QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, I.-W. Articles by Kuo, S.-M. Search for Related Content PubMed PubMed Citation Articles by Peng, I.-W. Articles by Kuo, S.-M.

---

Biochemical and Molecular Actions of Nutrients
Research Communication

Flavonoid Structure Affects the Inhibition of Lipid Peroxidation in Caco-2 Intestinal Cells at Physiological Concentrations

Department of Exercise and Nutrition Sciences, University at Buffalo, Buffalo, NY 14214

²To whom correspondence should be addressed. E-mail: smkuo{at}buffalo.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The antioxidant activity of flavonoids in cell-free systems has been studied extensively. We compared flavonoids with different structural features on their abilities to protect live Caco-2 intestinal cells from lipid peroxidation due to hydrogen peroxide and Fe²⁺ treatment. Flavonoids with o-dihydroxyl or vicinal-trihydroxyl groups, including quercetin, myricetin (flavonol), luteolin (flavone) and (-)-epigallocatechin gallate (EGCG; flavanol), when co-incubated with a mixture of 30 µmol/L H₂O₂ and 30 µmol/L FeSO₄, prevented the formation of malondialdehyde (MDA) at 1 or 10 µmol/L in at least one of two separate experiments. In experiments in which flavonoids were preincubated with cells but removed before the 30 µmol/L H₂O₂ and Fe²⁺ treatment, quercetin at 0.1 µmol/L, EGCG at 1 µmol/L and luteolin at 10 µmol/L exerted protective effects in at least one of two experiments. Kaempferol (flavonol) and the isoflavones, genistein and daidzein, did not prevent lipid peroxidation at 0.1–10 µmol/L in either co- or preincubation experiments. None of the flavonoids tested at 0.1–10 µmol/L increased H₂O₂ and Fe²⁺-induced lipid peroxidation after co- or preincubation. In summary, these observations support the importance of plant-based food items such as vegetables, fruits and teas in the diet.

KEY WORDS: • flavonoids • reactive oxygen species • iron • malondialdehyde • Caco-2 cells

Oxidative damage to various cellular components has been linked with the development of degenerative diseases (1). For example, unsaturated fatty acids in cell membranes can be oxidized in the presence of reactive oxygen species. The oxidation leads to the breakage of fatty acid chains and compromises the membrane integrity. In addition, the end product of lipid peroxidation, malondialdehyde (MDA), is cytotoxic (2). Greater intake of vegetables and fruits has been linked to a reduced incidence of diseases including cancer and cardiovascular diseases (3,4). The antioxidants in fruits and vegetables may contribute to the beneficial effects.

We investigated the ability of dietary flavonoids, a group of compounds found exclusively in plants, to prevent lipid peroxidation in live cells. Previous cell-free studies have demonstrated the ability of flavonoids to trap free radicals (5,6). In addition, flavonoids can prevent lipid peroxidation in microsomes and liposomes (5,7–9). The antioxidant potency of flavonoids in these studies depends on the arrangement of hydroxyl groups on the benzene ring (5–9). The first goal of this study was to examine whether in live cells that were incubated with reactive oxygen species, lipid antioxidant activity of flavonoids also followed the same structure-dependency as in the cell-free systems. Responses of live cells are complicated by the membrane permeability (10–12) and the intracellular metabolism of flavonoids (11,12). Nevertheless, experiments using live cells can provide more insight into the physiological importance of flavonoids. We used human intestinal Caco-2 cells as the model here because intestinal cells have the most exposure to dietary flavonoids under physiological conditions. Also, the transport, metabolism and biological activities of flavonoids have been characterized in Caco-2 cells (10–17).

