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Glucose determination in human aqueous humor with Raman spectroscopy.

It has been suggested that spectroscopic analysis of the aqueous humor of the eye could be used to indirectly predict blood glucose levels in diabetics noninvasively. We have been investigating this potential using Raman spectroscopy in combination with partial least squares (PLS) analysis. We have determined that glucose at clinically relevant concentrations can be accurately predicted in human aqueous humor in vitro using a PLS model based on artificial aqueous humor. We have further determined that with proper instrument design, the light energy necessary to achieve clinically acceptable prediction of glucose does not damage the retinas of rabbits and can be delivered at powers below internationally acceptable safety limits. Herein we summarize our current results and address our strategies to improve instrument design.

Algorithms↗

Glutamate levels in aqueous humor of patients with retinal artery occlusion.

PURPOSE: We analyzed the levels of glutamate and other amino acids in aqueous humor of patients with retinal artery occlusion (RAO) to determine whether glutamate is associated with retinal ischemia in RAO. METHODS: Aqueous humor samples were obtained from nine RAO patients by paracentesis performed as an emergent therapeutic intervention and from nine cataract patients without other ocular disease who served as controls. Aspartic acid, glutamate, taurine, and alanine concentrations were determined using high-performance liquid chromatography. RESULTS: The glutamate level in aqueous humor of patients with RAO (4.46 +/- 1.62 micromol/L) was significantly higher than that in controls (2.98 +/- 1.12 micromol/L) (P < 0.05). CONCLUSIONS: Elevated glutamate levels in aqueous humor may indicate diffusion of vitreous glutamate released from the damaged retina due to acute retinal ischemia in RAO. An increase in the extracellular glutamate level may play an important role in ischemic retinal damage in RAO.

Aged↗

Identification of a nonlysozymal bactericidal factor (beta lysin) in human tears and aqueous humor.

An antibacterial factor, identical with or closely related to beta lysin, was measured in human tears, in human aqueous humor, and in fractions of each fluid after an absorption step involving cellulose-asbestos filters. Antibeta lysin was used to help distinguish whether bactericidal activity was due to lysozyme or beta lysin. Beta lysin activity was found in both human tears and aqueous humor in higher amounts than were present in serum; appreciable amounts of lysozyme were found in human tears but not in aqueous humor. Beta lysin is a normal constituent of human tears and aqueous humor and its role in these fluids is that of an antibacterial agent that may also act in concert with lysozyme to destroy bacteria in tears.

Aqueous Humor↗

Fourth-generation fluoroquinolone penetration into the aqueous humor in humans.

PURPOSE: To compare the penetration and levels of the fourth-generation fluoroquinolones moxifloxacin 0.5% ophthalmic solution and gatifloxacin 0.3% solution in the aqueous humor (AH) in humans after topical application with published levels of other available fluoroquinolones under similar dosing conditions. DESIGN: Prospective, randomized, double-masked clinical trial. PARTICIPANTS: Forty-six patients undergoing cataract extraction. METHODS: Patients scheduled for routine phacoemulsification and intraocular lens implantation were provided either moxifloxacin 0.5% ophthalmic solution (n = 22) or gatifloxacin 0.3% ophthalmic solution (n = 24) to use 4 times daily the day before surgery plus 1 drop 1 hour before the surgical entry into the anterior chamber on the day of surgery. This regimen simulated a realistic postoperative dosing schedule. Aqueous humor samples were obtained and analyzed by high-pressure liquid chromatography. Aqueous humor fluoroquinolone concentrations were calculated by peak comparison with a known concentration peak for ciprofloxacin that was used as an internal standard. These values were compared with published concentrations of other available fluoroquinolones under similar dosing conditions. RESULTS: The mean age of the moxifloxacin 0.5% group was 67.8+/-9.7 years, whereas that of the gatifloxacin 0.3% group was 69.9+/-8.7 years. The moxifloxacin AH concentration was 1.86+/-1.06 microg/ml, and that of gatifloxacin was 0.94+/-0.72 microg/ml. This 2-fold difference was statistically significant (P = 0.001). CONCLUSIONS: Aqueous humor antibiotic concentrations achieved at the time of cataract surgery after topical application can serve as an effective surrogate for what can be achieved with typical postoperative topical dosing (e.g., 4 times daily). Both fourth-generation fluoroquinolones achieved a greater AH concentration after 4 times daily dosing relative to prior-generation fluoroquinolones. Moxifloxacin 0.5% ophthalmic solution achieved a 2-fold higher aqueous humor concentration than gatifloxacin 0.3% ophthalmic solution. The superior penetration of moxifloxacin into the AH may be attributed partially to its high degree of lipophilicity, greater solubility at neutral pH, and higher concentration in the commercial formulation. The enhanced penetration of moxifloxacin 0.5% ophthalmic solution may provide better protection against ocular infections.

