Potency and Tolerance of Calcitonin Stimulation with High Dose ...

J Clin Endocrin Metab. First published ahead of print June 2, 2009 as doi:10.1210/jc.2008-2403
BRIEF REPORT
Potency and Tolerance of Calcitonin Stimulation with High Dose Calcium versus
Pentagastrin in Normal Adults
Patricia Doyle, Christian Düren, Kai Nerlich, Frederik A. Verburg, Inge Grelle, Hanne Jahn,
Martin Fassnacht 1 , Uwe Mäder 2 , Christoph Reiners and Markus Luster
Department of Nuclear Medicine, 1 Dept. of Internal Medicine I, Endocrine and Diabetes Unit
and 2 Comprehensive Cancer Center, University Hospital, University of Würzburg, Würzburg,
Germany
Abbreviated title: Calcium is a more potent than pentagastrin
Key terms: human calcitonin, reference range, healthy adults, basal, stimulated, pentagastrin,
calcium, medullary thyroid cancer
Precis: High-dose calcium is a more potent and better-tolerated secretogogue of human
calcitonin than is pentagastrin in young thyroid-healthy human subjects
Word count excluding abstract, figure captions, and references: 1799
Correspondence and reprint requests to:
Christoph Reiners, MD, PhD
Department of Nuclear Medicine, University of Würzburg
Josef-Schneider-Strasse 2
97080 Würzburg GERMANY
+49-931-201-35868 (phone)
+49-931-201-35247 (fax)
reiners@nuklearmedizin.uni-wuerzburg.de
Disclosure:
P.D., C.D., K.N., F.A.V., I.G., H.J., M.F., U.M. and M.L. have nothing to declare.
C.R. Received Grant support for Logistical and editorial assistance on this paper from
Siemens Healthcare diagnostics GmbH
Copyright (C) 2009 by The Endocrine Society
1

Abstract
Objective: 1. To Compare pentagastrin- and calcium-stimulated serum hCT levels for non-
smoking healthy adults without evidence of thyroid disorders 2. Determine reference ranges
of basal and pentagastrin- and calcium-stimulated serum hCT levels.
Design: Healthy volunteer study including within-group and inter-group comparisons.
Setting: Tertiary referral center.
Subjects: Fifty healthy, non-smoking volunteers (25 female; ages 22 - 57 years) without
evidence of thyroid abnormality.
Interventions: hCT measurement using a Calcitonin two-site automated chemiluminescent
immunometric assay (the most common hCT assay in clinical practice) in serum samples
obtained before and 2 min, 5 min, and 15 min after intravenous stimulation using
pentagastrin, 0.5 µg/kg bodyweight, or calcium gluconate, 2.5 mg/kg.
Main outcome measures: Reference ranges for basal, unstimulated and pentagastrin- or
calcium-stimulated hCT; pentagastrin and calcium tolerability in healthy adults.
Results: Ninety-fifth percentile basal hCT values did not differ between males and females
(5.0 vs. 5.7 pg/mL). Ninety-fifth percentile maximal stimulated hCT values rose distinctly
after pentagastrin (peak: men, 37.8 pg/mL; women, 26.2 pg/mL) and even more so after
calcium (peak: men, 131.1 pg/mL, women, 90.2 pg/mL). No hCT increase was detected in
4/25 men and 12/25 women after pentagastrin versus 0/24 men and 2/18 women after
calcium. Calcium was associated with fewer and less intense adverse effects than was
pentagastrin.
Conclusion: High-dose calcium is a more potent and better-tolerated hCT stimulator than is
pentagastrin. The reference ranges for basal and stimulated hCT established via automated
chemiluminescent assay were lower than those reported for other assays.
2

Introduction
Human calcitonin (hCT) is a tumor marker essential to the diagnosis and follow-up of
medullary thyroid cancer (MTC ) (1-4). Current European consensus recommends hCT
measurement when initially evaluating thyroid nodules (5). If hCT is mildly elevated,
European centers commonly perform a confirmatory test with intravenous pentagastrin
stimulation (3, 6-8). A less popular alternative stimulation test uses a short intravenous
calcium infusion (3, 6). Stimulation testing also may aid early risk stratification in patients
with MTC (4).
For reasons including the unavailability of pentagastrin in the US, the American
Thyroid Association has not recommended routine hCT measurement (9). However, a recent
U.S. cost-effectiveness study (10), though not without flaws, provides data favoring such
testing .
Key to employing stimulated hCT measurement clinically are the reliability and
relevance to everyday practice of healthy adult reference values. Most published reference
values for stimulation tests were obtained in small series not rigorously screened for thyroid
abnormality (2, 6, 7, 11). Calcium stimulation was investigated in only a few studies (12)
using disparate regimens, e.g., 10% calcium gluconate dosages of 2.0-2.5 mg/kg body weight,
injection over 30 sec-5 min (3, 13-15).
Indeed, hCT reference ranges generally were obtained with competitive
radioimmunoassays and do not apply to automated non-competitive chemiluminescent
immunometric assays.
Differences in hCT measurement are evident even within the radioimmunoassay
class, emphasizing the need to re-evaluate reference ranges and cut-off values for each assay
individually (16).
We therefore conducted this study to define the normal ranges on a commonly used
chemiluminescent immunometric assay of serum hCT values in healthy adults before and
after pentagastrin or high-dose calcium stimulation,. We also sought to assess these
secretagogues’ tolerability in such subjects..
3

