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Original Studies |
Georgetown University (K.T.W.), Veterans Affairs Medical Center Surgery Service and Surgical Intensive Care Unit (K.T.W., J.C.W.), Veterans Affairs Medical Center Medical Service and Endocrinology Section (E.S.N., R.H.S., K.L.B.), and George Washington University (P.M.S., J.C.W., E.S.N., G.L.S., R.L.G., K.L.B.), Washington, D.C. 20422
Address all correspondence and requests for reprints to: Kevin T. Whang, M.D., University of California-Davis, 4860 Y Street, Suite 3100, Sacramento, California 95817. E-mail: ktwhang{at}yahoo.com
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Abstract |
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Twenty-nine patients meeting criteria for the systemic inflammatory response syndrome were studied daily in two intensive care units. Sera were collected, and APACHE II scores were determined until recovery or death. All patients had markedly elevated serum pre-CT. Prognostically, peak values were the most important. The highest values portended mortality, and a lower level could be ascertained below which all patients survived. Peak pre-CT levels were significantly higher in patients with infection documented by blood cultures than in those patients with no documented infection from any source (P < 0.05). Mature CT remained normal or only moderately elevated. Compared with the serum pre-CT levels, receiver operating characteristic curve analysis revealed that the APACHE II scores, although more cumbersome, were better overall predictors of mortality.
Thus, pre-CT is an important serum marker for systemic inflammatory response syndrome and is predictive of outcome. It also provides data concerning the presence of severe infection and may prove to be clinically useful for proactive patient care.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Mature calcitonin (CT) is a 32-amino acid peptide hormone involved in
skeletal homeostasis. The primary physiological role of CT appears to
be the maintenance of body calcium stores by attenuation of osteoclast
activity (3, 4). However, biologically relevant effects may also occur
in the kidney, the central nervous system, the respiratory tract, the
gastrointestinal tract, and the reproductive system (5, 6). The
prohormone precursor for CT is the 116-amino acid polypeptide,
procalcitonin (pro-CT), which contains three component peptides: 1) a
57-amino acid peptide at the amino-terminus, named aminoprocalcitonin
(aminopro-CT); 2) a centrally placed 33-amino acid immature CT; 3) and
a 21-amino acid CT carboxyl-terminus peptide-I (CCP-I; Fig. 1
) (6). The physiological activities of
these precursors are not well understood, but small amounts of pro-CT,
aminopro-CT, CCP-I, and the conjoined CT:CCP-I peptide are found in the
peripheral circulation of normal subjects (7). As none of the currently
used assays for these peptides are completely specific for any single
component of the pro-CT molecule, the term precalcitonin peptides
(pre-CT) refers to all moieties that are detectable by these assays
(7).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The clinical progress was followed, and serum was obtained daily from blood already taken for routine clinical care until each patient recovered or died. The APACHE II score was also determined at each time point that serum was obtained (19). The control group for pre-CT determinations consisted of 24 healthy volunteers, aged 21–45 yr, taking no medications.
A total of 300 serum samples were obtained, and the following immunoassays were performed.
Two-site assay (CCP-I capture antibody, CT tracer antibody)
This dual site sandwich immunochemiluminescent assay uses two monoclonal antibodies, one for the CCP-I portion and the other for the CT portion of the pro-CT molecule (Brahms Diagnostica, Berlin, Germany). It is performed in 3 h. The assay detects the intact pro-CT molecule as well as the free conjoined CT:CCP-I peptide; however, it does not distinguish between the two. The reliable sensitivity is 300 pg/mL. The serum pre-CT levels of normal subjects are below the sensitivity of this assay (7).
Aminopro-CT assay
This RIA uses a polyclonal antiserum, R2B7, which is specific for synthetic aminopro-CT. R2B7 is preincubated with standards or unknowns (10–100 µL) in 0.2 mL at 4 C for 16 h. [125I]Aminopro-CT and 50 µL goat anti-rabbit IgG bound to iron particles are added and incubated at room temperature in an orbital shaker (200 rpm) for 3 h. Bound and free hormone are separated with magnetic tube racks. The complete assay is performed in 19–22 h; however, a modified assay can be performed in considerably less time. It detects the free aminopro-CT, the intact pro-CT, and pro-CGRP molecules, but does not distinguish among them. The reliable sensitivity is 4 pg/mL, and the 50% bound/free ratio is 140 pg/mL. With this assay, serum pre-CT levels in normal subjects are usually detectable.
