Estimation of Gestational Age-Specific Reference Intervals for Coagulation Assays in a Neonatal Intensive Care Unit Using Real-World Data.

BACKGROUND: Interpretation of coagulation testing in neonates currently relies on reference intervals (RIs) defined from older patient cohorts. Direct RI studies are difficult, but indirect estimation may allow us to infer normative neonatal distributions from routinely collected clinical data. METHODS: We analyzed first-in-life coagulation testing results from all patients admitted to a level IV neonatal intensive care unit between 1/1/2018-1/1/2024. Results obtained after transfusion of any blood product were excluded. Indirect RIs were estimated across gestational age groups using refineR and compared to currently reported intervals for patients less than one year of age. RESULTS: Prothrombin times (PT) and international normalized ratios (INR) were available for 1,128 neonates, while activated partial thromboplastin times (aPTT) were available for 790 neonates. The indirect RI was 10-25s in preterm, 10-22s in term, and 10-24s in all neonates for PT, 0.7-2.1 in preterm, 0.8-1.8 in term, and 0.8-1.9 in all neonates for INR, and 25-68s in preterm, 25-58s in term, and 25-62s in all neonates for aPTT. Compared to our current intervals, the indirect RIs would flag 58% fewer PT, 43% fewer INR, and 17% fewer aPTT results as abnormal. CONCLUSIONS: Indirectly estimated RIs in neonates admitted to intensive care show substantial divergence from current, first-year-of-life RIs, leading to an abundance of abnormal flags. The associations between these flags and provider behavior, transfusion practice, or clinical outcomes is an area of future exploration.

Clinical Utility of LC-MS/MS for Blood Myo-Inositol in Patients with Acute Kidney Injury and Chronic Kidney Disease.

BACKGROUND: Diagnosing acute kidney injury (AKI) and chronic kidney disease (CKD) relies on creatinine, which lacks optimal diagnostic sensitivity. The kidney-specific proximal tubular enzyme myo-inositol oxygenase (MIOX) catalyzes the conversion of myo-inositol (MI) to D-glucuronic acid. We hypothesized that proximal tubular damage, which occurs in AKI and CKD, will decrease MIOX activity, causing MI accumulation. To explore this, we developed an LC-MS/MS assay to quantify plasma MI and assessed its potential in identifying AKI and CKD patients. METHODS: MI was quantified in plasma from 3 patient cohorts [normal kidney function (n = 105), CKD (n = 94), and AKI (n = 54)]. The correlations between MI and creatinine were determined using Deming regression and Pearson correlation and the impact of age, sex, and ethnicity on MI concentrations was assessed. Receiver operating characteristic curve analysis was employed to evaluate MI diagnostic performance. RESULTS: In volunteers with normal kidney function, the central 95th percentile range of plasma MI concentrations was 16.6 to 44.2 µM. Age, ethnicity, and sex showed minimal influence on MI. Patients with AKI and CKD exhibited higher median MI concentrations [71.1 (25th percentile: 38.2, 75th percentile: 115.4) and 102.4 (77, 139.5) µM], respectively. MI exhibited excellent sensitivity (98.9%) and specificity (100%) for diagnosing CKD. In patients with AKI, MI increased 32.9 (SD 16.8) h before creatinine. CONCLUSIONS: This study unveils MI as a potential renal biomarker, notably elevated in plasma during AKI and CKD. Plasma MI rises 33 h prior to serum creatinine, enabling early AKI detection. Further validation and exploration of MI quantitation in kidney disease diagnosis is warranted.

Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism.

