BACKGROUND

The present disclosure is directed to cellular oxidative stress treatment in mammals such as companion animals.

In people and animals, cells in homeostatic equilibrium are in a state of oxidative balance, namely, when there are sufficient cellular antioxidants (both small molecule radical scavengers and enzymes such as catalase and superoxide dismutase) to avoid a buildup of excess free radicals (oxidizing agents). However, when levels of oxidizing agents exceed antioxidants, the condition is termed oxidative stress (OS). OS is now known to trigger inflammatory responses and to be an underlying cause of most chronic diseases.

Extensive investigations of OS and chronic tissue inflammatory responses have established that they are major underlying risk factors that play key roles in the etiology of a range of human (and animal) diseases, including rheumatoid arthritis, cancers, diabetes, obesity, periodontal disease, neurodegenerative disorders, and cardiovascular diseases. The development and progression of these chronic diseases is influenced by a range of environmental, dietary, and lifestyle factors, and specific dietary components, that—along with exercise and some nutraceuticals'can significantly impact OS and inflammation.

Chronic inflammation resulting from increased free radical formation in OS, also called reactive oxygen species (ROS), occurs when tissues or organs receive inflammatory “mediator” messages that cause them to react as though a “trigger” such as a pathogen is present. Rather than repairing themselves, these cells can remain in an ongoing state of inflammation that can wax and wane for prolonged periods. Tissues thus become deficient in antioxidant mediators, such as glutathione, cysteine, ascorbic acid and other radical scavenging vitamins, as well as superoxide dismutase, catalase and other antioxidant enzymes, and these deficiencies are strongly correlated with poor clinical outcomes. For mammals in good health, ˜25% of oxygen intake gives rise to ROS, whereas this increases with age and poor health to as much as 75%.

When cells undergo damage due to OS and inflammation, the incidence and severity of infections, obesity cardiovascular disease and cancers increase. These damaged cells release molecules that can be used as biomarkers for OS and inflammation. Biomarkers of clinical relevance in people and pets include isoprostanes, malondialdehyde, and several cytokines including tumor necrosis factor (TNF), hydroxyoctadecadienoic acid (NODE), as well as certain microRNAs. Elevated levels of these biomarkers in biofluids (including plasma, urine and/or saliva) can be used to evaluate oxidative status and can addressed with diet and supplement changes to promote beneficial antioxidant effects. On the other hand, alternative biomarkers, such as glutathione, certain microRNAs, and some enzymes are indicative of a healthy antioxidant condition. Biomarkers can be analyzed assays periodically to assess the response and adjust lifestyle, nutritional and nutraceutical therapy, as needed.

Significant efforts have been made toward the dietary management of OS. In contrast to small molecule antioxidants (e.g., ascorbic acid) that can neutralize one ROS/molecule, recent studies have focused on the development of functional foods, e.g., those containing natural Nrf-2 activators since Nrf-2 upregulates expression of multiple antioxidant enzymes including superoxide dismutase, catalase, glutathione transferases and glutathione reductase. A single molecule of one of these enzymes can neutralize a huge number of ROS. Significant amounts of antioxidant radical scavengers and/or Hrf-2 activators are ingredients in many functional foods and supplements such as: turmeric (Curcuma longa); and its relative, ginger (Zingiber officiale); chili peppers (Capsicum annuum); green tea (Camellia sinensis, which contains tannins and polyphenol catechins, and other teas); soybeans (Glycine max); tomatoes (Solanum lycopersicum, rich in lycopenes); grapes (not for pets); honey (not for infants or very young animals); cranberries (Vaccinium macrocarpon, contains pro-anthocyanidins); licorice (Glycyrrhiza glabra); garlic (Allium sativum, in moderation for pets); milk thistle (Silybum marianum); cabbages and broccoli.

Some Primary Oxidative Stress Biomarkers

Isoprostanes are a series of prostaglandin-like compounds produced by non-enzymatic ROS-catalyzed peroxidation of arachidonic acid. The concentration of isoprostanes is considered the “gold standard” for quantifying OS in vivo in humans and certain animals.

MicroRNAs are small non-coding RNA molecules found mostly in the cells of plants, animals and some viruses. They function in RNA silencing and post-transcriptional regulation of gene expression. The function of microRNAs is in gene regulation.

Reduced glutathione (GSH) plays two key roles in the maintenance of redox balance. First, GSH is a major intracellular radical scavenger that can react with and neutralize ROS thus preventing ROS-mediated damage to macromolecules such as proteins and DNA.

GSH is also a co-substrate for Glutathione-S-Transferases (GSTs), a superfamily of isozymes best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to foreign substrates for the purpose of detoxification. https://en.wikipedia.org/wiki/Glutathione S-transferase—cite note-pmid15717864-1 are found in plants, animals, fungi, and some bacteria. In vertebrates there are over 18 distinct cytosolic GSTs which are expressed most tissues, but especially in liver, kidney, heart, lung, and brain tissues. High levels of GST are associated with resistance to the apoptosis (cell death) induced by a range of substances, including chemotherapeutic agents. The levels of specific GST isoforms in urine and serum are indicators of hepatocyte and renal tubular injury in transplantation, toxicity and viral infections of humans and rodents.

