Changes in ocular tone, pH, ascorbic acid and lactic acid concentration in the aqueous humor of rabbits treated with aceclidine

The Authors state the results of their research on the modification of the aqueous humor caused by the instillation of the Clonidina to the concentrations of 0,I25% in a group of ten rabbits. The results of these studies illustrate the variations of pH, the ocular tone, the concentration of lactic and ascorbic acid. In all the rabbits examined Clonidina was instillated in the RE while in the LE distilled water was instillated for the control. For two hours from the beginning of the experiment, every IO minutes measurements of the ocular tone in both eyes were taken. At the end of the two hours a sample of aqueous humor was taken from the RE and LE; It was found that: I) The drugs produced a very quick and prolonged decrease of the ocular tone. 2) There are no significant variations in the pH or in the concentration of lactic acid. 3) There was a notable reduction of the concentration of ascorbic acid.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.     

In vivo ocular availability of ketorolac following ocular instillations of aqueous,oil, and ointment formulations to normal corneas of rabbits: A technical note

Conclusions  In vivo ocular availability of ketorolac was evaluated following ocular instillation of aqueous, oil, and ointment formulations to normal corneas of rabbits and monitoring ketorolac concentration in aqueous humor by HPLC. Compared with aqueous drop, sesame and soybean oil drops of ketorolac provided higher ocular availability followed by ophthalmic ointment. The ointment formulation provided maximum sustained effect. Ketorolac aqueous drop with BAC and EDTA improved the rate of ocular absorption though not the extent. Published: October 24, 2005     

Ocular ascorbate transport and metabolism.

1. The concept is reviewed that the eye is subject to photo-oxidative damage through chemical free radical species that interact with sensitive tissue components. 2. The role of ascorbic acid may be to protect the eye by scavenging free radicals. 3. Ascorbic acid is present at a high concentration in various ocular compartments of diurnal animals, regardless of whether the animal synthesizes the compound or extracts it from the diet. 4. Ascorbic acid accumulates in the eye by active transport through the iris-ciliary body into aqueous humor, and subsequent transport into the lens and cornea. 5. Conservation of ascorbic acid occurs by reduction of dehydro-L-ascorbic acid and the ascorbate free radical through processes that appear to be enzymatic.     

Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork.

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.     

Ascorbic acid and copper in linoleate oxidation. II. Ascorbic acid and copper as oxidation catalysts

Both ascorbic acid and copper were strong prooxidants in the oxidation of linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C. Minimum concentrations at which catalytic activity was detected were 1.3 x 10(-7) m for copper and 1.8 x 10(-6) m for ascorbic acid. For concentrations up to 10(-3) m, the increase in rate of oxidation with increase in concentration of catalyst was greater for ascorbic acid than for copper. Ascorbic acid had maximum catalytic activity at 2.0 x 10(-3) m, but was still prooxidant at the highest concentration tested (5.0 x 10(-2) m). Dehydroascorbic acid was a weaker prooxidant than ascorbic acid. Further degradation products of ascorbic acid were not prooxidant. In early stages of the oxidation autocatalytic behavior was observed with copper, but not with ascorbic acid. Ascorbic acid functioned as a true catalyst, i.e., it accelerated the reaction but it was not oxidized simultaneously with the linoleate. It is proposed that the dehydroascorbic acid radical initiates the linoleate oxidation reaction.     

Stimulation of transepithelial sodium and chloride transport by ascorbic acid. Induction of Na+ channels is inhibited by amiloride

Ascorbic acid increases the short circuit current (I_sc) across the amphibian cornea when it is present at either surface of this epithelium. These effects were additive. The effect was greater when it was on the tear side. The response returned to baseline levels when the ascorbic acid was washed from the bathing media. The effect of ascorbic acid on I_sc when it was on the aqueous humor side of the cornea could be blocked by bumetanide but that due to the vitamin's presence on the tear side was unchanged. The ascorbic acid could enter the tissue and crossed the cornea at similar rates in either direction. When the cornea was bathed by a Cl^?-free solution or exposed to bumetanide, the rise in I_sc observed with ascorbic acid on the tear side was equivalent to an increased Na⁺ flux from the tear to the aqueous humor side. In normal (Cl^? present) Conway solution the rise in the I_sc seen with ascorbic acid on the aqueous humor side was equal to an increased flux of Cl^? from the aqueous to the tear surface. However, when ascorbic acid was present on the opposite, tear, side the increased I_sc reflected a rise in both Cl^? and Na⁺ transport, aqueous-to-tear side, and tear-to-aqueous side, respectively. Thiol reagents (tear side), including reduced glutathione (10^?5 M), blocked the effect of ascorbic acid (10^?3 M) providing they were added to the bathing solution prior to the vitamin. However, they had no effect once the response had been established. The effect of the reduced glutathione appeared to be of a non-competitive nature. Oxidized glutatione (10^?4 M) (and cystamine) blocked the effect of ascorbic acid (10^?3 M) when present on the tear side prior to the vitamin. However, they also increased the rate of decline of the response when added subsequently to the ascorbic acid. Amiloride (as low as 5·10^?9 M), on the tear side but not the aqueous humor side, prevented the response to ascorbic acid but could not reverse it, once it was established. The possible nature of the effect of ascorbic acid is discussed in relation to its pharmacological interactions with thiol and disulfide reagents and amiloride.     

The chemical composition and the osmotic pressure of the aqueous humor and plasma of the rabbit

Measurements were made of the osmotic pressure of plasma, and of aqueous humor taken from the anterior chamber of the right and left eyes and from the posterior chamber of unanesthetized rabbits. Aqueous humor from the anterior chamber was found to be hypertonic to the plasma by approximately 3 mM/liter equivalent of sodium chloride. The aqueous humor from the anterior and posterior chambers of the right and left eyes was isotonic. The concentration of chloride in the anterior and posterior chambers was the same. The concentration of all the major components of the aqueous humor and plasma has been determined by chemical analysis on fluid samples obtained from unanesthetized rabbits at approximately the same time. The calculated osmotic pressure of the total of these substances in terms of sodium chloride equivalent agrees to within better than 1 per cent of the total osmotic pressure as measured experimentally. The distribution of some individual anions and cations of the aqueous humor and plasma was determined. This distribution is widely different from that which would obtain at a state of equilibrium. The positive and negative charges carried by the ions in the aqueous humor were approximately equal. Sources of error in the experiments are discussed.     

Parasite-specific antibody profile in the aqueous humor of rabbits with ocular toxocariasis

Human ocular toxocariasis is diagnosed using ophthalmologic and immunologic examinations. Many researchers have suggested that intraocular parasite-specific antibody levels are indicative of ocular toxocariasis, but little is known about the time course of the changes in these levels. We therefore investigated the anti-Toxocara canis antibody profile in the aqueous humor in an animal model of ocular toxocariasis. We intravitreally injected T. canis larvae into the right eye of 4 rabbits; 2 rabbits were orally administered T. canis eggs. We collected serum, aqueous humor, and tear samples weekly and determined the serum and aqueous humor levels of anti-T. canis immunoglobulin (Ig)G, IgA, IgM, and IgE antibodies and the tear IgG antibody level by enzyme-linked immunosorbent assay (ELISA). The severity of vitreous opacity and the aqueous humor IgG levels (measured using optical density [OD]) changed concordantly in the larvae-injected eyes; the OD exceeded 0.1 from 2–4 weeks after infection and remained elevated during active intraocular inflammation. However, the aqueous humor IgG levels were also elevated in 6 out of 8 eyes without intraocular larvae in both groups, and were low in 1 eye with live intravitreal larvae. In contrast, the serum IgG and IgM levels and the tear IgG levels increased in all rabbits, regardless of the presence of intraocular inflammation. Vitreous opacity occurred in all intravitreally infected eyes, but significant histopathological evidence of retinal damage was not detected. Thus, besides the presence of intraocular larvae, some other factors in the host may be required for the development of retinal lesions.     

