High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

Experimental Medicine Study to Measure Immune Checkpoint Receptors PD-1 and GITR Turnover Rates In Vivo in Humans.

Development of monoclonal antibodies (mAbs) targeting immune-checkpoint receptors (IMRs) for the treatment of cancer is one of the most active areas of investment in the biopharmaceutical industry. A key decision in the clinical development of anti-IMR mAbs is dose selection. Dose selection can be challenging because the traditional oncology paradigm of administering the maximum tolerated dose is not applicable to anti-IMR mAbs. Instead, dose selection should be informed by the pharmacology of immune signaling. Engaging an IMR is a key initial step to triggering pharmacologic effects, and turnover (i.e., the rate of protein synthesis) of the IMR is a key property to determining the dose level needed to engage the IMR. Here, we applied the stable isotope labeling mass spectrometry technique using 13 C6 -leucine to measure the in vivo turnover rates of IMRs in humans. The 13 C6 -leucine was administered to 10 study participants over 15 hours to measure 13 C6 -leucine enrichment kinetics in 2 IMR targets that have been clinically pursued in oncology: GITR and PD-1. We report the first measurements of GITR and PD-1 median half-lives associated with turnover to be 55.6 and ≥ 49.5 hours, respectively. The approach outlined here can be applied to other IMRs and, more generally, to protein targets.

Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy.

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ T cells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response to combination therapy did not appear to be associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

Effects of CETP inhibition with anacetrapib on metabolism of VLDL-TG and plasma apolipoproteins C-II, C-III, and E.

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR.

Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted in-solution digestion in quantitative proteomics experiments.

RATIONALE: In quantitative analysis of protein biomarkers and therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS), it is a preferred and well-established approach to digest with proteolytic enzymes to produce smaller peptide fragments which are more suitable for LC/MS analysis than the intact protein. In-solution digestion is one widely used method for protein digestion. Proteolytically resistant proteins often require digestion times that extend beyond normal working hours and prohibit same day analysis. We evaluated the performance of an immobilized enzyme reactor (IMER) to determine if this technology could reduce method development time, digestion time and increase throughput. METHODS: We digested human plasma samples using a commercially available IMER, Flash Digest, and compared it to an in-solution digestion method for analysis of three different apolipoprotein biomarkers APOE, APOC2, and APOC3. The plasma digests were analyzed via LC/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM). Value assigned calibrators were selected over a relevant physiological concentration range for each protein of interest. Quality control samples (QCs) and 'unknown' human plasma samples were analyzed with both methods. RESULTS: Flash Digest significantly reduced digestion time for APOC3, the most proteolytically resistant of the three proteins, to 30 min compared with overnight used with in-solution digestion. The Flash Digest achieved comparable digestion efficiency with minimal method development and reduced sample preparation time. Both methods showed linearity over a physiologically relevant concentration range. Precision was evaluated and a percentage coefficient of variance (% CV) less than 8% was obtained during intra-day reproducibility evaluation for all three apolipoproteins with Flash Digest. Concentrations observed for QCs and unknown samples using Flash Digest were comparable to the in-solution method. CONCLUSIONS: An IMER such as Flash Digest may be a potential alternative to in-solution digestion to accelerate digestion of proteolytically resistant proteins in a quantitative proteomics experiments, reduce method development time and increase throughput. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of 13-Hour Hyperglucagonemia on Energy Expenditure and Hepatic Glucose Production in Humans.

Glucagon (GCG) acutely stimulates energy expenditure (EE) and hepatic glucose production (HGP) in humans, but whether these effects persist during hyperglucagonemia of longer duration is unclear. Using a prospective, randomized, single-blind, crossover study design, we therefore measured EE and rates of glucose appearance (glucose RA) during three separate infusion protocols in healthy lean males: A) 10-h overnight GCG infusion (6 ng/[kg × min]) followed by 3-h infusion of GCG, octreotide (OCT), and insulin (INS) for basal replacement; B) overnight saline (SAL) infusion followed by GCG/OCT/INS infusion; and C) overnight SAL infusion followed by SAL/OCT/INS infusion. Sleep EE, measured at 6 to 7 h of the overnight infusion, was increased 65-70 kcal/24 h in A compared with B and C. During the 3-h infusion, mean resting EE remained significantly increased in A versus C by ∼50 kcal/24 h; in B, resting EE increased with a statistical trend but was not significantly greater than in C. Glucose RA increased to comparable levels in A and B. We conclude that in healthy lean males, stimulation of EE and HGP is sustained during hyperglucagonemia of longer duration when insulin secretion is inhibited. The increase in EE at the present GCG dose was of marginal clinical significance.

