Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines

Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase $\text{in}$$\text{vitro}$. Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the $\text{in}$$\text{vivo}$ effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.     

Rapid purification of human placental aldose reductase

Sixty percent methanol is widely used for the extraction of nucleotides from lymphocytes for quantitation by high-performance liquid chromatography. In the course of such studies, we noted that these extracts analyzed on an anion-exchange column showed a major “unknown” uv-absorbing peak which eluted after the nucleosides and before the nucleotides. The material cochromatographed with and had the spectral properties of ascorbic acid. This compound was identified as ascorbic acid by chemical and enzymatic assays. The ascorbate content of human lymphocytes determined by high-performance liquid chromatography, $\text{42.2 ± 3.3 nmol}\text{10}{}^8$ cells (mean ± SEM), agreed closely with the levels obtained by standard less sensitive methodology. Evidence is presented that this technique can be used to determine the ascorbate content of lymphocytes where only scanty material or very low levels are found.     

Chromium and chronic ascorbic acid depletion effects on tissue ascorbate,manganese, and14C retention from14C-ascorbate in guinea pigs

Chromium (Cr) potentiates the effects of insulin and a role for insulin in ascorbic acid transport has been reported. Therefore, the effects of Cr and ascorbate depletion on tissue ascorbic acid and¹⁴C distribution and excretion after a¹⁴C ascorbate dose were investigated in guinea pigs. As utilization of dietary Cr is affected by interaction with other minerals, tissue manganese (Mn), zinc (Zn), copper (Cu), and iron (Fe) were examined. For 20 wk, 40 weanling animals were fed either a Cr-deficient (<0.06 μg Cr/g diet, ?Cr) or a Cr-adequate (2 μg Cr from CrCl₃/g diet, +Cr) casein-based diet and were given 1 mg ascorbate/d (?C) or 10 mg ascorbate/d (+C) for 20 wk. Animals fed the Cr-depleted diet had decreased weight at 20 wk (p<0.01). Six hours before necropsy, animals were dosed by micropipette with 1.8 μCi ofl-[carboxyl-¹⁴C] ascorbic acid and placed in metabolic cages. Ascorbate supplementation increased Fe concentrations in most analyzed tissues, hepatic¹⁴C, tissue ascorbate and Mn concentration in the adrenal and testes, but decreased the concentrations of Cu in the kidney and Mn in the spleen. Liver Mn concentration was higher and kidney Mn concentration was lower in +Cr animals. Interactions between Cr and ascorbic acid affected Mn concentrations in bone and brain. These results indicate that ascorbate and Cr may affect Mn distribution. Chromium supplementation decreased plasma cortisol, brain¹⁴C and the amount of¹⁴C expired as carbon dioxide. These findings suggest that dietary Cr may affect ascorbic acid metabolism and the metabolic response to stress.     

Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Inhibition of ascorbate autoxidation by a dialyzed,heat-denatured extract of plant tissues

Preparations obtained from various plant sources were analyzed for their effect on the autoxidation of ascorbic acid and norepinephrine. The former reaction was followed by spectro-photometric detection of ascorbic acid at 265 nm, the latter one by measuring the formation of noradrenochrome at 480 nm. Extracts were prepared from $\text{Philodendron}$ leaves and the edible portion and seeds of green peppers ($\text{Capsicum Annuum}$). The tissues were minced, homogenized in 10 volumes of 16 mM Na-phosphate buffer pH 7.4 and centrifuged at 35,000g for 30 min. The supernatant was dialyzed in 12,000 m.w. cut-off tubing, denatured in boiling water and centrifuged at 10,000g for 10min. Aliquots (5–50 ul) of the supernatant were assayed in 5 ml 16 mM Na-phosphate buffer pH 7.4 containing 100 uM ascorbate or norepinephrine. The denatured extracts had marked dose-dependent inhibitory effect on the autoxidation of ascorbic acid, with negligible influence on the formation of noradrenochrome. EDTA inhibited both reactions. The selectiveness of the extract toward the autoxidation of ascorbic acid makes it unlikely that the inhibitory effect is based on sequestering metal-ions.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

High-performance liquid chromatographic separation of ascorbic acid,erythorbic acid,dehydroascorbic acid,dehydroerythorbic acid,diketogulonic acid,and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH₂ analytical column, with acetonitrile: 0.05 m KH₂PO₄ (75:25, $\text{w}\text{w}$) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

The influence of NAD+-linked substrates on energy conservation at sites 2 and 3 in mitochondria treated with inorganic arsenate.

