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.

A toxic environment selects for specialist microbiome in poison frogs.

Shifts in microbiome community composition can have large effects on host health. It is therefore important to understand how perturbations, like those caused by the introduction of exogenous chemicals, modulate microbiome community composition. In poison frogs within the family Dendrobatidae, the skin microbiome is exposed to the alkaloids that the frogs sequester from their diet and use for defense. Given the demonstrated antimicrobial effects of these poison frog alkaloids, these compounds may be structuring the skin microbial community. To test this, we first characterized microbial communities from chemically defended and closely related non-defended frogs from Ecuador. Then we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of a single frog species. In both the field and lab experiments, we found that alkaloid-exposed microbiomes are more species rich and phylogenetically diverse, with an increase in rare taxa. To better understand the strain-specific behavior in response to alkaloids, we cultured microbial strains from poison frog skin and found the majority of strains exhibited either enhanced growth or were not impacted by the addition of DHQ. Additionally, stable isotope tracing coupled to nanoSIMS suggests that some of these strains are able to metabolize DHQ. Taken together, these data suggest that poison frog chemical defenses open new niches for skin-associated microbes with specific adaptations, including the likely metabolism of alkaloids, that enable their survival in this toxic environment. This work helps expand our understanding of how exposure to exogenous compounds like alkaloids can impact host microbiomes.

Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.

RATIONALE: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides. RESULTS: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine.

Implementing Home-Based Clinical Research for Caregivers and Persons with Stroke: Lessons Learned.

Conducting research in the home environment presents challenges related to setting, study participants, methods, and researchers. Researchers should be aware of potential challenges to ensure rigor and improve planning for future studies. This paper describes difficulties experienced and lessons learned when conducting a two-group, randomized pilot study (n = 32) of a web-based intervention (Carepartner and Constraint-Induced Therapy [CARE-CITE]) designed to foster positive carepartner engagement in home-based activities to improve upper extremity function in persons with stroke. Challenges and issues included: 1) recruitment and referral, 2) data collection in the home setting, 3) participants' understanding of the rationale for adhering to constraint-induced movement therapy principles (wearing mitt on the less-affected limb), 4) tracking adherence of upper extremity practice time, 5) participant-driven goal setting, 6) potentially unsafe participant practice activities, 7) home visit safety, 8) encouraging versus controlling-using autonomy support, 9) participant needs beyond study scope, and 10) ethical safeguards for addressing depressive symptoms. Researchers can incorporate suggested strategies to support methodological rigor and facilitate interventions engaging carepartners in the rehabilitation process when planning for research in the home environment.

Buprenorphine reduces somatic withdrawal in a mouse model of early-life morphine exposure.

The rising prevalence of early-life opioid exposure has become a pressing public health issue in the U.S. Neonates exposed to opioids in utero are at risk of experiencing a constellation of postpartum withdrawal symptoms commonly referred to as neonatal opioid withdrawal syndrome (NOWS). Buprenorphine (BPN), a partial agonist at the mu-opioid receptor (MOR) and antagonist at the kappa-opioid receptor (KOR), is currently approved to treat opioid use disorder in adult populations. Recent research suggests that BPN may also be effective in reducing withdrawal symptoms in neonates who were exposed to opioids in utero. We sought to determine whether BPN attenuates somatic withdrawal in a mouse model of NOWS. Our findings indicate that the administration of morphine (10mg/kg, s.c.) from postnatal day (PND) 1-14 results in increased somatic symptoms upon naloxone-precipitated (1mg/kg, s.c.) withdrawal. Co-administration of BPN (0.3mg/kg, s.c.) from PND 12-14 attenuated symptoms in morphine-treated mice. On PND 15, 24h following naloxone-precipitated withdrawal, a subset of mice was examined for thermal sensitivity in the hot plate test. BPN treatment significantly increased response latency in morphine-exposed mice. Lastly, neonatal morphine exposure elevated mRNA expression of KOR, and reduced mRNA expression of corticotropin-releasing hormone (CRH) in the periaqueductal gray when measured on PND 14. Altogether, this data provides support for the therapeutic effects of acute low-dose buprenorphine treatment in a mouse model of neonatal opioid exposure and withdrawal.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water.

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.

A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 µL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

Azetidine-2-carboxylic acid in the food chain.

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Validation of human ApoB and ApoAI immunoturbidity assays for non-human primate dyslipidemia and atherosclerosis research.

Emerging evidence suggests apolipoprotein B (apoB) and apolipoprotein AI (apoAI) are strong risk predictors for atherosclerosis. Non-human primates (NHP), including rhesus monkeys, cynomolgus monkeys, and African green monkeys, are important preclinical species for studying dyslipidemia and atherosclerosis as they more closely resemble humans in lipid metabolism and disease physiology compared to lower species such as rodents. However, no commercial assays are currently available for measuring apoB and apoAI in NHP. We therefore evaluated analytical methods for routinely measuring apoB and apoAI in our NHP dyslipidemia and atherosclerosis research. Since NHP apoB and apoAI sequences are likely highly similar to human, we focused on the clinically validated and widely utilized human apoB and apoAI immunoturbidity assays. We carried out technical validation of these assays with NHP samples and leveraged orthogonal technical platforms including mass spectrometry, independent ELISA assay, and absolute quantitation via SDS-PAGE for further characterization. Analysis of purified lipoproteins demonstrated that the immunoturbidity assays detect NHP apoAI and apoB, with good dilution linearity and spike recovery from NHP plasma. Orthogonal studies showed apoAI correlated with protein concentration and apoB levels correlated with LC/MS and an independent ELISA. NHP samples from a drug treatment study were analyzed with the immunoturbidity assays and levels of apoB and apoAI fit our understanding of biology and expectations from literature. These studies serve as important technical and biological validation of the immunoturbidity assays for NHP samples, and demonstrate that these assays provide a high-throughput, fully automated analytical platform for NHP samples. Our studies pave the way for future translational research in NHP for developing therapies for treating dyslipidemia and atherosclerosis.