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Evaluation of hepatic health, muscle strength and antioxidant response for therapeutic insight of ostarine in mice
*Corresponding author: Devinder Kumar Dhawan, Department of Biophysics, Panjab University, Chandigarh, India. dhawan@pu.ac.in
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Received: ,
Accepted: ,
How to cite this article: Sharma S, Saini A, Dhawan DK, Evaluation of hepatic health, muscle strength and antioxidant response for therapeutic insight of ostarine in mice. Indian J Physiol Pharmacol. doi: 10.25259/IJPP_243_2025
Abstract
Selective androgen receptor modulators (SARMs) are a family of small molecules that bind to androgen receptors to elicit therapeutic responses in age-related disorders. SARMs exert their beneficial effects by preventing muscle wasting and supporting bone regeneration, as well as helping in overcoming sarcopenia. Ostarine, a known SARM, improves muscle strength and helps in reversing osteoporosis. Preliminary studies have affirmed ostarine to be toxic at higher doses; however, extensive analysis remains to be done. We have undertaken this study for the first time to understand whether ostarine at higher dosage may cause adverse effects on mice liver. The cellular antioxidant framework, liver marker enzymes and histoarchitectural changes of hepatic tissue were investigated. Further, Rotarod and actophotometer tests were undertaken as markers, and mice treated with ostarine displayed increased muscular strength and locomotor activity. However, activities of liver marker enzymes namely, serum glutamic pyruvic transaminase and alkaline phosphatase, were significantly elevated in mice treated with ostarine. Further, ostarine treatment caused a significant increase in the levels of reactive oxygen species, lipid peroxidation and protein carbonyl content but revealed an appreciable decrease in glutathione levels. The activities of antioxidative enzymes were also significantly elevated following ostarine treatment. Further, ostarine treatment resulted in mitochondrial swelling and caused alterations in normal hepatic histoarchitecture. Therefore, the present study clearly indicates that ostarine may be an effective drug against locomotive disorders, but is toxic to the liver at higher dose.
Keywords
Liver
Ostarine
Sarcopenia
Selective androgen receptor modulator
Toxicity
INTRODUCTION
AAndrogen receptor (AR) is an important target for androgens, including testosterone, whose active metabolite, dihydrotestosterone, is not only required for sexual functions but also regulates diverse functions, including cellular differentiation.[1] The androgens act through binding to the AR, and drugs such as flutamide and enzalutamide are antagonists of ARs as they compete with androgens such as testosterone and dihydrotestosterone for binding to AR domains.[2] On the other hand, selective androgen receptor modulators (SARMs) are compounds that have affinity for AR and are selectively anabolic or catabolic in nature.[3] They have been shown to have a profound effect on bone density and hence have found their uses against old age diseases such as osteoporosis and sarcopenia.[4]
Ostarine, a popular SARM also known as Enobosarm or MK2866, works on enhancing muscle strength[5] and helps in the regeneration of osteoporosis and degenerative disorders.[6,7] However, it has been misused by athletes to increase their performance, as has been reported in an earlier study by Kintz et al. in 2020.[8] The liver is the primary organ responsible for metabolism and clearance of drugs and environmental pollutants from the body.[7] The system of flushing out toxic compounds makes our liver vulnerable to damage.[8] Ostarine is metabolised in the liver and then eliminated from the body through faeces and urine. However, information is lacking with regard to the adverse effects of ostarine on liver functions, except for one case report by Bedi et.al in 2021[9], which reported jaundice and scleral icterus in a healthy man who was taking enobosarm for 2 months. The present study was designed to elucidate the adverse effects of ostarine on mice liver by studying key indices which included liver markers, antioxidative enzymes and histoarchitecture.
MATERIALS AND METHODS
Chemicals
Ostarine was procured from Direct Sarms Pvt Ltd. (Orlando, USA). Chemicals such as 2,2- diphenyl-1-picrylhydrazyl, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid and Folin–Ciocalteu reagent for biochemical estimations were purchased from Sigma-Aldrich (St. Louis, MO, USA), BLD pharm and SRL, respectively. Liver profile test kits were purchased from Reckon Diagnostics Pvt. Ltd.
