Tumour, whole-blood, plasma and tissue concentrations of metformin in lung cancer patients.

AIM: Metformin is used for the management of type 2 diabetes mellitus (T2DM) and is being tested clinically as an anticancer agent. Metformin concentrations safely achievable in human solid tissues including tumours are unknown. This study was designed to determine metformin concentration in tissue compartments as a function of dose to inform rational dosing in preclinical models and interpretation of clinical results." METHODS: Subjects with solid tumours to be treated by resection and either (A) willingness to take metformin for 7-10 days before surgery or (B) taking metformin for T2DM were eligible. Whole blood, plasma, tumour, tumour-adjacent uninvolved tissue and subcutaneous adipose tissue were obtained for liquid chromatography with tandem mass spectrometry to measure metformin concentrations. RESULTS: All subjects had primary lung tumours. Metformin dose was significantly correlated with drug concentrations in all tissues analysed. Intersubject metformin concentrations varied by over two orders of magnitude. Metformin concentrations were significantly higher in tumour tissues and lower in adipose tissues compared to other tissues. Concentrations in blood and plasma were significantly correlated with concentrations in solid tissues. CONCLUSION: Metformin accumulates in cellular compartments. Concentrations observed in plasma, blood, lung and tumour tissues in subjects treated with US Food and Drug Administration-approved doses for T2DM are lower than those typically used in tissue culture studies. However, such tissue concentrations are in line with those found within cultured cells treated with supra-pharmacological doses of metformin. Given the large intersubject variability in metformin concentrations, it is imperative to determine whether there is an association between tissue metformin concentration and anticancer activity in humans.

Stealth Liposomes Encapsulating a Potent ACAT1/SOAT1 Inhibitor F12511: Pharmacokinetic, Biodistribution, and Toxicity Studies in Wild-Type Mice and Efficacy Studies in Triple Transgenic Alzheimer's Disease Mice.

Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer's Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.

Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication.

In many species, vocal communication is essential for coordinating social behaviors including courtship, mating, parenting, rivalry, and alarm signaling. Effective communication requires accurate production, detection, and classification of signals, as well as selection of socially appropriate responses. Understanding how signals are generated and how acoustic signals are perceived is key to understanding the neurobiology of social behaviors. Here we review our long-standing research program focused on Xenopus, a frog genus which has provided valuable insights into the mechanisms and evolution of vertebrate social behaviors. In Xenopus laevis, vocal signals differ between the sexes, through development, and across the genus, reflecting evolutionary divergence in sensory and motor circuits that can be interrogated mechanistically. Using two ex vivo preparations, the isolated brain and vocal organ, we have identified essential components of the vocal production system: the sexually differentiated larynx at the periphery, and the hindbrain vocal central pattern generator (CPG) centrally, that produce sex- and species-characteristic sound pulse frequencies and temporal patterns, respectively. Within the hindbrain, we have described how intrinsic membrane properties of neurons in the vocal CPG generate species-specific vocal patterns, how vocal nuclei are connected to generate vocal patterns, as well as the roles of neurotransmitters and neuromodulators in activating the circuit. For sensorimotor integration, we identified a key forebrain node that links auditory and vocal production circuits to match socially appropriate vocal responses to acoustic features of male and female calls. The availability of a well supported phylogeny as well as reference genomes from several species now support analysis of the genetic architecture and the evolutionary divergence of neural circuits for vocal communication. Xenopus thus provides a vertebrate model in which to study vocal communication at many levels, from physiology, to behavior, and from development to evolution. As one of the most comprehensively studied phylogenetic groups within vertebrate vocal communication systems, Xenopus provides insights that can inform social communication across phyla.

The effect of sonidegib (LDE225) on the pharmacokinetics of bupropion and warfarin in patients with advanced solid tumours.

