Back in the 1950s, Denham Harman presented his thoughts on the free radical theory of aging. In a nutshell, this theory posits that aging occurs because free radicals damage cellular membranes, DNA, RNA and proteins. This results in the death of the cell and ergo the individual due to oxidative damage. To this day, free radicals such as reactive oxygen species (ROS) are widely construed—and anthropomorphized—as malicious molecules with a vendetta, hell-bent on destroying everything in their paths.
Since Harman mounted his soapbox all those years ago, documents supporting the atrocities of free radicals snowballed. They overshadowed the fact that free radicals—ROS in particular—aren’t all bad. In fact, Ron Mittler, PhD, a professor in the Department of Agriculture, Food & Natural Resources and research professor in at the University of Missouri School of Medicine, asserts that ROS are actually good.
“Maintaining a basal level of ROS in cells is essential for life as they are necessary for basic biological processes,” Mittler explained.
Given this alternate and scientifically sound theory on free radicals and ROS, which we will explore in more detail here, one might then ask: Is the equine industry going overboard with the antioxidant supplements?
Reactive Oxygen Species (ROS) Overview
Of all the free radicals and reactive molecules produced during normal metabolic processes, reactive oxygen species (ROS) are frequently considered the vilest. ROS are derived from molecular oxygen either through redox (reduction-oxidation) reactions or electronic excitation. Examples include hydrogen peroxide (H2O2), singlet molecular oxygen (1O2), ozone (O3), superoxide anion radical (O2.-) and hydroxyl radical (.OH).
ROS are generated during aerobic metabolism, such as the mitochondrial electron transport chain, which is
the third phase of cellular respiration. Hydrogen peroxide, for example, is produced from molecular oxygen (O2) by NADPH oxidases and superoxide dismutases, as well as a long list of other cellular enzymes. In fact, Sies and Jones report that in humans, more than 40 enzymes capable of generating H2O2 and O2.- have been identified so far (Nat Rev Mol Cell Biol. 2020;21(7):363-383).
“As ROS are produced, enzymes and antioxidants remove them, ensuring that the ROS are maintained at a basal, nontoxic level in the cell. A fine balance exists between ROS production and ROS removal, requiring a fully functional ROS network,” described Mittler.
This ROS network includes all the enzymes and genes involved in ROS production, scavenging and transport in a cell. Mittler added, “In each different cell type, different parts of this network function. The overall outcome of the ROS network is that ROS are kept at a physiological, non-toxic level.”
The maintenance level of ROS is referred to as “oxidative eustress.” It is at these low cellular concentrations that ROS exert their beneficial effects.
Positive Actions of ROS
“ROS act as signaling molecules, regulating and maintaining normal physiological functions,” said Mittler.
A good example of how ROS are effective signaling molecules, said Mittler, is the interaction of H2O2 with cysteine residues of proteins. Peroxide can oxidize those cysteine residues, which in turn results in structural and therefore also functional changes in the protein.
“The function of many of these ROS-regulated proteins is essential for the control of cellular proliferation, development, growth and defense against pathogens,” explained Mittler.
Stem cells and immune cells are a good examples of cells that require ROS to conduct their function. However, according to Mittler, all cells need a basal level of ROS to regulate gene expression and maintain their functions.
“An increase in ROS production is required for the release of the proinflammatory cytokines interleukin-1b, tumor necrosis factor-a, and interferon-b that in turn are required for orchestrating an appropriate immune response,” said Mittler. “Low ROS levels therefore prevent immune-response activation and lead to immunosuppression.”
Mittler noted that gene regulation, cell-to-cell communication, hormonal regulation and other processes are either also linked to ROS levels or use ROS as messengers. But of course, the levels of ROS that a horse needs for optimal immunity, cell signaling, etc. have yet to be determined.
Exercise-Produced ROS Are Also Good
Strenuous, exhaustive or unaccustomed exercise in horses can induce oxidative stress. This is likely why antioxidant supplements are popular options for horses involved in competition. This is particularly true for endurance horses, show jumpers, dressage horses, Thoroughbred racehorses and Standardbred trotters, suggested a research team led by Peter Kruljc, dr.vet.med, Clinic for Reproduction and Large Animals, University of Ljubljana, Slovenia.
In addition to the important roles ROS play in normal cellular functions, Kruljc and colleagues relayed in their study that ROS are considered necessary for adaptation to exercise training. This reportedly occurs by modulating muscle contraction and activating the endogenous antioxidant system, including an increased expression of antioxidant enzymes.
In their 2021 article published in Antioxidants, the researchers relayed that the antioxidant defense systems—including enzymatic and non-enzymatic antioxidants—are capable of major adaptations during acute and chronic exercise. Moderate exercise could be considered an antioxidant because low to moderate concentrations of ROS act as signals that induce the expression of powerful endogenous antioxidant enzymes.
Measuring Oxidative Stress in Horses
“Lipid peroxidation is the most common consequence of exercise-induced oxidative stress. It can lead to the release of muscle enzymes into the systemic circulation,” said Kruljc.
