Authentic hSAA related with AA amyloidosis: New method of purification, folding and amyloid polymorphism.

The secondary amyloidosis of humans is caused by the formation of hSAA fibrils in different organs and tissues. Until now hSAA was thought to have low amyloidogenicity in vitro and the majority of SAA aggregation experiments were done using murine protein or hSAA non-pathogenic isoforms. In this work a novel purification method for recombinant hSAA was introduced, enabling to obtain monomeric protein capable of amyloid aggregation under physiological conditions. The stability and amyloid aggregation of hSAA have been examined using a wide range of biophysical methods. It was shown that the unfolding of monomeric protein occurs through the formation of molten globule-like intermediate state. Polymorphism of hSAA amyloids was discovered to depend on the solution pH. At pH 8.5, rapid protein aggregation occurs, which leads to the formation of twisted short fibrils. Even a slight decrease of the pH to 7.8 results in delayed aggregation with the formation of long straight amyloids composed of laterally associated protofilaments. Limited proteolysis experiments have shown that full-length hSAA is involved in the formation of intermolecular interactions in both amyloid polymorphs. The results obtained, and the experimental approach used in this study can serve as a basis for further research on the mechanism of authentic hSAA amyloid formation.

Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1.

Infection, sterile injury, and chronic inflammation trigger the acute phase response in order to re-establish homeostasis. This response includes production of positive acute phase proteins in the liver, such as members of the serum amyloid A (SAA) family. In humans the major acute phase SAAs comprise a group of closely related variants of SAA1 and SAA2. SAA1 was proven to be chemotactic for several leukocyte subtypes through activation of the G protein-coupled receptor FPRL1/FPR2. Several other biological activities of SAA1, such as cytokine induction, reported to be mediated via TLRs, have been debated recently. Especially commercial SAA1, recombinantly produced in Escherichia coli, was found to be contaminated with bacterial products confounding biological assays performed with this rSAA1. We purified rSAA1 by RP-HPLC to homogeneity, removing contaminants such as lipopolysaccharides, lipoproteins and formylated peptides, and re-assessed several biological activities attributed to SAA1 (chemotaxis, cytokine induction, MMP-9 release, ROS generation, and macrophage differentiation). The homogeneous rSAA1 (hrSAA1) lacked most cell-activating properties, but its leukocyte-recruiting capacity in vivo and it's in vitro synergy with other leukocyte attractants remained preserved. Furthermore, hrSAA1 maintained the ability to promote monocyte survival. This indicates that pure hrSAA1 retains its potential to activate FPR2, whereas TLR-mediated effects seem to be related to traces of bacterial TLR ligands in the E. coli-produced human rSAA1.

Purification, identification and profiling of serum amyloid A proteins from sera of advanced-stage cancer patients.

Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) is a powerful tool for screening potential biomarkers of various pathological conditions. However, low resolution and mass accuracy of SELDI-TOF-MS remain a major obstacle for determination of biological identities of potential protein biomarkers. We report here a refined workflow that combines ZipTip desalting, acetonitrile precipitation, high-performance liquid chromatography (HPLC) separation and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis for the profiling, purification and identification of the targeted serum proteins found by SELDI-TOF-MS. By using this workflow, we purified ten targeted proteins from the sera of patients with various types of advanced stage (stage III-IV) cancers. These proteins were identified as isoforms of the human serum amyloid protein A (SAA) family with or without truncations at their N-terminals. This was confirmed by Western blot analysis. Different SAA expression patterns were observed by MALDI-TOF-MS profiling. SAA has long been reported as a biomarker for various cancer types such as lung cancer, ovarian cancer, and breast cancer. However, in this study we found increased SAA expression in the sera of advanced-stage cancer patients with different cancer types. Our results suggest that maybe SAA should not be used alone as a biomarker for any specific cancer type.

Recombinant expression of goat milk serum amyloid A: preliminary studies of the protein and derived peptides on macrophage phagocytosis.