Development and validation of an IA-LC/MS method to quantitate active and total B-type natriuretic peptide in human plasma.

AIM: Patients with elevated levels of B-type natriuretic peptide (BNP) and/or NT-proBNP as measured by clinical tests have an elevated risk of heart failure (HF). Despite utility in large clinical studies, both assays are plagued by large biological variability and specificity issues. To address these concerns and further investigate BNP in the HF setting, we developed an LC/MS assay to characterize the ratio of active to total BNP. RESULTS: We have developed and validated a novel immunoaffinity LC/MS assay to measure BNP-derived fragments, as well as 'total BNP' in human plasma. The ratio of active BNP1-32 to total BNP in 11 HF subjects was found to be <8%, and the sum of detectable BNP fragments contributed approximately 20% of total BNP. CONCLUSION: We developed an assay with the specificity to measure the active form of BNP, which may aid in the accurate diagnosis and better management of HF.

An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.

BACKGROUND: For quantitative immunoaffinity IA-LC-MS, the utility of antibodies has been demonstrated many times but the utility of aptamers as affinity reagents is unproven. METHODS: Immunoaffinity reagents including a monoclonal antibody and an aptamer were coupled to magnetic beads and used as part of an enrichment strategy for PCSK9 quantitation in plasma. RESULTS: With limited method development, we have established a comparison of an anti-PCSK9 aptamer with an anti-PCSK9 monoclonal antibody. The background that results from a tryptic digest of affinity enrichment in plasma was demonstrated for each reagent using high-resolution full scan MS. The assay recovery was demonstrated for multiple concentrations of aptamer in plasma with different concentrations of PCSK9 protein. CONCLUSION: The aptamer achieved comparable enrichment to the antibody, but with lower peptide background, thus demonstrating the potential use of aptamers for IA-LC-MS.

The clinical utility of mass spectrometry based protein assays.

Reports of mass spectrometry based assays for peptides and proteins have become increasingly common in the literature. The growing interest of mass spectrometry for use in clinical laboratories has been primarily driven by the inherent selectivity of the platform relative to more traditional platforms such as immunoassays. However, the adoption of mass spectrometry for peptide and protein analysis in the clinic has been relatively slow compared its adoption in non-clinical laboratories such as in biomarker discovery efforts or within laboratories that support pharmaceutical and academic research. Here, we review some of the successful reports of MS based assays for human proteins in multiple stages of assay research, and describe how and why the platform was employed in order to demonstrate where and when mass spectrometry based assays will have value in the future.

An LC-MRM method for measuring intestinal triglyceride assembly using an oral stable isotope-labeled fat challenge.

AIM: A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids. RESULTS: Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG. This method was subjected to routine assay validation and meets typical requirements for an assay to be used to support clinical studies. CONCLUSION: Calculations for the fractional appearance rate of TG in plasma are presented along with the intracellular enterocyte precursor pool for 12 study participants.

Cholesteryl Ester Transfer Protein Inhibition With Anacetrapib Decreases Fractional Clearance Rates of High-Density Lipoprotein Apolipoprotein A-I and Plasma Cholesteryl Ester Transfer Protein.

OBJECTIVE: Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA)-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP. APPROACH AND RESULTS: Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II, and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, ANA treatment increased plasma HDL-C (63.0%; P<0.001) and apoA-I levels (29.5%; P<0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate. CONCLUSIONS: ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein.

Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.

BACKGROUND: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. METHODS: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. RESULTS: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. CONCLUSIONS: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.

Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects.

