Isolation and characterization of serum procalcitonin from patients with sepsis
Introduction
Levels of procalcitonin (PCT) and other calcitonin precursors are elevated in many conditions leading to systemic inflammatory response syndrome (SIRS), like bacterial infection [2], pancreatitis [4], [14], burns [12] or polytrauma [11]. Since PCT levels correlate with the severity of bacterial infection, PCT has been established over the last years as a useful marker for the diagnosis and therapy monitoring of sepsis, severe sepsis and septic shock of bacterial origin [5], [8], [15], [16]. The pathophysiological background of elevated PCT during severe bacterial infection is still unknown. Unpublished data from our laboratory show, that the rise in PCT concentration is due to an increased production in several non thyroidal tissues during sepsis (e.g. liver). The detection of PCT in the clinical routine is usually performed by a two-site immunometric chemiluminescence assay (ILMA), which has a functional assay sensitivity of 300 pg/ml.
In addition to this diagnostic value, experiments using a hamster model showed that increased PCT concentrations exacerbate mortality, whereas immunoneutralization employing an anti-calcitonin antibody increases survival [13]. Therefore it is of interest to characterise the structure of serum PCT from septic patients. This was done so far only by size exclusion chromatography and by binding of antibodies that have been raised against peptides representing the calcitonin-, katacalcin-, and N-terminal moiety of PCT [3]. Deduced from that it was suggested that PCT is encoded by the CALC-1 gene. Its cDNA sequence predicts a primary translational product of 141 amino acids, with potential processing by the signal peptidase to a final size of 116 amino acids. Recently we could demonstrate the in vitro cleavage of the first two N-terminal amino acids of recombinant PCT 1–116 by dipeptidyl peptidase IV (DPIV/CD26) [17].
While it has been speculated that PCT might be glycosylated [9] no data exist on post translational modifications of PCT [3]. It is also unknown, whether PCT contains an internal disulphide bond as does mature calcitonin.
It is important to elucidate the structure of PCT as a basis for i) understanding the function of PCT, ii) developing therapeutic intervention strategies and iii) developing novel diagnostic tests for PCT. We describe here the isolation of PCT from sera of septic patients and determination of the primary structure by mass spectrometry and N-terminal sequencing.
Section snippets
Serum samples
Sera containing high PCT-concentrations (>100 ng/ml) were collected from patients (n = 22) with severe sepsis according to the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference [1] following ethical guidelines, and all serum samples were frozen immediately at -20°C. Prior to analysis samples were pooled (final volume: 67.8 ml), filtered through a 0.45 μm filter and PCT immune reactivity (PCT-IR) was measured by using LUMItest PCT BRAHMS Diagnostica
Purification of serum PCT
Sera with high procalcitonin immune reactivity (PCT-IR) were collected from patients with severe sepsis. After pooling, a concentration of 12.7 μg PCT in 67.8 ml was obtained. The pooled sera were diluted, and after addition of protease inhibitors applied onto a CT21-immunoaffinity column. After washing the column, bound antigen was eluted by pH change and the fractions containing PCT-IR were pooled (2.5 ml containing 8.2 μg PCT-IR: 65% recovery). The affinity prepared material was further
Discussion
Our knowledge on the structure of circulating procalcitonin in septic patients is based so far mainly on hypotheses. For developing solid strategies to therapeutically neutralise endogenous PCT and to quantitate PCT, the structure of PCT must be known. Therefore we isolated PCT from a pool of sera from septic patients and determined its primary structure.
References (17)
- et al.
High serum procalcitonin concentrations in patients with sepsis and infection
Lancet
(1993) - et al.
CD26, let it cut or cut it down
Immunol Today
(1999) - et al.
Procalcitonin as a marker for the early diagnisis of neonatal infection
J Pediatr
(1996) - et al.
Procalcitonin is a glycoprotein
J Biol Chem
(1981) - et al.
Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections by dipeptidyl peptidase IV (DPIV)
FEBS Lett
(2000) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis
Crit Care Med
(1992)- et al.
Calcitoninstructure, molecular biology, and actions
- et al.
Early identification of biliary pancreatitis with procalcitonin
Am J Gastroenterol
(1998)
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Performance of serum procalcitonin as a biochemical predictor of death in hematology patients with febrile neutropenia
2021, Blood Cells, Molecules, and DiseasesCitation Excerpt :Several biomarkers are being studied to determine etiology and prognosis in this oncologic emergency. One of these is procalcitonin (PCT), a polypeptide initially described as a prohormone of calcitonin that appears in the onset of an acute inflammatory response [5,6]. PCT is important in the management of antibiotics in several conditions, like community acquired pneumonia, lower respiratory tract infection, sepsis and septic shock, and has also been used as a predictor of outcome in several conditions [7–10].
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2021, Clinica Chimica ActaCitation Excerpt :In these conditions, an increase up to 1100 folds of PCT concentration from the basal concentration was observed [32,33], and PCT exists in three different isoforms in the bloodstream: intact PCT (1-116), PCT (2-116) in which the first amino acid is cleaved, and PCT (3-116) in which the first two amino acids are cleaved. Among these forms, PCT (3-116) has been reported as the predominant form but little is known about the clinical significance and role of the different forms [34,35]. Interestingly, PCT concentration remains low in the case of viral infections and localized bacterial infections, which allows distinguishing between viral and bacterial infection [36,37].
Undue elevation of procalcitonin in pediatric paracetamol intoxication is not explained by liver cell injury alone
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C reactive protein and procalcitonin: Reference intervals for preterm and term newborns during the early neonatal period
2011, Clinica Chimica ActaCitation Excerpt :The entire literature on PCT has long assumed a size of 116 aminoacids, PCT 1–116, a molecule with two additional aminoacids (Ala-Pro) at the N-terminal which can be cleaved by dipeptidyl peptidase IV leading to N-terminal truncated PCT 3–116. In 2001, Weglöhner et al. showed by mass-spectrometric analysis that in sera from septic patients with high PCT immune reactivity, the truncated form PCT 3–116 was the major circulating form [30]. Nonetheless, the function of serum PCT 3–116 in septic and healthy status as well as the importance of the N-terminal truncation are still unknown.
Calcitonin peptides - The mediators in sepsis or just another fairy tale?
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