Urea cycle impairment and its relationship to obesity and inflammation remained elusive, partly due to the dramatic clinical presentation of classical urea cycle defects. We generated mice with hepatocyte-specific arginase 2 deletion (Arg2LKO) and revealed a mild compensated urea cycle defect. Stable isotope tracing and respirometry revealed hepatocyte urea and TCA cycle flux defects, impaired mitochondrial oxidative metabolism, and glutamine anaplerosis despite normal energy and glucose homeostasis during early adulthood. Yet during middle adulthood, chow- and diet-induced obese Arg2LKO mice develop exaggerated glucose and lipid derangements, which are reversible by replacing the TCA cycle oxidative substrate nicotinamide adenine dinucleotide. Moreover, serum-based hallmarks of urea, TCA cycle, and mitochondrial derangements predict incident fibroinflammatory liver disease in 106,606 patients nearly a decade in advance. The data reveal hierarchical urea-TCA cycle control via ARG2 to drive oxidative metabolism. Moreover, perturbations in this circuit may causally link urea cycle compromise to fibroinflammatory liver disease.

Accumulation of alkyl-lysophosphatidylcholines in Niemann-Pick disease type C1.

Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine [alkyl-LPC, also known as lyso-platelet activating factor (PAF)] species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPßCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This study identified alkyl-LPC species as secondary storage metabolites in NPC1 and indicates that LPC O-16:0 and LPC O-18:1, in particular, could serve as potential biomarkers for tracking treatment response in NPC1 patients.

Evaluation of an automated von Willebrand factor glycoprotein IbM activity assay compared with 3 alternative von Willebrand factor activity assays.

Background: To overcome deficiencies of the traditional von Willebrand factor (VWF) ristocetin cofactor activity assay (VWF:RCo), several automated assays for VWF platelet-binding activity have been developed. Information on the performance of these assays and their diagnostic utility remains limited. Objectives: To validate the VWF:glycoprotein IbM assay INNOVANCE VWF Ac and compare it with an automated VWF:RCo assay as well as with an automated assay and a manual VWF:Ab assay and to generate reference ranges and analyze reproducibility of the VWF:glycoprotein IbM assay. Methods: Clinical sites enrolled healthy subjects and patients representing the intended use population; VWF activity assays were performed, and results were analyzed. The performance of the INNOVANCE VWF Ac assay was also compared between the BCS XP System and the CS-2500 and CS-5100 analyzers. Results: The INNOVANCE VWF Ac assay correlated well with the VWF:RCo assay and the automated HemosIL VWF:Ab assay, with Pearson coefficients of >.9 and a predicted bias of ≤5.0 IU/dL at VWF levels of 30 IU/dL and ≤5.8 IU/dL at the levels of 50 IU/dL, but correlation and bias were not as good when compared with the REAADS manual VWF:Ab assay. Reference ranges observed for healthy subjects correlated well with previously published findings. Reproducibility of the INNOVANCE VWF Ac assay on the BCS XP System and the CS analyzers was excellent, as was correlation among devices. Conclusion: The characteristics of the INNOVANCE VWF Ac assay regarding comparability with other VWF activity assays, reference ranges, and precision support the use of this assay for evaluation of patients with concern for von Willebrand disease.

NF1 mutation-driven neuronal hyperexcitability sets a threshold for tumorigenesis and therapeutic targeting of murine optic glioma.

BACKGROUND: With the recognition that noncancerous cells function as critical regulators of brain tumor growth, we recently demonstrated that neurons drive low-grade glioma initiation and progression. Using mouse models of neurofibromatosis type 1 (NF1)-associated optic pathway glioma (OPG), we showed that Nf1 mutation induces neuronal hyperexcitability and midkine expression, which activates an immune axis to support tumor growth, such that high-dose lamotrigine treatment reduces Nf1-OPG proliferation. Herein, we execute a series of complementary experiments to address several key knowledge gaps relevant to future clinical translation. METHODS: We leverage a collection of Nf1-mutant mice that spontaneously develop OPGs to alter both germline and retinal neuron-specific midkine expression. Nf1-mutant mice harboring several different NF1 patient-derived germline mutations were employed to evaluate neuronal excitability and midkine expression. Two distinct Nf1-OPG preclinical mouse models were used to assess lamotrigine effects on tumor progression and growth in vivo. RESULTS: We establish that neuronal midkine is both necessary and sufficient for Nf1-OPG growth, demonstrating an obligate relationship between germline Nf1 mutation, neuronal excitability, midkine production, and Nf1-OPG proliferation. We show anti-epileptic drug (lamotrigine) specificity in suppressing neuronal midkine production. Relevant to clinical translation, lamotrigine prevents Nf1-OPG progression and suppresses the growth of existing tumors for months following drug cessation. Importantly, lamotrigine abrogates tumor growth in two Nf1-OPG strains using pediatric epilepsy clinical dosing. CONCLUSIONS: Together, these findings establish midkine and neuronal hyperexcitability as targetable drivers of Nf1-OPG growth and support the use of lamotrigine as a potential chemoprevention or chemotherapy agent for children with NF1-OPG.

