February, 1979 HSA-Coated XAD-7: I n Vivo Evaluation

Albumin-Coated Amberlite XAD-7 Resin for Hemoperfusion

Liver Failure in Acute

Part 11: I n Vivo Evaluation

Robin Hughes, Hsueh Yen Ton, Peter Langley, Michael Davies, M. Anwar Hanid, Peter Mellon, David B. A. Silk and Roger Williams

ABSTRACT Albumin-coated Amberlite XAD-7 h a s been pre- viously shown to be blood compatible in in vitro hemoperfusion experiments with human blood. In this study, the preliminary results are reported on single hemoperfusions with albumin-coated XAD-7 resin in four patients with acute liver failure. The mean platelet count was 116+ SE 16.3% of the initial arterial value and the mean white cell count was 96 ? SE 6.5% of initial at the end of four hours of hemoperfusion. Removal of bilirubin, phenols and substances in the middle molecular weight range by the resin was demonstrated.

These preliminary results suggest a lbumin- coated Amberlite XAD-7 resin to be blood com- patible and capable of removing protein-bound a n d middle molecular weight substances from patients with acute liver failure. Further clinical evaluation of repeated resin hemoperfusion is required to de- termine whether this treatment will be beneficial to patient survival.

acute liver failure, hemoperfusion, blood com- patibility, platelets, Amberlite resin

From The Liver Unit, KingS College Hospital and Medical School, Den- mark Hill, London, SE5, England.

This work is part of a continuing program to develop a n artificial liver support system o f the treatment o f fulminant hepatic failure and was supported bq funds f rom the Medical Research Council, the National Re- search and Ikvelopment Corporation and the Wolfson Foundation. The authors would like to thank Mr. T. Moore of the Pharmacy, the Department of Chemical Pathology and the s taf f o f the Liver Failure Unit for valuable help with this work and Linda Rimmer for editorial assistance.

This paper was presented, in part, at the “Hemoperfusion, Dialysate and Diafiltrate Purification” Symposium, September 11-13, 1978, Tutzing, Federal Reprrhlic of Germany.

INTRODUCTION The adverse effects of the Amberlite XAD resins (Rohm & Haas, Croyden, Surrey, England) on plate- lets’ have so far prevented their use in hemoper- fusion to adsorb protein-bound toxins from the circu- lation of patients with acute liver failure who have pre-existing abnormalities of platelet function and morphology.2 The authors have attempted to im- prove the blood compatibility of Amberlite XAD-7 by coating this resin with human serum albumin.:jj4

Blood compatibility was initially evaluated in a n in vitro hemoperfusion circuit where fresh heparinized h u m a n blood was perfused for two hours over small plastic columns containing the resin. Mean platelet losses were significantly less during perfusion of the albumin-coated resin (17%) compared to the uncoated resin (44%1).57~ These re- sults suggested tha t albumin coating successfully prevents the platelet losses associated with resin hemoperfusion.

I n this paper , t h e pre l iminary resu l t s of hemoperfusion over albumin-coated XAD-7 resin in four patients with fulminant hepatic failure are reported.

MATERIALS AND METHODS Pa tien ts

All four patients were in grade IV coma due to fulminant hepatic failure-a 27-year-old male and a n 18-year-old female as a result of paracetamol overdose, a 51-year-old male due to halothane hepa- titis, and a 21-year-old female due to acute viral hepatitis. Each patient had received between 1 and 5 four-hour periods of hemodialysis using the Rhane-Poulenc RP-6 (Paris, France) polyacryloni- trile membrane prior to resin hemoperfusion. Each patient was hemoperfused with resin at a blood flow rate of 150-170 ml/min on a single occasion for four

23

Artificial Organs Vol. 3, No. 1

hours. An acrylic plastic column with silicone rub- ber end pieces, containing 200 gm of albumin-coated Amberlite XAD-7 resin (8.26 mg HSA/Kg dry resin) prepared under sterile conditions, was connected to an arteriovenous shunt using standard dialysis lines.

Heparin anticoagulation (mucous; Leo Labora- tories Ltd., Hayes, Middlesex, England) was used during hemoperfusion, 15,000 units initially, as a loading dose, followed by a further 5,000 units after two hours.

