The multiple-indicator dilution technique for characterization of normal and retrograde flow in once-through rat liver perfusions

Abstract

The technique of normal and retrograde rat liver perfusion has been widely used to probe zonal differences in drug-metabolizing activities. The validity of this approach mandates the same tissue spaces being accessed by substrates during both normal and retrograde perfusions. Using the multiple-indicator dilution technique, we presently examine the extent to which retrograde perfusion alters the spaces accessible to noneliminated references. A bolus dose of 51Cr-labeled red blood cells, 125I-albumin, 14C-sucrose and 3H2O was injected into the portal (normal) or hepatic (retrograde) vein of rat livers perfused at 10 ml per min per liver. The outflow perfusate was serially collected over 220 sec to characterize the transit times and the distribution spaces of the labels. During retrograde perfusion, red blood cells, albumin and sucrose profiles peaked later and lower than during normal perfusion, whereas the water curves were similar. The transit times of red blood cells, albumin and sucrose were longer (p less than 0.005), whereas those for water did not change. Consequently, retrograde flow resulted in significantly larger sinusoidal blood volumes (45%), albumin Disse space (42%) and sucrose Disse space (25%) than during normal flow, whereas the distribution spaces for total and intracellular water remained unaltered. The distension of the vascular tree was confirmed by electron microscopy, by which occasional isolated foci of widened intercellular recesses and spaces of Disse were observed. Cellular ultrastructure was otherwise unchanged, and there was no difference found between normal and retrograde perfusion for bile flow rates, AST release, perfusion pressure, oxygen consumption and metabolic removal of ethanol, a substrate with flow-limited distribution, which equilibrates rapidly with cell water (hepatic extraction ratios were virtually identical: normal vs. retrograde, 0.50 vs. 0.48 at 6 to 7.4 mM input concentration). These findings suggest that the functional and metabolic capacities of the liver remain unperturbed during retrograde perfusion, rendering the technique suitable for the investigation of zonal differences in drug-metabolizing enzymes.

Assessment of the effect of malaria infection on hepatic clearance of dihydroartemisinin using rat liver perfusions and microsomes

“…Due to the reported changes to hepatic architecture in MI (Francis & Warrell, 1993;Garnham, 1966), we used the multiple indicator technique (St-Pierre et al, 1989) to determine cellular volume of the livers in the single-pass perfusions. We found that the cellular volume of the liver comprised 49% (controls) and 53% (MI) of liver wet weight.…”

“…The mean transit times of the tracers through the liver was calculated (Equations 4 ± 8; Appendix) and corrected for the mean transit time through the system without a liver (14.6+1.6 s; n=8). Cellular water space (functional volume) was determined from the dierence between total water space (obtained from [ 3 H]-H 2 O mean transit time) and the sinusoidal plus Disse volume (obtained from 14 C-sucrose mean transit time) (St-Pierre et al, 1989). At the completion of each perfusion, the livers werē ushed with chilled 0.9% sodium chloride solution, dried between absorbent towels and weighed.…”

Moment Analysis of Metabolic Heterogeneity: Conjugation of Benzoate with Glycine in Rat Liver Studied by Multiple Indicator Dilution Technique

“…The data, when compared with previous prograde data of Schwab et al (2001), revealed that the values for total water volumes (sum of both cellular and extracellular water spaces) were greater than unity (Table 4). The apparently high value is due to the distention of the vasculature during retrograde flow, as described by St-Pierre et al (1989) and Xu et al (1990). The MTTs for labeled red blood cells, albumin, and sucrose were all increased with retrograde flow, especially for data of the first injection, although changes in the second injection were attenuated.…”

“…In situ single-pass liver perfusion under retrograde flow was carried out as previously described (St-Pierre et al, 1989) with perfusate (12 ml/min) at 37°C entering via the hepatic vein and exiting via the portal vein; the hepatic artery was ligated. Perfusate consisted of bovine erythrocytes (20%), freshly obtained and washed (kind gift of Ryding-Regency Meat Packers, Toronto, ON, Canada), 5% bovine serum albumin, and 17 mM glucose (Baxter Travenol Laboratories, Deerpark, IL) in Krebs-Henseleit bicarbonate solution (pH 7.4).…”

“…A common method for probing enzyme zonation in the absence of a transport barrier is single-pass prograde/ retrograde perfusion of the rat liver preparation (Pang and Terrell, 1981;St-Pierre et al, 1989). With prograde perfusion, perfusate enters the portal vein and exits at the hepatic vein, first recruiting upstream and then downstream enzymes.…”