Prevalence of fatigue and cognitive impairment after traumatic brain injury.

BACKGROUND: Following traumatic brain injury (TBI) some patients develop lingering comorbid symptoms of fatigue and cognitive impairment. The mild cognitive impairment self-reported by patients is often not detected with neurocognitive tests making it difficult to determine how common and severe these symptoms are in individuals with a history of TBI. This study was conducted to determine the relative prevalence of fatigue and cognitive impairment in individuals with a history of TBI. METHODS: The Fatigue and Altered Cognition Scale (FACs) digital questionnaire was used to assess self-reported fatigue and cognitive impairment. Adults aged 18-70 were digitally recruited for the online anonymous study. Eligible participants provided online consent, demographic data, information about lifetime TBI history, and completed the 20 item FACs questionnaire. RESULTS: A total of 519 qualifying participants completed the online digital study which included 204 participants with a history of TBI of varied cause and severity and 315 with no history of TBI. FACs Total Score was significantly higher in the TBI group (57.7 ± 22.2) compared to non-TBI (39.5 ± 23.9; p<0.0001) indicating more fatigue and cognitive impairment. When stratified by TBI severity, FACs score was significantly higher for all severity including mild (53.9 ± 21.9, p<0.0001), moderate (54.8 ± 24.4, p<0.0001), and severe (59.7 ± 20.9, p<0.0001) TBI. Correlation analysis indicated that more severe TBI was associated with greater symptom severity (p<0.0001, r = 0.3165). Ancillary analysis also suggested that FACs scores may be elevated in participants with prior COVID-19 infection but no history of TBI. CONCLUSIONS: Adults with a history of even mild TBI report significantly greater fatigue and cognitive impairment than those with no history of TBI, and symptoms are more profound with greater TBI severity.

The altered TBI fecal microbiome is stable and functionally distinct.

Introduction: Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Methods: Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. Results: A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Discussion: Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Low growth hormone secretion associated with post-acute sequelae SARS-CoV-2 infection (PASC) neurologic symptoms: A case-control pilot study.

OBJECTIVE: To determine if patients that develop lingering neurologic symptoms of fatigue and "brain fog" after initial recovery from coronavirus disease 2019 (COVID-19) have persistent low growth hormone (GH) secretion as seen in other conditions with similar symptom etiology. DESIGN: In this case-control observational pilot study, patients reporting lingering neurologic post-acute sequelae of SARS-CoV-2 (PASC, n = 10) symptoms at least 6 months after initial infection were compared to patients that recovered from COVID-19 without lingering symptoms (non-PASC, n = 13). We compared basic blood chemistry and select metabolites, lipids, hormones, inflammatory markers, and vitamins between groups. PASC and non-PASC subjects were tested for neurocognition and GH secretion, and given questionnaires to assess symptom severity. PASC subjects were also tested for glucose tolerance and adrenal function. RESULTS: PASC subjects reported significantly worse fatigue, sleep quality, depression, quality of life, and gastrointestinal discomfort compared to non-PASC. Although PASC subjects self-reported poor mental resilience, cognitive testing did not reveal significant differences between groups. Neurologic PASC symptoms were not linked to inflammatory markers or adrenal insufficiency, but were associated with reduced growth hormone secretion. CONCLUSIONS: Neurologic PASC symptoms are associated with gastrointestinal discomfort and persistent disruption of GH secretion following recovery from acute COVID-19. (www. CLINICALTRIALS: gov; NCT04860869).

Efficient assessment of brain fog and fatigue: Development of the Fatigue and Altered Cognition Scale (FACs).

