Stability of fluorescein labeled dextrans in vivo and in vitro

MICROVASCULAR

Stability

RESEARCH, 11,33-39

(1976)

of Fluorescein

Labeled Dextrans In Vivo and In Vitro

U. SCHR~DER, K.-E. ARFORS AND 0. TANGEN Department of Experimental Medicine, Pharmacia AB, Uppsala, Sweden Received April 15, 1975 Fluorescein thiocarbamoyl dextrans (FITC-dextrans) were studied with respect to their potential use as tracer substances in circulatory research. It was found that the preparations did not contain any detectable free fluorescein. FITC-dextrans could conveniently be determined by fluorometry in the concentration range of 0.05-30 ~g/ml. Gelfiltrationexperiments demonstrated that the fluorescein label was evenly distributed in the molecular weight range of the dextran molecules, and the carbamoyl-dextran linkage was found to be perfectly stable both in in vitro and in vivo conditions. Intravenous infusion of FITC-dextran into rabbits resulted in plasma disappearance curves similar to those obtained with corresponding unlabeled dextran preparations. Consequently, FITC-dextrans should prove valuable as tracers for the study of many aspects of circulation.

INTRODUCTION Dextran has become a particularly useful tool in circulatory research in situations where water-soluble, stable, and nontoxic polymers with well-defined properties are needed. In order to make dextran easier to detect and analyze, fluorescein has been attached to the dextran molecule with a thiocarbamoyl binding, rendering dextran directly visible in the microcirculation by means of fluorescence microscopy. Furthermore, the strong fluorescence of the fluorescein dextran obviates the need for large samples for concentration determinations. It is possible to view the tracer substance directly in microcirculatory preparations and, at the same time, to withdraw very small samples from the interstitial tissue and from lymphatic capillaries. Consequently, quantitative information on the distribution of large molecules in the circulation and on subjects like transport across capillary walls can be obtained. The aims of this study were to investigate the stability of the thiocarbamoyl binding between the fluorescein and the dextran molecule in biological systems in vivo and in vitro and to evaluate the use of fluorescein dextrans as tracer substances in circulatory research.

MATERIAL

AND

METHODS

The fluorescein thiocarbamoyl dextrans (FITC-dextrans) were kindly made available by Dr. Anthony de Belder, Department of Chemistry, Pharmacia AB, Uppsala, Sweden. Table 1 shows the properties of the fractions used. The excitation wavelength is 490 nm and the emission peak is 515 nm for these FITC-dextrans. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

33

34

SCHRijDER,

ARFORS AND TANGEN

TABLE 1 FITC-DEXTRANS USED IN THIS STUDY AND PROPERTIESOF THE FLUORESCEINDEXTRAN

Abbreviations used in the text

nwa

mm”

A?,JA&

D.S.’

FITC-dx 64 FITC-dx145

64 000 145 500

39 800 93 300

1.61 1.56

1 0.5

a iii, is weight average molecular weight. b iii, is number average molecular weight. ’ D.S. is degree of substitution expressed as number of fluorescein groups per 100 glucose units.

Thin layer chromatography (TLC). The FITC-dextrans were subjected to TLC on glassplates coated with SephadexG 200 superfine to determine the presenceof any free fluorescein. The Sephadexwas allowed to swell in 0.3 % NaCl solution, and a 0.5mm thick layer was spread onto a glass plate (15 x 30 cm). Before use, the plates were thoroughly washed in a detergent solution and rinsed in distilled water to prevent unsatisfactory spreading of the gel. The plate was placed in a moist chamber, at a 15” angle to the horizontal plane, with the gel layer connected to the liquid containers by thick filter paper (Whatmann 3 MM) and equilibrated over 15 hr before application of the test sample. The run was stopped when the reference substance (FITC-dx 145) had reached three-fourths of the total length of the glass plate. This point was usually reached after about 4 hr. The eluant used was 0.3 % NaCl solution buffered to pH 7.40 with THAM-HCl to a final concentration of 0.154 M. Visualization of the chromatograms was made under UV light at 254 and 350 nm. Gelfiltration. SephadexLaboratory Columns K 15/75 (Pharmacia Fine Chemicals) equipped with flow adaptors were packed with Sepharose4B. The eluant was identical to that used for the TLC experiments, with the addition of 200 mg/liter of sodium azide to prevent bacterial growth. Gel filtration was performed at 25”, and an elution rate of 6 ml/hr maintained by means of a peristaltic pump. The optical density at 254 nm of the eluate was measured using a LKB 4701 Uvicord and LKB 6520 recorder. One milliliter of samplescontaining 0.1-5 mg of the FITC-dextrans was applied to the columns, and 2 ml fractions were collected and subjected to dextran analysis and fluorescencespectrophotometry. Dextran concentrations were determined by the anthrone method (Jenner, 1967). Fluorescence measurements wereperformed at pH 7.40using a Farrand spectrophotofluorometer model MK 1. A bandwidth of 5 nm was usedboth for the excitation as well as the emission monochromator. Dialysis of FITC-dextran in plasma and homogenized connective tissue was carried out using rotating dialysis chambers at room temperature according to Granath (personal communication). The chambers were fitted with Visking dialysis tubing