AIMS: We evaluated the potential effect of sonidegib at an oral dose of 800 mg once daily (QD) on the pharmacokinetics (PK) of the probe drugs warfarin (CYP2C9) and bupropion (CYP2B6). METHODS: This was a multicentre, open-label study to evaluate the effect of sonidegib on the PK of the probe drugs warfarin and bupropion in patients with advanced solid tumours. Cohort 1 patients received a single warfarin 15-mg dose on Day 1 of the run-in period and on Cycle 2 Day 22 (C2D22) of sonidegib administration. Cohort 2 patients received a single bupropion 75-mg dose on Day 1 of run-in period and on C2D22 of sonidegib administration. Sonidegib 800 mg QD oral dosing began on Cycle 1 Day 1 of a 28-day cycle after the run-in period in both cohorts. RESULTS: The geometric means ratios [90% confidence interval] for (S)-warfarin with and without sonidegib were: area under the concentration-time curve from time 0 to infinity (AUCinf ) 1.15 [1.07, 1.24] and maximum plasma concentration (Cmax ) 0.88 [0.81, 0.97]; and for (R)-warfarin were: AUCinf 1.10 [0.98, 1.24] and Cmax 0.93 [0.87, 1.0]. The geometric means ratios [90% confidence interval] of bupropion with and without sonidegib were: AUCinf 1.10 [0.99, 1.23] and Cmax 1.16 [0.95, 1.42]. Sonidegib 800 mg had a safety profile that was similar to that of lower dose sonidegib 200 mg and was unaffected by single doses of the probe drugs. CONCLUSIONS: Sonidegib dosed orally at 800 mg QD (higher than the Food and Drug Administration-approved dose) did not impact the PK or pharmacodynamics of warfarin (CYP2C9 probe substrate) or the PK of bupropion (CYP2B6 probe substrate).

Cross-Sectional Assessment of the Emotional Intelligence of Fourth-Year Veterinary Students and Veterinary House Officers in a Teaching Hospital.

Emotional intelligence (EI) is the recognition and management of emotions. This skill set is important to work relationships and professional success. In this cross-sectional, observational study, we investigated EI scores of 4th-year veterinary students, interns, and residents in a teaching hospital, using a psychometric tool with professional population norms. Participants' EI scores were compared with professional norms and between the sample groups. Scores were examined on the basis of demographics and residency program type. Twenty-four 4th-year students and 43 interns and residents completed the survey. Total, composite, and subscale scores for all groups were lower than professional means. We noted no statistically significant differences in EI scores between training levels, but evaluation of effect sizes showed a medium negative effect of higher training levels on Self-Perception Composite, Self-Regard, Emotional Expression, Interpersonal Composite, Flexibility, and Optimism and a medium positive effect of higher training levels on Impulse Control. Medium effects for residency type were found for Stress Tolerance, Flexibility, and Stress Management, with higher scores for residencies with heavy inpatient loads. Medium effects for residency type were found on Flexibility scores, with higher scores for residents in disciplines with a perceived high stress level. We found that baseline EI scores of 4th-year veterinary students, interns, and residents at a teaching hospital were similar to, but uniformly lower than, those of other professionals and did not increase with training level. These results may be used to build on strengths and address weaknesses associated with EI of students and house officers at this institution.

Premotor Neuron Divergence Reflects Vocal Evolution.