Housing of animals
Twelve healthy male Balb/c mice, 20 weeks old, weighing between 39 and 45 g were procured from the central animal house, Panjab University, Chandigarh, and were acclimated at an in-house animal facility of biophysics department. The mice were housed in acrylic cages and were maintained at a controlled temperature. They were fed with standard rodent pellets and water ad libitum. All the experimental protocols were approved by the Committee for Control and Supervision of Experiments on Animal (CCSEA), Panjab University, Chandigarh (PU/45/99/CPCSEA/IAEC/2023/856).
Experimental design
A total of 12 mice were divided randomly and equally into two groups.
Mice in Group 1 (normal control) were given a regular rodent pelleted diet and water ad libitum. Mice in Group 2 (Ostarine-treated) were administered ostarine orally every day at a dose of 10 mg/kg b.w. for a total duration of 20 days (sub-acute toxicity model) and also had free access to drinking water and diet.
Blood/serum collection
Blood samples were collected by puncturing the retro-orbital plexus using sterilised glass capillaries. The blood samples were then maintained at room temperature for a specified period and centrifuged at 2000 rpm for 10–15 min to separate the serum, which was subsequently used for the estimation of liver marker enzymes.
Homogenisation of hepatic tissue
The animals were sacrificed under anaesthesia by cervical dislocation after completion of 20 days. The livers were removed immediately, and one lobe was homogenised for biochemical estimations, and the other lobe was fixed in 10% formal saline (formalin in normal saline) for histoarchitectural examination. The liver tissues (10% w/v) were homogenised in 10 mM phosphate buffer saline and centrifuged at 2000 g for 10 min at 4°C to remove cell debris and nuclear pellets. The pellets were discarded, and the supernatants were centrifuged at 10,000 g for 30 min at 4°C. The final supernatants so collected were post-mitochondrial fractions (PMFs) and were used in most antioxidant assays.
Motor activity examination
Neuromuscular coordination assessment
The Rotarod test was used to assess motor coordination and maintenance of balance, and begins with a training trial of 3 min by placing the animal on a rotating bar at varying speeds of 10, 15, and 20 rpm.[10] The time of the fall of the animal was noted. After training at a 10 rpm speed of the rotating bar, the animal was allowed to rest for at least 5 min before taking the three test trials. The time of fall, as well as the speed at which the animal fell, was recorded, and a graph was plotted.
Locomotion analysis
To study the change in locomotive ability of the animal,[11] the actophotometer was used, which measures the activity of animals using a photocell. The interruption of a beam of light due to the movement of animals was recorded for a period of 5 min for each animal.
Liver marker enzymes
The biochemical estimations of marker enzymes were done in the serum of animals. The levels of alkaline phosphatase (ALP) were estimated in accordance with the procedure based on the recommendations by the German Society for Clinical Chemistry (GSCC) using a diagnostic kit.[12] Similarly, kits were used to study the levels of serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT).[13]
Protein estimation
Protein contents were estimated in both the crude extract and PMF of liver samples according to the method of Lowry et.al in 1968.[14] BSA was used as a standard at a concentration of 1 mg/mL.
Oxidative stress assessment
Reactive oxygen species (ROS)
ROS in liver samples were estimated using the method as described by Wang and Joseph, in 1999.[15] In this method, the conversion of the non-fluorescent dye 2,7-dichlorofluorescein diacetate to the fluorescent compound Dichlorofluorescein (DCF) was measured using a spectrofluorometer (Shimadzu, Japan) at an excitation of 485 nm and an emission of 535 nm.
Lipid peroxidation (LPO)
The levels of LPO were measured by using the method of Ohkawa et.al, 1979.[16] The method measures the reaction of TBA with malondialdehyde, which was measured spectrophotometrically at 532 nm.
Reduced glutathione (GSH)
The method used in the estimation of reduced GSH analysis was described by Ellman in 1959.[16] In this method, PMFs were used, and the proteins were precipitated using trichloroacetic acid (TCA) and centrifuged. The supernatants were mixed with Ellman’s Reagent to bring a light-yellow colour, which was measured at 412 nm.
Glutathione reductase (GR) and glutathione peroxidase (GPx)
Both enzymes are a part of the redox GSH cycle. The activity of GPx was measured using H202 as the substrate by following the method of Lawrence and Burk 1976.[17] GR activity was assessed using the method of Horn (1965)[18] in which the reduction of oxidised GSH to reduced GSH was monitored at a wavelength of 340 nm. The results were expressed as nmol NADPH oxidised/min/mg protein.