An additional goal of this study was to determine whether earlier flavonoid exposure helps to protect the cells from lipid peroxidation. The concentration of flavonoids fluctuates in the intestinal lumen and plasma depending on the pattern of ingestion. The nutritional importance of flavonoids will be greater if intracellular flavonoids can protect cells even after the extracellular flavonoids have been removed. Cell membranes are permeable to flavonoids (10–12). In addition, cellular retention of quercetin was demonstrated in our previous fluorescence imaging and gene expression experiments (13,14,17). A recent study on oxidative DNA damage reported an antioxidant effect of (-)-epigallocatechin gallate (EGCG) after extracellular EGCG was removed. EGCG was the only flavonoid examined and the mechanism of the protection was unknown (18). We hypothesized that the lipid protective effect will occur after flavonoids in the medium have been removed.

The plasma concentrations of flavonoids are usually <10 µmol/L even after a bolus dose, and the steady-state levels are mainly <1 µmol/L (19–22). The intestinal luminal concentrations of flavonoids, although higher, probably do not exceed 10 µmol/L from a normal diet (<1 mg flavonoid), an amount suggested by extrapolation from the bolus dose study (21). Our study thus focused on the physiologically achievable concentration range of 0.1–10 µmol/L. Because information on the physiological concentrations of flavonoids has only recently become available (19–22), most earlier studies on flavonoids and lipid peroxidation had not considered physiological concentrations.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

Quercetin, myricetin, kaempferol and luteolin (90–98% purity) were purchased from Sigma-Aldrich (St. Louis, MO). Genistein, daidzein and EGCG (purity > 95%) were purchased from Calbiochem (La Jolla, CA). Hydrogen peroxide and ferrous sulfate (purity > 99%) were obtained from Fisher Scientific (Pittsburgh, PA). 1-Methyl-2-phenylindole and malondialdehyde bis(dimethyl acetal) (99% pure) used for MDA analysis were from Sigma-Aldrich. All cell culture reagents with the exception of fetal bovine serum were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Hyclone (Logan, UT). Distilled deionized water or 100% ethanol was used for solution preparation. All other chemicals used in the study were of reagent grade with >99% purity.

Cell culture and treatment.

Human colon adenocarcinoma–derived Caco-2 cells were purchased from the American Type Culture Collection (Manassas, VA). These cells were cultured as described previously (13,15). Cells were seeded at 4 x 10⁴ cells/cm² in 6-well plates and used for the experiment on d 8 after seeding. Under these seeding conditions, Caco-2 cells were confluent on d 4 and started to express maltase, the differentiation marker of enterocytes after reaching confluence (15). All flavonoids were prepared as 10 or 25 mmol/L sterile ethanol stock solutions and kept at -20°C (15,16). In the co-incubation experiment, flavonoids were diluted to the desirable concentrations in HBSS immediately before the experiment. They were added to the wells containing attached cells incubated in HBSS. Solutions containing H₂O₂ and FeSO₄ (H₂O₂/Fe²⁺) were mixed in HBSS and added to the above wells 30 min later to initiate the oxidation reaction. The final concentrations of H₂O₂ and FeSO₄ were both 30 µmol/L. The concentrations of flavonoids used are indicated in Table 1. The cells were incubated for an additional 3 h before being lysed for the MDA assay. Hydrogen peroxide and ferrous sulfate solutions were freshly prepared for each experiment. All incubations were carried out inside the 37°C, 5% CO₂ cell culture incubator. Each treatment in an experiment was repeated three times with three independent wells of cells. Results from two independent experiments are shown.

Analytical methods.