Administration, Topical↗

[A study on cytokine levels and nitric oxide content in rabbit aqueous humor after lens extraction and intraocular lens implantation].

OBJECTIVE: To investigate the relationship among inflammatory reaction and cytokine levels, nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: Eighteen New Zealand rabbits were divided randomly into 3 groups (each 6 rabbits): (1) control group, (2) extracapsular cataract extraction group (ECCE) and (3) ECCE and posterior chamber intraocular lens implantation group (ECCE + IOL). The inflammation of all experimental rabbit eyes were observed by a zoom-photo slit-lamp microscope 0, 1, 3, 7, 14, 30 days postoperatively, including corneal edema and anterior chamber exudation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) count and classification, as well as for NO(2)(-)/NO(3)(-) and cytokine assays, including interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha). Statistics were taken by SPSS software. RESULTS: (1) The anterior chamber exudation was the most serious and monocyte/macrophage in aqueous humor were the highest in ECCE + IOL group in postoperative 7 - 14 days. (2) The levels of IL-2, TNF-alpha and the content of NO(2)(-)/NO(3)(-) in aqueous humor of ECCE + IOL group were higher than that in ECCE group and control group in the postoperative 1 - 14 days respectively, and they increased to their peak values at the postoperative 3 - 7 days and decreased gradually after postoperative two weeks. (3) The change regularity of IL-2, TNF-alpha, NO(2)(-)/NO(3)(-) and inflammatory reaction in each group were basically similar, i.e. the more serious the reaction, the higher the levels of the contents. CONCLUSION: The intraocular inflammation after intraocular lens implantation is closely related to the changes of cytokine levels and NO content in aqueous humor.

Animals↗

The flow of aqueous humor through micro-porous filters.

Flow resistance was measured as bovine and primate aqueous humor was passed through Nuclepore polycarbonate filters having flow dimensions similar to those found within the juxtacanalicular meshwork of the aqueous outflow network. The results indicate that aqueous humor has a greater flow resistance than isotonic saline; this greater resistance is attributable to proteins or glycoproteins in aqueous humor that obstruct the filters. If the same phenomenon is operative in the aqueous outflow network, it would help to explain discrepancies between calculated and measured aqueous outflow resistance.

Animals↗

Aqueous humor and serum immunoblotting for immunoglobulin types G, A, M, and E in cases of human ocular toxoplasmosis.

The purpose of this study was to compare the local and systemic Toxoplasma-specific humoral immune responses in individuals with ocular toxoplasmosis (OT). To this end, paired aqueous humor and serum samples from 46 individuals with active OT and from 30 individuals without inflammatory eye disease (controls) were analyzed by immunoblotting for anti-Toxoplasma immunoglobulin G (IgG), IgA, IgM, and IgE directed against 20- to 120-kDa antigens. The presence in the aqueous humor of a unique band, or of at least three bands that were at least three times more intense in aqueous humor than in serum, was taken as evidence of local antibody production. IgG bands were detected in 98% of the aqueous humor samples, while IgA bands were detected in 76%, IgM bands were detected in 8%, and IgE bands were not detected in any. Evidence of local production of specific antibodies was found in 32 cases (70%) (IgG in 23 [50%]; IgA in 16 [35%]). In 10 instances (22%), routine laboratory tests were not indicative of OT. In 14 cases (30%), no local antibody production was detected by immunoblotting; 3 of these cases yielded evidence of local antibody production according to the Goldmann-Witmer coefficient. Local antibody production was revealed for 7 of the 30 controls (23%). Hence, the sensitivity of immunoblotting for IgG and IgA is 70%, and the specificity is 77%. We conclude that immunoblotting for local specific IgG and IgA supports the clinical diagnosis of OT in 70% of cases. In 22% of these, the diagnosis is not confirmed by other laboratory tests. Hence, immunoblotting increases the sensitivity of routine laboratory tests and should be considered for samples that register negative by such tests.

Animals↗

[Change in the pH of aqueous humor after administration of anti-glaucoma agents in rabbits in vivo].

In this contribution the activity of antiglaucomatics--2% pilocarpine (pH 5.98), 0.5% timoptol (pH 6.58) and 0.005% xalatan (pH 6.83) is emphasized from the view of pH values and changes of the pH values of the rabbit aqueous humor in vitro after their application. The rabbit's aqueous humor pH is 7.59--similar to the human healthy eye (7.5-7.6). We observed that these antiglaucomatics decrease the aqueous humor pH to the acid levels in the following order: 2% pilocarpine > 0.5% timoptol > 0.005% xalatan. The most significant decrease was measured immediately after the application. From the view of speed of response and effectivity in time achieving the pH of the normal aqueous humor we constate the following order: 0.005% xalatan > 0.5% timoptol > 2% pilocarpine. The changes in pH of the aqueous humor after application of these antiglaucomatics are directly connect with their physiologic function in the eye. Moreover, they also point out the importance of the pH dependence of the antiglaucomatics applicated into the conjunctival sac in the treatment of the glaucoma disease.

