High concentrations of N-BNP are related to non-infectious severe SIRS associated with cardiovascular dysfunction occurring after off-pump coronary artery surgery.

BACKGROUND: Procalcitonin (PCT) blood concentrations are known to be an appropriate marker of severe systemic inflammatory response syndrome (SIRS) induced by coronary artery surgery with and without cardiopulmonary bypass. Pro-brain natriuretic peptide (N-BNP) is a newly described cardiac hormone considered to be an effective marker of severity and prognosis of acute coronary syndromes and congestive heart failure. We evaluated the perioperative time courses of PCT and N-BNP and investigated their role as early markers of severe SIRS (SIRS with cardiovascular dysfunction) induced by off-pump coronary artery bypass (OPCAB). METHODS: Sixty-three patients were prospectively included. The American College of Chest Physicians Classification was used to diagnose SIRS and organ system failure to define severe SIRS. Serum concentrations of PCT and N-BNP were determined before, during and after surgery. Receiver operating characteristic curves and cut-off values were used to assess the ability of these markers to predict postoperative severe SIRS. RESULTS: SIRS occurred in 25 (39%) patients. Nine of them (14%) showed severe SIRS. Significantly higher serum concentrations of N-BNP and PCT were found in patients with severe SIRS with peak concentrations respectively at 8887 pg ml(-1) (range 2940-29372 pg ml(-1)) for N-BNP and 9.50 ng ml(-1) (range 1-65 ng ml(-1)) for PCT. The area under the curve using N-BNP to detect postoperative severe SIRS was 0.799 before surgery (0.408 for PCT; P<0.01) and 0.824 at the end of surgery (0.762 for PCT; P<0.05). CONCLUSIONS: N-BNP may be an appropriate marker indicating the early development of non-infectious postoperative severe SIRS after OPCAB.

Increased plasma levels of pro-brain natriuretic peptide in patients with cardiovascular complications following off-pump coronary artery surgery.

OBJECTIVE: To compare N-terminal pro-brain natriuretic peptide (NT-pro-BNP), procalcitonin (PCT), and troponin I (Tn I) concentrations during and after coronary artery surgery in patients with or without cardiovascular complications. DESIGN AND SETTING: Prospective, comparative study of 12 months in the cardiovascular intensive care unit in a university hospital. PATIENTS: 60 adult patients undergoing coronary artery bypass grafting with the off-pump technique. MEASUREMENTS AND RESULTS: Plasma NT-pro-BNP, PCT, and Tn I levels were measured before and immediately after the end of operation and on PODs 1, and 2 and 3. We defined complicated postoperative course as myocardial infarction, cardiogenic shock, arrhythmias, congestive heart failure, and death occurring after the fourth postoperative hour. Receiver operating characteristic (ROC) curve cutoff values were used to assess the ability of the three markers to predict future cardiac events. The area under ROC curve (AUC) using NT-pro-BNP to detect a cardiovascular complicated course was 0.780 at the preoperative time and 0.850 at the end of surgery. A preoperative NT-pro-BNP value of 397 pg/ml had a sensitivity of 76%, specificity of 67%, and accuracy of 74% for predicting a subsequent cardiovascular complication. An immediate postoperative NT-pro-BNP value of 430 pg/ml had a sensitivity of 80%, specificity of 77%, and accuracy of 76%. Patients with preoperative NT-pro-BNP levels less than 275 pg/ml had an excellent postoperative prognosis. Other two markers were less appropriate. CONCLUSIONS: NT-pro-BNP levels measured before and immediately after off-pump coronary artery bypass seem to be predictive of postoperative cardiac events.

Thyroglobulin epitope recognition in a post iodine-supplemented Sri Lankan population.

