A Method of Determining Concentration of Albumin in the Living Eye

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1 Investigative Ophthalmology & Visual Science, Vol. 29, No. 1, January 1988 Copyright Association for Research in Vision and Ophthalmology A Method of Determining Concentration of Albumin in the Living Eye David C. Herman, Jay W. McLaren, and Richard F. Brubaker A noninvasive method of measuring the concentration of plasma protein in the anterior chamber is described. Fluorescein is applied to the cornea. After the aqueous humor becomes fluorescent, the polarization of fluorescence is measured. The concentration of plasma protein, mainly albumin, can be calculated from the polarization. Invest Ophthalmol Vis Sci 29:133137,1988 Several invasive methods are available for measuring the concentration of protein in aqueous humor. Observation of Rayleigh scattering is a noninvasive semiquantitative method which is used clinically. DysterAas and Krakau 1 and Krakau and Ohman 2 have described an apparatus which can quantify the backscatter from proteins in the anterior chamber. Polarization of fluorescence is a phenomenon which depends on the interaction of a small fluorescent molecule and a nonfluorescent colloid to which it binds; this property of fluorescence has been used to study the fluoresceinalbumin system. 3 The properties of the interaction between fluorescein and albumin permit the former to be employed as a probe to measure the concentration of the latter. This paper describes the technique of measuring polarization of fluorescence offluoresceinin the anterior chamber of the eye. We report preliminary results in rabbit and human eyes. Materials and Methods Fluorescein is wellsuited as a probe to measure albumin. Its rotational relaxation time in an aqueous solution at 37 C is approximately 0.4 nsec. The excited state lifetime offluoresceinis approximately 2.4 nsec (Fig. 1). For this reason, most of the light emitted during steadystatefluorescenceis unpolarized. However, when fluorescein is bound to albumin, its rotational relaxation time increases by a factor of 100. Because its motion is restricted by binding, the steadystate emission of bound fluorescein is polar From the Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota. Supported in part by NIH Grant EY00634, Research to Prevent Blindness, Inc., New York, New York and the Mayo Foundation, Rochester, Minnesota. Submitted for publication: August 26,1986; accepted August 19, Reprint requests: Richard F. Brubaker, MD, Mayo Clinic, Department of Ophthalmology, Rochester, MN ized. This polarization can be enhanced by excitation of the sample with polarized light. Measurement of polarization of fluorescence requires special equipment. For best results, the excitation beam must be linearly polarized. The emission detector must be fitted with a polarizing prism or a polarization filter which can be oriented parallel and perpendicular to the polarization of the excitation beam. Measurements through both polarizations must be made either simultaneously or in rapid succession. The best arrangement in vitro is a "Tconfiguration" fluorometer with parallel and perpendicular components measured simultaneously. Polarization is defined by the equation: p = In ~ (1) In + where \ is the intensity of emitted fluorescence polarized parallel to the polarization of the excitation light, and I ± is the intensity of emitted fluorescence polarized perpendicular to the polarization of the excitation light. To make measurements in vivo, we modified a scanning ocular fluorophotometer, previously described by McLaren and Brubaker. 4 This instrument employs an argon laser, which is highly polarized, as the excitation beam. The plane of polarization (plane of vibration of the electric field or evector) of the excitation beam was oriented horizontally and the polarization sharpened with a GlanTaylor prism, the last optical component before the beam reached the eye (Fig. 2). The emission detector contains a second Glan Taylor prism, placed between the objective lens and the field lens, where the light rays are parallel. The orientation of this prism could be snapped from horizontal (parallel to excitation polarization) to vertical (perpendicular to excitation polarization) by means of a computercontrolled solenoid. The scanning ocular fluorophotometer was programmed to make a 133

134 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988 Vol. 29 POLARIZATION OF FLUORESCENCE hi/, ex 2.4 nsec em 2.4 nsec Fig. 1. Principle of technique.

