Clinical Science (2008) 114, 123–130 - R.A. Assaly and others - Assessing capillary leak using fluorometry

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Clinical Science (2008) 114, (123–130) (Printed in Great Britain)

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Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis

Ragheb A. ASSALY*, Robert H. HABIB†‡, Mustafa AZIZI*, Joseph I. SHAPIRO* and J. David DIGNAM§

*Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A., †Saint Vincent Mercy Medical Center, Toledo, OH 43608, U.S.A., ‡Department of Surgery, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A., and §Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.

Key words: albumin, capillary leak, fluorophore, poly(ethylene glycol), redistribution phase, sepsis, vascular permeability.

Abbreviations: CLP, caecal ligation and puncture; FL, fluorescein; PEG, poly(ethylene glycol); PEG–Alb, albumin covalently linked to PEG; SIRS, systemic inflammatory response syndrome; t½, half-life; t_50%, time when the concentration reaches 50% of its baseline value; TER, transcapillary escape rate; TR, Texas Red.

Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n=11) and post-CLP (caecal ligation and puncture; n=14) Sprague–Dawley rats were injected with tracer amounts of albumin and PEG–Alb [albumin covalently linked to methoxy-poly(ethylene glycol)] labelled with fluorescein and Texas Red. Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined. The relative retention of the PEG–Alb and albumin was assessed via measurement of the TER (transcapillary escape rate; in %/h) and the t_50% estimate, defined as the time when the actual concentration reached 50% of its baseline. The concentration–time trends for both albumin and PEG–Alb tracers exhibited two-compartmental behaviour and were analysed using bi-exponential modelling. Retention times were significantly greater for PEG–Alb in both control and CLP rats. TER_PEG-Alb was significantly lower than TER_albumin for both control (8.1±5.6 compared with 14.8±7.1 %/h respectively; P<0.01) and CLP (14.8±6.6 compared with 22.5±7.3 %/h respectively; P<0.001) rats. The t_{50%[PEG–Alb]} was substantially greater than the corresponding t_50%[albumin] for both control (29.8±9.8 compared with 7.2±2.0 h respectively; P<0.001) and CLP (12.9±5.6 compared with 5.1±1.6 h respectively; P<0.001) rats. The result was similar irrespective of the fluorophore–protein combination, validating the multifluorophore technique. In conclusion, the double-fluorophore approach described in the present study may provide the future basis for a method to quantify capillary leak in disease.