Exposure of rat liver mitochondria to inorganic arsenate followed by reisolation and washing to remove the added arsenate results in uncoupled respiration with succinate and ascorbate ($\text{ADP}\text{0}\text{=0}$), but $\text{ADP}\text{0}$ and $\text{ATP}\text{0}$ values of 1.3 to 1.6 with 3-hydroxybutyrate or glutamate. $\text{ADP}\text{0}$ and $\text{ATP}\text{0}$ values greater than 1.0 with NAD⁺-linked substrates arise as a result of partial reactivation of coupling at sites 2 and 3 by these substrates. In the presence of rotenone, NAD⁺-linked substrates can still reactivate coupling with succinate or ascorbate at these sites. The extent of reactivation in the presence of rotenone by 3-hydroxybutyrate is decreased by simultaneous addition of acetoacetate. The results suggest that the coupling at sites 2 and 3 is amenable to control through changes in the reduction state of some specific components of the respiratory chain located remotely from these sites.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Selective fetal malnutrition: The effect of ethanol and acetaldehyde upon in vitro uptake of alpha amino isobutyric acid by human placenta

Uptake of alpha amino isobutyric acid was measured in human placental villus tissue exposed $\text{in vitro}$ to ethyl alcohol (ethanol) (0.3 g/dl–2 g/d1) or acetaldehyde (50 μM-20 mM). Ethanol and acetaldehyde significantly inhibited uptake of amino acid at higher, pharmacologic concentrations (2 g/dl and 2–20 mM respectively). Inhibition by 10 mM acetaldehyde was partially reversible. The results suggest that the human placenta is resistant to acute ethanol-associated effects upon amino acid transport $\text{in vitro}$. However, both ethanol and its major circulating metabolite, acetaldehyde, may still alter placental function during $\text{in vivo}$ chronic exposure.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand,DAla2DLeu5 enkephalin in mouse brain

The binding characteristics of the delta opioid receptor ligand, ³HDAla²DLeu⁵ enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for ³HDAla²DLeu⁵ enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate K_D value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The $\text{in}$$\text{vitro}$ addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect DAla²DLeu⁵ enkephalin binding characteristics. No alteration in the binding characteristics of ³H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a ³HDAla²DLeu⁵ enkephalin binding site, while the binding of ³H-naloxone was unchanged.     

The interaction of ethanol and exogenous arachidonic acid in the formation of leukotrienes and prostaglandin D2 in mastocytoma cells

The present studies were undertaken to examine the hypothesis that ethanol could effect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered $\text{in vivo}$ as well as during $\text{in vitro}$ experiments. The effects of ethanol which encompass these concentration ranges (200–1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20–50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandins D₂ (PGD₂) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 μg/ml), large increases in both PGD₂ and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD₂ formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments sugsests that ethanol affects metabolsim of arachidonic acid at reasonably low doses (200–400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, $\text{in vitro}$ experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500–1000 mg%) which are frequently utilized.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Ascorbic acid synthesis and concentrations in organs of copper-deficient and brindled mice