Glutathione-S-transferase (GST)
The method followed for GST enzyme estimation was described by Warholm et.al, in 1985.[19] In this test, the conjugation of 1-chloro-2,4-dinitrobenzene with reduced GSH was measured. This conjugation was accompanied by an increase in absorbance at 340 nm for 3 min.
Superoxide dismutase (SOD)
The method followed to assay SOD activity was originally described by Kono et.al.,[20] in 1978. The basic principle of the protocol was to check the inhibitory effect of SOD on the reduction of nitro blue tetrazolium chloride (NBT) dye by superoxide anions, which were generated by the photooxidation of hydroxylamine hydrochloride. The resultant product was measured at 560 nm at 30 s intervals for 2 min using a spectrophotometer.
Catalase (CAT)
The method of Aebi, 1974[21] was used for the estimation of CAT activity. The protocol required the post-mitochondrial fraction to be added to phosphate buffer at pH 7.0, and the initiation of the reaction required the addition of freshly prepared H2O2, and the rate of decomposition of H2O2 was checked by measuring absorbance for 2 min at 30 s interval at 240 nm.
Protein carbonyl content (PCC)
The method followed for the estimation of PCCs was performed as described by Levine et.al, in 1990. The proteins following precipitation with 20% TCA were treated with 2,4-dinitrophenylhydrazine for 1 h. Then the pellets were washed with ethanol and ethyl acetate in a ratio of 1:1. Finally, after washing, the pellets were dissolved in 6M guanidine, and the absorbance was measured at 370 nm.
Xanthine oxidase (XO)
XO enzyme activity was determined in the liver tissue using a fluorimetric method.[23] The frozen liver tissue samples were homogenised in 0.25 M sucrose, 10 mM DTT, 0.2 mM Phenylmethylsulfonyl fluoride, 0.1 mM Ethylene diamine tetraacetic acid, and 50 mM K-phosphate, pH 7.4. The homogenates were centrifuged, and the enzyme activity was measured by calculating the slope of the increase in fluorescence on adding pterin to the supernatants. Allopurinol was added to stop the reaction that measured the conversion of pterin to isoxanthopterin. The enzyme activity was expressed as nmol/min/mg of protein.
MITOCHONDRIAL SWELLING
To study the integrity of mitochondrial membranes, mitochondrial swelling and contraction were measured as described by Tedeshi and Harris (1955).[24] This method is based on measuring the increase in absorbance of mitochondria in a contracted state, resulting from cation influx, at a wavelength of 520 nm.
Histopathological examination
Liver tissue samples were fixed in 10% Formalin-saline. Then they were dehydrated in ascending grades of alcohol, cleared in benzene, and subsequently were embedded in paraffin wax at a temperature of 60 °C. The blocks were made, and 5 µm thin sections were obtained, which were then stained with haematoxylin and eosin dye for better examination under the light microscope for studying any histoarchitectural changes.
Statistics
The data were expressed as Mean ± S.D. The statistical significance (P < 0.5) of the results obtained for various comparisons was estimated using Student’s t-test.
RESULTS
Analysis of motor functions
Rotarod analysis
The motor performance of the animals was assessed by determining the time the animal remains on the rotating Rotarod before falling. The animals treated with ostarine showed an increase in muscular strength, as evidenced by increased time spent on the Rotarod rotating at different speeds as compared to controls. A percentage increase of 31.16% was seen in the performance of the Ostarine-administered group on Day 20 when compared to Day 0 [Figure 1a].
- (a) Depicts muscular coordination of mice using the Rotarod analysis at a speed of a1 (10 rpm), a2 (15 rpm) and a3 (20 rpm). Blue depicts Control, and Red depicts OST (Ostarine administered to mice at 10 mg/kg b.w.). (b) Graphical representation of locomotive activity of mice using an actophotometer. Values represented in Mean ± S.D (n = 6) with level of significance between Control and OST (10 mg/kg b.w. Ostarine administered mice) *P < 0.05.
Locomotion activity
The locomotor activity of ostarine-treated animals revealed a mere 7% increase in locomotive activity on day 10, whereas a significant increase up to 65.8% was seen on day 20 as compared to the control group. The trajectory of performance is similar in both groups [Figure 1b]. Paired t-tests revealed significant changes in locomotive activity within-group from Day 10 to Day 20 in both the OST-treated (mean difference = 100.33 ± 19.05, t(5) = 5.27, P < 0.01) and control groups (mean difference = 106.67 ± 39.52, t(5) = 2.70, P = 0.043). Unpaired t-tests showed no significant differences between the two groups on Day 10 and a significant difference on Day 20. These findings thus emphasise the primary alterations that occurred within and between groups over time.
