Chemical specificity in short-chain fatty acids and their analogues in increasing osmotic fragility in rat erythrocytes in vitro

We examined the role of the chemical specificity of short-chain fatty acids (SCFAs) and their derivatives in increasing osmotic fragility (OF) in rat red blood cells (RBCs). Except for formic acid, normal SCFAs with 2 to 8 carbons increased the OF in rat RBCs with increasing number of hydrocarbons in a dose-dependent manner. Replacement of the carboxylic group with sulfonic group inhibited, but did not abolish, the SCFA-mediated increase in OF. Introduction of another carboxylic group (dicarboxylic acids) completely abolished the SCFA-mediated increase in OF. Transformation of the hydrocarbon chains in SCFAs from straight to branched or cyclic chains affected the degree of the OF-increasing effect. Introduction of double- or triple-carbon bonds to the hydrocarbon chain in parent SCFAs did not affect the increase in OF. Both hydrophilic (carboxylic group) and hydrophobic elements (hydrocarbons) at opposite sides of a molecule were required to affect the RBC membrane, and the size and form of hydrophobic element were important factors in determining the SCFA-mediated increase in OF. The hydrocarbon chains probably enter the plasma membrane, with the hydrophilic carboxylic base remaining outside of the membrane, and interact with phospholipid in cell membrane and disturb the structure of lipid layer resulting in the increase in OF in the rat RBCs.     

Chemical specificity in short-chain fatty acids and their analogues in increasing osmotic fragility in rat erythrocytes in vitro

We examined the role of the chemical specificity of short-chain fatty acids (SCFAs) and their derivatives in increasing osmotic fragility (OF) in rat red blood cells (RBCs). Except for formic acid, normal SCFAs with 2 to 8 carbons increased the OF in rat RBCs with increasing number of hydrocarbons in a dose-dependent manner. Replacement of the carboxylic group with sulfonic group inhibited, but did not abolish, the SCFA-mediated increase in OF. Introduction of another carboxylic group (dicarboxylic acids) completely abolished the SCFA-mediated increase in OF. Transformation of the hydrocarbon chains in SCFAs from straight to branched or cyclic chains affected the degree of the OF-increasing effect. Introduction of double- or triple-carbon bonds to the hydrocarbon chain in parent SCFAs did not affect the increase in OF. Both hydrophilic (carboxylic group) and hydrophobic elements (hydrocarbons) at opposite sides of a molecule were required to affect the RBC membrane, and the size and form of hydrophobic element were important factors in determining the SCFA-mediated increase in OF. The hydrocarbon chains probably enter the plasma membrane, with the hydrophilic carboxylic base remaining outside of the membrane, and interact with phospholipid in cell membrane and disturb the structure of lipid layer resulting in the increase in OF in the rat RBCs.     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.     

Low-molecular-weight aliphatic carboxylic acids in soil solutions under different vegetations determined by capillary zone electrophoresis

Concentrations of low-molecular-weight aliphatic carboxylic acids in soil solution were determined by a newly developed capillary zone electrophoresis method. Soil solution samples were collected by centrifugation of soil from the A horizon of a Danish, homogeneous, nutrient-rich Hapludalf in adjacent forested and arable plots. The forested plots of 0.5 ha were 33-year old stands of beech (Fagus sylvatica L.), oak (Quercus robur L.), grand fir (Abies grandis Lindl.), and Norway spruce (Picea abies (L.) Karst.), while sugar beet (Beta vulgaris L.) and winter wheat (Triticum aestivum L.) were the agricultural crops this year. High variability in soil solution concentrations of metal cations (Al, Ca, K, Mg, Na), monocarboxylic acids (formic, acetic, lactic, and valeric acids), and di- and tricarboxylic acids (oxalic, malic, succinic, and citric acids) were found within each plot. Despite this short-range within-plot variability, higher concentrations of di- and tricarboxylic acids were found in the forested soils than in the arable soils. The vegetation seemed to favour some monocarboxylic acids, but the total monocarboxylic acid concentrations showed little relation to the vegetation. Probably due to much less soil water in the Norway spruce plot, the low-molecular-weight aliphatic carboxylic acid concentrations in the samples from that plot were much higher than those found in samples from the other plots. Carbon in low-molecular-weight aliphatic carboxylic acids only accounts for a few percent of dissolved organic carbon, and no general relation was found between carbon in low-molecular-weight aliphatic carboxylic acids and dissolved organic carbon, although the correlation between carbon in di- and tricarboxylic acids and dissolved organic carbon was significant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Purification, characterization, and mass spectrometric sequencing of a medium chain acyl-CoA synthetase from mouse liver mitochondria and comparisons with the homologues of rat and bovine