OBJECTIVE: We previously reported a high prevalence of raised thyroglobulin autoantibodies (TgAb) in apparently healthy Sri Lankan schoolgirls following salt iodination. To characterize these antibodies further we determined the epitopes on thyroglobulin (Tg) with which they react and compared these with serum obtained from both healthy subjects and established autoimmune thyroid disease (AITD) patients from the UK. To extend our study to a wider population within Sri Lanka, we in addition determined the epitopes recognized by a group of AITD patients selected from a thyroid clinic in Sri Lanka, as well as apparently healthy female Sri Lankan tea workers of distinct ethnicity from the schoolgirls and AITD patients. DESIGN: Sri Lankan schoolgirls (n = 282) and adult female tea estate workers (n = 208) were examined for thyroid autoimmune markers. Sera with high TgAb (> 98 kIU/l) were selected from these two groups (n = 36 and 45, respectively) to study epitope-binding patterns. We also examined the sera from 16 AITD patients attending a thyroid clinic in Colombo, 16 patients with AITD from the thyroid clinic at the University Hospital of Wales and 16 sera from healthy control UK women with no evidence of thyroid disease. To determine the epitopes on Tg recognized by the subjects' TgAb, we employed a panel of Tg mouse monoclonal antibodies labelled with alkaline phosphatase in a competitive enzyme-linked immunosorbent assay reaction with the subjects' serum. RESULTS AND CONCLUSIONS: A majority of the Sri Lankan schoolgirls did not react with the immunodominant epitopes and did not differ significantly from healthy subjects from the UK in their Tg epitope recognition pattern. On the other hand, tea estate workers and Sri Lankan AITD patients recognized typical autoimmune thyroid disease epitopes and, in addition, recognized a separate cluster not previously associated with either the autoimmune state or the healthy state. The significance of this cluster requires further clarification.

Hyperprocalcitonemia is related to noninfectious postoperative severe systemic inflammatory response syndrome associated with cardiovascular dysfunction after coronary artery bypass graft surgery.

OBJECTIVE: To investigate the role of 3 inflammatory parameters as early markers of severe systemic inflammatory response syndrome (SIRS) induced by coronary artery bypass graft surgery. DESIGN: Prospective study. SETTING: University hospital. PARTICIPANTS: Patients (n = 63) undergoing elective coronary artery bypass graft surgery with cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: The American College of Chest Physicians/Society of Critical Care Medicine classification was used to diagnose SIRS. Organ system failures were used to define severe SIRS. Serum concentrations of the inflammatory parameters (procalcitonin [PCT], C-reactive protein, leukocyte count) were determined before, during, and after surgery. SIRS occurred in 30 (47%) patients after surgery. Seven patients (11%) showed SIRS with greater-than-or-equal1 organ dysfunction (severe SIRS), whereas patients without SIRS had no organ dysfunction. Significantly higher serum levels of PCT were found in patients with severe SIRS from the 6th postoperative hour until the 3rd postoperative day with a peak level of 10.7 plus minus 13.2 ng/mL. No significant difference was detected between serum PCT of patients with SIRS but without any organ dysfunction and patients without SIRS. PCT levels of these patients remained lower than 1.7 ng/mL. Compared with PCT, plasma concentrations of C-reactive protein peaked later on the 2nd postoperative day and were not able to confirm the severity of SIRS. Leukocyte counts were not significantly modified. CONCLUSIONS: PCT seems to be an appropriate marker to identify the early development of noninfectious postoperative severe SIRS after coronary artery bypass graft surgery with cardiopulmonary bypass.

Multicenter study on TGPO autoantibody prevalence in various thyroid and non-thyroid diseases; relationships with thyroglobulin and thyroperoxidase autoantibody parameters.

OBJECTIVE: TGPO autoantibodies (aAbs) that bind simultaneously to thyroglobulin (Tg) and thyroperoxidase (TPO) are present in the serum of patients with autoimmune thyroid diseases (AITD) and have been found to differ from monospecific Tg and TPO aAbs. To obtain further insights on the prevalence defined as the rate of occurrence and significance of TGPO aAbs in a large population, we carried out a collaborative study involving 15 European teams. METHODS: Serum samples from 3122 patients with various thyroid and non-thyroid diseases and normal subjects were assayed using a novel TGPO aAb detection kit. This test was designed so that TGPO aAbs are trapped between the Tg-coated solid phase and the soluble TPO labeled with a radioiodinated monoclonal antibody. RESULTS: Only three out of the 220 normal subjects (prevalence of 1.4%) were found to have positive TGPO aAb levels, which were mainly observed in the patients with AITD: the group of patients suffering from Hashimoto's thyroiditis had a TGPO aAb prevalence of 40.5% (n=437 patients), those with Graves' disease, a prevalence of 34.6% (n=645) and those with post-partum thyroiditis, 16.0% (n=243). Among the non-AITD patients with positive TGPO aAb levels, the TGPO aAb prevalence ranged from 20.7% among those with thyroid cancer (n=246) to 0% among those with toxic thyroid nodules (n=47). Among the patients with non-thyroid diseases, the TGPO aAb prevalence ranged from 9.8% in the case of Biermer's pernicious anemia (n=78) to 0% in that of premature ovarian failure (n=44). It is worth noting that the groups showing the highest TGPO aAb prevalence also contained the patients with the highest TGPO aAb titers. Statistical comparisons between the TGPO aAb prevalences in the various groups showed that TGPO aAb could be used as a parameter to distinguish between the groups of Hashimoto's and Graves' patients and between the women with post-partum thyroiditis and the post-partum women with only Tg and/or TPO aAb established during early pregnancy. Unexpectedly, the correlations between TGPO aAbs and Tg and TPO aAbs were found to depend mainly on the assay kit used. CONCLUSION: High TGPO aAb titers are consistently associated with AITD but the reverse was not found to be true. TGPO aAbs are a potentially useful tool, however, for establishing Hashimoto's diagnosis, and would be worth testing in this respect with a view to using them for routine AITD investigations.

Interest of epitopic dissection in immunoanalysis of proteins and peptides: review of theoretical and practical aspects.

The literature abounds with reports showing discrepancies in immunoassays of proteins and peptides. Whereas the isomorphism and polymorphism of proteins remains largely hidden in immunoassays making use of polyclonal antibodies, the use of monoclonal antibodies uncovered the difficulty of accurately assaying microheterogeneous analytes. Indeed, most proteic hormones are not entities with unique structures but rather mixtures of molecular forms with slight differences in structure which may reflect large variations in biological and immunological activities; the monoclonal antibodies appeared clearly less suited than the polyclonal for testing a mixture of isoforms. Protein microheterogeneity also has an impact on assay standardisation, since reference preparations may contain several isoforms of the analyte. Using recombinant glycoprotein does not solve the problem. Regarding the problem of discrepancy in immunoanalysis of proteins and peptides, we could establish, in a previous work, that discrepancy among lutropin assay kits may be related to various causes: i) differences in standard preparation and calibration curves; ii) microheterogeneity of lutropin molecules leading to missing some isoforms due to the restricted epitopic specificity of the monoclonal antibodies used in the kits. The epitopic dissection we engaged in appeared thus instrumental in explaining these discrepancies. It allowed us to enumerate epitopes on the surface of lutropin molecules, to elucidate the immunological structure and, finally, to characterize monoclonal antibodies used in commercially available lutropin assay kits with regard to their epitopic specificity. This work allowed us to interpret the discrepancy in serum lutropin concentration which was related to the use of monoclonal antibody with given specificity. Epitopic dissection may thus be instrumental in explaining discrepancy among immunoassays of proteins and peptides and in improving the accuracy of kits.

[Hypercalcitoninemia in conditions other than medullary cancers of the thyroid]. / Les hypercalcitoninémies en dehors des cancers médullaires de la thyroïde.