Debilitating symptoms of fatigue and accompanying "brain fog" are observed among patients with various chronic health conditions. Unfortunately, an efficient and psychometrically sound instrument to assess these co-occurring symptoms is unavailable. Here, we report the development and initial psychometric properties of the Fatigue and Altered Cognition Scale (the FACs), a measure of self-reported central fatigue and brain fog. Traumatic brain injury (TBI) was chosen to model and develop the FACs due to research team expertise and established links between TBI and the symptom complex. Potential items were generated by researchers and clinicians with experience treating these symptoms, drawing from relevant literature and review of patient responses to measures from past and current TBI studies. The 20 candidate items for the FACs-ten each to assess altered cognition (i.e., brain fog) and central fatigue-were formatted on an electronic visual analogue response scale (eVAS) via an online survey. Demographic information and history of TBI were obtained. A total of 519 participants consented and provided usable data (average age = 40.23 years; 73% female), 204 of whom self-reported a history of TBI (75% reported mild TBI). Internal consistency and reliability values were calculated. Confirmatory factor analysis (CFA) examined the presumed two-factor structure of the FACs and a one-factor solution for comparison. A measurement invariance test of the two latent constructs (altered cognition, fatigue) among participants with and without TBI was conducted. All items demonstrated normal distribution. Cronbach's alpha coefficients indicated good internal consistency for both factors (α's = .95). Omega reliability values were favorable (α's = .95). CFA supported the presumed two-factor model and item loadings which outperformed the one-factor model. Measurement invariance found the two-factor structure was consistent between the two groups. Implications of these findings, study limitations, and potential use of the FACs in clinical research and practice are discussed.

Traumatic brain injury, abnormal growth hormone secretion, and gut dysbiosis.

The gut microbiome has been implicated in a variety of neuropathologies with recent data suggesting direct effects of the microbiome on host metabolism, hormonal regulation, and pathophysiology. Studies have shown that gut bacteria impact host growth, partially mediated through the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis. However, no study to date has examined the specific role of GH on the fecal microbiome (FMB) or the changes in this relationship following a traumatic brain injury (TBI). Current literature has demonstrated that TBI can lead to either temporary or sustained abnormal GH secretion (aGHS). More recent literature has suggested that gut dysbiosis may contribute to aGHS leading to long-term sequelae now known as brain injury associated fatigue and cognition (BIAFAC). The aGHS observed in some TBI patients presents with a symptom complex including profound fatigue and cognitive dysfunction that improves significantly with exogenous recombinant human GH treatment. Notably, GH treatment is not curative as fatigue and cognitive decline typically recur upon treatment cessation, indicating the need for additional studies to address the underlying mechanistic cause.

Is there a role for growth hormone replacement in adults to control acute and post-acute COVID-19?

The SARS-CoV-2 pandemic created a multitude of medical crossroads requiring real time adaptations of best practice covering preventative and interventional aspects of care. Among the many discoveries borne from efforts to address the myriad clinical presentations across multiple organ systems was a common impact on tissues with cells that express the ACE-2 receptor. The vast majority of acute infections began and often ended in the respiratory tract, but more recent evaluations have confirmed significant extrapulmonary manifestations including symptom clusters that extend beyond the acute phase of infection collectively referred to as "post-acute sequelae SARS-CoV-2 infection" (PASC) or more commonly as "long (-haul) COVID". Both acute SARS-CoV-2 infection and PASC are associated with gut microbiome dysbiosis and alterations in the gut-brain and HPA-axis in a subset of the infected. Mounting evidence suggests these extrapulmonary manifestations may ultimately lead to reduced growth hormone (GH) secretion as demonstrated following stimulation tests. Disrupted GH secretion could cause or exacerbate long lasting neuropsychological symptoms as seen in other similar manifesting conditions. Ongoing clinical research has shown promising improvement in PASC patients with fatigue and cognition complaints can be achieved via GH replacement therapy. GH stimulation testing should be considered in PASC workups and future research should delve deeper into the mechanistic effects of GH on acute COVID and PASC.

Impact of Adjunct Testosterone on Cancer-Related Fatigue: An Ancillary Analysis from a Controlled Randomized Trial.

