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Title: Studies on plasma proteins in liver disease using quantitative immunoelectrophoresis
Author: Murray-Lyon, I. M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1973
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#1. The history of knowledge on plasma proteins and the methods of separation are reviewed with particular reference to the development of the quantitative Immunoelectrophoresis technique used in the present work. The discovery of plasma protein abnormalities in liver disease and the evidence that the liver is the main site of synthesis is discussed. The mechanisms of plasma protein synthesis and cataholisss and their control are outlined. #2.A summary is given of the properties and functions of the 14 plasma proteins studied in this work (prealbumin, α₁-easily precipitable glycoprotein, α₁-antitrypsin, group component, α₂-macroglobulin, eaeruloplasmin, haptoglobin, haemopexin, transferrin, ß-lipoprotein, ß₁-A-C globulin (C3, third component of complement) and protein numbers 10, 16 and 18). #3. A quantitative modification (Clarke and Freeman) of Laurell's crossed Immunoelectrophoresis method using large plates is described. #4. Clarke and Freeman's method has been adapted for 5 x 5ess. plates (miniplates) with great saving in cost. This tfciniplate method was shown to give a reproducible and quantitative measure of plasma protein concentration. #5. Aliquots of sera were stored at -20°C. The concentration of flipoprotein fell by 30-50$ bwt the levels of other proteins were unchanged over a period of 12 months. #6.Using the miniplate method a normal range for 13 proteins was established in a control population of 70 healthy blood donors and laboratory personnel. Prealbumin was found to be significantly higher in males than females and α₂-macroglobulin and caeruloplasmin were higher in females. Haemopexin and easily precipitahle glycoprotein increased significantly with age. These findings are in agreement with other published studies. Repeated samples from normal individuals over a period of months showed there was little change in the concentration of any protein. As few pure protein standards are available, protein concentrations in this study have been expressed as a percentage of a standard serum. However, absolute values for 6 proteins were obtained by reference to a standard serum with known concentrations of these proteins. The normal ranges for these proteins in the control population agree well with other published results. #7. Sera from seven diagnostic groups of patients were examined. Large plates were used to study sera from patients with primary haemochromatosis (29), alcoholic cirrhosis (12), cryptogenic cirrhosis (12) and active chronic hepatitis (20). Miniplates were used for sera from patients with acute viral hepatitis (12), extrahepatic biliary obstruction (12), primary biliary cirrhosis (?0) and a further series of patients with haemochromatosis (28). Statistically significant changes in the concentration of at least one piasma protein were found in all diagnostic groups studied and the most striking and numerous changes occurred in the disease groups with the greatest abnormality of liver function tests. Prealbumin: Mean levels were low in each of the 4 groups of patients studied (viral hepatitis, extrahepatic obstructive jaundice, primary biliary cirrhosis and haemochromatosis). Easily precipitable glycoprotein: Mean levels were raised in acute viral hepatitis, extrahepatic obstructive jaundice, primary biliary cirrhosis and active chronic hepatitis but normal in the other diagnostic groups. Alpha-L-antitrypsin levels: Alpha-l-antitrypsin levels were raised in acute viral hepatitis, extrahepatic obstructive jaundice, andjsrimary biliary cirrhosis but normal in primary haemochromatosis. Group component: Mean concentrations were normal in all the seven diagnostic groups of patients with liver disease. Alpha-2-macroglobulin: The mean concentration was raised inly in those groups of patients with extensive hepatic fibrosis or cirrhosis. Caefruloplaamin: The concentration was normal in haemo chromato sis and cryptogenic cirrhosis but raised in the other 5 groups. Protein 10. The concentration was raised in acute viral hepatitis but normal in all other diagnostic groups. Haptoglobin: The concentration was low in acute viral hepatitis and active chronic hepatitis and no visible precipitation arc was seen in some cases. Levels were normal in the other groups. Haemopexin: The concentration was low in acute viral hepatitis, extraheptic obstructive jaundice, and primary biliary cirrhosis but normal in the other diagnostic groups. Transferrin: The concentration