Measurement of Midregional Proadrenomedullin in
Plasma with an Immunoluminometric Assay
Nils G. Morgenthaler,*Joachim Struck, Christine Alonso, and Andreas Bergmann
Background: Adrenomedullin (ADM) is a potent vaso-
dilatory peptide, and circulating concentrations have
been described for several disease states, including
dysfunction of the cardiovascular system and sepsis.
Reliable quantification has been hampered by the short
half-life, the existence of a binding protein, and physical
properties. Here we report the technical evaluation of an
assay for midregional pro-ADM (MR-proADM) that
does not have these problems.
Methods: MR-proADM was measured in a sandwich
immunoluminometric assay using 2 polyclonal antibod-
ies to amino acids 45–92 of proADM. The reference
interval was defined in EDTA plasma of 264 healthy
individuals (117 male, 147 female), and increased MR-
proADM concentrations were found in 95 patients with
sepsis and 54 patients with cardiovascular disease.
Results: The assay has an analytical detection limit of
0.08 nmol/L, and the interassay CV was <20% for values
>0.12 nmol/L. The assay was linear on dilution with
undisturbed recovery of the analyte. EDTA-, heparin-,
and citrate-plasma samples were stable (<20% loss of
analyte) for at least 3 days at room temperature, 14 days
at 4 °C, and 1 year at ?20 °C. MR-proADM values
followed a gaussian distribution in healthy individuals
with a mean (SD) of 0.33 (0.07) nmol/L (range, 0.10–0.64
nmol/L), without significant difference between males
or females. The correlation coefficient for MR-proADM
vs age was 0.50 (P <0.001). MR-proADM was signifi-
cantly (P <0.001) increased in patients with cardiovas-
cular disease [median (range), 0.56 (0.08–3.9) nmol/L]
and patients with sepsis [3.7 (0.72–25.4) nmol/L].
Conclusions: MR-proADM is stable in plasma of
healthy individuals and patients. MR-proADM mea-
surements may be useful for evaluating patients with
sepsis, systemic inflammation, or heart failure.
© 2005 American Association for Clinical Chemistry
Adrenomedullin (ADM)1is a 52-amino acid peptide de-
scribed as having a variety of physiologic functions. Its
strong vasodilatory activity has been described in several
studies (1–3). Quantification of ADM would be helpful in
the diagnosis, monitoring, and prognosis of various car-
diovascular diseases and sepsis, for which increased
plasma concentrations of ADM have been described (4–
7). However, the reliable measurement of ADM release in
the circulation is difficult. In addition to immediate bind-
ing of ADM to receptors in the vicinity of its production,
via autocrine and/or paracrine reactions, peripheral mea-
surement is also hampered by the existence of a binding
protein (8), the short half-life of ADM (22 min) (9), and
technical difficulties (10). ADM is derived from a larger
precursor peptide (preproADM; 185 amino acids) by
posttranslational processing (1) (Fig. 1). During the pro-
cessing of preproADM, other peptides are generated:
renomedullin N-terminal 20 peptide (PAMP) with a sug-
gested hypotensive effect (11), and 2 peptides flanking
ADM: one midregional part of proADM (proADM 45–92)
and the COOH terminus of the molecule (proADM 153–
We have recently identified this midregional proADM
(MR-proADM) in the plasma of patients with septic shock
(12). The affinity-purified material was characterized by
matrix-assisted laser desorption/ionization mass spec-
trometry and revealed no smaller peptides, indicating
that MR-proADM, in contrast to mature ADM and PAMP,
may be stable in human plasma (12). Although MR-
proADM might be functionally irrelevant, the lack of a
tight feedback control represents a highly interesting
diagnostic target. Because of its probable stoichiometric
Research Department, B.R.A.H.M.S AG, Biotechnology Centre Hennigs-
*Address correspondence to this author at: Research Department,
B.R.A.H.M.S AG, Neuendorfstrasse 25, D-16761 Hennigsdorf bei Berlin, Ger-
many. Fax 49-3302-883-451; e-mail firstname.lastname@example.org.