Subjects and Methods
Study sample
Table 1 summarizes study sample characteristics. The sample comprised 50 non-
smoking, non-pregnant adult volunteers without known chronic or serious acute illness or
evident thyroid abnormality after screening described below. Subjects could not take
prescription medication other than oral contraceptives (9/25 women). Any pre-study
laboratory abnormality (potassium, sodium, calcium, creatinine, liver enzymes) resulted in
exclusion. The University of Würzburg Ethics Committee approved the study; all subjects
gave written informed consent.
Screening for thyroid abnormalities
Pre-study, all subjects underwent thyroid ultrasonography with a Sonoline Elegra
system (Siemens, Erlangen, Germany) featuring a linear 7.5-MHz transducer. Thyroid
volume in mL was calculated using the algorithm, 0.5 x length x width x depth. Additionally,
subjects underwent free T3, free T4, and TSH measurement using IMMULITE ® 2000
automated chemiluminescent assays (Siemens Healthcare Diagnostics GmbH, Eschborn,
Germany). Thyroperoxidase and thyroglobulin autoantibody titers were determined using
enzyme immunoassays (Varelisa, Phadia GmbH, Freiburg, Germany).
Basal and Stimulated hCT testing
We averaged basal hCT measurements taken in the initial examination and before
each stimulation test. After pentagastrin stimulation, there was a wash-out period of at least
6 months before the calcium stimulation described below. Eight subjects (1 man, 7 women)
missed that calcium stimulation because they became pregnant (n = 1) or left our region (n =
7).
Pentagastrin (Pentagastrin Injection BP, Cambridge Laboratories, Wallsend, UK) was
administered as an intravenous injection of 0.5 µg/kg body weight over 10 sec and calcium
gluconate (Calcium Braun 10%, Braun Melsungen AG, Melsungen, Germany) as an
intravenous injection of 2.5 mg/kg at 10 mL/min. All subjects fasted ≥ 4 hr before
stimulation.
4

Serum hCT Assay
Blood samples were taken via in-dwelling intravenous cannula immediately before
and 2 min, 5 min, and 15 min after the intravenous pentagastrin or calcium injection.
Samples were placed in ice-water to prevent hCT degradation and transferred to the in-house
laboratory within 30 min of being drawn for centrifugation after clotting was complete. Serum
samples were assayed for calcitonin using the IMMULITE ® 2000 Calcitonin automated
chemiluminescent system (Siemens Healthcare Diagnostics GmbH, Eschborn, Germany), a
solid-phase, enzyme–labeled, two-site immunometric assay. The assay received regulatory
approval based on data showing an analytical sensitivity of 2 pg/ml. Subsequent testing by the
manufacturer has shown an analytical sensitivity of 0.34 pg/ml and a functional sensitivity of
0.7 pg/ml. Analytical sensitivity is defined as a concentration two standard deviations (SDs)
above the mean counts/sec of the zero calibrator, i.e., the 97.5 th percentile. Functional
sensitivity is defined as the lowest analyte level with an inter-assay coefficient of variation
(CV)

high percentage of women (80%) but not men (32%) had very low basal hCT concentrations
(

calcium-stimulated hCT levels were strongly correlated (Figure 1C, Spearmans’ rho = 0.758,
P < 0.001). Analysis of variance revealed that sex significantly influenced the magnitude of
the stimulated hCT levels (P =0.008), but age, weight, height, body mass index and thyroid
volume did not.
Tolerability of stimulation agents
Clinical side effects also are reported in Table 1. Due to a perception of more
unpleasant adverse effects of pentagastrin than of calcium, all subjects receiving both (n = 42)
declared a preference for the latter.
Discussion
Because of its relatively large sample size, rigorous exclusion of evidence of thyroid
abnormality, and inclusion of calcium stimulation testing, the present study provides the most
reliable and clinically relevant reference values for basal and stimulated serum hCT
concentrations in healthy adults obtained to date via automated chemiluminescent assay. Our
reference range for basal hCT agrees with Leboulleux et al.’s description of “normal” adult
circulating hCT levels as

