Diagnostic accuracy of silica clotting time method for lupus anticoagulant in a clinical population with various symptoms of antiphospholipid syndrome.

INTRODUCTION: Correct interpretation of lupus anticoagulant (LA) tests is crucial for diagnosis of antiphospholipid syndrome (APS). This study assessed diagnostic accuracy of the SCT method in a clinical population with various symptoms of APS. MATERIAL AND METHODS: Altogether 60 APS patients were consecutively recruited from a relevant clinical population. All cases had stable positivity of at least one of the reference tests (two other LA methods; anticardiolipin- and anti-ß2-glycoprotein-I antibodies). Controls (n = 62) with negative reference tests were also consecutively recruited from the same clinical population. RESULTS AND CONCLUSIONS: Receiver operator characteristic (ROC) analysis for the SCT test to identify the APS cases showed area under the curve of 0.82 (95% CI 0.75-0.90). The positive cut-off defined by a non-parametric method (99 percentile in a healthy population) had specificity of 92%, but low sensitivity of 53%. The optimal cut-off corresponded to the 97.5 percentile (67% sensitivity and 92% specificity). Combined sensitivity of the positive diluted Russell Viper Venom time (dRVVT) and SCT tests was 73%, while specificity remained 92%. The sensitivity of the SCT method varied in different clinical subgroups and was highest in patients with rheumatic diseases and in patients with triple positivity of three reference methods.

Kallikrein modification with affinity to IgG present in higher amounts than normal in plasma from patients with Crohn's disease.

This study presents immunological and functional evidence to suggest that the activation of prekallikrein (PK) in human plasma yields two modifications of kallikrein. One of these modifications showed amidolytic properties strongly deviating from those registered for the main part of the enzyme. The substrates were S-2302, Bz-Pro-Phe-Arg-pNA, S-2366 and S-2222. In PAGE immunoblots the PK heavy chain mAb 13G11 was found to detect the kallikrein 85 kD double band and bands with mol. weights of about 152 and 135 kD. Such a 152 kD band could be removed together with an IgG fraction on a Protein G column. In this study the kallikrein identity was confirmed by an estimation of the levels obtained in amidolytic assays of mixtures of normal plasma and plasma deficient in FXI, which in immunological assays showed a PK level of 140-150%, of normal. A comparison in amidolytic assays of normal plasma and plasma from patients with Crohn's disease showed that patients' plasma contained a significantly higher level than normal of modified kallikrein. An IgG removal procedure removed all modified kallikrein, and did not affect ordinary kallikrein levels.

Analysis of glyphosate residues in cereals using liquid chromatography-mass spectrometry (LC-MS/MS).

A fast and specific method for the determination of glyphosate in cereals is described. The method is based on extraction with water by ultrasonication. The samples are cleaned up and separated by high-performance liquid chromatography on a polystyrene-based reverse-phase column (clean-up) in series with an ion chromatography column (separation) using NaHCO(3) as eluent. A micro-membrane suppressor was inserted after the separator column to remove the Na(+) ions before detection by electrospray ionization mass spectrometry in the negative-ion mode. In MS/MS, mode the following transitions were monitored m/z 168--> 150 (glyphosate) and 170-->152 (internal standard 2-(13)C(15)N-glyphosate) for quantification. The mean recovery was 85% (n=32) at spiking levels from 0.03 to 0.33 mg kg(-1). From 1998 to 2001, from the analysis of about 50 samples per annum, a reduction in the glyphosate residues was observed owing to a Danish trade decision not to use grain with glyphosate residues for milling or bread production.

Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests.

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.

Removal of IgG from normal plasma and plasma from untreated patients with active Crohn's disease--effect on levels of contact factors.