Medium chain acyl-CoA synthetases catalyze the first reaction of amino acid conjugation of many xenobiotic carboxylic acids and fatty acid metabolism. This paper reports studies on purification, characterization, and the partial amino acid sequence of mouse liver enzyme. The medium chain acyl-CoA synthetase was isolated from mouse liver mitochondria. The purified enzyme catalyzes this reaction not only for straight medium chain fatty acids but also for aromatic and arylacetic acids. Maximal activity was found with hexanoic acid. High activities were obtained with benzoic acid having methyl, pentyl, and methoxy groups in the para- or meta-positions of the benzene ring. However, the enzyme was less active with valproic acid and ketoprofen. Salicylic acid exhibited no activity. The medium chain acyl-CoA synthetases from mouse and bovine liver mitochondria were subjected to in-gel tryptic digestion, followed by LC-MS/MS sequence analysis. The amino acid sequence of each tryptic peptide of mouse liver mitochondrial medium chain acyl-CoA synthetase differed from that from bovine liver mitochondria only in one or two amino acids. LC-MS/MS analysis provided the information about these differences in amino acid sequences. In addition, we compared the properties of this protein with the homologues from rat and bovine.     

Coupling of Inositol Phospholipid Metabolism with Excitatory Amino Acid Recognition Sites in Rat Hippocampus

Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, L-glutamate, and L-aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N-methyl-D-aspartate, kainate, quinolinate, and N-acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above-mentioned excitatory amino acids is potently and selectively antagonized by DL-2-amino-4-phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.     

Omega-Oxidation of Monocarboxylic Acids in Rat Brain

The accumulation of dicarboxylic acids is a prominent feature of inborn and toxin induced disorders of fatty acid metabolism which are characterized by impaired mental status. The formation of dicarboxylic acids is also a critical step in liver in the induction of intracellular fatty acid binding proteins and the proliferation of peroxisomes. In order to understand what potential roles dicarboxylic acids have in brain, we examined the extent of omega-oxidation in rat brain. Homogenates of rat brain catalyze the omega-oxidation of monocarboxylic acids with a specific activity of between 0.87 and 5.23 nmol/mg of post-mitochondrial protein/h, depending on the substrate. The activity is remarkably high, between one-fourth and 4 times the activity found in rat liver, depending on the chain length of the substrate. Specific activity increases with increasing chain length of the substrate. The omega-oxidation of palmitic acid is linear over a range of 0.125–3.0 mg of protein and 5–50 M substrate for up to 45 minutes of incubation. The product of omega-oxidation in brain is almost exclusively dicarboxylic acid. Cultured rat neurons, astrocytes, and oligodendrocytes all contain omega-oxidation activity. Western blots of rat brain homogenate demonstrate a protein that is recognized by antibody to rat liver CYP4A omega-hydroxylase. These results demonstrate that the omega-oxidative pathway is prominent in brain and could play a role in brain fatty acid metabolism.     

Stimulation of pollen tube growthin vitro by dicarboxylic acids

Summary Effect of eight dicarboxylic acids and three monocarboxylic acids on pollen growth ofCamellia japonica was tested. While monocarboxylic acids inhibited pollen germination and pollen tube elongation, dicarboxylic acids, namely oxalic, succinic, suberic, adipic, sebacic, traumatic cis-1,2-cyclohexane dicarboxylic, and 3,3-diethyl glutaric acids stimulated pollen tube elongation stronger than indoleacetic acid.     

