Lipoproteins and lipids in cow and human aqueous humor.

The aqueous humor of the cow and human was examined for the presence of lipids and lipoproteins. Whole aqueous humor collected from cow eyes within 30 min after slaughter contained about 1 micrograms/ml of cholesterol and phospholipid. Upon fractionation of bovine aqueous into various density ranges following sequential ultracentrifugations , about 99% of the total cholesterol was recovered at a density of greater than 1.063. Apolipoprotein A-I, the major apolipoprotein of high-density lipoprotein (HDL), was the major protein seen upon electrophoresis of the 1.063-1.21 fraction. Particles of about 80 A mean diameter were observed by electron microscopy in the 1.063-1.21 fraction. Using rocket immunoelectrophoresis, a concentration of about 2 micrograms/ml of apolipoprotein A-I was measured in cow aqueous humor and slightly less in aqueous humor from the adult human collected post-mortem (1-36 h). In conclusion, aqueous humor of cow and man appears to contain about 4 micrograms/ml of HDL and it is likely the sole lipoprotein in this fluid. The potential importance of this lipoprotein in supplying lipids to the lens is discussed.

The composition and optical rotary dispersion of bovine aqueous humor.

The concentration of over 20 biochemicals in bovine aqueous humor was determined by conventional means, and the contribution of each to the observed optical rotation was estimated. Of those studied, only glucose and the total proteins made a significant contribution. However, other unidentified chemicals apparently also contribute to the optical activity since it was not possible to reconstruct the actual aqueous humor optical rotation based on the chemicals included. Alternative experimental approaches that may yield more complete results are discussed. With further work, optical rotation might be used as the basis of a noninvasive sensor for measuring the glucose concentration of aqueous humor in the intact eye.

Noninvasive glucose monitoring of the aqueous humor of the eye: Part II. Animal studies and the scleral lens.

We have discussed the nature of a scleral lens that will allow us to follow changes in aqueous humor glucose levels in animals by a method based on optical rotation and a technique described in an earlier paper. We have shown how this lens can be micro-miniaturized and can be used in humans as a non-invasive glucose monitor. We have described preliminary experiments designed to show the correlation between the blood glucose assay (BGA) and the aqueous humor glucose concentration as determined by chemical assay (AGA) and by optical rotation determination (ARD). The last mentioned has been obtained by paracentesis directly into a microcell used in conjunction with instrumentation capable of measuring optical rotations as low as 0.0013 degrees (4.5") corresponding to 20 mg/dl glucose with a sensitivity of 0.0001 degrees (0.36"). The variability among normal rabbits as a function of individuality and diurnal changes is described, and the correlation between AGA and ARD shown to be essentially 1.0. Such rabbits are examined when undergoing very rapid decreases in BGA (insulin treatment) or very rapid increases in BGA (bolus of glucose). The AGA and ARD are shown to lag behind the BGA, and this is discussed in terms of the rate of change of BGA with respect to time and its concomitant change in AGA/ARD as well as a simple procedure that would materially reduce this lag.

Determination of trimethylpsoralen in blood, ophthalmic fluids, and skin.

Trimethylpsoralen (TMP) levels in the blood of vitiligo patients were determined through the use of a high-performance liquid chromatographic method. TMP was extracted from blood buffered at pH 9.0 with 95:5 (V/V) hexane-isopropanol mixture; evaporated to dryness, and reconstituted in 50 microliters of ethanol. A 10-microliters aliquot was injected into a Micropack MCH-10 column (Varian HPLC model #5000). The mobile phase consisted of a mixture of water and acetonitrile with a linear gradient. The retention time of TMP was 16.5 min. The calibration curve of the external standards was linear. Overall recovery of the internal stands was 75-92%, with the lower detection limit of TMP at 2 ng/ml. Peak blood levels as low as 140 ng/ml and as high as 800 ng/ml were obtained in vitiligo patients 1-2 hr following the oral administration of 30 mg of trioxsalen tablets (Paul B. Elder Co.). Blood TMP levels peaked consistently at 2 hr when patients were fasted for 8 hr prior to drug ingestion. These results are consistent with the clinical observation that maximum response due to phototherapy is obtained 1-2 hr after oral administration of the drug. Two hours after oral drug administration, TMP levels in the epidermis, dermis, and whole skin of the guinea pig (in ng per g tissue) were: epidermis, 226 +/- 15; dermis, 25 +/- 6; and whole skin 176 +/- 12. Also detected were TMP levels 244 +/- 17 ng/ml in aqueous humor and 63 +/- 6 mg/ml in vitreous humor. These results point to the fact that the eyes of patients must be protected from overexposure to sunlight after psoralen-ultraviolet treatment.

