Human Aqueous Humor Research Articles

Pharmacological and ocular hypotensive properties of topical carbonic anhydrase inhibitors.

There was a time gap of over 40 years between the demonstrated oral effectiveness of acetazolamide in lowering the intraocular pressure (IOP) of glaucoma patients and the introduction of a topical carbonic anhydrase (CA) inhibitor. This is due to the fact that CA-II, the isoenzyme which most likely plays an important role in the production of aqueous humor in humans, must be essentially inhibited by 100% to elicit a pharmacological response. The lack of success with earlier attempts to obtain a topical agent stems from an inability to attain and maintain a sufficiently high intraocular concentration of drug to achieve the required inhibition of CA. Dorzolamide and brinzolamide are two topical CA inhibitors which are currently available to treat ocular hypertension and/or glaucoma. Dorzolamide is a very potent inhibitor of CA-II and its site of action is local within the eye. Like oral CA inhibitors, topically applied dorzolamide lowers IOP by decreasing the production of aqueous humor. The drug is used in monotherapy as a 2% solution administered three times daily. Its ocular hypotensive effect is comparable to that of timolol at peak but is somewhat less at trough. The IOP lowering effect of timolol is enhanced by the twice daily administration of 2% dorzolamide either concomitantly or in combination. Topically applied dorzolamide is generally well tolerated and had a low drop-out rate in clinical studies. The most frequent ocular adverse experience is burning and/or stinging. Corneal and lenticular problems have generally not been encountered with long-term therapy with dorzolamide. Topically applied dorzolamide penetrates directly to the posterior segment of the eye and its presence is consistent with the initial report that dorzolamide increases retinal blood flow velocity in patients with normal tension glaucoma. The most frequent systemic adverse experience is a transient bitter taste. Biochemical changes indicative of the systemic inhibition of CA have not been observed in monotherapy studies lasting up to 2 years. This is in harmony with the inability of dorzolamide at steady-state to saturate CA in the red blood cell and the failure to detect its presence in plasma. A 1% suspension of brinzolamide is comparable to 2% dorzolamide in lowering IOP, both drugs being administered three times daily. Although brinzolamide has a lower incidence of burning/ stinging, it elicits more blurred vision.

Study of aqueous humour by 1H NMR spectroscopy

The aim of this work was to study the composition of the samples of human aqueous humour including the protein content. Using 1H NMR spectroscopy many compounds (proteins, glucose, lactate, citrate and other metabolites) can be identified and their concentrations evaluated using the internal standard. While the concentrations of non-proteins in aqueous humour were relatively stable, the amount of proteins differed much more. In most of the spectra, the signals of proteins were hardly distinguishable from the baseline. For some samples a significantly higher protein content (more than 1 mg/ml) was found. The total protein concentration expressed in albumin equivalents can be determined by comparing the spectra measured by S2PUL (standard measurement) and CPMG (protein suppression) pulse sequentions. For comparison, the spectra of rabbit and bovine aqueous humour are also given.

Sugars including erythronic and threonic acids in human aqueous humour

PURPOSE. Sugars in the aqueous humour of the eye serve both as a source of nutrients to the lens and other anterior ocular tissues, and potentially as an indicator of waste products from these tissues. In this work we intended to measure the levels of sugars in human blood and aqueous humour from cataract patients with and without diabetes. After initial results we decided to identify an unknown sugar component. METHODS. Sugars were measured by hplc. The unknown sugar peak was identified by gas chromatography/mass spectrometry RESULTS. Very little fructose and sorbitol were found. Glucose levels were higher in both blood and aqueous from diabetic patients. During these analyses we found a major component that did not correspond to any sugar reported previously in aqueous humour. This was identified as a mixture of threonic and erythronic acids. CONCLUSIONS. Glucose levels increase in human aqueous humour in diabetes without markedly raised levels of sorbitol or fructose. Erythronic and threonic acids are normal components of aqueous humour and blood. They may be derived from glycated proteins or from degradation of ascorbic acid.

Expression of muscarinic receptor subtypes (M1–M5) and adenylate cyclase subtypes II and IV in the human aqueous humor outflow pathway

Expression of muscarinic receptor subtypes (M1–M5) and adenylate cyclase subtypes II and IV in the human aqueous humor outflow pathway

Ocular levels of azithromycin.

