Is Zinc Concentration in Pancreatic Fluid

Surakit Pungpapong, James S Scolapio, Timothy A Woodward, Michael B Wallace,
Massimo Raimondo
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine.
Jacksonville, Florida, USA
ABSTRACT
Objectives An imbalance of free radicals and
scavenging processes has been implicated in
the pathogenesis of pancreatic tissue injury.
Gradual decreases in antioxidant enzyme
expression in pancreatic cells from normal
pancreas to chronic pancreatitis to pancreatic
cancer have been reported. Zinc is one of the
components of antioxidant enzymes and may
play a role in the pathogenesis of pancreatic
diseases. A recent report showed a significant
decrease in pancreatic zinc output in patients
with chronic pancreatitis and proposed that
zinc output can be used as an alternative to
enzyme output for the diagnosis. We aimed to
determine the zinc concentration in pancreatic
fluid of patients with chronic pancreatitis and
pancreatic cancer compared to normal
pancreas.
Patients Seventy-five consecutive patients
were prospectively evaluated. Chronic
pancreatitis was diagnosed in 20 patients by
ERCP (n=16) and histology (n=4). Pancreatic
cancer was diagnosed in 17 patients by
cytology from EUS-FNA (n=12) or surgical
pathology (n=5). Thirty-eight patients were
confirmed to have normal pancreas.
Interventions Patients underwent esophago-
gastroduodenoscopy following intravenous
secretin injection (0.2 µg/kg). Pancreatic fluid
collected for 10 minutes was immediately
frozen at -80 °C and stored until the assay was
performed.
Main outcome measure Zinc concentrations
were determined by inductively coupled
plasma optical emission spectroscopy.
Results Among the three groups, patients
with pancreatic cancer were significant older
(P<0.001) with a higher prevalence of
diabetes mellitus (P=0.002). Gender
distribution (P=0.098), body mass index
(P=0.925), and history of alcohol use
(P=0.997) were similar among groups. Zinc
concentrations in pancreatic fluid were not
significantly different among groups
(P=0.129).
Conclusions Zinc concentration in pancreatic
fluid was comparable in patients with chronic
pancreatitis and pancreatic cancer compared
to normal pancreas. Zinc concentration in
pancreatic fluid collected by our 10-minute
endoscopic method cannot reliably be used
for the diagnosis of chronic pancreatitis
and/or pancreatic cancer.
INTRODUCTION
Pancreatic cancer (PC) is the fourth leading
cause of cancer death, with more than 30,000

