Serum proteins in human disease

The assessment of serum proteins has become of great importance in the diagnosis and management of many human diseases. Two methods currently used for this purpose are electrophoresis and immunoelectrophoresis both of which separate serum proteins according to their charge. However, in recent years two simple techniques have become available which separate proteins principally according to their molecular size. These methods are sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and gel filtration chromatography. The aim of this thesis was to examine these methods and to see if they could be profitably applied to the analysis of serum proteins. In particular, emphasis was placed on the assessment of the molecular size of the serum immunoglobulins in certain diseases.
In Chapter one a general introduction was given about the serum proteins and several specific protein disorders were considered.
In Chapter two the methods of SDSPAGE and gel filtration were described. In addition, these methods were used in an analysis of various pathological sera. The sera of eight patients with multiple myeloma were found to contain paraproteins of atypical molecular size. These were considered to be paraproteins with heavy chain deletions or light chain dimers.
In Chapter three a study was undertaken of the clinical and immunological features of 25 patients with IgA myeloma. These patients were divided into two groups on the basis of the degree of paraprotein polymerization. The first group comprised those patients in whom the IgA paraprotein was greater than 50% polymerized, whilst in the second group the paraprotein was predominantly monomeric. No clinical or pathological differences were seen between the 'polymeric' and 'monomeric' groups of myeloma apart from that directly attributable to the physicochemical effect of the paraprotein polymerization. Thus, five patients out of 11 of the 'polymeric' group developed the hyperviscosity syndrome, whilst no patients in the 'monomeric' group developed this complication. The concentration of the paraprotein during the course of the disease was comparable in both groups.
Chapters four, five and six were concerned with the detection, biological properties and clinical significance of circulating immune complexes containing IgG. To detect these complexes the method of gel filtration was used. The rationale for using this method was based upon the fact that complexed IgG has a larger molecular size than non complexed or monomeric IgG and thus elutes in earlier fractions of the chromatogram. However, SDSPAGE is of no use in detecting complexed IgG as SDS (an anionic detergent) dissociates immune complexes.
In Chapter four a general review was undertaken of the implications of circulating immune complexes and the methods used for their detection. It was concluded that while immune complexes have been implicated in the pathogenesis of a large number of human diseases, there is no simple and sensitive method currently available to detect all forms of complexes.
In Chapter five IgG containing complexes were detected in the majority of the sera of 35 patients with rheumatoid arthritis. These complexes v/ere small, eluting between IgG and IgM, and were not seen in the sera of healthy subjects. A monomer to complexed IgG ratio was used for quantitation of the complexes. This ratio was derived from the IgG profile obtained by gel filtration of the serum. The quantity of complexes correlated significantly with inhibition obtained by the rheumatoid sera of cytolysis in vitro of IgG sensitised target cells by K cells from human peripheral blood. A significant inverse correlation was observed between the quantity of serurn complexes and the chemotactic index of the circulating polymorphonuclear leucocytes obtained from the same rheumatoid patient. This suggests that these complexes may be implicated in the suppression of polymorphonuclear leucocyte chemotaxis observed in patients with rheumatoid arthritis.
There was no correlation between the quantity of the complexes in the sera and other clinical, haematological and biochemical measurements. In addition to these rheumatoid patients the presence of complexes in the sera of patients with other joint diseases was also assessed. In some of these latter patients complexed IgG was found.
In Chapter six four groups of patients were studied for the presence of circulating immune complexes. The first group comprised 19 patients having newly diagnosed acute myeloid leukaemia. The IgG in the majority of these patients' sera eluted in a normal position and complexed IgG was not detected.
The second group was comprised of 11 patients with cutaneous vasculitis. Serum from these patients was examined for cryoglobulin and for complexed IgG. It was observed that complexed IgG was present in six of these sera and was generally associated with the presence of cryoglobulin and with a significant inhibition of K cell mediated cytotoxicity but not always so. In addition, in two patients active vasculitis was seen without the presence of either a cryoglobulin or complexed IgG. In the analysis of the cryoglobulins it was concluded that SDSPAGE might be a rewarding method.
The third group consisted of 26 patients with multiple sclerosis. The percentage inhibition of K cell mediated cytotoxicity by the serum of these patients was similar to that obtained by serum from patients with other neurological disorders. In addition no significant inhibition of cytotoxicity was obtained with any of the cerebrospinal fluid of these patients. Immune complexes are unlikely to be present in the serum or cerebrospinal fluid of these patients.
The fourth group consisted of 7 patients with a variety of diseases. One serum from a woman suffering from severe pre eclampsia was found to have complexed IgG.
In Chapter seven SDSPAGE was used to analyse the blood/CSF barrier in 27 patients with non demyelinating or non infectious neurological diseases. It was concluded that this barrier acts as an efficient molecular sieve to plasma proteins and that SDSPAGE of CSF was a simple means of assessing this barrier.
From the examples given above it was generally concluded that the assessment of the molecular size of serum proteins can provide information supplementary to that obtained using the current electrophoretic methods. It is hoped that now simple techniques are available, the assessment of protein molecular size will be more frequently performed in routine clinical practice.
The topic of Chapter eight deviated from the central theme. In this chapter an examination was undertaken of the cerebrospinal fluid immunoglobulins in various neurological diseases. An increase in the CSF IgG/albumin quotient was observed in 19/36 (53%) cases of definite multiple sclerosis, in 13/47 (28%) cases of probable or possible multiple sclerosis, in 6/9 cases of proven herpes simplex encephalitis, in 3/4 cases of neurosyphylis, in 1/1 case of subacute sclerosing panencephalitis, in 2/9 cases of other neurological infections and in 2/12 cases of polyneuritis when compared with a group of 236 patients having other neurological diseases. In many of these patients a comparison was also made between the IgG/albumin quotient and the CSF kappa/lambda ratio and the CSF and serum viral antibody titre. It was concluded that the measurement of CSF IgG/albumin quotient is a valuable adjunct in neurological diagnosis and can be supplemented by the measurement of the CSF kappa/lambda ratio or antibody titres to certain viruses.