2 134 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988 Vol. 29 POLARIZATION OF FLUORESCENCE hi/, ex 2.4 nsec em 2.4 nsec Fig. 1. Principle of technique. If rotational motion of the fluorophore occurs before thefluorescencephoton is emitted, the electric field of the emitted photon (hu em ) will have a different orientation relative to that of the excitation photon (hv ex ). If rotational motion is restricted by binding to a large molecule such as albumin, the emitted photon will have approximately the same orientation as the absorbed photon. series of 12 scans of 100 msec duration along a single axis through the cornea and the anterior chamber. The instrument was programmed to make three scans, pause to change the orientation of the polarizing prism, and make three more scans. This sequence was repeated twice in rapid succession. All 12 scans were completed in less than 2 seconds and all were made along the central axis of the eye. After capturing the 12 scans, the intensities Iy and I x for the cornea and the anterior chamber were obtained with the Fig. 2. Modification of fluorophotometer. The excitation beam was linearly polarized, with the evector oriented horizontally, and entered the eye 55 degrees below the optic axis. Fluorescent intensity was measured through a rotatable polarizing prism. Half of the axial scans were made with the evector parallel and half with the evector perpendicular to the evector of the excitation beam. aid of interactive video graphics programs. Equation (1) was used to determine polarization of fluorescence from the cornea and anterior chamber. To establish the relationship between concentration of albumin and polarization as measured above, a series of known mixtures offluoresceinand human serum albumin was prepared. Polarization of fluorescence was measured in three types of chambers. First, disposable spectrophotometer cuvettes were used to evaluate range and linearity of the polarization measurements. Second, a chamber was constructed from a pair of contact lenses (7.8 mm radius of curvature, 0.5 mm thick, 9 mm diameter) to simulate the dimensions of the human stroma and anterior chamber. This chamber was constructed to provide a 0.5 mm space between the lenses and a 3.0 mm deep chamber behind the lens. Typically, the 0.5 mm space was filled with a solution of fluorescein ten times more concentrated than the deeper section. Polarization was measured in a test solution in the deep chamber through the higher concentration in the 0.5 mm thick chamber, similar to measuring solutions in the anterior chamber through the cornea. Finally, the anterior chamber of an anesthetized albino rabbit was perfused with known solutions of albumin and fluorescein to determine the effect of the cornea on the measurement of polarization. The sensitivity of the technique in measuring pathologic changes of protein concentration in the anterior chamber was tested by measuring polarization in the anterior chamber of a pigmented rabbit after an iris lesion. The iris of one eye of a pigmented rabbit was photocoagulated with an argon laser photocoagulator (20 bursts at 300 mw, 200 nm diameter spot). This treatment is known to produce an immediate irritative response with an outpouring of protein into the aqueous humor. 56 Topical anesthesia (proparacaine, 0.5%) was applied to both eyes prior to treatment. Five hours prior to the laser treatment, 2% fluorescein had been applied topically to both eyes. Polarization offluorescencewas measured at 30 min intervals after the treatment, for 8 hr. This experiment conformed to the ARVO Resolution on the Use of Animals in Research. To determine normal values of polarization in the cornea and aqueous humor, measurements were made on 70 normal human subjects. Subjects ranged in age from 5 to 79 years and were undergoing fluorophotometry as part of another study. Three to four drops of a 2%fluoresceinsolution were applied topically to the cornea 5 to 9 hr prior to measurement of polarization. Polarization of fluorescence was also measured in eight patients who had clinical flare on slit lamp examination. These patients either had had recent cata

No. 1 INTRAOCULAR MEASUREMENT OF ALBUMIN / Herman er al. 135 ract extraction or cyclocryotherapy or had active uveitis. The flare was graded subjectively from I to V.

