The high-resolution mass spectrometry study of the protein composition of amyloid-like urine aggregates associated with preeclampsia.

The study of protein misfolding and post-translational processing abnormalities is a promising diagnostic approach for socially significant pathologies associated with the accumulation of abnormal forms of proteins. Recently, it was shown that amyloid-like aggregates can be observed in the urine of pregnant women with preeclampsia, which is the most severe hypertensive complication that can lead to fateful outcomes. The protein composition of urine aggregates may clarify the molecular mechanisms underlying the pathology and has not yet been studied in detail. Using a proteomic approach based on high-resolution mass spectrometry, we studied the protein composition of amyloid-like structures that aggregate in the presence of Congo red azo-dye in the urine of pregnant women with preeclampsia. Fragments of ß-sheets of α-1-antitrypsin, complement 3, haptoglobin, ceruloplasmin, and trypstatin were identified as most likely targets for Congo red binding.

Localized tongue amyloidosis: a single institutional case series.

OBJECTIVE: Review Mayo Clinic experience of localized tongue amyloidosis. STUDY DESIGN: Case series with retrospective chart review. SETTING: Academic medical center. SUBJECTS AND METHODS: Cases of localized tongue amyloidosis were identified from the dysproteinemia database at the Mayo Clinic in Rochester, Minnesota. Electronic records were reviewed with focus on presenting symptoms, laboratory results (ie, serum or urine immunoelectrophoresis, bone marrow biopsy, and fat aspirate analysis), treatment modality, and status of disease at follow-up. RESULTS: Six cases of localized tongue amyloidosis presented to the Mayo Clinic between 1969 and 2011. Mean patient age was 69 years (range, 43-90). Patients presented with asymptomatic tongue mass(es). Biopsy of the tongue mass in all patients showed amyloid on Congo red stains. Work-up for systemic amyloidosis, including bone marrow biopsy, fat aspiration, and serum and urine protein immunoelectrophoresis, was negative for all 6 patients, nor was there other organ involvement. Two patients underwent resection of the lesions, and the remaining patients elected for observation. Recurrence requiring repeat excision occurred in 1 of the patients that underwent resection. Repeat evaluation for systemic involvement was performed in 3 patients 1 to 3 years after the initial diagnosis. None of these patients went on to develop systemic involvement. CONCLUSIONS: Localized tongue amyloidosis remains a rare diagnosis and requires exclusion of systemic involvement. Localized lesions may be observed or resected; however, recurrence may occur with resection. Patients with localized tongue amyloidosis do not appear to be at increased risk of developing systemic involvement.

Evaluation of the serum-free light chain test in untreated patients with AL amyloidosis.

We evaluated the Serum Free Light Chain (FLC) test in a series of 133 untreated patients with systemic AL amyloidosis. The FLC test detected the monoclonal gammopathy in 87% compared with 92% for immunofixation of serum and urine in combination. However, both tests proved complementary. The FLC test was also a valuable tool in patients with advanced renal failure in spite of uninvolved light chain retention. Higher FLC levels were associated with higher bone marrow plasmocytosis, poorer Karnofsky index and heart involvement, and therefore reflected disease severity.

Urine cytology of localized primary amyloidosis of the ureter: a case report.

BACKGROUND: Morphologic findings of amyloid in urine cytology material have rarely been reported because amyloidosis of the urinary tract is a relatively uncommon disorder. We present a case of primary amyloidosis of the ureter, including catheterized urine cytologicfindings. CASE: A 78-year-old man had pollakiuria and dysuria for 5 years before admission after transurethral resection of the prostate. Clinical examination revealed left hydronephrosis and stricture of the lower part of the left ureter, and a malignant ureteral tumor was suspected clinically. In catheterized urine cytology, many clusters of epithelial cells, inflammatory cells and abundant, amorphous, waxy material were observed. The amorphous material stained light green by the Papanicolaou method and positive with direct fast scarlet (DFS), showing yellow-green birefringence under polarized light. Positivity with DFS staining was not affected by treatment with potassium permanganate. Immunocytochemically the material was AL-type amyloid protein. Atypia were absent from epithelial cells. The patient had no history of diseases that could cause secondary amyloidosis. The present case was considered to be primary amyloidosis localized to the left ureter because no particular morphologic change in the epithelial cells of the urinary tract was observed. CONCLUSION: Amyloid can be present in urine and should not be overlooked or confused with tumor diathesis when a malignant tumor is suspected clinically.