Received March 16, 2005; accepted July 18, 2005.
Previously published online at DOI: 10.1373/clinchem.2005.051110
1Nonstandard abbreviations: ADM, adrenomedullin; PAMP, proad-
renomedullin N-terminal 20 peptide; and MR-proADM, midregional proad-
Clinical Chemistry 51:10
generation, the released amounts of MR-proADM may
directly reflect those of ADM and PAMP.
Here we describe the technical characterization of a
new sandwich immunoassay for the measurement of
MR-proADM in human plasma, its reference interval in
healthy individuals, and the finding of increased plasma
concentrations in patients with cardiovascular disease or
Materials and Methods
Three peptides related to preproADM were purchased
from JERINI AG, where they were chemically synthe-
sized, purified, and quality controlled by standard proce-
dures. The peptides were SPCD19 (sequence CRPQDM-
preproADM plus an N-terminal cysteine residue), PSR13
83–94 of preproADM plus an N-terminal cysteine resi-
due), and proADM 45–92 (sequence ELRMSSSYPTGLAD-
resenting positions 45–92 of preproADM).
Sheep antisera containing antibodies directed against
peptides SPCD19 and PSR13 were generated by Micro-
pharm Ltd., according to standard procedures. Briefly,
peptides were conjugated with m-maleimidobenzoyl-N-
hydroxysuccinimide ester to keyhole limpet hemocyanin.
Sheep were initially immunized with 100 ?g of peptide in
its conjugated form and with 50 ?g in 4-weekly intervals
thereafter. Antisera were purchased from Micropharm
starting 3 months after initial immunization. For the
purification of peptide-specific antibodies, 5 mg each of
peptides SPCD19 and PSR13 were immobilized on Sul-
foLink gel (Pierce) according to the manufacturer’s in-
structions. Affinity purification was performed as follows:
50 mL of antiserum was diluted with 50 mL of binding
buffer [100 mmol/L potassium phosphate containing 1
mL/L Tween (pH 6.8) and 1 tablet of Complete Protease
Inhibitor (Roche) per 50 mL] and incubated according to
the manufacturer’s instructions. The gel was washed with
300 mL of binding buffer. Bound antibodies were eluted
with 50 mmol/L citric acid (pH 2.2) and neutralized with
50 mmol/L potassium phosphate (pH 7.4) and NAP
size-exclusion chromatography (Amersham) according to
the manufacturer’s instructions. The homogeneity of the
antibody preparations was confirmed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis, and the pro-
tein concentrations were measured by the bicinchoninic
acid method (Pierce).
A chemiluminescence sandwich immunoassay using
coated tubes was set up as follows. Purified anti-SPCD19
antibody (1 g/L) was labeled by incubation with a 1:2
molar ratio of MA70-Akridinium-NHS-Ester (1 g/L;
HOECHST Behring) for 15 min at room temperature. The
reaction was stopped by addition of a 1:10 volume of 1
mol/L Tris, and labeled antibodies were separated from
free label by size-exclusion chromatography on a Protein-
Pak SW300 HPLC column (Waters). Tracer was produced
by diluting the labeled antibody into assay buffer (300
mmol/L potassium phosphate, 50 mmol/L NaCl, 10 mM
sodium EDTA, 1 g/L bovine serum albumin, 1 g/L
unspecific sheep IgG, 1 g/L unspecific bovine IgG, 0.9
g/L sodium azide, pH 7.4) to achieve a concentration of
1 000 000 relative light units per 200 ?L, determined by a
(Greiner) were coated with anti-PSR13 antibody (per tube:
0.5 ?g in 0.3 mL of 100 mmol/L Tris, 50 mmol/L NaCl,
pH 7.8) overnight at room temperature, after which tubes
were blocked with 10 mmol/L sodium phosphate (pH
6.5) containing 3 mL/L Karion FP and 3 g/L bovine
serum albumin and lyophilized. Dilutions of peptide
proADM 45–92 in normal horse serum (Sigma) served as
calibrators. The immunoassay was performed by incubat-
ing 10 ?L of samples/calibrators and 200 ?L of tracer in
coated tubes under agitation (170–300 rpm) for 2 h at
room temperature (18–24 °C). Tubes were washed 4 times
with 1 mL of LUMItest wash solution (B ? R ? A ? H ? M ? S
AG), and bound chemiluminescence was measured for 1 s
per tube with a LB952T luminometer. The assay was
termed B ? R ? A ? H ? M ? S SEVADIL LIA®.