3 No. 1 INTRAOCULAR MEASUREMENT OF ALBUMIN / Herman er al. 135 ract extraction or cyclocryotherapy or had active uveitis. The flare was graded subjectively from I to V. Two percent fluorescein was applied topically, and polarization was measured 3 to 5 hr later. (Informed consent was obtained from each subject after the nature of the study had been explained and an eye examination had been performed.) Results Polarization of fluorescence of the test solutions increased as concentration of albumin in the solution increased (Fig. 3). The relation was similar when the measurement was made in a cuvette, the artificial anterior chamber, or the perfused rabbit eye, if the concentration of albumin was greater than 0.10 g/dl. In the rabbit eye polarization never dropped below In the artificial anterior chamber and cuvette, polarizations were as low as when albumin concentration was 0.05 g/dl, the normal concentration in aqueous humor. In the eye, a broad range of fluorescein concentrations may be encountered. As concentrations decrease, the uncertainty of measurement of fluorescence increases. To examine the sensitivity of polarization to concentration of fluorescein, polarization was measured in albumin solutions of 1.0 g/dl and fluorescein concentrations which ranged from 10 ng/ml to 200 ng/ml. Polarization did not change when the fluorescein concentration changed between 30 ng/ml and 200 ng/ml. At concentrations of fluorescein below 30 ng/ml polarization increased. Figure 4 shows polarization of cameral fluorescence in the rabbit during the 8 hr following photocoagulation. In the control eye, polarization increased slightly over the course of the experiment. Polarization in the treated eye steadily increased during the first one and a half hours. Polarization then gradually decreased. This experiment demonstrates the sensitivity of this method in measuring the time course of breakdown and repair of the bloodaqueous barrier. In the 70 normal human subjects, polarization of fluorescence in the fluorescein stained cornea had a mean of 0.18 ± (±SD) and ranged from to Polarization in the anterior chamber had a mean of ± (±SD) and ranged from 0.0 to These values correspond to equivalent albumin concentrations of 1.0 g/dl in the cornea and less than 50 mg/dl in the anterior chamber. Polarizations in the anterior chamber and corneas of eight patients are given in Table 1. In general, polarization was greatest in the anterior chamber of subjects with the greatest degree of flare. The only exception to this rule was one patient evaluated on the second postoperative day after cataract surgery. c o olarizati Q_ ~ D A A O A Q 0 m I I I I I Albumin concentration, gm/dl Fig. 3. Instrument calibration. The three methods of calibration, laboratory cuvette (O), double contact lens chamber (A), and perfused rabbit anterior chamber (D), (see text) were plotted. The relationship of log polarization to log albumin concentration was nearly linear in concentrations above 10 mg/dl for all methods. Although the surgeon stated in his note that the patient had +2 flare, polarization was low. Discussion Clinical studies of the bloodocular barrier are hampered by lack of a method of quantifying protein in the anterior chamber, except for the clinical grading of flare. The barrier function can be tested with systemically administered dyes such as fluorescein. 78 The advantage of measuring polarization of fluorescence is that the fluorophore can be administered B Time, minutes D Fig. 4. Polarization in anterior chamber of pigmented rabbit after 20 argon laser burns to iris. Polarization in the treated eye (solid line) peaked at 90 min and then fell to normal in the 8 hr period of measurement. Dashed line represents the fellow untreated eye.

4 136 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988 Vol. 29 Table 1. Polarization in subjects with clinical flare Flare grade Etiology Iridocyclitis Second day postcataract surgery Post cyclocryotherapy Glaucoma and chronic inflammation Anterior chamber polarization topically and albumin itself can serve as the barrier probe. With proper equipment the measurement can be made quickly and easily. Though the technique may not be as sensitive as observing flare, it is objective and permits more consistent quantification. Polarization of fluorescence is a function of the ratio of bound to unbound fluorescein, which is constant over a broad range of fluorescein concentrations, provided that the concentration of protein is much greater than the concentration fluorescein. This condition is met for the highest practical concentrations of fluorescein even at the lowest concentration of albumin that we measured. When the concentration of fluorescein is low, however, the measurement of polarization is limited by the uncertainty of measuring fluorescence in the eye. In practice, the lower limit offluoresceinconcentration can be determined by measuring polarization of solutions of varying concentrations of fluorescein mixed with albumin. In our experiments polarization was constant from 200 ng/ml down to 30 ng/ml fluorescein, but increased slightly at concentrations below this level. The most reliable measurements were made with concentrations offluoresceingreater than 30 ng/ml. In the cornea fluorescein encounters collagen and structural proteins as well as albumin and other soluble proteins. In such a complex system, the relation between protein concentration and polarization of fluorescence is probably not so simple. With our equipment the measured polarization in the central corneal stroma was This polarization is equivalent to a concentration of albumin of 1.0 g/dl in a simple system. Maurice and Watson 9 have found in the rabbit that the steadystate concentration of albumin in the central stroma is onetenth the concentration in the plasma, about 0.4 g/dl. Species differences notwithstanding, their results suggest that fluorescein binds to substances other than albumin in stroma, uncoupling the relation between polarization and albumin concentration. In the anterior chamber, polarization was near or below the lower limit of accuracy of this technique. The concentration of albumin corresponding to this polarization is less than 0.1 g/dl, in agreement with the published values of albumin concentration in the normal anterior chamber of about 60 mg/dl. 10 Albumin is not the only plasma protein to which fluorescein binds, 11 but it is quantitatively the most important. 3 In the normal eye, the concentration of protein in the aqueous humor is usually less than 50 mg/dl, and the predominant protein is albumin. The normal anterior chamber has a higher albumin to globulin (A/G) ratio than plasma. When the bloodocular barrier breaks down, the A/G ratio more closely resembles that of plasma, but albumin is still the predominant protein in the anterior chamber. High performance gel filtration chromatography of human aqueous humor has shown albumin to be present in quantities nearly 60 times that of immunoglobulins in the normal subject. It is 25 times more plentiful than immunoglobulins in rubeosis iridis where the anterior chamber protein concentration is greatly elevated. 10 Even though polarization of fluorescence does not specifically measure albumin, the conversion of polarization to equivalent albumin concentration is a potentially useful clinical technique. Because of species differences in albumin, the method must be calibrated specifically for each species. The optical properties of the cornea can affect the measurement of polarization inside the eye. The human cornea is birefringent and converts linearly polarized light to elliptically polarized light Bour and Cardozo 13 have shown that in the human, the direction of the extraordinary refractive index is approximately radial from the center of the cornea. In the vertical plane through the center of the eye (where our excitation beam enters the cornea) the plane of vibration of the linearly polarized excitation beam is perpendicular to the extraordinary refractive axis of the cornea. One would therefore expect little depolarization of the excitation beam in the normal eye. Indeed, polarization measured through the rabbit cornea, which is also birefringent, 14 was nearly identical to polarization measured in a cuvette when the concentration of albumin was greater than 0.1 g/dl. At concentrations of 0.1 g/dl and lower, polarization was higher when measured through the cornea of the perfused rabbit eye. If linear polarization of the excitation beam was reduced by the cornea, then polarization of fluorescence in the anterior chamber should have been less than in the cuvette. The reasons for the

