OXIDATIVE STRESS IS INCREASINGLY IMPLICATED in the pathogenesis of many diseases, perhaps most notably in liver disease. The liver is particularly vulnerable to oxidative stress due to its physiological role and anatomical placement, which make it susceptible to toxic, infectious and ischaemic insults. Oxidative stress is an important process in the pathology associated with both acute and chronic liver disease and it has been shown to play a direct role in sequelae of liver disease such as fibrosis and hepatic encephalopathy. Reactive oxygen species (ROS) are responsible for the damaging effects of oxidative stress. ROS are generated during aerobic metabolism and in response to certain pathological processes such as inflammation and cholestasis. Although they can have a role in normal physiology, ROS are also capable of causing extensive pathology; therefore the presence of ROS is regulated to maintain strict homoeostasis. Most ROS are free radicals that by their nature are unstable and highly reactive. Free radicals react with other substances to “steal” electrons in order to become more stable. However, in doing so this causes damage to the structure and function of the other substance. ROS can cause irreversible damage to DNA, RNA, lipids and proteins within cells that can lead to dysfunction of the cell and ultimately, cell death. ROS can also modulate intracellular signalling pathways and alter gene expression patterns, leading to events such as inflammation and apoptosis. A liver-specific effect of ROS is their ability to activate hepatic stellate cells, leading to the synthesis of collagen and extracellular-matrix that results in fibrosis of the liver. Healthy cells exist in a state of equilibrium between the generation of ROS and their neutralisation by antioxidants that ensures proper physiological function. Oxidative stress occurs when an imbalance arises between the generation of ROS and the neutralisation of ROS by antioxidants. Antioxidants can rapidly neutralise ROS before the ROS can exert a damaging effect, therefore playing a key role in protecting cells from ROS. However, an increase in ROS production or decrease in antioxidant availability can overwhelm a cell’s antioxidant capacity. This can lead to a vicious cycle in which ROS that are not neutralised exert a pro-inflammatory effect, increasing inflammation which in turn stimulates further production of ROS. Oxidative stress in liver disease is believed to be caused by both an increase in the production of ROS and by a decrease in antioxidants. Therefore, restoring the levels of antioxidants in the liver represents a potential therapeutic target in patients with liver disease.
Antioxidants in liver disease
Antioxidants can be described as enzymatic or non-enzymatic, and it is the latter that are the primary focus of supplementation in liver disease. One of the most important non-enzymatic antioxidants is glutathione, which is found at the highest concentration in the liver. Glutathione is synthesised in cells from s-adenosylmethionine (SAMe) and therefore is not required in the diet – indeed, glutathione has very poor oral bioavailability, so our current understanding is that it is unsuitable for oral supplementation. Other non-enzymatic antioxidants include vitamins C (ascorbic acid), E (tocopherol) and carotenoids (such as β-carotene), and in contrast to glutathione these antioxidants cannot be synthesised by the body, which means they must be provided through diet or supplementation. Dogs and cats with naturally-occurring hepatic disease have been shown to have a significant reduction in glutathione levels in the liver. A reduction in the synthesis of glutathione is believed to contribute to the depletion of glutathione in patients with liver disease, which arises through reduced activity of the enzyme SAMe synthetase that generates SAMe, the precursor to glutathione. This is caused by down-regulation of enzymatic activity in liver disease and a reduction in the availability of ATP, which is necessary to power the synthesis of SAMe.
Antioxidant supplementation
A number of antioxidant preparations are available for use in dogs and cats. The following represent the most common active ingredients used in patients with liver disease. Specific formulations may vary in the quantity, formulation and bioavailability of the active ingredient(s), so they should be assessed on an individual basis to ensure that an adequate dosage is biologically available.
- SAMe
Oral supplementation of SAMe has been associated with increased levels of glutathione in the liver in dogs. Studies of oral SAMe supplementation in dogs and cats have shown that SAMe is able to improve hepatic and erythrocyte redox status (the balance
between ROS and antioxidants), and it acts as a bile acid-independent choleretic. SAMe has been shown to protect against the damaging effects of paracetamol toxicity in cats. SAMe supplementation is likely to exert other beneficial effects that have not yet been identified, because of the importance of SAMe in several key biochemical pathways that contribute to liver health and the evidence that SAMe has antiinflammatory properties. SAMe is an unstable molecule which is easily oxidised to an inactive isomer. Therefore, it is vital that SAMe is protected from moisture
and oxygen by enteric coating or microencapsulation and administered on an empty stomach. Quality control is a serious issue surrounding SAMe supplements and a recent study comparing the levels of SAMe within commercially available products highlighted that some products contained as little as 39% of the levels stated on the packaging.
