LINUS PAULING INSTITUTE SPRING/SUMMER 2005 RESEARCH REPORT

Jan Frederik Stevens

Vitamin C Detoxifies Oxidized Fat

Jan Frederik Stevens, Ph.D.
LPI Principal Investigator

Summary: A study published in 2001 on reactions between vitamin C and lipid hydroperoxides, or rancid fat, caused concern that vitamin C might help generate DNA-damaging compounds from these lipid hydroperoxides, thereby increasing the risk of cancer. We discovered that, on the contrary, vitamin C reacts with breakdown products of rancid fats in the body to form harmless conjugates. This may help explain how vitamin C actually protects against DNA damage and cancer.

Vitamin C is an essential micronutrient that is involved in a variety of biochemical reactions. It is perhaps best known as a co-factor for the biosynthesis of the structural protein, collagen, thus adding strength and elasticity to skin, blood vessels, and bone. Vitamin C is also well known as an antioxidant that scavenges free radicals and reactive oxygen species to protect against oxidative damage to fatty acids, proteins, and DNA. Polyunsaturated fatty acids are especially susceptible to oxidation and, when oxidized, form lipid hydroperoxides, commonly known as rancid fats. Our body continuously produces low levels of these potentially harmful lipid hydroperoxides. Fortunately, these are largely converted into less toxic substances through a biochemical reaction that requires glutathione, an important antioxidant. A small fraction of lipid hydroperoxides, however, decomposes into a variety of breakdown products called lipid peroxidation (LPO) products. Some of these LPO products contain substances that can be chemically characterized as reactive aldehydes, meaning that they bind to biomolecules like proteins and DNA. We have discovered that vitamin C can bind to these toxic lipid-derived aldehydes and form unreactive conjugates, thus potentially preventing damage to proteins and DNA.

Studies performed by Blair and co-workers at the University of Pennsylvania and published in Science in 2001 showing that vitamin C can facilitate the breakdown of lipid hydroperoxides into reactive aldehydes attracted a lot of media attention. News reports sensationally reported that vitamin C could damage DNA and cause cancer. Because these aldehydes can damage DNA, thus potentially triggering early cancer-promoting events, Blair’s group argued that vitamin C can induce the formation of carcinogenic LPO products. However, Blair’s study was conducted in test-tube solutions that may not reflect the fate of lipid hydroperoxides in the body. Critics of Blair’s study have argued that traces of iron in the chemical solutions are more likely than vitamin C to be responsible for the degradation of lipid hydroperoxides. It is very difficult to remove all iron from solutions, and iron ions can initiate oxidizing reactions. On the other hand, iron in the body is tightly bound to proteins and is not available to react with lipid hydroperoxides. Like Blair’s colleagues, we have been careful in our test-tube experiments to remove all traces of iron. Despite this, we observed that vitamin C does seem to participate in the breakdown of lipid hydroperoxides. However, when the concentration of vitamin C exceeded that of lipid hydroperoxides by 15-fold, we observed that the initial increase in the formation of the reactive aldehyde, 4-hydroxy-2-nonenal (HNE), was followed by its disappearance. This striking observation led us to hypothesize that vitamin C is not only involved in the breakdown of lipid hydroperoxides, but also removes the resultant aldehydes by covalently binding to them. In subsequent test-tube experiments, we tested this hypothesis by exposing lipid hydroperoxides to a high concentration of vitamin C and monitoring the levels of HNE and ascorbylated HNE, which is what we call the vitamin C conjugate. We were pleasantly surprised when we saw that the disappearance of HNE was accompanied by the concomitant appearance of the vitamin C conjugate. These experiments provided clear evidence for a dual role of vitamin C in the removal of lipid hydroperoxides: 1) vitamin C facilitates the breakdown of lipid hydroperoxides into reactive LPO products, and 2) it binds to the reactive LPO products that it helped form. We subsequently determined the molecular structure of the vitamin C conjugate by a combination of nuclear magnetic resonance spectroscopy and mass spectrometry. We also detected two other ascorbylated LPO products in the reaction mixtures.

At this point we wondered why the Blair group only reported the vitamin C-induced decomposition of lipid hydroperoxides and not the subsequent ascorbylation reaction. The answer lies in the relative concentrations of lipid hydroperoxides and vitamin C. Both our group and Blair’s group used physiologically relevant concentrations of vitamin C, but the concentration of lipid hydroperoxides was much higher—non-physiological—in Blair’s study. When we incubated vitamin C with very high concentrations of lipid hydroperoxides, HNE, but not its vitamin C conjugate, was formed. This finding suggests that the amount of vitamin C must be much greater than the lipid hydroperoxide concentration for the ascorbylation reaction to occur. This is exactly what happens in the body. Because these studies were limited to chemical solutions in test tubes, the next step in our research was to determine whether the formation of ascorbylated LPO products has biological relevance.

Interactions of vitamin C with oxidzed lipidsTo determine the biological relevance of our newly discovered ascorbylation reaction, we developed a method for the selective and sensitive detection of the vitamin C conjugate of HNE in human plasma using liquid chromatography coupled to mass spectrometry. We were able to detect the vitamin C conjugate in the plasma of subjects with normal plasma vitamin C levels. The concentrations of the vitamin C conjugate were high compared to other LPO products, such as the F2-isoprostanes used as markers of oxidative stress, that are found in human plasma at concentrations at least 1,000-fold lower than the vitamin C conjugate. Therefore, we performed experiments to exclude the possibility that the vitamin C conjugate was formed in plasma after the blood samples had been drawn. These experiments verified that the vitamin C conjugate was formed in the body and not artificially. The results of this study were published in the December 28, 2004, issue of the Proceedings of the National Academy of Sciences U.S.A.

What, then, is the biological significance of this novel ascorbylation reaction? In view of the high plasma concentrations that we found for the ascorbylated LPO products, we hypothesized that ascorbylation represents an important pathway for the detoxification and elimination of toxic LPO products. When LPO products react with vitamin C, they lose their ability to modify DNA and induce mutations. Therefore, contrary to the speculation that emerged from the Blair study, it is conceivable that the vitamin C reactions we discovered may help prevent cancer. Ascorbylation may also help prevent inflammatory diseases that are exacerbated by LPO processes, such as atherosclerosis and autoimmune diseases like lupus. In the early stages of atherosclerosis, it appears that some LPO products make white blood cells stick to the arterial wall and start an inflammatory process that ultimately can lead to heart disease or stroke. A better understanding of that process and the role that micronutrients like vitamin C play in it may lead to specific health recommendations. Furthermore, the plasma levels of ascorbylated LPO products may provide useful information about disease states and the risk for developing a disease that has an underlying component of oxidative stress, such as Alzheimer’s. We now know that ascorbylated LPO products are formed in the body, and we need to find out what these vitamin C conjugates do and whether they have any value as an indicator, or biomarker, for people who may be at increased risk of developing chronic disease. We plan to study the role of this newly understood reaction between vitamin C and toxic lipids in atherosclerosis. We are also planning clinical studies to examine the blood chemistry of patients who have been diagnosed with coronary artery disease to see if ascorbylated LPO products are associated with this disease.

Last updated May, 2005


Micronutrient Research for Optimum Health


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