Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

Mitapivat, a pyruvate kinase (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Retrospective evaluation of postoperative joint immobilization using a temporary calcaneotibial screw for medial or lateral tarsocrural joint instability in dogs.

OBJECTIVE: To evaluate the use of a temporary calcaneotibial screw (CTS) to immobilize medial or lateral tarsocrural joint instability (TCI) in dogs. STUDY DESIGN: Retrospective study. ANIMALS: Twelve dogs (including five active working farm dogs) with TCI. METHODS: Medical records (January 2015-June 2023) were retrospectively reviewed for cases of TCI managed surgically including temporary joint immobilization using a CTS and external coaptation. Clinical data consisted of medical records and an online survey completed by the owner. RESULTS: Surgical techniques to address TCI included primary ligamentous repair, synthetic ligament reconstruction, or malleolar fracture repair. Immobilization with a CTS was employed for 6-8 weeks postoperatively. The online survey was completed for 10 dogs. All dogs exhibited good-to-excellent functional outcomes at the follow-up (median, 31 months; range, 4-66). All working farm dogs (5) were able to return to normal or substantial levels of their work. Four distinct complications were reported in three dogs including one CTS breakage and three bandage-related soft-tissue injuries. CONCLUSION: This retrospective study represents the first report of employing a temporary CTS for TCI in dogs. CLINICAL SIGNIFICANCE: A temporary CTS was effective in immobilizing the tarsocrural joint for dogs with TCI and the postoperative complication rate in this study was relatively low. A CTS screw and external coaptation is a viable alternative to previously reported methods of tarsocrural joint stabilization.

A retrospective evaluation of complications associated with forkless tibial tuberosity advancement performed in primary care practice.

OBJECTIVE: To report postoperative complications associated with forkless tibial tuberosity advancement (TTA) performed in primary care veterinary practice and to compare results with previous publications. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Three hundred seventy-four forkless TTAs in 329 dogs performed by six nonspecialist veterinarians. METHODS: Medical records of dogs treated with a standard forkless TTA (2013-2016) and with at least 12 months of postoperative follow-up were reviewed. Complications recorded by the referring practice or the operating veterinarian were classified as minor (medically treated) or major (surgically treated). RESULTS: Complications occurred in 57 of 374 (15.2%) TTAs; 28 (7.5%) complications were major, and 29 (7.7%) complications were minor. Postliminary meniscal injuries were documented in 12 of 374 (3.2%) TTAs (12/57 major complications) and were more common when the ratio of cage size to bodyweight was ≤0.25 (P = .019). Mean TTA (cage size) was greater in this population than what has been previously reported for a lower median bodyweight. CONCLUSION: The incidence of major complications was low and within the range previously reported for TTA in referral practice after adjusting for study design. The magnitude of advancement was greater, and the incidence of postliminary meniscal injury was lower than what has been previously reported, after accounting for dogs that had a preliminary meniscal injury or medial meniscal release. CLINICAL SIGNIFICANCE: Forkless TTA may be successfully performed by experienced veterinarians in primary care practice with a low rate of complications. The incidence of postliminary meniscal injury may be reduced by a greater degree of advancement of the tibial tuberosity.

Defining Metabolic and Nonmetabolic Regulation of Histone Acetylation by NSAID Chemotypes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for their effects on inflammatory gene expression. Although NSAIDs are known to impact multiple cellular signaling mechanisms, a recent finding is that the NSAID salicylate can disrupt histone acetylation, in part through direct inhibition of the lysine acetyltransferase (KAT) p300/CBP. While salicylate is a relatively weak KAT inhibitor, its CoA-linked metabolite is more potent; however, the ability of NSAID metabolites to inhibit KAT enzymes biochemically and in cells remains relatively unexplored. Here we define the role of metabolic and nonmetabolic mechanisms in inhibition of KAT activity by NSAID chemotypes. First, we screen a small panel of NSAIDs for biochemical inhibition of the prototypical KAT p300, leading to the finding that many carboxylate-containing NSAIDs, including ibuprofen, are able to function as weak inhibitors. Assessing the inhibition of p300 by ibuprofen-CoA, a known NSAID metabolite, reveals that linkage of ibuprofen to CoA increases its biochemical potency toward p300 and other KAT enzymes. In cellular studies, we find that carboxylate-containing NSAIDs inhibit histone acetylation. Finally, we exploit the stereoselective metabolism of ibuprofen to assess the role of its acyl-CoA metabolite in regulation of histone acetylation. This unique strategy reveals that formation of ibuprofen-CoA and histone acetylation are poorly correlated, suggesting metabolism may not be required for ibuprofen to inhibit histone acetylation. Overall, these studies provide new insights into the ability of NSAIDs to alter histone acetylation, and illustrate how selective metabolism may be leveraged as a tool to explore the influence of metabolic acyl-CoAs on cellular enzyme activity.