Liver markers
The activities of serum SGOT, SGPT and ALP were checked to analyse the structural integrity and functionality of the liver. The activities of all three liver marker enzymes were elevated, where SGPT and ALP showed a significant increase in serum of animals subjected to ostarine treatment as compared to the control group [Figure 2].
- Liver marker enzymes depicting levels of (a) SGOT (b) serum glutamic pyruvic transaminase and (c) alkaline phosphatase levels in Mean ± S.D. (n = 6) with level of significance between Control and OST (10 mg/kg b.w. Ostarine administered mice) *P < 0.05.
Oxidative stress
The animals treated with Ostarine showed increased oxidative stress, as indicated by an elevated ROS generation, as compared to the control [Figure 3a].
- Oxidant/antioxidant levels in Control and Ostarine administered mice (OST) where values of (a) Lipid peroxidation, (b) PCO, (c) glutathione, (d) ROS, (e) glutathione reductase, (f) glutathione peroxidase, (g) glutathione-S-Transferase, (h) superoxide dismutase, (i) xanthine oxidase and (j) catalase are depicted as Mean ± SD (n = 6) with the level of significance between control and OST (10 mg/kg b.w. ostarine administered mice) *P < 0.05, **P < 0.01, ***P < 0.001.
Oxidant/antioxidant status
The levels of LPO and PCC depicted a significant increase in animals treated with ostarine as compared to normal controls, thereby suggesting peroxidation of polyunsaturated fatty acids and proteins, respectively. On the contrary, a decrease in the enzyme activities of GR, GPx and CAT as well as GSH levels, was observed in the animals treated with ostarine as compared to the control group. Further, the activities of enzymes GST, SOD and XO were observed to be elevated significantly [Figure 3b].
Mitochondrial Swelling
An appreciable swelling of mitochondria was seen following ostarine treatment as compared to the normal control group [Figure 4].
- Graphical representation of mitochondrial swelling in control and ostarine-administered mice, where values are depicted as Mean ± S.D. (n = 6) with level of significance between Control and OST (10 mg/kg b.w. ostarine-administered mice) ** P < 0.01.
Histopathological analysis
The hepatic tissue of normal mice showed a regular structure of hepatocytes with intact lobules and a central vein. The portal triad, consisting of the bile duct, hepatic vein and hepatic artery, was in order and showed regular boundaries. In the group administered with ostarine, there were disruptions seen in the central vein and hepatic triad. The parenchyma was irregular with an increased number of Kupffer cells visible in the stroma [Figure 5]. A few regions containing focal necrotic changes in hepatic tissue, particularly in proximity to the disrupted central vein and portal triad structures, were also present, while fibrosis was not evident in the tissue section.
- Histopathological changes (20×; scale bar represents 50 micrometer) in (a-c) Control and (d-f) Ostarine administered mice: OST using H&E staining. The green arrows show the healthy histoarchitecture of Control hepatic tissue whereas the yellow arrow represents the disrupted portal triad with red arrows depicting increased Kupffer cells. The purple arrows show the loss of tissue integrity at multiple regions and the orange arrow represents that disformed central vein.
DISCUSSION
Ostarine, being a SARM, is a performance-enhancing drug that has the potential to combat old age maladies such as osteoporosis and sarcopenia. This study represents a sub-acute toxicity model to understand whether ostarine causes any adverse effects on the liver by examining hepatic and oxidative stress markers, as well as studying the changes in histoarchitecture. In the present study, muscular strength and locomotor activity of the animals were appreciably increased following treatment with ostarine, which indicates a positive influence of ostarine on cognitive functioning and motor coordination of the brain as evidenced by increased time spent on Rotarod rotating at different speeds as well as an increase in locomotor activity score of ostarine-treated animals. The plausible reason is understandably due to stimulation of the dopaminergic system as a consequence of ostarine treatment in animals.
