Short Term Response of
LDH Activity to H
poxia in
Fundulus heteroclitus
John H. Morton
June 9, 1995
Biology 175H
Advisors: Patricia Schulte and Dennis Powers
Acknowledgments:
would like to thank Trish Schulte for helping me with this
project. Without her infinite patience and incredible
teaching ability, I would never have completed it.
Abstract
Previous studies have shown that the activity of lactate
dehydrogenase (LDH) in liver of the killifish Fundulus heteroclitus
increases approximately two- fold after two weeks of hypoxia.
However, nothing is known about the early response of LDH activity
to hypoxia. In this study the effects of shorter durations of hypoxia
on LDH activities in Fundulus heteroclitus were examined. Hypoxia
was induced in aquaria by gassing with an air/ nitrogen mixture.
After 1, 2, 4, and 7 days of hypoxia fish were killed and livers
prepared for LDH assays. LDH activity in liver of Fundulus
heteroclitus increased two- fold after two days of hypoxia. This
finding demonstrates that the response to hypoxia in Fundulus
heteroclitus is much faster than previous research has suggested.
These results in F. heteroclitus are consistent with previous
findings that mRNA encoding LDH increases after 16 hours of hypoxia
in human cell cultures. Possible molecular mechanisms underlying
the hypoxia- induced increase in LDH activity are discussed.
Introduction
Hypoxia, or low oxygen concentration, can have profound effects on
organisms dependent on aerobic metabolism for a majority of their
energy production. One way in which organisms respond to hypoxia
is by altering the quantity or functioning of certain enzymes. One
enzyme of key interest in studies on hypoxia is lactate
dehydrogenase (LDH), which is the terminal enzyme in anaerobic
glycolysis. Glycolysis results in the breakdown of glucose with the
formation of 2 ATP and 2 NADH molecules. When oxygen is present
in the cell, the products of glycolysis, pyruvate and NADH, can be
used to produce more ATP through the Krebs cycle and electron
transport. When oxygen is not present LDH converts pyruvate to
lactate and oxidizes NADH to NAD+. Because NAD+ is a substrate of
other enzymes in glycolysis, its regeneration is essential for the
breakdown of glucose and production of energy in times of low
oxygen concentration in the cell.
Because LDH is important under anaerobic conditions, 1 hypothesized
that one response of cells exposed to hypoxia would be to produce
higher levels of LDH. Semenza et al., (1994) demonstrated that LDH
in human HeLa cell cultures is produced at 2 to 3 times normal
levels when they are exposed to hypoxia. Öther research confirms
this type of increase in mammalian cell cultures (Robin et al., 1984).
The purpose of the experiment presented here is to demonstrate the
effects of hypoxia on LDH production in the fish Fundulus
heteroclitus. Fundulus is a teleost fish which lives in estuarine
environments along the Atlantic coast from Newfoundland to Florida
(Powers et al., 1992). This animal was chosen because it has been
shown to have a high tolerance to hypoxic water and it has been the
subject of numerous biochemical physiological and genetic studies
(Greaney et al., 1978; Powers et al., 1992).
Previous studies (Greaney et al., 1980) demonstrated that LDH
activity in F. heteroclitus liver tissue was about 50 percent higher
than normoxic control levels after exposing the fish to two weeks of
hypoxia. Hypoxia was defined as 1.5- 2.5 ppm oxygen concentrations
in the water. Greaney et al. (1980) distinguished between short¬
term and long- term enzymatic/ hormonal responses to hypoxia in
Fundulus. The short-term response was an increase in the
production of LDH and other glycolytic enzymes in response to a drop
in oxygen concentration. The long- term response was an increase in
red blood cells as possibly caused by an increase in erythropoietin
hormone. As the amount of red blood cells increase, intracellular O2
concentrations also increase. This reduces the need for increased
glycolysis. Greaney et al. (1980) demonstrated that in the first 4
weeks of hypoxia, LDH activity remained at a relatively constant
level above the control and then dropped back to the control level by
the fifth week. An increase in hematocrit (red blood cell
concentration) was shown to parallel the increase in LDH activity
during the first 4 weeks. However, during the fifth week,
erythrocyte levels remained higher than control levels.
What is not accounted for by Greaney et al., (1980) is exactly when
the short- term response begins. Work by Semenaza et al., (1994)
nas shown that hypoxia induces an increase in transcription of mRNA
encoding LDH in HeLa and Hep3B cell cultures within 16 hours. From
these results, I hypothesized that Fundulus heteroclitus LDH
activity may respond to hypoxia much sooner than the first time
point of two weeks in Greaney's study, perhaps between 16 to 72
hours after the onset of hypoxic conditions.
Materials and Methods
Animals
Wild Fundulus heteroclitus were obtained from Woods Hole,
Massachusetts one month before onset of the experiment. This long
acclimation time was necessary to reduce potential handling effects
resulting from shipment and changes in water quality or
temperature between Woods Hole and Hopkins Marine Station. These
fish were placed in 10 gallon aquaria with filtered seawater at 20°0
and fed once daily.
Hypoxia Apparatus
An apparatus for producing hypoxic conditions in aquaria was
designed and built as shown in figure 1. Two 55 gallon aquaria were
placed next to each other. A submersible pump was used to pump
water from the lower aquarium through a canister filter and into the
upper aquarium. The second aquarium was raised so that water
would flow by siphon through a hose back into the lower aquarium.
Valves on the siphon hose and the pump outflow were adjusted to
maintain a steady level of water in both aquaria. Hypoxia was
induced in the aquaria by bubbling an air/ nitrogen gas mixture
through two air stones in each aquarium. This mixture was created
by feeding nitrogen into a 10 gallon gas mixing chamber with a
loosely fitting lid. Pure nitrogen at a flow rate of 1 liter per minute
mixed with air coming in at the top of the tank, and this mixture
was pumped at a constant rate of 1.3 liters per minute into the two
aquaria by a small air pump. Preliminary experiments showed these
flow rates produced hypoxic oxygen concentrations of generally
between 1.5 to 2.5 mg/ liter within the aquaria. High enough flow
rates were used to keep oxygen concentrations constant in spite of
removal of oxygen by fish respiration. Bubble wrap was placed over
the water in the two aquaria. This prevented ambient oxygen from
entering the water by simple diffusion. Dissolved oxygen
measurements were taken approximately every 30 minutes in order
to monitor the drop in oxygen level at the onset of hypoxia and were
taken once daily throughout the course of the experiment
A third 55 gallon aquarium was equipped with a separate filtration/
aeration device and maintained at about 7.5 mg/ I oxygen. This
aquarium was designated for normoxic control fish. All tanks were
filled with seawater from Monterey Bay, California at approximately
20°.
Experimental Design
Several days before the onset of the hypoxic treatment, fish were
moved into the three large aquaria. At the start of the experiment
there were 10 fish in the control aquarium, and 38 fish divided
between the two hypoxia aquaria. Five control fish were sampled
and bubbling of air/ nitrogen mixture was started,
Fish in the hypoxic tanks were taken out and dissected exactly one
day, two days, four days, and seven days after the nitrogen gas was
türned on. Six fish were taken at each time point. On the fourth day
the remaining four fish in the control tank were sampled.
Fish were quickly decapitated, weighed and their livers excised The
liver was divided in two parts and immediately frozen in liquid
nitrogen and stored at -70°c.
Frozen liver was weighed and then transfered to a small Dounce
homogenizer. Homogenization solution (50 mM NaHPOJ/NaH»POA, pH
7.00, and 1 mM EDTA (disodium salt) with 5mM 2-mercaptoethanol
added fresh) was added to the liver sample such that there was
always .025 milligrams of tissue per microliter of solution. The
tissue homogenate was then centrifuged at 4°C at 10,000 X g for 30
minutes. Supernatants were removed and stored on ice until
assayed.
Assay for LDH Activity
LDH activity was determined by measuring the rate of oxidation of
NADH to NAD- in the conversion of pyruvate to lactate. NADH
absorbs 340 nm light while NAD+ does not. As a result, NADH
oxidation results in a decrease in absorbance (Vassault, 1983). The
assay solution contained 0.1 M NaHPOY/NaHzPO4 (pH 7.5), 2.64 mM
pyruvate and 0.34 mM NADH (from Sigma Chemical Company)
(Greaney et al., 1980). 5 microliters of supernatant was added to 3
mi of assay solution and change in absorbance was recorded for two
minutes. All assays were done in a Beckman DU- 7
spectrophotometer at 25° C. Each assay was replicated 2-3 times,
Assay for Protein Concentration
Amount of protein in the supernatant was determined with the
Bradford method (Bradford, 1976). A microassay was done using 15
ul supernatant, 785 ul water and 200 ul of Bradford reagent (Bio¬
Rad Laboratories), according to the manufacturer's instructions.
Bovine Serum Albumin (BSA) was used as a standard.
Calculations
Units of LDH per gram fresh weight were calculated with the
following formula:
Units = [(AÖD per minute)X (assay volume (mi)))- [(.63 ml umol¬
mm- (NADH extinction coefficient)) X (10 mm (light path distance))
X(mass of sample (9)))
Units per milligram protein was calculated with the same formula
but expressed per milligram protein instead of grams liver.
Data Analysis
A fully nested ANÖVA with three levels was done on LDH activity per
gram wet weight of tissue. This would account for variance due to
difference within replicates. Replicate averages of LDH per ma
protein were analyzed for significant difference among treatments
with a one- way ANOVA. A posteriori comparisons between control
and each hypoxia treatment were made with Tukey's T test.
Results
Hypoxia in Aquaria
Figure 2a shows the decrease of dissolved oxygen in the two aquaria
as à function of time immediately after turning on air/ nitrogen
mixture. Time at 0 minutes marks the introduction of the air/
nitrogen mixture through air stones in the aquaria. This graph
demonstrates that fish experienced hypoxic concentrations of
oxygen (1.5- 2.5 mg/1) after 2 hours of bubbling with air/ nitrogen
mixture. Oxygen levels ranged from 1 to 2.6 mg/L throughout the
course of the experiment. Fluctuations in oxygen levels for all 7
days are seen in figure 2b.
LDH Assay Per Gram Wet Weight of Liver
Figure 3 and table 1 show that mean LDH activity per gram wet
weight of liver increases in fish under hypoxia as compared to that
of the fish in the normoxic control aquarium. There is a 50%
increase in LDH activity after one day of hypoxia and then a decrease
in activity in the day 2, 4, and 7 time points. These results are not
significant (p= .08) due to large amounts of variance in LDH activity
among the fish in each treatment,
LDH Assay per mg protein
A more accurate determination of LDH activity is the LDH activity
per milligram protein the supernatant. There are other components
in liver besides protein such as lipids and sugars (especially
glycogen) whose amounts can potentially differ in different livers,
These differences can be factored out by expressing LDH activity per
mg protein.
LDH activity per mg protein was calculated by dividing the mean
change in LDH optical density for the replicates by their mean
protein amount. These means are shown in figure 4 and table 2.
Mean LDH activities of hypoxic fish are consistently higher than the
controls. After 2 days of hypoxia LDH activity is 170% of the
control. Averages then drop in day 4 and 7 but remain slightly more
than the control. There is statistically significant difference among
all treatments (p « .05 as shown in table 4). Difference between the
normoxic control LDH activity and the average of all hypoxia time
points is significant at p= .012. When the mean LDH activity for
each day of hypoxia is compared to the control, tukey's T test
demonstrates significance in only that of the control ys. hypoxia day
Discussion
Short Term Response to Environmental Hypoxia
LDH activity in Fundulus heteroclitus liver increases in the first
days of exposure to a hypoxic environment. There is a statistically
significant two- fold increase in LDH activity per mg protein
relative to the control after 2 days of hypoxia.
In the study by Greaney et al. (1980) LDH activity per mg protien
rose from 2. 82 + .143 (mean + S.E.) in control fish to 3.95 + .274
after two weeks in hypoxia. These levels persisted until about 5
weeks when they dropped possibly due to a long-term erthrocyte
response. The present findings show, LDH activity increased by 2
days of hypoxia showing that the response is much quicker.
LDH activities then decreased back to almost the levels of the
control in days 4, and 7. Statistical tests demonstrate no
significant difference between hypoxia day 4, and 7 and the control.
Do LDH levels really increase after two days, decrease by day 7 and
then increase again by day 14 as suggested by combining our and
Greaney's data? It would follow that LDH levels increase after 2
days of hypoxia, and remain high until 5 weeks later when the long
term response eliminates the need for increased glycolytic activity
It could be that LDH activities in day 4 and 7 are truly higher than
the control, and that large amounts of variance within each
treatment resulted in the lack of statistically significant
differences between treatments.
There are a number of potential sources of error in this experiment
One potential source of variance was genetic variability between
fish. Fundulus heteroclitus is known to be made up of a genetically
distinct northern and southern type. The northern type has been
shown to have a liver LDH activity twice that of the southern
(Powers et al., 1994). F. heteroclitus from the Woods Hole area are
known to be about 75 percent northern type and 25 percent southern
type (Powers and Place, 1978). Because the exact type of each fish
in the experiment was not assessed, it is not known to what degree
different F. heteroclitus types effected the variance of the
treatment averages.
Öther possibly significant sources of error were stresses other than
hypoxia. One of these stresses, disease, may have had a significant
effect on LDH activity by possibly altering metabolic rates of the
fish. A second stress was human interactions and handling. Sending
fish by mail over long distances as well as being kept in a setting
characterized by a lot of human movement, may also have altered
metabolic rates and thus affected LDH activities (Fries, 1986).
A third source of error was the human error involved in making
homogenates and doing assays for LDH. An effort was made to
account for error in the LDH assay procedure by doing replicate or
triplicate assays for each fish. LDH was expressed as a function of
protein in an effort to rule out error caused by subtle differences in
the preparations of the homogenate.
Now that we have an idea of when LDH is produced, we may look at
the molecular level for possible factors which enhance LDH
production when hypoxic conditions are present. It has been shown
in rat and human cell cultures that a Hypoxia- inducible Factor (HIF)
promotes the transcription of mRNA which encodes for LDH.
According to this research, mRNA encoding LDH is transcribed 2- 3
times more in cell cultures after 16 hours of hypoxia. (Semenza et
al., 1994) It is also known that HIF triggers not only mRNA for LDH
and other glycolytic enzymes but also the erythropoietin hormone
involved in a long-term response to hypoxia (Goldberg et al., 1991).
An experiment looking for mRNA which encodes for LDH, other
glycolytic enzymes, and the erythropoietin hormone over several
time points from one day to several weeks under hypoxia would
compliment these findings and the findings of Greaney's research.
Fürther analysis of the DNA for HIF binding sites would help form
the big picture as to exactly what biochemical changes occur due to
hypoxia and if HIF induces them.
The relatively quick response to low oxygen environments seems to
indicate a beneficial adaptation of F. heteroclitus to withstand
areas of low oxygen in its natural habitat. Esturarine areas where
the fish lives are often characterized by slow-moving or stagnant
bodies of water containing large amounts of organic material and
potentially low levels of oxygen. If the fish is somehow trapped in a
hypoxic body of water due to, for example, lowering tides, it must
gear its biochemical machinery for the prevailing conditions in order
to survive.
Exactly at what time this response should be seen in Fundulus
depends on other factors than just a decrease in environmental
oxygen concentration. Fish behavior is of key importance to the
levels of oxygen that are found in blood and interstitial fluid in the
fish. Increased ventilation of the gills could result in stable levels
of blood oxygen concentrations even while outside environmental
concentrations drop. In addition physiological changes such as
vasodilation of arteries around the gills, increased heart rate and
expanded blood volume would all contribute to a stable oxygen
concentration in the tissues. These responses to hypoxia all delay
decreases in oxygen concentration within the cells until the
environmental oxygen concentration reaches a point where no
fürther continuation of these responses can maintain O» levels.
In conclusion, a definite response to hypoxia was found in LDH
levels. This response was found to be within 2 days after begining
the hypoxia treatment. Using these results as a starting point we
hope to elucidate the molecular mechanism which triggers increased
LDH production.
Literature Cited
Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein- dye binding. Analytiçal Biochemistry 72: 248-
254
Fries, C. R. 1986. Effects of environmental stressors and
immunosuppressants on immunity in Fundulus heteroclitus,
American Zoology 26: 271-282
Goldberg, M. A., Gaut, C. C., Bunn, H. F. 1991. Erythropoietin
messenger RNA levels are governed by both the rate of gene
transcription and posttranscriptional events. Blood. 77(2): 271-277
Greaney, G. S., Powers, D. A. 1978. Allosteric modifiers of fish
hemoglobins: in vitro and in vivo studies on the effect of ambient
oxygen and pH on erythrocyte ATP concentrations. Journal of
Experimental Zoology 203: 339-349.
Greaney, G. S., Place, A. R., Cashon, R. E., Smith, G., Powers, D. A.
1980. Time course changes in enzyme activities and blood
respiratory properties of killifish during long-term acclimation to
hypoxia. Physiological Zoology 53(2) :136-144.
Powers, D. A., Place, A. R., 1978. Biochemical genetics of Fundulus
heteroclitus (L.). I. Temporal and spatial variation in gene
frequencies of Ldh- B, Mdh- A, Gpi- B, and Pgm- A. Biochemiçal
Genetics 16: 593-607
Powers, D. A., Lauerman, T., Crawford, D., DiMichele, L. 1992. Genetic
mechanisms for adapting to a changing environment. Pages 629-659
in Campbell, A. (Ed.). Annual Beview of Genetics, Vol. 25. Annual
Reviews Inc., Palo Alto, USA.
Robin, E. D., Murphy, B. J., Theodore, J. 1984. Coordinate regulation
of glycolysis by hypoxia in mammalian cells. Journal of Cellular
Physiology 118: 287-290
Semenza, G. L., Roth, P. H., Fang, H. M., Wang, G. L. 1994.
Transcriptional regulation of genes encoding glycolytic enzymes by
Hypoxia- inducible Factor 1. The Journal of Biological Chemistyy
269: 23757-23763.
Vassault, A., 1983. Pages 118- 126 in H. U. Bergmeyer, ed. Methods
of Enzymatic Analysis. Verlag Chemie, Deerfield Beach, Florida.
Figure Legends
Figure 1:
Experimental Setup
Figure 2a: Expulsion of oxygen from aquaria after turning on air/
nitrogen mixture. Measurements were taken about
every 30 minutes for 9 hours.
Figure 2b:
Variation of oxygen levels in tank over a period of 7
days. Measurements taken once daily.
Figure 3:
Average LDH activities of liver tissue (per gram wet
weight). Error bars represent standard error. Six to nine
fish in each sample.
Figure 4:
Average LDH activities of liver tissue (per milligram
protein). Error bars represent standard error. Six to nine
fish in each sample.

4
3 —
100 200 300 400 500 600
Minutes
2.5
1
80.5
O 1 2
3 4 5 6 7
Days
20
Contro
Hypoxia
Day
Hypoxia Hypoxia
Day 4
Day
Hypoxia
Day 2
Treatment
Control
Hypoxia
Hypoxia
Day 1
Day 2
Treatment
Hypoxia
Day 2
Hypoxia
Day 7
Table 1: Average LDH activity in liver tissue
(per gram wet weight)
Treatment
Average activity in
Standard Error
units per gram wei
weight of liver
Control
262.66
38.094
Hypoxia
417.49
59.496
377.9
Hypoxia 2
46.870
Hypoxia 4
297.58
32.174
Hypoxia 7
335.20
24.465
Table 2: Average LDH activity in liver tissue with ANOVA analysis
(per milligram protein)
Treatment
Average activity in
Standard Error
units
per milligram
protein
Control
2.192
0.1790
Hypoxia 1
3.274
0.6023
Hypoxia 2
0.5331
3.782
Hypoxia 4
2.832
0.1920
Hypoxia
0.1403
2.694
Comparison
Degrees o
Sum of
F Value
P Value
Mean
Freedom
Squares
Square
Among
10.216
3.074
2.571
0.0322
Treatments
Within
28
23.418
0.836
Treatments
(Fish)
Orthogonal Comparsion Between Control and All Hypoxia Treatments
Degrees of
Mean Square
P Value
F- Value
Freedom
5.946
7.109
0126
28
8363
Tukey's T- Test for Minimum Significant Difference
MSD= 1.4154
Comparison
Difference Between Means
Control vs. Hypoxia Day
1.081
Control vs. Hypoxia Day 2
1.589
Control vs. Hypoxia Day 4
640
Control vs. Hypoxia Day
501