The Site of Bone Marrow Acquisition Affects the Myeloid to Erythroid Ratio in Apparently Healthy Dogs.

Bone marrow (BM) cytology and histopathology are complementary tools used to investigate hematological diseases. The purpose of this study was to determine if there are site-dependent differences in the diagnostic quality, myeloid to erythroid ratio (MER), and discordant findings in samples from different sites in the same dog. Eighteen apparently healthy dogs were used in the study. The sequence of sample acquisition was randomized according to a Latin square, and samples for BM cytology and histology were collected from both humeri and both ilial crests immediately after death. Board-certified clinical and anatomical pathologists read the cytology and histology, respectively. The data were analyzed using a mixed-effect model. The site of BM acquisition did not affect BM sample quality. The rate of discordant clinical findings between sites was 0.05 (95% confidence interval, 0.01-0.13). In general, by cytology, the MERs were slightly but significantly greater in samples from the ilial crests than from the humeri ( P = .01). The measured MER for histology was nearly twice that for cytology for all sites ( P < .001). In conclusion, there was a low-rate, site-dependent discordance in diagnostic findings in BM samples and differences in MER between the ilial crest and the humerus. A similar study is justified in sick dogs with hematological disease to determine the effect of sampling site on discordant findings between sites.

Evaluation of the rostral projection of the sacral lamina as a component of degenerative lumbosacral stenosis in German shepherd dogs.

OBJECTIVE: To determine the association between a greater rostral projection of the sacral lamina and clinical signs of cauda equina syndrome (CES) in German shepherd dogs (GSD) with presumptive degenerative lumbosacral disease (DLSS). STUDY DESIGN: Retrospective cohort study. SAMPLE POPULATION: One hundred forty-three GSD (125 police dogs and 18 pet dogs) presenting for either CES or prebreeding evaluation. Fifty-five were classified as affected by CES and diagnosed with DLSS, and 88 were classified as unaffected on the basis of clinical and imaging findings. METHODS: The position of the rostral edge of the sacral lamina was measured from radiographs and/or computed tomography (CT) scans. This position was compared between affected and unaffected dogs. In dogs that underwent both radiography and CT scanning, the agreement between sacral lamina localization using each imaging modality was determined. Owners/handlers were contacted to determine whether dogs subsequently developed clinical signs compatible with CES at a mean of 29 months (unaffected). RESULTS: The sacral lamina did not extend as far rostrally in affected dogs, compared to unaffected dogs (P = .04). Among the 88 dogs unaffected by CES at initial evaluation, 2 developed clinical signs consistent with CES at follow-up. CONCLUSION: Rostral projection of the sacral lamina, previously proposed as a potential risk factor in dogs with CES due to lumbosacral degeneration, was not associated with a diagnosis of DLSS in this study; the opposite was true. CLINICAL SIGNIFICANCE: Rostral projection of the sacral lamina may not be a predisposing factor in the development of CES due to DLSS in GSD.

Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine.

The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Evidence for Intramyocardial Disruption of Lipid Metabolism and Increased Myocardial Ketone Utilization in Advanced Human Heart Failure.

BACKGROUND: The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. METHODS AND RESULTS: We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic ß-hydroxybutyryl-CoA, in association with increased myocardial utilization of ß-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of ß-hydroxybutyrate and acetoacetate. CONCLUSIONS: These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart.

Metabolic tracing analysis reveals substrate-specific metabolic deficits in platelet storage lesion.

BACKGROUND: Storage of platelets (PLTs) results in a progressive defect termed PLT storage lesion (PSL). The PSL is characterized by poor PLT quality on a variety of assays. Metabolic defects are thought to underlie the PSL; thus this study was designed to quantitatively probe specific metabolic pathways over PLT storage. STUDY DESIGN AND METHODS: Relative incorporation of stable isotope-labeled substrates was quantified by isotopologue analysis of key acyl-coenzyme A (CoA) thioester products for fresh, viable (after collection, Days 2-5), and expired PLTs (after Day 5). We examined the incorporation of acetate, glucose, and palmitate into acetyl- and succinyl-CoA via liquid chromatography-tandem mass spectrometry. RESULTS: Storage-related defects in the incorporation of acetyl-CoA derived from acetate and palmitate were observed. Carbon derived from palmitate and acetate in succinyl-CoA was reduced over storage time. Glucose incorporation into succinyl-CoA increased in viable PLTs and then decreased in expired PLTs. Carbon derived from octanoate and pyruvate remained partially able to incorporate into acetyl- and succinyl-CoA in expired PLTs, with high variability in pyruvate incorporation. CONCLUSION: Isotopologue analysis is useful in probing substrate specific defects in the PSL.