On-line immunoaffinity-liquid chromatography-mass spectrometry for identification of amyloid disease markers in biological fluids.

Disease-specific alterations in proteins and peptides such as the appearance of new isoforms, changed relative concentrations of known isoforms, or changed catabolism characterize the group of protein precipitation disorders collectively known as amyloidoses. The goal of this study was to develop an approach for isolating and characterizing the pool of isoforms of a polypeptide of interest from biological fluids for use in development of diagnostic markers and elucidation of pathogenesis. For this purpose, we employed an on-line immunoaffinity-liquid chromatography-mass spectrometry (IA-LC-MS) modular approach using antibodies binding populations of protein isoforms. In this system, crude biological samples, e.g., serum, may be injected and subjected to fast hands-off analysis. The setup consists of an optional preclear column for removal of unspecific binding components, an immunoaffinity column, a short cartridgelike reversed-phase column, and an electrospray time-of-flight mass spectrometer. We have tested the system for the automated analysis of three amyloid-related polypeptides, serum amyloid P component, amyloid beta-peptide, and beta2-microglobulin, and we show the feasibility of detection of altered isoforms or determination of relative abundance of isoforms of the proteins from serum or cerebrospinal fluid samples. For each new protein investigated, the only change needed in the system is a new antibody or antibody mixture and the selection of a reversed-phase cartridge of appropriate hydrophobicity.

Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.

Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis.

Extension of A beta2M amyloid fibrils with recombinant human beta2-microglobulin.

In order to elucidate the pathogenesis of A beta2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of A beta2M amyloid fibril (fA beta2M) extension with native beta2microglobulin (n-beta2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant beta2M (r-beta2M) in vitro. n-Beta2M and r-beta2M were incubated with fA beta2M purified from synovial tissues excised from A beta2M amyloidosis patients. The fA beta2M extension reaction could be explained by a first-order kinetic model in both beta2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both beta2Ms. The fA beta2M extended with both beta2Ms assumed the similar helical filament structure, although the fibrils extended with r-beta2M were slightly wider than those extended with n-beta2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodified fA beta2M, we next extended fA beta2M repeatedly by the algorithmic protocol with r-beta2M. As the generation of the extended fibrils proceeded, the initial rate of the extension reaction increased The ultrastructure of fibrils was completely preserved throughout the repeated extension steps. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fA beta2M extended repeatedly with r-beta2M were composed solely of r-beta2M. The use of these r-beta2M and fA beta2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation of fA beta2M in vitro.

Abnormalities in thrombin-antithrombin pathway in AL amyloidosis.

Various pathogenic factors have been proposed to explain the abnormal hemostasis observed in AL amyloidosis. Since imbalance between clotting factors and inhibitors could play a pathogenic role in both hemorrhagic and thrombotic manifestations, we investigated the thrombin-antithrombin pathway in 35 patients with AL amyloidosis. Ten patients suffered from bleeding while 3 patients experienced deep venous thrombosis. Thrombin time was prolonged in 29 subjects, the mean values of antithrombin III activity (ATIII Act) were significantly lower than those of antithrombin III antigen (ATIII Ag) with loss of relationship between these two different techniques of ATIII detection, normally observed in healthy controls. In 19 patients increased levels of thrombin-antithrombin (TAT) complexes were present. Crossed immunoelectrophoresis of ATIII, performed in presence of heparin, evidenced ATIII forms with reduced binding capacity to heparin and TAT complexes of various electrophoretic mobilities. In conclusion, the impairment of the thrombin-antithrombin pathway, in association with the low ATIII biological activity, might play a pathogenic role in the hypercoagulable state reported in AL amyloidosis, despite the higher frequency of bleeding manifestations.

