Colin Lister

A NEW THEORY OF ENTERORECIRCULATION OF AMINO ACIDS AND ITS USE FOR DEPLETING UNWANTED AMINO ACIDS USING ORAL ENZYME-ARTIFICIAL CELLS, AS IN REMOVING PHENYLALANINE IN PHENYLKETONURIA.

Oral binders remove intestinal bile acid and prevent its reabsorption and recycling thereby lowering systemic cholesterol levels. The results in this paper demonstrate the presence of another extensive enterorecirculation for amino acids. Pancreatic and other glandular secretions into the intestine contain large amounts of proteins, enzymes and polypeptides. Tryptic digestion converts these into amino acids which are then reabsorbed back into the body as they pass down the intestine. This paper shows that this forms a large enterorecirculation of amino acids between the body and intestine. The dietary protein source of amino acids is negligible when compared to the endogenous source, since this paper shows that protein-free diet did not alter the intestinal amino acid concentration. This raises the possibility of using this for the selective depletion of specific body amino acids. In this paper we use a phenylketonuria (PKU) model in rats to test the use of this hypothesis. In PKU rats, artificial cells microencapsulated phenylalanine ammonia lyase (PAL) given orally is more effective than a phenylalanine-free diet. The enzyme artificial cells are more efficient in lowering PHE in the intestine, plasma and cerebrospinal fluid. Compared to PKU on PHE-free diet, this has resulted in better weight gain and general physical condition. Preliminary studies also show that artificial cells microencapsulated asparaginase, glutaminase and tyrosinase given orally can deplete the corresponding amino acid from the intestine.

Immunological Effects of Hemoglobin, Encapsulated Hemoglobin, Polyhemoglobin and Conjugated Hemoglobin Using Different Immunization Schedules

Repeated subcutaneous immunizing injections into rats of Freunds adjuvant containing rat hemoglobin or o-raffinose rat polyhemoglobin did not result in increase in IgG antibody titers. However, heterologous hemoglobin injected as above is antigenic (49.50 + 6.70 % CPM). Crosslinking heterologous hemoglobin into o-raffinose polyhemoglobin further increased its antigenicity (75.60 + 4.08). Thus, unlike homologous hemoglobin, cross-linking of heterologous hemoglobin increased its antigenicity. Liposome encapsulated homologous hemoglobin injected subcutaneously with or without Freunds adjuvant did not show antigenicity. Encapsulated heterologous hemoglobin resulted in a minimal increase in antibody titers (10.73 + 4.64) only with Freunds adjuvant, but no increase when injected without Freunds adjuvant. Conjugated heterologous hemoglobin (PEG-Hb) injected intravenously by itself at weekly intervals did not result in significant increase in antibody titers on the 5th week.

Clinical Evaluation of the Clearance Profiles of a Portable, Compact, Dialysate-Free System Incorporating Microencafsulated Charcoal Hemoperfusion for Blood Purification with Ultrafiltration for Fluid Removal

A total of 30 procedures have been carried out in two patients using a new portable, compact, dialysate-free system formed by combining 300 gms of albumin--cellulose nitrate microencapsulated activated charcoal (ACAC) in series with a small Amicon ultrafiltrator. Blood passing through the ACAC hemoperfusion system is purified of waste metabolites and toxins. Fluid removal is carried out with the hydrostatic pressure of the blood passing through the dialysate-free ultrafiltrator. Since ACAC hemoperfusion is much more efficient than hemodialysers for blood purification, the combined system with the ultrafiltrator results in a very efficient system for blood purification and fluid removal. Typical clearance data for the combined systems include: 75 ml/min for 2000--5000 MW; 112.7 ml/min for 300--1500 MW; 235 ml/min for creatinine; 240 ml/min for uric acid; 2500--2700 ml/2 hours for water removal; and 17--18 gm/2 hours for NaCl removal. Guanidines, mercaptans, and PTH are also cleared very efficiently.

