Sharon A. Center, DVM, DACVIM
Hepatic Antioxidant System
The pivotal involvement of the liver in cleansing blood from the gut (considered to be the richest source of oxidants and toxins in the body), it's central role in intermediary metabolism and detoxification processes, its large active Kupffer cell (macrophage) population, imposes high risk for exposure to injurious substances, infectious agents, toxic adducts, and oxidizing substrates. Consequently, the liver is endowed with diverse antioxidant/detoxification mechanisms. Many of the non-enzymatic antioxidants can be directly supplemented. Since glutathione (GSH) is involved in many antioxidant processes, there is tremendous interest in thiol (GSH) donors such as s-adenosyl-methionine (SAMe). SAMe functions in this capacity as well as participating in a large number of other critical cellular processes and biochemical pathways). An additional group of extracellular selenoproteins, thioredoxins, also possess antioxidant activity.
Antioxidative enzymes provide first line defense and whether or not these can be sufficiently supplemented remains controversial. Enzymatic antioxidants are localized in specific subcellular compartments where radicals are frequently generated (mitochondria, peroxisomes or microbodies, cell cytosol) or are present in the systemic circulation. Examples include superoxide dismutase (SOD), catalase, and GSH peroxidases (GSH-Px) a family of cytosolic, mitochondrial, and extracellular enzymes that detoxify lipid hydroperoxides and H2O2 by oxidizing GSH to GSSG. Several GSH-Px's require selenium. GSH reductase (requires riboflavin as a co-factor) regenerates reduced-GSH from its oxidized disulfide (GSSG).
There is great interdependence among the antioxidants such that supplementation with only a single agent may not provide an optimal response. At present, no one knows what "cocktail" is ideal for dogs or cats. Since nutritional balance is an important variable influencing antioxidant availability (e.g., vitamin E, trace metals, cysteine for GSH), nutritional support is essential in liver disease along with antioxidant supplementation. Protection by protein binding of toxic adducts or transition metals (copper or iron) to albumin, transferrin, ferritin, ceruloplasmin or metallothionein, provides another rapid venue for detoxication (liver and systemic circulation). While these cannot be directly supplemented (except albumin), their synthesis can be enhanced through nutritional support. Supplementing with supraphysiologic doses of single agents can produce oxidant injury. Examine the diagram above and note that excessive administration of vitamin C (ascorbate) or vitamin E (tocopherol) can result in accumulation of their respective oxidant radicals.
Combination Therapy: Antioxidant Agents with Immunomodulatory / Antifibrotic Medications
Advantageous combination therapy in necroinflammatory and cholestatic liver disorders may include glucocorticoids, other immunomodulatory or anti-inflammatory prescription drugs combined with various antioxidants. Since glucocorticoids are known to mediate inhibition of NF-kB (beneficially modulated by certain antioxidants such as thiols), adjunctive therapy with an effective thiol donor (e.g., NAC or SAMe) and vitamin E may optimize control of inflammatory mediator release and cell response as compared to pure immunomodulatory treatment or therapy limited to antioxidants. It is reasonable to expect, given the complexity of disease pathomechanisms, that a mixture of antioxidants (an antioxidant "cocktail" so to speak) will eventually be used in combination with other anti-inflammatory / antifibrotic agents for different types of liver disease and will necessitate unique recommendations for different species.
Thiols play a central yet cooperative role in the antioxidant network. (Figure adapted from: Sen CK, Packer L: Thiol homeostasis and supplements in physical exercise. Am J Clin Nutr 2000; 72(suppl): 653S-669S). Substances containing S-H bonds, HS-SH disulfide that have central importance in many biochemical and pharmacological reactions. Reduction of only a few essential SH-SH bonds in a biologic reactant can remarkably change its molecular properties. Thus, it is important that a system exists to protect against damage to these vulnerable bonds. Most thiols act as reducing agents (absorb emitted electrons in oxidant processes, thiol is lost). The importance for conserving thiol bonds is evident when one understands that incorporation of a cysteine moiety influences the tertiary structure of proteins (e.g., molecule configuration, folding) and can influence effects of certain drugs (e.g., insulin). A significant number of proteins involved in signaling have critical thiols (e.g., cell receptors, ubiquitinylation proteins, protein kinases, and some transcription factors). Metallothionein, the only known protein implicated in cellular zinc distribution, binds 7 zinc atoms via thiolated ligands. Binding sites lose function with oxidation of the thiol group; dogs with copper storage hepatopathy may benefit from antioxidants that preserve these bonds allowing zinc transport. Cysteine residues are among the most easily metabolized compounds, being easily oxidized by transition metals and participating in thiol-disulfide exchange reactions. The most important non-protein thiol source in intermediary metabolism is glutathione (GSH, containing a single cysteine moiety) and the small protein thioredoxin (containing two redox-active half-cysteine residues; a ubiquitous protein, important for gene expression) that we will not discuss. Each of these play important roles in antioxidant defense, protein folding, and signal transduction. In vivo, reductases recycle disulfides to thiols using cellular-reducing equivalents (e.g., NADH or NADPH) maintaining a favorable oxidoreductive (or redox) state in the cell and thiol conservation.