Differing organic Acid exudation pattern explains calcifuge and acidifuge behaviour of plants

Many vascular plant species are unable to colonize calcareous sites. Thus, the floristic composition of adjacent limestone and acid silicate soils differs greatly. The inability of calcifuge plants to establish in limestone sites seems related to a low capacity of such plants to solubilize and absorb Fe or phosphate from these soils. Until now, mechanisms regulating this differing ability of plants to colonize limestone sites have not been elucidated. We propose that contrasting exudation of low-molecular organic acids is a major mechanism involved and show that germinating seeds and young seedlings of limestone plants exude considerably more di- and tricarboxylic acids than calcifuges, which mainly exude monocarboxylic acids. The tricarboxylic citric acid is a powerful extractor of Fe, and the dicarboxylic oxalic acid a very effective extractor of phosphate from limestone soils. Monocarboxylic acids are very weak in these respects. The study is based on ten species from limestone soils and ten species from acid silicate soils.     

Cupric Oxide (CuO) Oxidation Detects Pyrogenic Carbon in Burnt Organic Matter and Soils

Wildfire greatly impacts the composition and quantity of organic carbon stocks within watersheds. Most methods used to measure the contributions of fire altered organic carbon–i.e. pyrogenic organic carbon (Py-OC) in natural samples are designed to quantify specific fractions such as black carbon or polyaromatic hydrocarbons. In contrast, the CuO oxidation procedure yields a variety of products derived from a variety of precursors, including both unaltered and thermally altered sources. Here, we test whether or not the benzene carboxylic acid and hydroxy benzoic acid (BCA) products obtained by CuO oxidation provide a robust indicator of Py-OC and compare them to non-Py-OC biomarkers of lignin. O and A horizons from microcosms were burned in the laboratory at varying levels of fire severity and subsequently incubated for 6 months. All soils were analyzed for total OC and N and were analyzed by CuO oxidation. All BCAs appeared to be preserved or created to some degree during burning while lignin phenols appeared to be altered or destroyed to varying extents dependent on fire severity. We found two specific CuO oxidation products, o-hydroxybenzoic acid (oBd) and 1,2,4-benzenetricarboxylic acid (BTC2) that responded strongly to burn severity and withstood degradation during post-burning microbial incubations. Interestingly, we found that benzene di- and tricarboxylic acids (BDC and BTC, respectively) were much more reactive than vanillyl phenols during the incubation as a possible result of physical protection of vanillyl phenols in the interior of char particles or CuO oxidation derived BCAs originating from biologically available classes of Py-OC. We found that the ability of these compounds to predict relative Py-OC content in burned samples improved when normalized by their respective BCA class (i.e. benzene monocarboxylic acids (BA) and BTC, respectively) and when BTC was normalized to total lignin yields (BTC:Lig). The major trends in BCAs imparted by burning persisted through a 6 month incubation suggesting that fire severity had first order control on BCA and lignin composition. Using original and published BCA data from soils, sediments, char, and interfering compounds we found that BTC:Lig and BTC2:BTC were able to distinguish Py-OC from compounds such as humic materials, tannins, etc. The BCAs released by the CuO oxidation procedure increase the functionality of this method in order to examine the relative contribution of Py-OC in geochemical samples.     

Antioxidant Status and Susceptibility of Sickle Erythrocytes to Oxidative and Osmotic Stress

The purpose of this study was to determine if differences in antioxidant status between the red blood cells (RBCs) of sickle cell anemia (SCA) patients and controls are responsible for the differential responses to oxidative and osmotic stress-induced hemolysis. Susceptibility to hemolysis was examined by incubating oxygenated and deoxygenated RBCs at 37°C with 73 mM 2,2' azobis (2-amidinopropane) HC1 (AAPH), a peroxyl radical generator, for up to 3.5 hours. The ability of RBCs to maintain membrane integrity under osmotic stress was determined over a range of diluted saline-phosphate buffer. Sickled RBCs showed a lesser degree of AAPH-induced hemolysis than control groups and were more resistant to osmotic stress-induced hemolysis. SCA patients had higher levels of RBC vitamin E and RBC lipids, but lower RBC GSH, plasma lipids and plasma carotenes than those of the hospital controls. No significant differences were observed in the levels of retinol, vitamin C, vitamin E, MDA and conjugated dienes in plasma, or the levels of MDA and conjugated dienes in RBCs. The results obtained suggest that the differences in antioxidant status between sickled RBCs and controls do not appear to be responsible for their different susceptibility to oxidative or osmotic stress-induced hemolysis observed.     

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit β-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit β-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO₂ using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO₂ to carboxylic acids.     