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fatal cases annually in the United States [1].
Several reports have demonstrated that
chronic pancreatitis (CP) may be one of the
most significant risk factors for pancreatic
cancer with the pathogenesis yet to be
identified [2, 3, 4, 5]. This could be due to
possible effects of reactive oxygen species
generated during episodes of acute and
chronic pancreatitis on tumor initiation and
transformation [5]. Previous studies have
shown that malignant transformation is
usually accompanied by lowered antioxidant
activities [6, 7]. In addition, increased cellular
reactive oxygen species are associated with
carcinogenesis, whereas antioxidants prevent
malignant transformation both in vitro and in
vivo studies [8, 9]. Therefore, an imbalance of
free oxygen species and scavenging processes
has been implicated in the pathogenesis of
pancreatic injury resulting in malignant
transformation.
Zinc is one of the major components of
antioxidant enzymes and may play a role in
the pathogenesis of CP and PC [5, 10]. A
recent report demonstrated a significant
decrease in level of pancreatic zinc output in
patients with CP [11]. Thus, the level of zinc
output was proposed as an alternative marker
to assess pancreatic function for the diagnosis
of CP [11]. However, this proposed method
requires a double-lumen tube placement with
the tip positioned close to the ligament of
Treitz for aspiration of fluid for 90 minutes.
Due to the cumbersome nature of prolonged
duodenal intubation resulting in patient’s
discomfort and the unavailability of this
procedure across the majority of community
hospital in United States, this method is likely
not applicable to general practice.
Recently, we reported our experiences with an
endoscopic pancreatic function test using a
standard
esophagogastroduodenoscopy
(EGD) following an intravenous injection of
secretin to collect fluid exiting from the
ampulla of Vater for 10 minutes [12]. This
method is simple and more practical to use in
clinical practice [12]. In the current study, we
aim to determine the role of zinc
concentration in pancreatic fluid to
differentiate normal pancreas from CP and PC
using our previously reported technique [12].
We hypothesize that patients with pancreatic
diseases, either CP or PC, have significantly
reduced zinc concentrations in pancreatic
fluid compared to controls with normal
pancreas.
PATIENTS AND METHODS
Between January 2003 and May 2004, 75
consecutive patients with clinical signs and/or
symptoms suggestive of pancreatic diseases
were prospectively evaluated at the pancreas
clinic of the Mayo Clinic Jacksonville,
Florida, USA.
The final diagnosis of CP was determined
according to the Cambridge classification at
endoscopic retrograde cholangiopancreato-
graphy (ERCP) [13] and by histology in
patients who underwent surgery. The
diagnosis of PC was established using
cytology results demonstrating malignant
cells by endoscopic ultrasonography (EUS)-
guided fine needle aspiration (FNA) and/or
final pathology in patients who had surgery.
All patients underwent EGD and ERCP under
standard conscious sedation (intravenous
midazolam and meperidine). The EGD and
ERCP were performed on different days but
within 60 days (median: 2 days; range: 0-53
days) of each other.
Secretion of pancreatic fluid was induced by
an intravenous injection of secretin
(SecreFlo
TM
; Repligen Corporation, Silver
Spring, MD, USA) at the dose of 0.2 µg/kg
immediately before an EGD [12]. A total of
10 mL of pancreatic fluid exiting from the
ampulla of Vater was collected from the
second portion of the duodenum using a
standard disposable washing aspiration
catheter (7 French size, 240 cm in length;
Wilson Cook, Winston-Salem, NC, USA)
immediately frozen in liquid nitrogen and
stored at -80 °C until the analysis was
performed.
Zinc concentrations were determined using
inductively coupled plasma optical emission
spectroscopy. Aqueous acidic calibrating
standards were diluted with a matrix plasma
or serum containing a normal concentration of

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zinc. Blanks were diluted with the aqueous
acidic diluent containing internal standards
but no serum matrix. Patient samples and
quality control specimens were also diluted in
an identical manner. In turn, all diluted
blanks, calibrating standards, quality control
samples, and patient samples were aspirated
into a pneumatic nebulizer and the resulting
aerosol directed to the hot plasma discharge
by a flow of argon. In the annular plasma, the
aerosol was vaporized, atomized, and then
ionized. Emission signals from zinc and the
internal standard were observed axially by the
emission spectrometer. Instrumentation
response was defined by using the linear
relationship of analyte concentration versus
the ration of the zinc emission signal ratioed
with the internal standard. After reagent blank
subtraction, unknown zinc concentration of
samples was calculated by entering the net
unknown zinc intensity ratio into the linear
calibration equation.
Patient characteristics and zinc concentrations
of pancreatic fluid from patients with CP and
PC were compared to control patients with
normal pancreas.
ETHICS
The study was reviewed and approved by the
Mayo Foundation Institutional Review
Boards and the protocol conforms to the
ethical guidelines of the World Medical
Association Declaration of Helsinki.
STATISTICS
Median range and frequencies were used as
descriptive statistics. All statistical analyses
were performed by SPSS (Statistical Package
for the Social Sciences. Chicago, IL, USA)
software version 13. Comparisons were
performed using non-parametric tests
(Kruskal-Wallis and Pearson chi-square test).
Multinomial logistic regression was further
performed to adjust for confounding factors.
Statistical significance was achieved for a P
value less than 0.05.
RESULTS
Of 75 patients enrolled in the study, 38
controls were confirmed to have normal
Table 1. Comparisons of patient demographics, body mass indexes, history of alcohol use, and prevalence of diabetes
mellitus.
Normal
pancreas
(38 patients)
Chronic
pancreatitis
(20 patients)
Pancreatic
cancer
(17 patients)
P value
Age, years; median (range)
49 (24-79)
58 (30-88)
73 (45-86)
<0.001d
Gender distribution:
- Males
- Females
11 (29%)
27 (71%)
9 (45%)
11 (55%)
10 (59%)
7 (41%)
0.098e
Body mass index, Kg/m2; median,, (range)
26.3 (17.0-35.6) 26.2 (18.1-33.0) 25.6 (17.9-31.5)
0.925d
History of alcohol use:
- No alcohol use a
- Social alcohol use b
- Active alcohol use c
21 (55%)
12 (32%)
5 (13%)
11 (55%)
6 (30%)
3 (15%)
9 (53%)
6 (35%)
2 (12%)
0.997e
Prevalence of diabetes mellitus
3 (8%)
1 (5%)
7 (41%)
0.002e
aNo alcoholic consumption within the past 6 months
bAverage alcohol consumption of less than 2 drinks per day in female patients and less than 3 drinks per day in male
patients
cAverage alcohol consumption of more than 2 drinks per day in female patients and more than 3 drinks per day in male
patients
dKruskal-Wallis test
ePearson chi-squared test

