AJEBAK 53 (Pt. 4) 333-336 (1975)

Brief Communication:

A METHOD FOR ASSESSING PHAGOGYTE GLEARANCE RATES IN MAN by VILLIS R. MARSHALL AND JOfIN LUDBROOK (From the Department of Surgery, The University of Adelaide. Adelaide, South Australia 5000,)

(Accepted for puhliration lutij 25. 1975.)

Summary. By comparing the clearance rates of ICG and "-'"'Tc-sulphur colloid from the blood stream, it has been possible to provide a method for assessing the pbagoc>'tic acti\'ity of the reticulo-endothelial system in man.

INTRODUCTION. It has been .shown in man that large single doses of heat aggregated albumin (10 mg/kg body weight) are cleared from the blood as an exponential function of time C = C^^IO-KT (Biozzi ('( al.. 1958). The rate constant K. or phagocytic index, varied inversely with the amount of colloid injected provided that a critical dose of 1 mg/kg was exceeded. These observations were supported by Iio and Wagner (1963) who suggested tbat the same information could Ix.' obtained by administering a range of intermediate-sized doses of tbe colloid and calculating the phagocytic index from the clearance data. A problem regarding interpretation and extrapolation from these studies is that the number of particles presented to the reticulo-endothelial system (RES) appears to be far greater than would be anticipated imder physiological or pathological conditions. It has been estimated that in bacterial infections in man 100,000 organisms per ml is an exceptionally high and rare blood level (Bennett and Beeson, 1954) and that in septicaemic patients the levels are usually closer to 500 per ml. Assuming a l)lood volume of 5 1, at any given time the maxinuun number of bacteria present in the blood stream would be between 2-5 x lO** and 5-0 x lO'*. On a weight basis this would correspond to a single dose of 0-001-0-05 mg/kg. Conseciuently, it is imlikely in man that the blood concentration of bacteria would ever approach the critical dose of particles proposed by Biozzi et al. (1958). This view has been supported by Martin and Kerby (1950) who described in rabbits that bacteria can still be removed from the splanchnic area at a normal rate during the course of fatal septicaemia. Donovan (1967) used small doses of particles to measure RES phagocytic activity but was unable to determine what eflect altered liver blood flow may have had on the interpretation of his data. In an attempt to overcome this problem, we have determined simultaneously the blood clearance rates of indocyanine green (ICG), wliich is cleared only by hepatocytes (Caesar et al., 1961), and """iTc-sulphur colloid which is cleared only by tbe cells of the RES. If there were normally a constant relationship between the two clearance rates over a range of rates of liver blood flow, then a comparison of these two measurements might provide information about the phagocytic activity of tlie RES under physiological and pathological situations.

334

VILLIS R. MARSHALL Axn JOHN LUDBROOK MATERIALS AND METHODS.

The subjects for this experiment were healthy male medical students aged 20-26 years, none of whom had a past history of liver disease. All subjects were fasted for 4 h prior to the experiment. Protocol 1.

While supine, each siiliject had a cannula, attached to a three way tap to facilitate blood sampling, inserted 3 cm into an antecubital or forearm vein. Three 8 0 ml blood samples were withdrawn and 0-2 ml of """'Tc-sulpbur colloid (0 004 mg/kg. radiation dose 3 mCi, particle size 0-5-3-0 nm) and ICG (0-03 mg/kg diluted to 8 ml) were injected intravenously into the other foreanu. Blood samples were taken at 1 miii intervals. Each sample, together ivith the preinjection samples, was divided into 5 0 ml and 3 0 ml lots. The 5-0 ml lots ivere allowed to clot at room temperature, centrifuged at 1,000 x g for 15 min and the optical density of the serum measured at the 805 nm absorption peak for ICG in a Zeiss PMQ2 speetrophotometer. The 3-0 ml lots of whole blood were counted in a Nuclear Chicago auto ganuna counter NM/EX/3. The log]^ of the optical density and the logn, of the counts per min thus determined were plotted against time after injection in min, and from the calculated regression lines the disappearance rate constants, kpy]|y,j and kJ^.^, were determined. Protocol 2.