The extent of lipid peroxidation was determined by measuring the cellular content of MDA. We used a colormetric assay (23) that is also available commercially as a kit for the determination of lipid peroxidation (#437634, Calbiochem). We followed the procedure of the kit with some modifications. Briefly, cells were rinsed twice with HBSS after the H₂O₂ + Fe²⁺ treatment. They were lysed in 20 mmol/L Tris buffer (pH 7.4) containing 5 mmol/L BHT through repeated freeze-thawing and a brief sonication. The cell lysate was then allowed to react with N-methy-2-phenylindole for 30 min at 65°C. MDA content in the cell lysate was determined on the basis of the OD₅₈₆ of samples and the MDA standard curve (Fig. 1A). The linear regression of all MDA standard curves was 0.99. MDA in every cell lysate sample was determined by duplicate measurements and the mean of the duplicate measurements represented the result of an independent sample. Results from three independent samples were used to compute the mean and SD of the triplicate samples. A standard MDA curve was determined in every experiment. MDA production in the absence of H₂O₂ + Fe²⁺ and flavonoids (baseline MDA) was also measured in every experiment. Baseline MDA was subtracted from the total MDA to obtain values of H₂O₂ + Fe²⁺–induced MDA production. The level of MDA was further normalized by the protein content of each sample as determined by the Lowry assay.

View larger version (23K): [in this window] [in a new window]   FIGURE 1 Malondialdehyde standard curve, n = 7 (panel A) and time-dependent production of malondialdehyde in Caco-2 cells after H2O2 and FeSO4 treatment (panel B). Values are means ± SD, of triplicate wells of cells. *Different from the 0-h time point, P < 0.05.

All results shown in the figures are means ± SD of three different wells of cells after subtracting the cellular basal MDA production measured in the absence of H₂O₂ + Fe²⁺ and flavonoid treatment. Statistical analyses were performed separately for two independent sets of data. One-way ANOVA was used followed by post-hoc Bonferroni-Dunn tests for comparisons with the respective control without flavonoid treatment. Differences from the control with P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Time-dependent generation of MDA from cells was measured after the H₂O₂ and Fe²⁺ treatment of Caco-2 cells (Fig. 1B). In a preliminary experiment, we compared 30 µmol/L each of H₂O₂ and Fe²⁺ and 100 µmol/L each of H₂O₂ and Fe²⁺. Under both conditions, the level of MDA increased rapidly during the first 2 h and then reached a plateau at 3 h. We thus decided to use a 3-h treatment for the rest of the study. Plasma H₂O₂ increased to 35 µmol/L (24). Serum total iron was within the range of 10–30 µmol/L although most iron is protein bound under normal conditions (25). To mimic physiological conditions to the greatest extent possible, we used 30 µmol/L each of H₂O₂ and Fe²⁺ to generate free radicals in the following experiments.

In the first part of the study, live cells were incubated simultaneously with flavonoids and 30 µmol/L each of H₂O₂ and Fe²⁺. Even in the presence of an intact cellular antioxidant protein defense system, quercetin, luteolin and EGCG inhibited MDA production when present at 1 or 10 µmol/L in at least one of two separate experiments (Table 1). Another flavonol, myricetin, exerted significant inhibition at 10 µmol/L (500 ± 84 vs. 844 ± 34 pmol MDA/mg in the control). We also tested the isoflavones, genistein and daidzein, and kaempferol, a flavonol without the o-dihydroxyl group. They did not inhibit lipid peroxidation under these experimental condition (results not shown).

We then examined the effect of flavonoid preincubation. Flavonoids in the medium were removed before the introduction of the 3-h oxidative stress. Preincubation of cells with 1 µmol/L EGCG significantly decreased the level of MDA in cells in both experiments (Table 1). Quercetin at 0.1 µmol/L and luteolin at 10 µmol/L each significantly decreased MDA levels in one of two experiments. Other flavonoids including genistein, daidzein, and kaempferol did not affect MDA levels (results not shown). None of the flavonoids tested enhanced lipid peroxidation in pre- or co-incubation experiments (Table 1). At the concentrations used (0.1–10 µmol/L), cell loss or morphological changes did not occur in either pre- or co-incubation experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our study is the first to examine the structure-function relationship of flavonoids as lipid antioxidants in live cells. We chose to study this panel of flavonoids because they represented four different subclasses of flavonoids and they are present in the human diet. Also, when these flavonoids were studied previously by other groups using cell-free systems (liposomes or microsomal preparations), they all appeared to have lipid antioxidant activity (6–9). In our live cell experiment, we found that quercetin, myricetin, luteolin and EGCG inhibited lipid peroxidation in cells (Table 1) whereas kaempferol, genistein and daidzein did not at concentrations < 10 µmol/L (data not shown). When we analyzed the structural features of the 7 flavonoids tested in our study, we concluded that the most important criterion in protecting live cells from lipid peroxidation is the adjacent hydroxyl groups (o-dihydroxyl or vicinal trihydroxyl) in the ring structure. Quercetin, myricetin, luteolin and EGCG meet this criterion, whereas this feature is absent in kaempferol, genistein and daidzein. Some previous studies using cell-free systems or oxidation potential estimation have also predicted kaempferol and isoflavones to be weaker antioxidants (6,26).

