Serum prealbumin and retinol-binding protein in the prealbumin-related senile and familial forms of systemic amyloidosis

In a series of 13 elderly patients with proven prealbumin-related senile systemic amyloidosis (SSA), depressed serum prealbumin values (110.7 +/- 14.1 micrograms/ml) were found as compared to an age-matched control group (175.1 +/- 20.3 micrograms/ml). As expected, there was a significant correlation between serum prealbumin and serum retinol-binding proteins in both groups of patients. Patients with reactive amyloid protein AA amyloidosis had slightly depressed serum prealbumin concentrations, whereas patients with prealbumin-related familial amyloidosis of Swedish type had prealbumin values within normal limits. Since the serum levels of the acute phase reactants, haptoglobin and amyloid-related serum protein AA, were higher in the group of patients with reactive amyloidosis than in patients with SSA, the depression of the prealbumin levels in SSA is not a result of inflammation. Since SSA is known to contain prealbumin, it is possible that a disturbed prealbumin metabolism in old age results in low prealbumin serum values and deposition of amyloid.     

Incidence and characterization of age related amyloid deposits in the human anterior pituitary gland

Summary To identify amyloid deposits in the anterior pituitary gland, we have immunohistochemical, histochemical and alkaline Congo red staining. The anti-human P component reacted positively with these amyloid deposits, while antisera against prealbumin, AA type amyloid fibril protein and various anterior pituitary hormones were negative. A combination of Congo red and anti-human P component staining was most sensitive and reliable for detection of amyloid in the anterior pituitary glands of 300 randomly autopsied patients. Amyloid deposits increased in parallel with the age of the patients, however, they appeared earlier and more frequently than heretofore reported. Deposition of amyloid was seen initially in the 3rd decade and the positivity rate of amyloid deposits was 73% in the 5th decade. The histochemical characteristics of these pituitary amyloid deposits differed from those of cerebral and systemic deposits, particularly those found in the amyloid of senile systemic amyloidosis.This study was supported in part by a grant from the Fundation for Advancement of Clinical Medicine and Ministry of Health and Welfare of Japan     

Novel histochemical approaches to the prealbumin-related senile and familial forms of systemic amyloidosis

The immunoperoxidase method, the autoclave method, and a newly developed alkaline-guanidine method were used to distinguish senile (SSA) and familial types (FAP) of prealbumin-related amyloidosis in formalin-fixed, paraffin-embedded tissue sections. Because all the amyloid deposits of SSA and FAP reacted positively with the antiprealbumin antiserum, a classification of the amyloid fibril proteins of FAP and SSA by immunohistochemistry, using polyclonal anti-prealbumin antisera, was not feasible. Both the senile and familial forms of amyloidosis showed unchanged Congophilia after prolonged autoclaving. In the alkaline-guanidine method, FAP amyloids were resistant to incubation for 2 hours. On the other hand, amyloid deposits of SSA lost the Congophilia and green birefringence with 2 hours' alkaline-guanidine treatment. Therefore, the autoclave method combined with the alkaline-guanidine method will considerably facilitate differentiation of SSA and FAP, without specific antisera.     

Amyloid localized to tenosynovium at carpal tunnel release. Immunohistochemical identification of amyloid type.

Thirty-five patients seen at the Mayo Clinic from 1968 to 1977 who had carpal tunnel syndrome and local deposition of amyloid without evidence of systemic amyloidosis were identified. The unlabeled immunoperoxidase method was used with antisera against purified amyloid proteins of the AA, A kappa, A lambda, AF/ASC1 (prealbumin) (transthyretin), and AB (beta 2-microglobulin) types. In 33 of the 35 patients, amyloid stained with antisera to transthyretin; in the remaining 2 patients, the amyloid did not stain with any antisera. Nine of the 35 patients had a monoclonal protein in the serum, and 2 had a monoclonal light chain in the urine. Systemic amyloidosis or multiple myeloma did not develop in any of these 11 patients. During follow-up, systemic amyloidosis developed in only 2 of the 35 patients: 1 had senile systemic amyloidosis and 1 had tissue that was inadequate for immunohistochemical staining. Amyloid localized to the tenosynovium consists of transthyretin, and systemic amyloidosis rarely develops.     