Animals↗

Voriconazole concentration in human aqueous humor and plasma during topical or combined topical and systemic administration for fungal keratitis.

Voriconazole (VRC) is an antifungal drug that effectively treats keratitis caused by yeasts and molds when administered orally. We retrospectively evaluated clinical outcomes and plasma and aqueous humor drug concentrations in five patients with fungal keratitis and one patient with posttraumatic endophthalmitis who were treated with VRC. VRC was administered either topically (1% eye drops every hour) or orally (400 mg twice a day). Plasma and aqueous humor samples from affected eyes were taken 12 h after oral administration or 1 h after eye drop application. The drug concentration was measured by liquid chromatography with UV or mass spectrometric detection. All six patients responded well to VRC treatment. The VRC concentration ranged from 2.93 to 3.40 mg/liter in the aqueous humor and from 3.20 to 4.20 mg/liter in the plasma after combined oral and topical treatment. Topical administration alone resulted in highly variable trough VRC concentrations of 0.61 to 3.30 mg/liter in the aqueous humor. VRC concentrations were above the MIC for Candida albicans Aspergillus fumigatus and clinical improvement was seen in all four patients with C. albicans and A. fumigatus keratitis. Combined orally and topically administered VRC resulted in aqueous humor drug concentrations of > or =2.93 mg/liter, which is above the VRC MIC for most fungi. Topical VRC treatment resulted in an aqueous humor drug concentration >0.61 mg/liter, which is above the MIC for most Candida species. The results from this small series of patients suggest that both topical and combined systemic and topical VRC therapy can be effective in treating fungal keratitis. Furthermore, the data provide preliminary support for initiation of VRC treatment with a combined topical and systemic administration until the causative fungus and its MIC are identified.

Administration, Oral↗

Insulin concentrations in aqueous humor after paracentesis and feeding of rabbits.

Although the lens has been shown to have the capacity to respond to insulin in vitro, little is known concerning the biochemical relationships of insulin to the lens in vivo. Therefore we have measured insulin in the aqueous humor of rabbits by a sensitive radioimmunoassay after paracentesis and feeding. The insulin concentration in aqueous humor was 3% of that in plasma. One hour after paracentesis the aqueous humor insulin concentration was increased sixfold, apparently due to breakdown of the blood-aqueous barrier, but 1 week after paracentesis it had returned to its original level, apparently because of restoration of the blood-aqueous barrier within that time. After feeding, the aqueous humor insulin concentration was increased by 30% compared to a 175% increase in plasma. Factors influencing the aqueous humor insulin concentration and the possibility of insulin influence on lens metabolism are discussed.

Animals↗

Determination of tamsulosin in human aqueous humor and serum by liquid chromatography-electrospray ionization tandem mass spectrometry.

A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.

Adrenergic alpha-Antagonists↗

Establishment of a leukemic lymphocyte culture from human aqueous humor.

We have recently developed new techniques for culturing chronic lymphocytic leukemia (CLL) lymphocytes from human aqueous humor. We used leukemic lymphocytes collected from the aqueous humor of a patient with CLL and leukemic glaucoma. We grew these leukemic cells in combination with a feeder cell layer and other technical refinements. Microscopy and immunoassay indicate success in obtaining a homogenous population of B-type CLL cells through the 12th passage of the culture. No significant effect on cell growth was found with either of two mitogens (PWM and PHA), or between culture with and without autologous serum. Our new techniques for culturing leukemic cells derived from the aqueous humor provide a reliable resource for the study of chronic lymphocytic leukemia lymphocytes and leukemic glaucoma.

Animals↗

Endothelin 1 levels in the aqueous humor of dogs with glaucoma.

PURPOSE: Endothelin 1 is a small peptide that is involved in regulation of intraocular pressure and modulation of ocular circulation. To investigate the role of endothelin 1 in canine glaucoma, the authors measured aqueous humor levels of endothelin 1 in healthy dogs and in dogs with hypertensive glaucoma. METHODS: Aqueous humor samples were obtained with general anesthesia from the eyes of healthy dogs (n = 5) and dogs with hypertensive glaucoma (n = 10). Measurements were made by enzyme immunoassay for endothelin 1. RESULTS: The endothelin 1 aqueous humor range was 1.12 - 3.63 pg/mL for healthy dogs and 1.97 - 14.97 pg/mL for glaucomatous dogs. The healthy and glaucomatous canine endothelin 1 aqueous levels (mean +/- SD) were 2.33 +/- 0.90 and 8.11 +/- 5.03 pg/mL, respectively. A two-way analysis of variance indicated that this difference was significant (P = 0.0084). The effect of age on endothelin 1 levels was not significant (P = 0.6283). The large variability found within the glaucomatous group could be explained by the degree of damage of the retina (P = 0.0006). There was no significant correlation between intraocular pressure and endothelin 1 aqueous humor levels within the glaucomatous group (P = 0.29). CONCLUSIONS: The aqueous humor of dogs with hypertensive glaucoma contains significantly higher levels of endothelin 1 than that of healthy dogs.