Effect of dorsal laminectomy and dorsal annulectomy with partial lumbosacral discectomy on the volume of the lateral intervertebral neuroforamina in dogs when the lumbosacral junction is extended.

OBJECTIVE: To determine the effect of dorsal annulectomy and partial discectomy on the volume of the lumbosacral lateral intervertebral neurovascular foramina (intervertebral foramina) in canine cadavers during extension of the lumbosacral junction. STUDY DESIGN: Ex vivo experiment. SAMPLE POPULATION: Lumbosacral specimens from 10 large breed dogs euthanatized for reasons unrelated to lumbosacral disease. METHODS: The lumbosacral specimens were clamped in a wooden jig and scanned using computed tomography (CT) with the lumbosacral junction in a neutral position and loaded in extension using a tensioning device. The 3-dimensional volumes of the lumbosacral intervertebral neurovascular foramina were measured and the extent of any disc degeneration was determined from the CT data. A limited dorsal laminectomy of S1 and a dorsal LS annulectomy and partial discectomy were then performed. The lumbosacral specimens were remounted into the jig and loaded into extension at the same tension and were re-scanned. Measurements of intervertebral foraminal volume were then repeated. RESULTS: The mean volume of the lumbosacral foramina (n = 20) was 381 mm3 in neutral (unloaded) positioning and 137 mm3 when loaded in extension. Following dorsal annulectomy, the mean volume was significantly reduced by a mean of 28% to 98 mm3 (P < .01). The foraminal volume was reduced in 19/20 lumbosacral foramen, with the post-annulectomy volume ranging from 31% to 97% of the pre-annulectomy volume (3%-69% reduction). CONCLUSIONS: This study suggests that a dorsal annulectomy with partial discectomy may induce further dynamic collapse of the lumbosacral articulation in the dog.

Computed tomographic evaluation of dynamic alteration of the canine lumbosacral intervertebral neurovascular foramina.

OBJECTIVE: To develop a computed tomographic (CT) method to measure the volume of the lumbosacral intervertebral neurovascular foramina (IVF) in dogs, and determine the effect of the range of motion of the lumbosacral (LS) junction on this measurement in German shepherd dogs (GSDs) with degenerative lumbosacral stenosis (DLSS) compared to unaffected controls. STUDY DESIGN: In vivo analysis and retrospective case series. SAMPLE POPULATION: Twenty-four working Police GSDs, 12 diagnosed with DLSS and 12 unaffected by DLSS were compared to 10 Greyhounds without DLSS. METHODS: Three-dimensional renderings of CT data were used to measure the lumbosacral foraminal volume of dogs positioned in dorsal recumbency with the LS junction alternately positioned in extension, neutral position, and flexion. RESULTS: Volumetric analysis of the IVF was found repeatable for the extended and neutral positions (interclass correlation coefficient of 0.89 and 0.8, respectively). The mean lumbosacral IVF volume was decreased by 74% between LS flexion and extension in Greyhounds, compared to 79 and 85% reductions in GSDs unaffected and affected by DLSS, respectively. The lumbosacral IVF volume was decreased by 23% when comparing extended to neutral LS positions in Greyhounds, 29% in unaffected GSDs, and 31% in affected GSDs. IVF volumes were smaller in affected GSDs compared to unaffected GSDs (P < .05) and Greyhounds (P < .01). CONCLUSIONS: Positioning the LS junction in full extension decreases the volume of the lumbosacral IVF. This dynamic narrowing was more pronounced in GSDs with signs of DLSS than in GSDs not overtly affected by DLSS.

Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism.

RATIONALE: Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role. METHODS: An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity. RESULTS: HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells. CONCLUSIONS: A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters.

Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.

Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid ß-oxidation, supporting acetyl-coenzyme A levels.

Rotenone is a naturally occurring mitochondrial complex I inhibitor with a known association with parkinsonian phenotypes in both human populations and rodent models. Despite these findings, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid metabolism in SH-SY5Y neuroblastoma cells exposed to rotenone. The absolute levels of acetyl-CoA were found to be maintained despite a significant decrease in glucose-derived acetyl-CoA. Furthermore, palmitoyl-CoA levels were maintained, whereas the levels of many of the medium-chain acyl-CoA species were significantly reduced. Additionally, using isotopologue analysis, we found that ß-oxidation of fatty acids with varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation, as has been found previously in other cell types. Finally, palmitoylcarnitine levels were increased in response to rotenone, indicating an increase in fatty acid import. Taken together, these findings show that alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone.

Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and ß-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.

Rotenone Stereospecifically Increases (S)-2-Hydroxyglutarate in SH-SY5Y Neuronal Cells.