Serum Amyloid A3 (SAA3) protein is a member of a complex group of acute phase and constitutive proteins which have been related to several immune functions. Bovine milk SAA3 (M-SAA3) has been described to have a unique N-terminal TFLK motif responsible for up regulating mucin expression in the intestine lumen and therefore a protective gastrointestinal role. cDNA sequences encoding the protein goat M-SAA3 were successfully cloned from milk, mammary gland tissue and liver, expressed despite observed toxicity and purified as a soluble protein. Sequence analyses of the milk and liver derived forms revealed a non mammary-restricted common N-terminal TFLR motif, unlike that described for bovine M-SAA3. Serum derived forms of SAA have been described to opsonize Gram-negative bacteria facilitating their phagocytosis by circulating macrophages or intestinal epithelial cells. However, no reports about a possible opsonic mechanism of the SAA3 isoforms have been described. Recombinant protein but not peptides encompassing the TFLR region increased blood and milk macrophage interaction and uptake of bacteria reported as number of bacteria per 100 macrophages and percentage of macrophages containing one or more bacteria. gMSAA3-derived peptides did not show any effect on phagocytosis. This would indicate that the TFLK-like region responsible for the up-regulation of mucins in the intestine is not the functional part of g-MSAA3 in promoting macrophage phagocytosis.

Evaluation of colostrum-derived human mammary-associated serum amyloid A3 (M-SAA3) protein and peptide derivatives for the prevention of enteric infection: in vitro and in murine models of intestinal disease.

In vitro experiments confirmed that a 10-mer peptide derived from human mammary-associated serum amyloid A3 (M-SAA3) protected intestinal epithelial cells from enteropathogenic Escherichia coli (EPEC) adherence. The entire 42-mer human M-SAA3 protein was even more effective, reducing EPEC binding by 72% relative to untreated cells (P<0.05), compared with 25% and 57% reductions for the human 10-mer and Lactobacillus GG, respectively. However, none of the M-SAA3 peptides reduced Salmonella invasion in vitro (P>0.05). Each of the M-SAA3 10-mer peptides and the 42-mer was then administered orally to mice at 500 mug day(-1) for 4 days before deliberate infection with either Citrobacter rodentium (mouse model of EPEC) or Salmonella Typhimurium. None of the peptides protected against Salmonella infection and the 42-mer may even increase infection, as there was a trend towards increased Salmonella counts in the liver and small intestine in 42-mer-treated mice compared with those in sodium acetate-treated control mice. Citrobacter counts were reduced in the caecum of mice administered the 42-mer relative to a scrambled 10-mer (P<0.05), but not compared with the sodium acetate control and no reductions were observed in the faeces or colon. Overall, although promising anti-infective activity was demonstrated in vitro, neither the 42-mer M-SAA3 protein nor a 10-mer peptide derivative prevented enteric infection in the animal models tested.

Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.

Monitoring changes in serum protein expression in response to acute events such as trauma, infection or drug intervention may reveal key proteins of great value in predicting recovery or treatment response. Concerted actions of many proteins are expected. Proteins sharing similar expression changes may function in the same physiological process. As a model we analyzed expression changes in serum of colon cancer patients, before, during, and after laparoscopic colon resection. Eight samples were taken from each of four patients before, during, and up to 5 days after surgery. Total serum and a low molecular weight fraction were analyzed by SELDI-TOF-MS. In total 146 masses were detected. A principal components analysis (PCA) illustrates the temporal variation in the postsurgery proteome. Time series for each mass could be clustered into four distinct groups based on similarity in expression pattern. Two masses of 11.4 and 11.6 kDa, part of a slow response cluster, were identified as forms of the acute phase protein serum amyloid A (SAA). Fourteen more proteins belong to this cluster and may also function in acute phase response. We present an approach to analyze temporal variation in the proteome. This approach may be useful to evaluate surgical, nutritional, and pharmacological interventions.

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS.

Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.

Purification of amyloid protein AA subspecies from amyloid-rich human tissues.

Protein AA, the major amyloid fibril protein in reactive (secondary) systemic amyloidosis is derived from the acute phase reactant liver-produced apolipoprotein serum AA (SAA) by proteolytic cleavage, usually in the C-terminal half of the 104 amino acid residues long precursor. The cleavage points in SAA vary between patients and the deposited protein AA is often quite heterogeneous. In this chapter, we describe methods to extract amyloid fibrils and to purify protein AA by sequential gel filtration. Further purification of subspecies of protein AA is best achieved by the use of differences in charge and chromatofocusing is described as the method of choice. Analytic methods include sodium dodecylsulfate polyacrylamide gel electrophoresis and analytic isoelectric focusing.

Serum amyloid A (apoSAA) expression is up-regulated in rheumatoid arthritis and induces transcription of matrix metalloproteinases.

The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1beta induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1beta induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases' transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium. A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.

A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA) -- a clinically useful inflammatory marker in the horse.