versa. Similar findings in healthy subjects were noted by Gharib et al. (14), who used a
different calcium infusion regimen (2mg/kg over 5 min) than ours and injected pentagastrin as
a bolus. However, in that study, pentagastrin provided more potent stimulation than did
calcium in a 12-person subgroup of thyroidectomized MTC patients (14). Additionally,
Costante et al. reported that a 30-second infusion of calcium gluconate, 2.5 mg/kg, was
equally effective as pentagastrin in identifying patients with C-cell hyperplasia or MTC (3).
Therefore the impact of the calcium regimen and the comparative efficacy of the two agents
in clinical practice remain unclear. Additional trials investigating hCT levels in patients with
thyroid abnormalities, such as multinodular goiter and autoimmune thyroiditis, and MTC
patients are necessary to resolve these issues and define appropriate cut-off values for disease.
In line with previous observations, virtually all subjects reported greater transient
discomfort of varying intensity after pentagastrin than after calcium (3, 17, 18).
This study had several limitations. Since all subjects underwent pentagastrin
stimulation before calcium stimulation, a sequencing effect was possible; however, the
months-long wash-out between stimulation tests renders this unlikely. The wash-out’s length,
however, may have contributed to the loss to follow-up of an appreciable portion (16%) of
subjects, which may have introduced bias into the comparison of the stimulation agents.
Additionally, the study included no elderly subjects or MTC patients.
Conclusions
In healthy young to middle-aged adults, calcium seems to be a more potent and
better-tolerated hCT stimulator than is pentagastrin. The reference ranges for basal and
stimulated hCT for healthy adults established via automated chemiluminescent assay were
lower than those reported for other assays. Gender-based cut-offs may be unnecessary for
unstimulated, but necessary for stimulated hCT testing.
Acknowledgements
We thank Petra Völcker, Werner Kühnel and Robert Marlowe for their assistance.
8

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10

Tables
Table 1. Subject characteristics at study entry and clinical side effects of pentagastrin and
calcium. FT3, free triiodothyronine; FT4, free thyroxine; SD, standard deviation;
TSH, thyroid-stimulating hormone
Characteristic Value
Females, n (%) 25 (50%)
Age, yr, mean ± SD (range) 33.1 ± 9.7 (23 - 57)
Body mass index, kg/m², mean ± SD (range) 22.8 ± 3.0 (18.1- 33.1)
Thyroid volume by ultrasound, mL, mean ± SD
(normal range)
11.0 ± 5.0 (5.0 - 24.0)
TSH, mIU/L, mean ± SD (normal range) 1.6 ± 0.6 (0.3 - 4.0)
FT3, pmol/mL, mean ± SD (normal range) 4.4 ± 0.7 (2.7 - 7.6)
FT4, pmol/mL, mean ± SD (normal range) 17.6 ± 2.1 (11.0 – 23.0)
Thyroperoxidase autoantibody titers, mean
(normal range)
Thyroglobulin autoantibody titers, mean (normal
range)
7.0 (0 - 100)
20.6 (0 – 100)
Calcium, mmol/L, mean ± SD (normal range) 2.4 ± 0.1 (2.0 - 2.7)
Pentagastrin side effects (N = 50), incidence, %
Retro/Substernal tightness / abdominal cramping
Extremity paresthesia
Feeling of warmth
Dizziness
Nausea, urge to micturate, salty metallic taste
Pentagastrin side effect duration
94%
74%
30%
12%
a few subjects each
1-2 min
11

Calcium side effects (n = 42), incidence, %
Temporary flushing, feeling of warmth
Facial paresthesia, altered gustatory sensation
98%
20%
Calcium side effects duration up to 15 min
12

Figure Legends
Figure 1. Serum calcitonin values in male versus female healthy adults at baseline (Panels A
and B), after pentagastrin stimulation (Panel A) (n = 50) or after calcium stimulation (Panel
B) (n = 42). The central box represents the values from the lower to upper quartile (25th to
75th percentile). The middle line represents the median. The vertical line extends from the
minimum to the maximum values, excluding outlier and far outlier values, which are
displayed as separate points. Outlier values are defined as being larger than the upper quartile
plus 1.5 times the interquartile range; far outlier values are defined as being larger than the
upper quartile plus 3 times the interquartile range. Panel C represents the relationship between
the maximum pentagastrin- and calcium-stimulated hCT levels (Spearmans’ rho = 0.758, P <
0.001).
13

Calcitonin (pg/ml)
A) Pentagastrin stimulation ― 95th percentile maximal stimulated hCT values (2 min*):
60
50
40
30
20
10
0
in men 37.8 pg/ml; in women 26.2 pg/ml
m f m f m f m f
basal 2 min * 5 min * 15 min *
* after pentagastrin-injection

Calcitonin (pg/ml)
B) Calcium stimulation ― 95th percentile maximal stimulated hCT values (2 min*):
260
240
220
200
180
160
140
120
100
80
60
40
20
0
in men 131.1 pg/ml (95.4 pg/ml); in women 90.2 pg/ml
m f m f m f m f
basal 2 min * 5 min * 15 min *
* after calcium-injection

C) Maximum calcium- vs. pentagastrin-stimulated hCT-levels