LINUS PAULING INSTITUTE RESEARCH REPORT


Recent Publications by LPI Scientists

Selected summaries by Stephen Lawson

A comprehensive list of all LPI scientific papers published in 2002 and 2003 can be found here. Summaries of some representative papers appear below. LPI scientists are identified in boldface.


Aging

BECKMAN JS. Nitric oxide, peroxynitrite and ageing. In: Critical Reviews of Oxidative Stress and Aging (Cutler RG and Rodriguez H, eds), vol. 1, pp. 54-83, 2002.

In this book chapter, Dr. Beckman discusses the biological role of peroxynitrite, which is a relatively stable and very damaging oxidant formed from the reaction between nitric oxide and the superoxide radical. Its formation can be attenuated by the endogenous anti oxidant enzyme superoxide dismutase. In fruit flies, the deletion of superoxide dismutase results in a 60% reduction in life span, which is corrected when super-oxide dismutase production is increased. Peroxynitrite serves an important function in the immune system, where it is generated by inflammatory cells as an antimicrobial defense. Peroxynitrite can damage proteins by nitrating the amino acid tyrosine, and nitration of structural proteins abundant in cells can result in loss of normal function. Since there are only about one million motor neurons in a human and they are not regenerated, damage to proteins in these neurons over time may be critical and result in some of the manifestations of aging. For instance, mutations to superoxide dismutase, the enzyme that inhibits the formation of peroxynitrite, are found in amyotrophic lateral sclerosis (ALS) patients. The abnormal superoxide dismutase loses its affinity for zinc and helps to generate, rather than suppress, the formation of peroxynitrite, which then damages the motor neurons. Peroxynitrite is a double-edged sword—small amounts generated by immune cells are beneficial, but residual or inappropriately generated peroxynitrite can damage biological molecules and vulnerable motor neurons, contributing to age-related decline.

HAGEN TM, MOREAU R, SUH JH, and VISIOLI F. Mitochondrial decay in the aging rat heart: evidence for improvement by dietary supplementation with acetyl-L-carnitine and/or lipoic acid. Ann. N.Y. Acad. Sci. 959: 491-507, 2002.

This paper summarizes a number of experiments with rats supplemented with acetyl-L-carnitine, a non-protein amino acid, or R-alpha lipoic acid. Heart muscle cells from old rats show both decreased oxygen consumption and increased production of damaging oxidants, or free radicals. Supplementation of old rats with acetyl-L-carnitine for one month had no effect on vitamin C levels or oxidant levels in cardiac cells, but reversed mito chondrial decay. Lipoic acid supplementation for two weeks increased vitamin C levels in cardiac cells in both young and old rats and restored the vitamin C status in cells from old rats to that of young, unsupplemented rats. These results suggest that the combined use of acetyl-L-carnitine and lipoic acid may help maintain myocardial function in aging mammals.

Photo of various journals

LIU J, HEAD E, GHARIB AM, YUAN W, INGERSOLL RT, HAGEN TM, COTMAN CW, and AMES BN. Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-L-carnitine and/or R-alpha-lipoic acid. Proc. Natl. Acad. Sci. USA 99: 2356-2361, 2002.

In this study, the authors examined the effect of supplemental acetyl-L-carnitine and/or R-alpha lipoic acid on memory function in old rats. They found increased oxidative damage in the hippocampus, a region of the brain associated with memory, in old rats, which may explain the decline in memory function with increasing age. Using two tests of memory performance—the Morris water maze and a time discrimination procedure—the investigators observed improved memory function in old rats supplemented with either acetyl-L-carnitine or lipoic acid. The greatest benefit was found when both supplements were given together, which also resulted in the reduction of oxidative damage in the hippocampus to levels seen in young rats.

MICHELS AJ, JOISHER N, and HAGEN TM. Age-related decline of sodium-dependent ascorbic acid transport in isolated rat hepatocytes. Arch. Biochem. Biophys. 410: 112-120, 2003.

The vitamin C concentration in liver cells from old rats is about 70% lower than that in young rats. Two forms of a sodium-dependent vitamin C transporter (SVCT) are found in cells—SVCT1 and SVCT2—that allow the uptake of vitamin C from extracellular fluids into cells. The authors found that the amount of SVCT1 in liver cells from old rats was only 55% of the amount in young rats, whereas the amount of SVCT2 remained unchanged with age. These results may help explain the decline in vitamin C status associated with aging, as has been observed in elderly humans, and suggest that higher plasma concentrations of vitamin C may be needed to offset age-related changes in SVCT1 in the elderly in order to maintain optimal vitamin C status.

Antioxidants and oxidative stress

DIETRICH M, BLOCK G, HUDES M, MORROW JD, NORKUS EP, TRABER MG, CROSS CE, and PACKER L. Antioxidant supplementation decreases lipid peroxidation biomarker F(2)-isoprostanes in plasma of smokers. Cancer Epidemiol. Biomarkers Prevent. 11: 7-13, 2002.

It is well established that smokers exhibit high levels of oxidative stress. The experiments in this paper were designed to evaluate whether antioxidants can lower oxidative stress in smokers. One hundred and twenty-eight male and female smokers from 20 to 78 years old were enrolled in the study and given daily either a placebo; 500 mg of vitamin C; or a combination of 500 mg of vitamin C, about 800 mg of total vitamin E as mixed tocopherols and tocotrienols, and 95 mg of lipoic acid. Plasma levels of F2-isoprostanes, markers of oxidative damage to lipids, were measured at baseline and after two months. In smokers with a body mass index (BMI) above the median, vitamin C decreased levels of F2-isoprostanes, but, surprisingly, the antioxidant combination did not. F2-isoprostanes were strongly associated with increasing BMI. No effect of antioxidants was observed in smokers with a BMI less than the median, suggesting that vitamin C can reduce oxidative stress only in smokers who are overweight.

HAGEN TM, LIU J, LYKKESFELDT J, WEHR CM, INGERSOLL RT, VINARSKY V, BARTHOLOMEW JC, and AMES BN. Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc. Natl. Acad. Sci. USA 99: 1870-1875, 2002.

The authors fed acetyl-L-carnitine and/or R-alpha lipoic acid to young and old rats and measured several parameters of metabolic function and oxidative stress. Young rats and old rats, which showed a three-fold decline in their activity level, were fed acetyl-L-carnitine and lipoic acid for one month. Both populations exhibited increased activity, although the increase was greatest in old rats. Oxygen consumption in liver cells from old supplemented rats also increased, reflecting improved metabolic function. Acetyl-L-carnitine plus lipoic acid also increased vitamin C levels in liver cells from both young and old rats. While acetyl-L-carnitine alone increased malondialdehyde (MDA), a marker of lipid oxidative damage, in liver cells from young and old rats, lipoic acid alone, as well as the combination of acetyl-L-carnitine and lipoic acid, lowered MDA levels in the livers of young and old rats.

LEONARD SW, TERASAWA Y, FARESE RV Jr, and TRABER MG. Incorporation of deuterated RRR- or all-rac-alpha-tocopherol in plasma and tissues of alpha-tocopherol transfer protein-null mice. Am. J. Clin. Nutr. 75: 555-560, 2002.

The vitamin E family has eight members—four tocopherols and four tocotrienols. The recommended dietary allowance (RDA) is based only on RRR-alpha-tocopherol, also known as natural or d-alpha-tocopherol, because this is the only form that the body retains. A protein synthesized in the liver, the alpha-tocopherol transfer protein (alpha-TTP), preferentially recognizes RRR-alpha-tocopherol, which it transfers into lipoproteins that circulate in the plasma. In the experiments reported in this paper, the investigators found that mice without the gene that codes for alpha-TTP accumulate alpha-tocopherol in the liver, resulting in the depletion of alpha-tocopherol in peripheral tissues. The results also demonstrate that the bioavailability of natural alpha-tocopherol is twice that of synthetic vitamin E, or d,l-alpha-tocopherol.