The production of GSH is dependent on the enzyme glutathione reductase (GSR), and the expression of GSR, as well as multiple cytosolic GSTs and other antioxidant enzymes including superoxide dismutase are upregulated by the transcription factor Nrf-2. Therefore, while direct measurement of Nrf-2 activation by diet, nutraceuticals, etc. is extremely difficult, the levels of glutathione in biological fluids can serve as a surrogate biomarker for Nrf-2 activation.

Malondialdehyde (MDA) is a key byproduct of the peroxidation of polyunsaturated fatty acids. It is very reactive and forms adducts with macromolecules, thereby altering or eliminating their function. MDA and/or MDA adducts are well established biomarkers for OS. However, some methods for the measurement of MDA, including the thiobarbituric acid reactive substance (TBARS) assay as it was traditionally performed do not provide consistent reliable results. Malondialdehyde is potentially mutagenic, and has been found in heated edible oils such as sunflower and palm oils. Corneas of human patients suffering from keratoconus and bullous keratopathy have increased levels of malondialdehyde, and this aldehyde also can be found in tissue sections of joints from human patients with osteoarthritis.

Tumor Necrosis Factor-Alpha (TNF-α), also called cachexin or cachectin, is a cell signaling protein involved in systemic inflammation and is one of the cytokines that play a primary role in the acute phase inflammatory response. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, natural killer (NK) cells, neutrophils, mast cells, eosinophils, and even neurons. https://en.wikipedia.org/wiki/Tumor necrosis factor alpha—cite note-ReferenceA-5

TNF is an endogenous pyrogen that can induce fever, apoptotic cell death, cachexia, inflammation, inhibit tumorigenesis and viral replication, and respond to sepsis via interleukin IL-1 and IL-6 producing cells. Dysregulation of TNF production has been implicated in a variety of human diseases such as Alzheimer's disease, cancer, depression, psoriasis and inflammatory bowel disease (IBD).

Hydroxyoctadecadienoic Acid (HODE) is a lipid peroxidase biomarker found to be elevated for early detection of periodontal disease, type-2 diabetes, rheumatoid arthritis, cataracts, Alzheimer's disease and hepatitis-B and -C in people.

Other Biomarker Enzymes include: Sorbitol Dehydrogenase, a cytosolic enzyme that converts sorbitol, the sugar alcohol form of glucose, into fructose; and 5′ Nucleotidase, which catalyzes the phosphorolytic cleavage of 5′ nucleotides, and is considered a maturation marker for T- and B-cells.

Measuring Biomarkers of Oxidative Stress

Traditionally, cellular biomarkers are measured in the serum and/or urine from humans and animals. However, collecting these samples especially from children and smaller animal species presents with difficulty and causes unnecessary stress.

Neither canine whole blood-, serum-, nor urine-based isoprostane quantitations are accurate, linear and predictive as a marker for tissue oxidative stress in the canine species. Similarly, there are no published studies of measuring HODE as a biomarker of OS in companion animals

There is a need for another form of OS testing in companion animals, especially canines.

Managing and Treating Elevated Biomarkers of Oxidative Stress. Use of treatments and preventive health measures including applicable drugs, diets, supplements and exercise.

SUMMARY

The disclosure includes management and treatment measures to alleviate elevated oxidative stress biomarkers and their clinical expression in companion animals such as dogs or cats, and other animal species.

According to the disclosure, measurement is affected in bodily fluids, selectively saliva.

Collection of saliva is noninvasive, painless, relatively inexpensive and convenient for the individual. Salivary testing of OS and/or inflammatory biomarkers can reveal the latent or pre-clinical form of developing OS.

There is a need to provide for practical and rapid screening or testing for OS to permit enhancement of the health of animals. Current methods and findings in humans and rodents measure OS biomarkers primarily in serum or urine. However, while one can measure OS biomarkers in healthy dog serum, neither serum nor urine has provided reliable quantitative and linear measurements in healthy dogs or especially in those with chronic diseases, where one would predict their elevation from tissue OS.

In accordance with this disclosure there is provided a diagnostic test system for OS stress assessment in animals, in particular companion animals, such as dogs, cats, rabbits, hamsters, and horses.

Saliva can be used as a diagnostic tool to assess the health or disease status of an animal. Saliva is easily collected, stored and shipped, and provides a non-invasive means of multiple or serial sampling for use as a diagnostic tool for a variety of conditions in animals.

The disclosure uses a species-specific test for companion animals such as dogs or cats, and other animal species, and the appropriate methods.

The disclosure is further described in detail.

