The effects of glucose on ascorbic acid uptake in heart endothelial cells: Possible pathogenesis of diabetic angiopathies

Glucose in concentrations of 20 mg% (or greater) significantly inhibited ¹⁴C-labelled ascorbic acid (1.25 mg%) uptake in endothelial cells in the presence of insulin (1600 ωU/ml). The absence of insulin also significantly reduced ascorbic acid uptake. Furthermore, this reduction could be exacerbated by glucose (40, 160 mg%) but not equimolar concentrations of fructose. Increased ascorbic acid concentrations (two-fold) in the absence of insulin (1) significantly enhanced uptake, and (2) reversed the inhibition by glucose. These findings support earlier reports that ascorbic acid uptake into the cell may be compromised by decreased insulin and/or increased extracellular glucose levels. Since previous animal studies have correlated experimental ascorbic acid deficiencies with atherogenic processes (presumably by altering glycosaminoglycan metabolism), the postulation that the “diabetic condition” (low insulin, hyperglycemia) accelerates the cellular changes leading to atherosclerosis by impairing ascorbic acid uptake into the vascular endothelium, may now be supported.     

Effect of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: mechanism of impaired ascorbate transport in diabetes

Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3H-2-deoxy-D-glucose (2.5 mg%) by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14C-L-ascorbic acid (1.25 mg%). The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus.     

Effect of vitamin C administration on blood cholesterol level in man

Vitamin C (1.0 g/day) was administered orally to 20 healthy males for 1 month under controlled conditions. The blood ascorbic acid level rose from 0.76 +/- 0.21 mg% to 1.24 +/- 0.19 mg% in young subjects (20-30 years), and from 0.74 +/- 0.29 mg% to 1.22 +/- 0.22 mg% in middle-aged ones (31-50 years). Simultaneously, the serum cholesterol levels decreased from 204 +/- 16 mg% to 177 +/- 21 mg% in the young and from 256 +/- 11 mg% to 225 +/- 36 mg% in the middle-aged, a statistically significant fall of 10-15%, on the average (P less than 0.01). The effect in normo-cholesteraemic subjects, in particular, supports the cholesterol-lowering action of vitamin C.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

L-Galactono-{gamma}-Lactone Dehydrogenase: Partial Characterization, Induction of Activity and Role in the Synthesis of Ascorbic Acid in Wounded White Potato Tuber Tissue

The total initial vitamin C content (23 mg%) of sliced whitepotato tuber tissue incubated at 20°C decreased slightlyduring the first 12 h and increased thereafter by 80% duringa 48-h incubation. The ascorbic acid (AsA) content (21 mg%)decreased slightly during the first 12 h and then increasedby 40% within 36 h and slowly decreased thereafter. The levelof dehydroascorbic acid (DHA) was low initially, being only2 mg% at 36 h it started to increase and reached 10 mg% at 48h. The activty of L-galactono-     

Preventable effect of L-threonate, an ascorbate metabolite, on androgen-driven balding via repression of dihydrotestosterone-induced dickkopf-1 expression in human hair dermal papilla cells

In a previous study, we recently claimed that dihydrotestosterone (DHT)-inducible dickkopf-1 (DKK-1) expression is one of the key factors involved in androgen-potentiated balding. We also demonstrated that L-ascorbic acid 2-phosphate (Asc 2-P) represses DHT-induced DKK-1 expression in cultured dermal papilla cells (DPCs). Here, we investigated whether or not L-threonate could attenuate DHT-induced DKK-1 expression. We observed via RT-PCR analysis and enzyme-linked immunosorbent assay that DHT-induced DKK-1 expression was attenuated in the presence of L-threonate. We also found that DHT-induced activation of DKK-1 promoter activity was significantly repressed by L-threonate. Moreover, a co-culture system featuring outer root sheath (ORS) keratinocytes and DPCs showed that DHT inhibited the growth of ORS cells, which was then significantly reversed by L-threonate. Collectively, these results indicate that L-threonate inhibited DKK-1 expression in DPCs and therefore is a good treatment for the prevention of androgen-driven balding.     

Effects of dietary factors on iron uptake from ferritin by Caco-2 cells

Biofortification of staple foods with iron (Fe) in the form of ferritin (Ft) is now possible, both by conventional plant breeding methods and transgenic approaches. Ft-Fe from plants and animals is absorbed well (25-30%) by human subjects, but little is known about dietary factors affecting its absorption. We used human intestinal Caco-2 cells and compared Fe absorption from animal Ft and FeSO4 to determine the effects of inhibitors and enhancers, such as phytic acid, ascorbic acid, tannic acid, calcium and heme. When postconfluent cells were coincubated with 59Fe-labeled (1 microM) FeSO4 and dietary factors, at different molar ratios of dietary factor to Fe (phytic acid:Fe, 10:1; ascorbic acid:Fe, 50:1; tannic acid:Fe, 50:1; calcium:Fe, 10:1 and hemin:Fe, 10:1), all inhibited uptake from FeSO4, except ascorbate, confirming earlier studies. In contrast, these dietary factors had little or no effect on Fe uptake from undigested Ft or Ft digested in vitro at pH 4, except tannins. However, results after in vitro digestion of Ft at pH 2 were similar to those obtained for FeSO4. These results suggest that Fe uptake occurs from both undigested as well as digested Ft but, possibly, via different mechanisms. The Fe-Ft stability shown here could minimize Fe-induced oxidation of Fe-supplemented food products.     