Clinical implications of inaccurate potassium determination in hemolyzed pediatric blood specimens.

BACKGROUND: Analysis of whole blood specimens is rapid and saves blood, but hemolysis may go undetected and compromise the accuracy of potassium measurement. We aimed to define the frequency and magnitude of error in whole blood potassium measurement. METHODS: 34 months of whole blood and plasma potassium data were extracted from patients aged less than 2 years at the time of sample acquisition. Hemolysis was detected using the plasma "H index." The magnitude of potassium bias was estimated from the difference between paired whole blood and plasma measurement separated by less than 2 h. RESULTS: 56,000 of the 105,000 data points were from plasma and 20 % of these had significant hemolysis. Rates of hemolysis (nearing 50 %) were greatest in the neonatal nursery. Of 662 proximal whole blood and plasma paired results, 8 % had elevated whole blood potassium with a normal plasma value and 4 % had a normal whole blood potassium with reduced plasma potassium. The bias between whole blood and plasma potassium ranged from -1.0 to 4.0 mmol/L. CONCLUSIONS: The use of whole blood analysis brings with it significant risk for error in potassium measurement. Better tools to detect hemolysis in these types of specimens are indicated.

High-sensitivity neonatal urine drug testing has similar positivity rates to meconium for detecting in utero exposure to methamphetamine and cocaine.

Current guidelines recommend universal screening for substance use disorders in obstetric patients, and neonatal drug testing is also frequently performed. Meconium is often the preferred specimen type to detect neonatal drug exposure due to a longer window of detection compared to urine, but most laboratories send out meconium testing to specialized reference laboratories, which can delay results for several days or more. Here, we evaluate a rapid and definitive liquid chromatography-tandem mass spectrometry method for neonatal urine drug testing and compare results obtained using this method to paired meconium drug testing in 1,424 neonates for amphetamines, cocaine, cannabinoids, opiates, oxycodone and phencyclidine. Urine testing showed equivalent sensitivity to current meconium methods for detecting in utero exposure to amphetamines and cocaine.

Biochemical characterization of patients with dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD; E3) oxidizes lipoic acid. Restoring the oxidized state allows lipoic acid to act as a necessary electron sink for the four mitochondrial keto-acid dehydrogenases: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain α-keto-acid dehydrogenase, and 2-oxoadipate dehydrogenase. DLD deficiency (DLDD) is caused by biallelic pathogenic variants in DLD. Three major forms have been described: encephalopathic, hepatic, and myopathic, although DLDD patients exhibit overlapping phenotypes. Hyperlactatemia, hyperexcretion of tricarboxylic acid cycle (TCA) metabolites and branched-chain keto acids, increased plasma branched-chain amino acids and allo-isoleucine are intermittent metabolic abnormalities reported in patients with DLDD. However, the diagnostic performance of these metabolites has never been studied. Therefore, we sought to systematically evaluate the diagnostic utility of these biomarkers for DLDD. We retrospectively analyzed the results of biochemical testing of six unrelated DLDD patients, including values obtained during both well visits and acute decompensation episodes. Elevation of branched-chain amino acid concentrations was not consistently observed. We found that five of six patients in our cohort had a maximum lifetime value of allo-isoleucine of 6 µmol/L, showing that alloisoleucine elevations even during illness may be subtle. Urine organic acid analysis (UOA) during acute decompensation episodes was abnormal in all cases; however, the pattern of abnormalities had high intersubject variability. No single biomarker was universally present, even in patients experiencing metabolic decompensation. We also observed novel biochemical associations: three patients had hyperexcretion of TCA cycle metabolites during crisis; in two patients, 2-ketoadipic and 2-hydroxyadipic acids, by products of lysine degradation, were detected. We propose that these result from 2-oxoadipate dehydrogenase deficiency, an underappreciated biochemical abnormality in DLD. Given the diversity of biochemical profiles among the patients with DLDD, we conclude that accurate biochemical diagnosis relies on a high index of suspicion and multipronged biochemical analysis, including both plasma amino acid and urine organic acid quantitation during decompensation. Biochemical diagnosis during the well state is challenging. We emphasize the critical importance of multiple simultaneous biochemical tests for diagnosis and monitoring of DLDD. We also highlight the under-recognized role of DLD in the lysine degradation pathway. Larger cohorts of patients are needed to establish a correlation between the biochemical pattern and clinical outcomes, as well as a genotype-phenotype correlation.

