The Effects of Acyl Chain Length on Antioxidant Efficacy of Mono- and Multi-Acylated Resveratrol: A Comparative Assessment.

Acylated derivatives of the dietary phenolic, resveratrol, were prepared via enzymatic and chemical transesterification modification with selected vinyl fatty acids to expand the potential application of resveratrol and its acylated derivatives in functional supplement, cosmetic/skincare, and pharmaceutical fields. The acylation was implemented using eight vinyl fatty acids with varying chain lengths (C2:0-C18:0). Eight monoesters enzymatically prepared, eight diesters and four triesters, chemically prepared, were isolated and purified and identified via MS (mass spectra) or/and NMR (nuclear magnetic resonance). The lipophilicity of resveratrol and its acylated derivatives was calculated using ALOGPS 2.1. Compared with related acylated products, resveratrol itself rendered higher antioxidant efficacy in all the antioxidant assays, namely DPPH, ABTS, FRAP, and ferrous chelation tests. Within various ester derivatives of resveratrol, short-chain fatty acid mono- and di-substituted resveratrols, especially the resveratrol monoacetate/diacetate, exhibited higher antioxidant efficacy in DPPH and ABTS assays than the rest of resveratrol derivatives, but the medium-chain monoesters of resveratrol, including caproate, caprylate, caprate, and laurate, showed a higher metal ion chelation ability compared to other acylated resveratrols. These results imply that resveratrol derivatives may be used in lipidic media as health-beneficial antioxidants.

Spectroscopic Characterization and Cytotoxicity Assessment towards Human Colon Cancer Cell Lines of Acylated Cycloartane Glycosides from Astragalus boeticus L.

In several European countries, especially in Sweden, the seeds of the species Astragalus boeticus L. were widely used as coffee substitutes during the 19th century. Nonetheless, data regarding the phytochemistry and the pharmacological properties of this species are currently extremely limited. Conversely, other species belonging to the Astragalus genus have already been extensively investigated, as they were used for millennia for treating various diseases, including cancer. The current work was addressed to characterize cycloartane glycosides from A. boeticus, and to evaluate their cytotoxicity towards human colorectal cancer (CRC) cell lines. The isolation of the metabolites was performed by using different chromatographic techniques, while their chemical structures were elucidated by nuclear magnetic resonance (NMR) (1D and 2D techniques) and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. The cytotoxic assessment was performed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in Caco-2, HT-29 and HCT-116 CRC cells. As a result, the targeted phytochemical study of A. boeticus enabled the isolation of three new cycloartane glycosides, 6-O-acetyl-3-O-(4-O-malonyl)-ß-d-xylopyranosylcycloastragenol (1), 3-O-(4-O-malonyl)-ß-d-xylopyranosylcycloastragenol (2), 6-O-acetyl-25-O-ß-d-glucopyranosyl-3-O-ß-d-xylopyranosylcycloastragenol (3) along with two known compounds, 6-O-acetyl-3-O-ß-d-xylopyranosylcycloastragenol (4) and 3-O-ß-d-xylopyranosylcycloastragenol (5). Importantly, this work demonstrated that the acetylated cycloartane glycosides 1 and 4 might preferentially inhibit cell growth in the CRC cell model resistant to epidermal growth factor receptor (EGFR) inhibitors.

Highly Sensitive, Simple, and Cost- and Time-Effective Method to Determine the Absolute Configuration of a Secondary Alcohol Using Competing Enantioselective Acylation Coupled with LC/MS.

The absolute-configuration determination of natural products and synthetic compounds with stereogenic centers is very important because stereoisomers dramatically and differentially affect many crucial properties, such as physical behaviors and biological functions. Despite several established methods for determining the absolute configuration, significant unmet needs for new methods still exist owing to the specific limitations of established methodologies. Here, we present a simple, optimized, new chemical-derivative method that utilizes competing enantioselective acylation followed by LC/MS analysis, and we demonstrate its successful application in determining the absolute configuration of a secondary alcohol in natural products with multiple reactive functional groups. This new development relies on the enantiomeric pair of homobenzotetramisole (HBTM) catalysts exhibiting adequate kinetic resolution for acylation of the secondary alcohol, and then the fast reaction was quantitatively confirmed via LC/MS as the characterization technique for the enantioselective transformations. Our new approach was successfully applied to determine the absolute configuration of one secondary alcohol in compound 1, which has other hydroxyl groups to be reacted. The identified stereocenter of 1 was verified by previously established methods including quantum chemical electronic-circular-dichroism (ECD) calculations, computational NMR-chemical-shift calculations followed by DP4+ calculations, and modified Mosher's method. In addition, our method was applied to five known naturally occurring compounds, which led to the successful verification of their absolute configurations. Our newly developed method using the HBTM catalyst provides a highly sensitive, simple, and cost- and time-effective approach and an applicable and convenient analytical method for determining the absolute configuration of one secondary alcohol in natural products.