A non-competitive chemiluminescence enzyme immunoassay for measuring serum amyloid A (SAA) in equine serum was developed. A polyclonal anti-equine-amyloid A antiserum specific for equine SAA was utilized, and the assay was standardized using highly purified equine SAA. An acute phase horse serum was calibrated against the purified SAA and was used as standard when running the assay. Serum SAA concentrations in the range of 3-1210 mg/l could be measured. The reference range of SAA in clinically healthy adult horses was <7 mg/l. The clinical validation of the assay comprised the SAA responses after surgery and experimentally induced aseptic arthritis, and those associated with viral and bacterial infections. The SAA response after surgery (castration) was consistent, with peak concentrations on day 2 and a return to normal SAA concentrations within eight days. The aseptic arthritis produced an SAA response with a pattern similar to that seen after surgery, with peak concentrations of SAA 36-48 h after induction. Seven horses showed a biphasic pattern, with a second rise in SAA concentrations on day 4 and 5. All animals had SAA levels <7 mg/l on day 15. All horses with viral and bacterial infections had SAA concentrations above 7 mg/l. The ranges of SAA concentrations following the different types of inflammation overlap, being consistent with the unspecific nature of the SAA response. This study revealed that SAA is a sensitive and unspecific marker for inflammation, and describes the dynamics of the SAA response after standardized and well defined tissue damage.

N-terminal sequence analysis of SAA-derivatives purified from murine inflammatory macrophages.

The pathogenesis of secondary amyloidosis in vivo is not well-understood. Experimental studies suggest that incomplete degradation of acute phase serum amyloid A (SAA), presumably endocytosed by activated monocytoid cells, may lead to intralysosomal formation of amyloid A (AA). To establish a possible link between these two events, we have carried out partial N-terminal sequence analysis of affinity purified SAA derivatives from peritoneal macrophages isolated at 4 weeks post-infection from alveolar hydatid cyst infected C57BL/6 mice. The macrophage lysates yielded five N-terminally intact SAA derivatives of approximately 5 to approximately 12 kDa which reacted with anti-mouse AA IgG, and contained a mixture of SAA1 and SAA2 isoforms. The SAA2:SAA1 ratio, evaluated from their proportion present in each M(r) SAA derivative, showed a decrease with the decreasing apparent mass of the N-terminally infected SAA material. These results not only confirm that both SAA1 and SAA2 are processed by activated monocytoid cells but, more importantly, establish a plausible link between N-terminally intact SAA derivatives and formation of AA within activated monocytoid cells.

A high resolution ultrastructural comparison of isolated and in situ murine AA amyloid fibrils.

There is an inconsistency between the ultrastructural organization of AA amyloid fibrils that have been isolated, which are composed of a slowly twisting set of two or more protofibrils, and those seen in situ, which are tubular entities with a tight helical substructure. In this study, the ultrastructure of fibrils isolated from experimental murine AA amyloid were observed at high resolution and compared with those seen in situ in the hope of clarifying the reason for this inconsistency. The fibrils in situ were composed of a microfibril-like 8-9 nm wide core covered by a layer of heparan sulfate proteoglycan (HSPG) to which 1 nm wide filaments, immunohistochemically identified as AA protein, were externally associated. Following isolation with the standard distilled water washing procedure, the HSPG layer and AA protein filaments detached from their core and dispersed into the water. The remaining denuded, variously loosened cores lost their typical appearance. In distilled water the detached 1 nm wide AA protein filaments became quite conspicuous and coiled themselves into 3 nm wide tight helices which in turn assembled into the characteristic slowly twisting sets of two parallel protofibrils similar to that previously reported as "isolated amyloid fibrils". The results emphasize that great caution must be taken in extrapolating amyloid fibril structure from isolated preparations to in situ tissue conditions.

The acute phase serum amyloid A protein (SAA) in the horse: isolation and characterization of three isoforms.

Serum amyloid A (SAA) from acute phase horse serum was isolated using hydrophobic interaction chromatography, gel filtration and ion exchange chromatography. Three SAA isoforms with different isoelectric points, i.e. SAA pI 8.0, SAA pI 9.0 and SAA pI 9.7, were identified by two-dimensional electrophoresis and further characterized with amino acid sequence analysis. These isoforms were found in similar concentrations in all animals investigated, with SAA pI 9.7 constituting about half of the total SAA content. Partial amino acid sequence analysis verified the previously published heterogeneous SAA sequence. SAA pI 8.0 was found to have isoleucine in Position 16, glutamine in Position 44 and glycine in Position 59. SAA pI 9.0 had leucine, glutamine and alanine in the corresponding positions. In SAA pI 9.7 leucine, lysine and alanine were detected. The three isoforms characterized in this study are all acute phase SAAs. SAA pI 9.0 and 9.7 correspond to amyloid A protein variants previously isolated from amyloid deposits of equine liver, while there are no reports on an amyloid A variant corresponding to SAA pI 8.0.