Although some flavonoids have the iron-chelating property noted earier (17,27–29), these experiments were carried out with higher concentration ratios of flavonoids to iron. In the current study, the iron concentration was 3- to 300-fold that of the flavonoids. It is thus unlikely that iron chelation contributed to the antioxidant activity observed here, especially in the experiments in which the medium flavonoids were removed before the addition of iron. It has been proposed, on the basis of an in vitro study, that flavonoids interact with the polar zone of phospholipid (30), and thus lipid solubility is not critical for the antioxidant activity. Our observation is consistent with this model. Among the flavonoids tested here, myricetin had the lowest lipid solubility and daidzein the highest (26), yet lipid antioxidant activity was detected for myricetin but not daidzein.

An interesting observation in our live cell experiment was the ability of some flavonoids to prevent simultaneous H₂O₂ and Fe²⁺ treatment-induced lipid oxidation even after flavonoids in the medium were removed. These are the same flavonoids that showed antioxidant activity in the short-term co-incubation experiments (Table 1). It is thus possible that the mechanism mediating the antioxidant activities in these two types of experiments is the same. EGCG prevented chromosomal damage in live cells in both co-incubation and preincubation experiments (18). Here, EGCG prevented lipid peroxidation in live cells in both types of experiments (Table 1). Future studies are required to determine whether quercetin can also prevent DNA damage under both conditions. Quercetin metabolites were shown to have antioxidant activities in cell-free experiments (31,32). They may also have contributed to the antioxidant activity of quercetin in our study because intestinal cells can metabolize quercetin (11).

Flavonoids have oxidation potential and thus can be oxidizing or reducing agents under different conditions (26). In cell-free studies, quercetin and myricetin were shown to have lipid antioxidant activities at lower concentration (<10 µmol/L), yet they increased hydroxyl radical production and oxidative damage of DNA at 100 µmol/L (33). EGCG increased H₂O₂ production at concentrations >30 µmol/L, although it protected DNA at more physiological concentrations of <10 µmol/L (18). In our live cell experiment with concentrations of flavonoids up to 10 µmol/L, no increase in the MDA level occurred even after prolonged incubation (Table 1). Our results are thus consistent with the study of DNA damage (18) that showed that flavonoids can act as antioxidants with no prooxidant activity.

In conclusion, we observed the lipid antioxidant activity of these flavonoids. Previous studies were carried out mainly in cell-free systems. We found that lipid antioxidant activities of flavonoids occur in live cells with intact antioxidant systems in the range of physiologically achievable concentrations. Exposure to certain flavonoids can reduce H₂O₂ and Fe²⁺-induced lipid peroxidation even after the extracellular flavonoids have been removed. The observations here complement earlier EGCG study and extend our knowledge of the prevention of oxidative damage (18). These observations further support the importance of plant-based food items such as vegetables, fruits and teas in the diet.

	FOOTNOTES

 
¹ Supported in part by National Institutes of Health grant DK54728.

³ Abbreviations used: EGCG, (-)-epigallocatechin gallate; H₂O₂ + Fe²⁺, solution containing H₂O₂ and FeSO₄; MDA, malondialdehyde.

