Fluorescein and fluorescein glucuronide in plasma

ACTA 0 P H T H A L M 0 L O G IC A

70 (1992) 447-453

Fluorescein and fluorescein glucuronide in plasma Lars L. Knudsen’, Thomas Olsen’ and Folmer Nielsen-Kudsk’ Department of Ophthalmology’, Arhus University Hospital, and Institute of Pharmacology2,Arhus University,Arhus, Denmark

Abstract. The evaluation of the blood-ocular barrier for fluorescein requires the measurement of free and unconjugated fluorescein in plasma. This study introduces a new and simple method for the determination of free fluorescein in plasma on the basis of determined total free plasma fluorescense and the free fraction of fluorescence. An excellent good correlation between differential spectrofluorophotometry and this new method is demonstrated. After intravenous administration of sodium fluorescein, the contribution of fluorescein glucuronide to total free plasma fluorescence was evaluated on basis of the areas under the plasma concentratiodtime curves for fluorescein and fluorescein glucuronide, respectively. After 1 h 8.2% of total free fluorescence in plasma was found to originate from fluorescein glucuronide and after 24 h 18.3%originated from this metabolite. It was concluded that although plasma fluorescein glucuronide measurements are important in the exact evaluation of the blood-ocular barrier, the contribution of fluorescein glucuronide to vitreous fluorescence after intravenous fluorescein administration seems to he of minor magnitude. Key words: vitreous fluorophotometry - fluorescein - fluorescein glucuronide - plasma fluorescence - pharmacokinetics - blood retinal barrier - permeability.

Vitreous fluorophotometry (VF) has been used to evaluate the blood-ocular barrier (BOB) since its introduction by Cunha-Vaz et al. (1975).An exact estimate of the BOB ‘permeability requires a knowledge of fluorescein in the blood as well as in the eye, and many studies have called attention to the necessity of measuring free plasma fluorescein (Lund-Andersen et al. 1982; Conway 1985; Mota &

Cunha-Vaz 1985; Blair et al. 1986), which is available for transport across the barrier. It has been shown that fluorescein is rapidly metabolised in the liver to fluorescein glucuronide, also a fluorophore (Webb et al. 1962; Chaha1 et al. 1985; Blair et al. 1986).Fluorescein glucuronide traverses the BOB with difficulty but is present in the plasma in considerable amounts (Blair et al. 1986).An exact estimation of the BOB permeability therefore requires determination of free plasma fluorescein excluding the interference of the fluorescence from fluorescein glucuronide. It is possible to determine both fluorescein and fluorescein glucuronide in plasma by differential spectrofluorophotometry (McLaren & Brubaker 1986; Lund-Andersen et al. 1987),which is a rather complicated technique. The present study introduces a new and simple method to determine fkee and unconjugated fluorescein in plasma without the use of differential spectrofluorophotometry. Based on determined pharmacokinetic parameters, the significance of fluorescein and its metabolite for the interpretation of vitreous fluorophotometry is discussed.

Material and Methods 1. Subjects .

Sodium fluorescein (14 mg/kg body weight) was administered intravenously to 4 normal subjects aged 29-52 years and 1 subject with giant cell arteritis aged 62 years. Plasma samples were 447

~ X I O - ~

[Fluorescein] (mol/l)

5x

used. In method 1 the primary filter for excitation was switched between 450 and 490 nm; the secondary fdter for emission was set at 515 nm. In method 2 the primary filter was at 490 nm and the secondary filter at 515 nm. In relation to the primary and secondary filters, slit width 1 and 2 were 2 111111.

4~10.~ 3~10.~ 2x10.~

1x10-5

3. Determination of free plasma fluorescein and fluorescein glucuronide

Method 1 Based on differential spectrofluorophotometry the concentration of free plasma fluorescein was calculated as described by McLaren & Brubaker (1986) and Lund-Andersen et al. (1987):

1x10-6

F 1x10-7

\

\

(hours)

'\ \ 1x10-8

0

2

4

6

8

10

12

14

16

18

20

22

Fig. 1. Free plasma fluorescein time course after a single iv injection of fluorescein (14 mg/kg bodyweight)in a normal subject.

drawn before and 7-9 times after fluorescein administration. The protocol was approved by the local Ethics Committee according to the Declaration of Helsinki 2. and informed consent was obtained before participation. 2. Plasma ultrafiltrate and fluorescence