Cataracts and hypertension in salt-sensitive rats. A possible ion transport defect.

In previous unrelated studies, we observed a 35 to 50% incidence of cataract formation in several groups of Dahl salt-sensitive hypertensive rats (DS) over a 4-year period. In the present study we evaluated longitudinal changes in blood pressure in DS in which cataracts eventually developed and those in which cataracts did not develop when all animals were maintained on a high sodium diet. Lenses were evaluated by slit-lamp microscopy to determine if cataractous lesions were similar among rats, to classify lesion types, and to define the age at which cataracts were detectable in DS. The possible participation of several cataractogenic risk factors as major influences on cataract formation also was evaluated. Finally, aqueous humor concentrations and lenticular content of sodium and potassium were determined to evaluate the possibility that a defect in ion transport at the lens epithelium and ciliary body might play a role in cataractogenesis in DS, since ion transport defects have been shown to lead to lens opacification in other models of genetic and experimental cataracts. Parallel studies were performed in Dahl salt-resistant control rats (DR). A high incidence of cataract formation was found in DS. Although systolic blood pressure was not consistently greater in adult DS with cataracts compared with values in age-matched DS without cataracts, the initial pressor response to a high salt diet was greatest in weanling DS in which cataractous lesions later developed. Slit-lamp analysis revealed that cataracts in this genetic model were cortical, with one mixed cortical, nuclear lesion. Posterior subcapsular lesions were not observed, suggesting that lesions were not steroid-induced.(ABSTRACT TRUNCATED AT 250 WORDS)

High postmortem levels of hypoxanthine in the vitreous humor of premature babies with respiratory distress syndrome.

To test whether or not premature babies at risk for retinopathy of prematurity have elevated hypoxanthine levels in the eye, the vitreous humor of 13 premature babies who died of severe respiratory distress syndrome and lung failure, was analyzed for hypoxanthine. Their hypoxanthine level was 459 +/- 171 mumol/L (mean +/- SD) compared with 54 +/- 71 mumol/L in seven newborn babies who died suddenly (P less than .001). In 53 adults who died suddenly, the hypoxanthine concentration was 136 +/- 119 mumol/L (P less than .001 when compared with babies with respiratory distress syndrome). Babies with respiratory distress syndrome underwent a significantly longer period with arterial PO2 levels less than 40 mm Hg (5.3 kPa) and they required supplementation with 100% oxygen significantly longer than control babies. The hypoxanthine concentration was correlated with the time during which the arterial PO2 was lower than 40 mm Hg (5.3 kPa) before death, and a significant positive correlation was found (R = .59, P less than .12). The study shows that high levels of hypoxanthine are found in vitreous humor of premature babies with respiratory distress syndrome. Because hypoxanthine is a potential oxygen radical generator and premature babies might have lower levels of antioxidants than full-term babies, it is suggested that the hypoxanthine accumulation in the eyes of premature babies with respiratory distress syndrome could play a pathogenetic role in the development of retinopathy of prematurity.

Rhesus monkey aqueous humor composition and a primate ocular perfusate.

