CAPÍTULO 2: ELEMENTOS DE LA HISTORIA DE LA INTEGRAL
2.5 LA INTEGRAL COMO OBJETO DE ESTUDIO MATEMÁTICO
2.5.1 Los inicios de un nuevo concepto de integral
3.6.1 Freeze Drying Process
Muscle samples of both injured and uninjured muscles, following 7 and 14 days of
treatment, previously stored at –172oC in liquid nitrogen were removed and cut in a
ooled cryostat. Approximately 5 mg of dry muscle is required for the extraction
rocess. The normal dry weight: wet weight ratio is approximately 1 to 4 (23%). Thus,
sufficient muscle (approximately 15mg) was c
p
the wet weight was measured to ensure
obtained. Holes were punctured in the top of eppendorf tubes which were placed in a
freeze dryer (Edwards Modulyo, Edwards High Vacuum, Britain, England). Samples
were freeze dried for a minimum of 36 hours.
At the completion of the freeze-drying, muscle samples were transferred to a dessicator
at room temperature (20-23oC). Muscle samples were weighed to determine dry weight
and enable the dry weight: wet weight ratio to be calculated. The benefit of freeze-
3.6.1.1 Metabolite Extraction Process
The metabolite extraction process was performed in accordance with the method of
Harris et al. (1974). Each muscle sample was powdered in a crucible for approximately
15–20 minutes. These are periodically observed under a microscope, with any
connective tissue or blood removed from the powder. The extraction process, performed
in on ice, began when 250µL of ice cold 0.5M perchloric acid (PCA)/1mM
ethylediniaminetetra-acetic acid (EDTA) was added to a labelled eppendorf tube
containing 2mg of powdered muscle. The perchloric acid is a strong oxidizing agent
and extracts all of the acid-soluble metabolites, at the same time denaturing the proteins
nd causing them to precipitate out. The suspension was vortexed and tapped
ove fibres from the wall of the vessel. The samples
2.1M ice cold KHCO3- was added to each tube, and left
stand on ice for 5 minutes. The mixtures neutralize, with the potassium and
g another precipitate. The samples were again centrifuged at
28,000RPM for 2 minutes. The supernatant was removed with pasteur pipettes, placed
a labelled cryule, and stored in the ultra-freezer (–172oC) for subsequent metabolite
nd Cr.
. 6. 2 ATP and PCr Analysis
he extract was analysed using a 3-step enzymatic process requiring readings in a
intermittently for 10 minutes to rem
were then placed in a pre-cooled (0-2oC) centrifuge and spun at 28,000 RPM for 2
minutes. Once finished, the eppendorf tubes were placed back in the ice. 200µL of the supernatant was removed by an aliquot, without disturbing the pellet, and placed into a
second eppendorf tube. 50µL of to
perchloride ions formin
in
analysis of ATP, PCr, a
3
T
triplicate on a fluorometer (Turner Fluorometer model 112, Sequoia-Turner
Corporation, USA). ATP and PCr levels were determined according to the method of
Principle of analysis:
creatine kinase
ATP + Glucose ADP + Glucose-6-P
glucose dehydrogenase
+
P-Creatine + ADP e + ATP
h
heexxookkiinnaassee
Glucose-6-phosphate + NADP Gluconolactone + NADPH
g everything with the exception of hexokinase and
e ADP/creatine kinase solution. The presence of excess ADP and creatine kinase
reaction 2.
his reaction produces glucose-6-phosphate, which is the only thing required for
ples are left to run for 30 minutes, which is enough time
proceeding, the amount of NADPH gives the value of ATP
oncentration in the muscle sample.
Creatin
6-P-
An NADH standard curve was recorded on a UV-visible spectrophotometer at 340 nm.
The change in fluorescence of the internal ATP, CP standards was then checked against
the NADH standard curve (refer to Appendix D for detailed methodology).
Blanks, ATP, CP and NADH standards and samples are analysed in triplicate. A
cocktail reagent was made containin
th
insures that the reaction moves from left to right. The first reading (R1) taken gives an
indication of any residual endogenous NADPH that may be present.
Dilute hexokinase solution was added to each tube. This was the catalyst for
T
reaction 3 to proceed. The sam
for equilibrium to be reached. The second reading (R2) is recorded. R1 subtracted from
R2 gives the change in NADPH concentration. As all of the NADPH from R2 has come
from reaction 2
However, there is sti CK/ADP solution is added and this runs the
action producing ATP. The dilute hexokinase is still present in solution and it
he NADPH
produced in this step can be d mount of PCr present.
.6.3 Creatine (Cr) Analysis
Creatine + ATP + P-Creatine
Pyruvat-kinase
Pyruvate + NADH + H+ + NAD+
lanks and CP standards were analysed in triplicate, samples were analysed in
reagent made contained everything with the exception of ll PCr left. The
re
catalyses the two-step process of NADPH. A third reading (R3) is taken. T
irectly attributed to the a
3
The extract was analysed using a 3-step enzymatic process (Turner Fluorometer model
112). Creatine (Cr) levels were determined according to the method of Lowry and
Passoneau (1972).
Principle of analysis:
Creatine Kinase
ADP + P-Pyruvate ADP + Pyruvate
Lactate dehydrogenase
ADP
Lactate
A 15 mM NADH standard was recorded on a UV-visible spectrophotometer at 340 nm.
The change in fluorescence of the internal CP standards was then checked against the 15
mM NADH standard (refer to Appendix E for detailed methodology).
B
duplicate. The cocktail
creatine kinase. The presence of excess creatine kinase ensures that the reaction moves
from left to right. The first reading (R1) is taken after 15 minutes incubation. This gives
A creatine kinase/0.05% BSA solution is then added to each tube. The samples are left
to run for 60 minutes. This is the catalyst for reaction 2. This reaction produces
pyruvate; pyruvate is the catalyst for reaction 3. The second reading (R2) is recorded.
R1 subtracted from R2 gives the change in NADH concentration. As all of the NADH
from R2 has come from reaction 2 and 3 proceeding, thus, the amount of NADH will
ive a value of creatine concentration in the muscle sample.
s means + g
3.7 Statistical Analysis
All values are reported a SEM. Statistical evaluation for each muscle group
DL and soleus) and recovery time point (day 7 and day 14) was accomplished by
sing a two-way analysis of variance (ANOVA) with one between groups factor
upplementation) and one repeated factor (damage vs contralateral. Where an
interaction was found, the location of the difference was determined by a one-way
ANOVA. Difference in animal morphology characteristics between groups was
assessed by students’ t-test. Supplementation protocol between groups was assessed by
chi-square test. A P value of less then 0.05 was accepted for statistical significance.
While the animal studies will be reported separately in the subsequent chapters to align
them with the human studies, the data was compared together as both studies utilized
the same control group. (E
u