Graefe's Archive Ophthalmology

Graefe's Arch Clin Exp Ophthalmol (1985) 222:202-205

for Clinical and Experimental

© Springer-Verlag 1985

Factors affecting vitreous fluorescence M.A. Mosier Department of Ophthalmology, University of California, Irvine, California, USA

Abstract. A variety of mechanical (intensity of the light source, the width and the angle of the exciting source) and physiological factors (retinal pigmentation, vitreous fluorescence) and many systemic factors affect the results of vitreous fluorophotometry. These factors are discussed with respect to identifying those that are inconsequential and those that are significant. Methods to compensate for the important factors are described.

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Introduction

The technique of vitreous fluorophotometry has already provided important information about the early breakdown of the blood-retinal barrier in diabetes and its restoration with improved metabolic control, and promises to reveal much more about ocular physiology and disease. Certain aspects of the method, however, make interpretation of data difficult. I would like to raise some of these aspects for consideration. David Schoch quotes H.L. Menken who said, " T o every problem there is an answer that is simple, direct and wrong." Our goal is to examine those mechanical and physiological features of the technique that may lead us to erroneous conclusions. We would like to dismiss those factors found to be inconsequential and to correct for those that are significant.

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Fig. 1. Schematic diagram representing an idealized curve of fluorescein concentration overlying a saggital section of the eye: R, retina; V1, posterior vitreous; V2, midvitreous; V3 anterior vitreous; L, crystalline lens; AC, anterior chamber

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Mechanical factors

Until recently, ocular fluorophotometrists were obliged to work with instruments constructed from components from different manufacturers. We found that a number of factors made a difference in the signal (Fig. 1). Now most of these mechanical problems have been eliminated in the new Fluorotron from the Coherent company in Palo Alto, California. The intensity of the light source is very important. A halogen lamp gives improved results. T h e width of the exciting beam of light should be as narrow as possible; we found ] 50 gm satisfactory. The angle between the exciting beam of light and the detector probe determines the depth of the "slice" of ocular tissue under examination: the greater the angle, the shorter the anterior-posterior dimension of the "focal triangle" and the lower the signal

Fig. 2. Diagram showing the effects of different angle" beam of exciting light and detector probe

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(Fig. 2). Photometer sensitivity varies and eventually decays, greatly decreasing the readings and requiring repeated standardization. Even the speed with which the scan through the eye is made affects reproducibility. Also, there is a little difference between in vivo and in vitro measurements. There is gradual increase in transmission of the ocular media for wavelengths between 400 and 550 nm, as reported by Boettner and Wolter (1962).

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Fig. 4. Curve demonstrating decreasing fluorescence of fluorescein with increasing concentrations of protein (BSA = bovine serum albumin), or °' quenching" phenomenon

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Physiological factors are the more challenging and include those within the eye and those having to do with blood and other tissues. Ocular tissues are permeable to fluorescein to different degrees and these relationships change with disease. There is already early evidence that patterns of change in permeability relationships characterize certain diseases. Reliable methods of differentiating among the contributions to vitreous fluorescence made by the retinal vasculature, retinal pigment epithelium, ciliary body, and iris are not yet available (Fig. 3). Decrease of retinal pigmentation increases internal light reflection and scatter. Changes in the crystalline lens affect both transmission and detection of light of certain wavelengths. Boettner and Wolter (1962) found that the young lens maximally transmits at 450 mn, while the older lens maximally transmits at 540 mn. The older lens shows a larger variation in the amount of transmitted radiation and also increases light scattering. For those who work with rabbits and monkeys, it is of interest that the transmittance of the human eye does not vary appreciably from these laboratory animals. The level of fluorescence also affects the signal. Nagataki (1975) found a 12% over-estimation of fluorescence when aqueous fluorescence was 2.5 x 10 -7 g/ml. Vitreous fluorescence may be reduced by the active transport of fluorescein from the vitreous by the retina, as described by Cunha-vaz and Maurice (1969). Systemic factors include everything that happens to fluorescein once it enters the bloodstream. Most of it is quickly bound to albumin by relatively weak nonspecific mecha-

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Fig. 5. Decay curve of level of fluorescein in blood of a healthy female subject following intravenous injection of dye. Venipuncture was performed at each time interval to avoid artifact resulting from an indwelling catheter nisms, including hydrophobic and electrostatic forces. Binding results in partial polarization of emitted light, providing a technique by which the amount of bound fluorescein can be measured. Also, binding to albumin causes a red shift in the fluorescence, as shown by Anderson et al. (1971) and by Laurence (1952). The level of plasma albumin may change in certain disease states. Decreased blood fluorescence on the basis of reduced serum albumin may need to be taken into account in the normalization of vitreous fluorescence readings. In this connection, it is worth noting that fluorescence of a low concentration of fluorescein (1 x 10-v g/ml) drops with increasing amounts of protein (Fig. 4). At concentrations of bovine serum albumin (BSA) between 0 and 4 mg%, there is a sharp decrease in levels of fluorescence. The normal concentration of human serum albumin is 3-5.5 rag%. This quenching phenomenon can be expected to occur in the vitreous and aqueous humors, depending on the amount of protein present, a major variable in inflammatory states and diabetes. Laurence (1952) found that the fluorescence of bound fluorescein is reduced from that of the free dye by a factor of 0.26. A study done by Nagataki (1975), using a level of serum protein of 250 mg/100 ml, found 10%