New Perspectives on Acyl Glucuronide Risk Assessment in Drug Discovery: Investigation of In vitro Stability, In situ Reactivity, and Bioactivation.

BACKGROUND: Acyl glucuronides of xenobiotics have been a subject of wide interest from the pharmaceutical industry with respect to biochemical reactivity, hepatic disposition, and enterohepatic circulation. The reactivity and lack of stability of an acyl glucuronide for a clinical candidate could pose major developability concerns. To date, multiple in vitro assays have been published to assess the risk associated with acyl glucuronides. Despite this fact, the translation of these findings to predicting clinical safety remains poor. METHODS: In the present investigation, we aimed to provide simplified in vitro strategy to understand the bioactivation potential of acyl glucuronides of 10 commercial, carboxylic acid containing drugs that have been categorized as "safe," "warning," or "withdrawn" with respect to their marketed use. Acyl migration was measured as a function of the number of peaks observed in LC-MSn analysis. In addition, we carried out reactive intermediate trapping studies with glutathione and methoxylamine to identify the key intermediates in the transacylation bioactivation and glycation pathways, respectively. We also conducted reaction phenotyping with recombinant UDP-glucuronosyltransferase (UGT) Supersomes® to investigate if the formation of acyl glucuronides could be linked to specific UGT isoform(s). RESULTS: Our results were in line with reported values in the literature. Our assay could be used in discovery research where half-life calculation completely eliminated the need to chemically synthesize the acyl glucuronide standard for risk assessment. We captured our results for risk assessment in a flow chart to simplify the various complex in vitro techniques historically presented. CONCLUSION: While the compounds tested from "withdrawn" and "warning category" all formed the glutathione adduct in buffer, none from "safe" category formed the glutathione adduct. In contrast, none of the compounds tested from any category formed methoxylamine conjugate, a reaction with putative aldehyde moiety formed via acyl migration. These results, highly favor the nucleophilic displacement as a cause of the reactivity rather than the acyl migration via aldehyde formation. The workflow presented could also be applied in the discovery setting to triage new chemical entities of interest.

Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation.

Facile synthesis of C-terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid-phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C-terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

While simple O- (ether-linked) and N-glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates.

Acyl glucuronide metabolites: Implications for drug safety assessment.

Acyl glucuronides are important metabolites of compounds with carboxylic acid moieties and have unique properties that distinguish them from other phase 2 metabolites. In particular, in addition to being often unstable, acyl glucuronide metabolites can be chemically reactive leading to covalent binding with macromolecules and toxicity. While there is circumstantial evidence that drugs forming acyl glucuronide metabolites can be associated with rare, but severe idiosyncratic toxic reactions, many widely prescribed drugs with good safety records are also metabolized through acyl glucuronidation. Therefore, there is a need to understand the various factors that can affect the safety of acyl glucuronide-producing drugs including the rate of acyl glucuronide formation, the relative reactivity of the acyl glucuronide metabolite formed, the rate of elimination, potential proteins being targeted, and the rate of aglucuronidation. In this review, these factors are discussed and various approaches to de-risk the safety liabilities of acyl glucuronide metabolites are evaluated.

ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.

Rapid Vortex Fluidics: Continuous Flow Synthesis of Amides and Local Anesthetic Lidocaine.

Thin film flow chemistry using a vortex fluidic device (VFD) is effective in the scalable acylation of amines under shear, with the yields of the amides dramatically enhanced relative to traditional batch techniques. The optimized monophasic flow conditions are effective in ≤80 seconds at room temperature, enabling access to structurally diverse amides, functionalized amino acids and substituted ureas on multigram scales. Amide synthesis under flow was also extended to a total synthesis of local anesthetic lidocaine, with sequential reactions carried out in two serially linked VFD units. The synthesis could also be executed in a single VFD, in which the tandem reactions involve reagent delivery at different positions along the rapidly rotating tube with in situ solvent replacement, as a molecular assembly line process. This further highlights the versatility of the VFD in organic synthesis, as does the finding of a remarkably efficient debenzylation of p-methoxybenzyl amines.

Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC-MS/MS.

PURPOSE: Polyesters with hydrophilic domains, i.e., poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(L-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis. METHODS: Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts. RESULTS: Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender. CONCLUSIONS: Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

Mechanistic studies of malonic acid-mediated in situ acylation.

We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules.