Measuring markers of lipid peroxidation (such as malondialdehyde concentration) as well as the activities of antioxidant enzymes in blood samples could give a reasonable idea of how much oxidative stress is occurring in a horse at a specific time. Increased lipid peroxidation might be associated with the leakage of muscle enzymes into the circulation. Measuring muscle enzymes (creatine kinase and aspartate aminotransferase) can easily be accomplished in any setting. Measuring oxidative stress is limited to research settings.
According to Alenka Nemec Svete, a co-author on the study, it should be noted that “measuring creatine kinase and aspartate aminotransferase on their own is not a good indicator of oxidative stress. These two enzymes may be increased after exercise due to higher permeability of muscle cells. On the other hand, the activity of these two enzymes might be elevated due to high extent of lipid peroxidation—a consequence of increased oxidative stress—that affects muscle membrane permeability.”
Oxidative Stress Parameters
In the study by Kruljc et al., various oxidative stress parameters were measured in untrained Standardbred horses subject to acute moderate exercise:
- Total antioxidant capacity (TAC) of serum or plasma to measure the uptake and/or production of antioxidants and their consumption during normal or increased oxidative stress
- SOD and GPX, endogenous enzymatic antioxidants
- Malondialdehyde levels, a marker of lipid peroxidation
“Malondyladehyde is a reliable and commonly used marker for lipid peroxidation and oxidative stress in a research setting,” said Kruljc.
Unfortunately, those tests are not currently available through veterinary diagnostic laboratories for everyday use in horses. This void in methods for assaying lipid peroxidation and oxidative stress in general therefore leaves horse owners and veterinarians unsure as to which horses require antioxidant supplementation. In many cases, horses end up being offered antioxidant supplements in a “better safe than sorry” manner, despite the fact that ROS are beneficial.
Antioxidant Supplement Research
Kruljc and colleagues noted that vitamin E is one of the most-studied and widely used antioxidant supplements in horses. Some studies suggest that vitamin E, a potent free-radical scavenger, can reduce the concentration of lipid peroxidation products. Therefore, it can protect horses from oxidative stress. “However, there are inconsistent results regarding the effects of vitamin E supplementation on exercise-induced oxidative stress,” said Kruljc.
Those inconsistencies could be related to the amount, duration, form and frequency of vitamin E supplementation. They could be related to the type and timing of exercise used in the studies assessing the exercise-induced oxidative stress. They could also be related to and the vitamin E status of the subjects prior to the studies, as well as the methods used to assess oxidative damage.
“Even in human athletes there is limited evidence to support the use of vitamin E as an antioxidant,” said Kruljc.
Similar conflicting results have been obtained for other antioxidant supplements (e.g., coenzyme Q10). Many supplements contain antioxidants or ingredients that purportedly have antioxidant properties that have not been studied in horses.
“I am a firm believer [in] supplementing vitamin E for horses without access to pasture that are exercising heavily. I have seen the conflicting research but still think there is enough benefit to not worry about any detriment. As for other antioxidants, I have not seen any concrete evidence of benefit,” said Carey Williams, PhD, an equine extension specialist and professor from Rutgers.
Antioxidants and Vitamin E in Horses
“As we can see, some ROS are good, but too many are not. So, where is that line drawn, and how do we figure that out in the real world?” Williams asked. Kruljc and colleagues used a multitude of assays to evaluate oxidation in the horses in their study. However, those tests are largely restricted to a research setting.
“Clinically, there aren’t many options to measure ROS in horses,” said Carrie Finno, DVM, PhD, DACVIM, the Gregory L. Ferraro Endowed Director of the Center for Equine Health at the University of California, Davis. “However, we can measure serum vitamin E and a balance of ROS, which has been shown to be important across species.”
Several veterinary diagnostic laboratories offer vitamin E testing for horses. This could be a valuable tool. A recently published study found that despite some pasture access or being offered some form of vitamin E supplementation, over one-third of horses had suboptimal plasma vitamin E levels (Pitel et al. 2020). In general, horses with low vitamin E levels had <6 hours/day pasture access and received <500 IU of supplemental vitamin E/day. The authors stated, “Furthermore, >20% of horses with apparently adequate vitamin E supplementation and pasture access were found to have deficient or marginal plasma alpha-tocopherol concentrations.”
Factors Contributing to Suboptimal Vitamin E Levels in Horses
Possible factors contributing to suboptimal vitamin E levels in supplemented horses and those with pasture access could be due to the amount. It could also be due to the source or quality of vitamin E, or even failure to ingest the product. These results suggest that intermittent evaluation of plasma vitamin E levels might be valuable for heavily exercising horses to ensure their vitamin E status is optimal.
Testing for ROS in horses might be available sooner than later. Rossi et al. (2021) described a commercially available test for measuring antioxidant stress in horses. This test was evaluated in five Standardbred trotter horses at various times of training and racing. The research showed: “The KRL test may represent a valid method to determine oxidative stress in athletic horses.”
Final Thoughts on ROS
No one is suggesting that horses shouldn’t be offered antioxidants. Instead, this article simply highlights the beneficial features of ROS. It opens the door for us to ask: What we are hoping to gain from supplementing with antioxidants? This can be a challenging question since we can’t easily determine which horses are suffering oxidative stress.