5 No. 1 INTRAOCULAR MEASUREMENT OF ALBUMIN / Herman er ol. 137 measured difference at low concentrations of albumin are unclear. Polarization of fluorescence is a simple measurement to help evaluate the bloodaqueous barrier. It can provide a method of quantifying albumin in the anterior chamber without disturbing the structural integrity of the eye. The usefulness of this system is illustrated by the experiment represented in Figure 4. The increase in polarization during the first 100 min and subsequent return toward normal is consistent with flare as measured by scattering after a similar iris lesion. 2 With repeated measurements, this technique can be used to study the kinetics of breakdown of the bloodaqueous barrier, the severity of pathologic states, or the efficacy of antiinflammatory drugs. Key words: fluorophotometry, fluorescein,fluorescencepolarization, albumin, aqueous flare References 1. DysterAas HK and Krakau CET: A photoelectric instrument for measuring the aqueous flare in the intact eye. Ophthalmologica 146:48, Bengston E, Krakau CET, and Ohman R: The inhibiting effect of indomethacin on the disruption of the blood aqueous barrier in the rabbit eye: Measurement of the aqueousflare.invest Ophthalmol 14:306, Anjou CIN and Krakau CET: A photographic method for measuring the aqueous flare of the eye in normal and pathologic conditions. Acta Ophthalmol 38:178, Brubaker RF, Penniston JT, Grotte DA, and Nagataki S: Measurement offluoresceinbinding in human plasma using fluorescence polarization. Arch Ophthalmol 100:625, McLaren JW and Brubaker RF: A twodimensional scanning ocular fluorophotometer. Invest Ophthalmol Vis Sci 26:144, Neufeld AH, Jampol LM, and Sears ML: Aspirin prevents the disruption of the bloodaqueous barrier in the rabbit eye. Nature 238:158, Nagataki S: Aqueous humor dynamics of human eyes as studied usingfluorescein.jpn J Ophthalmol 19:235, Araie M, Sawa M, Nagataki S, and Mishima S: Aqueous humor dynamics in man as studied by oralfluorescein.jpn J Ophthalmol 24:346, Maurice DM and Watson PG: The distribution and movement of serum albumin in the cornea. Exp Eye Res 4:355, Saari KM, Aine E, and Parviainen MT: Determination of protein content in ocular fluids by highperformance gel nitration chromatography. In Advances in Immunology and Immunopathology of the Eye, O'Connor GR and Chandler JW, editors. New York, Masson Publishing USA, Inc., 1982, pp Li W and Rockey JH: Fluorescein binding to normal human serum proteins demonstrated by equilibrium dialysis. Arch Ophthalmol 100:484 : Stanworth A and Naylor EJ: Polarized light studies of the cornea. J Exp Biol 30:160, Bour LJ and Cardozo NJL: On the birefringence of the living human eye. Vision Res 21:1413, Maurice DM: The structure and transparency of the cornea. J Physiol 136:263, 1957.

A Comparison of the Laser Flare Cell Meter and Fluorophotometry in Assessment of the Blood-Aqueous Barrier

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