Ascorbic acid and copper in linoleate oxidation. I. Measurement of oxidation by ultraviolet spectrophotometry and the thiobarbituric acid test

The UV absorption method and the thiobarbituric acid (TBA) test for oxidation of an aqueous suspension of linoleate were compared. The absorption method depends on the formation of hydroperoxides having conjugated double bonds that absorb strongly at 233 nm. The absorption at 233 nm increased markedly during oxidation of linoleate catalyzed by either ascorbic acid or cupric ions. The concentration of ascorbic acid in the reaction mixture was also measured by UV absorption at 265 nm and pH 7.0. Color development in the TBA test also increased markedly with the extent of oxidation of linoleate. When ascorbic acid was the catalyst, UV absorption detected early stages of oxidation with greater sensitivity than the TBA test. The reverse was true when Cu(++) was the catalyst. In general, the relation between the two procedures will depend on whether copper is present when the TBA test is made.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Simultaneous quantification of cefotaxime, desacetylcefotaxime, ofloxacine and ciprofloxacine in ocular aqueous humor and in plasma by high-performance liquid chromatography

Cefotaxime, given intravenously, is currently used as a broad-spectrum antibiotic for prophylaxis of intra- and postoperative infections in ocular lens surgery. A proposed therapeutic and economic alternative is the use of orally active fluoroquinolone ofloxacine as prophylactic agent. A HPLC method was developed for determination of both antibiotics in ocular aqueous humor and plasma in order to optimize dosage for safe surpassing minimal inhibitory concentration in the humor compartment. For plasma determinations a solid-phase extraction procedure was used with ciprofloxacine as internal standard. Detection limits for direct HPLC-analysis of ocular aqueous humor was 0.08 μg/ml for all compounds, whereas in plasma 0.31 μg/ml could be determined after solid-phase extraction.     

Inhibition of the ocular effects of sodium arachidonate by anti-inflammatory compounds

Topical administration of aqueous solutions of sodium arachidonate to the eyes of rabbits increases intraocular pressure, constricts the pupil and increases both the protein and prostaglandin content of aqueous humor. Arachidonic acid itself dissolved in arachis oil is less effective than sodium arachidonate, although addition of polysorbate mono-oleate greatly increases the effects produced by arachidonic acid. Pretreatment with topically applied non-steroidal anti-inflammatory agents prevents the ocular effects of sodium arachidonate, indomethacin being 2–4 times as potent as either indoxole or pirprofen. Dexamethasone was without effect in these experiments. The results suggested that non-steroidal anti-inflammatory agents deserve serious consideration for topical use in the treatment of ocular inflammation.     

Characteristics of the transport of ascorbic acid into leucocytes

The degree and the mode of association of [14C]-ascorbic acid with leucocytes are examined. The degree of association of ascorbic acid with polymorphonuclear leucocytes (1-3%) is dependent on cell type, extracellular concentration of ascorbic acid, incubation temperature, intactness of the cells and the extracellular pH. All experiments are performed according to strict protocols as these compounds are labile in aqueous solutions. Further it is noticed that in all experiments an outward gradient of leucocyte endogenic ascorbic acid exists. The results suggest that the association process comprises at least one saturable pathway. The activation of polymorphonuclear leucocytes by phorbol myristate acetate increases the accumulation of ascorbic acid threefold.     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Effect of ascorbic acid and Vitamin E supplementation on semen quality and biochemical parameters of male rabbits

The objective of this study was to determine the effects of supplementation of ascorbic acid, Vitamin E (Vit. E) and their combination in drinking water on sperm characteristics, lipid peroxidation (LPO) and seminal plasma enzymes of mature male rabbits. Twenty-four male New Zealand White rabbits (5 months old) were given drinking water supplemented with ascorbic acid (1.5 g/l), Vit. E (1.0 g/l) and ascorbic acid+Vit. E (1.5+1.0 g/l) for 12 weeks. Vitamin supplementation in drinking water increased feed intake, but body weight gain was not significantly affected. Concentrations of thiobarbituric acid-reactive substances (TBARS) were significantly (P<0.05) reduced in seminal plasma of treated groups compared with the control. Treatment with ascorbic acid, Vit. E, and their combination significantly (P<0.05) increased lipido (reaction time), ejaculate volume, sperm concentration, total sperm output, sperm motility index, total motile sperm, packed sperm volume, initial hydrogen ion concentration (pH), and semen initial fructose concentration. Abnormal and dead sperm were significantly (P<0.05) decreased in treated animals. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were significantly (P<0.05) decreased, whereas glutathione S-transferase (GST) showed a significant increase in seminal plasma of treated animals compared with the controls. The results from this study indicated that supplementation of drinking water with antioxidant ascorbic acid, Vit. E and their combination reduced the production of free radicals and can improve rabbit semen quality, but the greater improvement seemed to be from Vit. E.     