Reexamining the Minimum Sweat Rate Requirement for Sweat Chloride Testing.

BACKGROUND: Guidelines for sweat chloride testing endorse a minimum sweat rate for reporting results. Bilateral sweat collection is recommended, but if both sites fail to meet the minimum rate (quantity not sufficient, QNS), the test should be repeated. In this study, we examine the correlation between sweat rate and sweat chloride concentration ([Cl-]), assess the accuracy of specimens collected at suboptimal rates, and investigate the use of pooled bilateral specimens for chloride measurement. METHODS: Pearson correlation was employed to analyze the relationship between sweat rate and chloride concentration, [Cl-], in 674 macroduct collections. Weighted kappa was evaluated to determine cystic fibrosis (CF) diagnostic classification concordance for 18 tests with paired arms above vs below the minimum sweat rate. Deming regression was applied to compare [Cl-] from pooled bilateral specimens vs neat specimens in 27 collections with residual volume available after clinical testing. RESULTS: Pearson correlation of sweat rate vs [Cl-] was minimal (r = -0.0735) across specimens with varying rates and [Cl-]. There was substantial agreement in CF diagnostic classification between arms for bilateral collections with discordant sweat rates. Regression analysis of [Cl-] in pooled vs nonpooled specimens revealed a slope of 0.984 and an intercept of 0.796. CONCLUSIONS: Negligible correlation of sweat rate and [Cl-] suggests the minimum sweat rate for macroduct collectors may be overly stringent. Reporting of [Cl-] in specimens with ≥10 µL (rate ≥0.3 µL/min) may reduce QNS rates without compromising diagnostic accuracy. Preliminary data suggests pooling of bilateral collections may be a feasible option to achieve the required volume for testing.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Raising the Dead Volume: Analysis of Microsamples Diluted and Corrected with Near Infrared Tracer.

BACKGROUND: Sample processing robotics require large liquid volumes to operate efficiently. Robotics are impractical in settings that deal in small specimen volumes such as pediatric laboratories. Short of manual sample handling, remedies for the current state include a redesign of current hardware or specialized adaptation for submilliliter specimens. METHODS: We blindly increased the volume of plasma specimens with diluent containing a near infrared dye, IR820, to assess the change to the original specimen volume. Diluted specimens were analyzed using a variety of assay formats/wavelengths (sodium, calcium, alanine aminotransferase, creatine kinase, cholesterol, HDL cholesterol, triglyceride, glucose, total protein, creatinine), and results were compared to neat specimens. Recovery of analyte in the diluted specimens vs neat was the primary outcome measure. RESULTS: Mean analytic recovery from the diluted specimens across all assays ranged from 93% to 110% after correction using IR820 absorbance. Absorbance correction compared favorably to mathematical correction using known volumes of specimens and diluents (93%-107%). Pooled mean analytic imprecision across all assays ranged from 2% using the neat specimen pool to 8% when plasma pool was diluted to 30% of its original concentration. No interference from dye addition was noted, indicating the diluent was broadly applicable and chemically inert. The greatest variability in recovery was observed when respective analyte concentrations were present near the lower limits of assay detectability. CONCLUSIONS: Addition of a chemically inert diluent containing a near-infrared tracer is a feasible way to raise specimen dead volume and potentially automate processing and measurement of clinical analytes in microsamples.

