There was a good agreement with nominal values of each analyte in an external quality assessment scheme. In patients prescribed either 400 mg/d of itraconazole (n = 46) or 600-800 mg/d of posaconazole (n = 28) only 24% and 7% of samples, respectively, had serum itraconazole or posaconazole concentrations above the target threshold suggested in published guidelines. CONCLUSIONS: A simple, sensitive high-performance liquid chromatographic method has been developed for the analysis of itraconazole, hydroxyitraconazole, and posaconazole in serum/plasma. Few of the samples measured from patients participating in the clinical study attained concentrations of the drug/metabolite in serum that have been recommended for effective antifungal therapy.

LC-MS in analytical toxicology: some practical considerations.

Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the influence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology.

A rapid and simple assay for lamotrigine in serum/plasma by HPLC, and comparison with an immunoassay.

Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average.

The Influence of Donor and Recipient Complement C3 Polymorphisms on Liver Transplant Outcome.

Despite early reports of an impact of complement C3 polymorphism on liver transplant patient and graft survival, subsequent evidence has been conflicting. Our aim was to clarify the contributions of donor and recipient C3 genotype, separately and together, on patient and graft outcomes and acute rejection incidence in liver transplant recipients. Eight donor/recipient groups were analyzed according to their genotype and presence or absence of C3 F allele (FFFS, FFSS, FSFF, FSFS, FSSS, SSFF, SSFS, and SSSS) and correlated with clinical outcomes of patient survival, graft survival, and rejection. The further impact of brain death vs. circulatory death during liver donation was also considered. Over a median 5.3 y follow-up of 506 patients with clinical information and matching donor and recipient tissue, five-year patient and graft survival (95% confidence interval) were 90(81-91)% and 77(73-85)%, respectively, and 72(69-94)% were rejection-free. Early disadvantages to patient survival were associated with donor C3 F variant, especially in brain-death donors. Recipient C3 genotype was an independent determinant of graft survival by Cox proportional hazards analysis (hazard ratio 0.26, P = 0.04), and the C3 F donor variant was again associated with worse liver graft survival, particularly in brain-death donors. C3 genotype did not independently determine rejection incidence, but a greater proportion of recipient C3 F carriers were rejection-free in the circulatory death, but not the brain-death cohort. Cox proportional hazards analysis revealed significant effects of acute rejection on patient survival (hazard ratio 0.24, P = 0.018), of retransplantation on rejection risk (hazard ratio 6.3, P = 0.009), and of donor type (circulatory-death vs. brain-death) on rejection incidence (hazard ratio 4.9, P = 0.005). We conclude that both donor and recipient complement C3 genotype may influence patient and graft outcomes after liver transplantation but that the type of liver donor is additionally influential, possibly via the inflammatory environment of the transplant.

Agm1/Pgm3-mediated sugar nucleotide synthesis is essential for hematopoiesis and development.

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

Plasma clozapine and norclozapine in patients prescribed different brands of clozapine (Clozaril, Denzapine, and Zaponex).

To investigate the bioequivalence of the three clozapine brands licensed in the United Kingdom, we compared plasma clozapine and norclozapine in therapeutic drug monitoring samples from patients switched from Clozaril to either Denzapine or Zaponex tablets. For Clozaril/Denzapine, the median prescribed clozapine dose was 450 mg/day (range, 125-850 mg/day) (n = 66) and the median time between samples was 16 weeks. The Clozaril/Zaponex comparison (n = 57) was not dose-controlled; the median Clozaril dose was 450 mg/day (range, 150-900 mg/day) and the median Zaponex dose 400 mg/day (range, 100-850 mg/day). The median time between samples was 19 weeks. There was no significant difference in mean plasma clozapine and norclozapine concentration before and after switching in either case, although some individual results showed clinically relevant concentration differences. Plasma norclozapine showed greater reproducibility between samples than clozapine. The different brands of clozapine available in the United Kingdom show bioequivalence. Nevertheless, careful monitoring of mental state, smoking habit, adherence, and of possible life-threatening adverse effects is mandatory if the drug is to be used safely.

Plasma clozapine, norclozapine, and the clozapine:norclozapine ratio in relation to prescribed dose and other factors: data from a therapeutic drug monitoring service, 1993-2007.

Therapeutic drug monitoring of plasma clozapine and of its principal plasma metabolite N-desmethylclozapine (norclozapine) (predose or "trough" sample) can help in monitoring adherence, in dose adjustment, and in minimizing the risk of toxicity. To obtain data to assist in the interpretation of analytical results, the results from a clozapine therapeutic drug monitoring service, 1993-2007, have been audited. There were 104,127 samples from 26,796 patients [18,750 (70%) men aged at time of first sample (median, range) 34 (10-89) years, and 7763 (30%) female aged 38 (12-90) years]. Clozapine was not detected (plasma concentration <0.01 mg/L) in 1.5% of samples (prescribed clozapine dose up to 900 mg/d). Plasma clozapine was either below 0.35 mg/L or greater than 0.60 mg/L in 42.5% and 28.4% of samples, respectively; in 0.4% samples plasma clozapine was 2.0 mg/L or more. Although plasma clozapine was broadly related to prescribed dose, there was much variation: 1.2% of samples had plasma clozapine >1.0 mg/L at prescribed clozapine doses up to 150 mg/d (76.2% < 0.35 mg/L), whereas 23.3% of samples had plasma clozapine < 0.35 mg/L at doses of 850 mg/d and over (18.0% > 1.0 mg/L). The highest plasma clozapine and norclozapine concentrations encountered were 4.95 and 2.45 mg/L, respectively. Although the median plasma clozapine:norclozapine ratio was 1.25 at plasma clozapine concentrations < 0.35 mg/L, the median ratio was 2.08 at plasma clozapine concentrations > 1.0 mg/L. Data (median, 10th-90th percentile) for both clozapine and norclozapine by prescribed clozapine dose band are useful in assessing partial adherence. Analysis of the plasma clozapine:norclozapine ratio by clozapine concentration provides clear evidence that clozapine N-demethylation becomes saturated at higher plasma clozapine concentrations and adds urgency to the requirement for dose adjustment should smoking habit change. A clozapine:norclozapine ratio greater then 2 suggests either a nontrough sample, or that clozapine N-demethylation has become saturated.