Hepatic GSH in Liver Disease
Low hepatic GSH concentrations develop in dogs and cats with substantial liver injury (necroinflammatory, cholestatic disorders). Contributing factors include: reduced nutritional intake (proteins, essential amino acids, competition between amino acid transporters, vitamin insufficiency [riboflavin: GSSG reductase, nicotinic acid: NADPH, pyridoxine (B6): SAMe pathway], mitochondrial dysfunction: ATP deficiency, down regulation SAMe synthase, oxidation of SAMe pathway enzymes, enhanced GSH utilization: conjugation reactions (toxins, drugs), and overwhelming oxidative challenge.
N-Acetylcysteine (NAC), S-Adenosylmethionine (SAMe), Whey Protein Extract: (limited scientific evidence of efficacy), Lipoic Acid: (lethal feline toxicity proven)
An acetylated variant of L-cysteine can provide SH groups. A direct thiol donor given IV when supplementation urgent (e.g., oxidant challenge / fulminant hepatic failure). Metabolized into products that stimulate GSH synthesis and promote detoxification pathways. Functions directly as a free radical scavenger (circulation). Dose: severe oxidant injury / hepato-toxicosis, IV administration of saline diluted NAC (1:2 for 10% or 1:4 for 20% NAC solution). Administer via non-pyrogenic filter. Protocol as for acetaminophen toxicity: 140 mg/kg IV initially: slow bolus not constant rate infusion (ammonia toxicity) then 70 mg/kg IV or PO x 3-6 (or more) treatments given at 8 to 12 hour intervals. Treatment tailored to condition urgency/severity. Chronic PO treatment provided with SAMe. NAC Toxicity: Oral dose LD50 approximates 6-8 gm/kg in rodents. Infrequent allergic reactions (IV administration) in humans and dogs (rash, pruritus, facial swelling, tachycardia, gastrointestinal signs). Note: doses as low as 1.2 g daily in humans might confer a pro-oxidant effect, lowering GSH and increasing the GSH/GSSG ratio.
Plays a complex role in metabolism, functioning as a methyl group donor (transmethylation reactions), precursor of sulphur-containing compounds (transsulfuration pathway), and in production of polyamines. Note important products of the transmethylation pathway and transsulfuration pathway shown. The polyamine pathway influences cell replication / regeneration, DNA synthesis, and apoptosis and yields an important metabolite methylthioadenosine (plays a pivotal role in hepatocyte function and gene transcription). Since 85% of transmethylation reactions and up to 48% of methionine metabolism occurs in the liver, hepatic SAMe availability is essential. SAMe has been shown to provide a benefit in a variety of liver disorders (modeled in experimental animals, in humans, and in dogs and cats), the molecular basis of SAMe's protective effects have been demonstrated in vitro using hepatocyte cell cultures. Oral SAMe in cirrhotic humans replenishes hepatic GSH and improves resistance against free radical / reperfusion injury. By sustaining appropriate DNA methylation, SAMe has a broad range of potential effects on cell repair, inflammatory mediator release, and the pathobiology of ongoing immune-mediated liver injury. Studies in dogs with glucocorticoid induced vacuolar hepatopathy, cats with spontaneous liver disorders, and healthy cats, has proven both hepatic and RBC protective effects. We documented bioavailability and biologic effects of a stable salt of SAMe (1,4-butanedisulfonate salt, Nutramax Laboratories, Nutramax Laboratories, USA) in healthy cats, cats with asymptomatic cholangiohepatitis, and dogs with glucocorticoid-induced vacuolar hepatopathy. In each circumstance, this stable SAMe salt was absorbed, significantly increased plasma SAMe concentrations, hepatic GSH concentrations, and improved tissue redox status. Beneficial effects on RBCs also were shown. How SAMe accesses hepatocytes has not been clarified but it is proven to become rapidly incorporated within cell membrane phospholipids, to influence intracellular SAMe pathways and products and to readily cross into mitochondria.
Dose: 17-20 mg/kg is advised for dogs and of 200 mg/day in cats using bioavailable enteric coated tablets given on an empty stomach. We only use a form of SAMe proven to be bioavailable that contains an excess of the biologically active S'S SAMe isomer. Selecting an appropriate SAMe product depends on specific information provided by the manufacturer regarding shelf-life, bioavailability, and S'S isomer content.
Toxicity: Adverse effects to SAMe are rare but may include inappetence, vomiting, lethargy, and agitation after initial treatment. Effects may self-resolve, or abate with a reduced dose, gradually increasing back to the therapeutic target. In rodents, a single dose LD50 > 4.6 gm/kg SAMe exits. Chronic studies in rats given 200 mg/kg body weight per day for 104 weeks, dogs given 20 mg/kg PO x 84 days, cats given 40-65 mg/kg for 118 days did not produce any signs of toxicity (blood testing, liver histology).
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