Predictors of survival following liver transplantation for pediatric hepatoblastoma and hepatocellular carcinoma: Experience from the Society of Pediatric Liver Transplantation (SPLIT).

Management of unresectable pediatric hepatoblastoma (HB) and hepatocellular carcinoma (HCC) remains challenging. The Society of Pediatric Liver Transplantation (SPLIT) database was used to study survival predictors in pediatric liver transplantation (LT) for HB and HCC. Event-free survival (EFS), associated risk factors, and postoperative complications were studied in children requiring LT for HB/HCC at 16 SPLIT centers. Three-year EFS was 81% for HB (n = 157) and 62% for HCC (n = 18) transplants. Of HB transplants, 6.9% were PRETEXT II and 15.3% were POST-TEXT I/II. Tumor extent did not impact survival (p = NS). Salvage (n = 13) and primary HB transplants had similar 3-year EFS (62% versus 78%, p = NS). Among HCC transplants, 3-year EFS was poorer in older patients (38% in ≥8-year-olds vs 86% <8-year-olds) and those with larger tumors (48% for those beyond versus 83% within Milan criteria, p = NS). Risk of infection (HR 1.5, 95% CI 1.1-2.2, p = .02) and renal injury (HR 2.4, 95% CI 1.7-3.3, p < .001) were higher in malignant versus nonmalignant LT. Survival is favorable for pediatric HB and HCC LT, including outcomes after salvage transplant. Unexpected numbers of LTs occurred in PRE/POST-TEXT I/II tumors. Judicious patient selection is critical to distinguish tumors that are potentially resectable; simultaneously, we must advocate for patients with unresectable malignancies to receive organs.

Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.

Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared with a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarities. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability to perform fast analyses without sacrificing sensitivity or metabolic information suggests that this CZE-MS method is ideal for metabolomics-inclusive, high-throughput PK profiling.

Preliminary Evaluation of a Novel Point of Care Diagnostic Device for Sports-Related Concussion.

OBJECTIVE: Visinin-like protein 1 (VILIP-1) is a neuron-specific calcium sensor protein rapidly released into blood after mild traumatic brain injury (mTBI) and may be a suitable biomarker for identification of sports-related concussion (SRC). The objective of the study is to test if quantification of a specific post-translationally modified (ubiquitinated) form of VILIP-1 (ubVILIP-1) from a fingerstick blood sample using a point of care (POC) lateral flow device (LFD) can be used to rapidly identify athletes with SRC. DESIGN: Prospective cohort study. SETTING: Side-line blood collection at football, soccer, and volleyball games/practices. PARTICIPANTS: Division I athletes with/without SRC. MAIN OUTCOME MEASURES: Blood ubVILIP-1 concentrations. RESULTS: Data collected over 2 athletic seasons from non-SRC athletes (controls) show a small but statistically significant elevation of ubVILIP-1 over an individual season for male athletes (P = 0.02) dependent on sport (P = 0.014) and no significant changes in ubVILIP-1 levels between seasons. For SRC athletes, the data show ubVILIP-1 levels substantially increase above baseline as soon as 30 minutes postdiagnosis with peak concentrations and times postinjury that vary based on injury severity. CONCLUSION: Results of the study suggest quantification of blood ubVILIP-1 levels measured using an LFD may provide an objective identification of athletes with SRC, setting the stage for further study with a larger number of SRC patients.

Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood.

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

Outcomes of Severe Seronegative Hepatitis-associated Aplastic Anemia: A Pediatric Case Series.

OBJECTIVES: Hepatitis-associated aplastic anemia (HAAA) is a potentially life-threatening diagnosis without clear treatment guidelines. The goal of the study was to characterize the presentation, evaluation, histopathology, and outcomes of therapy in children with HAAA to guide future research and to develop standardized care guidelines for this rare disease. METHODS: Retrospective chart review of 4 patients with HAAA who presented to Children's Hospital Colorado between 2016 and 2019 was conducted. Patient presentation, evaluation, bone marrow and liver pathology, interventions, and clinical course were collected. Immunohistochemistry of liver biopsies was performed. RESULTS: We treated 4 patients with HAAA without liver failure. All had evidence of systemic hyperinflammation and CD8+ T cell predominant liver tissue infiltration. One had a genetic mutation predisposing him to immune-mediated disease, but all other genetic testing was negative. In 3 of the 4 patients, hepatitis was poorly responsive to standard therapy with steroids, azathioprine, or tacrolimus; however, sustained biochemical remission of hepatitis was induced after more aggressive immunosuppressive therapies including Anti-Thymocyte Globulin (ATG) at standard immunosuppressive therapy (IST) dosing for severe Aplastic Anemia (sAA). Two patients underwent hematopoietic stem cell transplant (HSCT); 1 as first line therapy and 1 for refractory sAA. CONCLUSIONS: We found that ATG-based IST induced remission of hepatitis in patients with steroid-refractory HAAA. This is also an appropriate initial treatment for severe Aplastic Anemia, though may not prevent the need for HSCT. We propose that equine ATG based IST at standard dosing regimen for sAA is a therapy that in select cases can be considered early on in the treatment course and could lead to a sustained remission of both hepatitis and sAA. This should be considered in collaboration with a pediatric hematologist.

