Ascorbic acid in human neutrophils

Washko, Philip W.; Rotrosen, Daniel; Levine, Mark

doi:10.1093/ajcn/54.6.1221s

Cited by 169 publications

(190 citation statements)

References 36 publications

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“…The affinity of the putative transporter for AH seemed to be low and in the millimolar range. Various cells contain a low-affinity AH transporter (K m 2-7 mM) that has been primarily described for AH influx [36,[38][39][40]. In addition, cells accumulate millimolar levels of AH intracellularly [23,38,41].…”

Section: Discussionmentioning

confidence: 99%

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Upston

Karjalainen

Bygrave

et al. 1999

Biochemical Journal

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Ascorbate (AH, the reduced form of vitamin C) is an important radical scavenger and antioxidant in human plasma; the resulting ascorbyl radical can disproportionate to AH and dehydroascorbic acid (DHA). Here we address potential maintenance mechanism(s) for extracellular AH by examining the ability of cells to convert extracellularly presented DHA to AH. DHA was rapidly transported into human liver (HepG2), endothelial and whole blood cells invitro by plasma membrane glucose transporters and reduced intracellularly. Liver cells displayed the highest capacity to release the intracellularly accumulated AH. The proteins responsible for DHA uptake and AH release could be distinguished by inhibitor studies. Thus, unlike DHA uptake, AH efflux was largely insensitive to cytochalasin B and thiol-reactive agents but was inhibited by phloretin, 4,4′-di-isothiocyanostilbene-2,2′-disulphonate and isoascorbate. Efflux of AH from cells was temperature-sensitive and saturable with a low affinity (millimolar, intracellular) for AH. In addition to isolated liver cells, perfusion of intact rat and guinea-pig liver with DHA resulted in AH in the circulating perfusate. Our results show that hepatocytes take up and reduce DHA and subsequently release part of the AH formed, probably via a membrane transporter. By converting extracellular DHA to extracellular AH, the liver might contribute to the maintenance of plasma AH, a process that could be important under conditions of oxidative stress.

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Section: Discussionmentioning

confidence: 99%

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Upston

Karjalainen

Bygrave

et al. 1999

Biochemical Journal

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“…Neutrophils were isolated from heparinized whole blood collected from healthy volunteers as described previously [3] and suspended in Hanks' balanced salt solution without calcium, magnesium, or Phenol red (mHBSS) (pH 7.4) at a final concentration of 1 x lO'/ml. Neutrophils were routinely >95% pure and >95% viable as revealed by Wright's staining and exclusion of Trypan blue, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…High-performance liquid chromatography was used to measure total intracellular ascorbic acid, as previously described [3]. Where applicable, ["Clascorbic acid elution profiles of the samples were obtained by this method.…”

Section: Methodsmentioning

confidence: 99%

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Ascorbic acid accumulation in plated human neutrophils

1990

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Ascorbic acid uptake was investigated in isolated, plated human neutrophils using high-performance liquid chromatography with coulometric electrochemical detection. Freshly isolated neutrophils contained 1.3 mM ascorbic acid and accumulated significantly greater amounts when physiologic concentrations of the vitamin were present in the extracellular buffer. In several different butlers uptake was dependent on the presence of cakium and magnesium. Under these conditions, ~intillation spectrometry of ~4C]ascorbic acid in conjunction with hip-~rfo~an~ liquid chromatography was suited for measuring ascorbic acid transport.

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“…Once inside the cell, DHA is reduced to AA through enzymatic processes that involve glutathione, NADH or NADPH reductants (Deutsch, 2000). While tissues vary widely in AA/DHA content, the concentrations are often in the low millimolar range (Ek et al, 1995;Grunewald, 1993;Rice and Russo-Menna, 1998;Washko et al, 1993), with the highest concentrations in the pituitary and adrenal glands, and the brain (Grunewald, 1993). Thus, the same tissues that contain NOS enzymes have high levels of AA and DHA.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

Rubakhin

Sweedler

2008

Journal of Neuroscience Methods

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Spatial measurements of nitric oxide (NO) production are important to understand the function and metabolism of this molecule. The reagent, 4,5-diaminofluorescein (DAF-2) and several structurally similar probes are widely used for detection and imaging of NO. However, DAF-2 also reacts with dehydroascorbic acid (DHA) in biological samples, with both products having nearly indistinguishable fluorescence spectra. Measurements using fluorimetry and fluorescence microscopy cannot easily differentiate NO-related fluorescent signals from DHA-related signals. While DAFs and the structurally related diaminorhodamines (DARs) both react with NO and DHA, they do so to different extents. We report a multiderivatization method to image NO and DHA simultaneously by using both DAF and DAR. Specifically, DAF-2 and DAR-4M are used to image NO and DHA concentrations; after reaction, the solutions are excited, at 495 nm to measure fluorescence emission from DAF-2, and at 560 nm to measure fluorescence emission from DAR-4M. Using the appropriate calibrations, images are created that depend either on the relative NO or the relative DHA concentration, even though each probe reacts to both compounds. The method has been validated by imaging NO production in both undifferentiated and differentiated pheochromocytoma cells.

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Ascorbic acid in human neutrophils

Cited by 169 publications

References 36 publications

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Ascorbic acid accumulation in plated human neutrophils

Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

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