The composition of rhesus monkey aqueous humor has been studied in large-volume, pooled samples. Replicate determinations of the concentrations of a number of constituents have been carried out for both aqueous humor and serum from large veins by means of automatic analyzing equipment. Since aqueous humor has been obtained by anterior chamber paracentesis, it is a mixture of anterior and posterior chamber aqueous. When compared to serum, the pooled aqueous contains an excess of chloride, bicarbonate, ascorbate, lactate, uric acid, and several neutral amino acids. Rhesus monkey aqueous humor is deficient in calcium, urea nitrogen, phosphates, glucose, protein, creatinine, iron, bilirubin, cholesterol, triglycerides, a number of serum enzymes, acidic and basic amino acids, and several neutral amino acids. Sodium, potassium, magnesium, and two neutral amino acids (cysteine and valine) are of equal concentration in aqueous humor and serum. Glutathione concentration is very low in both aqueous humor and serum. Pooled rhesus monkey aqueous humor and serum are isosmolar, with measured osmolality being about 303 mOsm. Based upon the chemical analysis, a new solution has been formulated to substitute for primate aqueous humor during anterior ocular perfusion. This new solution causes very little change in the physiologic integrity of the outflow pathways during prolonged, repeated perfusion. In this respect, its effects are very similar to those of pooled rhesus monkey aqueous humor during perfusion of rhesus monkey eyes. In contrast, perfusion of rhesus monkey eyes with glutathione-bicarbonate-Ringer's solution has been shown to cause progressive increase of the total facility. To minimize physiologic alterations during operative procedures, a solution similar to this new one could be formulated for irrigation of the inside of the human eye.

Tissue plasminogen activator in the trabecular endothelium.

By quantitative analysis of tissue plasminogen activator (TPA) in the trabecular endothelium, corneal endothelium, and iris in the eyes of monkeys and dogs, we found significant levels of TPA activity. In a [125I]fibrin-coated well assay, the levels for the dog and monkey were, respectively: trabecular endothelium, 0.2 and 0.5; corneal endothelium, 0.8 and 0.5 IU per mg protein. The iris tissue showed high TPA activity, but its protein content could not be measured with the techniques employed. Activity in the aqueous humor was not detectable. By the ELISA technique, the values (in ng TPA/mg tissue protein) for the dog and monkey were, respectively: trabecular endothelium, 0.16 and 0.44; corneal endothelium, 0.48 and 0.92. Again, iris tissue showed high TPA activity, whereas the aqueous humor showed low activity (0.86 ng/ml). The data obtained with the two methods showed a reasonable consistency, although a direct comparison was not possible because two separate standards were used. The presence of TPA in the trabecular endothelium, corneal endothelium, and iris may be important in modulating the resistance to aqueous outflow under normal conditions as well as those of hyphema.

Distributions of 35S-sulfate and 3H-glucosamine in the angular region of the hamster: light and electron microscopic autoradiography.

The distribution of 35S-sulfate and 3H-glucosamine in the angular region of the hamster was studied by light and electron microscopic autoradiography following intraperitoneal injection of these compounds to hamsters. Exposed silver grains of 35S-sulfate were concentrated in the trabecular meshwork, sclera, and cornea, and grains of 3H-glucosamine were localized in the trabecular region. The radioactivity of both isotopes was observed in the Golgi apparatuses of the endothelial cells of the angular aqueous plexus and the trabecular meshwork. The grains were noted over the entire cytoplasm, except for the nucleus, and then were incorporated into the amorphous substance and collagen fibers in the region adjacent to the angular aqueous sinus. These results suggest that endothelial cells in the angular region synthesize and secrete the sulfated glycosaminoglycans and hyaluronic acid.

Ocular penetration of 5-fluorocytosine.

Ocular penetration of 5-fluorocytosine (5FC) was studied in uninfected rabbits after subconjunctival and oral administration. With oral administration, 5FC achieved therapeutic levels in both the vitreous and aqueous humors. The use of a pharmacokinetic model permitted objective comparison of kinetic events within the eye chambers and the serum. The rates of entry and elimination in the vitreous were found to be slower than those in the aqueous, but the mean concentration over 24 hr was the same. The therapeutic levels achieved in the aqueous after subconjunctival administration were of shorter duration, and no detectable levels occurred in the vitreous. Oral administration is clearly therapeutically superior to subconjunctival administration in this model.

The continuous and quantitative observation of permeability changes of the blood-aqueous barrier in allergic inflammation of the eye.

