Antioxidant Effects of 1,5-Anhydro-D-fructose, a New Natural Sugar, in vitro

• Abstract
• Introduction
• Materials and Methods
• Materials
• Scavenging of DPPH radicals
• Cell culture
• Superoxide anion assay
• H₂O₂ assay
• LDL preparation and oxidation
• ELISA
• Results
• Radical trapping activity of 1,5-AF
• Effect of 1,5-AF on PMA stimulation induced superoxide production in THP-1 cells
• Effect of 1,5-AF on PMA stimulation induced H₂O₂ production in THP-1 cells
• Effect of 1,5-AF on the copper-mediated LDL oxidation
• Discussion
• Acknowledgements
• References

Abstract

The antioxidant effects of 1,5-anhydro-D-fructose (1,5-AF), a unique anhydrohexulose, were studied in 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, in human cells along with lipid peroxidation of low-density lipoprotein (LDL). We have confirmed that 1,5-AF scavenges DPPH radicals directly in solution and inhibits the formation of hydrogen peroxide and superoxide anion, typical reactive oxygen species (ROS), induced by phorbol myristate acetate (PMA) in a dose-dependent manner in THP-1 cells. We also observed the dose-dependent antioxidant effects of 1,5-AF on copper-mediated LDL oxidation. These findings suggest that 1,5-AF might play a role in reducing the risk of atherosclerosis and may help prevent coronary heart disease.

Introduction

It is well known that ROS play an important role in many biological processes. Although ROS production is usually well controlled under physiologic conditions, ROS damage the cell membrane and biological molecules when they overwhelm the endogenous antioxidant system [1]. They are also responsible for the development of many diseases such as cancer, arteriosclerosis, and ischemia. Most ROS are scavenged by the endogenous antioxidant defense systems such as superoxide dismutase (SOD), the glutathione peroxidase/glutathione system, catalase, and peroxidase but these systems are not completely efficient [2], making it desirable to remove exogenous antioxidants from the diet.

It has been reported that dietary antioxidants scavenge excess ROS and inhibit LDL oxidation. Oxidative modification of LDL has been suggested to play an important role in the development of human atherosclerosis [3]. Thus, protecting LDL from oxidation effectively retards or prevents the progression of the disease.

We used 1,5-AF, a tautomer of 2-hydroxyglucal, derived from glycogen starch through an α-1,4-glucan lyase reaction (EC 4.2.2.13), in these experiments [4] (Fig. [1]). 1,5-AF has a unique structure in which its carbonyl group undergoes no hemiacetal bonding, but it is fully hydrated in aqueous solution [5] so that it may play an active role metabolically. It has been reported that 1,5-AF exists in rat livers [5], fungi [6], and algae [4], but its biological functions are still obscure.

1,5-Anhydro-D-glucitol (1,5-AG), the deoxy form of glucopyranose that is used as a diagnostic marker for diabetes [7], originates from glycogen in animal cells through two enzymatic steps [8]. First, glucose residues are eliminated from such non-reducing terminals of glycogen as 1,5-AF. Second, 1,5-AF is reduced to 1,5-AG. These two reactions have been suggested to constitute the third glycogenolytic pathway, along with hydrolysis and phosphorolysis [8]. Little is known, however, regarding the physiological importance of this alternative glycogen-degrading route in mammals.

In the present study, we examined the radical trapping activity of 1,5-AF by using the stable free radical DPPH [9]. We also examined the effects of 1,5-AF on typical ROS, hydroxyperoxide, and superoxide anion in the human monocyte/macrophage cell line THP-1 stimulated by PMA, and on copper-mediated LDL oxidation using a monoclonal antibody against oxidized lipoprotein by enzyme-linked immunosorbent assay (ELISA).

