Immunonutrition with Omega-3 Fatty Acid Supplementation in Severe TBI: Retrospective Analysis of Patient Characteristics and Outcomes.

Early evidence-based medical interventions to improve patient outcomes after traumatic brain injury (TBI) are lacking. In patients admitted to the ICU after TBI, optimization of nutrition is an emerging field of interest. Specialized enteral nutrition (EN) formulas that include immunonutrition containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been developed and are used for their proposed anti-inflammatory and proimmune properties; however, their use has not been rigorously studied in human TBI populations. A single-center, retrospective, descriptive observational study was conducted at the LAC + USC Medical Center. Patients with severe TBI (sTBI, Glasgow Coma Scale score ≤ 8) who remained in the ICU for ≥2 weeks and received EN were identified between 2017 and 2022 using the institutional trauma registry. Those who received immunonutrition formulas containing n-3 PUFAs were compared with those who received standard, polymeric EN with regard to baseline characteristics, clinical markers of inflammation and immune function, and short-term clinical outcomes. A total of 151 patients with sTBI were analyzed. Those who received immunonutrition with n-3 PUFA supplementation were more likely to be male, younger, Hispanic/Latinx, and have polytrauma needing non-central nervous system surgery. No differences in clinical markers of inflammation or infection rate were found. In multivariate regression analysis, immunonutrition was associated with reduced hospital length of stay (LOS). ICU LOS was also reduced in the subgroup of patients with polytrauma and TBI. This study identifies important differences in patient characteristics and outcomes associated with the EN formula prescribed. Study results can directly inform a prospective pragmatic study of immunonutrition with n-3 PUFA supplementation aimed to confirm the biomechanistic and clinical benefits of the intervention.

Synthesis of site-specific Fab-drug conjugates using ADP-ribosyl cyclases.

Targeted delivery of small-molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full-length antibodies are mainly used as drug-carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small-molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP-ribosyl cyclase-enabled fragment antigen-binding (Fab) drug conjugates (ARC-FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+ )-based drug linker at its active site, CD38 genetically fused with Fab mediates robust site-specific drug conjugations via enzymatic reactions. Generated ARC-FDCs with defined drug-to-Fab ratios display potent and antigen-dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site-specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

A ribose-functionalized NAD+ with versatile activity for ADP-ribosylation.

An NAD+ featuring an adenosyl 4'-azido functions as a general substrate for poly-ADP-ribose polymerases. Its derived mono- and poly-ADP-ribosylated proteins can be adequately recognized by distinct ADP-ribosylation-specific readers. This molecule represents the first ribose-functionalized NAD+ with versatile activities across different ADP-ribosyltransferases and provides insight into developing new probes for ADP-ribosylation.

Immunonutrition with Omega-3 Fatty Acid Supplementation in Severe TBI: Retrospective Analysis of Patient Characteristics and Outcomes.

Background: Early evidence-based medical interventions to improve patient outcomes after traumatic brain injury (TBI) are lacking. In patients admitted to the ICU after TBI, optimization of nutrition is an emerging field of interest. Specialized enteral nutrition (EN) formulas that include immunonutrition containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been developed and are used for their proposed anti-inflammatory and pro-immune properties; however, their use has not been rigorously studied in human TBI populations. Methods: A single-center, retrospective, descriptive observational study was conducted at LAC + USC Medical Center. Patients with severe TBI (sTBI, Glasgow Coma Scale score ≤ 8) who remained in the ICU for ≥ 2 weeks and received EN were identified between 2017 and 2022 using the institutional trauma registry. Those who received immunonutrition formulas containing n-3 PUFAs were compared to those who received standard, polymeric EN in regard to baseline characteristics, clinical markers of inflammation and immune function, and short-term clinical outcomes. Results: A total of 151 patients with sTBI were analyzed. Those who received immunonutrition with n-3 PUFA supplementation were more likely to be male, younger, Hispanic/Latinx, and have polytrauma needing non-central nervous system surgery. No differences in clinical markers of inflammation or infection rate were found. In multivariate regression analysis, immunonutrition was associated with reduced hospital length of stay (LOS). ICU LOS was also reduced in the subgroup of patients with polytrauma and TBI. Conclusion: This study identifies important differences in patient characteristics and outcomes associated with the EN formula prescribed. Study results can directly inform a prospective pragmatic study of immunonutrition with n-3 PUFA supplementation aimed to confirm the biomechanistic and clinical benefits of the intervention.

