Plasma elimination of indocyanine green in the intact pig after bolus injection and during constant infusion: comparison of spectrophotometry and high-pressure liquid chromatography for concentration analysis.

Indocyanine green is used to estimate liver blood flow rate and hepatic intrinsic clearance. However, its use as a test substance for studies of liver function has been limited by two puzzling kinetic observations: a biexponential plasma decay after bolus injection with an extremely slow late phase and an apparently steadily decreasing clearance value during constant infusion. These observations have been made with spectrophotometric concentration analysis. In anesthetized 30- to 40-kg pigs, we examined plasma concentration curves of indocyanine green after intravenous bolus injection and during long-term infusion. We compared spectrophotometry with high-pressure liquid chromatography for measurement of plasma indocyanine green concentration. In freshly prepared commercially available indocyanine green, high-pressure liquid chromatography could separately measure two fractions, the genuine indocyanine green (97% to 99% of total) and an in vitro degradation product (1% to 3%). Because their spectra were nearly identical, these fractions could not be distinguished by spectrophotometry. After intravenous administration both fractions were identified in the plasma by high-pressure liquid chromatography. In the first series (n = 6) 25 mg of indocyanine green was injected intravenously for 5 min. When analyzed by high-pressure liquid chromatography, the genuine indocyanine green plasma concentration decay was biexponential with rate constants 0.196 +/- 0.021 (mean +/- S.E.M., n = 6) and 0.0372 +/- 0.0064 min-1. The degradation product of indocyanine green decayed almost monoexponentially, with a rate constant of 0.0093 +/- 0.0002 min-1. With spectrophotometry a biexponential decay was observed with rate constants 0.130 +/- 0.012 and 0.0095 +/- 0.0001 min-1. The biexponential decay of indocyanine green after spectrophotometry was the result of codetermination of the two fractions: genuine indocyanine green was responsible for initial phase, and the degradation product of indocyanine green was responsible for the late phase. In the second series (n = 9), indocyanine green was administered as a constant intravenous infusion. From 90 to 240 min the intrinsic hepatic clearance of genuine indocyanine green did not change detectably with time. In contrast, the degradation product of indocyanine green never reached steady-state concentrations. Because of code-termination of these two indocyanine green fractions, the apparent intrinsic hepatic clearance of indocyanine green estimated from spectrophotometry was steadily decreasing by 8.9% +/- 1% per hour of its initial value. At the same time estimation of liver plasma flow rate based on Fick's principle was not affected by the choice of analytical methodology. These observations indicate that high-pressure liquid chromatography is superior to spectrophotometry for kinetic analysis of indocyanine green elimination.