For the highest calibrator (S5), chemically synthesized
proADM 45–92 peptide was added to horse serum at a
concentration of 25 nmol/L. This was diluted to prepare
calibrators (S1 to S4) with final concentrations of 0.2, 0.8,
2.5, and 10.0 nmol/L. As controls, horse sera containing
1 nmol/L (control I) and 5 nmol/L (control II) of calibra-
tor peptide were added at the beginning and end of each
run. A typical calibration curve is shown in Fig. 2A.
Plasma samples (EDTA) from healthy individuals were
collected from the members of a local health club. Partic-
ipants had to be without clinical evidence of acute disease
or a history of chronic illness. Of ?900 regular members,
326 showed interest in participation, of whom 62 had to
be excluded because of a history of cardiovascular dis-
ease, diabetes, autoimmune disease, cancer, or infections
within the last 3 months. Written consent was obtained
from the remaining 264 participants. After peripheral
Fig. 1. Sequence of preproADM.
Numbers indicate amino acids. Signal, signal peptide. The assay principle for
MR-proADM is shown. Tracer, labeled antibody; solid phase, antibody coated on
tubes. Single letter amino acid code for MR-proADM is shown. Bold font indicates
Morgenthaler et al.: Measurement of MR-proADM
venipuncture, all blood samples were centrifuged and
frozen in aliquots at ?20 °C within 1 h.
Samples from patients with cardiovascular disease,
sepsis, severe sepsis, or septic shock [as defined by the
American College of Chest Physicians/Society of Critical
Care Medicine consensus conference (13)] were collected
from intensive care unit wards according to ethics guide-
lines and were stored at ?20 °C until further use. Cardiac
patients were scheduled for coronary artery bypass oper-
ations or mitral or aortic valve replacement and had
ejection fractions ?50%. Patients with coronary artery
disease had stable angina and were not suffering from
acute myocardial infarction.
All statistical analyses were performed with Graph Pad
Prism 4.0. Comparisons of parametric data were done
with an unpaired t-test or one-way ANOVA. Nonpara-
metric data were compared with Kruskal–Wallis ANOVA
and the Dunn post test. Correlations were calculated as
Pearson correlations. P values ?0.05 were considered
The lower detection limit, as determined with horse
serum (mean relative light units of 20 determinations plus
2 SD), was 0.08 nmol/L. The intraassay imprecision (CV)
was determined by measuring 16 human EDTA plasma
samples covering the range 0.08–14.7 nmol/L in 10 par-
allel measurements. The intraassay CV was ?5% in all
samples except for the highest sample, for which it was
7% (Fig. 2B). The interassay imprecision was determined
by measuring 30 samples (range, 0.10–14.1 nmol/L) on 10
different days by alternating operators on 2 different
luminometers (Fig. 2C). The functional assay sensitivity,
defined as the MR-proADM concentration with an inter-
assay CV of 20%, was 0.12 nmol/L, and the MR-proADM
concentration for which the interassay CV was 10% was
0.4 nmol/L. A high-dose hook effect was seen when
MR-proADM concentrations ?500 nmol/L were added to
plasma samples. However, this concentration is 20-fold
higher than the highest calibrator and would still be
found as highly positive in the assay.
Fig. 2. Measurement range and precision of MR-proADM assay.
(A), representative calibration curve of MR-proADM assay. RLU, relative light
units. (B), intraassay imprecision for 16 EDTA-plasma samples. The intraas-
say CV of all samples was ?10% over the entire range of the calibration curve.