MOYER RA, HUMMER KE, FINN CE, FREI B, and WROLSTAD RE. Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: Vaccinium, Rubus, and Ribes. J. Agric. Food Chem. 50: 519-525, 2002.

Many fruits contain a variety of antioxidants like vitamin C and other substances called phytochemicals, some of which, known as flavonoids, also function as antioxidants. In this investigation, the authors measured the antioxidant capacity and anthocyanin (a dark pigment) content of various cultivars of blueberries, blackberries, and black currants. The antioxidant capacity was found to be related more to total flavonoid content than to anthocyanin content. Blueberries exhibited the highest anthocyanin content, followed by black currants and blackberries. While one test of antioxidant capacity found blueberries to have the highest value, a different test found higher values in blackberries, although there was a lot of variability among the specific cultivars. These results show that these fruits are an important source of antioxidants and that selective breeding of particular cultivars may enhance these properties.

DEVARAJ S and TRABER M. Gamma-Tocopherol, the new vitamin E? Am. J. Clin. Nutr. 77: 530-531, 2003.

In this editorial, the authors call for urgent clinical studies to determine the respective protective functions of the different tocopherols in platelet aggregation, inflammation, and heart disease. Although the body preferentially retains the d-alpha-tocopherol form of vitamin E, gamma-tocopherol has been found in some experiments to exhibit greater anti-inflammatory and antioxidant properties. Unlike alpha-tocopherol, gamma-tocopherol scavenges reactive nitrogen species, such as peroxynitrite. Alpha-tocopherol has a more pronounced effect than gamma-tocopherol on the antiatherogenic behavior of vascular smooth muscle cells. Dose and duration of use, as well as monitoring oxidative stress, inflammation, plasma levels of vitamin E, and relevant clinical endpoints, are important factors to consider when designing effective clinical studies to assess the relative merits and functions of the different tocopherols.

SUH J, ZHU B-Z, and FREI B. Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide. Free Radic. Biol. Med. 34: 1306-1314, 2003.

The potential pro-oxidant function of vitamin C in vivo has been controversial. The experiments reported in this paper demonstrate that vitamin C functions as an antioxidant even in circumstances in which it might be expected to be pro-oxidant. The authors depleted human plasma of vitamin C or added vitamin C to other samples. Iron or copper salts were then added to the plasma samples. In classical chemical experiments, vitamin C becomes pro-oxidant when treated with these transition metal ions in solution. However, in this study, vitamin C strongly protected against lipid oxidation in plasma, as measured by hydro-peroxide and malondialdehyde formation. Lipid oxidation was promoted only in the plasma that had been depleted of vitamin C. Vitamin C also did not increase protein oxidation, as measured by protein carbonyl formation.

Cancer

FRANKE AA, CUSTER LJ, COONEY RV, TANAKA Y, XU M, and DASHWOOD RH. Inhibition of colonic aberrant crypt formation by the dietary flavonoids (+)-catechin and hesperidin. Adv. Exp. Med. Biol. 505: 123-133, 2002.

Heterocyclic amines are mutagens formed in protein-rich food, such as meat, by high-temperature cooking. When rats are fed these cooked-meat mutagens, they develop precancerous lesions in the intestine called aberrant crypts, which are presumed to be reliable biomarkers for human colon cancer. In this study, the investigators tested the effects of hesperidin, which is a flavonoid from citrus fruit, and catechin, a flavonoid from tea, on the inhibition of colonic aberrant crypts in rats treated with cooked-meat mutagens. Catechin and hesperidin inhibited the development of aberrant crypts, suggesting that they may be effective chemopreventive substances against colon cancer. Working in the lab

KRUEGER SK, WILLIAMS DE, YUEH M-F, MARTIN SR, HINES RN, RAUCY JL, DOLPHIN CT, SHEPHARD EA, and PHILLIPS IR. Genetic polymorphisms of flavin-containing monooxygenase (FMO). Drug Metab. Rev. 34: 523-532, 2002.

