Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
2001-05-09
2003-04-01
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S074000, C436S084000
Reexamination Certificate
active
06541265
ABSTRACT:
BACKGROUND OF THE INVENTION
The consumption of foods rich in antioxidant compounds is well known to reduce the incidence of many chronic disease states, such as cancer. Moreover, the intake of certain antioxidants, such as vitamins C (ascorbic acid) and E (&agr;-tocopherol), is essential for survival in humans and most mammals, because these compounds are not biosynthesized.
Aerobic organisms are partially protected against oxidative challenges by sophisticated antioxidant defense systems. The importance of antioxidant defense systems in humans is demonstrated by the essential in vivo presence of both enzymatic and non-enzymatic antioxidant components. Oxidative stress and resultant oxidative damage may occur as a result of oxidative challenges such as air pollution or the “oxidative burst” associated with activated neutrophils mediated by the immune response. A constant source of oxidative stress results from formation of superoxide anions via “electron leakage” in the mitochondria during production of adenosine triphosphate (ATP). Although a superoxide anion is not exceedingly reactive in and of itself, it can initiate a cascade of events that eventually results in the formation of highly reactive free radicals and other oxidants. If these reactive oxygen species are not controlled by enzymatic and/or non-enzymatic antioxidant systems, in vivo oxidation of critical cellular components such as membranes, DNA, and proteins will result, eventually leading to tissue damage and dysfunction.
Inflammation can be induced by acute exercise in untrained individuals (Jenkins, R. R. et al. [1993
] Med Sci Sports Exerc
25:213-7). Strenuous exercise increases oxygen consumption and causes disturbance of the intracellular homeostasis between pro-oxidants and antioxidant, resulting in oxidative stress. Reactive oxygen species pose a serious threat to the cellular antioxidant defense system, such as by diminishing the reserve of antioxidant vitamins and glutathione, and increasing tissue susceptibility to oxidative damage. However, enzymatic and non-enzymatic antioxidants have demonstrated great adaptation to acute and chronic exercise. The delicate balance between pro-oxidants and antioxidants suggests that supplementation of antioxidants may be desirable for physically active individuals under certain physiological conditions by providing a larger protective margin.
In vitro experiments have shown that certain substances, including some antioxidant vitamins, such as vitamin C, are reactive with free iron and can cause oxidative damage to biomolecules. Under normal physiological conditions, metals are found bound to circulating proteins and rendered redox-inactive (Halliwell, B. et al. [1999
] Free radicals in Biology and Medicine
. Oxford N.Y.: Clarendon Press, Oxford University Press; Winterboum, C. C. [1981
] Biochem J
198:125-31). However , levels of free metals, such as iron, may be elevated during acute inflammation and become redox-active. Others have shown that under several physiological and pathophysiological conditions, increases in free iron in the presence of vitamin C results in oxidative stress.
Inflammation stimulates polymorphonuclear leukocytes and macrophages that produce large amounts of superoxide (O
2
•−
) and hydrogen peroxide (H
2
O
2
) (Babior, B. M. et al. [1973
] J Clin Invest
52:741-744; Halliwell, B., et al. [1999] supra). The detrimental effects of these radicals may be amplified in the presence of iron and the subsequent formation of other reactive intermediates, such as the hydroxyl radical (HO
•
). NADPH oxidase, a membrane-associated electron transport chain protein, becomes activated during inflammation and directly reduces O
2
to O
2
•−
(Equation 1). Superoxide can then be dismutated by superoxide dismutase to produce H
2
O
2
(Equation 2).
NADPH+2O
2
→2O
2
•−
+NADP
+
+H
+
(Equation 1)
O
2
•−
+O
2
•−
+2H
+
→H
2
O
2
+O
2
(Equation 2)
Superoxide can reduce transition metals, including ferric iron (Fe
3
+
), to ferrous iron (Fe
2
+
) (Equation 3). The reduced metal ion can then react with H
2
O
2
to generate the highly oxidizing HO
•
radical species (Equation 4).
Fe
3
+
+O
2
•−
→O
2
+Fe
2
+
(Equation 3)
Fe
2
+
+H
2
O
2
→HO
•
+OH
−
+Fe
3
+
(Equation 4)
The hydroxyl radical has been widely postulated to cause significant damage to several biomolecules in vivo. Although the relevance of the hydroxyl radical in biology has been questioned because of the requirement of redox-active free iron, Biemond and colleagues have shown iron release from ferritin during inflammation (Biemond, P. et al. [1984
] J Clin Invest
73(6): 1576-9).
In vitro vitamin C can exert pro-oxidant effects. The reduction potentials of Fe
3
+
(−0.4 V) and ascorbate (−0.17 V) easily allow the formation of the ascorbate radical and Fe
2
+
iron (Equation 5).
Fe
3
+
+ascorbate→Fe
2
+
+ascorbate
•
(Equation 5)
In addition, the formation of ferrous iron increases the possibility of the production of HO
•
by reacting with H
2
O
2
(Equation 4). Thus, it is feasible that the release of iron and the presence of vitamin C during acute inflammation characterized by high fluxes of oxidants could lead to HO
•
and ascorbate generation.
The reduction potentials of Fe
3+
(−0.4 V) and ascorbate (−0.17 V) easily allow the formation of the ascorbate radical and Fe
2+
iron (Equation 5). Therefore, in vitro vitamin C can exert pro-oxidant effects, by converting Fe
3+
into Fe
2+
, which reacts with H
2
O
2
to generate HO
•
(Halliwell, B. et al. [1999] supra; Roginsky, V. A. et al. [1994
] Free Radic Biol Med
17:93-103). Iron-ascorbate mixtures have been shown to stimulate free-radical damage to DNA, lipids, and proteins in vitro (Halliwell, B. et al. [1990
] Methods Enzymol
186:1-85). In vivo iron (Kadiiska, M. B. et al. [1995
] J Clin Invest
96:1653-7) supplementation and ascorbate-copper supplementation (Kadiiska, M. B. et al. [1992
] Mol Pharmacol
42:723-9) to rats have been reported to stimulate HO
•
generation. Thus, it is feasible that the release of iron and the presence of vitamin C during acute inflammation could lead to HO
•
and ascorbate
•
generation.
Until recently, there has been no investigation as to whether antioxidant supplements can act as pro-oxidants, causing oxidative stress in humans or animals under acute inflammatory conditions characterized by increases in levels of redox-active metal ions. Long-term supplementation with these compounds could have detrimental effects in patients suffering from acute inflammation and inflammatory conditions accompanied by increased levels of free iron. However, these supplements could also have detrimental effects on patients with certain disease conditions characterized by increased levels of free iron without the presence of inflammation. Such disease conditions include, for example, Alzheimer's, Parkinson's, atherosclerosis, diabetes, and hemachromotosis (Gerlach, M. et al. [1994
] J Neurochem
63(3):793-807; Halliwell, B. et al. [1985
] Mol Aspects Med
8(2):89-193). One study protective effect of vitamin C in the plasma in the presence of high levels of free iron in vitro, without the presence of inflammation (Berger, T. M. et al. [1997
] J Biol Chem
272: 15656-60). The Berger study (Berger, T. M. et al. [1997] supra) investigated if the naturally occurring high levels of ascorbic acid in pre-term infants, along with the presence of detectable levels of iron, would increase oxidative stress. The Berger study showed no concurrent increases in either lipid hydroperoxides or protein carbonyls in the
Saliwanchik Lloyd & Saliwanchik
University of Florida
Wallenhorst Maureen M.
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