Mutations in the accessory subunit NDUFB10 result in isolated complex I deficiency and illustrate the critical role of intermembrane space import for complex I holoenzyme assembly.

An infant presented with fatal infantile lactic acidosis and cardiomyopathy, and was found to have profoundly decreased activity of respiratory chain complex I in muscle, heart and liver. Exome sequencing revealed compound heterozygous mutations in NDUFB10, which encodes an accessory subunit located within the PD part of complex I. One mutation resulted in a premature stop codon and absent protein, while the second mutation replaced the highly conserved cysteine 107 with a serine residue. Protein expression of NDUFB10 was decreased in muscle and heart, and less so in the liver and fibroblasts, resulting in the perturbed assembly of the holoenzyme at the 830 kDa stage. NDUFB10 was identified together with three other complex I subunits as a substrate of the intermembrane space oxidoreductase CHCHD4 (also known as Mia40). We found that during its mitochondrial import and maturation NDUFB10 transiently interacts with CHCHD4 and acquires disulfide bonds. The mutation of cysteine residue 107 in NDUFB10 impaired oxidation and efficient mitochondrial accumulation of the protein and resulted in degradation of non-imported precursors. Our findings indicate that mutations in NDUFB10 are a novel cause of complex I deficiency associated with a late stage assembly defect and emphasize the role of intermembrane space proteins for the efficient assembly of complex I.

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

A novel method for the rapid detection of post-translationally modified visinin-like protein 1 in rat models of brain injury.

BACKGROUND: Although elevated serum levels of visinin-like protein 1 (VILIP-1), a neuron-specific calcium sensor protein, are associated with ischaemic stroke, only a single study has evaluated VILIP-1 as a biomarker of traumatic brain injury (TBI). The current proof-of-concept study was designed to determine whether serum VILIP-1 levels increase post-injury in a well-characterized rat unilateral cortical contusion model. METHODS: Lateral flow devices (LFDs) rapidly (< 20 min) detected trace serum levels (pg/mL) of VILIP-1 in a small input sample volume (10 µL). Temporal profiles of serum levels at baseline and post-injury were measured in male Sprague Dawley rats subjected to very mild-, mild unilateral-cortical contusion, or naïve surgery and in male Sprague Dawley rats following a diffuse TBI or sham surgery. RESULTS: Mean serum levels were significantly elevated by 0.5 h post-injury and remained so throughout the temporal profile compared with baseline in very mild and mild unilateral contusions but not in naïve surgeries. Serum levels were also elevated in a small cohort of animals subjected to a diffuse TBI injury. CONCLUSIONS: Overall, the current study demonstrates that the novel LFD is a reliable and rapid point-of-care diagnostic for the detection and quantification of serum levels of UB-VILIP-1 in a clinically relevant time frame.

Multiregional analysis of global 5-methylcytosine and 5-hydroxymethylcytosine throughout the progression of Alzheimer's disease.

Epigenetic modifications to cytosine are known to alter transcriptional states and deregulate gene expression in cancer, embryonic development, and most recently in neurodegeneration. To test the hypothesis that global levels of cytosine modification are altered throughout the progression of Alzheimer's disease, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were quantified using gas chromatography/mass spectrometry (GC/MS) and stable labeled internal standards of cytosine, 5-mC, and 5-hmC. Cytosine modifications were quantified in DNA extracted from tissue specimens of four brain regions (cerebellum, inferior parietal lobe, superior and middle temporal gyrus, and hippocampus/parahippocampal gyrus) of cognitively normal control (NC) subjects and subjects with mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), late onset Alzheimer's disease, frontotemporal lobar degeneration (FTLD) and dementia with Lewy bodies (DLB). Repeated measures analyses of the data show significant alterations in 5-mC and 5-hmC in early stages of Alzheimer's disease (PCAD and MCI), as well as FTLD and DLB subjects, across multiple regions of the brain. These data suggest alterations in epigenetic regulation of genes may play an early role in the progression of AD as well as other types of neurodegeneration.

