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Isoprostane concentrations in the urine of cats from smoking households compared to cats from non-smoking households

Jennifer Ryan Walls BA, Maureen McMichael* DVM Diplomate ACVECC, Craig Ruaux** BVSc PhD MACVSc

*Department of Small Animal Medicine and Surgery,
**Gastrointestinal Laboratory, College of Veterinary Medicine
Texas A&M University

Cigarette smoking promotes serious health problems from both direct inhalation and second hand exposure.  Many resulting health issues may be caused by the oxidants in smoke, which result in cellular oxidative damage. The potential risk of diseases caused by smoking has sparked numerous studies of oxidative stress.

Oxidative stress is defined as an excess of reactive oxygen species (ROS) over endogenous antioxidants and/or a decrease in endogenous antioxidants. The effects of oxidative stress are widespread and potentially detrimental. Reactive oxygen species continuously form in vivo as products of aerobic metabolism,1 therefore cell membranes and organelles are equipped with control mechanisms to limit damage. These control mechanisms are termed antioxidants and the body has an abundance of natural antioxidants to combat the normal production of ROS. If oxidative molecules overwhelm the cell systems, a cascade of detrimental effects ensues. Three key players are involved in ROS damage to biological membranes. Hydrogen peroxide and superoxide anion are produced and create limited damage. In the presence of free iron, specifically ferrous iron, the hydroxyl radical is formed. This is the most potent free radical known and can produce damage to all biological membranes.2 Several pathologic processes such as reperfusion injury, cardiovascular disease, spinal cord injuries,1 and smoking involve ROS mediated tissue injury.

Lipid peroxidation of cell membranes, initiated by the hydroxyl radical, provides a major source of ROS byproducts.2 Assessment of ROS mediated injury includes measurement of ROS themselves, measurement of reaction products/byproducts produced during ROS mediated injury, or measurement of endogenous antioxidants. Two known byproducts of ROS damage are malondialdehyde (MDA) and isoprostanes. Earlier studies focused on measuring MDA. Unfortunately, the methodology was inconsistent between laboratories, the samples varied (i.e., tissue biopsies, blood, plasma, urine, CSF, etc.) and the results were inconsistent. One consistent problem resulted from ex-vivo lipid peroxidation occurring in blood samples.3 

The action of ROS on arachidonic acid produces byproducts called isoprostanes, which are now considered to be one of the most accurate markers for oxidative stress in vivo. Isoprostanes circulate in plasma and are excreted in urine.4 Non-invasive urine collection allows for easy application of isoprostane measurements. Isoprostanes are being used as a biomarker of lipid peroxidation in humans for many diseases such as heart failure, diabetes, hepatic cirrhosis, COPD, and asthma.5 The nomenclature of the isoprostanes is confusing. There are three different nomenclature systems in use all identifying an individual isoprostane by different names. Originally they were called iso-prostaglandins. The term isoprostane was chosen to differentiate them from prostaglandins, which are produced by the enzyme cyclooxygenase'action on arachidonic acid. This means that compounds that act on the cyclooxygenase enzyme (e.g., NSAIDS, glucocorticoids) do not affect the formation of isoprostanes.6 Eight-iso-prostaglandin F2 F2t-isoprostane and iPF2 are the same isoprostane. This is the isoprostane most commonly tested and the one that our study assessed.  In vivo, 8-iso-prostaglandin F2 a exhibits biological activity as a vasoconstrictor in renal and pulmonary vasculature while also acting as a mitogen in vascular smooth muscle cells.4 Therefore, finding elevated concentrations of this isoprostane may be both a marker for oxidative stress and may, itself, be a cause of biological injury.

Rats have been the animal model used to show increased levels of isoprostanes following induced oxidant stress in many research studies.Although there is ample data on clinical studies of humans, little clinical research has been done to study oxidant stress and correlating isoprostane levels in domestic animals. Cigarette smoking was one of the first of many conditions that demonstrated elevated isoprostane levels.5 

The purpose of this study was to assess whether isoprostanes were elevated in the urine of cats from smoking households compared to cats from non-smoking households.