In vitro modulation of AL-amyloid formation by human mesangial cells exposed to amyloidogenic light chains.

We have shown in vitro AL-amyloid formation by human mesangial cells (HMCs). AL-amyloid formation may require lysosomal processing of the light chains (LCs) by HMCs for amyloidogenesis to occur. Chloroquine inhibits lysosomal activity. TGF-beta mediates extracellular matrix formation in many glomerulopathies. Thrombospondin (TSP) has been proposed as a mediator of cell proliferation and a marker of early fibrosis. We investigated amyloid formation by HMCs exposed to AL-LCs in the absence of amyloid enhancing factor (AEF). The effects of TGF-beta, TSP and chloroquine on in vitro amyloid formation were studied. HMCs were incubated with two AL-LCs, a light chain deposition disease (LCDD)-LC, or one of two tubulopathic LCs (T-LCs). Additional cells were treated with an AL-LC and chloroquine, TGF-beta, or TSP. Amyloid formation was evaluated microscopically using hematoxylin and eosin, Congo red and Thioflavin-T stains, as well as ultrastructurally. Amyloid was formed only when HMCs were incubated with AL-LCs. Addition of TSP significantly enhanced amyloid formation. In contrast, exogenous TGF-beta and chloroquine significantly attenuated amyloid formation. These findings show that some AL-LCs do not require AEF for amyloidogenesis to occur, and that chloroquine, TGF-beta and sTSP modulate in vitro AL-amyloidosis.

Renal elimination of islet amyloid polypeptide.

Six healthy volunteers showed significantly higher plasma islet amyloid polypeptide levels following an oral glucose tolerance test compared to fasting levels. The urine IAPP concentration before and after the OGTT was comparable to that in plasma. Reverse phase HPLC and radioimmunoassay analysis of urine samples revealed a single IAPP-immunoreactive peak. Before hemodialysis, the plasma levels of IAPP and C-peptide, but not of insulin, were significantly elevated in eight fasting patients with chronic renal failure, compared to eight healthy matched control subjects. After hemodialysis, there was a tendency for decreased IAPP levels compared to before dialysis. In summary, elevated levels of plasma IAPP were found in patients with chronic renal failure and the peptide is eliminated by hemodialysis. Furthermore, immunoreactive IAPP is normally present in the urine. These results suggest that IAPP is, at least in part, renally eliminated from the plasma by excretion (glomerular filtration and/or tubular secretion.

[Organ-specific nodular pulmonary amyloidosis of the A lambda type in Sjogren syndrome]. / Organ-limitierte noduläre pulmonale Amyloidose vom Typ A lambda bei Sjögren-Syndrom.

The exclusive involvement of the lungs with A lambda-type amyloidosis in nodular dispositions in a case of Sjögren's syndrome is very rare. An immunoglobulin lambda-type light chain, benign, monoclonal gammopathy has been verified as the ethological cause. The urine concentration of paraproteins was below the detection limit of the common examination methods and could only be found immunoelectrophoretically in urine concentrated 100-200 fold. The question of a possible relationship with Sjögren's syndrome is being discussed.

Lack of islet amyloid polypeptide/amylin-immunoreactivity in urine collected from healthy volunteers after ingestion of a carbohydrate-rich meal.

Islet amyloid polypeptide (IAPP), or amylin, is synthesized by beta cells in the islets of Langerhans of the pancreas. Plasma IAPP levels are highly elevated in patients with advanced renal failure. To investigate the involvement of the kidney in the clearance of IAPP, the response of plasma and urinary IAPP to a carbohydrate-rich meal was investigated in 14 healthy volunteers. Although plasma IAPP levels increased severalfold after the meal, no IAPP-immunoreactivity was detected in the urine samples up to 4 hours after the meal. This might be due to the fact that urinary IAPP levels are under the detection limit of the assay or, assuming the presence of IAPP in the primary urine, immunoreactive IAPP molecules may be processed by renal mechanisms in such a way that they are no longer recognized by the antibodies used in the radioimmunoassay. Processed IAPP molecules may be reabsorbed in the proximal tubules of the kidney and/or excreted.