Eicosanoids and other oxylipins in liver injury, inflammation and liver cancer development.

Liver cancer is a malignancy developed from underlying liver disease that encompasses liver injury and metabolic disorders. The progression from these underlying liver disease to cancer is accompanied by chronic inflammatory conditions in which liver macrophages play important roles in orchestrating the inflammatory response. During this process, bioactive lipids produced by hepatocytes and macrophages mediate the inflammatory responses by acting as pro-inflammatory factors, as well as, playing roles in the resolution of inflammation conditions. Here, we review the literature discussing the roles of bioactive lipids in acute and chronic hepatic inflammation and progression to cancer.

Eicosanoid lipidome activation in post-mortem brain tissues of individuals with APOE4 and Alzheimer's dementia.

BACKGROUND: Chronic neuroinflammation is one of the hallmarks of late-onset Alzheimer's disease (AD) dementia pathogenesis. Carrying the apolipoprotein ε4 (APOE4) allele has been associated with an accentuated response to brain inflammation and increases the risk of AD dementia progression. Among inflammation signaling pathways, aberrant eicosanoid activation plays a prominent role in neurodegeneration. METHODS: Using brains from the Religious Order Study (ROS), this study compared measures of brain eicosanoid lipidome in older persons with AD dementia to age-matched controls with no cognitive impairment (NCI), stratified by APOE genotype. RESULTS: Lipidomic analysis of the dorsolateral prefrontal cortex demonstrated lower levels of omega-3 fatty acids eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and DHA-derived neuroprotectin D1 (NPD-1) in persons with AD dementia, all of which associated with lower measures of cognitive function. A significant interaction was observed between carrying the APOE4 allele and higher levels of both pro-inflammatory lipids and pro-resolving eicosanoid lipids on measures of cognitive performance and on neuritic plaque burden. Furthermore, analysis of lipid metabolism pathways implicated activation of calcium-dependent phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and soluble epoxide hydrolase (sEH) enzymes. CONCLUSION: These findings implicate activation of the eicosanoid lipidome in the chronic unresolved state of inflammation in AD dementia, which is increased in carriers of the APOE4 allele, and identify potential therapeutic targets for resolving this chronic inflammatory state.

Supra-lacrimal protein-based carriers for cyclosporine A reduce Th17-mediated autoimmunity in murine model of Sjögren's syndrome.

Sjögren's syndrome (SS) is a multifactorial autoimmune disease with principal symptoms including inflammation and loss of function of lacrimal glands (LG) and salivary glands. While glandular infiltrates includes both B- and T-cells, CD4+ T cells are strongly implicated. Utilizing the male non-obese diabetic (NOD) mouse model of SS, this work: 1) identifies clinically-relevant elevations in cytokines (IL-17A, IL-2) in LG-derived CD4+ T cells; and 2) explores tissue-specific immunosuppression of SS using a novel protein-based drug carrier to concentrate cyclosporine A (CsA) directly in the LG. As a potent immunosuppressant, topical ophthalmic CsA is approved for dry eye disorders; however, it cannot effectively resolve inflammation due to limited accumulation in the LG. Systemic CsA has dose-limiting side effects that also limit its ability to block LG inflammation. Using elastin-like polypeptides (ELPs) fused genetically to cyclophilin, the intracellular cognate receptor of CsA, this manuscript reports a sustained-release formulation of CsA that maintains therapeutic drug concentrations in the LG and extends intervals between doses. This formulation blocked both in vitro Th17 cell differentiation and IL-17A secretion. In vivo treatment significantly decreased the abundance of Th17.1 cells, a helper cell population sharing phenotypes of both Th17 and Th1, in the LG of diseased NOD mice. Treatment with even a single dose of the sustained-release formulation was effective enough to improve basal levels of tear production. Thus, this sustained-release formulation suppressed local LG inflammation driven through IL-17 dependent pathways, while improving ocular surface function.

Discovery of an NAD+ analogue with enhanced specificity for PARP1.

Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD+). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD+ featuring an adenosyl 3'-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoform-specific modulators.

NAP1051, a Lipoxin A4 Biomimetic Analogue, Demonstrates Antitumor Activity Against the Tumor Microenvironment.