To identify mechanisms of behavioral evolution, we investigated the hindbrain circuit that generates distinct vocal patterns in two closely related frog species. Male Xenopus laevis and Xenopus petersii produce courtship calls that include a fast trill: trains of ∼60 Hz sound pulses. Although fast trill rates are similar, X. laevis fast trills have a longer duration and period than those of X. petersii To pinpoint the neural basis of these differences, we used whole-cell patch-clamp recordings in a key premotor hindbrain nucleus (the Xenopus parabrachial area, PBX) in ex vivo brains that produce fictive vocalizations, vocal nerve activity corresponding to advertisement call patterns. We found two populations of PBX neurons with distinct properties: fast trill neurons (FTNs) and early vocal neurons (EVNs). FTNs, but not EVNs, appear to be intrinsically tuned to produce each species' call patterns because: (1) X. laevis FTNs generate longer and slower depolarizations than X. petersii FTNs during their respective fictive vocalizations, (2) current steps in FTNs induce burst durations that are significantly longer in X. laevis than X. petersii, and (3) synaptically isolated FTNs oscillate in response to NMDA in a species-specific manner: longer and slower in X. laevis than in X. petersii Therefore, divergence of premotor neuron membrane properties is a strong candidate for generating vocal differences between species.SIGNIFICANCE STATEMENT The vertebrate hindbrain includes multiple neural circuits that generate rhythmic behaviors including vocalizations. Male African clawed frogs produce courtship calls that are unique to each species and differ in temporal patterns. Here, we identified two functional subtypes of neurons located in the parabrachial nucleus: a hindbrain region implicated in vocal and respiratory control across vertebrates. One of these neuronal subtypes exhibits distinct properties across species that can account for the evolutionary divergence of song patterns. Our results suggest that changes to this group of neurons during evolution may have had a major role in establishing novel behaviors in closely related species.

A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death.

The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p < 0.0001). We demonstrate here the identification of a variant in the titin gene highly associated with the disease in this spontaneous canine model of dilated cardiomyopathy. This large animal model of familial dilated cardiomyopathy shares many similarities with the human disease including mode of inheritance, clinical presentation, genetic heterogeneity and a pathologic variant in the titin gene. The dog is an excellent model to improve our understanding of the genotypic phenotypic relationships, penetrance, expression and the pathophysiology of variants in the titin gene.

Probing forebrain to hindbrain circuit functions in Xenopus.

The vertebrate hindbrain includes neural circuits that govern essential functions including breathing, blood pressure and heart rate. Hindbrain circuits also participate in generating rhythmic motor patterns for vocalization. In most tetrapods, sound production is powered by expiration and the circuitry underlying vocalization and respiration must be linked. Perception and arousal are also linked; acoustic features of social communication sounds-for example, a baby's cry-can drive autonomic responses. The close links between autonomic functions that are essential for life and vocal expression have been a major in vivo experimental challenge. Xenopus provides an opportunity to address this challenge using an ex vivo preparation: an isolated brain that generates vocal and breathing patterns. The isolated brain allows identification and manipulation of hindbrain vocal circuits as well as their activation by forebrain circuits that receive sensory input, initiate motor patterns and control arousal. Advances in imaging technologies, coupled to the production of Xenopus lines expressing genetically encoded calcium sensors, provide powerful tools for imaging neuronal patterns in the entire fictively behaving brain, a goal of the BRAIN Initiative. Comparisons of neural circuit activity across species (comparative neuromics) with distinctive vocal patterns can identify conserved features, and thereby reveal essential functional components.

Evolution of vocal patterns: tuning hindbrain circuits during species divergence.

The neural circuits underlying divergent courtship behaviors of closely related species provide a framework for insight into the evolution of motor patterns. In frogs, male advertisement calls serve as unique species identifiers and females prefer conspecific to heterospecific calls. Advertisement calls of three relatively recently (∼8.5 Mya) diverged species - Xenopus laevis, X. petersii and X. victorianus - include rapid trains of sound pulses (fast trills). We show that while fast trills are similar in pulse rate (∼60 pulses s-1) across the three species, they differ in call duration and period (time from the onset of one call to the onset of the following call). Previous studies of call production in X. laevis used an isolated brain preparation in which the laryngeal nerve produces compound action potentials that correspond to the advertisement call pattern (fictive calling). Here, we show that serotonin evokes fictive calling in X. petersii and X. victorianus as it does in X. laevis As in X. laevis, fictive fast trill in X. petersii and X. victorianus is accompanied by an N-methyl-d-aspartate receptor-dependent local field potential wave in a rostral hindbrain nucleus, DTAM. Across the three species, wave duration and period are strongly correlated with species-specific fast trill duration and period, respectively. When DTAM is isolated from the more rostral forebrain and midbrain and/or more caudal laryngeal motor nucleus, the wave persists at species-typical durations and periods. Thus, intrinsic differences within DTAM could be responsible for the evolutionary divergence of call patterns across these related species.