Materials and Methods

Materials

RPMI 1640, penicillin, fetal bovine serum (FBS), and Hanks’ balanced salt solution were purchased from GIBCO BRL, Co. (New York, U.S.A.). PMA, copper sulfate, DPPH, and α-tocopherol were purchased from Wako Pure Chemical Co. (Osaka, Japan). Superoxide dismutase, LDL from human plasma, bovine serum albumin (BSA), and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was purchased from LAMBA (Graz, Austria). A partially purified murine monoclonal antibody, FOH1a/DLH3, was generously provided by Dr. H. Itabe (Teikyo University, Kanagawa, Japan). The alkaline phosphatase (ALP)- conjugated goat anti mouse Ig (G+M) antibody was purchased from Biosource International (Camarillo, CA, USA). 1,5-AF, used for this experiment, was obtained from Nihon Denpun Kogyo Co. (Kagoshima, Japan). 1,5-AF was prepared from waxy maize starch by an enzymatic reaction of α-1,4-glucan lyase, which was purified from red seaweed, Gracilaria verrucosa. Maize starch in 50 mM acetate buffer (pH 5.5) and α-1,4-glucan lyase solution mixture was incubated for 48 h at 35 °C. The mixture was purified by gel-exclusion chromatography. The purity of 1,5-AF was 98.7 % by HPLC [10].

Scavenging of DPPH radicals

The scavenging of DPPH radicals by test compounds was determined from the decrease in the optical absorbance of DPPH at 517 nm due to scavenging of its stable free radicals. 1,5-AF (25 μg/ml-2.5 mg/ml) and α-tocopherol (25 μg/ml) were added to 3 ml of ethanol containing 100 μM DPPH and 2 ml of 50 mM MES buffer at pH 6.5 and pH 7.4. DPPH radical-scavenging activity was monitored after incubation for 2 hours at room temperature [9], [11].

Cell culture

THP-1 cells, a human monocytic cell line established from a patient with acute monocytic leukemia, were obtained from ATCC (Rockville,. MD. U.S.A.). Cells were cultured in RPMI1640 medium supplemented with 10 % fetal bovine serum and 2 % penicillin/streptomycin at 37 °C in 5 % CO₂ atmosphere.

Superoxide anion assay

Cells (1 × 10⁶ cells/ml) were suspended in Hanks balanced salt solution. Superoxide anion was induced by the addition of PMA (8 μM), followed by the simultaneous addition of 1,5-AF (0 - 50 μg/ml) and cytochrome c (160 μM), and incubated at 37 °C for 20 minutes. A reaction mixture without cells was used as a blank, and superoxide dismutase (300 U/ml = 120 μg/ml) was used as a positive inhibitor. Cytochrome c reduction was measured at 550 nm [12].

H₂O₂ assay

Monolayers of the cells, cultured at a density of 1 × 10⁵ cells/ml per well in 500 μl in a 24-well plate were stimulated by PMA (8 μM) in the absence or presence of 1,5-AF (5 - 50 μg/ml). Ascorbic acid (1 mM = 180 μg/ml) was used as a positive inhibitor. DCFH-DA (5 μM) was added to the monolayers and incubated at 37 °C for 20 minutes. Fluorescence was measured in a cytofluor plate reader (Fluoroscan II, Labosystem Genesis, Helsinki, Finland) with excitation and emission wavelengths of 485 nm and 538 nm, respectively.

LDL preparation and oxidation

LDL from human plasma was dissolved in 0.15 M sodium chloride, 1 mM EDTA, pH 7.4, for conservation. Prior to the oxidation reaction, EDTA was removed by dialysis. Lipoprotein (25 μg) in a final volume of 165 μl of phosphate-buffered saline (PBS) was incubated in the presence of 30 μM copper sulfate with the addition of 1,5-AF (0.25 - 25 μg/ml) for 4 hours at 37 °C. BSA (50 μg/ml) was used as a positive inhibitor.

ELISA

We investigated the effects of 1,5-AF on copper-mediated LDL oxidation determined by ELISA using the monoclonal antibody FOH1a/DLH3, which was produced by immunizing against the homogenates of human atheroma and selecting the hybridomas by reactivity to the copper-mediated oxidized LDL. A 100-μl sample of PBS containing 1 μg of antigen protein was placed in each well of a 96-well microtiter plate and incubated for 16 hours at 4 °C. After removing the antigen fluid, the wells were blocked with PBS containing 1 % BSA for 2 hours at 37 °C. After washing the wells three times with Tris-HCl-buffered saline (TBS)-Tween, the monoclonal antibody was allowed to react for 2 hours at 37 °C, followed by the addition of alkaline phosphatase conjugated goat anti-mouse Ig (G+M) antibody allowed to react for 2 hours at 37 °C. Finally, the remaining alkaline phosphatase activity was determined using p-nitrophenyl phosphate as a substrate. The results were read spectrophotometrically as optional density on an ELISA plate reader with the filter at 405 nm [13]. In the preliminary experiments, we confirmed that the same concentration of 1,5-AF alone did not affect the results.