Many cancer patients undergoing treatment experience cancer-related fatigue (CRF). Inflammatory markers are correlated with CRF but are not routinely targeted for treatment. We previously demonstrated in an NIH-funded placebo-controlled, double-blind, randomized clinical trial (NCT00878995, closed to follow-up) that seven weekly injections of 100 mg adjunct testosterone preserved lean body mass in cancer patients undergoing standard-of-care treatment in a hospital setting. Because testosterone therapy can reduce circulating proinflammatory cytokines, we conducted an ancillary analysis to determine if this testosterone treatment reduced inflammatory burden and improved CRF symptoms and health-related quality of life. Randomization was computer-generated and managed by the pharmacy, which dispensed testosterone and placebo in opaque syringes to the administering study personnel. A total of 24 patients were randomized (14 placebo, 10 testosterone), and 21 were included in the primary analysis (11 placebo, 10 testosterone). Testosterone therapy did not ameliorate CRF symptoms (placebo to testosterone difference in predicted mean multidimensional fatigue symptom inventory scores: -5.6, 95% CI: -24.6 to 13.3), improve inflammatory markers, or preserve health-related quality of life and functional measures of performance in late-stage cancer patients.

Changes in Northern Elephant Seal Skeletal Muscle Following Thirty Days of Fasting and Reduced Activity.

Northern elephant seals (NES, Mirounga angustirostris) undergo an annual molt during which they spend ∼40 days fasting on land with reduced activity and lose approximately one-quarter of their body mass. Reduced activity and muscle load in stereotypic terrestrial mammalian models results in decreased muscle mass and capacity for force production and aerobic metabolism. However, the majority of lost mass in fasting female NES is from fat while muscle mass is largely preserved. Although muscle mass is preserved, potential changes to the metabolic and contractile capacity are unknown. To assess potential changes in NES skeletal muscle during molt, we collected muscle biopsies from 6 adult female NES before the molt and after ∼30 days at the end of the molt. Skeletal muscle was assessed for respiratory capacity using high resolution respirometry, and RNA was extracted to assess changes in gene expression. Despite a month of reduced activity, fasting, and weight loss, skeletal muscle respiratory capacity was preserved with no change in OXPHOS respiratory capacity. Molt was associated with 162 upregulated genes including those favoring lipid metabolism. We identified 172 downregulated genes including those coding for ribosomal proteins and genes associated with skeletal muscle force transduction and glucose metabolism. Following ∼30 days of molt, NES skeletal muscle metabolic capacity is preserved although mechanotransduction may be compromised. In the absence of exercise stimulus, fasting-induced shifts in muscle metabolism may stimulate pathways associated with preserving the mass and metabolic capacity of slow oxidative muscle.

Altered Fecal Microbiome Years after Traumatic Brain Injury.

Patients with chronic traumatic brain injury (TBI) requiring long-term, permanent care suffer a myriad of clinical symptoms (i.e., impaired cognition, fatigue, and other conditions) that persist for years beyond the acute brain injury. In addition to these comorbid clinical symptoms, chronic TBI patients exhibit altered amino acid and hormonal profiles with distinct cytokine patterns suggesting chronic inflammation. This metabolic link suggests a role of the gut-brain axis in chronic TBI. Thus, we utilized a two-site trial to investigate the role of the gut-brain axis in comorbidities of chronic TBI. The fecal microbiome profile of 22 moderate/severe TBI patients residing in permanent care facilities in Texas and California was compared to 18 healthy age-matched control subjects working within the participating facilities. Each fecal microbiome was characterized by 16S(V4) ribosomal RNA (rRNA) gene sequencing and metagenomic genome sequencing approaches followed by confirmatory full 16S rRNA gene sequencing or focused tuf gene speciation and specific quantitative polymerase chain reaction evaluation of selected genera or species. The average chronic TBI patient fecal microbiome structure was significantly different compared to the control cohort, and these differences persisted after group stratification analysis to identify any unexpected confounders. Notably, the fecal microbiome of the chronic TBI cohort had absent or reduced Prevotella spp. and Bacteroidies spp. Conversely, bacteria in the Ruminococcaceae family were higher in abundance in TBI compared to control profiles. Previously reported hypoaminoacidemia, including significantly reduced levels of l-tryptophan, l-sarcosine, ß-alanine, and alanine, positively correlated with the reduced levels of Prevotella spp. in the TBI cohort samples compared to controls. Although the sequelae of gut-brain axis disruption after TBI is not fully understood, characterizing TBI-related alterations in the fecal microbiome may provide biomarkers and therapeutic targets to address patient morbidity.