Resolving tumor-associated inflammation in the tumor microenvironment (TME) may promote antitumor effects. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. Here, a biomimetic of LXA4, NAP1051, was shown to have LXA4-like in vitro properties and antitumor activity in colorectal cancer xenograft models. NAP1051 inhibited neutrophil chemotaxis toward fMLP and dose-dependently promoted dTHP-1 efferocytosis which was equipotent to aspirin-triggered lipoxin A4 (ATLA). In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT similar to formyl peptide receptor 2 (FPR2/ALX) agonists. In two mouse xenograft colorectal cancer models, NAP1051 significantly inhibited tumor growth when given orally at 4.8 to 5 mg/kg/day. Flow cytometric analyses showed that NAP1051 reduced splenic and intratumoral neutrophil and myeloid-derived suppressor cell populations, which correlated to the antitumor effect. In addition, NAP1051 reduced NETosis in the TME while stimulating T-cell recruitment. Overall, these results show that NAP1051 possesses key lipoxin-like properties and has antitumor activity against colorectal cancer via modulation of neutrophils and NETosis in the TME.

Site-specific antibody-drug conjugates with variable drug-to-antibody-ratios for AML therapy.

Random conjugations of chemotherapeutics to monoclonal antibodies result in heterogeneous antibody-drug conjugates (ADCs) with suboptimal pharmacological properties. We recently developed a new technology for facile generation of homogeneous ADCs by harnessing human CD38 catalytic domain and its dinucleotide-derived covalent inhibitor, termed ADP-ribosyl cyclase-enabled ADCs (ARC-ADCs). Herein we advance this technology by designing and synthesizing ARC-ADCs with customizable drug-to-antibody ratios (DARs). Through varying numbers and locations of CD38 fused to an antibody targeting human C-type lectin-like molecule-1 (hCLL-1), ARC-ADCs featuring DARs of 2 and 4 were rapidly generated via a single step with cytotoxic monomethyl auristatin F (MMAF) as payloads. In contrast to anti-hCLL-1 ARC-ADC carrying 2 drug molecules, anti-hCLL-1 ARC-ADC with a DAR of 4 shows highly potent activity in killing hCLL-1-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. This work provides novel ADC candidates for combating AML and supports ARC-ADC as a general and versatile approach for producing site-specific ADCs with defined DARs.

Intralacrimal Sustained Delivery of Rapamycin Shows Therapeutic Effects without Systemic Toxicity in a Mouse Model of Autoimmune Dacryoadenitis Characteristic of Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease associated with severe exocrinopathy, which is characterized by profound lymphocytic infiltration (dacryoadenitis) and loss of function of the tear-producing lacrimal glands (LGs). Systemic administration of Rapamycin (Rapa) significantly reduces LG inflammation in the male Nonobese Diabetic (NOD) model of SS-associated autoimmune dacryoadenitis. However, the systemic toxicity of this potent immunosuppressant limits its application. As an alternative, this paper reports an intra-LG delivery method using a depot formulation comprised of a thermoresponsive elastin-like polypeptide (ELP) and FKBP, the cognate receptor for Rapa (5FV). Depot formation was confirmed in excised whole LG using cleared tissue and observation by both laser-scanning confocal and lightsheet microscopy. The LG depot was evaluated for safety, efficacy, and intra-LG pharmacokinetics in the NOD mouse disease model. Intra-LG injection with the depot formulation (5FV) retained Rapa in the LG for a mean residence time (MRT) of 75.6 h compared to Rapa delivery complexed with a soluble carrier control (5FA), which had a MRT of 11.7 h in the LG. Compared to systemic delivery of Rapa every other day for 2 weeks (seven doses), a single intra-LG depot of Rapa representing 16-fold less total drug was sufficient to inhibit LG inflammation and improve tear production. This treatment modality further reduced markers of hyperglycemia and hyperlipidemia while showing no evidence of necrosis or fibrosis in the LG. This approach represents a potential new therapy for SS-related autoimmune dacryoadenitis, which may be adapted for local delivery at other sites of inflammation; furthermore, these findings reveal the utility of optical imaging for monitoring the disposition of locally administered therapeutics.

Synthesis of site-specific antibody-drug conjugates by ADP-ribosyl cyclases.

Most of the current antibody-drug conjugates (ADCs) in clinic are heterogeneous mixtures. To produce homogeneous ADCs, established procedures often require multiple steps or long reaction times. The introduced mutations or foreign sequences may cause high immunogenicity. Here, we explore a new concept of transforming CD38 enzymatic activity into a facile approach for generating site-specific ADCs. This was achieved through coupling bifunctional antibody-CD38 fusion proteins with designer dinucleotide-based covalent inhibitors with stably attached payloads. The resulting adenosine diphosphate-ribosyl cyclase-enabled ADC (ARC-ADC) with a drug-to-antibody ratio of 2 could be rapidly generated through single-step conjugation. The generated ARC-ADC targeting human epidermal growth factor receptor 2 (HER2) displays excellent stability and potency against HER2-positive breast cancer both in vitro and in vivo. This proof-of-concept study demonstrates a new strategy for production of site-specific ADCs. It may provide a general approach for the development of a novel class of ADCs with potentially enhanced properties.

A poly-ADP-ribose polymer-based antibody-drug conjugate.

Protein poly-ADP-ribosylation (PARylation) plays vital roles in many aspects of physiology and pathophysiology. This posttranslational modification is catalyzed by poly-ADP-ribose polymerases (PARPs) through additions of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to protein residues, forming linear or branched poly-ADP-ribose (PAR) polymers. In this study, we explored a new concept of utilizing functionalized PAR polymers for targeted drug delivery. This was achieved by rapid and efficient generation of auto-PARylated PARP1 with 3'-azido ADP-riboses and subsequent conjugations of anti-human epidermal growth factor receptor 2 (HER2) antibodies and monomethyl auristatin F (MMAF) payloads. This designed PARylated PARP1-antibody-MMAF conjugate could potently kill HER2-expressing cancer cells in high specificity. This proof-of-principle work demonstrates the feasibility of production of PAR polymer-based antibody-drug conjugate and its application in targeted delivery. The PAR polymer-based conjugates may lead to new types of therapeutics with potentially improved physicochemical and pharmacological properties.

A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation.

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3'-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD+ provides an important tool for studying cellular PARylation.

Genome-wide association study of circulating folate one-carbon metabolites.

Experimental, observational, and clinical trials support a critical role of folate one-carbon metabolism (FOCM) in colorectal cancer (CRC) development. In this report, we focus on understanding the relationship between common genetic variants and metabolites of FOCM. We conducted a genome-wide association study of FOCM biomarkers among 1,788 unaffected (without CRC) individuals of European ancestry from the Colon Cancer Family Registry. Twelve metabolites, including 5-methyltetrahydrofolate, vitamin B2 (flavin mononucleotide and riboflavin), vitamin B6 (4-pyridoxic acid, pyridoxal, and pyridoxamine), total homocysteine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, and creatinine were measured from plasma using liquid chromatography-mass spectrometry (LC-MS) or LC-MS/MS. For each individual biomarker, we estimated genotype array-specific associations followed by a fixed-effect meta-analysis. We identified the variant rs35976024 (at 2p11.2 and intronic of ATOH8) associated with total homocysteine (p = 4.9 × 10-8 ). We found a group of six highly correlated variants on chromosome 15q14 associated with cystathionine (all p < 5 × 10-8 ), with the most significant variant rs28391580 (p = 2.8 × 10-8 ). Two variants (rs139435405 and rs149119426) on chromosome 14q13 showed significant (p < 5 × 10-8 ) associations with S-adenosylhomocysteine. These three biomarkers with significant associations are closely involved in homocysteine metabolism. Furthermore, when assessing the principal components (PCs) derived from seven individual biomarkers, we identified the variant rs12665366 (at 6p25.3 and intronic of EXOC2) associated with the first PC (p = 2.3 × 10-8 ). Our data suggest that common genetic variants may play an important role in FOCM, particularly in homocysteine metabolism.

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease.

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.

Circulating angiotensin peptides levels in Acute Respiratory Distress Syndrome correlate with clinical outcomes: A pilot study.