Results

Radical trapping activity of 1,5-AF

The deep violet of DPPH is due to the vibration of unpaired fades when radical scavengers are present, and this coloring is used for determination of the antioxidant. The absorbance of DPPH at 517 nm is highly stable between pH 5.0 and 6.5 [11]. However, it is stable for a short time, approximately 2 hours, at physiological pH 7.5. The radical trapping activity of 1,5-AF was then examined at pH 6.5 and 7.4 (Fig. [2]). It was found that 2.5 mg/ml of 1,5-AF scavenged DPPH radicals at both pH 6.5 and 7.4 as effectively as α-tocopherol (20 μM = 8.6 μg/ml).

Effect of 1,5-AF on PMA stimulation induced superoxide production in THP-1 cells

Superoxide production in THP-1 induced by PMA (8 μM) was sufficiently inhibited by 1,5-AF (Table [1]). A 5-μg/ml solution of 1,5-AF inhibited 48 % of superoxide production and for a 50 μg/ml of 1,5-AF solution the scavenging activity was higher than that of SOD (120 μg/ml).

Effect of 1,5-AF on PMA stimulation induced H₂O₂ production in THP-1 cells

We also examined whether 1,5-AF inhibits the production of H₂O₂ in response to PMA stimulation by THP-1 cells. The fluorescent probe DCFH-DA was used to measure the intracellular production of H₂O₂ in a cytofluor plate reader. DCFH-DA rapidly diffuses through the cell membrane into the cytoplasm and is hydrolyzed to DCFH in the cytoplasm. DCFH can be oxidized to a highly fluorescent product, DCF, when H₂O₂ is generated. The cells were then incubated in the absence or presence of 1,5-AF (5 - 50 μg/ml) and stimulated with PMA. Unstimulated cells had a low level of cellular fluorescence, while there was a marked increase in cellular fluorescence in response to the PMA stimulation. However, co-incubation of 1,5-AF inhibited H₂O₂ production on THP-1 cells in a dose-dependent manner (Table [1]). These results suggest that a 50 μg/ml of 1,5-AF antioxidant activity for H₂O₂ is much higher than that of ascorbic acid (1mM = 176.1 μg/ml). Treatment of the cells by 1,5-AF (5 - 50 μg/ml) had no visible effect on the cell viability as judged by a trypan blue exclusion test.

Effect of 1,5-AF on the copper-mediated LDL oxidation

The antibody FOH1a/DLH3 recognizes the oxidized products of phosphatidylcholine (PC) in oxidized LDL and does not react to malondialdehyde-treated LDL, acetylated LDL, or native LDL. The addition of 1,5-AF to the oxidation mixture significantly suppressed the copper-mediated LDL oxidation in a dose-dependent manner, with complete inhibition observed at 25 μg/ml (Fig. [3]).

Discussion

In the present study, we examined the radical scavenging activity of 1,5-AF by using the stable free radical DPPH. In DPPH radical scavenging, α-tocopherol takes effect immediately after its addition at both pH 6.5 and 7.4, whereas the effect of 1,5-AF is much slower at pH 6.5 (Fig. [2]). However, 50 μg/ml of 1,5-AF showed a much higher antioxidant activity in the biological conditions of our experiments.

ROS that currently are known to be produced in the body include superoxide anion, hydrogen peroxide, and hydroxyl radical. Although superoxide anion does not have the strongest toxicity of these species, it plays an important role as an initiator of the generation of various types of ROS. In the present study, we demonstrated the effects of 1,5-AF on typical ROS, superoxide anion, and hydrogen peroxide, in the human monocyte/macrophage cell line THP-1 cells and its antioxidant activity was higher than that of the other known antioxidants. It has been suggested that the plasma membranes of K-562 cells are permeable to 1,5-AF, and it is believed that 1,5-AF penetrates the cells by simple diffusion and/or pinocytosis [14]. The present data indicates that 1,5-AF is incorporated into the cells by transporting proteins as an analog of a natural sugar [15].