Copper deficiency was studied in mice to investigate an interaction between copper and ascorbic acid. Twelve-day-old mutant brindled mice that exhibited signs of copper deficiency were compared to their normal brothers as well as to age-matched suckling mice that were copper deficient (-Cu) because their dams were consuming a copper-deficient diet throughout gestation and lactation, and a fourth group of copper-supplemented ( + Cu) suckling mice that served as dietary controls. Dietary copper deficiency was also produced in older mice by beginning the treatment at birth and continuing for 7 wk. Organ ascorbate levels were determined by high performance liquid chromatography with electrochemical detection. Differences caused by diet and genetics were evident but age-dependent. Compared to controls, liver and kidney ascorbate levels did not change remarkably in young or old copper-deficient mice. Cardiac ascorbate levels were higher in 7-wk-old - Cu mice and lower in 12-d-old - Cu mice, despite hypertrophy in both cases. Spleen ascorbate levels were lower in older -Cu mice and higher in 12-d-old mice, but total spleen ascorbate reflected the hypertrophic and atrophic size in the older and younger -Cu mice, respectively. Brindled mutants had an extremely low level of ascorbate in spleen. Plasma ascorbate was lower in 7-wk-old - Cu mice. Reasons for the alterations in ascorbate levels are not known. Synthesis in liver from D-glucuronate was not altered by dietary copper deficiency in 7-wk-old mice. Synthesis was lower in livers from 12-d-old - Cu and brindled mice compared to control values. However, the difference correlated better with body weight of the mice rather than with degree of copper deficiency. Consequences of the altered organ levels of ascorbate in copper-deficient mice are not completely known.     

A new type of enzyme electrode: The ascorbic acid eliminator electrode

A new type of enzyme electrode has been developed for $\text{in}$$\text{vivo}$ electrochemical measurements which allows discrimination between ascorbic acid and catecholamines and their metabolites. The electrode employs the enzyme ascorbic acid oxidase held between the voltammetric electrode and solution by a dialysis membrane or immobilized on the outer surface of serous membrane from rat small intestine. The electrode gives linear calibration curves for all catecholamines and metabolites independent of any ascorbic acid concentrations significant in physiological measurements. The electrode has been tested in brain slice measurements and shown to respond to releases of catecholamines initiated by potassium ion stimulation.     

Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Ethanol directly inhibits Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity in rat testis interstitial cells

Acute ethanol exposure has been demonstrated to inhibit testosterone synthesis both $\text{in vivo}$ and $\text{in vitro}$; however, the precise step(s) affected is controversial. Using intact collagenase-dispersed interstitial cells or 10,000xg supernatants of interstitial cell homogenates, studies were undertaken to determine whether ethanol specifically inhibited Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity. In both cellular preparations, varing concentrations of ethanol (2.2 – 652 mM) inhibited this enzyme activity. Because alcohol dehydrogenase activity was identified specifically in Leydig cells and because the inhibition of Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity by concentrations of ethanol normally observed in circulation of alcoholic men (2.2 – 65 mM) could be reversed by saturating concentrations of NAD⁺ (0.2 mM) or by 4-methylpyrazole (2 mM), these results suggest that the mechanism of this inhibition is by limitation of available cofactor.     

Radiation Chemistry of Foods

In food irradiation, water-soluble food constituents undergo the attack of ${\text{e}}_{\text{aq}}^{?}$ and OH at the neutral region.

Reaction rate constants of some food constituents with ${\text{e}}_{\text{aq}}^{?}$ and OH were measured by competition methods using nitrous oxide and ³H-formate as competitors, respectively. High selectivity was observed among the reactions with ${\text{e}}_{\text{aq}}^{?}$ and the ${\text{e}}_{\text{aq}}^{?}$-reaction rate constants are 10¹⁰ M^?1 sec^?1 (cysteine), 10⁹ M^?1 sec^?1 (methionine, ascorbic acid, and histidine), and much smaller (sugars, and the other types of amino acids). The OH-reaction rate constants range from 10⁹ M^?1 sec^?1 (cysteine, histidine, methionine, aromatic amino acids, and ascorbic acid) to 10⁸ M^?1 sec^?1 (sugars and the other amino acids), indicating that the reactions with OH are less selective. Selective destructions of cysteine and ascorbic acid in food irradiation may be partly ascribed to their selective reactivities with some less reactive species which are produced by reactions of ${\text{e}}_{\text{aq}}^{?}$ or OH with oxygen or the other constituents as well as their higher reactivity with ${\text{e}}_{\text{aq}}^{?}$.