Permeability changes of the blood-aqueous barrier were studied in the eyes of rabbits subjected to immunologic inflammation. The changes were investigated by slit-lamp microphotometers. The leakage of fluorescein-labeled rabbit serum albumin into the anterior chamber was observed in eyes inflamed by reverse passive Arthus reactions. The permeability of the blood-aqueous barrier changed in biphasic pattern in allergic inflammations; the first phase began 5 min after the antigen challenge and lasted for 1 hr, followed by the late phase at which the dye concentration reached a peak 2 hr after the challenge and then gradually decreased.

Glutathione in the aqueous humor of human and other species.

Glutathione was measured in the aqueous humon of rabbit, dog, monkey, baboon, and man. The average concentrations were 17, 6, 6, 2, and 2 mumol/L of fluid, respectively. Except in the case of the monkey, where delay in aspiration of the aqueous apparently led to anomalous values, nearly all the glutathione was in the reduced state, the range being from 85% in the rabbit to over 99% in the human. Possible sources of the glutathione in the aqueous humor are discussed and also its significance for the physiology of the cornea.

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.

Rapid detection of experimental E. coli endophthalmitis by the Limulus lysate test.

Experimental E. coli endophthalmitis was produced in rabbits. The Limulus lysate test was applied to aqueous and vitreous samples at various intervals after the intravitreal injection of E. coli organisms. Results indicated that this test is feasible using vitreous and aqueous samples. The Limulus test was positive for E. coli endotoxin within hours after infection, requiring only 1 hr to determine the presence of endotoxin after sampling. This test may have some value in the rapid diagnosis of gram-negative endophthalmitis.

Complement inhibitors in normal cornea and aqueous humor.

C1 inhibitor, beta 1H and C3b inactivator are important inhibitory proteins that regulate the complement system. These inhibitor proteins were detected by gel double diffusion in eluates from normal corneas, but not in normal aqueous humor. Functional tests of C1 inhibitor and C3b inactivator showed low-to-absent levels of these inhibitory proteins in normal aqueous humor. On the other hand, the mean activities of C1 inhibitor and C3 inactivator in corneas were nearly as high as those in sera, and there were no statistically significant differences between the values. The lower molecular weights of complement inhibitors, relative to other complement components, may account for their higher levels in normal cornea and may tip the balance in favor of inhibition of complement activation in the noninflamed cornea.

In vivo aqueous humor oxygen tension--as estimated from measurements on bare stroma.

Aqueous humor oxygen tension of the in vivo rabbit eye was estimated by a relatively atraumatic procedure. The anterior corneal surface of the eye was first scraped free of its epithelial layer. A polarographic oxygen electrode was then used to measure the oxygen tension at the bare stromal surface. Aqueous humor oxygen tension can be estimated from this measured steady-state stromal surface oxygen tension by correcting the measured oxygen tension for the tension drop across the stroma and endothelium. When animals breathed room air (155 mm Hg oxygen tension), the oxygen tension of the aqueous humor was 13 mm Hg; this rose to 150 mm Hg when the inspired oxygen tension was 713 mm Hg.

Identification of heavy-molecular-weight soluble protein in aqueous humor in human phacolytic glaucoma.

Aqueous humor was obtained by paracentesis at the time of cataract surgery from six patients with phacolytic glaucoma, diagnosed on the basis of acute unilateral open-angle glaucoma associated with an apparently leaking hypermature or mature cataract, and from six control patients with immature cataracts. Three of the latter had primary open-angle glaucoma. Quantities of heavy-molecular-weight (HMW) protein (MW greater than 150 X 10(6)) sufficient to obstruct aqueous outflow were identified in all six phacolytic aqueous humor specimens but in none of the controls. Three of the hypermature cataractous lenses from the cases of phacolytic glaucoma were also examined and were found to have 14-fold greater quantities of HMW protein in their liquefying cortex than were present in the cortex of immature cataractous lenses. These findings, correlated with experimental HMW protein perfusion studies in excised human eyes that we have already reported, strongly suggest that direct obstruction of the aqueous outflow channels by liberated HMW soluble lens protein may be a significant and previously unappreciated factor in the pathogenesis of phacolytic glaucoma.

Monocyte chemotactic activity induced by intravitreal endotoxin.