was low in extrahepatie obstructive jaundice, haemochromatosis and alcoholic cirrhosis but raised in active chronic hepatitis. Beta lipoprotein. The concentration was raised in acute viral hepatitis and reduced in extrahepatic obstructive jaundice but normal in the other groups. Protein 16. The concentration was raised in extrahepatic obstructive jaundice and primary biliary cirrhosis but normal in acute viral hepatitis and haemochromatosis. Protein 18. The mean concentration was reduced in extrahepatic obstructive jaundice and the protein was undetectable in several cases. Levels were normal in acute viral hepatitis, primary biliary cirrhosis and haemochromatosis. Beta 1-AC globulin (C3): was measured only in the large plate studies and was raised in cryptogenic and alcoholic cirrhosis and active ohronic hepatitis. The pattern of changes seen was very similar to the non-specific "acute phase reaction". When there was severe parenchymal liver disease the pattern appeared to he modified by the reduced hepatic capacity for protein synthesis. ThiB was clearly seen in cases of active chronic hepatitis where only after liver function had improved with treatment was the synthetic capacity of the liver increased sufficiently to egress the acute phase reaction fully. The major differences from the acute phase reaction as normally seen were normal or low concentrations of haptoglobin thought to he due at least in part to shortened red-cell life span and raised levels of alpha-2-ciacroglohulin which were found only in liver disease in which there was extensive fibrosis or established cirrhosis. #8.The previously reported finding of raised concentrations of haemopexin and other proteins in primary haemochromatosis was not confirmed in further studies using the rainiplate technique, and detailed examination of the discrepancy suggested that a technical error accounted for the results of the earlier study. Serial venesection in 6 patients with haemochromatosis did not significantly effect the concentration of any protein with the exception of transferrin which rose progressively in 3 cases. Haemopexin concentrations were also normal in close relatives of patients with haemochromatosis and in patients with secondary iron overload. The serum transferrin concentration was significantly reduced in the relatives of patients with haemochromatosis. #9. There was no evidence for in vivo conversion of the third component of complement (C3) in a small number of patients with active chronic hepatitis and primary biliary cirrhosis. #10. The changes in plasma protein concentration following orthotopic transplantation of the liver were followed in 4 patients. TJie same pattern of changes was seen post-operatively with rejection of the graft, biliary obstruction and sarcinomatosis and the pattern was similar to the "acute phase reaction". In one case a precipitous fall in C3 occurred at the onset of an acute rejection episode suggesting consumption of complement. #11. In the sera from 7 patients with severe deficiency of alpha-1-antitrypsin deficiency the concentration of the other proteins was normal apart from a rise in α₂-macroglobulin and protein 10. #12. No change occurred in the concentration of the 13 proteins studied over a 7-day period in two normal subjects during hepatic microsomal enzyme induction. In 15 epileptic patients receiving long-term anti-convulsant therapy, in whom raised urinary glucaric acid excretion indicated hepatic enzyme induction, there were significant increases in the concentration of easily precipitable glycoprotein, α₁--antitrypsin, group component, α₂-macroglobulin, caeruloplasiain and protein 16. #13. The acute phase reaction and the factors controlling its expression are reviewed. The changes in the concentration of the individual proteins in the different categories of liver disease are discussed in relation to the factors known to control their synthesis and catabolism. #14. The diagnostic value of the plasma proteins measured by immunoelectrophoresis was assessed using a linear stepwise discriminant function and a sequential Bayesian. function to reclassify the cases inkseven diagnostic groups. The overall error using both methods lay between 12 and 34%. No protein was of particular diagnostic value and the error in reclassification was least when the values of all proteins measured were used. The error in reclassification of 85 cases in seven diagnostic groups using data derived only from the cellulose acetate electrophoresis strip was 51% Diagnostic accuracy was greatest when all the data from Immunoelectrophoresis and cellulose acetate electrophoresis was combined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available