- Silybin (also known as silibinin)
Silybin is the major active flavonoid of silymarin, the extract from milk thistle seeds (Silybum marianum). The oral bioavailability of silybin is significantly increased (by around 10 fold) by complexing it with phosphatidylcholine, which is also an anti-fibrotic. Silybin has been shown to have a protective effect against cell membrane
damage and to act synergistically with SAMe to increase glutathione levels in the liver. It has also been demonstrated to have anti-inflammatory and choleretic properties. Research in dogs has shown that oral silymarin supplementation attenuates
the increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators of liver damage in toxic liver injury. The combination of SAMe and silybinphosphatidylcholine
complex (Denamarin) has been shown to reduce the hepatotoxic effects of the chemotherapeutic agent lomustine (CCNU) in dogs with neoplastic disease.
- Vitamin E (tocopherol)
Vitamin E is a fat-soluble vitamin that can exert multiple beneficial effects in patients with liver disease, including antioxidant, anti-inflammatory and anti-fibrotic effects. The most biologically-active form of vitamin E is α-tocopherol. Absorption of vitamin E is likely to be reduced in patients with liver disease because it is a fat-soluble vitamin and fat malabsorption is a common feature of liver disease, secondary to cholestasis. Care should be taken to adhere to the recommended dose of vitamin E, as excessive supplementation can lead to functional vitamin K deficiency and vitamin E can act as a pro-oxidant when in excess.
- N-acetylcysteine
Like SAMe, N-acetylcysteine is a glutathione precursor. N-acetylcysteine has poor oral bioavailability, so it is most commonly administered parentally in cases of acute hepatotoxicity.
- Other antioxidants
Vitamin C depletion has not been associated with liver disease in dogs and cats, which means it is not routinely supplemented in these patients. Furthermore, vitamin C supplementation is not recommended in animals with copper storage hepatopathy or chronic hepatitis (which tends to be associated with iron sequestration) as vitamin C can augment oxidative injury associated with the accumulation of transition metals. Carotene supplementation in animals with liver disease has not been investigated.
Conclusion
The liver is particularly susceptible to oxidative injury and antioxidants represent an important facet in the management of liver disease; there is evidence to suggest a beneficial effect of antioxidants in specific paradigms of liver disease. Further investigation into antioxidant supplementation in patients with liver disease would provide valuable insight into the clinical implications of this strategy.
References and further reading
Center, S. A. (2017) Canine Chronic Hepatitis – Veterinary Manual.
MSD Vet. Man. URL http:// www.msdvetmanual.com/en-gb/ digestive-system/hepatic-diseasein- small-animals/canine-chronichepatitis (accessed 5.15.17). Center, S. A., Warner, K. L. and Erb, H. N. (2002) Liver glutathione concentrations in dogs and cats with naturally occurring liver disease. Am J Vet Res 63: 1187-1,197. Center, S. A., Warner, K. L., McCabe, J., Foureman, P., Hoffmann, W. E. and Erb, H. N. (2005) Evaluation of the influence of S-adenosylmethionine on systemic and hepatic effects of
prednisolone in dogs. Am J Vet Res 66: 330-341. Mata-Santos, H. A., Dutra, F. F., Rocha, C. C., Lino, F. G., Xavier, F. R., Chinalia, L. A., Hossy, B. H., Castelo-Branco, M. T. L., Teodoro, A. J., Paiva, C. N. and dos Santos Pyrrho, A. (2014) Silymarin reduces profibrogenic cytokines and reverses hepatic fibrosis in chronic murine schistosomiasis. Antimicrob Agents Chemother 58: 2,076-2,083. doi: 10.1128/AAC.01936-13. Medina, J. and Moreno-Otero, R.
(2005) Pathophysiological basis for antioxidant therapy in chronic liver disease. Drugs 65: 2,445-2,461. Sanchez-Valle, V., Chavez-Tapia, N. C., Uribe, M. and Mendez- Sanchez, N. (2012) Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem 19: 4,850-4,860. doi: 10.2174/092986712803341520. Skorupski, K. A., Hammond, G. M., Irish, A. M., Kent, M. S., Guerrero, T. A., Rodriguez, C. O. and Griffin, D. W. (2011) Prospective Randomized Clinical Trial Assessing the Efficacy of Denamarin for Prevention of CCNU-Induced Hepatopathy in Tumor-Bearing Dogs. J Vet Intern Med 25: 838-845. doi: 10.1111/j.1939- 1676.2011.0743. Sturgess, C. P. (2014) Measurement of the S-adenosyl methionine (SAMe) content in a range of commercial veterinary SAMe supplements. J Small Anim Pract 55: 447-450. doi: 10.1111/ jsap.12244. Viviano, K. R., Vanderwielen, B. (2013) Effect of n-acetylcysteine supplementation on intracellular glutathione, urine isoprostanes, clinical score, and survival in hospitalized ill dogs. J Vet Intern Med 27: 250-258. doi: 10.1111/jvim.12048.