Comprehensive Determination of Amino Acids for Diagnosis of Inborn Errors of Metabolism.

Analysis of clinically relevant amino acids using ion exchange chromatography coupled to photometric/fluorescent detection has been an indispensable component in the detection of inborn errors of metabolism for six decades. Detection of amino acids using mass spectrometry offers advantages in speed and analytic specificity. Employing methanol extraction and controlled butylation, C8 reversed-phase chromatography, and MS/MS detection, 32 amino acids are quantified in 20 min with clinically appropriate imprecision in plasma, urine, and cerebrospinal fluid (CSF). Quantitation is linear to 2500 µM, and limits of detection are at least 1.0 µM. Important isobaric amino acids are distinguished by chromatography or by unique patterns of fragmentation following collision-induced dissociation (CID). The technique employs commercially available reagents and may be expanded and customized for specific clinical or research settings.

Comparison between BNP and NT-proBNP in pediatric populations.

BACKGROUND: B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are essential biomarkers for the evaluation of cardiac pathologies. However, pediatric reference intervals for BNP and NT-proBNP are not well defined and concordance between them in the evaluation of pediatric patients has been poorly described. METHODS: Paired BNP and NT-proBNP testing was performed on 311 specimens representing 175 pediatric patients. Pediatric BNP and NT-proBNP reference intervals derived from the literature were used to evaluate concordance of results based on age group and cardiac pathology. RESULTS: Deming regression analysis of BNP and NT-proBNP results revealed a slope of 13.63 (95% CI, 10.35-16.92) and y-intercept of -977.8 (-2063-107.2) with a positive Spearman correlation (r = 0.91). By age group, concordance kappa between BNP and NT-proBNP was 1.0 for 0-10 days, 0.23 (0-0.62) for 11-30 days, 0.82 (0.67-0.97) for 31 days-1 year, 0.81 (0.57-1.0) for 1-2 years and 0.73 (0.64-0.86) for 2-18 years. The ratio of NT-proBNP to BNP was lowest in heart transplant patients (ratio, 6.5 [95% CI, 5.1-8.1]) relative to those with heart disease (10.5 [8.8-13.7]) and pulmonary hypertension (14.2 [11.3-16.0]) but no differences in concordance were observed. For serial specimens, 21% displayed inverse, discordant changes in BNP and NT-proBNP results. Review of discordant serial results revealed that kinetics of changes was comparable and unlikely to be clinically significant. CONCLUSIONS: There is positive correlation and moderate concordance between BNP and NT-proBNP in the pediatric population studied.

Current State of Pediatric Reference Intervals and the Importance of Correctly Describing the Biochemistry of Child Development: A Review.

Importance: Appropriately established pediatric reference intervals are critical to the clinical decision-making process and should reflect the physiologic changes that occur during healthy child development. Reference intervals used in pediatric care today remain highly inconsistent across a broad range of common clinical biomarkers. Observations: This narrative review assesses biomarker-specific pediatric reference intervals and their clinical utility with respect to the underlying biological changes occurring during development. Pediatric reference intervals from PubMed-indexed articles published from January 2015 to April 2021, commercial laboratory websites, study cohorts, and pediatric reference interval books were all examined. Although large numbers of pediatric reference intervals are published for some biomarkers, very few are used by clinical and commercial laboratories. The patterns, extent, and timing of biomarker changes are highly variable, particularly during developmental stages with rapid physiologic changes. However, many pediatric reference intervals do not capture these changes and thus do not accurately reflect the underlying biochemistry of development, resulting in significant inconsistencies between reference intervals. Conclusions and Relevance: There is a need to correctly describe the biochemistry of child development as well as to identify strategies to develop accurate and consistent pediatric reference intervals for improved pediatric care.