The second series of estimations were performed from 4 days to 6 weeks after the first. The experimental method was identical except that after the insertion of the cannula the subjects were tilted (foot-down) 65° from the horizontal on a tilt table and left in this po.sition for 30 min prior to the injection of the test substances. Tilting was performed in an attempt to alter liver blood flow, sc) that if a correlation existed between the two disappearance rate constants it could be tested at dilferent flow rates in the same indi\idual. Fifteen individuals were studied: in 10, observations were carried out both supine and foot-down. Of the remaining 5, 3 fainted while they were tilted and the ejq^eriment was .stopped, and in the remaining 2, studies were not performed in the foot-down posture. Standard statistical methods were used to analyse the data (cf. Armitage, 1971). The symbol ± has been used to indicate the standard error.

RESULTS. Among the 10 subjects for whom there were paired observati{ms k,,.,, was found to be unaltered by posture (supine 0-082 ± 0-007; foot-down 0-084 ± 0-008). Nor was \..,U,M affected by posture (supine 0-086 ± 0-004; foot-down 0-079 ± 0-008). Foot-down tilting evidently failed to produce in our subjects the fall in splanchnic blood flow reported by Culbertson et al. (1951). When the paired and unpaired data from the 15 snbjects were examined, the calculated regression of k,,,,,!,^;,, on kic; was altered by postnre, but covariant analysis for slope and elevation revealed that this difference was not significant. Thus, the data from both postures were pooled, the calculated regression line being l^,.,,iioi,i = 0-648 kj,,^, + 0-027 with r = 0-790. Fig, 1 shows the regression line and its 95% confidence limits. Also shown are the 95% confidence limits within which a value of kp^j,,,||,, would be expected to lie for a single measured value of ^H^Q in an individual drawn from the same populatitm as our subjects.

BLOOD CLEARANCE RATES IN MAN

335

DISCUSSION. In Fig. 1 it can be seen that there is a positive intercept of k,.,^,[,,;,j. This is consistent with previously reported studies (cf. Caesar et al, 1961) in whieh it has beeri shown that ICG is almost exclusively removed from the blood stream liy the liver, whereas colloid.s can be taken up elsewhere, particularly by the spleen (Mundsehenk, Flromec and Fischer, 1971). Extra-hepatic uptake of colloid is likely to be further enhanced by any condition that causes obstruction to the portal vein, with resultant diversion of the splanchnic blood flow away from the liver. The slope of the rejir(\ssinn line in Fin. 1 implies that over the physiologieal range of

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ICG Fig. 1. Plot of kp,,,i,,.,| against kj,,,,. Closed circles: supine. Open circles: foot-down. Heavy line: calculated regression line of k,.^!,^,,, on k,,-.,.. Dense shading: 95^ confidence limits of regression line. Light shading: area within which the value of k^,,,,,,,,,, should He with 95^ confidence, for a single \'ahie of kj,.,;. hepatic blood flow """'Te-sulphur colloid is less efficienlly cleared from the blood stream by the liver than ICG, and this is presumably due to the lower hepatic extraction ratio for colloids (Vetter, Falkner and Neuniayr, 1954) than for ICC (Reemtsma et al. 1960). The outer confidence limits drawn in Fig. 1 represent a prediction of k,,,.,;,,,,, given k,,.p, with kj,-,,> providing an index of liver blood fiow. Logieally, this predietive function can be used onlv if the individual under test has an ICC clearance within the ranye exhibited by the.se nonnal subjeets. Thus, a prediction is only valid within the 95% confidence range of values for kj,.,, (0-039-0'13). Within these limitations a value of k^,o,],,ij above these boundaries indicates enhanced, and below correspondingly reduced, whole body phagocytic function. This comparison between the simultaneous measurements of Hvcr blood How by a method that is independent of phagocyte activity and one that is entirely dependent on phagocyte activity appears to provide a means of assessing liver phagoeyte function under

336

VILLIS R. MARSHALL AND yOHN LUDBROOK

physiological circumstances. This method eliininatt-s the need to swamp the phagocytic system with particles prior to attempting tii assess the ability of the system to clear particles from the blood. The dose of "''"'Tc-sulphur colloid of 0-004 mg/kg is well below the critical dose proposed by Biozzi et al. (1958), and is more likely to be representative of the number of particles presented to the RES under real-life circumstances. The dose of radioactivity that has been used is about half that used for a routine* liver scan, and can therefore be repeated on .several occasions if sequential tests of function are to be carded out. Acknowtedfiements. We are indebted to the medical students who volunteered to participate in this study, to Dr. P. M. Ronai and Dr. M. Marion for assistance with the colloid studies and to Hynson, Westcott and Dunning Inc. for providing the indocyanine green.

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