Protein G columns were used to remove IgG from human plasma, and the effect on levels of factor XII, factor XI and prekallikrein was studied in functional tests. IgG was detected in PAGE immunoblot experiments with Fc-specific antibodies. Removal of the bulk of IgG in a procedure based on a low plasma dilution (1+2.5) allowed the passage of an IgG fraction along with the contact factors. This fraction was found to be present in higher amounts in plasma from patients with Crohn's disease (n=5) than in control plasma (n=12). In a previous study, PAGE immunoblot experiments showed that part of the prekallikrein was removed along with IgG when a higher plasma dilution (1+10.8) was used (Scand J Clin Lab Invest 1999; 59: 55-64). This observation was supported by results in the present work based on parallel assays with the peptide substrates S-2302 and Bz-Pro-Phe-Arg-pNA. The prekallikrein fraction removed was present in a functional state differing from the main part of prekallikrein by yielding kallikrein with a significantly increased activity against the substrate S-2366. This prekallikrein fraction was present in higher amounts in patient plasma than in control plasma. Part of the corresponding amidase activity was blocked by lima bean trypsin inhibitor, suggesting its presence in association with factor XI. The results also indicated that prekallikrein activator activity was connected with this fraction. With the high dilution procedure an extensive removal of IgG from the patient plasma was obtained compared to the control plasma.

Part of prekallikrein removed from human plasma together with IgG--immunoblot experiments and functional tests.

With the present study, evidence is provided that prekallikrein (PK) in human plasma might be present in two different states, one of them removed along with IgG on Protein G columns. At a plasma dilution of 1 + 2.5, small amounts of an IgG fraction were left in plasma along with all of the PK. At a dilution of 1 + 11, nearly all IgG was removed. The removal in parallel of part of the PK was shown in immunoblot experiments and confirmed in amidase assays. One monoclonal antibody against PK (13G11) and two preparations of polyclonal antibodies were used for the immunoblot experiments. Different peptide substrates (S-2302, S-2222, Bz-Pro-Phe-Arg-pNA), along with protease inhibitors (soybean trypsin inhibitor, corn trypsin inhibitor, lima bean trypsin inhibitor) were used for the amidase assays. The amidase assays indicated that factors XII and XI were reduced by Protein G columns. In all experiments with extensive removal of IgG, protein recognized by the factor XII light chain mAb C6B7 was removed at the same time. This antibody preparation did not detect purified contact factors, but it did recognize a preparation of purified beta-FXIIa, and also significant amounts of protein present in plasma deficient in factor XII and not detectable in plasma deficient in PK. This protein accordingly seems to be connected with the PK fraction removed with IgG.

Significance of IgG for the activity of factor XII measured in human plasma.

The plasma level of factor XII (FXII) was measured in samples from healthy young men. The activated contact factor was assayed as prekallikrein activator (PKA), as S-2222 amidase, and in radial immunodiffusion tests. By removing the bulk of IgG on protein G columns before the activation procedure, the functional activities increased to about 135%. In such test preparations, PAGE immunoblot experiments with polyclonal antibodies against FXII showed, in addition to FXIIa (80 kD), a double band with a molecular weight of about 46 kD. This protein could also be detected with a light-chain-specific monoclonal antibody to FXII, but not with such an antibody directed against its heavy chain. The 46-kD band was also observed in plasma deficient in FXII. The amidase assays indicated that the minor part of FXIIa was present in some kind of association with another protease. To obtain a correct estimation of total FXIIa in the amidase assays a sufficiently high level of FXI was required compared to that of FXII. The PKA assays were generally carried out with a prekallikrein (PK) substrate containing IgG. By replacing this substrate by PK free from IgG additional PKA activity was observed, the activity appearing also in plasma deficient in FXII.

Amidolytic assay of factor XI in human plasma--significance of kallikrein for the activity measured.

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper (Thromb. Res. 74, 477-485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibitor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at -70 degrees C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

Contact factors in plasma from women on oral contraception--significance of factor XI for the measured activity of factor XII.

The plasma levels of contact activation factors were measured in women using a low estrogen dose oral contraceptive (OC). Basic values for factor XII (FXII), factor XI (FXI), prekallikrein (PK), and high molecular weight kininogen (HK) were obtained in immunoassays by comparing with control plasma. The plasma levels of FXII and PK were significantly increased in OC plasma, to 147% and 146% respectively, whereas no significant increase could be registered for FXI (106%) or for HK (107%). Functional assays carried out with different peptide substrates (S-2222 for FXIIa, and S-2222, S-2302 and Bz-Pro-Phe-Arg-pNA for kallikrein) showed increases in OC plasma to about 150% for both proteases, in accordance with results obtained in radial immunodiffusion (RID). However, when FXIIa was measured with the high molecular weight substrate PK, no significant increase could be registered. Further experiments suggested this result to be due to the low level of FXI present in OC plasma, as compared to the levels of FXII and PK. Assays were carried out in mixtures of test plasma (OC or control plasma) and plasma deficient in FXI or FXII. The results obtained suggested that FXIa was present in some kind of association with part of FXIIa and part of kallikrein present. At low concentrations of FXI the functional activity of FXIIa was reduced, and the assay data indicated that an appropriate level of FXI was required to obtain maximum rate of hydrolysis of prekallikrein by FXIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of an association between factor XII and kallikrein in human plasma--significance of storage of plasma and the functional state of plasma kallikrein.