The flavin-containing monooxygenase (FMO) system comprises a number of genes that code for enzymes that metabolize drugs and other foreign compounds (xenobiotics) in mammals. Mutations in these genes are associated with various diseases, including trimethylaminuria, in which the affected patient excretes trimethylamine, a substance with a potent fishy odor, in urine and sweat. Specific forms of FMO are found in different tissues, and the form found in the lung is called FMO2. FMOs exist in different populations in a number of closely related but different forms called polymorphisms. This study reports the finding that one variant form, or polymorphism, of FMO2 is present in a significant percentage of African Americans and Hispanics, which may affect drug metabolism or xenobiotic toxicity in the lung and has implications for determining dosage in drug therapy.

SANTANA-RIOS G, ORNER GA, XU M, IZQUIERDO-PULIDO M, and DASHWOOD RH. Inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced colonic aberrant crypts in the F344 rat. Nutr. Cancer 41: 98-103, 2001.

This paper reports experiments showing that white tea—the least processed type of tea—strongly inhibited the formation of colonic aberrant crypts in rats treated with a cooked-meat mutagen known as PhIP. White tea is prepared by steaming and drying the buds and young leaves but without withering. It has higher levels of polyphenols called catechins than green or black tea. Tea was prepared as it is for humans and given to rats for 8 weeks, along with five exposures to PhIP, resulting in a 75% decrease in the number of aberrant crypts compared to rats given water instead of tea. Further studies revealed that white tea altered cytochrome P450 enzymes that metabolize drugs and xenobiotics, leading to enhanced metabolism of PhIP and increased excretion of its metabolites in the urine. Additionally, in another test of mutagenicity, nine substances extracted from white tea and then combined were less effective than whole tea in inhibiting mutagenicity.

BLUM CA, XU M, ORNER GA, DÍAZ GD, LI Q, DASHWOOD W-M, BAILEY GS, and DASHWOOD RH. Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis, cell proliferation, and beta-catenin/Tcf signaling. Mutat. Res. 523-524: 217-223, 2003.

This paper reports that chlorophyll and chlorophyllin, a derivative of chlorophyll, had differential effects on the development of aberrant crypts in the rat colon induced by carcinogens, including the cooked-meat mutagen IQ and the chemical carcinogen 1,2-dimethylhydrazine (DMH). Chlorophyll or chlorophyllin were fed to rats in their diet or given in drinking water after the rats had been exposed to, or initiated with, either of the carcinogens. Post-initiation treatment with low-dose chlorophyllin promoted the formation of aberrant crypts by DMH only, whereas high-dose treatment was inhibitory against IQ-induced aberrant crypts. In another experiment, an intermediate dose of natural chlorophyll suppressed aberrant crypts induced by exposure to IQ or a metabolite of DMH. Further experiments are planned to understand the mechanisms responsible for these effects more thoroughly.

DASHWOOD RH and XU M. The disposition and metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline in the F344 rat at high versus low doses of indole-3-carbinol. Food Chem. Toxicol. 41: 1185-1192, 2003.

The authors examined the effect of a range of doses of indole-3-carbinol (I3C), a substance found in cruciferous vegetables that has anticancer properties, on the metabolism of a cooked-meat mutagen known as IQ in rats. At very low doses of I3C, the metabolism of IQ by liver enzymes was unfavorably altered, whereas larger doses induced the enzymes that detoxify IQ, leading to decreased DNA damage. However, the specific liver enzyme induced by I3C is also known to activate certain carcinogens, such as polycyclic aromatic hydrocarbons that are found in tobacco smoke and grilled meats. Therefore, I3C may not only inhibit cancer, but depending on the dosage and timing, may also enhance certain types of cancer.

WILLIAMS DE, BAILEY GS, REDDY A, HENDRICKS JD, OGANESIAN A, ORNER GA, PEREIRA CB, and SWENBERG JA. The rainbow trout (Oncorhynchus mykiss) tumor model: recent applications in low-dose exposures to tumor initiators and promoters. Toxicol. Pathol. 31 (Suppl): 58-61, 2003.