In order to clarify the factors responsible for the cellular infiltrate characteristic of anterior uveitis, the authors have induced inflammation in rabbits by the intravitreal injection of 100 ng of Escherichia coli or Salmonella endotoxin (ET). A 2% concentration of aqueous humor 18 to 24 hr after ET consistently induced monocyte migration as measured in modified Boyden chambers. Activity was significantly greater in these samples than in aqueous after saline injection or 3 hr after endotoxin injection (prior to cellular infiltrate). Using either sephadex G-75 molecular sieve chromatography or a cibacron blue column, the vast majority of migratory activity co-eluted with albumin. Serum albumin, however, at a comparable concentration did not induce migration. Activity was largely heat- and acid-stable and was maximal in the presence of a concentration gradient, indicating that it was chemotactic rather than chemokinetic. A second peak of activity eluted from the G-75 column just prior to a marker with molecular weight of 427 and was also present in eluates from normal aqueous humor. Chloroform:methanol extraction, radioimmunoassay, and high performance liquid chromatography indicated that a small portion of the chemotactic activity could be ascribed to lipid including leukotriene B4. In contrast to the prominence of complement (C5a) derived chemotactic activity resulting from intravenous ET, C5a was not a major contributor to aqueous chemotactic activity subsequent to local ET. These observations demonstrate that leukocyte migration factors in aqueous humor can be characterized and compared. This approach can be used to test the hypothesis that subsets of anterior uveal inflammation might be distinguished on the basis of associated chemotactic factors.

Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.

The permeability of the blood-retinal and blood-aqueous barriers to fluorescein (F) and the rate of aqueous flow can be estimated by measurements of F in the vitreous, aqueous, and plasma after systemic administration. F is commonly measured by fluorescence, but fluorescein glucuronide (FG), a metabolite of F, also fluoresces. To assess the influence of FG on the quantitation of F by fluorescence, we studied the pharmacokinetics of F and FG for 38 hr in the plasma of five normal subjects given 14 mg/kg of sodium fluorescein intravenously. The plasma and the plasma ultrafiltrate were measured by fluorescence and by high performance liquid chromatography. In our fluorophotometer, FG was 0.124 times as fluorescent as F. F was rapidly converted to FG, and within 10 min the concentration of unbound FG exceeded that of unbound F. The terminal half-lives of F and FG in the plasma ultrafiltrate were 23.5 and 264 min, respectively, so that FG contributed almost all of the plasma fluorescence after 4-5 hr. Because FG was less bound in the plasma than F, the ratio of the fluorescence of the plasma ultrafiltrate to that of the plasma increased with time. The greatest proportion of the total F available to penetrate into the ocular compartments occurred shortly after injection. We concluded that FG is an important contributor to the fluorescence of the plasma ultrafiltrate after intravenous injection and that accurate quantitation of physiologic parameters calculated from the plasma F requires taking this factor into account.

Conjunctival goblet cell frequency after alkali injury is not accurately reflected by aqueous tear mucin content.

Goblet cell counts have been used to evaluate the suitability of conjunctiva as a source of ocular surface epithelial cells. However, since tear mucin content can be determined without tissue excision, it seemed that the concentration of those compounds might be a useful indicator of conjunctival vitality. To test the extent to which aqueous tear composition reflects conjunctival goblet cell frequency, goblet cell frequency and aqueous tear mucin content were measured after alkali injury in rabbits. Mild alkali injury (0.1 N NaOH for 30 sec) caused a transient but substantial decrease in goblet cells (to 25% of normal at day 7) with a return to normal by six weeks. Tear mucin content was decreased to a lesser degree, from a normal value of 6.4 +/- 0.47 nmol oligosaccharide per microliter (n = 10) to a minimal value of 4.7 +/- 0.64 (n = 7) (73% of normal) at day 7, returning to normal 4 weeks after injury. Thus, the direction of the change was the same, but the magnitudes were quite different. These results suggest that conjunctival goblet cell frequency is not accurately reflected by aqueous tear mucin content, and therefore, that tear mucin content cannot be used directly as an indicator of conjunctival health.