BACKGROUND: Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib. METHODS: We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR). RESULTS: Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR. CONCLUSION: These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance. TRIAL REGISTRATION: ClinicalTrials.gov NCT00990808. FUNDING: Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Development and fit-for-purpose validation of a LC-MS/MS assay for fibrinogen peptide A quantitation in human plasma.

BACKGROUND: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma. The lower limit-of-quantitation (LLOQ) of this assay was determined to be 0.16 nM. The inter- and intra-day%CV was <15%. Freeze-thaw stability of FPA was ±30% up to 3 cycles and linear response of FPA was observed for plasma dilution up to 16-fold. CONCLUSION: The assay was validated and the biological variability of FPA in plasma was established (1-30 nM).

Quantitation of human peptides and proteins via MS: review of analytically validated assays.

Since the development of monoclonal antibodies in the 1970s, antibody-based assays have been used for the quantitation of proteins and peptides and, today, they are the most widely used technology in routine laboratory medicine and bioanalysis. However, in the last couple of decades, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) techniques have been adopted in the quantitation of small molecules, and more recently have made significant contributions in the quantitation of proteins and peptides. In this article, we will review clinical MS-based assays for endogenous peptides, proteins, and therapeutic antibodies, for which validated methods exist. We will also cover the measurement of protein turnover and the unique solutions that MS can offer in this field.

Applications of low-flow LC-SRM for the analysis of large molecules in pharmaceutical R&D.

Although ligand-binding assays are frequently employed to measure large molecules, the use of LC-SRM assays is increasingly popular due to the inherent selectivity advantage and the ability to operate without exquisitely selective antibodies. Until recently LC-SRM assays have been unable to compete with ligand-binding assays in terms of sensitivity. However, the use of low-flow chromatography prior to mass spectrometry has played a crucial role in increasing the sensitivity of LC-SRM platforms and enabling measurements of large molecules that had previously been unmeasurable. In this article, we highlight some technical advances, describe strategies for employing low-flow chromatography, and review recent literature that describes implementation of low-flow LC-SRM to support large-molecule analysis in pharmaceutical R&D.

Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.

RATIONALE: Apolipoprotein(a) is a polymorphic glycoprotein covalently bound to apoB100 in Lp(a) particles and has been described to be both atherogenic and prothrombotic, although its exact mechanism of action is not well defined. Apolipoprotein(a) is routinely measured by immunoassays. Unfortunately, the accuracy of the measurement can be affected by the apolipoprotein(a) size (number of kringles) polymorphism in Lp(a) particles. Here we describe an ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) assay that is capable of measuring apolipoprotein(a) concentrations while simultaneously determining the number of kringles present per protein. METHODS: Plasma samples were diluted and proteins de-lipidated with deoxycholate prior to tryptic digestion. Distinct tryptic peptides from different regions of apolipoprotein(a) were measured to determine both concentration and the number of kringles present per protein. Separation and quantitation of tryptic peptides is carried out at 700 µL/min using a 1.7 µm C18 column (2.1 × 100 mm) coupled to a Thermo Vantage triple quadrupole (QQQ) mass spectrometer with a heated electrospray ionization (HESI) source. RESULTS: This method was compared to established methods for measuring concentration (monoclonal antibody based ELISA) and size (gel-electrophoresis) using 80 plasma samples proved by NWLRL. The slope and r(2) value for the correlation of concentrations were determined to be 0.96 and 0.98, demonstrating excellent agreement of absolute values between the UPLC/MS and ELISA methods. As measured by UPLC/MS, the average kringle number or size is smaller than determined by the electrophoretic method. CONCLUSIONS: A single UPLC/MS method was developed capable of measuring apolipoprotein(a) concentration and size (by measuring the number of kringles per protein). This assay passes criteria required for 'fit for purpose' assays including sensitivity, intra and interday reproducibility and freeze/thaw stability. While the agreement between UPLC/MS and ELISA is excellent for concentration and may provide researchers with additional tools for studying apolipoprotein(a), the dissimilarities between UPLC/MS and the electrophoretic method may also be exploited for understanding apolipoprotein(a) structure and function.

Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates.