1. My sincerest thanks go to Dr. William Karasov for welcoming me
into the Karasov lab and supporting me in all aspects of my
research. I also thank Dr. Enrique Caviedes-Vidal, Katie Rott, and
Cherry Brown for their constant support and guidance throughout
the summer. I am grateful to Lisa Wachtel and Carmen Lombard,
the leaders of the MMSD Science Research Internship program, as
well as Rachel Eagan, for organizing this wonderful opportunity for
students with an interest in the sciences.
Sources Cited
• Pawel Brzek, Kevin Kohl, Enrique Caviedes-Vidal, and William H.
Karasov,
(2009).
• Douglas J. Levey, Allen R.Place, Pedro J.Rey, Carlos Martinez del
Rio, (1999). An Experimental test of dietary enzyme modulation
in Pine Warblers Dendroica Pinus.
Acknowledgements
Pancreatic Lipase Activity in Nestling House Sparrows Passer domesticus
Fiona Rambo with Dr. William Karasov and Katie Rott
University of Wisconsin-Madison Department of Forest and Wildlife Ecology
Results
Conclusion
Introduction Materials & Methods
Omnivorous birds consume a range of different
foods based on their nutritional needs and the
availability of food throughout the seasons. This
study aimed to explore how changes in the diet of
house sparrows Passer domesticus affect the
production of pancreatic lipase. It was hypothesized
that the amount of pancreatic lipase activity would
increase in tandem with the amount of lipid
substrate in the bird’s diet (Brzek et al. 2009),
eliminating the wasteful production of enzymes that
are not being used. This hypothesis is supported by a
study on dietary enzyme modulation in Pine
Warblers that found that birds fed a high-lipid insect
and seed diet displayed higher levels of lipase
activity than birds fed the high-carbohydrate fruit
diet (Levey et al. 1999).
Overall, the goal of this research was to improve
predictions about birds’ abilities to adapt to changes
in their environment, promote the gastrointestinal
tract as an archetype of phenotypic flexibility, and to
enhance the limited, current knowledge about
passerine birds and the regulation of digestive
enzymes. However, conclusions cannot be made
about the relationship between diet and pancreatic
lipase activity because the assays did not yield
reliable or valid data.
Body temperatures of the birds were recorded daily,
birds were weighed three times a day before
feedings, and the tarsus bone was measured at the
end of the day to track growth. Every hour from
6:30am until 8:30pm, the birds were fed one of the
three assigned diets with a syringe. After being fed
for three days on one diet, some birds were
switched to a different diet for the next three days,
while other birds were fed the same diet for the
whole treatment period.
All % dry mass
C=carbohydrate(starch), P=protein(casein),
L=lipid(corn oil), i= inert ingredient (grit & cellulose)
Assay
All assays were conducted using materials from the
Sigma Aldrich Lipase Activity Assay Kit MAK046.
Frozen pancreas samples were homogenized and
transferred to a centrifuge. Supernatant from the
centrifuged homogenates was pipetted into a 96-
well plate along with buffer. A glycerol calibration
curve was created on the plate by pipetting
increasing volumes of a 1 mM glycerol standard
solution over 6 wells. Buffer was added to the
glycerol calibration curve to bring each well up to
50μL volume. One well of the plate was dedicated
to a lipase positive control consisting of a sample of
lipase provided in the kit and buffer. A master
solution of buffer, peroxidase, and enzyme mix was
added to all wells in order to bring the volume up to
97μL. Finally, additional buffer was added to the
wells containing sample blanks, and lipase substrate
was added to all other samples and glycerol
standards. Plates were mixed using a horizontal
shaker and incubated at 40°C for 3 minutes. The
absorbance measurements were read every 5
minutes at 570nm using a Wallace Victor 1420
multi-label plate reader and incubated at 40°C
between reads.
In order to correct for these possible faults related
to the assay, two adjustments to the method were
tried. First, the pancreatic tissue was homogenized
using a mortar and pestle with liquid nitrogen to
better separate the lipase from other components
of the tissue. The absorbance signal of the samples
continued to show a downward trend, showing that
the mortar and pestle method did not improve
lipase extraction. Second, calcium chloride was
added to the assay buffer to test whether the assay
reaction was calcium dependent. There were no
indications that this was the case, because the data
maintained the same pattern.
Because the lipase assays did not yield reliable data,
no conclusions can be made about the effect of
high-lipid diets on the production of lipase in Passer
domesticus using these methods. However, future
research should test whether the freezing of
pancreas samples denatures the lipase, thereby
rendering the assay unviable. To test whether lipase
is destroyed by freezing, an assay should be
performed using fresh pancreas tissue that has
never been frozen. Additionally, need remains to
establish a reliable method for assaying pancreatic
lipase.
Treatment
Diet Carb Protein Lipid e i kj/g
C 50 15 8 10 17 14.8
P 5 60 8 10 17 14.8
L 5 15 25 10 45 13.4
Lipase activity in the pancreas samples was to be
quantified by analyzing the absorbance
measurements of the samples over a period of
time. As time passes, the amount of glycerol
freed by the lipase enzyme should increase in the
presence of the substrate, therefore increasing
the absorbance measurement of the samples.
Below is a graph of time versus the absorbance
measurement of a pancreas sample. As with all of
the assays, the absorbance measurement of the
lipase positive control, provided by the assay kit,
increased over time. However, the absorbance
measurement of the real pancreas samples
decreased. These results show that the activity of
the lipase in the pancreas samples was not
properly being detected.
-0.060
-0.040
-0.020
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0 20 40 60 80
AbsorbanceMeasurement–PancreasSample
(570nm)
Time (m)
Pancreas Sample
Lipase Pos. ControlMaterials & Methods
Treatment
House Sparrow nestlings Passer domesticus were
collected three days post-hatch from locations on
the UW campus. Nests were numbered and checked
daily in order to monitor the number of eggs and
nestlings. At three days post-hatch, birds were
retrieved from their nests and transported to the
Karasov Lab. The birds were weighed, assigned one
of three diets, and added (in their nest container) to
the humidified, incubated tub with the other
subjects
In the first assay trials, the samples had absorbance
measurements much higher than the highest glycerol
standard, making it impossible to determine how
much lipase they contained.
After diluting the samples to fit the glycerol standard
curve, a graph of the absorbance measurements
versus time showed a downward trend in each
sample. In the presence of a substrate, lipase is
expected to release increasing amounts of glycerol
over time. The downward trend that was observed
would seem to indicate that lipase was either being
improperly extracted from the tissue samples or that
the glycerol was going undetected by the assay.
Results
Time v. Absorbance measurement