ANALELE ŞTIINŢIFICE ALE UNIVERSITĂŢII “AL. I. CUZA” IAŞI
Tomul III, s. Biofizică, Fizică medicală şi Fizica mediului 2007
ANALYSIS METHODS OF OXYGEN CONSUMPTION
IN BIOLOGICAL SYSTEMS
Magda Aflori 1, I. Neacşu 2, V. Melnig3
KEYWORDS: oxygen consumption, Clark method, Warburg method, Winkler
method
Three usual methods to determine the oxygen consumption: electrode Clark potentiometric
method, Warburg micromanometric method and Winkler titrimetric method were described
in this paper.
1.
INTRODUCTION
The potentiometric Clark oxygen electrode method is used for evaluation of
oxidative respiratory process, fermentation processes, etc. The Warburg
micromanometric method allows the estimation of the photosynthesis process intensity
by the measurement of CO2 consumption and/or O2 consumption in respiratory
processes. The Winkler method permits to appreciate the O2 consumption, for aquatic
organisms, from the estimation of O2 quantity dissolved in liquid environment.
2.
EXPERIMENTAL SETUP
2.1. Oxygen electrode method (Clark)
When an electrode of noble metal, such as platinum or gold, is polarized within
0.6 - 0.8 V, negative in respect to a suitable reference electrode, such as Ag/AgCl or a
calomel electrode (Fig. 1) [1, 2], in a neutral KCl solution, the oxygen dissolved in the
liquid is reduced at the surface of the noble metal following the next electrodes’
reactions:
Chatodic reaction:
(the electrons are boiled off
of the platinum electrode)
1
O2 + 2H2O + 2e- → H2O2 + 2OHH2O2 + 2e- → 2OH-
“Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania
Faculty of Biology, “Al. I. Cuza” University, Iasi, Romania
3 Faculty of Physics, “Al. I. Cuza” University, Iasi, Romania
2
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Magda Aflori, I. Neacşu, V. Melnig
Anodic reaction:
(the electrons complete
the circuit)
Overall reaction:
Ag + Cl- → AgCl + e-
(I)
4Ag + O2 + 2H2O + 4Cl- → 4AgCl + 4OH-
Current flows from the silver electrode to the platinum electrode as electrons
boil off into solution from the latter. Removal of electrons from solid silver produces
silver ions. The silver ions combine with chloride ions in solution to precipitate silver
chloride on the surface of the silver electrode. This leaves potassium ions behind,
however since OH- ions are produced by the chatodic reactions the charge remains
balanced.
Fig. 1: Experimental set-up for the oxygen reducing
at the surface of the noble metal.
Two principal pathways were proposed for reduction of oxygen at the noble
metal surface. One is a 4-electron pathway, where the oxygen in the bulk diffuses to
the surface of the cathode and is converted to OH- via H2O2 (path a in Fig. 2). The
other is a 2-electron pathway, where the intermediate H2O2 diffuses directly out of the
cathode surface into the bulk liquid (path b in Fig. 2). The oxygen reduction path may
change depending on surface condition of the noble metal. This is probably the cause
for time-dependent current drift of polarographic probes. Since the hydroxyl ions are
constantly being substituted for chloride ions as the reaction starts, KCl or NaCl has to
be used as the electrolyte. When the electrolyte is depleted of Cl-, it has to be
replenished.
Fig. 2: Alternative pathways of oxygen reduction at cathode surface.
ANALYSIS METHODS OF OXYGEN…
7
This phenomenon can be observed from a current-voltage diagram - called a
polarogram - of the electrode, as shown in Fig. 3.a. When the negative voltage applied
to the noble metal electrode (cathode) is increased, the current increases initially but
soon it becomes saturated. In this plateau region of the polarogram, the reaction of
oxygen at the cathode is so fast that the rate of reaction is limited by the diffusion of
oxygen to the cathode surface. When the negative bias voltage is further increased, the
current output of the electrode increases rapidly due to other reactions, mainly, the
reduction of water to hydrogen. If a fixed voltage in the plateau region is applied to
the cathode, for example, - 0.6V, the current output of the electrode can be linearly
calibrated to the dissolved oxygen (Fig. 3.b). It has been noted that the current is
proportional with the activity or equivalent partial pressure of dissolved oxygen,
which is often referred to as oxygen tension. A fixed voltage between -0.6 and -0.8 V
is usually selected as the polarization voltage when using Ag/AgCl as the reference
electrode.
Fig. 3: Polarogram diagram of the oxygen reduced at the surface of the noble metal (a)
and intensity vs. O2 concentration calibrating diagram (b).
When the cathode, anode, and the electrolyte are separated from the
measurement medium by a polymer membrane, which is permeable to the dissolved
gas but not to most of the ions and other species, and when most of the mass transfer
resistance is confined in the membrane, the electrode system can measure oxygen
tension in various liquids. This is the basic operating principle of the membrane
covered polarographic probe (Fig. 4).
In principle an oxygen analyzer consist in a current-tension circuit converter
amplifier that convert the 0.1μA sensor current order to voltage as shown schematically in
Fig. 5.
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Magda Aflori, I. Neacşu, V. Melnig
Fig. 4: Section sketch of the membrane covered polarographic probe.
Fig. 5: Circuit for current to voltage conversion and application of polarization voltage.
2.2. Warburg micromanometric method
The Warburg micromanometric method [3, 4] consists in determination of oxygen
consumption at constant respiration volume.
The Warburg device (Fig. 6) contains a thermostatic water bath with a stirrer
system; a Warburg vessel attached to a manometer made from a capillary tube (A) with
Brodie liquid; the reaction vessel (B) which has a main compartment that contains the
sample for analyzing, a central one (e) which contains KOH for CO2 retention and a lateral
branch (f) closed with a faucet (g); a rubber tube (a), tank for the Brodie liquid; a faucet
(h) which allows the Brodie liquid up and down in the manometer; a faucet (c) for closing
the system.
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ANALYSIS METHODS OF OXYGEN…
Fig. 6: Sketch of the Warburg manometer.
The total respiration container volume represents the air volume limited by the
reaction vessel and the surface of Brodie liquid in the capillary tube. The volume’s
constant, C, of Warburg vessel is done by the total respiration container volume,
following the next relation:
C=
T0
+ V1 a
T
,
P0
(Vt − V1 )
(1)
where: Vt - the total volume (in mm3) of respiration container; Vl - the liquids’ volume
from respiration container; T0 = 273,15 oC; T – the working temperature in K; a – the
Bunsen solubility coefficient of O2 and P0 – represents the atmospheric pressure
expressed in mm col Brodie liquid (10,000 mm col Brodie liquid = 1 atm).
The oxygen consumptions, XOxigen (in mm3 normal volume), by tissue sample is
determined by the equation:
(2)
X Oxigen = h ⋅ C ,
where h (in mm col Brodie liquid) represents the length of Brodie liquid column that
compensates the depression from respiration volume, determined by consumption of
oxygen, at constant respiration volume.
2.3.The Winkler method [5]
The oxygen dissolved in the liquid environment is fixed with MnCl2 in
presence of sodium hydroxide and potassium iodide, forming manganiferous
hydroxide, which, in the presence of oxygen is transformed in precipitated manganic
hydroxide.
The precipitate dissolves in concentrated hydrochloric acid releasing the iodine
(KI) that can be highlighted by blue coloring with 2-3 drops of 0,1% starch solution
and then titrated with 0,02N sodium tiosulfate solution until the color disappears. In
short, the O2 concentration is proportional with the sodium tiosulfate mass implied in
colorimetric titration.
The concentration of oxygen, C, from water is calculated with the formula:
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Magda Aflori, I. Neacşu, V. Melnig
(
)
C mg Oxygen L =
Vtiosulphat × f tiosulphat × 1000 × 0.08
(
VWinkler − V MgCl2 + V KI inNaOH
)
,
(3)
where: ftiosulphate – the tiosulphate solution factor; 1,000 represents the volume
conversion factor from liters, L, to millilitres, mL, and 0.08 – the oxygen quantity, in
mg, titrated by 1 mL 0.01 N tiosulphate solution. If the oxygen concentration is
expressed in mLOxigen/L the 0.08 value from equation (3) must by replaced with 0.056;
all the volumes from equation (3) are in mL.