Assessment of acylation routes and structural characterisation by liquid chromatography/tandem mass spectrometry of semi-synthetic acyl ester analogues of lipophilic marine toxins.

RATIONALE: Esterification is one of the most important metabolic routes of lipophilic marine toxins in shellfish. In this work we assessed several chemical acylation reactions aimed at obtaining acyl ester analogues via partial synthesis from the free toxins. The procedures developed including sensitive and selective methods based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be applied to obtain reference materials that may be used as analytical standards (internal/external) for method development and calibration, as well as to perform toxicological in vitro and in vivo studies. METHODS: Acylation systems involved both anhydrous and non-anhydrous fatty acid or acid halides as a source of the acyl radical, and several catalysers of the reaction. A series of mass spectrometric experiments involving product ion scans and multiple reaction monitoring (MRM) were used to confirm the identity and to elucidate the fragmentation pathways of the synthesised products. RESULTS: Reaction yields regarding reaction time and temperature were examined at sub-nmol scale for the acylation system consisting of palmitic anhydride and 4-(dimethylamino)pyridine (DMAP) in anhydrous pyridine, showing the best conditions at 75 °C for 60 min, 75 °C for 120 min and 100 °C for 270 min for cyclic imines, azaspiracid-1 and pectenotoxin-2, respectively. The esterification approach was verified at a larger scale for the esterification of gymnodimine-A (GYM-A), which kept a good yield >90% for the synthesis of 10-O-palmitoyl-GYM-A. CONCLUSIONS: Acyl ester analogues of lipophilic marine toxins have been synthesised and their structure elucidated by LC/MS/MS. For acyl ester analogues identical to natural metabolites, the procedures developed have potential to be applied for the semi-synthesis of metabolites in a sustainable, scalable and controlled way, avoiding extensive and tedious isolation and purification procedures from naturally contaminated shellfish. For the semi-synthetic esters structurally different than those found in shellfish, they may have applicability as internal standards for accurate quantifications of natural metabolites present in complex matrices.

Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.

Accelerated, microwave-assisted, and conventional solvent extraction methods affect anthocyanin composition from colored grains.

Anthocyanins are important dietary components with diverse positive functions in human health. This study investigates effects of accelerated solvent extraction (ASE) and microwave-assisted extraction (MAE) on anthocyanin composition and extraction efficiency from blue wheat, purple corn, and black rice in comparison with the commonly used solvent extraction (CSE). Factorial experimental design was employed to study effects of ASE and MAE variables, and anthocyanin extracts were analyzed by spectrophotometry, high-performance liquid chromatography-diode array detector (DAD), and liquid chromatography-mass spectrometry chromatography. The extraction efficiency of ASE and MAE was comparable with CSE at the optimal conditions. The greatest extraction by ASE was achieved at 50 °C, 2500 psi, 10 min using 5 cycles, and 100% flush. For MAE, a combination of 70 °C, 300 W, and 10 min in MAE was the most effective in extracting anthocyanins from blue wheat and purple corn compared with 50 °C, 1200 W, and 20 min for black rice. The anthocyanin composition of grain extracts was influenced by the extraction method. The ASE extraction method seems to be more appropriate in extracting anthocyanins from the colored grains as being comparable with the CSE method based on changes in anthocyanin composition. The method caused lower structural changes in anthocaynins compared with the MAE method. Changes in blue wheat anthocyanins were lower in comparison with purple corn or black rice perhaps due to the absence of acylated anthocyanin compounds in blue wheat. The results show significant differences in anthocyanins among the 3 extraction methods, which indicate a need to standardize a method for valid comparisons among studies and for quality assurance purposes.

Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids.

Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P < 0.05) increased emulsifying properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW < 3 kDa), 7S- and 11S-peptides were improved compared to native peptides. 7S peptides acylated with long chain fatty acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW < 3 kDa and APP peptides. Fluorescence spectra studies revealed structural conformation of acylated soy peptides as compared to native peptides. This study shows that chemical modification with fatty acids can further affect functional properties of soy proteins.

Controlled release of octreotide and assessment of peptide acylation from poly(D,L-lactide-co-hydroxymethyl glycolide) compared to PLGA microspheres.