A plasma ultrafiltrate was obtained by 1 h centrifugation at 1500 r/min of 1 ml plasma contained in a visking tube (8/32 from Union Carbide). The ultrafiltrate volume was less than 0.1 ml. Non-leakage of plasma proteins through the tube was ensured by albumin measurements in the ultrafiltrate using a RIA technique (Christensen & 0rskov 1984). The ultrafiltrate was assayed for fluorescein and fluorescein glucuronide on a SPF 125 Aminco Spectrofluorometer set a different wavelengths of excitation and emission according to the two methods 448

=

(I(b)* fg(a) - I(a) * fg(b))/(fg(a)* f(b)- fg(b) j: f(a))

where F is the free fluorescein concentration in the ultrafiltrate sample: I(a) and I(b) are the fluorescence intensities at 450 and 490 nm, respectively; f(a) and f(b) are the molar fluorescence coefficients of fluorescein at 450 and 490 nm, respectively;fg(a) and f&b) are the molar fluorescence coefficients of fluorescein glucuronide at the same wavelengths. Free fluorescein glucuronide in plasma was determined in a similar way. Method 2 Both fluorescein and fluorescein glucuronide have been found to be bound to albumin at a constant fraction within a wide concentration range. Several investigators have observed an increase in the free fraction of plasma fluorescence after injection (Araieet al. 1980; Lund-Andersen et al. 1982; Nagatoki & Matsunaga 1985). This effect is due to the fact that fluorescein is metabolised to fluorescein glucuronide, which is less effectively bound to albumin (Blair et al. 1986). If one assumes both fluorescein and fluorescein glucuronide to be bound at a constant fraction, it is possible to calculate free unconjugated fluorescein from the change in free fraction of plasma fluorescence on the basis of the following equation, which is valid when both compounds are present:

F(T) = fpf(T) * ff(O)/ff(T) where F(T) is the free plasma fluorescein concentration at time T after injection of fluorescein, ff(0) is the free fraction of total fluorescencejust after injection of fluorescein (4-6 min), ff(T) is the free fraction of total fluorescenceat time T and fpf(T)is

2x10-5

1x10-5

1x10-6

Plasma fluorescein Wmolll) (method I)

(hours)

'\

1x10-7

\\

2

0

4

6

8

10

12

14

16

18

Fzg. 3. Linear correlation between free plasma fluorescein determined by the previously described method 1 (bicromatic) and the new method 2 (free fraction) in 5 subjects. The correlation coefficient r = 0.997 (p < 0.00001). The remession line slope is 0.928, and y-axis intercept is 0.328, which does not deviate significantly from zero. Y

20

22

24

Fig. 2. Free plasma fluorescein glucuronide time course after a single iv injection of fluorescein (14 mg/kg bodyweight) in a normal subjects.

an uncorrected concentration value of free fluorescein in plasma calculated directly from free total plasma fluorescence at time T. The expression ff(O)/ff(T) represents the ratio between fluorescence from fluorescein and free total plasma fluorescence from both fluorescein and fluorescein glucuronide. On the assumption that all the fluorescence arises either from fluorescein or fluorescein glucuronide, determination of the plasma level of free fluorescein glucuronide FG(T) can then be calculated by subtraction of F(T) from fpf(T),i.e. FG(T) = fpf(T)* (1 - ((ff(O)/ff(T)))

data was performed, from which A, B, a and p and coefficients of determination were found. The total area under the plasma fluorescein concentration curve from zero time to infiiity (AUC(F))was determined as: AUC(F) = A/a

+ (B/B).

B. Plasmafluorescein glucuronide (Fig 2) The time course of free fluorescein glucuronide in plasma after iv administration of fluorescein could be described by another biexponential curve (Blair et al. 1986) representing the function: CFG=

c * e-Kft- C * e-Ke

t

The area under the free fluorescein glucuronide concentration curve in plasma was calculated as: AUC(FG)

=

C/Kf - (C/Ke)

4. Pharmacokinetic analysis

A. Plasmafluorescein (Fig.I ) It was found that the time course of free fluorescein in plasma could be described by a biexponential decay curve (Conway 1985; Blair et al. 1986). C

=

A

h e'