Animals↗

Pharmacological study on the effects of some ocular hypotensive drugs on aqueous humor formation in the arterially perfused enucleated rabbit eye.

The effects of pilocarpine, isoproterenol, epinephrine, timolol, acetazolamide, ouabain, and dinitrophenol on aqueous humor production were investigated in enucleated rabbit eye. The metabolic inhibitors ouabain and dinitrophenol at a concentration of 10(-5) M produced a 50% inhibition in aqueous humor formation. Acetazolamide at the same concentration was without effect, but at 10(-4) M concentration lowered aqueous production by 40%. The autonomic drugs epinephrine and isoproteranol at a concentration of 10 micrograms/ml medium reduced aqueous humor formation by 64 and 52% respectively. Timolol, even at a concentration of 0.05 microgram/ml, suppressed aqueous humor formation by approximately 30%. Pilocarpine on the other hand was ineffective. Except for pilocarpine, these responses are similar to those reported to occur in the cat both in vivo and in vitro.

Acetazolamide↗

Bremazocine increases C-type natriuretic peptide levels in aqueous humor and enhances outflow facility.

A relatively selective agonist of kappa opioid receptors (KOR), bremazocine (BRE), lowers intraocular pressure in rabbits, in part, by increasing natriuretic peptide levels in aqueous humor and by enhancing total outflow facility (TOF). Natriuretic peptide (NP) levels [atrial NP (ANP), brain NP (BNP), and C-type NP (CNP)] were measured in aqueous humor of rabbits either by radioimmunoassay or enzyme immunoassay. TOF was determined in rabbits by two-level constant pressure perfusion of the anterior chamber. Experimental regimens included topical treatment with BRE in the presence or absence of KOR antagonist (norbinaltorphimine), protein kinase C inhibitor (chelerythrine), and natriuretic peptide receptor antagonist (isatin). The rank order of basal NP levels in aqueous humor of rabbits was BNP CNP > ANP. Topical administration of BRE (1-100 microg) caused dose-related elevations of CNP levels in aqueous humor that were inhibited by topical pretreatment with either norbinaltorphimine (100 microg, bilaterally) or chelerythrine (10 microg, bilaterally). Topically administered BRE (100 microg) also elevated levels of ANP and BNP in aqueous humor and evoked an 80% increase in TOF. The increase in TOF was antagonized by topical pretreatment with either norbinaltorphimine (100 microg, bilaterally) or isatin (100 microg, bilaterally). Bremazocine induced an increase in NP (ANP, BNP, and CNP) levels and TOF in rabbits by activating KOR. The increase in CNP levels elicited by BRE was inhibited by norbinaltorphimine and chelerythrine; therefore, this event is most likely mediated by a KOR-linked activation of protein kinase C. These data provide evidence that the increase in TOF elicited by BRE was mediated by a KOR-activated paracrine effect of NPs on tissues within ocular outflow tract(s).

Analgesics↗

Pigment epithelium-derived factor in cataractous aqueous humor and lens epithelial cells.

PURPOSE: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. METHODS: The PEDF concentration in the aqueous humor was measured by enzyme-linked immunosorbent assay in senile (130 cases) and congenital (18 cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System III was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10 cases) and congenital (10 cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium-derived factor (PEDF). RESULTS: The mean aqueous level of PEDF was (178.9 +/- 87.5) ng/ml, and there was negative linear correlation of PEDF level and age (r = 0.811, P < 0.001) . In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r = 0.447, P < 0.01), and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01). PEDF immunostaining was detected in LEC of all capsular specimens. CONCLUSION: The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.

Aged↗

Ciliary ganglion stimulation. I. Effects on aqueous humor inflow and outflow.

Stimulation of the ciliary ganglion in an enucleated, arterially perfused cat eye preparation produced a sustained increase in aqueous humor formation and an increase in the rate of aqueous humor outflow. The increased aqueous humor formation induced by ciliary ganglion stimulation has been found to be pressure-dependent and therefore suggests that ultrafiltration may be the underlying mechanism of action. No change in capillary permeability of the ciliary body could be demonstrated.

Acetylcholine↗