The water from the piscicultural basins is poor in oxygen if this value becomes
lower then 4 mg O2/L.
3.
RESULTS AND DISCUSSION
The purpose of this practical is to investigate the respiratory chain by using
these techniques and to increase the understanding of the respiratory chain and the
flow of electrons through the major enzyme complexes.
The following experiments will be done:
1. Determination of ADP:O quotient and respiratory control;
2. Effect of different inhibitors on the respiratory chain.
3.1. Clark method
Oxygen electrode (oxygraph) recordings
If mitochondria are incubated in an oxygraph apparatus (oxygen electrode) in
an isotonic medium containing substrate and phosphate, then addition of ADP causes
a sudden burst of oxygen uptake as the ADP is converted into ATP.
The actively respiring state is sometimes referred to as "state 3" respiration,
while the slower rate respiring state, after all the ADP has been phosphorylated to
form ATP, is referred to as "state 4". State 4 respiration is usually faster than the
original rate before the first addition of ADP because some ATP is broken down by
ATPase activities contaminating the preparation, and the resulting ADP is then rephosphorylated by the intact mitochondria.
The ratio [state 3 rate] : [state 4 rate] is called the respiratory control index and
indicates the tightness of the coupling between respiration and phosphorylation. With
isolated mitochondria the coupling is not perfect, probably as a result of mechanical
damage during the isolation procedure. Typical RCI values range from 3 to 10,
varying with the substrate and the quality of the preparation. Coupling is thought to be
better in vivo, but may still not achieve 100%. It is possible to calculate an ATP:O
ratio (the relationship between ATP synthesis and oxygen consumption) by measuring
the decrease in oxygen concentration during the rapid burst of state 3 respiration after
adding a known amount of ADP. It is necessary to subtract the basal respiration due to
imperfect coupling and the recycling of ATP, as shown in the graph from figure 7.
The concentration change must then be multiplied by the chamber volume, so that the
answer (in micro-atoms of oxygen) can be related to the quantity of ADP added. The
ANALYSIS METHODS OF OXYGEN…
11
quantity of oxygen in the chamber is calculated from published oxygen solubility data
at the appropriate temperature.
Example: Considering a decreasing in [O2] = 0.135 mM (Fig. 7); Vchamber =
2.5ml, oxygen atoms consumed in state 3 = 0.135 * 2 * 2.5 = 0.68 micro-atoms (note
the factor of 2, allowing for 2 atoms in an oxygen molecule); injected ADP (20
microlitres of a 50mM solution) = ATP formed = 1 micromole in total, are obtained:
ATP:O = [ATP formed] : [oxygen consumed] = 1.48 (for succinate oxidation);
NAD-linked substrates give consistently higher values for the ATP:O ratio
(about 2.5) compared with succinate (about 1.5).
These results indicate that electrons from relatively poor reducing agents such
as succinate (also acyl CoA and glycerol phosphate) enter part of the way along the
respiratory chain, by-passing the first coupling site where energy is captured for ATP
synthesis.
The sharp changes in slope after exhaustion of ADP and again after all the
oxygen has been used up imply that mitochondria must have very high affinities for
ADP and oxygen. The concentration of both these compounds is very low in most
healthy cells, since they are efficiently scavenged by the mitochondria. The addition of
an uncoupling agent (such as dinitrophenol or CCCP) leads to a permanently high rate
of respiration in the absence of ADP, until all the oxygen has been consumed.
Many natural and synthetic poisons block mitochondria respiration. If
mitochondria are incubated in an oxygraph experiment with substrates and inorganic
phosphate, the interactions between inhibitors and uncouples allow two major types of
inhibition to be distinguished: those that prevent respiration by blocking the
respiratory chain itself, and those that inhibit the ADP phosphorylation system, so it
only blocks respiration in coupled mitochondria.
Fig. 7: Sketch of a mitochondria respiration experiment diagram.
Preparation of mitochondria [2, 6]
1. Sacrifice a chicken and transfer the liver to a beaker containing ice chilled
sucrose buffer.
2. Cut 6 g of the liver in small pieces in a beaker containing 10 ml of sucrose
buffer. Decant the sucrose solution and rinse again. Transfer the liver pieces with 25
ml sucrose buffer to a Potter- Elvenhjem homogeniser.
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Magda Aflori, I. Neacşu, V. Melnig
3. Homogenize the liver sample by moving the pistil up and down until the pistil
reaches the bottom of the homogeniser. After that, move the pistil up and down 3 more times.
4. Dilute the homogenate to 10% (w/v) with sucrose buffer.
5. Centrifuge at 600 g (2400 rpm) during 10 min at 4 °C.
6. Transfer the supernatant, very carefully, to new ice chilled centrifuge tubes.
7. Centrifuge the supernatant at 11,000 g (9000 rpm) for 10 min at 4 °C.
8. Remove the supernatant and the "fluffy" layer and make a suspension of the
mitochondria (the pellet) by carefully pipetting the sucrose buffer up and down in the
pipette (50% of the homogenate volume).
9. Centrifuge the mitochondria suspension at 11,000 g (9000 rpm) during 10 min at 4
°C. Remove the supernatant and suspend the mitochondria as described according to point 8.
10. Repeat as point 9. Suspend the mitochondria in about 5 ml sucrose buffer.
In figure 8 is suggestively presented the upper experimental protocol.
Fig. 8: The schematic representation of isolation mitochondria protocol.
ANALYSIS METHODS OF OXYGEN…
13
Determination of ADP:O quotient and respiratory control [7-10]
Oxygen is consumed when suitable substrates are oxidized by the mitochondria
electron transport system. This decrease in oxygen concentration in a system closed
from the atmosphere can be measured with an oxygen electrode, which is in effect an
oxygen polarograph. In figure 9 is presented the sketch of experimental set-up used in
Clark electrode method.
In "coupled mitochondria", electron transport and the synthesis of ATP from
ADP and Pi are mutually dependent processes, i.e., in addition to an oxydizable
substrate; the presence of both ADP and inorganic phosphate is required for oxygen
uptake to occur.
The amount of consumed oxygen linked to substrate oxidation can be
determined following the next protocol:
1. Determine the volume of the reaction vessel (the reaction vessels have
different volumes). Calculate how much of the incubation media that has to be added
to fill the reaction vessel with the other substances added afterwards. The incubation
media should be kept in an Erlenmeyer flask placed in a thermostated water bath (30
°C) and it should be gassed with a continuous air flow.
2. Add the calculated amount of incubation media to the reaction vessel.
3. Add 100 μL mitochondria suspension after about 5 minutes and mark on the
protocol paper which additions are made in consecutive order.
4. Follow the graph for 1-2 min and add 20 μL substrate (start with glutamate)
and observe what happens after the addition.
5. After about 1-2 min registration add 10 μL ADP via a Hamilton syringe and
record the O2-concentration until all ADP has been consumed.
6. Finally, when all ADP is consumed, add 20 μL of DNP. The DNP addition
shall be done only with one of the substrates e.g. with glutamate.
7. Repeat the experiment by using succinate and ascorbate/TMPD as substrate.
When ascorbate/TMPD is used add only 5 μL ADP (instead of 10 μL).
8. Calculate the ADP:O quotient and the respiratory control ratios for all three
substrates used. Indicate the theoretical ADP:O values for the three substrates.
Calculate the respiratory control ratio (the slope with ADP/without ADP) for each
substrate.
Fig. 9: Schematic sketch of experimental set-up used in Clark electrode method.
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Magda Aflori, I. Neacşu, V. Melnig
Effect of different inhibitors on the respiratory chain
By following the oxygen consumption range we can estimate the ADP:O ratio
by referring the consumed ADP quantity to the O2 quantity consumed for his
phosphorilation.
Different inhibitors block the electron flow through the respiratory chain. In
our experiment we used inhibitors like rothenone, antimycin and azida. From this
experiment conclusions can be drawn about which enzyme complex is blocked by
which respective inhibitor. The respiration diagram for the used inhibitors is presented
in figure 10. From the figure there one can evaluate the intensity of blocking effect of
rothenone, antimycin and azida. The antimycin has a strong effect followed by
rothenone and azida.
The conclusion to be drawn from this type of experiment is that our inhibitors
only block respiration in coupled mitochondria compared with cyanide, that prevents
respiration by blocking the respiratory chain itself; cyanide is effective whether or not
ADP or uncouplers are added.
Fig. 10: Respiration diagram for rothenona, antimicina and azida used as inhibitors.
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ANALYSIS METHODS OF OXYGEN…
3.2. Warburg method [2, 4]
The respiratory reactivity is a function of the tissue type and the experimental
length.
Oxygen consumption (mg O2/ g tissue /hour)
The respiration process can be observed on tissular samples from frog Rana
ridibunda, Pall. consisting in liver, striated muscle Sartorius or smooth stomach
muscle fragments. The O2 consumption (mgO2/g tissue/hour) was determined for 90
min time, 20ºC constant temperature and constant volume. The results are presented in
figure 11.
The respiratory intensity value is high to the striated muscle, medium for the
liver and lower to the stomach tissue.
Liver
Striated muscle
Stomach muscle
2,4
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
10
20
30
40
50
60
70
80
90
100
Time (min)
Fig. 11: The O2 consumption (mgO2/g tissue/hour) determined for 90 min time, 20 ºC
constant temperature and constant volume for samples figured in legend.
3.3.Winkler method [5]
In this experiment it has been observed the influence of water pollution on the
fishes respiratory intensity. Five fish (Carassius auratus gibelio) were placed in closed
respiratory vessels with water from three Prut stations (Prisacani): in station 1 with
clean water; in station 2 a lower quantity of dissolved oxygen and a bigger quantity of
organic substance (moderate pollution); in station 3 a certain pollution degree which
cause oxidative stress to fish, a lower concentration of dissolved oxygen and a bigger
content of organic substance which maintains reduction processes with oxygen
consumption.
In figure 12 is presented the dependence of respiratory intensity vs. mass
weight of fishes and pollution water degree parameter.
Considering 100% respiration percentage for station 1 the following
conclusions can be done:
Station 1: the respiratory intensity is in general inverse proportional with fish
weight;
Station 2: all fish have a bigger respiratory intensity compared with station 1
(106.15%);
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Magda Aflori, I. Neacşu, V. Melnig
Oxygen consumption (mg O2/kg/hour)
Station 3: the intensity of respiration becomes as bigger as the pollution has
increased (120.80%).
In all experiments it has been observed a decrease of oxygen consumption with
increasing of fish mass weight.
Station 1 (100%)
Station 2 (106,15%)
Station 3 (120,80%)
80
75
70
65
60
55
50
45
40
55
60
65
70
75
80
Fish weight (g)
Fig. 12: Oxygen consumption vs. fishes mass weight and pollution water
degree parameter.
4.
CONCLUSIONS
This paper describes the usual methods used to determine the oxygen
consumption in respiratory processes.
The methods presented have particularities that influence precision of the
results and/or in particularly experiments can be used exclusively [11].
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Benga Gh., “Indrumar pentru lucrarile practice de biologie celulara”, Ed. IMF Cluj-Napoca,
1982.
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Nuta GH., Busneag C., Biochemical Investigations, Ed. Didactica si Pedagogica, Bucharest,
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5.
Mustata Gh., “Lucrari practice de hidrobiologie”, Ed. Univ. Al. I. Cuza Iasi, 1992.
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Lehninger A.L., The Mitochondrion: Molecular Basis of Structure and Function, W.A. Benjamin,
New York, 1964.
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Racker E., Mechanisms in Bioenergetics, Academic Press, New York, 1965.
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Mitchell P., Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn
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Mitchell P., Chemiosmotic Coupling and Energy Transduction, Glynn Research, Bodmin, 1968.
10. Mitchell P., Moyle J., Biochemistry of Mitochondria, Academic Press, London,53, 1967 c.
11. Park J.H., Kaplan N.O., Kennedy E.P., Current Aspects of Biochemical Energetics, Academic
Press, New York, 299, 1966.