Blood samples were taken with syringes from injection sites in the inlet and outlet lines of the column initially and hourly during hemoperfusion for hematological and biochemical measurements. Hematology

Platelet and white cell counts were determined after collection into EDTA (1 mg/ml blood) using a Coulter counter model ZF (Coulter Electronics, Har- lesden, Essex, England) fitted with a 70 pm orifice, The hematocrit was measured in micro-capillary tubes using a micro-hematocrit centrifuge. The screen filtration pressure of the blood was deter- mined to detect the presence of cellular aggregates in blood using the apparatus as described by Swank.7 Platelet aggregation due to ADP in vitros was measured before and after hemoperfusion. Where appropriate the results in this study were corrected for hematocrit. Biochemistry

Plasma total and conjugated bilirubin were measured by the method of Michael~son.~ Plasma total free phenols were measured using a modifica- tion of the method of Muting.10 Other biochemical parameters and electrolytes were measured by the routine chemical pathology laboratory.

After hemoperfusion of the patient, the resin was eluted with 50% aqueous ethanol; 100 ml of this was concentrated by freeze-drying and was chro- matographed on Sephadex G-25 (1000-5000 fraction- ation range, Pharmacia Fine Chemicals, London, England) in phosphate-buffered saline, pH 7.2. Five- ml fractions were collected and the optical density continuously monitored at 280 mpm (LKB Instru- ments Ltd., Croydon, Surrey, England).

RESULTS AND DISCUSSION In all four cases, four hours of hemoperfusion over resin were carried out without apparent adverse ef- fects to the patients. In fact, three out of four of the patients recovered consciousness two to five days post-hemoperfusion, though each patient was also treated by dai ly RP-6 hemodialysis. l l Unfor- tunately, all four patients subsequently died.

The mean initial arterial platelet count was

'"T ..

Initial Count

1

0 Arterial

A Venous

1 2 3 L

Perfusion Time Ihrl

FIG. 1. Mean platelet levels during hemoperfusion over resin, expressed as a percentage of the initial arterial value (mean f SEM, n = 4).

I T

Initial Count

Arterial

A Venous

1 2 3 L Perfusion l ime (hr)

FIG. 2. Mean white cell levels during hemoperfusion over resin, expressed as a percentage of the initial arterial value (mean? SEM, n = 4).

135+SE 24 x 109/L and there was no significant change in platelet levels during hemoperfusion (Fig. 1). In fact there was a slight increase in mean arterial platelet count (to 1632 50 x 109/L at 4 hours) but this was influenced considerably by one patient in whom there was a 50% increase in platelet count. There was no significant arterio-venous extraction of platelets by the column (Fig. 1).

Similarly, there was no significant loss or arterio-venous extraction of white blood cells during four hours of hemoperfusion with resin (Fig. 2). The results are surprising, as marked white blood cell

24

February, 1979

losses (55%) were observed in i n vitro biocompati- bility studies of the albumin-coated r e ~ i n ~ . ~ using normal human blood. It is known that neutrophil function is depressed in liver failure12 and, as a re- sult, these cells possibly no longer adhere to the resin surface.

The blood hematocrit dropped from an initial arterial value of 352 1.9% to 31f 1.7% during hemo- perfusion, which probably reflects dilution due to the priming solution in the column and blood lines a n d i n f u s i o n of dex t rose s o l u t i o n d u r i n g hemoperfusion.

The results of the Swank screen filtration pres- sure measurements are shown in Figure 3. There were no changes in screen filtration pressure during hemoperfusion, which suggests that no cellular ag- gregates were formed in the blood. This is further substantiated as there was no significant change in initial platelet response to ADP post-hemoperfusion.

The results of the biochemical measurements in which changes occurred during hemoperfusion are shown in the Table. Other routine biochemical para- meters, plasma electrolytes, blood gases and serum protein electrophoresis were unchanged. The removal of bilirubin, which consisted of equal a- mounts of conjugated and unconjugated bilirubin, and phenols indicates the resin is capable of re- moving protein-bound toxins. The loss of calcium could be prevented by the administration of calcium chloride during hemoperfusion. The loss of albumin and total protein during hemoperfusion might seem surprising. However, in vitro studies 3j4 have shown that the amount of albumin coating on the resin used in these patients does not fully saturate the resin. Thus, further albumin and other proteins are bound from the large amounts available in the cir- culating blood.

The initial results of the investigation of middle molecular weight substances accumulating in blood in acute liver failure show that the albumin-coated Amberlite XAD-7 (Fig. 4) binds substances occur- ring in this molecular weight range (1000-5000 daltons). Further studies are in progress to identify the significance and nature of these compounds that have been adsorbed from the blood.