35

FLUORESCEIN LABELED DEXTRANS

membranes, and the sampleswere dialyzed against plasma. Samplesfor spectrophotofluorometric measurementswere collected at 0, 6,48, and 168hr from both sidesof the membrane. Sodium azide (200 mg/liter) was used to prevent bacterial growth. Incubation in vitro. To test their chemical stabilities, the FITC-dextrans wereincubated to a final concentration of 0.1% wt/vol for 24 hr at 37” in rabbit plasmas adjusted to pH 5.5, 7.4, and 9 by addition of l/IO volume of 1.54 A4 THAM-HCI buITer. The incubations were carried out both with and without free divalent cations, using heparin and trisodium citrate, respectively, as anticoagulants. Samples of 1 ml taken before and after 24 hr of incubation were subjected to gel filtration as described earlier. Experiments in vivo. Healthy rabbits weighing between 2.0-4.0 kg were injected with

FITC dextran 145 (500 mg/kg body wt iv). Blood samples were obtained through a cannula (Portex 60) inserted under local anaesthesiainto the central ear artery, and catheter samples of urine were collected from conscious animals at 2-hr intervals. At that time the central ear artery was cannulated in a distal direction, and the ear was amputated, perfused with 0.9 % NaCl, and homogenized in an Omni-mixer (Sorvall) at 16,000rpm for 5 min. The suspension was centrifuged at 30,000g for 45 min on a MSE High Speed 18 centrifuge, and the supernatant was analyzed for free fluorescein. RESULTS No free fluorescein could be detectedin the two FITC-dextran preparations as tested with TLC. In addition, these batches were gel filtered on Sepharose4B, and the eluant

;_

I

lo-’

I

10-3

I

10-Z Concencrarton

I

10.’ (#/ml)

I I

I 10

FIG. 1. --ais standard curve for FITC, linear between 5 x 10-5-0.1 ,ug/ml. -A standard curve for FITC-dx 145, linear between 0.05-30 &ml.

36

SCHRijDER,

ARFORS AND TANGEN

fractions were measured on the spectrophotofluorometer. No free fluorescein could be detected. The emitted energy of the FITC-dextran was found to be proportional to concentration between 0.05-30 pg/ml FITC-dextran and for fluorescein between 5 x 10d5 and 0.1 pg/ml (Fig. 1). The plasma blank is negligible if concentrations of FITC-dextrans above 0.1 pg/ml are measured. Figure 2a shows that upon gel filtration, free fluorescein was eluted as a well-defined peak after the plasma proteins and together with the low molecular components of plasma. This demonstrates that any free fluorescein formed during the incubation experiments is not protein bound to the extent that it is eluted in association with the plasma proteins. Figure 2b showsthe gel filtration pattern of a plasma samplecontaining FITC-dextran 145analyzed both for dextran (anthrone method) and with spectrophotop.2

% g -4 .s .g s E 3: h

0 20 10 60 80 1w i -

g o3n, zo2 LO$ 60e 80F b-- loo-

50

60

70

80

90

100

ll0c-d efuak

FIG. 2a. Elution diagram of gel filtration on Sepharose 4B of a mixture of plasma and free FITC.

represents spectrophoto-.-.represents transmission of the eluate at 254 nm, whereas __ fluorometric determination of FITC. FIG. 2b. Elution diagram of gel filtration on Sepharose 4B of a mixture of plasma and FITC-dx 145. -.-.-represents transmission at 254 nm, whereas determinations of FITC-dx 145 were performed by means of the anthrone method (. . . . . .) and by spectrophotofluorometry (-->.

fluorometry. The identical patterns obtained by the two different analytical procedures show that at these FITC-dextran concentrations, the methods can be used interchangeably in the presenceof plasma. No evidence of breakage of the carbamoyl-dextran linkage during incubation in plasma at pH 5.5, 7.4, and 9.0 could be found (Fig. 3). The presenceof the divalent cations did not alter these results. When free FITC is infused, it is cleared from plasma and excreted in the urine and could easily be detectedby TLC. After infusion of FITC-dextran 145,no free fluorescein could be detected in the urine when examined by means of TLC. The plasma disappearancecurves of FITC-dextran 64 and unlabeled dextran 70 are shown in Fig. 4. The somewhat faster elimination of the FITC dextran is due to its slightly smaller molecular weight.

37

FLUORESCEIN LABELED DEXTRANS B 2

12

;

10

a E -z P

..“.. :

pH 5.0

8 6 4 2

= 0 i;;‘i_ T .5060708090

fWml eluate

is 12 30 10LL!!lL ’c

86

I 5 % ?\a

4

.:’......

/YH 9.0 \

2

0

50

60

70

80

90

100 ml eiuafe

FIG. 3. Elution diagrams after gel filtration on Sepharose 4B of FITC-dx 145 incubated in citrate plasma and analyzed by the anthrone method (.. . . . .) and spectrophotofluorometrically (---). Incubation time was 24 hr at 37”, and three different pH were tested.