Compound heterozygosity for a frame shift mutation and a likely pathogenic sequence variant in the planar cell polarity­ciliogenesis gene WDPCP in a girl with polysyndactyly, coarctation of the aorta, and tongue hamartomas.

We report on a young girl with polysyndactyly, coarctation of the aorta, and tongue hamartomas. These features are similar to those reported in individuals with variant forms of orofaciodigital syndrome known as congenital heart defects, hamartomas of the tongue and polysyndactly (CHDHTP: OMIM 217085) [Örstavik et al., 1992] and orocardiodigital syndrome [Digilio et al., 1996]. Whole exome sequencing revealed that she is a compound heterozygote for a frame shift mutation and a likely pathogenic sequence variant in WDPCP, a gene that regulates planar cell polarity and ciliogenesis. Results of genotyping in her parents and unaffected siblings were consistent with autosomal recessive inheritance of the mutation and the WDPCP variant. These results suggest that disruption of planar cell polarity and ciliogenesis may result in this unusual form of orofaciodigital syndrome.

Biomarkers of lipid peroxidation in Alzheimer disease (AD): an update.

Increasing evidence suggests that free radical-mediated oxidation of biological substrates is a key feature of Alzheimer's disease (AD) pathogenesis. While it has long been established that biomarkers of lipid peroxidation (LPO) are elevated in AD brain as well as ventricular CSF postmortem, more recent studies have demonstrated increased LPO biomarkers in postmortem brain from subjects with mild cognitive impairment, the earliest clinically detectable phase of dementia and preclinical AD, the earliest detectable pathological phase. Furthermore, multiple LPO biomarkers are elevated in readily accessible biological fluids throughout disease progression. Collectively, these studies demonstrate that LPO is an early feature during disease progression and may be considered a key pathway for targeted therapeutics as well as an enhancer of diagnostic accuracy for early detection of subjects during the prodromal phase.

Cavernous Hemangiomas of the Pediatric Calvaria.

This series reports 2 pediatric cases of calvarial cavernous hemangioma (cavernoma, cavernous malformation) treated surgically at Children's Hospital Colorado between 2008 and 2010. Both cases presented as painless bony masses which enlarged over time. Both patients underwent surgical resection without complication and have remained recurrence free since surgery. Because so few cases have been reported among pediatric populations, little is known regarding the epidemiology and prognosis of calvarial cavernous hemangiomas in children. These cases represent interesting additions to the small body of literature on these rare tumors.

Nucleic acid oxidation: an early feature of Alzheimer's disease.

Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age-matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late-stage AD, and non-AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ≤ 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general. Levels of oxidized nucleic acids in nDNA and mtDNA were found to be significantly elevated in mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), late-stage AD (LAD), and a pooled diseased control group (DC) of frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB) subjects compared to normal control (NC) subjects. Nucleic acid oxidation peaked early in disease progression and remained elevated. The study suggests nucleic acid oxidation is a general event in neurodegeneration.

Cervical bronchogenic cysts: case report and review of the literature.

Pediatric cervical masses can present a diagnostic dilemma given their broad differential diagnosis. We present a 3-year-old girl with a midline anterior neck mass found to have histopathologic findings consistent with a bronchogenic cyst. Although rare, bronchogenic cysts should be considered in the differential diagnosis in both lateral and anterior pediatric cervical masses as their pathophysiology and embryogenesis differ considerably from more common cervical masses. Imaging is an important aspect in the pre-operative work-up, although diagnosis is only made after histopathologic analysis. Complete surgical excision is the definitive treatment.

Protein biomarkers GDF15 and FGF21 to differentiate mitochondrial hepatopathies from other pediatric liver diseases.

BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.

Toll-like receptor 4-dependent Kupffer cell activation and liver injury in a novel mouse model of parenteral nutrition and intestinal injury.