The authors of this study have developed trout as a model for human carcinogenesis. The use of trout has a number of advantages: a vast number of small, relatively inexpensive animals can be used for high statistical power, trout are sensitive to human carcinogens and metabolize them similarly to humans, and they have a low incidence of spontaneous tumors. These attributes allow dose-response studies in trout that reliably evaluate low doses of potential carcinogens. In one experiment, trout were exposed to the liver carcinogen aflatoxin B1, produced by molds on grains, and then fed diets containing increasing amounts of indole-3-carbinol (I3C), an anticancer substance in cruciferous vegetables. I3C significantly increased tumor incidence and multiplicity (number of tumors per animal) at levels in the diet of 500 ppm and higher, corresponding to a daily intake of 14-28 mg I3C/kg of body weight. Therefore, the use of some commercial I3C supplements that achieve this range of intake in humans may pose risks. The second experiment found that the relationship between the amount of the carcinogen dibenzo[a,l]pyrene (DBP), a polycyclic aromatic hydrocarbon found in charred meat and tobacco smoke, administered to trout and the resultant tumor incidence was not linear at low doses, i.e., very low doses of DBP may not be as carcinogenic as previously thought.

AUBORN KJ, FAN S, ROSEN EM, GOODWIN L, CHANDRASKAREN A, WILLIAMS DE, CHEN D, and CARTER TH. Indole-3-carbinol is a negative regulator of estrogen. J. Nutr. 133: 2470S-2475S, 2003.

Epidemiological studies suggest that the consumption of cruciferous vegetables decreases the risk of breast cancer, and animal studies support the role of indole-3-carbinol (I3C), an anticancer substance in these vegetables, in preventing estrogen-enhanced cancer, such as cancers of the breast, cervix, and uterus. This study investigated the effect of diindolylmethane (DIM), which is an I3C metabolite, and genistein, which is the major flavonoid in soy, on the growth of estrogen-sensitive human breast cancer cells in culture. At physiologically relevant concentrations, DIM and genistein had slight effects on cell death, but when combined, they dramatically increased cell death by increasing the amount of certain proteins that stimulate programmed cell death, increasing the expression of a tumor suppressor gene, and interfering with estrogen signaling.

Heart disease

FANG JC, KINLAY S, BELTRAME J, HIKITI H, WAINSTEIN M, BEHRENDT D, SUH J, FREI B, MUDGE GH, SELWYN AP, and GANZ P. Effect of vitamins C and E on progression of transplant-associated arteriosclerosis: a randomised trial. Lancet 359: 1108-1113, 2002.

This clinical study reported that daily supplementation with 1,000 mg of vitamin C and 800 IU of vitamin E for one year after cardiac transplantation significantly retarded the progression of arteriosclerosis in the coronary arteries. In the placebo group of 21 subjects, the intimal index—a measure of atherosclerotic plaque—increased about 8%, but did not increase at all in the 19 patients treated with vitamins C and E. The first few years following transplantation is the period in which thickening of the arterial wall, which predicts clinical outcome, proceeds most rapidly. The authors speculated that the benefit of vitamins C and E observed in this study may be applicable to patients undergoing other organ transplants.

Working in the lab

VISIOLI F, SMITH A, ZHANG W, KEANEY JF Jr, HAGEN T, and FREI B. Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status. Redox Rep. 7: 223-227, 2002.

Abnormal dilation or stiffness of the arteries is associated with hypertension and heart disease. A number of clinical studies have found that vitamin C promotes relaxation of the arteries (vasodilation), probably through its effect on the generation of nitric oxide, a signaling molecule that induces the relaxation of vascular smooth muscle cells and also inhibits platelet aggregation. In this study, the authors reported that lipoic acid and vitamin C enhanced nitric oxide production in cultured vascular endothelial cells, but had no effect on the levels of another antioxidant, glutathione, suggesting that depletion of glutathione is not involved in vascular dysfunction. Vitamin C enhances nitric oxide synthesis by stabilizing and increasing the amount of a molecule critical to the synthesis of nitric oxide, tetrahydrobiopterin, but the mechanism by which lipoic acid stimulates the production of nitric oxide is unknown.