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Fig. 6. Demonstration of mildly autofluorescent nature of protein (BSA, bovine serum albumin) in saline solution

• quenching. It should be pointed out that this is much higher than the physiologic level of serum protein. In diabetes, the mean intravascular mass of albumin was found by Brochner-Mortensen et al. (1979) to be 1.94 g/kg body weight in healthy girls 8-14 years old, compared to 1.64 g/kg in girls who had been diabetic for 7-14 years. The rate of disappearance of fluorescein from blood may change in certain disease states. Decreased renal function and albuminuria may alter the clearance rate. The phenomenon of transcapillary escape of albumin in diabetics, described by Brochner-Mortensen et al. (1979) may also alter the disappearance rate of fluorescein. A study of a healthy, young adult females, injected with 14 mg/kg body weight in fluorescein, yielded the curve of disappearance rate seen in Fig. 5. There is a very sharp drop in blood concentration during the 1st h, as fluorescein is excreted by the kidneys. By 12 h, it is down to the preinjection level. Another factor may be important in rapidly reducing the level of blood fluorescence after intravenous injection. Sawa et al. (1981) have reported that at 5 h, 96% of fluorescein has been converted by the liver to glucuronide. He found that glucuronide of fluorescein has approximately 1/24 the fluorescence of fluorescein. Glycosylation of serum albumin in diabetics is enhanced, as described by Guthrow et al. (1979), Dolhofer and Weiland (1979), and by McFarland et al. (1979). An unanswered question is whether this conjugation affects the fluorescence of circulating fluorescein. A footnote to the albumin story is that serum albumin has a fluorescence of its own, albeit a very low level. This natural fluorescence increases with the concentration of protein, as might be expected (Fig. 6). Other factors affecting fluorescence include p H of the protein solution. As plasma pH increases from 6.95 to 7.60, Delori et al. (1978) found that fluorescence increases by 15%. As pH rises above 9 in in vitro experiments, Anderssen et al. (1971) found that protein binding to fluorescein diminishes considerably. Another possible cause of changes in apparent fluores-

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cence is the binding of fluorescein to substances other than albumin or the reduction of protein binding sites in vivo. As the fluorescein concentration in plasma drops below 1 x 10-5 g/ml, Delori et al. (1978) showed that the efficacy of fluorescence varies linearly. We found that the fluorescence of fluorescein in 4 rag% bovine serum albumin varies with the concentration of fluorescein (Fig. 7). At each concentration, fluorescence has been reduced from 100% fluorescence of the standard solution in buffered saline to the noted value. Fluorescence is lowest in the middle range of 10- 6 and 10- 7 g of fluorescein/ml, rising at the low end of the scale and again at the higher end. No such paradoxical effects are seen with fluorescein in buffered saline, where fluorescence predictably increases with increasing concentrations (Fig. 8).

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References Andersson L-O, Rehnstr6m A, Eaker DL (1971) Studies on "nonspecific" binding: the nature of the binding of fluorescein to bovine serum albumin. Eur J Biochem 20:371-380 Boettner EA, Wolter JR (1962) Transmission of the ocular media. Invest Ophthalmol 1 : 776-783 Brochner-Mortensen J, Ditzel J, Mogensen CE, Rodbro P (1979) Microvascular permeability to albumin and glomerular filtration rate in diabetic and normal children. Diabetologia 16:307-311 Cunha-vaz J, Maurice D (1969) Fluorescein dynamics in the eye. Doc Ophthalmol 26 : 61-72 Delori FC, Castany MA, Webb RH (1978) Fluorescence characteristics of sodium fluorescence in plasma and whole blood. Exp Eye Res 27:417-425

Dolhofer R, Weiland OH (1979) Increased glycosylation of serum albumin in diabetes mellitus. Diabetes 29:417-422 Guthrow CE, Morris MA, Day, JF, Thorpe SR, Baynes JW (1979) Enhanced nonenzymatic glucosylation of human serum albumin in diabetes mellitus. Proc Natl Acad Sci 76:4258-4261 Laurence D JR (1952) A study of the adsorption of dyes on bovine serum albumin by the method of polarization of fluorescence. Biochem J 51:168-180 McFarland KF, Catalano EW, Day JF, Thorpe SR, Baynes JW (1979) Nonenzymatic glucosylation of serum proteins in diabetes mellitus. Diabetes 28:1011-1014 Nagataki S (1975) Aqueous humor dynamics of human eyes as studied using fluorescein. Jpn J Ophthalmol 19:235-249 Sawa M, Araie M, Nagataki S (1981) Permeability of the human corneal endothelium to fluorescein. Jpn J Ophthalmol 25: 60-68