A SCREENING TEST FOR MODIFIED HEMOGLOBIN BLOOD SUBSTITUTES BEFORE CLINICAL USE : BASED ON C3a COMPLEMENT ACTIVATION IN HUMAN PLASMA

Modified hemoglobin preparations may potentially cause hypersensitivity and anaphylactic reactions, antibody-antigen reactions and other problems. Unfortunately response in animal safety studies may not reflect the same response in human. The next best test before clinical trial in human may the use of human plasma in-vitro. This paper present an in-vitro procedure based on complement activation (C3a) of human plasma. The procedure involves collecting heparinised blood and separating the plasma and freezing the heparinised plasma at -70 degrees C until use. Each 100 lambda of control or test samples is added to 400 lambda of this plasma. This is incubated at 37 degrees C, 60 rpm for 1 hour, then added to EDTA saline to stop the reaction and stored at -70 degrees C until analysed by standard radioimmunoassay for C3a. (C3 measurement is not sensitivity enough). Using the screening test procedure described above, C3a levels (ng/ml) in plasma were: control, 1,980 +/- 280; Zymosan, 20,000; Hemoglobin preparation A, 2,227 +/- 617; Hemoglobin preparation B, 4,967 +/- 153; A 75% + B 25%, 3,967 +/- 270; A 50% + B 50%, 4,553 +/- 517; A 25% + B 75%, 4,920 +/- 430. Hemoglobin preparation A did not cause significant increases in C3a complement activation. Hemoglobin preparation B caused significant increase in C3a complement activation. Serial dilution of Hemoglobin preparation B in Hemoglobin preparation A continued to cause the same degree of C3a complement activation. This is not due to C3 exhaustion because Zymosan resulted in C3a of greater than 20,000ng/ml. This appears to show that this screening test can detect complement activation even at low concentrations of the hemoglobin preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Finger-Prick Human Blood Samples as a More Convenient Way for in-Vitro Screening of Modified Hemoglobin Blood Substitutes for Complement Activation: a Preliminary Report

Safety test of modified hemoglobin in animals does not always reflect safety in human. We have earlier reported an in-vitro preclinical screening test based on in-vitro complement activation of human plasma In this test, modified hemoglobin is added to human plasma in a test tube. Complement activation is followed by the C3a levels. Since this directly measures the effect of modified hemoglobin on human plasma, this could be an important bridge before clinical use in patients. The use of plasma is suitable for research, development and industrial applications. However, there are many extra steps involved and may not be convenient for population or patient screening on a large scale bases. The present study shows that it is possible to use small sample of whole blood obtained directly from finger pricks and use immediately for analysing complement activation using an ELISA enzyme immunoassay method.

A Preclinical Screening Test for Modified Hemoglobin to Bridge the Gap Between Animal Safety Studies and Use in Human

The infusion of large amount of modified hemoglobin as blood substitute can potentially result in hypersensitivity and anaphylactic reactions, antibody-antigen reactions and others. Animal safety studies are important. However, response in animals may not be the same as in human. Before injecting into human, we may need to use an in-vitro screening procedure. One approach is based on testing the effects of modified Hb on complement activation (C3a) of human plasma. This paper describes this screening test. It also discusses how this may potentially be used. For instance using this to test for contamination from trace membrane fragments with blood group antigen or lipids, antibody-antigen complexes, endotoxin, trace fragments of microorganisms, residual amounts of some polymers, emulsifying agents, and organic solvents. There is also the possibility of obtaining plasma from a very large human population and analyse each of these to study the epidemiology of adverse reactions in different groups and types of patients.

Assessments of Two Rat Models of Fulminant Hepatic Failure for Testing Artificial Liver Devices

Research in the treatment of fulminant hepatic failure is hampered by the lack of a suitable experimental animal model. We have looked into two animal models which can be conveniently used in large numbers for statistical analysis of effects of hemoperfusion on survival. One is carbon tetrachloride induced fulminant hepatic failure (1); another is the galactosamine induced fulminant hepatic failure rat model (2,3,4). These models seem to fulfill many of the requirements such as severe fulminant hepatic failure, reversibility and safety for research personnel. However, these animal models have not yet been used for the assessment of the effectiveness of treatment regimes for fulminant hepatic failure. Hemoperfusion has been used for patients with hepatic coma (5–13). The original finding (5,6) of hemoperfusion resulting in improvements of consciousness in grade 4 hepatic coma patients have now been supported by all centers. However, the effects on hemoperfusion in the improvements of long-term survival is not yet conclusive. For this, studies of a large series including rigid control studies is required. This is not too feasible in acute fulminant hepatic failure because of relatively small numbers of patients and the variations due to etiology, age, grade of coma and other factors.