The α-ketoglutarate metabolite, 2-hydroxyglutarate (2-HG), has emerged as an important mediator in a subset of cancers and rare inherited inborn errors of metabolism. Because of potential enantiospecific metabolism, chiral analysis is essential for determining the biochemical impacts of altered 2-HG metabolism. We have developed a novel application of chiral liquid chromatography-electron capture/atmospheric pressure chemical ionization/mass spectrometry, which allows for the quantification of both (R)-2-HG (D-2-HG) and (S)-2-HG (L-2-HG) in human cell lines. This method avoids the need for chiral derivatization, which could potentially distort enantiomer ratios through racemization during the derivatization process. The study revealed that the pesticide rotenone (100 nM), a mitochondrial complex I inhibitor, caused a significant almost 3-fold increase in the levels of (S)-2-HG, (91.7 ± 7.5 ng/10(6) cells) when compared with the levels of (R)-2-HG (24.1 ± 1.2 ng/10(6) cells) in the SH-SY5Y neuronal cells, a widely used model of human neurons. Stable isotope tracers and isotopologue analysis revealed that the increased (S)-2-HG was derived primarily from l-glutamine. Accumulation of highly toxic (S)-2-HG occurs in the brains of subjects with reduced L-2-HG dehydrogenase activity that results from mutations in the L2HGDH gene. This suggests that the observed stereospecific increase of (S)-2-HG in neuronal cells is due to rotenone-mediated inhibition of L-2-HG dehydrogenase but not D-2-HG dehydrogenase. The high sensitivity chiral analytical methodology that has been developed in the present study can also be employed for analyzing other disruptions to 2-HG formation and metabolism such as those resulting from mutations in the isocitrate dehydrogenase gene.

ACCURACY OF RADIOGRAPHIC DETECTION OF THE CRANIAL MARGIN OF THE DORSAL LAMINA OF THE CANINE SACRUM.

An elongated sacral lamina has been described as one of the contributing factors for dogs with cauda equina syndrome due to degenerative lumbosacral stenosis (DLSS); however, published evidence is lacking on the accuracy of radiographic screening for the presence of this lesion. Objectives of this prospective, cross-sectional cadaver study were to describe the accuracy and repeatability of detection of the cranial sacral lamina margin on plain lateral radiographs of the lumbosacral junction in dogs. Twenty-five medium and large breed canine cadavers were radiographed before and after placement of a radiopaque hook in the cranial margin of the sacral lamina. Three independent evaluators placed digital markers at the perceived margin on preinterventional radiographs. The distance from perceived location to the true location on postinterventional radiographs was recorded for each dog and observer. A discordance threshold (distance between perceived and actual margin) of 1.5 mm was subjectively defined as clinically relevant. The three evaluators demonstrated good repeatability, although the accuracy for margin detection was only fair (mean discordance 1.7 mm). Evaluators demonstrated greater accuracy in identifying the landmark in juveniles (1.4 mm) vs. adults (1.8 mm; P < 0.01). Results of this study indicated that observer repeatability is good and accuracy is fair for correctly identifying the radiographic cranial margin of the sacral lamina in dogs. This should be taken into consideration when interpreting elongation of the sacral lamina in radiographs of dogs with suspected DLSS, especially adults.

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.

RATIONALE: Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. METHODS: Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. RESULTS: Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. CONCLUSIONS: This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

Comparison between a novel bovine xenoimplant and autogenous cancellous bone graft in tibial tuberosity advancement.

OBJECTIVE: To compare radiographic healing and clinical outcome of a frontal-opening wedge osteotomy of canine tibiae when the osteotomy site is packed with either a novel bovine xenograft or standard autogenous cancellous bone graft (ACBG). STUDY DESIGN: Cohort study. ANIMALS: Dogs (n = 82) with partial or complete rupture of the cranial cruciate ligament that had tibial tuberosity advancement (TTA). METHODS: In 48 dogs, the osteotomy was packed with a novel bovine xenograft and in 34 dogs, ACBG was used. Eight week postoperative radiographs from both groups were graded for osteotomy healing using a 0-4-point scale. Data were analyzed using a Mann-Whitney test with significance set at P < .05. RESULTS: Thirty-four dogs (39 stifles) with xenoimplants had complete records and radiographic follow-up at 8 weeks. No significant differences between xenografting and autografting were identified in grading of osteotomy fill, osteointegration, or healing of the distal osteotomy. Significant differences were noted in grading of osteotomy healing proximally (autograft > xenoimplant) and of opacity in the osteotomy site (xenoimplant > autograft). CONCLUSIONS: Radiographic evidence of healing of the xenoimplanted portion of the TTA osteotomy was equivalent to results with ACBG. Healing of the proximal osteotomy site (above the cage) was improved when ACBG was used as the graft.