In a previous study evidence was provided that zymogen FXII might associate with part of the kallikrein generated by acetone treatment of human plasma in the presence of benzamidine (Thromb. Res. 61, 123-133, 1991). Some results also suggested an increase in such a complex formation upon storage of plasma, and two questions were raised in the present study: Does kallikrein activated by acetone-treatment of plasma exist in modifications with different abilities to associate with FXII? And will -70 degrees storage of plasma increase the liability to complex formation? S-2302 amidase assays carried out in mixtures of normal plasma and plasma genetically deficient in prekallikrein (PK) suggested an inhomogeneity of the kallikrein generated. A minor and unstable part of it could be blocked by corn trypsin inhibitor, thus indicating the presence of an association with FXII. In fractions from gel filtration of acetone-activated plasma, kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassay, and FXII, PK and HK were studied in PAGE immunoblot experiments. When freshly collected plasma was used, an amidase double peak (mol. wts. 400 and 300 kD) indicated an inhomogeneity of the kallikrein present, HK being observed in both peaks. FXII eluted separately over a gel. mol. wt. range of 90-55 kD. When plasma was stored at -70 degrees for 10 months before use, the more low-molecular part of the kallikrein double-peak had disappeared and was recovered, in a highly unstable state, adsorbed to the column material together with HK and FXII. Accordingly both functional assays and the results of immunoblot experiments indicated an inhomogeneity of the kallikrein present, and also a tendency of the minor part of it to associate with FXII, a tendency increased upon storage of plasma at -70 degrees.

Contact activation factors in plasma from women using oral contraceptives--increased levels of factor XII, kinin-free high molecular weight kininogen and acetone-activated kallikrein.

The plasma levels of FXII, prekallikrein (PK) and high- and low molecular weight kininogens (HK and LK) were measured in women on low estrogen dose (30-40 micrograms ethinylestradiol) oral contraceptives (OC) and in controls. FXIIa was assayed in acetone-treated citrated plasma (CPL) with PK and the tetrapeptide S-2222 as substrates, and in acetone-treated citrated plasma with benzamidine (BPL) with PK as substrate. The level of FXII was found to be about 20% higher in OC-plasma than in control plasma. Kallikrein was assayed in CPL with S-2222 as substrate and in BPL with the tripeptide S-2302 as substrate. No difference in PK-level was observed in the CPL-based method, whereas an increase in kallikrein activity of about 30% was registered in BPL. The levels of HK and LK were estimated both in rocket immunoassay and in bioassay of released kinin. No difference in HK-level could be registered in immunoassay, whereas the bioassay revealed a HK-level in OC-plasma of 40% of the control level. Immunoblot studies showed that a substantial part of HK in OC-plasma was present as kinin-free protein (mol. wt. 103 KD), assumed to possess a higher cofactor potency than that of native HK. Both in bioassay and immunoassay the level of LK was found to be 60% higher in OC-plasma than in control plasma. Considered together the observations on contact factors made in this study provide support for the assumption of an increased readiness for contact activation in plasma from women using OC.

Contact activation factors in plasma from pregnant women--increased level of an association between factor XII and kallikrein.

The plasma levels of FXII, prekallikrein (PK), and high- and low molecular weight kininogens (HK and LK) were studied in pregnant women in the last trimester and in non-pregnant controls. FXIIa and plasma kallikrein were assayed in acetone-treated citrated plasma (CPLa) with the tetrapeptide S-2222 as substrate, using soybean trypsin inhibitor and corn inhibitor to exclude kallikrein and FXIIa respectively. No difference in PK-level could be registered for the two kinds of plasma, but the level of FXII had increased to about 150% in the pregnancy plasma. No difference in HK-level was observed, whereas the LK-level was significantly higher in pregnancy plasma, about 250% and 160% in rocket immunoassay and bioassay respectively. In fractions from gel filtration of plasma acetone-activated in the presence of benzamidine (BPLa), kallikrein was assayed as S-2302 amidase, HK and LK were measured in rocket immunoassay, and HK and FXII were studied in PAGE immunoblot experiments. In contrast to previous results obtained upon gel filtration of CPLa, not only kallikrein and HK, but in addition also FXII now appeared together in the same fractions and as two separate peaks. One peak eluting in early fractions (gel mol. wt. 300-400 KD), and one late eluting peak of proteins adsorbed to the gel material. The first peak was notably marked in pregnancy plasma. The results provide support for the assumption of an association in plasma between the three contact activation factors studied.

Functional correlation between kallikrein and factor XII activated in human plasma.

Plasma kallikrein and FXIIa were assayed in acetone-treated human citrated plasma (CPLa) with the chromogenic peptide Bz-Ile-Glu-Gly-Arg-pNA (S-2222) as substrate. In end point assays with short incubation periods (1-10 min.) nearly all kallikrein present could be blocked by a low concentration of soybean trypsin inhibitor (STI). In 30 min. assays the main part of the kallikrein was recovered in a functional state not inhibited by STI, and at the same time the level of FXIIa (as amidase activity blocked by corn inhibitor, C.I.) was reduced to about 2/3 of the initial value. The formation of an association between FXIIa and kallikrein is suggested. In fractions from gel filtration of CPLa kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassays, and HK and FXII were studied in PAGE immunoblot experiments. Kallikrein appeared as one peak together with HK (gel mol. wt. 300 KD), about 40% of HK was free (220 KD), and no FXII was observed in the kallikrein or HK peaks, but in two areas corresponding to 78-79 KD and 39-42 KD. When experiments, however, were carried out with plasma acetone-activated and gel filtered in the presence of benzamidine (5 mM), part of the amidase activity present in kallikrein peak fractions was blocked by C.I. This observation supports the above suggestion of an association between FXIIa and kallikrein.

Assay of factor XII in human plasma using prekallikrein or the chromogenic peptide S-2222 as substrates--significance of the functional state of plasma kallikrein.

Factor XII was assayed in acetone-treated and kaolin-activated human citrated plasma (total plasma dilution 1.0 + 0.3 v/v during activation with kaolin, 1.8 mg/ml incubate). Measurements were performed with the tetrapeptide Bz-Ile-Glu-Gly-Arg-pNa (S-2222) and with prekallikrein as substrates. The correlation of both methods to another S-2222 based method recently described was good, r = 0.90 and 0.85 for the two methods respectively. Under the assay conditions used, FXIIa was present as a S-2222 amidase that could be blocked by corn inhibitor, whereas plasma kallikrein was found to be present partly as an amidase blocked by a low concentration of soybean trypsin inhibitor, and partly in a functional state not inhibited and adding to the measured level of FXII. The presence of benzamidine 0.7 to 2.1 mM during acetone treatment increased the measured level of FXII assayed both as prekallikrein activator and as S-2222 amidase.

Rocket immunoassay of high and low molecular weight kininogens in human plasma.

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) in human plasma could be rapidly (36 min.) separated on a DEAE-Sepharose Fast Flow column (1.0 x 5 cm and 0.50 ml plasma) by applying a NaCl step gradient. Quantification was then carried out by the Laurell rocket method with an antiserum raised against HMWK. Standard preparations for the assays were (I) crude HMWK and (II) crude LMWK prepared by the one-step procedure mentioned. In disc PAGE (8% with 0.1% SDS) immunoblot showed two main bands in I, migrating to apparent mol.wts. of 180,000 and 120,000. The 180,000 band predominated in native plasma. Purified HMWK (spec.act. 14 micrograms bradykinin/A 280) yielded in addition a band corresponding to a mol.wt. of 100,000. Immunoblot of II showed one broad zone over the mol.wt. range 65-70,000. The average assay values obtained in human plasma specimens from 10 males were 85 micrograms/ml for HMWK (range 65-130) and 174 micrograms/ml for LMWK (range 164-183). HMWK occasionally lost immunoreactivity during purification without a corresponding loss of kinin. Such a loss of immunoreactivity seemed to run parallel with a reduced release of kinin induced by hog pancreas kallikrein.