(C), interassay imprecision for 30 plasma samples measured on 10 different
days by different operators on 2 different luminometers. The interassay CVs
at 10% and 20% are indicated by dotted lines.
Clinical Chemistry 51, No. 10, 2005
We assessed assay linearity by dilution experiments;
we also performed pooling and recovery studies. Linear
dilutions (up to 1:32) of 8 EDTA-plasma samples were
tested. Measured concentrations were multiplied by the
dilution factor and compared with the original undiluted
concentrations. None of the 8 samples showed a deviation
during dilution ?20% of the original value. Pooling of 5
plasma samples with low MR-proADM concentrations
with 5 plasma samples with high MR-proADM concen-
trations in 10 different combinations gave a mean mea-
sured concentration that was 98% of the expected concen-
tration (range, 93%–104%). We performed recovery
experiments by adding the calibrator peptide in 2 concen-
trations to 5 different plasma samples. Recovery of the
analyte was 89%–105% of the calculated value in all 10
We measured the interference of several biological
substances by adding the potential interferents to sam-
ples, according to Clinical and Laboratory Standards
Institute (formerly NCCLS) guidelines. The assay was not
influenced by albumin concentrations up to 10 g/L,
bilirubin up to 0.4 g/L, hemoglobin up to 5.46 g/L,
triglycerides up to 6.34 g/L, or heparin up to 8000 IU/L.
The SD between the samples without added interferents
and those with added interferents was always within the
?1 SD range of the interassay precision profile, and the
deviation of the values for samples without added inter-
ferents was always ?20%.
We evaluated the stability of the analyte at room
temperature in citrate-, EDTA-, and heparin-plasma sam-
ples from 5 different patients. All samples for stability
studies contained only endogenous MR-proADM and
contained no added peptide. The analyte was stable in all
3 matrices for at least 72 h at room temperature. Mean
measured concentrations were between 92% and 104% of
the original value (Fig. 3). We evaluated the stability of
MR-proADM at 4 °C in 11 different EDTA-plasma sam-
ples containing endogenous MR-proADM (range, 1.5–11.3
nmol/L) without added MR-proADM peptide. After 14
days of storage, measured MR-proADM values were
between 78% and 129% of the original values (mean,
98%). The same 11 samples were subjected to 4 freeze–
thaw cycles, which had no influence on the analyte. The
mean measured concentration after the first freeze–thaw
cycle was 101% of the original value (range, 80%–115%),
and that after the fourth cycle was also 101% of the
original value (range, 77%–122%). The mean measured
concentration for 11 samples stored for 12 months at
?20 °C was 99% of the original value.
Matrices other than EDTA plasma were tested with 50
matched samples obtained from healthy controls. Serum
consistently gave values that were ?30% lower than those
obtained with EDTA plasma, and a high percentage of
healthy control samples had values below the functional
assay sensitivity (interassay CV ? 20%). The assay there-
fore is not suitable for measurement of MR-proADM in
serum samples. Heparin plasma gave higher values
(mean of 10% higher); however, a few heparin-plasma
samples had very low values compared with the matched
EDTA-plasma sample. We therefore do not recommend
use of heparin plasma. Citrate plasma gave consistently
lower values (mean of 7% lower) than EDTA plasma. It
therefore is possible to use citrate plasma if the laboratory
defines reference intervals for this specimen type. All
values presented in the remainder of this study were
obtained with EDTA plasma.