Prealbumin in Swedish Patients with Senile Systemic Amyloidosis and Familial Amyloidotic Polyneuropathy

A prealbumin (PA)-like protein was demonstrated in amyloid fibrils both from a patient with senile systemic amyloidosis (SSA) and from a patient in northern Sweden with familial amyloidotic polyneuropathy (FAP). The investigated properties of this protein were similar in the two types of fibrils. The protein had molecular weight, antigenic determinants, and at least one cysteinyl residue in common with the subunit of normal PA. In contrast to normal PA, it contained disulphide-linked subunits and was to some extent bound to the fibril via the cysteinyl residues. The noncovalent forces between its subunits were weaker than in normal PA. It constituted part of the previously described AScl protein of the SSA fibrils. Proteins with lower molecular weight than the PA monomer were major proteins in both SSA and FAP fibrils. These proteins had similar properties in the two kinds of fibril and may be derived from PA. PA in serum from Swedish patients with FAP and SSA was normal with regard to the isoelectric pH of the monomers and tetramers.     

Histopathological diagnosis of amyloidosis

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.     

Amyloid in familial amyloidosis, Finnish type, is antigenically and structurally related to gelsolin.

Immunohistochemical studies of six patients with familial amyloidosis, Finnish type, showed that their amyloid deposits did not react with polyclonal antibodies against the amyloid proteins of other, established forms of systemic or cerebral amyloidosis. However, strong immunoreactivity was observed with rabbit antiserum raised against a low molecular weight purified amyloid subunit isolated from one of the patients. This immunoreactivity was abolished by absorption with the low molecular weight amyloid fraction. The amino terminal sequence of the amyloid protein subunit was homologous to gelsolin, an actin-modulating protein, and the amyloid deposits in tissues reacted with a monoclonal antibody against gelsolin. These studies show that the amyloid protein in familial amyloidosis, Finnish type, is not related to previously identified forms of amyloid, including prealbumin (transthyretin) variants, but represents a novel amyloidogenic protein related to gelsolin, a plasma and cytoplasmic protein.     

Prealbumin: its association with amyloid.

In recent years prealbumin has been shown to be a major component of two forms of systemic amyloid, senile systemic amyloid (SSA), and familial amyloidotic polyneuropathy (FAP). Despite the fact that the amyloid fibril proteins associated with these two forms of amyloid, designated ASc1 and AF, respectively, share many similarities the clinical features of the two diseases are remarkably different. To understand better this paradox the clinical, histochemical, immunological and biochemical features of SSA and FAP were reviewed.     

Amyloid deposits in lymph nodes: A morphologic and immunohistochemical study

In a series of approximately 80,000 lymph nodes, amyloid deposition was found in 18; 12 of those nodes were selected, on the basis of availability of specimens, for investigation by immunohistochemical typing to identify the protein of origin and by correlation with morphologic criteria and clinical information. Four patterns of amyloid deposition were identified: lymph node vessel involvement, follicular deposition, diffuse deposition, and a combination of follicular and diffuse deposition. All cases were classified immunohistochemically with the amyloid type-specific antisera anti-AA, anti-A lambda, anti-A kappa, anti-ASc1, and anti-AF. Immunoglobulin-derived protein (AL) in lymph nodes was found in every case of isolated amyloidosis, lymphoplasmacytic/lymphoplasmacytoid immunocytoma, plasmacytoma, and idiopathic amyloidosis. Among the cases of AL amyloidosis were nine of A lambda and one of the A kappa type. AA protein was present in two cases of reactive systemic amyloidosis. There was no useful morphologic correlation with the immunohistochemically identified amyloid types.     

