A method for noninvasive measurement of Hb O2 saturation (SO2) in retinal blood vessels by digital imaging was developed and tested. Images of vessels were recorded at O2-sensitive and O2-insensitive wavelengths (600 and 569 nm, respectively) by using a modified fundus camera with an image splitter coupled to an 18-bit digital camera. Retinal arterial SO2 was varied experimentally by having subjects breathe mixtures of O2 and N2 while systemic arterial SO2 was monitored with a pulse oximeter. Optical densities (ODs) of vascular segments were determined using a computer algorithm to track the path of reflected light intensity along vessels. During graded hypoxia the OD ratio (ODR = OD600/OD569) bore an inverse linear relationship to systemic SO2. Compensation for the influence of choroidal pigmentation significantly reduced variation in the arterial SO2 measurements among subjects. An O2 sensitivity of 0.00504 +/- 0.00029 (SE) ODR units/%SO2 was determined. Retinal venous SO2 at normoxia was 55 +/- 3.38% (SE). Breathing 100% O2 increased venous SO2 by 19.2 +/- 2.9%. This technique, when combined with blood flow studies in human subjects, will enable the study of retinal O2 utilization under experimental and various disease conditions.

The retinal oximeter is reliable, easy to use, and sensitive to changes in SO(2) when concentration of O(2) in inhaled air is changed.

We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% +/- 3% bleaching flash was used as a criterion of photochemical activity at 28 degrees C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6 omega 3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol% docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (L alpha) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a L alpha to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.

To gain a better understanding of the biological roles of polyunsaturated phospholipids, deuterium (2H) NMR studies have been conducted of 1 -perdeuteriopalmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, an asymmetric or mixed-chain saturated-polyunsaturated phospholipid, in the liquid crystalline (La) phase. The palmitoyl (16:O) chain at the glycerol sn-1 position was labeled with 2H by perdeuteration, whereas the polyunsaturated, docosahexaenoyl (22:6w3) chain at the sn-2 position was unlabeled, Le., protiated. The 2H NMR results were compared to studies of 1,2-diperdeuteriopalmitoyl-snglycero-3-phosphocholine, in which both the sn-1 and sn-2 palmitoyl chains were perdeuterated, as well as l-palmitoyl-2perdeuteriopalmitoyl-sn-glycero-3-phosphocholine, in which only the sn-2 chain was perdeuterated. Multilamellar phospholipid dispersions containing 50 wt % H 2 0 were employed, and 2H NMR spectra were obtained using quadrupolar echo methods at a magnetic field strength of 8.5 T. The experimental 2H NMR spectra were numerically deconvoluted (de-Paked) to yield subspectra corresponding to the parallel bilayer orientation with respect to the main applied magnetic field. The increased resolution of the de-Paked subspectra enabled profiles of the segmental order parameters of the individual C-2H bonds, denoted by lScD(i)1, to be derived as a function of chain position. Significant differences in the 2H N M R spectra and derived JScD!i)l profiles of the per-*H-16:0 chains of the polyunsaturated and saturated bilayers were found. Based on simplified statistical mechanical theories, the differences can be interpreted in terms of an increase in the configurational freedom of the palmitoyl chains in the polyunsaturated bilayer, relative to bilayers of phosphatidylcholines with two identical saturated chains. The increased configurational freedom may correspond to an increase in the equilibrium area per chain in the case of the polyunsaturated bilayer. Possible further interpretations of the results in terms of the thickness of the hydrocarbon region and the presence or lack of interdigitation of the polyunsaturated and saturated acyl chains are also briefly discussed. We conclude that the configurational properties of the acyl chains of polyunsaturated bilayers are significantly different from those of saturated phospholipid bilayers.

A trend of increasing retinal oxygen saturation was found from controls to NDR group to increasing levels of diabetic retinopathy, though significance was only reached for the comparison of controls to severe-NPDR, PDR, and all-DR groups.

Hyperspectral imaging can be adapted to measure and map relative oxygen saturation in retinal structures and the ONH in nonhuman primate eyes.

No abstract

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)