Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1.

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.

Purification of serum amyloid A and other high density apolipoproteins by hydrophobic interaction chromatography.

A chromatographic procedure is described for the purification of apolipoprotein components of high density lipoprotein from serum. Hydrophobic interaction chromatography (HIC) using phenyl- or octyl-Sepharose was used to purify the high density lipoprotein (HDL)-associated acute phase reactant serum amyloid A (SAA). The purification of SAA is described in detail and it is shown how the main components of normal HDL, apolipoproteins AI and AII (Apo-AI, Apo-AII), can also be purified. Serum was applied at a low salt concentration and apolipoproteins were eluted with a gradient into 4 M guanidine hydrochloride, 30% ethanediol, and 10 mM NaOH. This method was also used to partially purify the low density lipoprotein component apolipoprotein B. Apolipoproteins are purified free from lipid in one rapid chromatographic procedure rather than several ultracentrifugation steps and delipidation with organic solvents. The apolipoproteins from HIC chromatography are already partially separated and can be purified to homogeneity using conventional chromatographic methods under dissociating conditions.

Antibodies to amyloid A protein in rheumatic diseases.

Circulating autoantibodies against amyloid A protein (AA) were demonstrated by enzyme immunoassay in 18/62 patients with rheumatoid arthritis (RA) and in 9/27 patients with systemic lupus erythematosus (SLE). In the subset of RA patients who had developed amyloid, the frequency of antibodies to AA was lower than in those without amyloid (P less than 0.05). The antibody levels showed some variation in serial serum samples during follow-up (1-4 years) of patients with amyloidosis or SLE, but did not correlate with disease activity. In contrast to the patients with rheumatic diseases, patients with inflammatory bowel disease and acute bacterial peritonitis had antibody levels within the range of the healthy control subjects. The results show that autoantibodies to protein AA may occur in rheumatic diseases; their occurrence does not, however, identify subjects with tissue amyloid deposits. Absorption experiments suggest that the antibodies may be directed to circulating amyloid A protein.

Dexamethasone modulation of LPS, IL-1, and TNF stimulated serum amyloid A synthesis in mice.

Three secretory products of the macrophage, interleukin 1 (IL-1), tumor necrosis factor/cachectin (TNF) and hepatocyte stimulating factor/interleukin 6 (IL-6) modulate liver protein synthesis during the acute phase response. Induction of serum amyloid A (SAA) synthesis is one of the most notable acute phase changes in liver proteins, with maximal SAA concentrations varying over a thousand-fold range in proportion to the amount of tissue injury and cell necrosis. Exogenous IL-1 and TNF induce SAA synthesis in vivo and in vitro, while exogenous IL-6 is a far less potent stimulus of in vivo SAA gene expression. Dexamethasone (DEX), a potent inhibitor of macrophage IL-1, TNF and IL-6 synthesis, was utilized to analyze the endogenous mediators of SAA synthesis in mice injected with lipopolysaccharide (LPS). DEX, although itself exhibiting the capacity to stimulate SAA synthesis to a limited extent, significantly reduced LPS induced SAA production. However, DEX did not reduce, but rather potentiated, IL-1 and TNF stimulated SAA production, indicating that these monokines do not require macrophage products to mediate their in vivo SAA inducer activity. SAA synthesis was observed in adrenalectomized mice, following administration of LPS, IL-1 and TNF, indicating that SAA induction by monokines is not secondary to corticosteroid release.

Characterization of amyloid proteins AA and SAA as apolipoproteins of high density lipoprotein (HDL). Displacement of SAA from the HDL-SAA complex by apo AI and apo AII.

An AA-like protein with a molecular weight of 8600 complexed to high-density lipoprotein (HDL) was demonstrated in several acute-phase sera with high levels of SAA. The protein 'apo AA' (to distinguish it from tissue AA) was isolated by elution from sodium dodecyl sulphate (SDS)-polyacrylamide gel, and showed antigenic identity with purified tissue protein AA in double immunodiffusion. Normal HDL was shown to bind purified tissue AA in vitro. When the in vitro-associated HDL-AA complexes were given intravenously to mice during induction of amyloidosis, human AA was incorporated in the amyloid fibrils. Both apo AI and apo AII were shown to displace SAA from acute phase HDL when added to HDL-SAA complexes in vitro. This might be of importance in amyloidogenesis, as the liver and the small intestine, which are the main sites for AI and AII synthesis, are also sites of early amyloid deposition.

The effect of gold salts on casein-induced macrophage activation and serum amyloid protein SAA levels.

Previous work from our laboratory has demonstrated a marked inhibitory activity of Auranofin (Au) and Gold Sodium Aurothiomalate (GST) on monocyte-macrophage function and an important role for macrophages in the pathogenesis of casein-induced experimental amyloidosis. In the present study we have looked at the in vivo effect of Au and GST on the inhibition of casein-induced macrophage activation and serum SAA levels. Au and GST markedly inhibited casein-induced macrophage activation while only Au reduced serum SAA levels to a significant degree. The present data raises the possibility that Au may be helpful in the treatment of secondary amyloid disease.

Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.

Serum amyloid A protein (apo-SAA), an acute phase reactant, is an apolipoprotein of high density lipoproteins (HDL), in particular the denser subpopulation HDL3. The structure of HDL3 isolated from humans affected by a variety of severe disease states was investigated with respect to density, size, and apolipoprotein composition, using density gradient ultracentrifugation, gradient gel electrophoresis, gel filtration, and solid phase immunoadsorption. Apo-SAA was present in HDL particles in increasing amounts as particle density increased. Apo-SAA-containing HDL3 had bigger radii than normal HDL3 of comparable density. Purified apo-SAA associated readily with normal HDL3 in vitro, giving rise to particles containing up to 80% of their apoproteins as apo-SAA. The addition of apo-SAA resulted in a displacement of apo-A-I and an increase in particle size. Acute phase HDL3 represented a mixture of particles, polydisperse with respect to apolipoprotein content; for example, some particles were isolated that contained apo-A-I, apo-A-II, and apo-SAA, whereas others contained apo-A-I and apo-SAA but no apo-A-II. We conclude that apo-SAA probably associates in the circulation of acute phase patients with existing HDL particles, causing the remodeling of the HDL shell to yield particles of bigger size and higher density that are relatively depleted of apo-A-I.

Serum amyloid A protein in acute myocardial infarction.

Tissue injury including myocardial infarction leads to a variety of changes in plasma proteins commonly referred to as "the acute phase response". In this report the concentrations of serum amyloid A protein (SAA) were measured serially in 6 patients with myocardial infarction and 4 with angina. SAA was found to be increased in all patients with infarction, but in no patients with angina. Significantly increased SAA levels were detected 12 hours after the peak level of creatine kinase, and the concentrations of SAA seemed to correlate to the amount of damaged tissue. The SAA-response was both faster and more extensive than the response of C-reactive protein (CRP), but the correlation between SAA and CRP was very good.

Serum amyloid A protein and C-reactive protein in systemic amyloidosis.

In 106 patients with systemic amyloidosis (56 primary, 27 secondary, and 23 familial), serum amyloid A protein (SAA) was measured by solid-phase radioimmunoassay and C-reactive protein (CRP) was measured by rate nephelometry. SAA and CRP concentrations were highly correlated (r = 0.75, P less than 0.001) throughout the normal and abnormal concentration ranges. In systemic amyloidosis, SAA was more sensitive than CRP as an indicator of the acute-phase response, particularly in secondary amyloidosis. Acute-phase proteins are only occasionally increased during the course of familial amyloidosis. The overlap of acute-phase protein levels does not permit reliable separation of primary amyloidosis from secondary amyloidosis solely on the basis of such studies despite the significantly higher SAA and CRP levels in the latter.

Serum amyloid A protein during the treatment of rheumatoid arthritis with second-line drugs.

Serum amyloid A protein (SAA), serum C-reactive protein (CRP) and the ESR were measured in 19 patients with rheumatoid arthritis before treatment and during therapy with gold, penicillamine or sulphasalazine for a mean period of 14.8 months (range 6-23 months). All three measurements decreased significantly; however, only 7% of SAA values fell to within the normal range (18-44 mg/l), compared to 38% measurements of serum CRP (less than 10 mg/l) and 32% of the ESR (less than 25 mm/h). In 8 (42%) of the 19 patients, SAA remained high (greater than 400 mg/l) for 3 months or more whilst serum CRP was depressed below 20 mg/l; this discrepancy was not related to particular drugs. We conclude that during treatment of rheumatoid arthritis with gold, penicillamine or sulphasalazine, SAA concentrations can be high when serum CRP and ESR are suppressed. SAA may be a more sensitive index of disease activity.

Posttransplantation monitoring of high-density lipoprotein-associated serum amyloid A protein: a new diagnostic aid in the detection of renal allograft rejection.

The concentrations of serum amyloid A protein, a high-density lipoprotein-associated protein synthesized in the liver, were monitored in 66 recipients of cadaveric renal allografts. Acute graft rejection episodes were associated with dramatic elevations of serum amyloid A. Electrofocusing and immunoblotting of rejection sera showed a polymorphic serum amyloid A pattern similar to that obtained with control (apo) serum amyloid A isolated from the high-density lipoprotein fraction of plasma. Rejections in patients receiving cyclosporine alone as posttransplantation immunosuppressive medication were characterized by significantly higher serum amyloid A levels than in those receiving cyclosporine in combination with methylprednisolone. Due to the dramatic rejection-induced serum amyloid A elevation, limit values could be used which combined high sensitivity (87-96%) with reasonably high predictive value of a positive test (82%). The serum amyloid A test was applicable also in patients with initially nonfunctioning or poorly functioning grafts. It is concluded that monitoring of the serum amyloid A concentrations offers a valuable noninvasive aid in the early diagnosis of acute renal allograft rejection, including patients with acute tubular necrosis of the graft.