‘0

20-l

1

FIG. 4. Elimination of dx 70 (. . . . . .) and FITC-dx 65 () from rabbit. Plasma concentrations are shown as percentage of the initial concentration after iv infusion of 100 mg/kg body wt.

The homogenated ear and the plasma containing FITC-dextran 145 that were dialyzed for 168 hr showed an initial change in fluorescenceon both sides of the membrane due to volume changescausedby the initial osmotic imbalance. After equilibrium was reached, no exchange of fluorescent material across the membrane could be detected.

38

SCHRijDER,

ARFORS

AND

TANGEN

These results are taken as evidence that the thiocarbamoyl binding between FITC and dextran is not destroyed under normal in vioo conditions. DISCUSSION Tracers in circulatory researchhave beenusedfor a long time, but most of thesehave been dyes that are reversibly bound to plasma proteins when added to the circulation. Fluorochromes have come into use in the last few years in investigations of the microcirculation. In the use of fluorochromes, two methods have been employed : (1) making a covalent bond betweenthe fluorochrome and a specificprotein in vitro with subsequent infusion or (2) infusing the fluorochrome and allowing a reversible protein binding to occur in vivo. FITC, for example, is bound to albumin, and brilliantsulphoflavin is bound to /I-globulin and albumin. Hauck and Schroer (1969) have used this last technique with FITC and brilliantsulphoflavin in investigations of the “gradient of vascular permeability.” In this study we have mainly used FITC dextran 145 as a model substance for the FJTC-dextrans-the main reason being to use a substance that stays for as long as possible in the circulation. The FITC-dextran 145has a relatively narrow li;l, distribution, with only about 5 % of the molecules under ii?w- 50,000.Arturson and Wallenius (1964) have shown that, in man, dextran with lower iii, than 50,000is excreted rapidly by the kidneys. Except for C14-dextran, the analysis of dextran by spectrophotofluorometry using FITC coupled to dextran, has been found to be more sensitive than any other method now available. C14-dextranis, however, rather expensive,and its useis time-consuming compared to FITC-dextrans. The standard curves in Fig. 1 varies from time to time depending on the spectrophotofluorometer. Highly standardized conditions must be used, therefore, to obtain reproducible results. This means,first of all, that the standard solution must be changed frequently in the cuvettes because of the fading of the fluorescencewhen exposedto strong light. In casethe FITC-dextrans were degradedin the body, one might expect free FITC to be found in either the intravascular or extravascular compartments. In none of these, however, was any FITC found when connective tissue was testedby dialysis and plasma and urine were tested by dialysis and TLC, respectively. Jonsson et al. (1969) have shown, using FITC-dextrans in experiments in the rabbit ear chamber, that transport of FITC-dextran from blood vesselsto the tissue is delayed with increasing m,,,. FITC-dextrans with Mw under 30 000 are very rapidly distributed through all the tissue of the ear chamber. After 4-5 hr a higher concentration of FITC-dx 145 was reached in the lymphatic vesselsin the ear chamber than in the blood vessels when compared directly by fluorescencemicroscopy. The results in this study using FITC, irreversibly bound to dextran, make conclusions regarding vascular permeability possible, and the persistenceof tracer molecules in an organ can be measured. In addition, the FITC-dextrans can be viewed directly in the microcirculation. A further advantage of the FITC-dextrans is the possibility to vary their molecular weights from 3000upwards. With gel filtration techniques it is possible to isolate very sharp fractions of high a,,,-dextrans with an mw to i@” ratio practically equal to 1.

FLUORESCEIN LABELED DEXTRANS

39

The combination of the stability of the FITC-dextrans and the possibility of using narrow molecular weight fractions indicates that FITC-dextrans will prove to be a valuable tool in microvascular research. REFERENCES G., AND WALLENIUS, G. (1964). The renal clearance of dextran of different molecular sizes in normal humans. &and. J. Clin. Lab. Invest. 16,81-86. FISCHER, L. (1969). Laboratory Techniques in Biochemistry and Molecular Biology. North-Holland Publishing Company, Amsterdam. HAUCK, G., AND SCHR~ER,H. (1969). Die transmurale Passagevon Proteinen und der Endostrombahn in Abhlngigkeit vom “Gradient of Vascular Permeability” (Proc. 5th Eur. Conf. Microcirculation, Gothenburg, 1968). Bibl. Anat. 10,225-227. JENNER,H. (1967). Automated determination of molecular weight distribution of dextran: Automation in analytical chemistry. Technicon Symposia 2,203. JONSSON, J. A., ARFORS,K.-E., AND HINT, H. C. (1969). Studies on microcirculatory communications between blood and lymphatic system. Acta Physiol. Stand. Suppl. 330,103. WALLENIUS,G. (1953). Some procedures for dextran estimation in various body fluids. Acta Sot. Med. Upsal. 59, 69.

ARTURSON,