Transthyretin-its function and pathogenesis

Transthyretin (TTR) is a transport protein for retinol-binding protein and thyroxin, and works as a rapid turnover protein. Recently, it has been used as a nutrition assessment protein in the assessment of the acute phase nutritional status in various diseases because it contains four tryptophans in the tetramer of the protein and its plasma half life is 1.9 days. However, the wild-type protein and its mutated form become a precursor protein of amyloid fibrils in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy(FAP), respectively. Recent biochemical and pathological studies revealed that instability of the terameric form of TTR by mutation and post-translational modifications leads to amyloid formation in the tissues of SSA and FAP. In the process of TTR amyloid formation, misfolding of TTR, the trigger of amyloid formation, is also induced. For these amyloid formation mechanisms, Cr3+ administration, BSB(FSB) therapy, gene therapy, and antibody therapy are now on-going therapeutic projects for FAP and SSA.     

Diagnosis and immunohistochemical classification of systemic amyloidoses

 Fourty-three cases of systemic amyloidosis were identified in an unselected autopsy series from our institute (6305 autopsies between 1979 and 1993) and classified immunohistochemically by means of a panel of antisera directed against five major amyloid fibril proteins. Amyloid A (AA) amyloidosis was the most common type, being found in 21 cases (48.8%). Transthyretin-derived (ATTR) amyloidosis was present in 11 cases (25.6%), and immunoglobulin light chain-derived (AL) amyloidosis in 10 cases (23.3%). A single case (2.3%) contained deposits of more than one type of systemic amyloid. AA amyoloidosis was associated with chronic inflammatory or infectious diseases (81%), malignant tumours (19%) or both (9.5%). Immunoglobulin light chain-derived amyloidoses were associated with myeloma (50%) or primary (idiopathic; 50%). In AA and AL amyloidosis the kidney was the organ most frequently involved. ATTR amyloid affecting mostly the heart and lungs presented as senile systemic amyloidosis. Systemic amyloidosis was the cause of death in 5 cases (12%) and caused symptoms in 17 cases (39%). Our results suggest that most cases can be classified by using a panel of sensitive and specific antibodies against five major amyloid fibril proteins. This technique may make amyloid type-specific therapy possible for AL amyloid patients who do not have evidence of an underlying plasma cell dyscrasia. Received: 9 December 1997 / Accepted: 2 February 1998     

Splenic amyloidosis: correlations between chemical types of amyloid protein and morphological features

Sixty-one autopsy cases of splenic amyloidosis were reviewed to assess the relationship between the morphological patterns and chemical types of amyloid protein. On the basis of immunohistochemical reactions of amyloid protein, the cases were classified into 34 cases of AA and 27 of AL amyloidosis. Amyloid deposition in the spleen was divided into three major sites: the red pulp, the white pulp, and blood vessels. Red pulp involvement by amyloid was noted in 52% of the AL cases but in none of the AA cases. White pulp amyloid deposition was found in 70% of the AL and 35% of the AA cases. This difference was statistically significant (P less than 0.001). On the other hand, vascular deposition of amyloid was invariably noted in all cases with AA or AL amyloidosis, affecting the AA cases rather severely. These results strongly suggest that the widely held concept of deposition of amyloid as predominantly vascular in AL amyloidosis and parenchymal in AA amyloidosis requires revision. Our findings indicate that parenchymal, especially the red pulp, involvement is a consistent feature of AL amyloidosis, whereas vascular involvement is a finding common to both types of systemic amyloidosis.     

Amyloid fibril protein in familial amyloidosis with cranial neuropathy and corneal lattice dystrophy (FAP type IV) is related to transthyretin