Alveolar hydatid cyst (AHC): inflammation-induced reactive gastrointestinal (GL) amyloidosis in AHC-infected mice and chemical characterization of the GL amyloid.

A high incidence of GI amyloidosis has been described in patients with various forms of systemic amyloidosis but its evolution and progression in different subregions of the GI tract are not well documented. These aspects including the chemical nature of GI amyloid were examined in the AHC mouse model of inflammation-associated reactive amyloidosis. C57BL/6 mice were infected intraperitoneally with 250 AHC. Paraffin sections from the stomach and the small and large intestines of AHC mice were stained at different time intervals with Congo red or immunocytochemically with monospecific RAA. The submucosal blood vessels at 1 week postinfection were found to be the first target of amyloid deposition. With time the amyloid deposits extended to the mucosa and the Peyer's patches and immunoreacted with RAA; ileum was the most severely affected region. Amyloid was extracted from the GI tract and purified by size exclusion chromatography using 5 M guanidine-formic acid, pH 3. The purified amyloid was identified by Western blotting using RAA and by partial N-terminal microsequencing up to 10 cycles. The GI amyloid showed homology with murine SAA2, although SAA2 mRNA is not expressed in murine GI tract. These results shows that (a) the GI amyloid is derived, similar to that of splenic/hepatic amyloid, from circulating SAA2 and (b) the GI tract submucosal blood vessels are the first target of AA deposition. The data also suggest that AA-mediated damage to the submucosal blood capillaries may lead to SAA leakage followed by cascading of AA deposition in other layers of the GI tract.

Both murine SAA1 and SAA2 yield AA amyloid in alveolar hydatid cyst-infected mice.

Amyloid susceptible C57BL/6 and partially amyloid resistant A/J mice, infected intraperitoneally with 250 alveolar hydatid cyst (AHC), the larval stage of a cestode parasite Echinococcus multilocularis, develop multiple organ amyloid deposits at approximately 1 and 4 weeks post infection (p.i.), respectively. Pooled spleens and livers from each mouse strain, at 8 and 10 weeks p.i., were used for the purification of protein AA utilizing a HiLoad Superdex 200 column equilibrated with 5 M guanidine-HCl. Protein AA from each mouse strain was separated on 16% Tris-tricine SDS-PAGE gels and immunoblotted with monospecific rabbit anti-mouse AA IgG; five and six immunoreactive AA subspecies were detected in the C57BL/6 and A/J materials, respectively. N-Terminal amino acid sequence analysis was performed on the bulk column-purified protein AA as well as on the electroblotted AA subspecies from each mouse strain. The results show a mixture of serum amyloid A1 (SAA1) and (SAA2)-derived AA protein from each mouse strain; SAA1-derived AA, although alluded to, has never been demonstrated as tissue deposits in mice. These findings suggest that the intense and persistent inflammatory processes in AHC-infected mice may have induced conversion of weakly amyloidogenic SAA1 to AA. This conversion could be detected by amino acid sequencing of electrophoretically separated AA subspecies.

Quantification and mapping of antigenic determinants of serum amyloid A (SAA) protein utilizing sequence-specific immunoglobulins and Eu3+ as a specific probe for time-resolved fluorometric immunoassay.