Preferential expression of human lambda-light-chain variable-region subgroups in multiple myeloma, AL amyloidosis, and Waldenström's macroglobulinemia.

We have compared the distribution of lambda-light-chain variable-region (V lambda) subgroups among Ig lambda molecules found in the serum of normal individuals with that of monoclonal Ig lambda components obtained from patients with plasma cell and related immunoproliferative disorders. A panel of monoclonal antibodies specific for each of the major human V lambda subgroups--V lambda I, V lambda II, V lambda III, V lambda IV, V lambda VI, and V lambda VIII--was used in a highly sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate each of these populations. The mean distribution of Ig lambda I, Ig lambda II, Ig lambda III, Ig lambda IV, Ig lambda VI, and Ig lambda VIII molecules in serum specimens collected from 20 normal adults was approximately 40, 3, 43, 5, 5, and 3% of the total Ig lambda population, respectively. In contrast, that of monoclonal IgG, IgA, and IgD proteins and Bence Jones proteins obtained from patients with multiple myeloma and related gammopathies (n = 196) was approximately 27, 28, 39, 5, 0, and 1%, respectively. The percentage of monoclonal Ig lambda II components found in individuals with AL lambda amyloidosis (n = 41) was comparably increased to that seen in multiple myeloma and was even higher in patients with Waldenström's macroglobulinemia (n = 16), in whom 63% of the IgM lambda proteins were of the V lambda II subgroup. Also evidenced were differences in the distribution of other V lambda subgroups in the disease states: Most striking was the predominance (41%) of the V lambda VI subgroup among monoclonal lambda chains obtained from patients with AL amyloidosis and that this subgroup was found exclusively on amyloidosis-associated proteins. No Ig lambda VI-type myeloma- or macroglobulinemia-related proteins were identified. The observed alterations in V lambda subgroup distribution among "pathologic" monoclonal Igs were attributed to the particular disease and not related to the heavy-chain class. Our finding that certain V lambda subgroups are nonstochastically expressed in lambda-type multiple myeloma, AL amyloidosis, and Waldenström's macroglobulinemia provides evidence for abnormal VL gene usage in these disorders and, thus, furnishes new insight into their pathogenesis.

Amyloid urinary-tract calculi in patients on chronic dialysis.

Urinary calculi found in 4 patients on chronic hemodialysis or continuous ambulatory peritoneal dialysis (CAPD) were identified as protein calculi by infrared spectroscopic analysis. Positive Congo red staining and immunological assessment revealed that the calculi were composed of amyloid protein derived from beta 2-microglobulin. A comparison of the patients who excreted calculi with 10 patients on chronic dialysis without urinary calculi showed no significant differences in the urinary and serum levels of beta 2-microglobulin. The mechanism of amyloid calculus formation may involve factors independent of the concentration of beta 2-microglobulin in urine or serum. Urinary calculi found in patients on chronic hemodialysis or CAPD were composed of amyloid protein derived from beta 2-microglobulin.

Radioimmunoassay for urinary amyloid P component.

A sensitive double-antibody radioimmunoassay for the measurement of amyloid P component (AP) in urine is described. The method is linear with AP concentrations in the range 5 to 2500 micrograms/L, the lower limit of the assay being 5 micrograms/L. Intra-assay and interassay coefficients of variation ranged from 2.2% to 10.3% and from 3.0% to 9.8%, respectively. The mean urinary excretion of AP in 20 normal subjects was 37 micrograms/24 hr, range 5 to 102 micrograms/24 hr. Compared with normal subjects, patients with rheumatic disease had an increased urinary output of AP (mean 98 micrograms/24 hr, range 5 to 218 micrograms/24 hr; P less than 0.005). In patients with reactive (secondary) amyloidosis the diurnal excretion of AP was markedly increased (mean 615 micrograms/24 hr), but the urinary AP/albumin ratio did not significantly differ from that in patients with nonamyloid kidney disease who were matched with the amyloid group with respect to the degree of proteinuria and impairment of renal glomerular filtration rate.