Effect of high-fat diet on peripheral blood mononuclear cells and adipose tissue in early stages of diet-induced weight gain.

Subcutaneous adipose tissue (scAT) and peripheral blood mononuclear cells (PBMC) play a significant role in obesity-associated systemic low-grade inflammation. High-fat diet (HFD) is known to induce inflammatory changes in both scAT and PBMC. However, the time course of the effect of HFD on these systems is still unknown. The aim of the present study was to determine the time course of the effect of HFD on PBMC and scAT. New Zealand white rabbits were fed HFD for 5 or 10 weeks (i.e. HFD-5 and HFD-10) or regular chow (i.e. control (CNT)-5 and CNT-10). Thereafter, metabolic and inflammatory parameters of PBMC and scAT were quantified. HFD induced hyperfattyacidaemia in HFD-5 and HFD-10 groups, with the development of insulin resistance in HFD-10, while no changes were observed in scAT lipid metabolism and inflammatory status. HFD activated the inflammatory pathways in PBMC of HFD-5 group and induced modified autophagy in that of HFD-10. The rate of fat oxidation in PBMC was directly associated with the expression of inflammatory markers and tended to inversely associate with autophagosome formation markers in PBMC. HFD affected systemic substrate metabolism, and the metabolic, inflammatory and autophagy pathways in PBMC in the absence of metabolic and inflammatory changes in scAT. Dietary approaches or interventions to avert HFD-induced changes in PBMC could be essential to prevent metabolic and inflammatory complications of obesity and promote healthier living.

A randomized trial of adjunct testosterone for cancer-related muscle loss in men and women.

BACKGROUND: Cancer cachexia negatively impacts cancer-related treatment options, quality of life, morbidity, and mortality, yet no established therapies exist. We investigated the anabolic properties of testosterone to limit the loss of body mass in late stage cancer patients undergoing standard of care cancer treatment. METHODS: A randomized, double-blind, placebo-controlled phase II clinical trial was undertaken to assess the potential therapeutic role of adjunct testosterone to limit loss of body mass in patients with squamous cell carcinoma of the cervix or head and neck undergoing standard of care treatment including chemotherapy and chemoradiation. Patients were randomly assigned in blocks to receive weekly injections of either 100 mg testosterone enanthate or placebo for 7 weeks. The primary outcome was per cent change in lean body mass, and secondary outcomes included assessment of quality of life, tests of physical performance, muscle strength, daily activity levels, resting energy expenditure, nutritional intake, and overall survival. RESULTS: A total of 28 patients were enrolled, 22 patients were studied to completion, and 21 patients were included in the final analysis (12 placebo, nine testosterone). Adjunct testosterone increased lean body mass by 3.2% (95% confidence interval [CI], 0-7%) whereas those receiving placebo lost 3.3% (95% CI, -7% to 1%, P = 0.015). Although testosterone patients maintained more favourable body condition, sustained daily activity levels, and showed meaningful improvements in quality of life and physical performance, overall survival was similar in both treatment groups. CONCLUSIONS: In patients with advanced cancer undergoing the early phase of standard of care therapy, adjunct testosterone improved lean body mass and was also associated with increased quality of life, and physical activity compared with placebo.

Sex-dependent difference in the relationship between adipose-tissue cholesterol efflux and estradiol concentrations in young healthy humans.