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JOP. Journal of the Pancreas – http://www.joplink.net – Vol. 6, No. 5 –September 2005. [ISSN 1590-8577]
428
pancreas by normal ERCP while 20 and 17
patients were determined to have CP and PC,
respectively. Of 20 patients with CP, 16
patients were diagnosed by ERCP and the
remaining 4 patients were diagnosed by
pathology. Of 17 patients with PC, 12 patients
had positive cytology results from EUS-FNA
and the remaining 5 patients were diagnosed
by pathology. Fourteen of these patients
(82%) had pancreatic ductal adenocarcinoma,
1 patient (6%) had acinar cell carcinoma, and
2 patients (12%) had intraductal papillary
mucinous neoplasm with invasive carcinoma.
The alternative diagnoses of 38 controls were:
peptic ulcer diseases (n=3), non-ulcerative
dyspepsia (n=11), irritable bowel syndrome
(n=16), symptomatic cholelithiasis (n=2), and
radiating neuropathic pain from spine
abnormalities (n=6).
Patient demographics (age and gender
distribution), body mass index, history of
alcohol use, and prevalence of diabetes
mellitus were compared among groups and
demonstrated in Table 1. Patients with PC
were significantly older compared to patients
with CP and normal pancreas with the median
of 73, 58, and 49 years, respectively
(P<0.001). Gender distribution (P=0.098),
body mass index (P=0.925), and history of
alcohol use (P=0.997) were similar among
groups. Prevalence of diabetes mellitus was
significantly different among the 3 groups
(P=0.002). Every patient consumed a regular
western diet and none was taking zinc
supplements. Serum amylase and lipase levels
were normal at the time of pancreatic fluid
collection.
Comparison of zinc concentrations in
pancreatic fluid among groups was performed
and demonstrated in Figure 1. The median of
zinc concentrations in patients with normal
pancreas was 0.98 µg/mL (range: 0.26-11.10)
compared to 1.02 µg/mL (range: 0.29-3.24) in
patients with CP and 1.35 µg/mL (range:
0.52-5.43) in patients with PC. No statistically
significant differences were demonstrated
among groups (P=0.129). In addition,
multinomial logistic regression analysis
adjusting for age and prevalence of diabetes
mellitus was further performed and confirmed
no significant difference of zinc concentration
in pancreatic fluid among groups (P=0.678).
DISCUSSION
Chronic pancreatitis is a well-defined disease
based on histopathology, but for clinical
purposes, diagnosis is generally not based on
a histological examination. It remains
challenging especially in the early stage of
disease because of an inability to obtain a
tissue sample due to a deep-seated
retroperitoneal location of pancreas and a
high incidence of complications after core
tissue biopsy [14]. To date, direct and
invasive tests of pancreatic exocrine function
using intravenous hormonal stimulants
(cholecystokinin and/or secretin) infusion and
duodenal drainage analysis are considered the
gold standard for diagnosis. Unfortunately,
these tests are only available in a selected
number of tertiary care referral centers in the
United States. Their limitations include the
need for a specialized gastroenterology
laboratory for fluid analysis, the cumbersome
nature of prolonged duodenal intubation (at
least 90 minutes) resulting patient’s
discomfort and time consumption, and lack of
a universal laboratory standard. Thus, the
concept of a simple and fast direct pancreatic
exocrine function test that can be performed
concurrently with an EGD has been proposed
Figure 1. Comparison of zinc concentration in
pancreatic fluid. Data is illustrated in box-and-whisker
plot. Box represents the median and interquartile range.
Whisker line represents the maximal and minimal
distributions of data set. (mcg = µg)