1-Aminocyclopentane-1,2,4-tricarboxylic acids screening on glutamatergic and serotonergic systems

Enantiopure constrained 1-aminocyclopentane-1,2,4-tricarboxylic acids containing the glutamic acid skeleton were prepared as two diastereomers characterized by having the carboxylic groups in position two and four cis-oriented to each other and trans with respect to 1-carboxylic group and all cis-oriented carboxylic groups, respectively. A biochemical screening of activity of the above amino acids was investigated on glutamate and 5-HT receptors to find a possible metabotropic agonist, acting on the serotoninergic system.     

A Model of Piezo1-Based Regulation of Red Blood Cell Volume

A red blood cell (RBC) performs its function of adequately carrying respiratory gases in blood by its volume being ～60% of that of a sphere with the same membrane area. For this purpose, human and most other vertebrate RBCs regulate their content of potassium (K⁺) and sodium (Na⁺) ions. The focus considered here is on K⁺ efflux through calcium-ion (Ca²⁺)-activated Gárdos channels. These channels open under conditions that allow Ca²⁺ to enter RBCs through Piezo1 mechanosensitive cation-permeable channels. It is postulated that the fraction of open Piezo1 channels depends on the RBC shape as a result of the curvature-dependent Piezo1-bilayer membrane interaction. The consequences of this postulate are studied by introducing a simple model of RBC osmotic behavior supplemented by the dependence of RBC membrane K⁺ permeability on the reduced volume (i.e., the ratio of cell volume to its maximal possible volume) of RBC discoid shapes. It is assumed that because of its intrinsic curvature and strong interaction with the surrounding membrane, Piezo1 tends to concentrate in the dimple regions of these shapes, and the fraction of open Piezo1 channels depends on the membrane curvature in that region. It is shown that the properties of the described model can provide the basis for the formation of the negative feedback loop that interrelates cell volume and its content of potassium ions. The model predicts the relation, valid for each cell in an RBC population, between RBC volume and membrane area, thus explaining the large value of the measured membrane area versus the volume correlation coefficient. The mechanism proposed here for RBC volume regulation is in accord with the loss of this correlation in RBCs of Piezo1 knockout mice.     

Swelling in Bean Shoot Mitochondria Induced by a Series of Potassium Salts of Organic Anions

A series of potassium salts of organic anions were examined for their effect on the volume change of bean shoot mitochondria as measured by spectrophotometric light scatterings. A passive osmotic swelling (substrate independent) as well as an active osmotic swelling (substrate dependent) was shown with a series of organic anions. Both oxidizable substrates and non-oxidizable substrates induce swelling. The monocarboxylic acids including acetate, β-OH-butyrate, propionate, and pyruvate induce active swelling which is partially inhibited by the presence of an ATP generating system or the uncoupler 2,4-dinitrophenol (DNP). Dicarboxylic acids produce less extensive rates and amounts of active swelling. Moreover, the swelling induced by dicarboxylic acids is inhibited less completely by an ATP generating system or by DNP. Metabolizable substrates including citrate, pyruvate, glutarate, and α-oxo-glutarate induced swelling despite their poor rates or lack of oxidation. It was concluded that with these anions, penetration across the inner membrane as measured by osmotic swelling of isolated mitochondria is not the rate limiting step in their metabolism.     

Oxidation-induced calcium-dependent dehydration of normal human red blood cells

Phenazine-methosulphate (PMS) is a strong oxidant that induces reactive oxygen species (ROS) formation in cells. Though it has been shown that PMS increases the red blood cell (RBC) membrane permeability to K⁺, the hypotheses on the mechanism of PMS-induced effects are contradictory and there are no data on volume changes induced by this oxidant. Therefore, the influence of the PMS + ascorbate oxidative system on the volume of normal human RBCs was studied. In a Ca^{2 +} -containing medium, PMS + ascorbate caused dehydration (shrinking) of RBCs judged by: (1) changes in the density and osmotic resistance distributions of RBCs, and (2) a decrease in their low-angle scattering assessed by FACS analysis. The dehydration resulted from activation of the Gardos channels, was PMS and ascorbate concentration-dependent, was associated with broadening of the density and osmotic resistance distributions of the RBCs, and decreased in the presence of the taxifolin and rutin antioxidants. These findings contribute to a better understanding of the physiology and pathology of oxidatively-modified RBCs and may be of practical significance in estimating the antioxidant activity of various substances.