In 264 healthy individuals (117 male, 147 female)
MR-proADM values followed a gaussian distribution
(Fig. 4A) with mean (SD) values of 0.33 (0.07) nmol/L
(95% confidence interval of the mean, 0.32–0.34 nmol/L)
and a range of 0.10–0.64 nmol/L. The 99th percentile of
the healthy population was 0.52 nmol/L, the 97.5th per-
centile was 0.49 nmol/L, 2.5th percentile was 0.17
nmol/L, and the 1st percentile was 0.14 nmol/L (based on
a gaussian distribution). There was no significant differ-
ence in mean MR-proADM values between males and
females (0.32 nmol/L for males; 0.32 nmol/L for female;
Fig. 4B). Further stratification of the population by age is
shown in Table 1. The mean (SD) increased from
0.29 (0.04) nmol/L in the age group ?25 years of age, to
0.41 (0.08) nmol/L in the group 55–64 years of age, and to
0.41 (0.06) nmol/L in the age group ?65 years of age (P
?0.001, ANOVA). Post hoc analysis by the Newman–
Keuls multiple comparison test revealed significant dif-
ferences among all groups except between the 18–24 and
25–34 groups, between the 25–34 and 35–44 groups, and
between the 2 oldest groups. Correlation analysis re-
vealed a significant correlation of MR-proADM concen-
trations and age (r ? 0.50; 95% confidence interval,
0.34–0.58; P ?0.001).
Fig. 3. Stability of the MR-proADM assay.
Stability of MR-proADM in citrate-, EDTA-, and heparin plasma at room tempera-
ture. Shown is the mean (SD) MR-proADM concentration as a percentage of the
original value in 5 different samples over 72 h at room temperature.
Morgenthaler et al.: Measurement of MR-proADM
To evaluate intraday variations in healthy individuals,
we monitored 6 participants (3 male, 3 female) from 0800
to 1700, taking 15 consecutive samples for MR-proADM
over this period. At the first blood sample, all participants
had been without water or food for 14 h. Although values
among individuals varied between 0.16 and 0.28 nmol/L,
the values for each participant remained very stable (SD
of 15 measurements, 0.01–0.05 nmol/L) over the observa-
tion period and were not influenced by water (1 L) or food
intake (standardized lunch of ?1200 kcal) at defined time
points (Fig. 4C).
MR-proADM was increased in patients undergoing
heart surgery because of an underlying cardiac disease
and in patients in the Intensive Treatment Unit with
Fig. 4. MR-proADM in healthy individuals and patients.
(A), gaussian distribution of the MR-proADM values in all 264 healthy individuals. (B), distribution of MR-proADM values in 117 male and 147 female healthy individuals.
There was no significant difference (NS) in mean values (unpaired t-test), as indicated by the horizontal bracket. (C), proADM values for 4 of 6 representative healthy
individuals over a 9-h period. Arrows indicate the intake of water (1 L within 5 min) and a standardized meal (?1200 kcal over 45 min). ?, 33-year-old male, body mass
index ? 21 kg/m2; ?, 44-year-old male, body mass index ? 24 kg/m2; Œ, 50-year-old female, body mass index ? 23 kg/m2; F, 31-year-old male, body mass index ?
30 kg/m2. (D), distribution of MR-proADM values in 54 patients with cardiac disease and 95 patients with sepsis compared with the 264 healthy individuals. The
median value for each group is indicated by a line. Differences between all 3 groups are significant by Kruskal–Wallis ANOVA.
Table 1. MR-proADM in 264 healthy blood donors stratified by age.
0.29 (0.04) 0.31 (0.06)
Age of blood donors, years
Mean (SD), nmol/L
No significant differencea
aAll differences are significant (P ?0.05, ANOVA with Newman–Keuls post test), except for column pairs indicated by letters b to d.
Clinical Chemistry 51, No. 10, 2005
sepsis, severe sepsis, or septic shock. Cardiac patients
were scheduled for coronary artery bypass surgery or
mitral or aortic valve replacement and had ejection frac-
tions ?50%. Median (range) values were 0.56 (0.08–3.9)
nmol/L for cardiac patients and 3.7 (0.72–25.4) nmol/) for
sepsis patients. The difference, compared with healthy
individuals, was significant for both groups (P ?0.001,
Kruskal–Wallis ANOVA with Dunn post test). The data
distributions in all 3 groups are shown in Fig. 4D.
We describe the technical characterization of a new MR-
proADM sandwich immunoassay. The assay has a func-
tional assay sensitivity (defined as an interassay CV
?20%) of 0.12 nmol/L and allows the measurement of
MR-proADM in a range between 0.12 and 25 nmol/L.