Serum amyloid A (SAA) protein, the most prominent amongst acute-phase proteins, is the specific precursor protein of secondary reactive amyloidosis. The fact that SAA once released into the circulation as a 'free' protein rapidly associates with lipoproteins of the high-density range indicates a specific role in lipoprotein metabolism. In this study a new sensitive assay for quantification of human SAA protein in biological specimens using affinity-purified polyclonal antibodies and Eu3+ as a specific probe for time-resolved fluorometric immunoassay is presented. Both purified SAA and SAA-rich high-density lipoprotein particles served as reliable standards in the indirect and the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). The detection limit of the DELFIA technique presented was 4-10 ng after sample dilution of 1/2500. The intra-assay coefficient of variation averaged 4.3% whereas the inter-assay coefficient of variation averaged 6.2%. Comparison with the nephelometric assay, a widely and commonly used assay for SAA quantification in plasma, revealed correlation coefficients of 0.9428. In addition to polyclonal anti-human SAA antibodies sequence-specific antibodies raised against synthetic peptides corresponding to region; 1-17, 14-30, 27-44, 40-63, 59-72, 68-84, 79-94, and 89-104 of the human SAA amino acid sequence were studied. Sequence-specific antibodies raised against epitopes 27-44, 59-72, 68-84, and 89-104 recognize human SAA protein in the DELFIA assay whereas antibodies raised against epitopes 1-17, 14-30, 40-63 and 79-94 failed to recognize the corresponding epitopes. Results obtained from these studies indicate that the N-terminal domain (1-30) as well as epitopes 40-63 and 79-94 of human SAA are apparently masked by the environment of the lipoprotein particle. From our studies it is proposed that the epitopes 31-39, 64-78, and 95-104 may be responsible for the interaction of SAA-rich high density lipoprotein particles with peripheral cells.

The primary structure of rabbit serum amyloid A protein isolated from acute phase serum.

Serum amyloid A (SAA) protein, a sensitive acute phase protein and the precursor of protein AA in secondary amyloid, was purified from pooled acute phase rabbit serum using two different methods: isolation of protein SAA directly by octyl-Sepharose chromatography of total serum, and dissociation and isolation of apoSAA from acute phase high density lipoprotein (HDL). The protein SAA fraction obtained was further purified using gel filtration and ion exchange chromatography. Rabbit protein SAA has 104 amino acid residues, like human SAA, and has a partially blocked N terminus. The highly conserved region from position 33 to position 63 found in SAA from all species studied was confirmed also in rabbit SAA. No microheterogeneities were observed. The amino acid sequence showed extensive N-terminal homology with the rabbit amyloid A protein, except for the microheterogeneity in position 12 in protein AA. It also showed identical amino acid sequence with that deduced from the rabbit cDNA clone pSAA 55. Complete homologies were found with clone SAA 2, except for positions 22 and 78, clone SA8-1, except for positions 22 and 79 and clone SA7-3, except for position 22. This pSAA 55/SA7-3/SA8-1/SAA2-like protein was the only SAA isotype found both in total serum and in the HDL fraction. Isotypes corresponding to other SAA-like genes could not be found in this pool of acute phase rabbit sera.

[Amyloidosis as cause of severe abdominal symptoms in a young Somali immigrant?]. / Amyloidosis als oorzaak van ernstige buikklachten bij een jonge Somalische immigrante?

Extensive deposition of amyloid was detected in the digestive tract of a Somali woman aged 20 yr with abdominal pain, diarrhoea and cachexia. Immunohistochemical characterisation showed that the amyloid protein involved was of the AA type. Elevated levels were also found for serum amyloid A (SAA), an acute-phase protein which is the precursor of AA amyloid. The underlying inflammatory disease was peritoneal tuberculosis. The normalisation of the SAA levels and the recovery of the small intestine during tuberculostatic therapy showed that the tuberculosis was the cause of the enteropathy. This case report highlights the importance of early detection and effective treatment of the underlying inflammatory disease in case of AA amyloidosis.

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis.

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2-76 (or 75) of SAA2 alpha and the other corresponded to positions 2-76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1 alpha has valine and alanine and SAA1 beta has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA152,57Ala.

Serum amyloid A isoforms in inflammation.

Serum amyloid A protein (SAA) was extracted from serum using hydrophobic interaction chromatography and four or six isoforms were separated by isoelectric-focusing. These represented three pairs of isoforms, each with and without an N-terminal arginine. SAA1 (pI 6.1), SAA1 des-arg (pI 5.9), SAA2 alpha (pI 6.9) and SAA2 alpha des-arg (pI 6.6) were found to be present in all individuals from Europe and the USA. A minority of these individuals (11 of 56) expressed SAA2 beta (pI 7.1) and SAA2 beta des-arg (pI 6.8). Serum from patients in Papua New Guinea and Malawi both showed a much higher frequency of SAA2 beta. There was no indication of altered isoforms in regions with high incidence of reactive AA amyloidosis. In sequential serum samples, concentrations of des-arg isoforms were found to reach a maximum 0-24 h later than isoforms with an arginine. Concentrations of the isoform SAA1 decreased faster in five of six patients (16 +/- 7.5 h to decrease 50%) than SAA1 des-arg (22 +/- 11 h to decrease 50%). Variations in the handling of N-terminal arginine may be important for the formation-susceptibility of amyloid deposits.