We have witnessed a significant increase in the enzyme activity of ALP following ostarine treatment. Alkaline phosphatase is a loosely bound lysosomal enzyme and is released into systemic circulation on the disintegration of lysosomal membranes. Since we have also seen an increase in LPO, which is also a consequence of disruption of membranous integrity and therefore validates our observation of increased ALP activity. Further, transaminases are enzymes that are specific to liver functions and interconnect the metabolism of carbohydrates and proteins. The results of our study also reveal an increase in the levels of ALT and AST after treatment with ostarine, which arguably is due to damage inflicted on hepatocyte membranes, thereby resulting in the release of these enzymes from cytosol, and also indicating its adverse effects on liver function. The cellular level alterations were also visible in the histopathological study. Our results revealed an increase in PCC after treatment of rats with ostarine. PCC is considered a marker to ascertain oxidative damage due to protein oxidation, and our observed increase in PCC is a reflection of an increase in ROS, which can remove protons from methylene groups present in amino acids and result in the release of carbonyls that may, in turn, cause oxidation of sulfhydryl groups and result in loss of protein functions.
ROS have long been implicated in the pathophysiology of acute liver injury. The results of the present study showed a significant increase in ROS following ostarine treatment, which is a reasonable consequence of oxidative stress in mitochondria. An increase in oxidative stress leads to cellular damage that culminates in cell death. The oxidant-antioxidant balance in a cell is maintained between the production and scavenging activity of free radicals. The shift in balance, if not checked, results in irreversible damage contributing to tissue necrosis. The antioxidant status of the cells was studied to check the scavenging activity of enzymes against free radical production. An increase in ROS is also related to Nrf2 overexpression in damaged cells. The transcription factor, Nrf2, is present in the cytoplasm and translocates to the nucleus to maintain redox homeostasis in mitochondria. The gene helps in the regulation of numerous cell defence genes and protects cells against the harmful effects of chemical-induced oxidative stress. Nrf2 status is checked to predict the susceptibility of a given drug or chemical toxicity in the human body. Therefore, future studies are required to study the effect of ostarine on the levels of Nrf2 in cells.
Further, in the present study, ostarine treatment of rats resulted in a marked increase in LPO levels. LPO is an outcome of the oxidative degradation of polyunsaturated fatty acids, impairing membrane function and structural integrity, as well as inactivating membrane-bound enzymes.[24] LPO is generally associated with depletion in GSH levels, and we have also witnessed a marked decrease in GSH levels in our study, which seemingly is due to depleted GSH stores, as GSH is consumed by GR and GPx to combat peroxides generated due to increased LPO. Since antioxidative enzymes play a crucial role in combating the ROS and limit the adverse effects of oxidant molecules in a cell and so are very effective in the defence against oxidative damage and tissue injury. In the present study, we have seen a marked increase in the enzyme activity of SOD but a concomitant decrease in the activities of CAT and GPx in the ostarine-administered group as compared to the control group. The antioxidant enzymes SOD, GPx and CAT provide strong defences as free radical scavengers and therefore contain the onslaught of oxidant radicals on tissues. Further, the increased activity of GST in case of ostarine administered animals portrays the drug-induced perturbations that affect the biological homeostasis and is seen as a sign of major cellular detoxification against xenobiotics. XO plays an essential role in purine catabolism and is a marker of antioxidant health. The increase in XO levels following ostarine treatment reveals the toxic and antioxidant status of the liver and validates that ostarine does disrupt the first line of defence - GSH, SOD and CAT, and thus makes the cells vulnerable to oxidative damage.
The oxidative and antioxidative stress is known to affect mitochondrial channels, thereby damaging the mitochondrial Permeability Transistion Pore and the inner mitochondrial membrane. Besides, mitochondria are the epicentre of anti-oxidant homeostasis, and any alteration in proper functioning leads to alterations in mitochondrial membrane functions. The alterations in mitochondrial functions pinpoint their unhealthy status, which affects the integrity of hepatic tissue that gets validated by a few disruptions in the hepatic triad and an increased number of Kupffer cells, which are visible in the stroma following ostarine treatment.
The present study thus concludes that Ostarine at a higher dose adversely affects the hepatic functions by targeting the antioxidant status of hepatic cells. However, Ostarine does improve the muscular strength and locomotor activity of the animals, which indicates its positive influence on cognitive functioning and motor coordination of the brain. It is safe to say that even though ostarine improves the locomotive performance of old mice, but is toxic at higher dosages.
Hence, future studies should aim at standardising the dose of ostarine at a chronic level (> 90 days) to harness its full potential, being more effective and simultaneously nontoxic in nature.
CONCLUSION
Our study underscores the potential dual effects of Ostarine, a SARM, demonstrating its efficacy in enhancing muscular strength and locomotor activity while highlighting its hepatotoxicity at higher doses. These findings emphasise the importance of cautious consideration regarding the therapeutic use of ostarine and the necessity for further research to elucidate its optimal dosage and long-term safety profile.