Regulation of PGC1α Downstream of the Insulin Signaling Pathway Plays a Role in the Hepatic Proteotoxicity of Mutant α1-Antitrypsin Deficiency Variant Z.

BACKGROUND & AIMS: Insulin signaling is known to regulate essential proteostasis mechanisms. METHODS: The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. RESULTS: We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator-activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. CONCLUSIONS: These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies.

Development and Implementation of One-Step, Broad-Spectrum, High-Sensitivity Drug Screening by Tandem Mass Spectrometry in a Pediatric Population.

BACKGROUND: Drug screening by immunoassay is common in pediatric populations. However, false-positive and -negative results due to antibody cross-reactivity and dilute urine are frequent and underappreciated. Accurate ascertainment of drug exposure in children has significant clinical and medico-legal consequences. DESIGN AND METHODS: We developed and characterized an LC-MS/MS drug screening assay to supplant immunoassay and detect 38 compounds at the lowest concentrations distinguishable from analytic noise. Once implemented, we conducted a retrospective analysis of 3985 pediatric urine drug screens performed a year before (n = 1663) and after (n = 2322) implementation to examine the frequency and breadth of drug detection in our pediatric population. RESULTS: Using immunoassay, 23% (293/1269) of samples from the general pediatric and 37% (147/394) of nursery populations had presumptively positive results. Of the presumptive positive compounds, 85% (288/338) from the general pediatric population and 40% (65/162) from the nursery cohort were confirmed by mass spectrometry. After LC-MS/MS implementation, 31% (628/2052) of general pediatric, and 18% (48/270) of the nursery samples were positive for 1 or more compounds. In the nursery population, immunoassays over-detected the presence of THC but under-detected exposure to cocaine. CONCLUSION: A broadly targeted, analytically sensitive LC-MS/MS drug screening assay detects a larger number and variety of compounds in a single step compared to a screen-then-confirm approach initiated by immunoassay in our pediatric population. Rapid delivery of accurate results enables timely, appropriate disposition of patients in a variety of settings including the emergency department and labor/delivery.

GATA6 modulates the ductular reaction to bile duct ligation.

BACKGROUND: GATA6, a transcription factor expressed in cholangiocytes, has been implicated in the response to liver injury. In biliary atresia, a disease characterized by extrahepatic bile duct obstruction, liver expression of GATA6 increases with pathological bile duct expansion and decreases after successful Kasai portoenterostomy. The aim of this study was to garner genetic evidence that GATA6 is involved in ductular formation/expansion. METHODS: The murine Gata6 gene was conditionally deleted using Alb-cre, a transgene expressed in hepatoblasts (the precursors of hepatocytes and cholangiocytes) and mature hepatocytes. Bile duct ligation (BDL) was used to model biliary obstruction. RESULTS: Alb-Cre;Gata6flox/flox mice were viable and fertile. Cre-mediated recombination of Gata6 in hepatocytes had little impact on cellular structure or function. GATA6 immunoreactivity was retained in a majority of biliary epithelial cells in adult Alb-Cre;Gata6flox/flox mice, implying that surviving cholangiocytes were derived from hepatoblasts that had escaped biallelic Cre-mediated recombination. Although GATA6 immunoreactivity was preserved in cholangiocytes, Alb-cre;Gata6flox/flox mice had a demonstrable biliary phenotype. A neutrophil-rich infiltrate surrounded newly formed bile ducts in neonatal Alb-Cre;Gata6flox/flox mice. Foci of fibrosis/necrosis, presumed to reflect patchy defects in bile duct formation, were observed in the livers of 37% of adult Alb-cre;Gata6flox/flox mice and 0% of controls (p < 0.05). Most notably, Alb-cre;Gata6flox/flox mice had an altered response to BDL manifest as reduced survival, impaired bile ductule proliferation, increased parenchymal necrosis, reduced fibrosis, and enhanced macrophage accumulation in the portal space. CONCLUSIONS: GATA6 orchestrates intrahepatic biliary remodeling and mitigates liver injury following extrahepatic bile duct obstruction.