The natural histidine-containing dipeptide Nalpha-acetylcarnosine as an antioxidant for ophthalmic use

The naturally occurring compound Nalpha-acetylcarnosine is proposed as a prodrug of L-carnosine that is resistant to enzymatic hydrolysis by carnosinase. Eyes of rabbits were treated with 1% Nalpha-acetylcarnosine, L-carnosine, or placebo and extracts of the aqueous humor from the anterior eye chamber were analyzed for imidazole content by reverse-phase analytical high performance liquid chromatography (HPLC) and thin-layer (TLC) and ion-exchange chromatographic techniques. Topical administration of pure L-carnosine to the rabbit eye did not lead to accumulation of this compound in the aqueous humor over 30 min in concentration exceeding that in the placebo-treated matched eye. Nalpha-Acetylcarnosine showed dose-dependent hydrolysis in its passage from the cornea to the aqueous humor, releasing L-carnosine after l5-30 min of ocular administration of the prodrug in a series of therapeutic modalities: instillation < or = subconjunctival injection < or = ultrasound-induced phoresis. Different treatment techniques showed excellent toleration of 1%Nalpha-acetylcarnosine by the eye. Once in the aqueous humor, L-carnosine might act as an antioxidant and enter the lens tissue when present at effective concentrations (5-l5 mM). The advantage of the ophthalmic prodrug Nalpha-acetylcarnosine and its bioactivated principle L-carnosine as universal antioxidants relates to their ability to give efficient protection against oxidative stress both in the lipid phase of biological membranes and in aqueous environments. Nalpha-Acetylcarnosine is proposed for treatment of ocular disorders that have a component of oxidative stress in their genesis (cataracts, glaucoma, retinal degeneration, corneal disorders, ocular inflammation, complications of diabetes mellitus, and systemic diseases).     

Enhanced isomer purity of lactic acid from the non-sterile fermentation of kitchen wastes

In order to improve the purity of lactic acid isomers, the effects of pH, temperature, fermentation time and their interactions on l(+) or d(-)-lactic acid production were evaluated during lactic acid fermentation of the non-sterile kitchen wastes. The results showed that l(+)-lactic acid was the main isomeric form. The isomer purity was much higher at acidic or alkalic pH (non-controlled pH, pH 5 and pH 8) than neutral pH (pH 6 and pH 7). Increasing the fermentation temperature from 35 degrees C to 45 degrees C at pH 7 enhanced the isomer purity from 60:40 to 83:17. The optimal fermentation time for the purity of lactic acid isomers was found to depend on the corresponding pH and temperature. From the response surface analysis, the optimized combination of pH and temperature could obviously increase the l(+)-isomer concentration. It is confirmed that the variation of the isomer purity with pH, temperature and fermentation time change resulted from the substitution of microbial community composition. The lactic acid bacteria and Clostridium sp. dominated the fermentation of non-sterile kitchen wastes, and the emergence and disappearance of lactic acid bacteria which produced l(+)-isomer and Clostridium sp. resulted in the variations of the isomer purity.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

Lactic acid extraction with trioctyl amine

Lactic acid extraction was carried out with trioctyl amine (TOA) in three diluents. The effect of initial lactic acid concentrations on the extraction efficiency was investigated. It was observed that although the percentage extraction remained constant or decreased but the loading ratio was increased in all the cases. The overloading was observed in the case of TOA in methyl isobutyl ketone (MIBK). The extraction of lactic acid was favored at a lower aqueous pH?in all the diluents. The improvement of the extraction efficiency at a higher aqueous pH?(=?6) was achieved by using the modified TOA (treated with HCl) in MIBK. However, the recovery of lactic was very poor in the case of modified TOA in MIBK, although the complete recovery was obtained for untreated TOA.