Manuscript received 5 February 2003. Initial review completed 11 March 2003. Revision accepted 30 April 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Ames, B. N., Shigenaga, M. K. & Hagen, T. M. (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90:7915-7922.[Abstract/Free Full Text]

2. Ji, C., Rouzer, C. A., Marnett, L. J. & Pietenpol, J. A. (1998) Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde. Carcinogenesis 19:1275-1283.[Abstract/Free Full Text]

3. Ness, A. R. & Powles, J. W. (1997) Fruit and vegetables, and cardiovascular disease: a review. Int. J. Epidemiol. 26:1-13.[Abstract/Free Full Text]

4. Terry, P., Terry, J. B. & Wolk, A. (2001) Fruit and vegetable consumption in the prevention of cancer: an update. J. Intern. Med. 250:280-290.[Medline]

5. Cotelle, N., Bernier, J.-L., Catteau, J.-P., Pommery, J., Wallet, J.-C. & Gaydou, E. M. (1996) Antioxidant properties of hydroxyl-flavones. Free Radic. Biol. Med. 20:35-43.[Medline]

6. Cao, G., Sofic, E. & Priori, R. (1997) Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic. Biol. Med. 22:749-760.[Medline]

7. van Acker, S., van den Berg, D.-J., Tromp, M., Griffioen, D. H., van Bennekom, W. P., van der Vijgh, W. & Bast, A. (1996) Structural aspects of antioxidant activity of flavonoids. Free Radic. Biol. Med. 20:221-251.

8. Arora, A., Nair, M. G. & Strasburg, G. M. (1998) Antioxidant activities of isoflavones and their biological metabolites in a liposomal system. Arch. Biochem. Biophys. 356:133-141.[Medline]

9. Arora, A., Nair, M. G. & Strasburg, G. (1998) Structure-activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radic. Biol. Med. 24:1355-1363.[Medline]

10. Walgren, R. A., Walle, U. K. & Walle, T. (1998) Transport of quercetin and its glucoside across human intestinal epithelial Caco-2 cells. Biochem. Pharmacol. 55:1721-1727.[Medline]

11. Murota, K., Shimizu, S., Chujo, H., Moon, J.-H. & Terao, J. (2000) Efficiency of absorption and metabolic conversion of quercetin and its glucosides in human intestinal cell line Caco-2. Arch. Biochem. Biophys. 384:391-397.[Medline]

12. Murota, K., Shimizu, S., Miyamoto, S., Izumi, T., Obata, A., Kikuchi, M. & Terao, J. (2002) Unique uptake and transport of isoflavone aglycones by human intestinal Caco-2 cells: comparison of isoflavonoids and flavonoids. J. Nutr. 132:1856-1961.

13. Kuo, S.-M. (1996) Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells. Cancer Lett 110:41-48.[Medline]

14. Kuo, S.-M., Leavitt, P. & Lin, C.-P. (1998) Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells. Biol. Trace Elem. Res. 62:135-153.[Medline]

15. Kuo, S.-M. & Leavitt, P. S. (1999) Genistein increases metallothionein expression in human intestinal cells, Caco-2. Biochem. Cell Biol. 77:79-88.[Medline]

16. Kameoka, S., Leavitt, P., Chang, C. & Kuo, S.-M. (1999) Expression of antioxidant proteins in human intestinal Caco-2 cells treated with dietary flavonoids. Cancer Lett 146:161-167.[Medline]

17. Kuo, S.-M., Huang, C.-T., Blum, P. & Chang, C. (2001) Quercetin cumulatively enhances copper induction of metallothionein in intestinal cells. Biol. Trace Elem. Res. 84:1-10.[Medline]

18. Sugisawa, A. & Umegaki, K. (2002) Physiological concentrations of (-)-epigallocatechin-3-o-gallate (EGCg) prevent chromosomal damage induced by reactive oxygen species in WIL2-NS cells. J. Nutr. 132:1836-1839.[Abstract/Free Full Text]