BACKGROUND: Impaired adipose tissue function and lower levels of high density lipoprotein cholesterol (HDL-C) have been implicated in the development of vascular dementia, and metabolic diseases such as hypertension, atherosclerosis, type 2 diabetes (T2D) and metabolic syndrome. Interestingly, both the substrate fluxes in adipose tissue and HDL-C concentration differ between men and women. Moreover, adipose tissue cholesterol efflux has been implicated in modulation of HDL-C levels. Thus, we aimed to determine if the association between serum estradiol levels and adipose tissue cholesterol efflux is sex-dependent. METHOD: We evaluated the serum estradiol levels and adipose tissue cholesterol efflux in young healthy men (n=5) and women (n=3). Adipose tissue cholesterol efflux was determined using subcutaneous microdialysis probes. Linear regression analyses were used to determine the relationship between the parameters, p<0.05 was considered as statistically significant. RESULTS: Our data demonstrated that serum estradiol levels directly associated with adipose tissue cholesterol efflux; however, the relationships may be sex-dependent. We discussed our results in the context of currently available data regarding sex-dependent variability in adipose tissue function and HDL-C metabolism as a potential contributor to higher rates of vascular dementia in men. Further research is required to understand the sex-dependent and -independent variabilities in adipose tissue metabolism to determine novel targets for interventions to prevent the development of vascular dementia.

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O2. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism

Glucocorticoids increase skeletal muscle NF-κB inducing kinase (NIK): links to muscle atrophy.

Glucocorticoids (GC) are a frontline therapy for numerous acute and chronic diseases because of their demonstrated efficacy at reducing systemic inflammation. An unintended side effect of GC therapy is the stimulation of skeletal muscle atrophy. Pathophysiological mechanisms responsible for GC-induced skeletal muscle atrophy have been extensively investigated, and the ability to treat patients with GC without unintended muscle atrophy has yet to be realized. We have reported that a single, standard-of-care dose of Methylprednisolone increases in vivo expression of NF-κB-inducing kinase (NIK), an important upstream regulatory kinase controlling NF-κB activation, along with other key muscle catabolic regulators such as Atrogin-1 and MuRF1 that induce skeletal muscle proteolysis. Here, we provide experimental evidence that overexpressing NIK by intramuscular injection of recombinant human NIK via adenoviral vector in mouse tibialis anterior muscle induces a 30% decrease in the average fiber cross-sectional area that is associated with increases in mRNA expression of skeletal muscle atrophy biomarkers MuRF1, Atrogin-1, myostatin and Gadd45. A single injection of GC induced NIK mRNA and protein within 2 h, with the increased NIK localized to nuclear and sarcolemmal locations within muscle fibers. Daily GC injections induced skeletal muscle fore limb weakness as early as 3 days with similar atrophy of muscle fibers as observed with NIK overexpression. NIK overexpression in primary human skeletal muscle myotubes increased skeletal muscle atrophy biomarkers, while NIK knockdown significantly attenuated GC-induced increases in NIK and Atrogin-1. These results suggest that NIK may be a novel, previously unrecognized mediator of GC-induced skeletal muscle atrophy.

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism.

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O2. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.

Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.

Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40 to 4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melon-headed whale, which had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives.

Myoglobin extraction from mammalian skeletal muscle and oxygen affinity determination under physiological conditions.

An accurate determination of myoglobin (Mb) oxygen affinity (P50) can be difficult due to hemoglobin (Hb) contamination and autoxidation of Mb to metMb which is incapable of binding oxygen. To reduce Mb autoxidation, P50 is often measured at refrigerated temperatures. However, the temperature dependent shift in Mb oxygen affinity results in a greater oxygen affinity (lower P50) at colder temperatures than occurs at physiological temperature (ca. 37-39°C) for birds and mammals. Utilizing the temperature dependent pH shift of Tris buffer, we developed novel methods to extract Mb from vertebrate muscle samples and remove Hb contamination while minimizing globin autoxidation. Cow (Bos taurus) muscle tissue (n=5) was homogenized in buffer to form a Mb solution, and Hb contamination was removed using anion exchange chromatography. A TCS Hemox Blood Analyzer was then used to quickly generate an oxygen dissociation curve for the extracted Mb. The oxygen affinity of extracted bovine Mb was compared to commercially available horse heart Mb. The oxygen affinity of extracted cow Mb (P50=3.72±0.16 mmHg) was not statistically different from commercially prepared horse heart Mb (P50=3.71±0.10 mmHg). With high yield Mb extraction and fast generation of an oxygen dissociation curve, it was possible to consistently determine Mb P50 under physiologically relevant conditions for endothermic vertebrates.