Determination of L-threonate in human plasma and urine by high performance liquid chromatography-tandem mass spectrometry

A fast and selective HPLC-MS-MS method was established to determine L-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC J'Sphere C(18) column with methanol-acetonitrile-10mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and L-threonate was quantified by monitoring the ion transition of m/z 134.5-->74.7. The linear calibration curves of L-threonate in plasma and urine were obtained over the concentration range of 0.25-50 microg/ml and 2.5-500 microg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 microg/ml, respectively. Accuracy was within 85-115%, and intra- and inter-batch precision (R.S.D.%) were within +/-15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of L-threonate in Chinese healthy subjects.     

Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.     

Ascorbic acid-dependent GLUT3 inhibition is a critical step for switching neuronal metabolism

Intracellular ascorbic acid is able to modulate neuronal glucose utilization between resting and activity periods. We have previously demonstrated that intracellular ascorbic acid inhibits deoxyglucose transport in primary cultures of cortical and hippocampal neurons and in HEK293 cells. The same effect was not seen in astrocytes. Since this observation was valid only for cells expressing glucose transporter 3 (GLUT3), we evaluated the importance of this transporter on the inhibitory effect of ascorbic acid on glucose transport. Intracellular ascorbic acid was able to inhibit (3)H-deoxyglucose transport only in astrocytes expressing GLUT3-EGFP. In C6 glioma cells and primary cultures of cortical neurons, which natively express GLUT3, the same inhibitory effect on (3)H-deoxyglucose transport and fluorescent hexose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was observed. Finally, knocking down the native expression of GLUT3 in primary cultured neurons and C6 cells using shRNA was sufficient to abolish the ascorbic acid-dependent inhibitory effect on uptake of glucose analogs. Uptake assays using real-time confocal microscopy demonstrated that ascorbic acid effect abrogation on 2-NBDG uptake in cultured neurons. Therefore, ascorbic acid would seem to function as a metabolic switch inhibiting glucose transport in neurons under glutamatergic synaptic activity through direct or indirect inhibition of GLUT3.     

Ascorbic acid and aging in the rat. Uptake of ascorbic acid by teeth and concentration of various forms of ascorbic acid in different organs

1. The uptake of ascorbic acid in vitro by the teeth of rats showed a gradual decrease with age, indicating that the uptake may be related to collagen synthesis as in bone. 2. The concentration of total free ascorbic acid in various organs declined with age, but the rate of decline was different in different organs. In the spleen, however, it increased until maturity and then declined. 3. This decrease may be due to one or both of the following reasons: (a) the permeability of different tissues may decrease at different rates for ascorbic acid, or (b) the requirement for ascorbic acid may decrease at different rates. 4. The bound ascorbic acid declined with age in the skin, kidney, liver and brain after the age of 10-12 weeks, and in the spleen after the age of 26 weeks. 5. The concentration of dehydroascorbic acid and dioxogulonic acid declined with age in the skin.     

Pulmonary bioavailability of ascorbic acid in an ascorbate-synthesising species,the horse

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.     

Therapeutic potentials of combined use of DMSA with calcium and ascorbic acid in the treatment of mild to moderately lead intoxicated mice

The aim of this study was to explore the therapeutic efficacies of combined use of meso-2,3-dimercaptosuccinic acid (DMSA) with calcium and ascorbic acid in the treatment of mild to moderately lead-intoxicated mice. Female albino mice were exposed to lead by drinking water contaminated with 0.1% (moderate lead exposure) or 0.05% (mild lead exposure) lead acetate. After the cessation of lead exposure, mice were supplemented by gavage with saline solution, 50 mg/kg body weight (b.w) DMSA, 100 mg/kg b.w DMSA, calcium and ascorbic acid, or 50 mg/kg b.w DMSA and calcium as well as ascorbic acid, respectively. Atomic absorption spectrophotometric method was used to analyze lead levels in blood, bone, liver, kidney and brain. Activities of blood δ-aminolevulinic acid dehydratase (ALAD) were determined by colorimetric method. DMSA supplemented alone could reduce lead levels in both soft tissues and bone and reverse lead-inhibited activities of blood ALAD in mild to moderately lead-intoxicated mice. On the other hand, combined use of DMSA with calcium and ascorbic acid achieved better therapeutic efficacies in mobilizing lead in blood, liver and kidney, and reversing lead-inhibited activities of blood ALAD in moderately lead intoxicated mice than DMSA supplemented alone. Moreover, the better therapeutic efficacies were also found in mildly lead intoxicated mice in mobilizing lead in blood and bone achieved by combined use of DMSA with calcium and ascorbic acid. Combined use of DMSA with calcium and ascorbic acid seems to be the better choice in the treatment of mild to moderate lead-intoxication.     