FREI B. To C or not to C, that is the question! J. Am. Coll. Cardiol. 42: 253-255, 2003.

In this editorial, Dr. Frei briefly reviewed the evidence on the role of vitamin C in the prevention of heart disease. Specifically, he discussed a recent paper that showed an impressive 28% reduction in the risk of heart disease in women taking vitamin C supplements. Unlike a number of other studies, dietary vitamin C intake, mainly from fruits and vegetables, was not associated with protection against heart disease, suggesting that vitamin C itself is beneficial. The dietary range of intake in the study was 61-209 mg/day of vitamin C, while the supplement users had a median daily intake of 672 mg. Recent pharmacological data indicate that a daily intake of 400 mg is necessary to fully saturate circulating cells in women, which is compatible with these results. Dr. Frei also noted that the study did not reveal any increased risk of heart disease attributable to vitamin C. The mechanism by which vitamin C affords protection against heart disease is still under investigation and may be related to improved endothelial cell function and antioxidant defense.

Neurodegenerative disease

CASSINA P, PELUFFO H, PEHAR M, MARTINEZ-PALMA L, RESSIA A, BECKMAN JS, ESTEVEZ AG, and BARBEITO L. Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J. Neurosci. Res. 67: 21-29, 2002.

Astrocytes are star-shaped cells that nurture neurons, the specialized cells in the nervous system that conduct nerve impulses. In the cell culture experiments reported in this paper, spinal cord astrocytes became morphologically altered after exposure to peroxynitrite, a powerful oxidant formed in the body from reactions between nitric oxide and the superoxide radical. Additionally, the altered astrocytes became toxic to motor neurons and induced apoptosis (programmed cell death). Peroxynitrite scavengers protected motor neurons from death.

TENG R-J, YE Y-Z, PARKS DA, and BECKMAN JS. Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration. Free Radic. Biol. Med. 33: 1243-1249, 2002.

Nitrative stress caused by the highly reactive peroxynitrite is associated with many pathologies. Peroxynitrite and its metabolites damage proteins by a process known as nitration. In Parkinson’s disease, tyrosines in certain enzymes are nitrated, resulting in loss of function. In amyotrophic lateral sclerosis (ALS), neurofilaments are damaged by nitration. In this study, the investigators examined the effect of various substances, including urate, vitamin C, and sulfur-containing (thiol) antioxidants, on the nitration of proteins in rat heart and brain. Vitamin C and thiols had little effect on nitration by peroxynitrite in either the heart or brain, but urate, which accumulated in the heart to levels higher than those in the brain, protected against protein nitration. Urate is formed naturally in the body as a by-product of DNA metabolism and can also be formed from dietary inosine.

ISCHIROPOULOS H and BECKMAN JS. Oxidative stress and nitration in neurodegeneration: cause, effect, or association? J. Clin. Invest. 111: 163-169, 2003.

In this perspective paper, the authors offered the view that oxidative and nitrative processes that generate reactive oxygen radicals and reactive nitrogen radicals are central to the death of motor neurons in neurodegenerative diseases like Parkinson’s and ALS. Oxidants like peroxynitrite, formed from the reaction between nitric oxide and the superoxide radical, as mentioned above, damage proteins, resulting in abnormal protein aggregation, compromised integrity of the blood-brain barrier, and, indirectly, the necrosis or apoptosis (programmed cell death) of motor neurons. In models of Parkinson’s disease, certain environmental toxins generate reactive species that produce cellular dysfunction and death. In ALS, mutations in an important endogenous antioxidant enzyme, superoxide dismutase, have been linked to muscle degeneration. While maintaining or improving antioxidant status may prove important in preventing or treating these diseases, other metabolic dysfunctions, such as failures in energy production, changes in the regulation of metals like iron, copper, and zinc, and the inability to repair damaged proteins, also contribute to the pathologies.

Last updated November, 2003


Micronutrient Research for Optimum Health


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