To assess azithromycin levels in human serum, aqueous humor, tear fluid, and conjunctival tissue specimens after administration of a single 1-g oral dose of azithromycin. Sixty patients undergoing cataract surgery were included in this analysis. Serum, aqueous, and tear specimens were collected 3, 6, and 12 hours and 1, 2, 3, and 4 days after azithromycin administration. Conjunctival tissue biopsy specimens were collected 1, 2, 3, 4, 6, 8, 10, 12, and 14 days after azithromycin administration. All specimens were subjected to analysis by high-performance liquid chromatography-mass spectrometry. Azithromycin concentration ranges during the specified sampling times were as follows: serum, 21 to 974 ng/mL; tear, 82 to 2892 ng/mL; aqueous, 10 to 69 ng/mL; and conjunctival, 0.7 to 32 micrograms/g. Levels above the 90% minimal inhibitory concentration (MIC90) for Chlamydia trachomatis were detected after 4 days in all tear samples and after 14 days in all conjunctival tissue specimens following oral azithromycin administration. We demonstrated prolonged high levels of azithromycin in drug-targeted ocular tissue. Prolonged high concentrations of azithromycin in conjunctival tissue make this drug suitable for treatment of conjunctivitis caused by chlamydiae and other susceptible organisms.

Molecular identification and coexpression of galanin and GalR-1 galanin receptor in the human ocular ciliary epithelium: differential modulation of their expression by the activation of alpha2- and beta2-adrenergic receptors in cultured ciliary epithelial cells.

Here we report the coexpression of the neuropeptide galanin and GalR-1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM-2) derived from the NPE cells. Stimulation of ODM-2 cells with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or forskolin resulted in an up-regulation (two- to threefold) of galanin mRNA expression. Procaterol, a selective beta2-adrenergic agonist, and the catecholamine isoproterenol exerted a long-term down-regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective beta2-adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin-like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM-2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down-regulation in the level of galanin or galanin-like product accumulated in the medium of cultured ODM-2 cells to levels even lower than those induced by beta2-adrenergic agonists. This effect is best explained by the concomitant up-regulation (four- to fivefold) of GalR-1 galanin receptor transcripts induced through the activation of alpha2-adrenergic receptors. These findings support the view that pathways elicited by the activation of alpha2- and beta2-adrenergic receptors influence the expression of galanin and GalR-1 galanin receptors in ciliary epithelial cells.

Comparison of protein mixtures in aqueous humor by membrane preconcentration - capillary electrophoresis - mass spectrometry.

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.

Simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor with HPLC and electrochemical detection

A sensitive and selective bioanalytical method for simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor using reversed-phase HPLC and electrochemical detection is described. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 μm; 150×4.6 mm I.D.). This support is coated with a hydrophilic polyoxyethylenepolymer. It allows protein-containing samples to be injected directly onto the column. The electrochemical detector permit a detection limit of 500 pg diclofenac per ml (daily relative standard deviation 6.3%) and 50 ng oxybuprocaine per ml (daily R.S.D. 2.6%), respectively. Results of administered and measured drug-concentrations in time dependent decrease are presented.

Brain natriuretic peptide: identification of a second natriuretic peptide in human aqueous humour

AIMS/BACKGROUNDTo measure aqueous humour levels of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in humans. To compare peptide levels in glaucomatous and control eyes to test the hypothesis...

How an Increase in the Carbon Chain Length of the Ester Moiety Affects the Stability of a Homologous Series of Oxprenolol Esters in the Presence of Biological Enzymes

β-Blockers including timolol and propranolol are administered in eye-drops for the treatment of glaucoma. Due to high incidence of cardiovascular and respiratory side-effects, their therapeutic value is limited. As a result of poor ocular bioavailability, many ocular drugs are applied in high concentrations, which give rise to both ocular and systemic side-effects. Therefore, some methods have been employed to increase ocular bioavailability such as (a) the development of drug delivery devices designed to release drugs at controlled rates, (b) the use of various vehicles that retard precorneal drug loss, and (c) the conversion of drugs to biologically reversible derivatives (prodrugs) with increased corneal penetration properties, from which the active drugs are released by enzymatic hydrolysis. A series of structurally related oxprenolol esters were synthesized and investigated as potential prodrugs for improved ocular use. The stability of each ester was studied in phosphate buffer (pH7.4), also in the presence of (a) 30% human plasma, (b) aqueous humor, and (c) corneal extract at pH7.4 and at 37°C. An account is given of how the stability of a homologous series of oxprenolol esters in the presence of biological enzymes is affected by an increase in the carbon chain length of the ester moiety.

Effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells.

The effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells were studied. The melanoma cells were plated into 24-well plates. Melatonin, its 6-hydroxy or 6-chloro derivative, serotonin, tryptophan or kynurenine was added to the medium in concentrations of 0.001 to 1000 nM. After 5 days the cells were detached, counted, and compared with the controls. Melatonin inhibited the growth of uveal melanoma cell lines in a dose-dependent manner (0.1-10 nM). This growth inhibition occurred at concentrations of melatonin (2 nM) found in human aqueous humour. The melatonin derivatives also inhibited the growth of uveal melanoma cells; 6-chloromelatonin was more potent than melatonin and 6-hydroxymelatonin was the least active (6-chloromelatonin > melatonin > 6-hydroxymelatonin). The precursors of melatonin (tryptophan and serotonin) and the abnormal metabolite of tryptophan (kynurenine) did not inhibit the growth of the melanoma cells, indicating that changes to the metabolic processes of melatonin may play a role in the pathogenesis of uveal melanoma.

Evaluation of Serotonin Levels in Human Aqueous Humor

Purpose: Serotonin is biochemically present in the iris and ciliary body of animals and humans. Controversial findings are reported about the concentrations of serotonin in aqueous humor with respect to plasma in humans. The aim of this study was to evaluate the levels of serotonin both in aqueous humor and plasma in human subjects. Methods: In 50 patients with glaucoma or cataract, plasma and aqueous humor serotonin levels were measured by HPLC with electrochemical detection. Serotonin plasma levels were also measured in 25 healthy subjects as controls. Results: In all patients with cataract or glaucoma, the aqueous humor serotonin concentration is significantly lower than that in plasma [1.14±0.29 (SEM) vs. 5.33±1.03 ng/ml, p < 0.01]. Furthermore, in the same patients and in 25 healthy controls, serotonin plasma levels were similar. Conclusion: Our study shows that serotonin is present in human aqueous humor and its concentration is 4 times lower than in plasma.

Detection of CSF Leakage by Isoelectric Focusing on Polyacrylamide Gel, Direct Immunofixation of Transferrins, and Silver Staining

Leakage of cerebrospinal fluid (CSF) from the subarachnoid space into the nasal or aural cavity creates a pathway for life-threatening central nervous system infection. Detection of the leakage can be cumbersome (1). In former days the identification and localization of CSF leakage was almost entirely dependent upon the clinical history and methods involving intrathecal injection of radioactive material or a dye. Furthermore, various chemical tests, such as determination of glucose, protein, and electrolytes, performed on the secreted material have been advocated for the differentiation between CSF leakage and other secretions (2). The injection methods are risk-bearing for the patient and the above methods yield a considerable chance of false-positive or false-negative results (3). Nowadays O -sialotransferrin, also named β2-transferrin, asialotransferrin, or the τ fraction, is an accepted marker protein for the detection of CSF in excretions from the nose or the ear, or from head or neck wounds. O -Sialotransferrin is not strictly unique to CSF. The protein can also be demonstrated in human aqueous humor (4) and in perilymph fluid (5)(6)(7). It is also found in the blood of patients with chronic liver diseases (8)(9), with an inborn error of glycoprotein metabolism (10), and with a genetic variant of transferrin (8)(11)(12). The protein can be detected by direct immunofixation of transferrins after agarose electrophoresis (high-resolution electrophoresis, HRE) (3)(13) or after blotting (1)(14)(15). The method we used up to now was the agarose/immunofixation method according to Zaret et al. (3). We used the Paragon High Resolution Electrophoresis kit (HRE kit, Beckman Instruments) and an anti-transferrin reagent (Beckman Array® System, Beckman Instruments). One disadvantage of this fast method is the high …

PURIFICATION of an antimicrobial peptide from rabbit aqueous humour

To identify an antimicrobial factor previously demonstrated in rabbit aqueous humour. Rabbit aqueous humour was fractionated by a multi-stage process involving anion-exchange and size-exclusion liquid chromatography. The antimicrobial effect of aqueous humour fractions upon Staphylococcus aureus were evaluated in an in vitro model. The components of aqueous humour fractions mediating an antimicrobial effect were investigated by SDS-PAGE. A single peptide of molecular weight approximately 8 kDa was identified which mediated an antimicrobial effect upon Staphylococcus aureus. Attempts to identify the peptide have been unsuccessful. Rabbit aqueous humour contains an unidentified peptide that mediates an antimicrobial effect upon Staphylococcus aureus. If such a peptide is present in human aqueous humour it may contribute to the apparent resistance to bacterial infection manifest in the anterior chamber.