Changes in the blood parameters of an air-breathing fish during different respiratory conditions.

Some of the blood parameters recorded in an air-breathing eel, Amphipnous cuchia under normal respiratory condition during non-breeding period (September-April) are haemoglobin (Hb) concentration 19.26%, haematocrit value 56.16%, RBC number 1.71 million/mm3, RBC size 18.86 X 9.70 mum, mean corpuscular haemoglobin (MCH) 113.4 ng, mean corpuscular haemoglobin concentration (MCHC) 34.2%, blood sugar 77 mg% and ascorbic acid 0.435 mg%. The higher concentration of haemoglobin (19.26%) appears to be related to its obligatory air breathing habit and habitat in a water of low oxygen content. Though a definite trend of increase in the haemoglobin and haematocrit concentration with an increase in the body weight of the fish was lacking, variations were clearly marked related to intrinsic activity of the fish connected with different respiratory conditions. Asphyxiation in a submerged but continuous flow of water (liter/h) for 5 1/2 h resulted in an increase in the above-mentioned parameters to an appreciable extent. These increases were 0.23 million/mm3 in the number of erythrocytes, 6.16% in haemoglobin concentration, 10% in haematocrit value, 20% in blood sugar and 35% in ascorbic acid content. The mean corpuscular haemoglobin showed a decline of 6.2%. Exclusive aerial breathing for 5 1/2 h also caused 7.4% increase in haemoglobin concentration, 9.4% in haematocrit value, 0.14 million/mm3 in RBC number, 20% in blood sugar level, 9% in ascorbic acid content but almost no change in mean corpuscular haemoglobin. The average surface area for diffusion of gases appeared to have reduced by 6.8 mum2 per RBC.     

Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-Dihydroxycholecalciferol

Lysosomes in chick intestinal absorptive cells from rachitic (vitamin D-deficient) and vitamin D-replete animals were studied utilizing transmission electron microscopic histochemistry and ultrastructural morphometry. Absorptive cells from rachitic animals, serum calcium = 7.3±0.3 mg%, contained an average of 4.0±0.3 supranuclear lysosomes. In rachitic chicks sacrificed 9 hr post-injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D, the values for both serum calcium, 9.8 ± 0.2 mg%, and the number of apical absorptive cell lysosomes, 12.9±0.6, were increased over non-injected or vehicle-only injected animals. Lysosomes in vitamin D-replete absorptive cells were characterized by their intense staining with pyroantimonate, indicative of their high calcium content. The same organelles also produced a positive reaction for acid phosphatase. Rachitic lysosomes, also acid phosphatase positive, were only lightly stained with pyroantimonate. The lysosomal proliferation apparently induced by 1,25-dihydroxycholecalciferol may be a further indication that these organelles play a role in intestinal calcium transport and/or intracellular calcium homeostasis within the absorptive cell.     

Effect of cooking and legume species upon calcium, iron and zinc uptake by Caco-2 cells

An in vitro system, consisting of simulated gastrointestinal digestion and Caco-2 cell culture, was used to estimate the uptake of calcium, iron and zinc from white beans, chickpeas and lentils, and the effect of cooking upon uptake, with the ultimate aim of evaluating legumes as a dietary source of the aforementioned minerals. In raw products, differences were observed in the uptake percentages by Caco-2 cells of a same mineral from different legumes, although these were not related to the total mineral content. In the three elements studied, the highest uptake values corresponded to chickpeas. Traditional cooking significantly (p<0.05) increased the uptake (%) of calcium, iron and zinc from white beans, and of calcium from lentils. This effect can be partially ascribed to the conversion of inositol hexaphosphate to its lower phosphate forms. When mineral uptakes from raw, traditionally cooked, and ready-to-eat lentils were compared, the highest uptake values corresponded to the ready-to-eat product, which could be attributed to the combined effect of EDTA soaking, the cooking under pressure process, and citric and ascorbic acid addition.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.     

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

Age-dependent changes in uptake and recycling of ascorbic acid in erythrocytes of Beagle dogs

Uptake and recycling of ascorbic acid (AA) were studied in erythrocytes of 1- to 12-month-old Beagle dogs. At 1 month, both AA uptake and recycling capacity were high. Ascorbic acid entered erythrocytes mainly in the oxidized form with elevated activity of Glut 1 glucose transporter. However, this trait of erythrocytes was rapidly lost in the course of postnatal growth. At 3 months, ascorbic acid uptake and recycling capacity decreased to almost adult levels. Thereafter, AA was transported mainly in the reduced form, and its uptake and recycling capacity became one-third the levels of 1-month-old dogs. Postnatal anemia and recovery were indicated by changes in hemoglobin and packed cell volume levels at 1 and 3 months. Glutathione reductase (GR) activity was twice as high as in adults in 1-month-old dogs, allowing efficient reduction of oxidized ascorbic acid, which enters cells in large amounts due to elevated activity of the Glut 1 glucose transporter. One-month-old dogs need high levels of AA for antioxidant protection and skeletal development. The high AA recycling capacity of erythrocytes is considered to balance the expenditure of AA in young Beagle dogs.