Studies using either pooled samples or addition of the
chemically synthesized analyte as well as dilution studies
showed good performance. In contrast to mature ADM,
MR-proADM is stable in plasma at room temperature for
at least 72 h, at 4 °C for at least 14 days, and at ?20 °C for
at least 12 months. Although the choice of anticoagulant
had no influence on the stability of the analyte after the
sample was taken, the use of heparin as anticoagulant
gave, in a few matched samples, lower MR-proADM
values compared with EDTA (?50% of EDTA value). We
therefore do not recommend the use of heparin plasma or
serum for this assay, whereas citrate plasma can be used.
The released amounts of MR-proADM may directly
reflect those of ADM and PAMP, and measurement of the
latter molecules is technically challenging because they
can be cleared rapidly as a result of their autocrine/
paracrine effects; i.e., because their receptors are located
very close to the site of release (2, 3, 14, 15). In addition,
circulating ADM could be influenced by a binding protein
(complement factor H), making it less accessible for
immunometric analysis (8). It has also been reported that
ADM tends to stick to surfaces and thus might not be
quantitatively recovered in an immunoassay (10). In a
study comparing different ADM gene-derived peptides
(16), MR-proADM had no physiologic effect, whereas all
other peptides (ADM, PAMP, and adrenotensin) exhib-
ited activity. The apparent stability may thus be attribut-
able to this lack of function because only bioactive sub-
stances require tight regulation by proteolysis.
Because of a lack of readily available routine assays for
the measurement of ADM, we could not demonstrate a
direct correlation between MR-proADM and mature
ADM. Although it is fair to speculate on a stoichiometric
release of both peptides, there are reports concerning
ADM and PAMP in which this stoichiometric release was
found in patients with heart failure (17) but not in
patients on hemodialysis (18). At present it is unclear
whether this discrepancy is attributable to the respective
assays or whether it reflects a distinct release pattern of
the 2 peptides.
In 264 healthy individuals, MR-proADM followed a
gaussian distribution with a mean (SD) of 0.33 (0.07)
nmol/L. There was no difference between the male and
female cohort, and all healthy individuals had detectable
MR-proADM. There was a significant trend to higher
MR-proADM values in older individuals, whereas in
individual volunteers, values were very stable during the
day and not influenced by either food or water intake. The
concentration of MR-proADM found in this study is more
than 1000-fold higher than that reported for mature ADM
in healthy individuals (2.7–10.1 pmol/L) (10).
The concept of replacing the problematic measurement
of a bioactive, rapidly cleared peptide by measuring a
nonfunctional, stable peptide derived from the cognate
precursor is well known and has been applied with great
success for the A- and B-type natriuretic peptides (19–23).
Our finding of such a kind of peptide derived from the
ADM precursor opens the door to better assessment of the
actual release of ADM gene products under pathologic
conditions involving dysfunctions of the cardiovascular
system and, thus, to improving the diagnosis, monitoring,
and prognosis of these diseases.
One practical example in this study is the measurement
of MR-proADM in patients with sepsis, severe sepsis, or
septic shock. Although it is well known that ADM is
increased in these conditions (7), the previously reported
concentrations of ADM (median, 0.194 nmol/L) are much
lower than the MR-proADM concentrations reported here
(median, 3.7 nmol/L). The ADM concentration in a pa-
tient may be of relevance for clinical intervention (24);
therefore, the actual true release of ADM should be
known. Because the influences on ADM measurement
may vary considerably among individuals and among
different pathologies, measurements of mature ADM may
underestimate the true release rate of this potent vasoac-
tive peptide in patients. It will be up to further studies to
evaluate whether MR-proADM has technical and clinical
advantages over the measurement of mature ADM and
whether this peptide is influenced by a certain medica-
We thank Marco Talke, Detlef Hintzen, Johanna Hetzel,
Angelina Herzberg, Anne Schmiedel, and Barbara Scha ¨f-
fus for excellent technical assistance.
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