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in several studies [13, 15, 16, 17]. With the
similar concept of using an intravenous
hormonal injection to stimulate pancreatic
exocrine function for measurement of
pancreatic enzymes and/or bicarbonate
concentrations in pancreatic fluid, the
feasibility of the tests were demonstrated with
estimated sensitivity and specificity ranging
from 60-92% and 70-95%, respectively [13,
15].
Because of several previous reports
confirming possible correlations between
antioxidant activities and pathogenesis of
pancreatic tissue injury resulting in CP and
possibly PC, an idea of identifying the other
marker, which is a major component of these
antioxidants to accurately diagnose CP and
PC was attempted. We have recently reported
a lack of correlation of selenium
concentration in pancreatic fluid and the
presence of chronic pancreatitis [18]. A
recently published study demonstrated that
pancreatic zinc output following an
intravenous infusion of secretin could be
accurately used in diagnosis of CP with the
sensitivity and specificity of 97% and 91%,
respectively [11]. However, this method still
requires a prolonged duodenal intubation with
a double-lumen tube for 90 minutes that may
result in the similar patient’s discomfort and
low tolerability, similar to the standard
pancreatic exocrine function tests.
In the current study, we report our experience
in using an EGD with a 10-minute collection
of pancreatic fluid to determine zinc
concentration in pancreatic fluid following an
intravenous injection of secretin in patients
with a suspicion of pancreatic diseases. The
results demonstrated that zinc concentration
in pancreatic fluid was similar in patients with
CP compared to patients with normal
pancreas. Thus, it is interesting that the results
of our study did not confirm findings from the
previous study and zinc concentration in
pancreatic fluid cannot be used reliably to
support the diagnosis of CP. In addition to
CP, our study also demonstrated that there
was no significant difference in zinc
concentration in pancreatic fluid from patients
with PC compared to patients with normal
pancreas or CP. Thus, zinc concentration in
pancreatic fluid cannot be reliably used to
differentiate PC from normal pancreas and
CP.
The discrepancy of our results compared to
findings from the previous study remained
unclear. However, it may probably be
explained by significant differences in
methodology and patient population being
studied. In the previous study, approximately
80% of patients with CP being evaluated had
either moderate or severe CP based on
bicarbonate output while only 25% and 20%
of patients in our study had moderate and
severe CP defined by the Cambridge
classification at ERCP, respectively. It seems
logical that the patients with more severe CP
would have higher degree of impaired
pancreatic exocrine functions leading to more
pronounced difference when compared to
patients with normal pancreas. Besides, the
methodology of pancreatic fluid collection
was also dissimilar. Due to a short duration of
pancreatic fluid collection by our method, a
total quantification of zinc output cannot be
performed and zinc concentration was
therefore instead used in our analyses. The
zinc concentration of pancreatic fluid may
vary over time. Thus, this may be another
important factor resulting in the disagreement
of our results compared to the previous study.
In summary, our study demonstrated that zinc
concentration in pancreatic fluid is similar in
patients with CP and PC compared to patients
with normal pancreas. Using a simple EGD
and a 10-minute direct endoscopic collection
of pancreatic fluid immediately after an
intravenous infusion of secretin, our results
did not confirm findings from the previous
study. Thus, until proven in a larger, multi-
center study, zinc concentration in pancreatic
fluid should not be reliably used to
differentiate patients with CP and/or PC from
those without pancreatic diseases.
Received May 12
th
, 2005 - Accepted June
30
th
, 2005

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430
Keywords
Pancreatic Function Tests;
Pancreatic Neoplasms; Pancreatitis; Secretin;
Zinc
Abbreviations CP: chronic pancreatitis;
EGD: esophagogastroduodenoscopy; ERCP:
endoscopic retrograde cholangiopancreato-
graphy; EUS: endoscopic ultrasound; FNA:
fine needle aspiration; PC: pancreatic cancer
Presentation This study is an oral
presentation in distinguished abstract plenary
sessions at Digestive Disease Week, May 14-
19, 2005, Chicago, Illinois
Correspondence
Massimo Raimondo
Mayo Clinic College of Medicine
4500 San Pablo Road
Jacksonville, FL 32224
USA
 
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