Sex differences and endocrine regulation of auditory-evoked, neural responses in African clawed frogs (Xenopus).

Mating depends on the accurate detection of signals that convey species identity and reproductive state. In African clawed frogs, Xenopus, this information is conveyed by vocal signals that differ in temporal patterns and spectral features between sexes and across species. We characterized spectral sensitivity using auditory-evoked potentials (AEPs), commonly known as the auditory brainstem response, in males and females of four Xenopus species. In female X. amieti, X. petersii, and X. laevis, peripheral auditory sensitivity to their species own dyad-two, species-specific dominant frequencies in the male advertisement call-is enhanced relative to males. Males were most sensitive to lower frequencies including those in the male-directed release calls. Frequency sensitivity was influenced by endocrine state; ovariectomized females had male-like auditory tuning while dihydrotestosterone-treated, ovariectomized females maintained female-like tuning. Thus, adult, female Xenopus demonstrate an endocrine-dependent sensitivity to the spectral features of conspecific male advertisement calls that could facilitate mating. Xenopus AEPs resemble those of other species in stimulus and level dependence, and in sensitivity to anesthetic (MS222). AEPs were correlated with body size and sex within some species. A frequency following response, probably encoded by the amphibian papilla, might facilitate dyad source localization via interaural time differences.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Species-specific loss of sexual dimorphism in vocal effectors accompanies vocal simplification in African clawed frogs (Xenopus).

Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species.

The Xenopus amygdala mediates socially appropriate vocal communication signals.

Social interaction requires that relevant sensory information is collected, classified, and distributed to the motor areas that initiate an appropriate behavioral response. Vocal exchanges, in particular, depend on linking auditory processing to an appropriate motor expression. Because of its role in integrating sensory information for the purpose of action selection, the amygdala has been implicated in social behavior in many mammalian species. Here, we show that two nuclei of the extended amygdala play essential roles in vocal communication in the African clawed frog, Xenopus laevis. Transport of fluorescent dextran amines identifies the X. laevis central amygdala (CeA) as a target for ascending auditory information from the central thalamic nucleus and as a major afferent to the vocal pattern generator of the hindbrain. In the isolated (ex vivo) brain, electrical stimulation of the CeA, or the neighboring bed nucleus of the stria terminalis (BNST), initiates bouts of fictive calling. In vivo, lesioning the CeA of males disrupts the production of appropriate vocal responses to females and to broadcasts of female calls. Lesioning the BNST in males produces an overall decrease in calling behavior. Together, these results suggest that the anuran CeA evaluates the valence of acoustic cues and initiates socially appropriate vocal responses to communication signals, whereas the BNST plays a role in the initiation of vocalizations.

A multidimensional gas chromatography method for the analysis of hydrogen sulfide in crude oil and crude oil headspace.

Two-dimensional heart-cutting gas chromatography is used to analyze dissolved hydrogen sulfide in crude samples. Liquid samples are separated first on an HP-PONA column, and the light sulfur gases are heart-cut to a GasPro column, where hydrogen sulfide is separated from other light sulfur gases and detected with a sulfur chemiluminescence detector. Heart-cutting is accomplished with the use of a Deans switch. Backflushing the columns after hydrogen sulfide detection eliminates any problems caused by high-boiling hydrocarbons in the samples. Dissolved hydrogen sulfide is quantified in 14 crude oil samples, and the results are shown in this work. The method is also applicable to the analysis of headspace hydrogen sulfide over crude oil samples. Gas hydrogen sulfide measurements are compared to liquid hydrogen sulfide measurements for the same sample set. The chromatographic system design is discussed, and chromatograms of representative gas and liquid measurements are shown.

Courtship behavior: Resurrecting an undead song.

The fly Drosophila yakuba has lost an ancestral component of the male courtship song: this is due to ontogenetic death of effector neurons in the ventral nerve cord, a result of the D. yakuba sex-determining gene dsx producing a male isoform, dsxM, with cell-death-promoting activity similar to that of the female isoform, dsxF, in D. melanogaster.