Immunocytochemical methods were used to study the nature of the amyloid deposits in the Finnish type-familial amyloid polyneuropathy (FAP) type IV, which is characterized by cranial neuropathy and corneal lattice dystrophy. Commercial antisera to human plasma transthyretin (prealbumin) did not stain the amyloid deposits, but in every case a positive staining was obtained with antibodies raised against transthyretin-related amyloid fibril whole protein isolated from the myocardium of a patient with familial amyloid polyneuropathy from the state of New York. The FAP type IV amyloid deposits stained also with antiserum to serum amyloid P component, but did not stain with antisera to retinol-binding protein, amyloid A protein, gamma-trace protein, beta 2-microglobulin, or immunoglobulin light chains. The serum level of serum transthyretin was significantly decreased in FAP type IV patients (256 +/- 75 (SD) mg/L, n = 15) as compared with Finnish control subjects (360 +/- 56 mg/L, n = 30, P less than 0.001), whereas the level of retinol-binding protein was within the normal range. The results of this study strongly suggest that the amyloid fibril protein in FAP type IV amyloidosis is related to transthyretin.     

Fibronectin and basement membrane components in renal amyloid deposits in patients with primary and secondary amyloidosis.

Kidney biopsies from one patient with primary (AL) and three with secondary (AA) amyloidosis were used for an ultrastructural study of the collocalization of basement membrane proteins and the extracellular matrix protein fibronectin within amyloid deposits. Antibodies against amyloid P component, laminin, and heparan sulphate proteoglycan core protein all reacted with the basement membranes and the amyloid depositions in AA and AL amyloidosis. Monoclonal and polyclonal antibodies against collagen type IV reacted only with the basement membranes. Anti-fibronectin reaction was found in association with the basement membranes in all four cases, while labelling of amyloid depositions was found only in one of the AA amyloid cases and in the AL amyloid depositions. It is concluded that basement membrane components may be of importance for the formation of amyloid fibrils.     

Characterization of different amyloids with immunological techniques

We studied 71 cases of amyloidosis from the autopsy material of our institute. 17 cases were secondary amyloidosis of which 7 had initially been diagnosed as primary amyloidosis; all cases reacted positively with an anti-substance A antibody; 13 showed a suppression and 4 a strong reduction of the Congo red staining following tissue incubation in KMnO4. 25 cases were identified as primary amyloidosis of which 7 had initially not been recognized as such. In 16 cases amyloid deposits reacted positively with antibodies specific for the light chains of the immunoglobulins (3 X kappa, 13 X lambda). A monoclonal plasmacytic cell proliferation in the bone marrow was seen in 14 cases. In all cases deposits were KMnO4 resistant, but 1 case showed a slight reduction of staining intensity. 27 cases were cardio-vascular (senile) amyloidosis; in 20 cases at least 3 organs showed deposits; 12 cases had deposits in 5 and more organs. 2 cases were heredofamilial amyloidosis. In those 29 cases deposits reacted positively with an anti-prealbumin antibody, but were negative for AA and the light chains of the immunoglobulins; the Congo red staining remained strong in all cases when previously incubated in KMnO4. KMnO4-Congo red staining and antisera specific for AA, L-chains and prealbumin proved of value for classification of amyloidosis and for its organ distribution.     

Histomorphometric analysis of intramyocardial vessels in primary and senile amyloidosis: epicardium versus endocardium.

BACKGROUND: The literature is unclear as to the frequency and degree of vascular involvement among the various types of cardiac amyloidosis, specifically the senile type. METHODS: We analyzed the intramyocardial vascular involvement, both qualitatively and quantitatively, in 18 patients with autopsy-proven cardiac amyloidosis [11 with primary amyloidosis (AL) and 7 with senile systemic amyloidosis (SSA)]. RESULTS: AL patients (10/11) showed focally transmural vascular involvement by amyloid with thickening of the wall and impingement on the lumens. In SSA patients (6/7), the amyloid deposition was concentrated largely in the adventitia and external media. Histomorphometric analysis revealed that all patients with AL, SSA, and heart failure without amyloidosis had greater luminal areas than normal controls. In addition, the median of the ratio of subendocardial to subepicardial vascular luminal areas was 0.59 in AL patients, 1.17 in SSA patients, 0.94 in heart failure without amyloidosis patients, and 0.89 in normal controls (P=.034). CONCLUSIONS: This study shows that the degree of vascular involvement and wall thickening with subsequent impingement on the lumen is much greater in AL than in SSA. All patients with heart failure, with and without amyloidosis, displayed greater luminal areas than normal controls. Moreover, a twofold dilatation of the vessels in the subepicardium compared to those in the subendocardium was noted only in patients with AL. This may be due to transmural vascular involvement by amyloid in these patients, which concomitantly decreases the transmural ventricular perfusion pressure, leading to compensatory expansion of the subepicardial vessels.     

