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Ascorbate transport and recycling in human neutrophils: Potential roles in oxidant defense
ASCORBATE TRANSPORT AND RECYCLING IN HUMAN NEUTROPHILS: POTENTIAL ROLES IN OXIDANT
DEFENSE MR. Cinicnl
Dnruwala, 1. Son , S.C. Rumsey, W.S. Koh, P. Eck. Molecular and Nutrition Section, NI 6 DK, NIH, Bethesda,MD 20892.1372 USA
Ascorbate accumulation in human neutrophils is controlled by two mechanisms. The first mechanism is via a sodium-dependent ascorbate transporter. Two isoforms of human ascorbate transporters were cloned and expressed in Xenopus laevis ooctyes. The second mechanism is ascorbate recycling, a three-step process consisting of oxidation of extracellular ascorbate to dehydroascorbic acid, transport of dehydroascorbic acid, and its intracellular reduction to ascorbate. Glucose transporters GLUT 1,3, and 4 mediate dehydroascorbic acid transport. The new ascorbate analog [125IJ-6-deoxy-6-iodo-L-ascorbic acid was transported by cellular ascorbate transporters but not by the dehydroascorbic acid transporters GLUT 1 and GLUT 3. Conversely, analogs that transport dehydroascorbic acid but not ascorbate are being synthesized. Once inside neutrophils, intracellular dehydroascorbic acid is immediately reduced by the protein glutaredoxin with glutathione. Gram-positive pathogenic bacteria, gram-negative pathogenic bacteria and the fungal pathogen Candida albicans stimulate ascorbate recycling in neutrophils. Induction of recycling results in as high as a 30-fold increase in intracellular ascorbate compared to neutrophils not exposed to microorganisms. Microorganisms themselves do not perform ascorbate recycling, because they are impermeant to both ascorbic and dehydroascorbic acids. These data suggest that ascorbate recycling may be a specific mammalian defense mechanism against oxidants. Near maximal stimulation of ascorbate recycling occurs with 75 mM external vitamin C, which is similar to the plasma concentration achieved in humans with ingestion of 200 mg of ascorbate daily.
IP-101 Vitamin E Understanding Absorption, Regulation and Tissue Distribution for Determination of Requirements Maret G. Traber Linus Pauline Institute, Oregon State University, Corvallis, OR 97331-6512,~USA. [email protected] _ Vitamin requirements are traditionally established by determining the deficiency symptom and the minimum amount of the nutrient needed to reverse it. Vitamin E does not fit this pattern because its deficiency symptom in humans is a peripheral neuropathy that can take 50 years to become apparent. However, neuropathy may not be the most sensitive marker for a suboptimal vitamin E intake. Epidemiologic and intervention studies have suggested that pharmacologic vitamin E intakes are associated with a decreased risk of some chronic diseases, notably heart disease. Does a chronic low intake of vitamin E result in inadequate cellular defense against lipid peroxidation? Are there are specific cellular functions accomplished only by RRR-alpha-tocopherol, the biologically most active form of vitamin E, that are compromised by suboptimal intakes. Although the various vitamin E forms have nearly similar antioxidant activities, they are quite different in their abilities to prevent vitamin E deficiency symptoms in experimental animals, Studies in humans using vitamin E forms labeled with deuterium have demonstrated that a- and y-tocopherols, and different stereoisomers of cc-tocopherol (e.g. RRR- and SRR-) are equally well absorbed, but only RRR-cc-tocopherol is maintained in the plasma. The critical factor appears to be the hepatic c(tocopherol transfer protein (TTP) because it is required to maintain normal plasma concentrations of a-tocopherol; humans with a genetic defect in TTP become vitamin E-deficient. Metabolism of vitamin E may also be important; non-a-tocopherol forms of vitamin E are readily metabolized and excreted into the urine. Thus, the requirement for vitamin E in humans seems to depend both on the function of TTP and upon vitamin E metabolism.
56
OXYGEN
I
9
9
DEFENSE MR. Cinicnl
Dnruwala, 1. Son , S.C. Rumsey, W.S. Koh, P. Eck. Molecular and Nutrition Section, NI 6 DK, NIH, Bethesda,MD 20892.1372 USA
Ascorbate accumulation in human neutrophils is controlled by two mechanisms. The first mechanism is via a sodium-dependent ascorbate transporter. Two isoforms of human ascorbate transporters were cloned and expressed in Xenopus laevis ooctyes. The second mechanism is ascorbate recycling, a three-step process consisting of oxidation of extracellular ascorbate to dehydroascorbic acid, transport of dehydroascorbic acid, and its intracellular reduction to ascorbate. Glucose transporters GLUT 1,3, and 4 mediate dehydroascorbic acid transport. The new ascorbate analog [125IJ-6-deoxy-6-iodo-L-ascorbic acid was transported by cellular ascorbate transporters but not by the dehydroascorbic acid transporters GLUT 1 and GLUT 3. Conversely, analogs that transport dehydroascorbic acid but not ascorbate are being synthesized. Once inside neutrophils, intracellular dehydroascorbic acid is immediately reduced by the protein glutaredoxin with glutathione. Gram-positive pathogenic bacteria, gram-negative pathogenic bacteria and the fungal pathogen Candida albicans stimulate ascorbate recycling in neutrophils. Induction of recycling results in as high as a 30-fold increase in intracellular ascorbate compared to neutrophils not exposed to microorganisms. Microorganisms themselves do not perform ascorbate recycling, because they are impermeant to both ascorbic and dehydroascorbic acids. These data suggest that ascorbate recycling may be a specific mammalian defense mechanism against oxidants. Near maximal stimulation of ascorbate recycling occurs with 75 mM external vitamin C, which is similar to the plasma concentration achieved in humans with ingestion of 200 mg of ascorbate daily.
IP-101 Vitamin E Understanding Absorption, Regulation and Tissue Distribution for Determination of Requirements Maret G. Traber Linus Pauline Institute, Oregon State University, Corvallis, OR 97331-6512,~USA. [email protected] _ Vitamin requirements are traditionally established by determining the deficiency symptom and the minimum amount of the nutrient needed to reverse it. Vitamin E does not fit this pattern because its deficiency symptom in humans is a peripheral neuropathy that can take 50 years to become apparent. However, neuropathy may not be the most sensitive marker for a suboptimal vitamin E intake. Epidemiologic and intervention studies have suggested that pharmacologic vitamin E intakes are associated with a decreased risk of some chronic diseases, notably heart disease. Does a chronic low intake of vitamin E result in inadequate cellular defense against lipid peroxidation? Are there are specific cellular functions accomplished only by RRR-alpha-tocopherol, the biologically most active form of vitamin E, that are compromised by suboptimal intakes. Although the various vitamin E forms have nearly similar antioxidant activities, they are quite different in their abilities to prevent vitamin E deficiency symptoms in experimental animals, Studies in humans using vitamin E forms labeled with deuterium have demonstrated that a- and y-tocopherols, and different stereoisomers of cc-tocopherol (e.g. RRR- and SRR-) are equally well absorbed, but only RRR-cc-tocopherol is maintained in the plasma. The critical factor appears to be the hepatic c(tocopherol transfer protein (TTP) because it is required to maintain normal plasma concentrations of a-tocopherol; humans with a genetic defect in TTP become vitamin E-deficient. Metabolism of vitamin E may also be important; non-a-tocopherol forms of vitamin E are readily metabolized and excreted into the urine. Thus, the requirement for vitamin E in humans seems to depend both on the function of TTP and upon vitamin E metabolism.
56
OXYGEN
I
9
9