Distinct neural and neuromuscular strategies underlie independent evolution of simplified advertisement calls.

Independent or convergent evolution can underlie phenotypic similarity of derived behavioural characters. Determining the underlying neural and neuromuscular mechanisms sheds light on how these characters arose. One example of evolutionarily derived characters is a temporally simple advertisement call of male African clawed frogs (Xenopus) that arose at least twice independently from a more complex ancestral pattern. How did simplification occur in the vocal circuit? To distinguish shared from divergent mechanisms, we examined activity from the calling brain and vocal organ (larynx) in two species that independently evolved simplified calls. We find that each species uses distinct neural and neuromuscular strategies to produce the simplified calls. Isolated Xenopus borealis brains produce fictive vocal patterns that match temporal patterns of actual male calls; the larynx converts nerve activity faithfully into muscle contractions and single clicks. In contrast, fictive patterns from isolated Xenopus boumbaensis brains are short bursts of nerve activity; the isolated larynx requires stimulus bursts to produce a single click of sound. Thus, unlike X. borealis, the output of the X. boumbaensis hindbrain vocal pattern generator is an ancestral burst-type pattern, transformed by the larynx into single clicks. Temporally simple advertisement calls in genetically distant species of Xenopus have thus arisen independently via reconfigurations of central and peripheral vocal neuroeffectors.

Influence of sex on renin-angiotensin-aldosterone system metabolites and enzymes in Doberman Pinschers.

BACKGROUND: Estrogen modulates the renin-angiotensin-aldosterone system (RAAS) in women, but sex differences have not been fully explored in dogs. OBJECTIVE: We hypothesized that the RAAS profile of intact female (IF) Doberman Pinschers (DP) would differ from spayed female (SF) and intact male (IM) DP. ANIMALS: Eighteen healthy DP (6 IF, 6 SF, 6 IM). METHODS: Absolute and indexed RAAS metabolites, angiotensin-converting enzyme (ACE) and ACE2 activities, and genotypes (pyruvate kinase dehydrogenase 4, titin, and ACE variants) were compared among sex groups using Kruskal-Wallis or chi-square tests, and linear regression controlling for age. Data are expressed as median (minimum, maximum) and P < .05 was considered significant. RESULTS: The ACE activity was higher in IF DP (656 pmol/L; 436, 784) compared to SF DP (411 pmol/L; 287, 451; P = .01) and IM DP (365 pmol/L; 276, 1200; P = .04) after controlling for age. Angiotensin II, angiotensin I, and plasma renin activity marker (PRA-S) were higher in IF DP compared to SF DP, but not significantly (P ≤ .25). After controlling for age, angiotensin 1-7/angiotensin I was lower in IF DP compared to SF DP (P = .01). Genotypes did not differ among groups. Most DP (94%) were ACE variant positive. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Sex and reproductive status influenced the RAAS of DP, with IF DP showing genotype-independent higher ACE activity. These findings hold implications for sterilization practices in female dogs, and support sex and reproductive status as a source of variability in RAAS studies. Additionally, the frequency of the ACE gene variant was very high in this group of DP.

Beyond Angiotensin-Converting Enzyme Inhibitors: Modulation of the Renin-Angiotensin-Aldosterone System to Delay or Manage Congestive Heart Failure.

The renin-angiotensin-aldosterone system (RAAS) consists of bioactive angiotensin peptides, enzymatic pathways, receptors, and the steroid hormone aldosterone. The RAAS regulates blood pressure, sodium, and electrolyte homeostasis and mediates pathologic disease processes. Within this system is an alternative arm that counterbalances the vasoconstrictive, sodium and water retentive, and pro-fibrotic and inflammatory effects of the classical arm. Improved biochemical methodologies in RAAS quantification are elucidating how this complex system changes in health and disease. Future treatments for cardiovascular and kidney disease will likely involve a more nuanced manipulation of this system rather than simple blockade.