Serum calcitonin (CT) assays are the most useful tumoral marker for the diagnosis and follow up of medullary thyroid carcinoma (MTC). Since 1988 the sensitivity and specificity of CT assays have been considerably improved. Normal basal and pentagastrin (Pg) stimulated CT ranges remain to be established and it appears necessary to determine the pathological circumstances which may be responsible for hypercalcitoninemia in addition to MCT. By reviewing literature and data from the "Groupe d'Etude des Tumeurs à Calcitonine": a/we compared basal and Pg stimulated CT values obtained with two commercially available immunometric CT assays and we observed that CT values measured by the CT-EASIA MEDGE-NIX kit were three fold the values obtained by suing the hGH ELSA CIS BIOINDUSTRIE Kit; b/we determined that hypercalcitoninemia may be observed in isolated C Cell Hyperplasia (HCC) surrounding either lymphocytic thyroiditis or follicular thyroid carcinoma loci, in chronic renal failure on maintenance hemodialysis, and in various neuroendocrine tumors. Surprisingly, the hypercalcitoninemia related to HCC has been found in genetically unaffected members (without any identified gene RET mutation) of both a Multiple Endocrine Neoplasia type 2A and isolated familial hereditary MTC.

Abnormal calcitonin basal levels and pentagastrin response in patients with chronic renal failure on maintenance hemodialysis.

Hypercalcitoninemia has been reported in renal failure. Using a specific monomeric calcitonin (CT) immunoassay, we measured CT levels in 154 hemodialyzed patients. The relationship between CT and serum intact parathyroid hormone (PTH), gastrin, alkaline phosphatases, phosphate and calcium was studied. The pentagastrin test was performed in 26 patients exhibiting basal hypercalcitoninemia. Basal CT levels over 5.7 pmol/l (20 ng/l) were found in 25.3% of the patients and values higher than 26 pmol/l (90 ng/l) in 7.8%. Although CT is cleared by hemodialysis, post-dialysis CT levels either were unchanged or increased as compared with pre-dialysis values. This suggests that hypercalcitoninemia is not related to a decreased renal clearance, and that hemodialysis induces a specific regulatory pathway. None of the parameters studied were found to explain high CT levels. Of the patients with hypercalcitoninemia, 11.5% exhibited abnormal CT response to pentagastrin but no relationship between CT and phosphate, calcium and PTH levels was evidenced. Our findings confirm high CT monomer levels in renal failure. As there was no correlation with parameters classically involved in CT regulation, its physiological significance remains unclear. Abnormal CT response to pentagastrin raises the problem of its specificity as a tumoral marker with regard to medullary thyroid carcinoma.

Hormone formation in the isolated fragment 1-171 of human thyroglobulin involves the couple tyrosine 5 and tyrosine 130.

The 22 kDa fragment (Asn1-Met171) purified from iodine-poor human thyroglobulin (hTg) is capable by itself to synthesize thyroxine at Tyr5, the preferential hormonogenic acceptor site of the protein, after iodination in vitro. To identify the corresponding donor site in this model we studied the fate of the six Tyr residues present in the 22 kDa peptide after in vitro hormone synthesis. Structural studies of the tyrosyl peptides showed that Tyr5 was the only thyroxine-forming site, the other tyrosines (29, 89, 97 and 107) were noniodinated and Tyr130 was recovered in alanine form after CNBH4 treatment of the Tyr130-containing peptide. Taking into account that alanine could arise from aminoreduced pyruvate species, these results showed that in the 22 kDa fragment (1) hormone formation involves the couple Tyr5 (acceptor)-Tyr130 (donor), and (2) dehydroalanine, the resultant product of donor tyrosine after hormone synthesis, has evolved in pyruvoyl form. To test whether Tyr130 could also act as donor in hTg hormone synthesis, the 22 kDa peptide was isolated from hTg iodinated under conditions leading to iodotyrosine formation followed or not by hormone formation and the tyrosyl peptides were analyzed. After hTg iodination and before coupling (i.e. hormone synthesis) only Tyr5 and Tyr130 were recovered in iodotyrosine form; after coupling thyroxine was found at Tyr5 whereas Tyr130 disappeared. Taken together these results, correlated with the previously reported cleavage of hTg chain at Tyr130 occurring during in vivo hormone synthesis, support the theory that the couple Tyr5 (acceptor)-Tyr130 (donor) would be the preferential hormonogenic site in human Tg.

Hormone synthesis in human thyroglobulin: possible cleavage of the polypeptide chain at the tyrosine donor site.

At moderate iodination levels (20 iodine atoms/mol) human thyroglobulin (hTg) produces after reduction a hormone-rich peptide of 26 kDa which contains the preferential hormonogenic 'acceptor' tyrosine (Tyr 5) of the protein. The site of cleavage of the hTg chain was demonstrated by analysis of the 26 kDa tryptic hydrolysis products. It consistently yielded the peptide Gln-82-Val-129 which consequently made it possible to localize the hTg chain cleavage at tyrosine residue 130. Evidence for tyrosine involvement in hTg cleavage during thyroid hormone formation supports the hypothesis that peptide bond cleavage would occur at the 'donor' tyrosine residue and suggests that tyrosine 130 would be the donor site reacting with the major hormone-forming acceptor site (Tyr 5) of hTg.

Characterization of a hormonogenic domain from human thyroglobulin.

A polypeptide domain of molecular mass near 22 kDa was purified from CNBr-digest of iodine poor human thyroglobulin (hTgb). This fragment represents the N-terminal part of the hTgb molecule and consequently contains the preferential hormonogenic tyrosine 'acceptor' of the protein. This fragment could correspond to the non-iodinated and unreduced form of the thyroxinyl-containing 26 kDa peptide previously purified from reduced and iodinated hTgb. This 22 kDa fragment is capable by itself, i.e. independently of the remaining hTgb molecule, of synthesizing thyroxine with a high efficiency after in vitro iodination. Its study should constitute a valuable way to identify at least one of the hormonogenic tyrosine 'donor' residues of hTgb.

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin.

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.

[Comparative study of radioimmunologic and immunoenzyme methods for the determination of alpha feto-protein]. / Etude comparative des méthodes radio-immunologiques et immunoenzymologiques de dosage de l'alpha foeto-protéine.

Estimation of alpha-protein (AFP) in human serum using three types of immunoenzymatic kits has given similar results. A comparative study concerning 60 sera has shown statistically significant correlations between enzyme immunoassays and radioimmunoassay (RIA): r = 0,98, p less than 10(-6). AFP estimations by EIA and RIA seem to be equally used.

[Relation between the iodination of human thyroglobulin and the cleavage of the hormone peptide 26K N-terminal]. / Relation entre l'iodation de la thyroglobuline humaine et le clivage du peptide hormonogénique 26K N-terminal.

At moderate iodination levels (about 20 iodine atoms/mol) human thyroglobulin yields after reduction and alkylation a hormone (T4)-containing N-terminal peptide of 26K. Further iodination of the thyroglobulin in vitro results in the cleavage of this part of the molecule into smaller peptides of 22K and 18K. A precursor-product relationship between the 26K peptide segment and the latter was established by showing an identical N-terminal T4-containing sequence in the 3 peptides. Cleavage of peptide bonds in the 26K segment to give the smaller fragments could possibly be related to the formation of another hormone residue.

[Comparison of quantity of thyroxine in thyroglobulin and the peptide hormones derived from chromatography and immunoenzyme assay]. / Comparaison du dosage de la thyroxine dans la thyroglobuline et les hormonopeptides dérivés par chromatographie et par immunoenzymologie.

An evaluation of the thyroxine content in thyroglobulin and different derived thyroxine-containing peptides by enzyme immunoassay is presented. Results correlate very well with those obtained by ion-exchange chromatography which served as a reference method: r = 0.997, p less than 10(-6). Enzyme immunoassay is more rapid and sensitive than ion-exchange chromatography and then suitable for routine use. Furthermore, this paper shows, for the first time, that enzyme immunoassay can be used to perform direct thyroxine estimation in small peptide (Mr less than 2,500) without previous total enzymatic hydrolysis.

In vitro and in vivo iodination of human thyroglobulin in relation to hormone release.

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.