Various biological factors are involved in the generation and progression of atherosclerosis; however, it has been widely accepted that oxidatively modified LDL plays a crucial role in the development and progression in atherosclerosis. As the first step, oxidized LDL modifies endothelial functions and down-regulates thrombomodulin expression with up-regulation of a tissue factor. Secondly, oxidized LDL stimulates the endothelial cells and expresses cell adhesion molecules (VCAM-1). Through this VCAM-1, circulating monocytes invade subendothelial vessel-wall and uptake oxidized LDL expressing various biologically active factors. Thus, oxidized LDL plays a very important role for the pathomechanism of atherosclerosis.

In the present study, 1,5-AF showed dose-dependent antioxidant effects on copper-mediated LDL oxidation. To exclude the possibility of a redox-inactivation of copper (e. g., by chelation) by 1,5-AF, we compared the reduction of copper by α-tocopherol and 1,5-AF in the presence of bathocuproine disulfonic acid (BCSA), a selective chelator for Cu⁺, in 50 % ethanol [16]. α-Tocopherol (20 μM) was observed to completely reduce copper within 10 seconds, while the reduction of copper by 1,5-AF (50 μg/ml) was not complete within 60 min (data not shown), indicating that there is no redox-inactivation of copper by 1,5-AF.

1,5-AF has a unique structure in that the carbonyl group does not participate in any hemiacetal bonding, as other reducing sugars do. 1,5-AF exists in aqueous solution in an equilibrium mixture in the 2,3-enediol form, the 2-enol form, keto-, and hydrated forms [17]. Its antioxidant power thus seems to be due to the presence of the enediol form [18], and this power is lost in response to the reduction of 1,5-AF to 1,5-AG in the LDL oxidation experiment (Fig. [3]). In addition, 1,5-AG has no double bonds, and these double bonds may be important to its antioxidant power.

Recently, Ahren et al. have suggested that 1,5-AF augments endogenous glucagon-like peptide-1 (GLP-1) secretion and at the same time increases glucose tolerance and insulin secretion when 1,5-AF is given enterally, but not parenterally, in mice [19]. These findings suggest that the dietary intake of 1,5-AF may prevent not only the oxidative damage caused by various oxidative stresses and reduce the risk of atherosclerosis, but may also be effective in the treatment of diabetes. More experiments are needed to elucidate the inhibitory mechanism of 1,5-AF before its clinical application, and such experiments are currently underway in our laboratory.

Acknowledgements

We are thankful to Dr. Miyata, Department of First Internal Medicine of the Kagoshima University, for his helpful advice in the LDL oxidation studies. We would also like to thank Dr. Itabe, Teikyo University, for providing a monoclonal antibody against oxidized lipoprotein and Dr. Muroya, Nihon Denpun Kogyo Co., for providing 1,5-AF. This study was supported in part by funds for the project entitled ”High and Ecological Utilization of Regional Carbohydrates”, through Special Coordination Funds for Promoting Science and Technology (Leading Research Utilizing Potential of Science and Technology) of the Science and Technology Agency of Japan, 1997.