Use of serum osmolality to identify heart disease stage in dogs and relationship to mathematical chloride correction.

BACKGROUND: Heart failure-associated hypochloremia can be depletional from diuretics or dilutional from water retention. Serum osmolality reflects water balance but has not been evaluated in dogs with heart disease. HYPOTHESIS: To determine if serum osmolality is related to heart disease stage and amount of mathematical correction of serum chloride (Cl- ) concentrations in healthy dogs and dogs with myxomatous mitral valve degeneration (MMVD). ANIMALS: Seventy-seven dogs (20 healthy, 25 Stage B MMVD, 32 Stage C/D MMVD). METHODS: Serum Cl- concentrations were mathematically corrected. Osmolality was calculated (calOsm) and directly measured by freezing point depression (dmOsm) and compared by Bland-Altman analysis. Biochemical variables and osmolality were compared among healthy, Stage B, and Stage C/D dogs. Correlations were explored between osmolality and biochemical variables. Median and range are presented. P < .05 was considered significant. RESULTS: The calOsm was different among groups (P = .003), with Stage B (310 mOsm/kg; 306, 316) and C/D dogs (312 mOsm/kg; 308, 319) having higher calOsm than healthy dogs (305 mOsm/kg; 302, 308). Osmolality methods were moderately correlated (P < .0001, rs = .46) but with proportional bias and poor agreement. The amount of Cl- correction was negatively correlated with calOsm (P < .0001, rs = -.78) and dmOsm (P = .004, rs = -.33). Serum bicarbonate concentration was negatively correlated with Cl- (P < .0001, rs = -.67). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with Stage B and Stage C/D heart disease had higher calOsm than healthy dogs. Osmolality was inversely related to the amount of Cl- correction, which supports its use in assessing relative body water content. Poor agreement between calOsm and dmOsm prevents methodological interchange.

Angiotensin-converting enzyme 2 in dogs with Dirofilaria immitis.

BACKGROUND: Infection by the canine heartworm, Dirofilaria immitis, causes significant cardiopulmonary disease, with progression impacted by increasing parasite numbers and duration of infection. The renin-angiotensin-aldosterone system (RAAS) is an important mediator of cardiac and pulmonary disease. Angiotensin-converting enzyme 2 (ACE2) mitigates the maladaptive effects of angiotensin II by converting it to angiotensin (1-7). We hypothesized that circulating ACE2 activity would be altered in dogs with high heartworm infection intensities relative to dogs without heartworms. METHODS: Frozen serum samples (-80 °C) from 30 dogs euthanized at Florida shelters were analyzed for ACE2 activity using liquid chromatography-mass spectrometry/mass spectroscopy and a kinetics approach with and without an ACE2 inhibitor. A convenience sample of 15 dogs without heartworms (HW0) and 15 dogs with > 50 heartworms (HW>50) was included. Heartworm number and microfilariae presence were determined at necropsy. The effects of heartworm status, body weight, and sex on ACE2 were evaluated using regression analysis. Values of P < 0.05 were considered significant. RESULTS: All HW0 dogs were D. immitis microfilariae-negative and all HW>50 dogs were D. immitis microfilariae-positive with a median adult worm count of 74 (minimum = 63, maximum = 137). The ACE2 activity of HW>50 dogs (median = 28.2 ng/ml; minimum = 13.6, maximum = 76.2) was not different from HW0 dogs (median 31.9 ng/ml; minimum = 14.1, maximum = 139.1; P = 0.53). The ACE2 activity was higher in dogs with high body weight (median 34.2 ng/ml minimum = 14.1, maximum = 76.2) than in dogs with low weight (median 27.5 ng/ml; minimum = 16.4, maximum = 139.1; P = .044). CONCLUSIONS: Heartworm infection did not impact ACE2 activity in shelter dogs with or without heartworms, but heavier dogs had higher ACE2 activity compared to lighter dogs. Comprehensive RAAS evaluation and additional clinical information would aid in understanding how ACE2 activity relates to the entire cascade and clinical status in dogs with heartworm disease.