Amyloid localized to tenosynovium at carpal tunnel release. Natural history of 124 cases

One hundred fifty-two patients with amyloid in the tenosynovium who had carpal tunnel release were identified. Twenty-eight patients were excluded because of systemic amyloidosis: primary systemic amyloidosis (AL) in 24, secondary amyloidosis (AA) in 3, and familial amyloidosis (AF) in 1. The remaining 124 patients (82%) had carpal tunnel syndrome with local deposition of amyloid and no evidence of systemic amyloidosis. Median survival of the 124 patients from diagnosis of amyloidosis was 12 years. Only two patients had systemic amyloidosis develop--9 and 10 years after recognition of tenosynovial amyloid. Of particular interest were 12 patients who had an M-protein in the serum or urine. None of the 12 patients have had evidence of systemic amyloidosis or multiple myeloma during the median follow-up of 14 years. The authors conclude that amyloid may be localized to the tenosynovium and that systemic amyloidosis rarely develops during long-term follow-up.     

Hepatic amyloidosis. A histopathologic analysis of primary (AL) and secondary (AA) forms.

The liver is a major site of amyloid deposition. The spectrum of histopathologic changes in the liver was studied in 38 patients with systemic amyloidosis (25 with primary or myeloma-associated amyloidosis [AL] and 13 with secondary, reactive [AA] amyloidosis). Overall architectural distortion, alterations of portal triads, as well as predilection for topographic deposition in the parenchyma and/or blood vessel walls were noted. Significant histopathologic differences in AL or AA amyloid liver involvement included 1) portal fibrosis, seen in 7 of 25 (28%) AL patients and 8 of 13 (62%) AA patients (P = 0.05), 2) parenchymal amyloid deposition in 25 of 25 (100%) AL amyloid and 10 of 13 (77%) AA amyloid patients (P = 0.04), and 3) vascular amyloid deposition found in 17 of 25 (68%) with AL amyloid and 13 of 13 (100%) patients with AA amyloid (P = 0.02). These data vary from the widely held concept that deposition of amyloid is predominantly vascular in the AL form and parenchymal in amyloid AA. Clearly, however, in individual cases significant overlap occurred, and characterization of amyloid types based on morphologic distribution of amyloid deposits may be possible in only a minority of cases. In most cases, differentiation of amyloid AL and amyloid AA forms requires clinical, histochemical, immunochemical, and sometimes more elaborate laboratory amino acid sequence studies for accurate identification.     

Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology

The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild-type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub-endocardium, sub-epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly approximately 79-residue C-terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR-associated amyloidoses.     

A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo

We synthesized (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) and used this compound to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial prion disease, and systemic amyloidosis, such as familial amyloidotic polyneuropathy, amyloid A (AA) amyloidosis, light chain (AL) amyloidosis, and dialysis-related amyloidosis. BSB showed reactions in all Congo red-positive and immunoreactive regions of the samples examined in the study, and some amyloid fibrils in the tissues could be detected more precisely with BSB than with the other methods. In the mouse model of AA amyloidosis, injected BSB reacted with amyloid in all regions in the serial sections in which Congo red staining was positive. A highly sensitive 27-MHz quartz crystal microbalance analysis revealed that BSB showed a significant affinity for amyloid fibrils purified from familial amyloidotic polyneuropathy and dialysis-related amyloidosis samples and suppressed formation of transthyretin amyloid in vitro. These results suggest that BSB may become a valuable tool for detection of amyloid deposits in amyloidosis and of the mechanism of amyloid formation.