References

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Kazuyo Yamaji

Department of Laboratory and Molecular Medicine

Faculty of Medicine

Kagoshima University, 8-35-1

Sakuragaoka

Kagoshima City, 890-8520

Japan

Telefon: +81-99-275-5437

Fax: +81-99-275-2629

eMail: yamaji@m3.kufm.kagoshima-u.ac.jp

References

• 1 Halliwell B, Gutteridge J MC. Role of free radicals and catalytic metal ions in human disease.  Methods in Enzymology. 1990;  186 1-85
  PubMedSuche in Google Scholar
• 2 Jacob R A, Burri B J. Oxidative damage and defense.  The American Journal of Clinical Nutrition. 1996;  63 985S-90S
  PubMedSuche in Google Scholar
• 3 Witztum J L, Steinberg D. Role of oxidized low-density lipoprotein in atherogenesis.  The Journal of Clinical Investigation. 1991;  88 (6) 1785-92
  CrossrefPubMedSuche in Google Scholar
• 4 Yu S, Kenne L, Pederson M. Alpha-1,4-glucan lyase, a new class of starch/glycogen degrading enzyme. I. Efficient purification and characterization from red seaweeds.  Biochimica et Biophysica Acta. 1993;  1156 313-20
  PubMedSuche in Google Scholar
• 5 Kametani S, Mizuno H, Shiga Y, Akanuma H. NMR of all-carbon-13 sugars : an application in development of an analytical method for a novel natural sugar, 1,5-anhydrofructose.  Journal of Biochemistry. 1996;  119 180-5
  PubMedSuche in Google Scholar
• 6 Baute M A, Baute R, Deffieux G. Fungal enzymic activity degrading 1,4-α-glucans to 1,5-D-anhydrofructose.  Phytochemistry. 1988;  27 3401-3
  CrossrefPubMedSuche in Google Scholar
• 7 Yamanouchi T, Minoda S, Yabuuchi M, Akanuma Y, Akanuma H, Miyashita H, Akaoka I. Plasma 1,5-anhydro-D-glucitol as new clinical marker of glycemic control in NIDDM patients.  Diabetes. 1989;  38 723-9
  PubMedSuche in Google Scholar
• 8 Kametani S, Shiga Y, Akanuma H. Hepatic production of 1,5-anhydrofructose and 1,5-anhydroglucitol in rat by the third glycogenolytic pathway.  European Journal of Biochemistry. 1996;  242 832-8
  CrossrefPubMedSuche in Google Scholar
• 9 Marsden S B. Antioxidant determinations by the use of a stable free radical.  Nature. 1958;  181 1199-200
  PubMedSuche in Google Scholar
• 10 Yoshinaga K, Fujisue M, Abe J, Hanashiro I, Takeda Y, Muroya K, Hizukuri S. Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme.  Biochimica et Biophysica Acta. 1999;  1472 (3) 447-54
  PubMedSuche in Google Scholar
• 11 Kogure K, Goto S, Abe K, Ohiwa C, Akasu M, Terada H. Potent antiperoxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine : the amine moiety is responsible for its pH-dependent radical scavenge activity.  Biochimica et Biophysica Acta. 1999;  1426 133-42
  PubMedSuche in Google Scholar
• 12 Pick E, Mizel D. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader.  Journal of Immunological Methods. 1981;  46 211-26
  CrossrefPubMedSuche in Google Scholar
• 13 Itabe H, Takeshima E, Iwasaki H, Kimura J, Yoshida Y, Imanaka T, Takano T. A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions. Complex formation of oxidized phosphatidylcholines and polypeptides.  The Journal of Biological Chemistry. 1994;  27 15 274-9
  PubMedSuche in Google Scholar
• 14 Suzuki M, Kametani S, Uchida K, Akanuma H. Production of 1,5-anhydroglucitol from 1,5-anhydrofructose in erythroleukemia cells.  European Journal of Biochemistry. 1996;  240 23-9
  CrossrefPubMedSuche in Google Scholar
• 15 Suzuki M, Akanuma H, Akanuma Y. Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells.  Journal of Biochemistry. 1988;  104 956-9
  PubMedSuche in Google Scholar
• 16 Lynch S M, Frei B. Reduction of copper, but not iron, by human low-density lipoprotein (LDL). Implications for metal ion-dependent oxidative modification of LDL.  The Journal of Biological Chemistry. 1995;  270 (10) 5158-63
  CrossrefPubMedSuche in Google Scholar
• 17 Taguchi T, Haruna M, Okuda J. Effects of 1,5-anhydro-D-fructose on selected glucose-metabolizing enzymes.  Biotechnology and Applied Biochemistry. 1993;  18 275-83
  PubMedSuche in Google Scholar
• 18 Yu S, Olsen C E, Marcussen J. Methods for the assay of 1,5-anhydro-D-fructose and α-1, 4-glucan lyase.  Carbohydrate Research. 1998;  305 73-82
  PubMedSuche in Google Scholar
• 19 Ahren B, Holst J J, Yu S. 1,5-Anhydro-D-fructose increases glucose tolerance by increasing glucagon-like peptide-1 and insulin in mice.  European Journal of Pharmacology. 2000;  397 219-25
  CrossrefPubMedSuche in Google Scholar

Kazuyo Yamaji

Department of Laboratory and Molecular Medicine

Faculty of Medicine

Kagoshima University, 8-35-1

Sakuragaoka

Kagoshima City, 890-8520

Japan

Telefon: +81-99-275-5437

Fax: +81-99-275-2629

eMail: yamaji@m3.kufm.kagoshima-u.ac.jp