Mature CT assay
This immunochemiluminescent assay using a biotin-coupled antibody directed against CT (amino acids 11–23) and an acridine ester-labeled tracer antibody directed against CT (amino acids 21–32) is performed in 4 h. Avidin beads are used for separation of bound and free tracer antibody. The assay is performed according to the manufacturer’s instructions (Nichols Institute Diagnostics, San Juan Capistrano, CA). It detects only mature CT and does not cross-react with any of the immature CT precursors or fragment peptides. The reliable sensitivity is 1 pg/mL. The manufacturer reports that normal men have serum levels less than or equal to 11.5 pg/mL (n = 72) with this assay, which we have confirmed.
Serum chemistry tests were performed in the Washington V.A. Medical Center diagnostic chemistry laboratory using a sequential multichannel analyzer (Hitachi 717, Boehringer Mannheim, Indianapolis, IN), and hematology tests were performed by the hematology laboratory using an automated cell counter (Coulter STAK-S, MAX-M, Ft. Lauderdale, FL).
The SigmaStat statistical and SigmaPlot graphing packages (Jandel Scientific, San Rafael, CA) were used to analyze and plot the experimental data. Receiver operating characteristic (ROC) curves were generated using software from MedCalc (Belgium). Among statistical tests applied were Student’s t test, Wilcoxon signed rank test, and Mann-Whitney rank sum test (when normality was violated). P < 0.05 was considered statistically significant. Values are expressed as the mean ± SEM, except when otherwise indicated.
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Results |
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). However, these three patients with
undetectable levels by the two-site assay had serum levels above the
upper limit of normal when determined by the aminopro-CT assay.
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The categories of systemic inflammation in this patient population were pancreatitis (n = 12), pneumonia (n = 8), and sepsis of nonpulmonary origin (n = 9).
With the two-site assay, there were no deaths for those patients whose
maximal serum pre-CT levels were below 2100 pg/mL (165 fmol/mL). In
contrast, the mortality rate was 65% during hospitalization for those
patients with a peak pre-CT level above 2100 pg/mL. With this assay,
evaluation of the ROC curve confirms that the best cut-off level for
predicting mortality is 2060 pg/mL (162 fmol/mL; Fig. 3A). At this value the sensitivity is
100%, and the specificity is 56%. Using, the aminopro-CT assay, the
cut-off value was 1980 pg/mL (320 fmol/mL), corresponding to a
sensitivity for mortality of 77%, and a specificity of 75% (Fig. 3B).
The similarity in results between the two-site and aminopro-CT assays
is not surprising, as there is a near-perfect correlation between the
two assays (Fig. 4).
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). Overall, the ROC curves for APACHE II and the serum pre-CT levels showed that APACHE II was the best predictor of mortality. The area under the curve (AUC) for APACHE II was 0.94 ± 0.05 compared to 0.77 ± 0.09 (AUC ± SE) for serum pre-CT measured by the two-site assay and 0.74 ± 0.10 measured by the aminopro-CT assay.
Using the aminopro-CT assay, the serum level in patients with a positive bacterial culture from any source was 3600 ± 1300 pg/mL, whereas the serum level in those patients who had no documented infection was 180 ± 60 pg/mL (P = 0.01, by Wilcoxon test). When considering only those patients with positive blood cultures, the serum pre-CT determined by the aminopro-CT assay was 5700 ± 2600 pg/mL compared to 1300 ± 600 pg/mL in those patients without positive blood cultures (P = 0.03, by Wilcoxon test). Of the nine patients with blood culture growth, five had Gram-negative bacteria, three had Gram-positive bacteria, and one had Candida albicans. ROC curve evaluation was also applied to the aminopro-CT assay for detection of infection; the best cut-off value was 680 pg/mL (110 fmol/mL), with a sensitivity of 67% and a specificity of 80%. For the two-site assay, the best cut-off value was 1080 pg/mL (85 fmol/mL), with sensitivity and specificity nearly identical to those of the aminopro-CT assay.
The correlation between the length of hospitalization of survivors and the serum pre-CT level determined by the two-site assay was nonsignificant (r = 0.47; P = 0.07). However, when determined by the more sensitive aminopro-CT assay, there was a strong, positive correlation with the length of the hospital stay (r = 0.81; P = 0.0001).
The time-course curves for the serum pre-CTs and the APACHE II scores
followed similar patterns, peaking simultaneously in the select
patients represented in Fig. 5. These
patients had excellent correlations between pre-CT levels and APACHE II
scores, as their peaks were discrete (r = 0.9); this correlation
held true for both of the pre-CT assays. However, this observation was
not obvious in those patients who did not have clearly distinguishable
peaks for either parameter.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Using a commercially available two-site kit, in our studies the assay sensitivity for serum pre-CT is unreliable below the level of 300 pg/mL. However, we have determined that normal serum levels of individual or combined pre-CT peptides are well below this level (7). In contrast, normal values of pre-CT are readily determined with our aminopro-CT assay. Thus, the development of a more rapid aminopro-CT assay should make it useful as a screening tool for the detection of early systemic inflammation/sepsis at a time when serum pre-CT levels are only moderately elevated above the normal range.
Studies by other investigators found that levels of serum pre-CT are related to the severity of illness when measured only once at admission (16). However, it was rare that a patient in this study manifested the highest serum level on the day of admission. The most important value was the peak level, and this correlated best with prognosis.
In this study the peak APACHE II score, although cumbersome, was a better overall predictor of mortality. Nevertheless, serum pre-CT levels provided rapid complementary and confirmatory data concerning prognosis. For example, an APACHE II score over 24 was associated with a 100% mortality, whereas pre-CT levels less than 2100 pg/mL determined by the two-site assay were associated with 100% survival. Thus, both parameters used together provided excellent prognostic information.
In addition, the serum pre-CT levels often reflected the patient’s daily clinical progress, suggesting improvement or deterioration in select patients who demonstrated discrete peaks in the serum levels. This relationship was based on a close parallelism with the APACHE II score. However, this could not be demonstrated in patients who had only modest elevations of serum pre-CTs without distinguishable peaks.
Most of the patients in this study had bacterial infections. Patients with viral infection commonly, but not always, have considerably lower serum pre-CT values (11, 15). This probably is related to the lower incidence of associated severe systemic inflammation in viral infection.
There was a pronounced difference in the levels of serum pre-CT between infected and noninfected patients in the present study. This has important implications in the decision to institute empiric antibiotic therapy. The highest serum pre-CT levels were found in bacteremic patients. The difference was most notable with the aminopro-CT assay, although the less sensitive two-site assay produced very similar results. Furthermore, the aminopro-CT assay was sensitive enough to detect milder inflammation of a noninfectious nature.
Another advantage of the aminopro-CT assay over the two-site assay was the better correlation of serum levels with length of hospitalization. Here, the lack of significant correlation using the two-site assay was due to its relative insensitivity. In contrast, the aminopro-CT assay was sensitive enough to detect the patients who had relatively small elevations of serum pre-CT in mild inflammation. These patients were inherently less ill and recovered more quickly, thus decreasing their hospitalization time.
The factors inciting the remarkable hypersecretion of CT prohormone in severe systemic inflammation and sepsis are not readily apparent. Despite the markedly increased levels of serum pre-CT, the mature CT hormone remained normal or only minimally elevated. Nevertheless, a positive correlation existed between the serum pre-CT value and mature CT. One possible explanation for this disparity of serum levels relates to the intracellular processing of pre-CT to the mature CT. Hypothetically, either the massively increased biosynthesis of the prohormone overwhelms the cellular posttranslational capacity, or the processing enzymes are dysfunctional, deficient, or absent. The cellular source of the enormous levels of serum pre-CTs in systemic inflammation and sepsis is also unknown. Although the thyroid C cells are an important source of CT, other neuroendocrine cells contain and/or secrete CT (20, 21). Furthermore, increased serum levels of pre-CT have occurred in a septic thyroidectomized patient (15).
It is conceivable that the bulk of serum pre-CT seen in inflammation, infection, and/or sepsis originates from nonneuroendocrine cells. Normal cells presumably possess cell type regulatory mechanisms, limiting expression of pro-CT messenger ribonucleic acid. A cytokine-induced stimulation of synthesis in such cells would preferentially favor pre-CT secretion because these cells lack the appropriate enzymatic mechanisms for efficient prohormonal processing.
Unlike the irregular and often transient appearance of proinflammatory cytokines in sepsis (22), the present study demonstrates that serum pre-CT is a novel and clinically relevant marker, has a consistent and sustained elevation in systemic inflammation/sepsis, often increases or decreases proportionally to clinical course, and may be predictive of final outcome.
Recently, we reported that immunoneutralization of pro-CT improves survival in septic animals (23). If such therapy should prove efficacious in humans, pre-CT markers may facilitate the selection of those who would benefit.
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Footnotes |
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Received October 16, 1997.
Revised March 27, 1998.
Revised June 2, 1998.
Accepted June 12, 1998.
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