UNLABELLED: Infants with intestinal failure who are parenteral nutrition (PN)-dependent may develop cholestatic liver injury and cirrhosis (PN-associated liver injury: PNALI). The pathogenesis of PNALI remains incompletely understood. We hypothesized that intestinal injury with increased intestinal permeability combined with administration of PN promotes lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR4) signaling dependent Kupffer cell (KC) activation as an early event in the pathogenesis of PNALI. We developed a mouse model in which intestinal injury and increased permeability were induced by oral treatment for 4 days with dextran sulphate sodium (DSS) followed by continuous infusion of soy lipid-based PN solution through a central venous catheter for 7 (PN7d/DSS) and 28 (PN28d/DSS) days. Purified KCs were probed for transcription of proinflammatory cytokines. PN7d/DSS mice showed increased intestinal permeability and elevated portal vein LPS levels, evidence of hepatocyte injury and cholestasis (serum aspartate aminotransferase, alanine aminotransferase, bile acids, total bilirubin), and increased KC expression of interleukin-6 (Il6), tumor necrosis factor α (Tnfα), and transforming growth factor ß (Tgfß). Markers of liver injury remained elevated in PN28d/DSS mice associated with lobular inflammation, hepatocyte apoptosis, peliosis, and KC hypertrophy and hyperplasia. PN infusion without DSS pretreatment or DSS pretreatment alone did not result in liver injury or KC activation, even though portal vein LPS levels were elevated. Suppression of the intestinal microbiota with broad spectrum antibiotics or ablation of TLR4 signaling in Tlr4 mutant mice resulted in significantly reduced KC activation and markedly attenuated liver injury in PN7d/DSS mice. CONCLUSION: These data suggest that intestinal-derived LPS activates KC through TLR4 signaling in early stages of PNALI.

Rosai-Dorfman-Destombes Disease in the Pediatric Head and Neck.

BACKGROUND: Rosai-Dorfman-Destombes disease (RDD), or sinus histiocytosis with massive lymphadenopathy, is a rare form of non-Langerhans cells histiocytosis. It has a wide-ranging variability in presentation since first described in 1969 but much of its characteristics in children remain unknown. METHODS: A retrospective chart review of children diagnosed with RDD at a tertiary care children's hospital was conducted from 2000 to 2021. RESULTS: Twelve RDD patients were identified, with an average age of 7 years (SD 4.3). Males comprised 58% of the cohort, and African American ethnicity was most common (42%). Nodal RDD was found in 7 patients (58%). Nine patients (75%) presented RDD within the head and neck, 6 of whom had nodal RDD. The most common presentation was cervical lymphadenopathy, which most often involved levels V (67%), II (56%), III (44%), and I (11%), in order of frequency. Recurrence and persistence of disease after initial treatment was common, with 5 (42%) being disease free at the time of the last follow up. Fifty-eight percent (7/12) developed recurrence or had persistent disease and 4 required adjuvant systemic treatment with corticosteroids and/or chemotherapy. One patient succumbed after developing treatment related acute myelodysplastic leukemia (t-AML) from chemotherapy used to treat recurrent RDD. CONCLUSION: Pediatric RDD presents at a young age and most commonly involving cervical lymphadenopathy. Ongoing surveillance in the setting of persistence or recurrence without clearly defined prognostic risk factors is important.

Why so low? An unusual case of myositis in a child.

BACKGROUND: Sarcoidosis is characterized by non-caseating epithelioid granulomas in various tissues throughout the body, most commonly the lung. Non-caseating granulomas may be seen in skeletal muscle, though typically asymptomatic and under-recognized. While rare in children, there is a need to better characterize the disease and its management. Here we present a 12-year-old female with bilateral calf pain who was ultimately found to have sarcoid myositis. CASE PRESENTATION: A 12-year-old female presented to rheumatology with significantly elevated inflammatory markers and isolated lower leg pain. MRI of the distal lower extremities demonstrated extensive bilateral myositis with active inflammation, atrophy, and to a lesser extent fasciitis. This distribution of myositis in a child garnered a broad differential requiring a systematic evaluation. Ultimately, muscle biopsy revealed non-caseating granulomatous myositis with perivascular inflammation, extensive muscle fibrosis, and fatty replacement of the muscle with a CD4+ T cell predominant, lymphohistiocytic infiltrate consistent with sarcoidosis. Review of histopathology from age 6 of an extraconal mass resected from her right superior rectus muscle further confirmed the diagnosis. She had no other clinical symptoms or findings of sarcoidosis. The patient improved significantly with methotrexate and prednisone, though flared again after self-discontinuation of medications and was subsequently lost to follow-up. CONCLUSION: This is the second reported case of granulomatous myositis associated with sarcoidosis in a pediatric patient, and the first to present with a chief complaint of leg pain. Increased knowledge of pediatric sarcoid myositis within the medical community will enhance recognition of the disease, improve the evaluation of lower leg myositis, and advance outcomes for this vulnerable population.

Glycerol as a precursor for hepatic de novo glutathione synthesis in human liver.

BACKGROUND: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis. METHODS: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U-13C3]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2-0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy. RESULTS: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14-19]; BMI 47.4 kg/m2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U-13C3]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [13C2]glycine or [13C2]glutamate derived from the [U-13C3]glycerol drink were readily detected, and 13C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U-13C3]glycerol trended to be lower in obese adolescents with liver pathology. CONCLUSIONS: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver.