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Journal of Clinical Endocrinology and Metabolism Vol. 79, No. 5
Copyright 0 1994 by The Endocrine Society Printed in U.S.A.

Procalcitonin Increase after Endotoxin Injection in

Normal Subjects
PARESH DANDONA, DAVID NIX, MICHAEL F. WILSON, AHMAD ALJADA,
JOHN LOVE, MARCEL ASSICOT, AND CLAUDE BOHUON
Departments of Medicine (P.D., M.F. W., A.A., J.L.) and Clinical Pharmacokinetics (D.N.), Millard Fillmore
Hospitals, State University of New York, Buffalo, New York 14209; and Department de Biologie Clinique,
Institut Gustave-Roussy (M.A.), Villejuif 94805 Cedex, France

ABSTRACT at 8 h. The procalcitonin concentration, which was undetectable (<lo

As procalcitonin concentrations have been shown to be elevated in pg/mL) at 0, 1, and 2 h, was detectable at 4 h and peaked at 6 h,
patients with septicemia and gram-negative infections in particular, we maintaining a plateau through 8 and 24 h (4 ng/mL). There was no
proceeded to investigate the effect of endotoxin, a product of gram- elevation of calcitonin concentrations, which remained below 10 pg/
negative bacteria, on procalcitonin concentrations in normal human mL, the lowest sensitivity of the assay. Procalcitonin was measured by
volunteers. Endotoxin from Escherichia coli 0113:H10:k, was injected a two-antibody immunoradiometric assay specific for this peptide, with
iv at a dose of 4 mg/kg BW into these healthy volunteers. Blood no cross-reactivity with calcitonin, katacalcin, or calcitonin gene-re-
samples were obtained before and 1, 2, 4, 6, 8, and 24 h after injection lated peptide. We conclude that endotoxin induces the release of
of the endotoxin. Each patient’s cardiovascular and overall clinical procalcitonin systemically, that this increase is not associated with an
status was monitored over this period. The patients developed chills increase in calcitonin, and that the increase in procalcitonin associated
and rigors, myalgia, and fever between l-3 h. Tumor necrosis factor-a with septicemia in patients may be mediated through the effect of
levels increased sharply at 1 h and peaked at 90 min, reaching the endotoxin described here. Whether procalcitonin participates in the
baseline concentration thereafter by 6 h. Interleukin-6 levels increased mechanisms underlying inflammation remains to be investigated. (J
more gradually, peaking at 3 h and reaching the baseline concentration Clin Endocrinol Metab 79: 1605-1608, 1994)

C ALCITONIN and other peptide products of the calci-

tonin gene are known to be elevated in medullary
thyroid carcinoma (1, 2) and several other systemic diseases,
the parafollicular cell metabolism is unlikely. Therefore, it
would appear that the parafollicular cellsof the thyroid gland
are not the source of procalcitonin in these patients. The
including systemic infections (3-6). One constituent of tissue source of procalcitonin in such patients is not clear. In
amyloid deposited in medullary thyroid carcinoma is procal- addition, the temporal relationship between the onset or
citonin (7). Although amyloid is also deposited during sepsis control of infection and procalcitonin concentrations is not
in various organs, the possibility that it may contain procal- known, nor is the mechanism underlying the induction of
citonin has not been investigated. Procalcitonin, a propeptide procalcitonin after an infection.
of calcitonin that undergoesposttranslational proteolysis into To assesswhether the increase in plasma procalcitonin
its hormonal derivative, has recently been shown to increase concentrations associatedwith infection is mediated by endo-
markedly in patients with severe systemic infections, septi- toxin, we measuredplasma procalcitonin concentrations se-
cemia, and local infections (8). The magnitude of the increase quentially before and after the injection of endotoxin in
in procalcitonin concentrations after systemic viral and lo- normal subjects. Our data demonstrate clearly that although
calized bacterial infections is much smaller than that after procalcitonin is not detectable in the plasma of normal sub-
systemic infections with bacteremia (8). Antibiotic therapy is jects, it consistently increasesafter endotoxin injection at 3-
associatedwith a fall in procalcitonin concentrations (8). An 4 h in all subjects and then rises rapidly to a plateau at 6 h
infection-associated increase in procalcitonin has also been and remains elevated until at least 24 h.
shown to occur in a thyroidectomized patient with septicemia
(8). Furthermore, in such infected patients the increase in
procalcitonin concentrations is not associated with an in- Subjects and Methods
creasein calcitonin concentrations (8). This observation sug- Seven normal male subjects (age range, 21-35 yr; weight range, 74-
geststhat procalcitonin-producing cells in sepsisare unable 90.3 kg; mean + SD, 83.1 + 5.5 kg) were included in this study. They
to processthis precursor form to the mature hormone. Al- were admitted to the Clinical Research Center 1 day before the investi-
gation and infused with 60 mL/h physiological (0.9%) saline overnight.
though it cannot be excluded that sepsismight alter hormone After the overnight rest, they had an iv cannula inserted into the anterior
processingin parafollicular cells, such a complete defect in cubital vein, which was kept open with heparinized saline for the
removal of blood samples. The subjects remained supine for at least 8 h
Received November 18, 1993. Revision received May 26, 1994. Ac- during this experiment. All subjects were injected iv with 4 nalke
cepted July 18, 1994. reference standard endotoxin from Escherichih coli 0113:HlO:k 6.g
Address all correspondence and requests for reprints to: Dr. Paresh FDA, Bethesda, MD). Each subiect’s cardiovascular status was continu-
Dandona, Department of Medicine, Millard Fillmore Hospitals, State ously monitored; p&e, blood pressure, and temperature were measured
University of New York, 3 Gates Circle, Buffalo, New York 14209. at least every 30 min, and cardiac output and peripheral vascular

1605

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1606 DANDON A ET AL. JCE & M. 1994
Vol79.No5

resistance were measured at baseline and 1, 2, 4, 6, and 24 h. Blood even at 24 h. No elevation of calcitonin levels was detectable
samples were obtained at 0, 1, 2, 4, 6, 8, and 24 h. Blood samples were during the course of the experiment. Calcitonin remained
processed for full blood count, electrolyte, urea, and creatinine measure-
ments. consistently undetectable (<lo pg/mL). Serum calcium con-
Tumor necrosis factor-o (TNFol) and interleukin-6 (IL-6) concentra- centrations fell significantly (P < 0.05) at 4 h (9.0 + 0.19 mg/
tions in serum were measured by specific enzyme-linked immunoassays, dL) and remained below the baseline values (9.4 + 0.39 mg/
using kits obtained from Boehringer Mannheim Biochemical (Indianap- dL) until 24 h (8.9 * 0.39 mg/dL). Plasma albumin concen-
olis, IN). von Willebrand Factor (vWF) was assayed in plasma from
titrated blood samples as an index of endothelial cell damage by the
trations did not change significantly (Fig. 1).
method described by Ramsey and Evatt (9). In this assay, vWF activity
is measured by comparing the potencies of unknown plasma samples Discussion
with those of standards (provided by Bio-data, Horsham, PA), and the
values are expressed as a percentage of the standards. Procalcitonin was These data demonstrate for the first time that although
measured by the specific and ultrasensitive immunoluminometric assay
of Ghillani et al. (IO). This assay uses 2 monoclonal antibodies, 1 directed
procalcitonin is not detectable in serum from normal subjects,
against residues around 96-106 of procalcitonin as the capture antibody it increases consistently and rapidly after the injection of
and 1 directed against the residues 70-76, as the tracer antibody. The endotoxin. It is first detected at 4 h and then plateaus at 6-
sequence 70-76 is a part of the calcitonin molecule, whereas the se- 8 h, remaining high even at 24 h. This increase is independent
quence 96-106 is a portion of the 21-amino acid long C-terminal region of a concomitant increase in calcitonin, katacalcin, or N-
(this sequence is part of the katacalcin molecule). Synthetic procalcitonin
(116 amino acid residues) was used as the standard. This assay, which procalcitonin peptide concentrations, as the two monoclonal
is specific for procalcitonin, has a limit of detection of 10 pg/mL. The antibodies used in this immunoradiometric assay are directed
linear portion of the standard curve ranged from lo-60 pg/mL. Inter- against sequences 96-110 (contained in the katacalcin resi-
and intraassay variations at both low and high concentrations were less due) and 70-76 (contained in the calcitonin residue). This
than 8% and 7%, respectively.
Serum calcitonin was measured with a monoclonal immunoradiome-
increase, therefore, is analogous to that observed in patients
tric assay (CIS Biointemational, Gif sur Yvette, France). This assay is with systemic and local infections and septicemia. These data
specific for mature calcitonin and has a sensitivity limit of 10 pg/mL. also show that the association of severe infection with hy-
The assay for calcitonin uses antibodies directed against two separate perprocalcitoninemia is probably due to endotoxin and pos-
sequences in the calcitonin molecule. The sequence 26-32 bearing the sibly other bacterial, viral, or inflammatory products.
C-terminal carboxamide group is reactive only in mature calcitonin and
is nonreactive on the precursor, procalcitonin. The use of this antibody In view of the modulatory effect of serum calcium concen-
ensures that procalcitonin in which this epitope, 26-32, is not reactive trations on calcitonin secretion, we examined the possible
is not in this assay. The specificity of this assay and that of procalcitonin correlation between sequential serum concentrations and
in terms of cross-reactivity has previously been confirmed with the use procalcitonin levels. There was a significant fall in plasma
of specific monoclonal antibodies (11).
Oxygen free radical generation by leukocytes was determined by
calcium concentrations at 4 h, and this was still maintained
measuring chemiluminesence in diluted (10%) whole blood in response at 24 h. As an increase in serum calcium concentrations is a
to zymosan in a Chronolog aggregometer-luminometer. This assay is trigger, and a fall in calcium an inhibitor of calcitonin con-
similar to that described by Tosi and Hamedani (12). Serum calcium centrations, it is unlikely that the serum calcium concentra-
concentrations were measured by routine laboratory techniques. This tion had a role in triggering procalcitonin secretion after
study protocol was approved by the Millard Fillmore Hospitals and State
University of New York at Buffalo’s Institutional Review Board, and endotoxin injection.
written informed consent was obtained from the patients. The fact that TNFa and IL-6 peaked before the appearance
of procalcitonin in plasma suggests that these cytokines may
have a role in inducing the release of procalcitonin from a
Results
putative target cell. Data demonstrating a rapid increase in
All subjects felt ill within 1 h of injection of endotoxin and procalcitonin after an injection of IL-2 have also been re-
became febrile between l-2 h postinjection. They had chills, ported (8). The fact that the peak increase in vWF coincides
rigors, and myalgias between l-3 h. Thereafter, they grad- with that in procalcitonin suggests that the endothelial cell
ually improved and improved markedly by 8 h (13). Fever may be a potential target cell for the action of endotoxin,
and tachycardia often persisted after 8 h. They had all TNFa, and/or IL-6, resulting in procalcitonin release. How-
recovered totally by 24 h, except for mild fatigue in a few ever, preliminary studies with endothelial cells in vitro do
subjects. The clinical and cardiovascular status of these sub- not reveal procalcitonin secretion. Further studies are re-
jects will be described in detail elsewhere. quired to establish the source of procalcitonin in patients
TNFa levels increased sharply at 1 h and peaked at 2 h, with infection, septicemia, and endotoxemia.
declining thereafter and reaching baseline concentrations at The other possible target cells for endotoxin action that
6 h (14) (see Table 1). IL-6 levels increased more gradually, participate in inflammatory processes and are, therefore, a
peaking at 3 h, declining thereafter, reaching baseline con- possible source of procalcitonin are the leukocyte and the
centrations at 8 h (see Table 1). vWF increased at 2 h, peaked macrophage. There are rapid changes in leukocyte counts
at 4 h, and plateaued thereafter (see Table 1). It was still after the injection of endotoxin, and their activation is shown
markedly elevated at 24 h (14). Zymosan-induced oxygen by increases in TNFa and IL-6. In our experiments we also
free radical generation increased at 2 h, peaked at 8 h, and observed an increase in oxygen free radical generation by
was still elevated by 250% over the basal value at 24 h (15). leukocytes 2 h after endotoxin injection. Endotoxin acts on
Procalcitonin was not detectable at 0, 1, or 2 h. It was leukocytes, and the cytokines released have a further stim-
consistently detected at 4 h and increased sharply by 6 h, ulating effect on leukocytes and other cells that may be
remaining elevated at 8 and 24 h with no evidence of decline putative sites of procalcitonin secretion. Whether stimulated

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 PROCALCITONIN INCREASE AFTER ENDOTOXIN INJECTION 1607

TABLE 1. TNFa, IL-6, vWF, and calcium concentrations before and sequentially after endotoxin injection

0 1 1.5 2 2.5 3 4 6 8 24
TNFa
Mean 33.9 289.4” 770.3 827.5" 567.1" 298.1” 105.8” 54.1” 62.3" ND
k30.3 2151.5 f303.0 ck471.3 f308.7 f148.2 f69.0 f39.6 k66.9 ND
IL?
Mean 6.39 19.47” 304.11” 1601.70” 2099.09” 2257.01” 2071.33” 245.20" 75.44" ND
SD k4.27 k8.96 +11s.os k787.61 k1418.27 +1918.30 k3463.56 k372.83 k122.06 ND
Calcium
Mean 2.35 ND ND 2.33 ND ND 2.25" 2.25" 2.25" 2.23“
SD +O.lO ND ND f0.06 ND ND rto.05 kO.05 kO.06 +0.10
vWF (%)
Mean 85 82 ND 255" ND ND 452" 402" 350 280"
SD +12 +11 ND +32 ND ND +51 +48 +51 *35
TNFa and IL-6 are expressed as picograms per mL, calcium as millimoles per L, and vWF as a percentage of the activity of the standard. ND,
Not done.
0 P < 0.01 (by t test for paired data) compared with readings before endotoxin injection.

FIG. 1. Serial procalcitonin concentra-

tions in plasma of normal subjects in-
jected with endotoxin (4 rig/kg BW) at
time zero. Note that procalcitonin was
not detectable between O-2 h. It became
detectable in all subjects at 4 h. Results
are expressed as the mean f SEM (nan-
ograms per mL). The mean values at 4
h and thereafter are significantly greater
than those at 0, 1, and 2 h (P < 0.01).
There was only one reading at 12 h;
hence, there is no SEM.

0 2 4 6 a 10 12 14 16 18 20 22 24 26 20 30

TIME (~~OURC)

leukocytes release procalcitonin is currently being investi- various peptides in responseto endotoxin and cytokines (17).
gated in our laboratory. However, it is possible that there It is of interest that endotoxin has been shown to induce the
may be other mediators of procalcitonin increase,becausein expression of PTH-related peptide in the spleen (22). This
conditions such as viral infections and Crohn’s disease, effect of endotoxin is mediated by TNFQ and IL-l. Another
which are characterized by leukocyte activation and cytokine product of the calcitonin gene, calcitonin gene-related pep-
release,procalcitonin concentrations are only marginally el- tide (CGRP), has been shown to be increased in systemic
evated (8). It is alsonoteworthy that conditions characterized infections (23). Plasma CGRP is mainly derived from peri-
by macrophage activation, e.g. sarcoidosis, are associated vascular nerves. Although the magnitude of the increase in
with increased secretion of angiotensin-converting enzyme CGRP in infections is much smaller than that in procalci-
(16) and the expression of la-hydroxylase in the macro- tonin, it would be of interest to determine whether it, too,
phages (17). It is also possiblethat procalcitonin releasemay increasesafter the injection of endotoxin. It is also possible
occur from other organs that respond to endotoxin and that the increase in CGRP is due to pro-CGRP, rather than
cytokines, i.e. kidney (18), spleen (19), liver (20), and lung CGRP itself, or to a combination of both, as the assay
(21). The spleen, liver, and lung have macrophagesof their methodology used for CGRP, a simple RIA, would not
own, whereas medullary cells in the kidney may express exclude cross-reactivity with pro-CGRP in this study.

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1608 DANDONA ET AL. JCE & M .1994
Vol79.No5

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activity using formalin fixed platelets and microtitration technique.
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