RATIONALE: We propose renin angiotensin system (RAS) peptides are critical in wound reparative processes such as in acute respiratory distress syndrome (ARDS). Their role in predicting clinical outcomes in ARDS has been unexplored; thus, we used a targeted metabolomics approach to investigate them as potential predictors of outcomes. METHODS: Thirty-nine ARDS patients were enrolled within 24 hours of ARDS diagnosis. Plasma RAS peptide levels were quantified at study entry and 24, 48 and 72 hours using a liquid chromatography-mass spectrometry based metabolomics assay. RAS peptide concentrations were compared between survivors and non-survivors, and were correlated with clinical and pulmonary measures. MEASUREMENTS AND MAIN RESULTS: Angiotensin I (Ang-I or A(1-10)) levels were significantly higher in non-survivors at study entry and 72 hours. ARDS survival was associated with lower A(1-10) concentration (OR 0.36, 95% CI 0.18-0.72, p = 0.004) but higher A(1-9) concentration (OR 2.24, 95% CI 1.15-4.39, p = 0.018), a biologically active metabolite of A(1-10) and an agonist of angiotensin II receptor type 2. Survivors had significantly higher median A(1-9)/A(1-10) and A(1-7)/A(1-10) ratios than the non-survivors (p = 0.001). Increased A(1-9)/A(1-10) ratio suggests that angiotensin converting enzyme II (ACE2) activity is higher in patients who survived their ARDS insult while an increase in A(1-7)/A(1-10) ratio suggests that ACE activity is also higher in survivors. CONCLUSION: A(1-10) accumulation and reduced A(1-9) concentration in the non-survivor group suggest that ACE2 activities may be reduced in patients succumbing to ARDS. Plasma levels of both A(1-10) and A(1-9) and their ratio may serve as useful biomarkers for prognosis in ARDS patients.

Exploratory metabolomic study to identify blood-based biomarkers as a potential screen for colorectal cancer.

INTRODUCTION: colorectal cancer (CRC) continues to be difficult to diagnose due to the lack of reliable and predictive biomarkers. OBJECTIVE: to identify blood-based biomarkers that can be used to distinguish CRC cases from controls. METHODS: a workflow for untargeted followed by targeted metabolic profiling was conducted on the plasma samples of 26 CRC cases and ten healthy volunteers (controls) using liquid chromatography-mass spectrometry (LCMS). The data acquired in the untargeted scan was processed and analyzed using MarkerView™ software. The significantly different ions that distinguish CRC cases from the controls were identified using a mass-based human metabolome search. The result was further used to inform the targeted scan workflow. RESULTS: the untargeted scan yielded putative biomarkers some of which were related to the folate-dependent one-carbon metabolism (FOCM). Analysis of the targeted scan found the plasma levels of nine FOCM metabolites to be significantly different between cases and controls. The classification models of the cases and controls, in both the targeted and untargeted approaches, each yielded a 97.2% success rate after cross-validation. CONCLUSION: we have identified plasma metabolites with screening potential to discriminate between CRC cases and controls.

Simultaneous quantitation of folates, flavins and B6 metabolites in human plasma by LC-MS/MS assay: Applications in colorectal cancer.

An analytical method using electrospray ionization and high- performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify the vitamin B metabolites found in the folate one-carbon metabolism, using 50 µL of human plasma. Analytes in plasma were extracted using protein precipitation after being stabilized in 1% ascorbic acid. The analytes were separated using a Kinetex 2.6 µm Pentafluorophenyl (2.1 × 30 mm) column utilizing a gradient mobile phase system of 0.1% formic acid in water and 100% acetonitrile in a 13.2 min run. The MS detector run using a positive multiple reaction monitoring with parameters optimized for each analyte's ion pair. The assay was selective and linear for all analytes at defined dynamic ranges. The recoveries were generally above 80% except for the folate metabolites whose recoveries dipped possibly due to the drying process. The inter-day precision (%coefficient of variation) and accuracy (%calculated concentration of the nominal concentrations) for six replicates of all quality control samples were ≤14% and within 12.2%, respectively. The lower limit of quantification ranged from 0.2 to 3.9 nM. No significant instability was observed after repeated freezing and thawing or in processed samples. The LC-MS/MS assay was found applicable for sensitive, accurate and precise quantitation of vitamin B metabolites in plasma of healthy volunteers and colorectal cancer patients.

Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin.

Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin. In this study, it is determined whether adipocytes clear daunorubicin from the tumor microenvironment (TME). Intracellular daunorubicin concentrations were evaluated using fluorescence. Daunorubicin and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of daunorubicin-metabolizing enzymes, aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3), in human adipose tissue, were queried using public databases and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized daunorubicin to daunorubicinol, reducing its antileukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of daunorubicin to daunorubicinol in vivo Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human noncancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated daunorubicin metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. As AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases.Implications: Adipocyte absorption and metabolism of chemotherapies can reduce cytotoxicity in cancer microenvironments, potentially contributing to poorer survival outcomes. Mol Cancer Res; 15(12); 1704-13. ©2017 AACR.