CONCLUSIONS These preliminary results suggest that albumin- coated ‘XAD-7 resin is blood compatible when used in hemoperfusion of patients with acute liver failure, confirming the authors’ in vitro Secondly, perfusion with resin was capable of re- moving protein-bound and middle molecular weight

150

100

mmHg

50

HSA-Coated XAD-7: I n Vivo Evaluation

Mean 2 SEM [ n = L )

1 I 1 I

1 2 3 L

Perfusion Time ( h r )

FIG. 3. Swank screen filtration pressure measurements during hemoperfusion with resin.

TABLE THE RESULTS OF BIOCHEMICAL MEASUREMENTS PERFORMED IN ARTERIAL PLASMA PRE AND POST

RESIN HEMOPERFUSION ( m e a n t SE, n = 4)

Pre Post Total bilirubin, pmol/L 409 ? 104 352 t 89 Total phenols, pg/ml 74 i: 49* 68 t 49 Calcium, mmol/L 2.21 t 0.17 2.03 5 0.12 Total protein, gm/L 64 t 5 56 ? 4 Albumin, gm/L 35 5 3 30 t 2

*One patient had values ten times greater than the other three:

20 LO 60 80 1m 120 110

Froction No 15rn i l

FIG. 4. Column chromatography on Sephadex G-25 (1000-5000 daltons MW fractionation) of material eluted with aqueous etha- nol from resin column after patient hemoperfusion.

25

Artificial Organs Vol. 3, No. 1

substances from the blood of these patients. Further clinical evaluation with repeated perfusion in the same patient is required to determine whether this treatment will be beneficial to patient survival.

References 1. WESTON, M. J., GAZZARD, B. G., BUXTON, B. H., WINCH, J.,

MACHADO, A. L, FLAX, H., WILLIAMS, R. Effects of haemo- perfusion through charcoal or XAD-2 resin on a n animal model of fulminant hepatic failure. Gut, 15:482, 1974.

2. RUBIN, M. H., WESTON, M. J., BULLOCK, G., ROBERTS, J., JANGLEY, P. G., WHITE, Y. S., WILLIAMS, R. Abnormal plate- let function and ultrastructure in fulminant hepatic failure. Q J Med, 46339, 1977.

3. TON, H. Y., HUGHES, R. D., SILK, D. B. A., WILLIAMS, R. Ad- sorption of human serum albumin to Amberlite XAD-7 resin. J Biomed Mater Res (in press).

4. TON, H. Y., HUGHES, R. D., SILK, D. B. A., WILLIAMS, R. Albumin-coated Amberlite XAD-7 resin for hemoperfusion in acute liver failure. Part I: adsorption studies. Artif Organs, 3( 1):20, 1979.

5. HUGHES, R. D., TON, H. Y., LANGLEY, P. G., SILK, D. B. A,, WILLIAMS, R. Blood compatibility of albumin-coated XAD-7 resin for use in artificial liver support systems. Proc Eur Soc Artif Organs (in press).

6. HUGHES, R. D., TON, H. Y., LANGLEY, P. G., SILK, D. B. A,, WILLIAMS, R. The use of a n in uitro hemoperfusion circuit to evaluate the blood compatibility of albumin coated Amberlite XAD-7 resin. Int J Artif Organs, 1:129, 1978.

7. SWANK, R. Screen filtration pressure method in platelet re- search. Significance and interpretation. Ser Haematol, 1:146, 1968.

8. BORN, G. V. R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 194:927, 1962.

9. MICHAELSSON, M., NOSSLIN, B., SJOLIN, S. Plasma bilirubin determination in the newborn infant. Pediatrics, 35925, 1965.

10. MUTING, D., KELLER, H., KRAUS, W. Quantitative determina- tion of free phenols in serum and urine of healthy adults using modified diazo reaction. Clin Chim Acta, 27:177, 1970.

11. SILK, D. B. A., HANID, M. A., TREWBY, P. N., DAVIES, M., CHASE, R. A., LANGLEY, P. G., MELLON, P. J., WHEELER, P. G., WILLIAMS, R. Treatment of fulminant hepatic failure by polyacrylonitrile membrane haemodialysis. Lancet, 2:1, 1977.

12. BAILEY, R. J., WOOLF, I. L, CULLENS, H., WILLIAMS, R. Met- abolic inhibition of polymorphonuclear leucocytes in fulmi- nant hepatic failure. Lancet, 1:162, 1976.

26