PURPOSE: To investigate the in vitro release of octreotide acetate, a somatostatin agonist, from microspheres based on a hydrophilic polyester, poly(D,L-lactide-co-hydroxymethyl glycolide) (PLHMGA). METHODS: Spherical and non-porous octreotide-loaded PLHMGA microspheres (12 to 16 µm) and loading efficiency of 60-70% were prepared by a solvent evaporation. Octreotide release profiles were compared with commercial PLGA formulation (Sandostatin LAR(®)); possible peptide modification with lactic, glycolic and hydroxymethyl glycolic acid units was monitored. RESULTS: PLHMGA microspheres showed burst release (~20%) followed by sustained release for 20-60 days, depending on the hydrophilicity of the polymer. Percentage of released loaded peptide was high (70-90%); > 60% of released peptide was native octreotide. PLGA microspheres did not show peptide release for the first 10 days, after which it was released in a sustained manner over the next 90 days; > 75% of released peptides were acylated adducts. CONCLUSIONS: PLHMGA microspheres are promising controlled systems for peptides with excellent control over release kinetics. Moreover, substantially less peptide modification occurred in PLHMGA than in PLGA microspheres.

Assessment of 6'- and 6'''-N-acylation of aminoglycosides as a strategy to overcome bacterial resistance.

Amongst the many synthetic aminoglycoside analogues that were developed to regain the efficacy of this class of antibiotics against resistant bacterial strains, the 1-N-acylated analogues are the most clinically used. In this study we demonstrate that 6'-N-acylation of the clinically used compound tobramycin and 6'''-N-acylation of paromomycin result in derivatives resistant to deactivation by 6'-aminoglycoside acetyltransferase (AAC(6')) which is widely found in aminoglycoside resistant bacteria. When tested against AAC(6')- or AAC(3)-expressing bacteria as well as pathogenic bacterial strains, some of the analogues demonstrated improved antibacterial activity compared to their parent antibiotics. Improvement of the biological performance of the N-acylated analogues was found to be highly dependent on the specific aminoglycoside and acyl group. Our study indicates that as for 1-N-acylation, 6'- and 6'''-N-acylation of aminoglycosides offer an additional promising direction in the search for aminoglycosides capable of overcoming infections by resistant bacteria.

Catalytic acylation of amines with aldehydes or aldoximes.

The simple nickel salt NiCl(2)·6H(2)O catalyzes the coupling of aldoximes with amines to give secondary or tertiary amide products. The aldoxime can be prepared in situ from the corresponding aldehyde. The use of (18)O-labeled oximes has allowed insight into the mechanism of this reaction.

Scalable synthesis of a prostaglandin EP4 receptor antagonist.

The evolution of scalable, economically viable synthetic approaches to the potent and selective prostaglandin EP4 antagonist 1 is presented. The chromatography-free synthesis of multikilogram quantities of 1 using a seven-step sequence (six in the longest linear sequence) is described. This approach has been further modified in an effort to identify a long-term manufacturing route. Our final synthesis involves no step requiring cryogenic (< -25 degrees C) conditions; comprises a total of four steps, only three of which are in the longest linear synthesis; and features the use of two consecutive iron-catalyzed Friedel-Crafts substitutions.

The effects of acylation stimulating protein supplementation VS antibody neutralization on energy expenditure in wildtype mice.

BACKGROUND: Acylation stimulating protein (ASP) is an adipogenic hormone that stimulates triglyceride (TG) synthesis and glucose transport in adipocytes. Previous studies have shown that ASP-deficient C3 knockout mice are hyperphagic yet lean, as they display increased oxygen consumption and fatty acid oxidation compared to wildtype mice. In the present study, antibodies against ASP (Anti-ASP) and human recombinant ASP (rASP) were tested in vitro and in vivo. Continuous administration for 4 weeks via osmotic mini-pump of Anti-ASP or rASP was evaluated in wildtype mice on a high-fat diet (HFD) to examine their effects on body weight, food intake and energy expenditure. RESULTS: In mature murine adipocytes, rASP significantly stimulated fatty acid uptake (+243% vs PBS, P < 0.05) while Anti-ASP neutralized the rASP response. Mice treated with Anti-ASP showed elevated energy expenditure (P < 0.0001), increased skeletal muscle glucose oxidation (+141%, P < 0.001), reduced liver glycogen (-34%, P < 0.05) and glucose-6-phosphate content (-64%, P = 0.08) compared to control mice. There was no change in body weight, food intake, fasting insulin, adiponectin, CRP or TG levels compared to controls. Interestingly, HFD mice treated with rASP showed the opposite phenotype with reduced energy expenditure (P < 0.0001) and increased body weight (P < 0.05), cumulative food intake (P < 0.0001) and liver glycogen content (+59%, P < 0.05). Again, there was no change in circulating insulin, adiponectin, CRP or TG levels, however, plasma free fatty acids were reduced (-48%, P < 0.05). CONCLUSION: In vitro, Anti-ASP effectively neutralized ASP stimulated fatty acid uptake. In vivo, Anti-ASP treatment increased whole body energy utilization while rASP increased energy storage. Therefore, ASP is a potent anabolic hormone that may also be a mediator of energy expenditure.