Ethical approval:
The research/study was approved by the Institutional Review Board at Panjab University, number PU/45/99/CPCSEA/IAEC/2023/856, dated 14th November 2023.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflict of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: University Grants Commission, New Delhi, India.
References
- Androgens, brain, and behavior. Am J Psychiatry. 1996;153:974-84.
- [CrossRef] [PubMed] [Google Scholar]
- Enzalutamide inhibits androgen receptor-positive bladder cancer cell growth. Urol Oncol Semin Orig Investig. 2016;34:432.e15-23.
- [CrossRef] [PubMed] [Google Scholar]
- Selective androgen receptor modulators: The future of androgen therapy? Transl Androl Urol. 2020;9:S135-48.
- [CrossRef] [PubMed] [Google Scholar]
- Prevention of body weight loss and sarcopenia by a novel selective androgen receptor modulator in cancer cachexia models. Oncol Lett. 2017;14:8066-71.
- [CrossRef] [PubMed] [Google Scholar]
- Nonsteroidal selective androgen receptor modulator OstarineTM in cancer cachexia. Future Oncol. 2009;5:1211-20.
- [CrossRef] [PubMed] [Google Scholar]
- Identification of S22 (ostarine) in human nails and hair using LC-HRMS. Application to two authentic cases. Drug Test Anal. 2020;12:1508-13.
- [CrossRef] [PubMed] [Google Scholar]
- Tissue-level modeling of xenobiotic metabolism in liver: An emerging tool for enabling clinical translational research. Clin Transl Sci. 2009;2:228-37.
- [CrossRef] [PubMed] [Google Scholar]
- Drug-induced liver injury from enobosarm (ostarine), a selective androgen receptor modulator. ACG Case Rep J. 2021;8:e00518.
- [CrossRef] [PubMed] [Google Scholar]
- A note on a simple apparatus for detecting neurological deficit in rats and mice. J Am Pharm Assoc Am Pharm Assoc. 1957;46:208-9.
- [CrossRef] [PubMed] [Google Scholar]
- Ameliorating effect of piperine on behavioral abnormalities and oxidative markers in sodium valproate induced autism in BALB/C mice. Behav Brain Res. 2014;270:86-94.
- [CrossRef] [PubMed] [Google Scholar]
- Alkaline phosphatase in the blood serum; method for its appraisal. Hospital (Rio J). 1958;54:547-53.
- [Google Scholar]
- Serum glutamic pyruvic transaminase SGP-T in hepatic disease: A preliminary report. Ann Intern Med. 1956;45:801-11.
- [CrossRef] [PubMed] [Google Scholar]
- Influence of sucrose on protein determination by the Lowry procedure. Anal Biochem. 1968;24:337-9.
- [CrossRef] [PubMed] [Google Scholar]
- Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999;27:612-6.
- [CrossRef] [PubMed] [Google Scholar]
- Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351-8.
- [CrossRef] [PubMed] [Google Scholar]
- Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun. 1976;71:952-8.
- [CrossRef] [PubMed] [Google Scholar]
- Glutathione reductase In: Methods of Enzymatic Analysis. Cambridge: Academic Press; 1965. p. :875-9.
- [CrossRef] [Google Scholar]
- Glutathione transferases from human liver. Methods Enzymol. 1985;113:499-504.
- [CrossRef] [PubMed] [Google Scholar]
- Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys. 1978;186:189-95.
- [CrossRef] [PubMed] [Google Scholar]
- Catalase In: Methods of enzymatic analysis. New York: Academic Press Inc; 1974. p. :673-84.
- [CrossRef] [Google Scholar]
- Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464-78.
- [CrossRef] [PubMed] [Google Scholar]
- Effect of xanthine oxidase-generated extracellular superoxide on skeletal muscle force generation. Am J Physiol Regul Integr Comp Physiol. 2010;298:R2-8.
- [CrossRef] [PubMed] [Google Scholar]
- The osmotic behavior and permeability to non-electrolytes of mitochondria. Arch Biochem Biophys. 1955;58:52-67.
- [CrossRef] [PubMed] [Google Scholar]
- Lipid peroxidation: Mechanisms, inhibition, and biological effects. Biochem Biophys Res Commun. 2005;338:668-76.
- [CrossRef] [PubMed] [Google Scholar]