Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

Grilo, Luís F.; Martins, José Luı́s; Cavallaro, Chiara H; Nathanielsz, Peter W.; Oliveira, Paulo J.; Pereira, Susana P.

doi:10.1016/j.tiv.2020.104996

Cited by 18 publications

(8 citation statements)

References 61 publications

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“…Catalase activity was determined by measuring hydrogen peroxide decomposition by following the 240 nm absorbance decrease [ 55 ]. Cells were seeded at a concentration of 15,000 cells/cm 2 in tissue-culture dishes, with a final volume of 10 ml and allowed to proliferate for 24 h. Then, 25 μM of AntiOxCIN 4 was added to cells for 48 h. In the last 24 h of the experiment, cell culture medium was changed for OXPHOSm.…”

Section: Methodsmentioning

confidence: 99%

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A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients

et al. 2021

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Parkinson's Disease (PD) is a neurodegenerative disorder affecting more than 10 million people worldwide. Currently, PD has no cure and no early diagnostics methods exist. Mitochondrial dysfunction is presented in the early stages of PD, and it is considered an important pathophysiology component. We have previously developed mitochondria-targeted hydroxycinnamic acid derivatives, presenting antioxidant and iron-chelating properties, and preventing oxidative stress in several biological models of disease. We have also demonstrated that skin fibroblasts from male sporadic PD patients (sPD) presented cellular and mitochondrial alterations, including increased oxidative stress, hyperpolarized and elongated mitochondria and decreased respiration and ATP levels. We also showed that forcing mitochondrial oxidative phosphorylation (OXPHOS) in sPD fibroblasts uncovers metabolic defects that were otherwise hidden. In this work, we tested the hypothesis that a lead mitochondria-targeted hydroxycinnamic acid derivative would revert the phenotype found in skin fibroblasts from sPD patients. Our results demonstrated that treating human skin fibroblasts from sPD patients with non-toxic concentrations of AntiOxCIN 4 restored mitochondrial membrane potential and mitochondrial fission, decreased autophagic flux, and enhanced cellular responses to stress by improving the cellular redox state and decreasing reactive oxygen species (ROS) levels. Besides, fibroblasts from sPD patients treated with AntiOxCIN 4 showed increased maximal respiration and metabolic activity, converting sPD fibroblasts physiologically more similar to their sex- and age-matched healthy controls. The positive compound effect was reinforced using a supervised machine learning model, confirming that AntiOxCIN 4 treatment converted treated fibroblasts from sPD patients closer to the phenotype of control fibroblasts. Our data points out a possible mechanism of AntiOxCIN 4 action contributing to a deeper understanding of how the use of mitochondria-targeted antioxidants based on a polyphenol scaffold can be used as potential drug candidates for delaying PD progression, validating the use of fibroblasts from sPD patients with more active OXPHOS as platforms for mitochondria-based drug development.

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Section: Methodsmentioning

confidence: 99%

“…The maximal catalase activity was determined using the initial linear part of this fitting curve. Results are expressed in enzyme units (U) obtained directly from the decomposition of hydrogen peroxide using the Beer-Lambert law with l = 0.691 cm and Ɛ = 43.6 M −1 cm −1 [ 55 ].…”

Section: Methodsmentioning

confidence: 99%

A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients

et al. 2021

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“…Measurement of Catalase activity —The rate of the decomposition of H 2 O 2 by catalase was calculated following the method proposed by Grilo et al [ 41 ]. Briefly, 10 mg of frozen liver tissue was homogenized in 50 mM phosphate buffer, pH 7.8.…”

Section: Methodsmentioning

confidence: 99%

Brassica oleracea Var italica by-Products Prevent Lipid Accumulation and Cell Death in a Liver Cell Model of Lipid Toxicity

et al. 2023

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Obesity, a rising concern in the Eastern world, encompasses several co-morbidities, namely non-alcoholic fatty liver disease (NAFLD). Potential natural-based interventions to decrease the burden of obesity complications are being investigated. Many of the edible parts of plants are not sold for consumption and end up as massive waste, losing nutritional potential. In fact, a sizeable amount of waste is generated within the different steps of the food supply chain, representing a massive loss of both plant material and natural resources. A good example is Brassica by-products (BBPs). The objective of this work was to investigate the effect of three different extracts from broccoli (Brassica oleracea var italica) by-products in an in vitro model of free fatty acid (FFA)-induced lipotoxicity using human hepatoma HepG2 cells. Broccoli leaf, stalk, and inflorescence extracts induced a dose-dependent decrease in the cell viability of HepG2 cells. However, the maximal non-lethal concentrations of leaves, stalks, and inflorescences (10 μg/mL) did not compromise mitochondrial function or neutral lipid accumulation in HepG2 cells. The extracts significantly decreased FFA-induced lipid accumulation in HepG2 cells either in a co-incubation or pre-incubation strategy. The broccoli extracts’ capacity to prevent the FFA-induced decrease in catalase activity in HepG2 may explain the observed effects.

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“…The expression of relevant transcripts associated with mitochondrial biogenesis, stress responses, oxidative phosphorylation, and auto(mito)phagy will also be evaluated [23]. This includes enzymes such as superoxide dismutase, catalase [38], glutathione reductase, parkin and PINK1 proteins, the mitochondrial transcription factor A (TFAM), the regulator of mitochondrial biogenesis (such as PGC1-α), and metabolism (such as AMPK-α) [37]. The evaluation of these parameters aims to unravel the molecular mechanism through which PA promotes mitochondrial metabolism remodeling in PD patients.…”

Section: Methods and Analysismentioning

confidence: 99%

Investigating the impact of physical activity on mitochondrial function in Parkinson’s disease (PARKEX): Study protocol for a randomised controlled clinical trial

Magaña,

Deus,

Baldellou

et al. 2023

Preprint

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Parkinson's disease (PD) is characterised by the progressive dopaminergic neuron degeneration, resulting in striatal dopamine deficiency. Mitochondrial dysfunction and oxidative stress are associated with PD pathogenesis. Physical activity (PA) has been shown to ameliorate neurological impairments and to impede age-related neuronal loss. In addition, skin fibroblasts have been identified as surrogate indicators of pathogenic processes correlating with clinical measures. The PARKEX study aims to compare the effects of two different PA programs, analysing the impact on mitochondrial function in patients' skin fibroblasts as biomarkers for disease status and metabolic improvement. Early-stage PD patients (n=24, H&Y stage I to III) will be randomised into three age- and sex-matched groups. Group 1 (n=8) will undergo basic physical training (BPT) emphasising strength and resistance. Group 2 (n=8) will undergo BPT combined with functional exercises (BPTFE), targeting the sensorimotor pathways that are most affected in PD (proprioception-balance-coordination) together with cognitive and motor training (Dual task training). Group 3 (n=8) will serve as control (sedentary group; Sed). Participants will perform three sessions per week for 12 weeks. Assessment of motor function, quality of life, sleep quality, cognitive aspects and humour will be conducted pre- and post-intervention. Patient skin fibroblasts will be collected before and after the intervention and characterised in terms of metabolic remodelling and mitochondrial bioenergetics. Ethical approval has been given to commence this study. This trial is registered at clinicaltrials.gov (NCT05963425)

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Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

Cited by 18 publications

References 61 publications

A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients

A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients

Brassica oleracea Var italica by-Products Prevent Lipid Accumulation and Cell Death in a Liver Cell Model of Lipid Toxicity

Investigating the impact of physical activity on mitochondrial function in Parkinson’s disease (PARKEX): Study protocol for a randomised controlled clinical trial

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