19. de Vries, J., Hollman, P., Meyboom, A., Buysman, M., Zock, P., vanStaveren, W. & Katan, M. (1998) Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Am. J. Clin. Nutr. 68:60-65.[Abstract]

20. Pietta, P., Simonetti, P., Gardana, C., Brusamolino, A., Morazzoni, P. & Bombardelli, E. (1998) Relationship between rate and extent of catechin absorption and plasma antioxidant status. Biochem. Mol. Biol. Int. 46:895-903.[Medline]

21. Scalbert, A. & Williamson, G. (2000) Dietary intake and bioavailability of polyphenols. J. Nutr. 130:2073S-2085S.[Abstract/Free Full Text]

22. Watanabe, S., Arai, Y., Haba, R., Uehera, M., Adlercruetz, H., Shimoi, K. & Kinae, N. (2000) Dietary intake of flavonoids and isoflavonoids by Japanese and their pharmacokinetics and bioactivities. Shahidi, F. Ho, C. T. eds. Phytochemicals and Phytopharmaceuticals 2000:164-174 AOCS Press Champaign, IL. .

23. Janero, D. R. (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol. Med. 9:515-540.[Medline]

24. Halliwell, B., Clement, M. V. & Long, L. H. (2000) Hydrogen peroxide in the human body. FEBS Lett 486:10-13.[Medline]

25. McMorrow, M. E. & Malarkey, L. (1998) Laboratory and Diagnostic Tests 1998 W. B. Saunders Philadelphia, PA.

26. Yang, B., Kotani, A., Arai, K. & Kusu, F. (2001) Estimation of the antioxidant activities of flavonoids from their oxidation potentials. Anal. Sci. 17:599-604.[Medline]

27. van Acker, S.B.E., van Balen, G. P., van den Berg, D.-J., Bast, A. & van der Vijgh, W.J.F. (1998) Influence of iron chelation on the antioxidant activity of flavonoids. Biochem. Pharmacol. 56:935-943.[Medline]

28. Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P. & Cillard, J. (1993) Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45:13-19.[Medline]

29. Soczynska-Kordala, M., Bakowska, A., Oszmianski, J. & Gabrielska, J. (2001) Metal ion-flavonoid associations in bilayer phospholipid membranes. Cell. Mol. Biol. Lett. 6:277-281.[Medline]

30. Ratty, A. K., Sunamoto, J. & Das, N. P. (1988) Interaction of flavonoids with 1, 1-diphenyl-2-picrylhydrazyl free radical, liposomal membranes and soybean lipoxygenase-1. Biochem. Pharmacol. 37:989-995.[Medline]

31. Manach, C., Morand, C., Crespy, V., Demigné, C., Texier, O., Regerat, F & Rémésy, C. (1998) Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett 426:331-336.[Medline]

32. Da Silva, E. L., Piskula, M. K., Yamamoto, N., Moon, J.- & K. & Terao, J. (1998) Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma. FEBS Lett 430:405-408.[Medline]

33. Laughton, M. J., Halliwell, B., Evans, P. J. & Hoult, J.R.S. (1989) Antioxidant and pro-oxidant action of the plant phenolics, quercetin, gossypol and myricetin. Biochem. Pharmacol. 38:2859-2865.[Medline]

This article has been cited by other articles:

				R. Soundararajan, A. D. Wishart, H. P. V. Rupasinghe, M. Arcellana-Panlilio, C. M. Nelson, M. Mayne, and G. S. Robertson Quercetin 3-Glucoside Protects Neuroblastoma (SH-SY5Y) Cells in Vitro against Oxidative Damage by Inducing Sterol Regulatory Element-binding Protein-2-mediated Cholesterol Biosynthesis J. Biol. Chem., January 25, 2008; 283(4): 2231 - 2245. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, I.-W. Articles by Kuo, S.-M. Search for Related Content PubMed PubMed Citation Articles by Peng, I.-W. Articles by Kuo, S.-M.