Purification of an antimicrobial peptide from rabbit aqueous humour

Purpose. To identify an antimicrobial factor previously demonstrated in rabbit aqueous humour.Methods. Rabbit aqueous humour was fractionated by a multistage process involving anion-exchange and size-exclusion liquid chromatography. The antimicrobial effect of aqueous humour fractions upon Staphylococcus aureus were evaluated in an in vitro model. The components of aqueous humour fractions mediating an antimicrobial effect were investigated by SDS-PAGE.Results. A single peptide of molecular weight approximately 8 kDa was identified which mediated an antimicrobial effect upon Staphylococcus aureus. Attempts to identify the peptide have been unsuccessful.Conclusions. Rabbit aqueous humour contains an unidentified peptide that mediates an antimicrobial effect upon Staphylococcus aureus. If such a peptide is present in human aqueous humour it may contribute to the apparent resistance to bacterial infection manifest in the anterior chamber. Curr. Eye Res. 17: 783–787, 1998.

Penetration of Ofloxacin in Human Aqueous and Vitreous Humors Following Oral and Topical Administration

Penetration of Ofloxacin in Human Aqueous and Vitreous Humors Following Oral and Topical Administration

Human Hepatocyte Growth Factor (HGF) in the aqueous humor

Using an enzyme-linked immunosorbent assay, we measured the concentration of hepatocyte growth factor (HGF) in human aqueous humor and serum from 36 eyes of 33 patients and analyzed the relationship between HGF and several parameters of corneal endothelial cells. Aqueous HGF concentrations ranged from 0.020-0.194 ng/mL (average: 0.101 ± 0.044 ng/mL) and was correlated positively with corneal endothelial cell density ( r = 0.343, P = 0.04). The aqueous HGF level did not correlate with other corneal endothelial cell parameters or the serum HGF level. The HGF receptor, c-Met, was found in the corneal endothelial cell membranes. This study suggests that the aqueous humor HGF is a factor which maintains an integrity of corneal endothelial cells.

Ascorbate Stimulates Type I and Type III Collagen in Human Tenon??s Fibroblasts

To better understand wound healing after glaucoma filtration surgery by measuring the production of type I and type III collagen in cultured Tenon's fibroblasts and determine the effect of ascorbic acid on collagen subtype production. An ELISA-type dot blot assay was used to directly measure the production of types I and III collagen by subconfluent cultures of fibroblasts from human Tenon's capsule. Because ascorbic acid is both high in aqueous humor and necessary for the production of collagen, we measured the dose response of type I and type III collagen production to ascorbic acid. Ascorbic acid stimulated an increase in collagen production that reached a maximum level at 100 micrograms/ml. This is approximately half of the ascorbic acid concentration found in human aqueous humor. Unlike previous reports, we found no toxic effects from ascorbic acid at concentrations as high as 250 micrograms/ml over a 24-hour period. The lack of toxicity may result from the use of serum-free media in the assay. This culture system will be useful for exploring factors that may alter collagen production and could potentially affect wound healing.

Crocheted ETFE-reactor for on-line post-column photoderivatization of diclofenac in high-performance liquid chromatography

A sensitive and selective bioanalytical method for diclofenac using reversed-phase HPLC and fluorescence detection is described. Diclofenac was detected as its fluorescent derivative after on-line post-column photoderivatization. Irradiation with UV light of diclofenac in aqueous solutions leads to the sequential loss of both chlorine substituents and ring closure. The major product, carbazole-1-acetic acid, was detected by a fluorescence detector using an excitation wavelength of 286 nm and an emission wavelength of 360 nm. The self-made reactor was a crocheted ethylene and tetrafluoroethylene (ETFE, named TEFZEL) capillary, 20 m in length, wound directly around a 253.7 nm UV lamp. The capillary was crocheted in order to overcome peak widening. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 μm; 150 mm×4.6 mm i.d.) and a LiChrospher 100 RP-18 (5 μm) guard column from E. Merck. The detection limit was 1 ng ml −1 at an injection volume of 20 μl. Daily relative standard deviations (RSD) were 5.5% (73 ng diclofenac/ml, n=9), and 5.1% (405 ng diclofenac/ml, n=6), respectively. Chromatograms of human aqueous humor and human serum containing diclofenac, and figures showing the time dependent increase/decrease of the photoderivatization product, are shown.

Diffuse-reflectance infrared Fourier transform spectroscopy: new technique of sample preparation

A new technique of measurement of the diffuse-reflectance infrared FT spectra, based on the preparation of a cylinder from the mixture of the sample and powdered KBr under pressure of about 5.85 MPa, has been tested. During the measurement, the axis of the formed cylinder is perpendicular to the direction of the incident light. A repeatability of the measurement of selected bands and also of the background was investigated for hydroquinone, nicotinamide, silica gel, rice, tea and also lyophilized human aqueous humour. The relative standard deviations of log( 1 R ) showed a dependence on the character of the measured compound, but in general were comparable or slightly better than those obtained by the standard method of loosely packed cups. The values were better than 1.5% in the most cases. The main advantage of the proposed technique lies in its simplicity and rapidity of obtaining statistically significant data.