Lowered prealbumin levels in patients with familial amyloid polyneuropathy (FAP) and their non-affected but at risk relatives.

Amyloid fibrils in familial amyloid polyneuropathy, the familial (AF) form of systemic amyloidosis, are composed of the monomeric unit (14,000 MW) of prealbumin molecules. By radioimmunoassay, the serum level of prealbumin was measured in 25 patients from 12 different kinships with this dominantly inherited form of amyloidosis and 56 unaffected, but at risk, relatives from two of the kinships. Results were compared to prealbumin levels in normal individuals and patients with primary (AL) and secondary (AA) forms of systemic amyloidosis. Significantly lowered prealbumin levels were found in the AF patients (149.2 micrograms/ml) and their at risk relatives (169.0 micrograms/ml) when compared to normal individuals (232.9 micrograms/ml), AL patients (221.9 micrograms/ml) and AA patients (211.7 micrograms/ml). No abnormality was found in levels of retinol binding protein (RBP), which is carried by prealbumin, in the serum of either the AF patients or their relatives. The depressed prealbumin levels may indicate a structural variant molecular form, an extra hepatic synthesis or an abnormality in catabolism of this protein that is present prior to the clinical or histopathologic onset of the AF disease.

Trauma, high density lipoproteins, and serum amyloid protein A.

Several apoproteins not readily detectable in normal serum lipoproteins were found in markedly increased amounts in the lipoprotein density interval 1.125-1.21 g/ml (HDL3) of serum obtained from victims of severe trauma. Following electrophoretic separations, they were shown to be isotypes of the acute-phase protein serum amyloid A (SAA) by immunoreaction with antiserum to the structurally related tissue amyloid protein A (AA). The two principal apoSAA isotypes in the HDL3 of trauma patients appear to be identical to the two principal isotypes (apoSAA1 and apoSAA2) previously isolated from the HDL3 of pooled serum representing an unselected patient population. Concentrations of apolipoproteins A-I and A-II in the HDL3 of the trauma patients were significantly lower than in the HDL3 of normal serum. Evidence is presented that several recently described 'new' families of HDL apoproteins, all of an acute-phase nature, are SAA apoproteins, which are emerging as sensitive indicators of tissue damage.

Three assays for the characterization and quantitation of human serum amyloid A.

Two different radioimmunoassays (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation and antigenic characterization of amyloid A (AA) and serum amyloid A (SAA) proteins, and the three assays were evaluated and compared with each other. Sensitivity, reproducibility, effect of denaturation and storage of serum and range of determination were considered. All three assays were found useful, but for different purposes. The most suitable method for the determination of SAA in whole serum was a second antibody precipitation RIA with purified SAA as labelled tracer and standard, and polyclonal rabbit anti-SAA as first antibody. This assay provided SAA concentrations in absolute amounts (mg/l) and acceptable reproducibility without need for prior denaturation of serum. Both advantages and disadvantages of ELISA using monoclonal antibodies to SAA and a solid-phase RIA using AA, SAA, anti-AA and anti-SAA were observed. The three assays were found suitable for antigenic studies of AA and SAA.

Amyloid A fibril degrading activity in serum in liver disease--relation to serum acute phase and other protein levels.

Human serum contains amyloid A degrading activity. This activity is decreased in patients with reactive systemic amyloidosis. To study the specificity of this finding, we evaluated the effect of liver disease per se on the amyloid degrading activity in serum as well as its relation to serum amyloid A (SAA), the putative precursor of amyloid A fibrils, and other serum protein levels in alcoholic and non-alcoholic non-malignant liver diseases without signs of amyloidosis. The amyloid A-degrading activity was significantly decreased in liver cirrhosis. The lowest activity was seen in patients with advanced cirrhosis. There was a positive correlation between the degradative activity and indices of hepato-cellular synthetic function (serum albumin, r = 0.77; serum prealbumin, r = 0.65). Most of the patients with liver disease had a detectable SAA level; 77% had a level higher than 5 mg/l (compared to 12% among blood donors). However, the SAA increase was generally only slight, e.g. in alcoholic liver cirrhosis the median SAA level was 15 mg/l. The results show that liver dysfunction per se decreases amyloid A-degrading activity in serum. A reduced activity cannot therefore be regarded as specific for reactive amyloidosis. Since reactive amyloidosis is extremely rare in liver cirrhosis, it seems obvious that a reduced amyloid degrading activity alone, in the absence of a markedly elevated SAA level, does not predispose, at least in patients with liver disease, to amyloidosis.

Quantitative radial immunodiffusion assay for serum amyloid A protein.

A radial immunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1% agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37 degrees C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples.