ESTUDIO DE LOS SECADORES Y DEL GRANO DE MAÍZ
1.2 SECADORES DE ALIMENTOS
1.2.1 SECADORES DE CALENTAMIENTO DIRECTO
during inertial sprint cycling and MNPR in young and elderly subjects.
2. To examine the relationship between the mechanical output variables and
the MHC isoform composition of the quadriceps in young and elderly
4.2 METHODS
Subjects
A ll subjects w ere m ales, the groups con sisted o f a ‘y o u n g ’ group (n = 7) and an
‘eld erly ’ group (n = 7). The physical characteristics are presented in T able 4.1. All
subjects w ere classified as healthy u sin g the health criteria as described by G reig
et al (1 9 9 4 ). A ll participating subjects w ere untrained Prior to testing, all subjects
w ere inform ed o f the testing procedures and sign ed a consent form agreeing to
take part in the study. The study had the approval o f the R oyal Free H ospital
E thics C om m ittee.
Group A g e (yrs) W eigh t (k g) H eight (cm )
Y ou n g 2 9 .4 ± 1.8 83.3 ± 1.1 180.1 ± 1.8
Elderly 73.8 ± 1.6 7 4 .6 ± 2 .2 176.3 ± 2 . 7
Table 4.1 P h ysical characteristics o f subjects. A ll table values sh ow n as mean ± SE.
Anthropometry
Lower limb volume was estimated from anthropometric measurements
comprising segmental circumferences and lengths as described by Jones and
Pearson (1969). Skinfold measurements were made at four sites; anterior,
posterior mid thigh and lateral, medial mid calf using skin callipers (John Bull
Ltd). Skinfold corrections were made using the following regression equations
(Personal communication Professor P. R. M. Pearson), anterior thigh
{y = 1.0142 +0.557 X skinfold value), posterior thigh (y = 1.368+0.532 x skinfold
value), medial calf (j^ = 0.985+ 0.499 x skinfold value) and lateral calf
(0.8701 + 0.3926 x skinfold value), where y represents the corrected skinfold value
used in the calculations.
Equipment
See methods section Chapter 3.
Sprint cycle protocol
See methods section Chapter 3. In contrast to the protocol in Chapter 3, 3
exertions were carried out at each of the five different inertial loads, ranging from
Cycling mechanical variables - calculation of optimal velocity and torque
See methods section for details of calculations (Chapter 3). A value of maximal
power can then be calculated from the values of optimal velocity. For the MNPR
data a similar procedure was used, however a second order polynomial was used
as this best fitted the data.
The torque at maximal power for both the cycling and MNPR was calculated
using a linear fit of the torque - velocity data as this method gave better r^ values
than the polynomial method.
Figure A. Diagram showing positioning of subject for each protocol.
Habituation
Due to the ‘skilled’ nature of cycling the groups were habituated (HAB) on the
cycle. This consisted of two exertions at three inertial loads (0.024, 0.29 and 0.54
kg m^). The habituation was carried out on the same day as the MNPR testing.
MNPR protocol
All exertions of the preferred lower limb were carried out with the subjects seated
as previously described (See Chapter 1 section 2). A total of 15 exertions were
recorded, 3 trials at each of 5 inertial loads ranging in order from 0.024 - 0.54 kg
m^. The exertion at which the highest value of power occurred within the 3 trials
at each inertial load was subsequently used for further analysis. Testing was
carried out at identical inertial loads to those for the sprint cycling. Values of
maximal power, optimal velocity and torque were calculated using the polynomial
method as described.
Muscle tissue sampling
Each subject had a biopsy of muscle tissue taken from the middle of the belly of
the vastus lateralis. Prior to the biopsy procedure the site was cleaned with
chlorhexidine and a local anaesthetic was administered (2 % lignocaine). An
incision was made through the skin and muscle fascia to allow the biopsy needle
to pass into the muscle belly. The biopsy was taken using a 5 mm diameter
Bergstrom needle with additional suction. The sample weight ranged from 30 -60
mg. The muscle sample taken was immediately frozen in liquid nitrogen and then
approximately 10 minutes and pressure applied to the site to prevent further
bleeding. The biopsy site was then closed with steri strips and an adhesive
dressing.
Electrophoretic separation of mvosin isoforms
The relative proportions of the three MHC isoforms (MHC-I, HA and XIX),
contained in the vastus lateralis biopsy samples from each subject was determined
through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS -
PAGE) (Andersen and Aagaard 2000).
i) Preparation of solutions and samples for SDS - PAGE
a) Sample buffer stock solution
A 50ml stock solution of sample buffer consisted of 6.25ml of 0.5 M Tris (pH
6.8), 10 ml of 10% SDS, 10 ml glycerol and 2.5 ml beta-mercaptoethanol. The
solution was then made up to 50 ml with distilled water. Bromophenol (25mg),
was added in order to stain the solution (Fry, Allmeir and Staron 1994).
b> Muscle tissue preparation
The muscle was homogenised manually with a pestle and mortar prior to adding
the stock solution. Each muscle sample had 500 X of sample buffer added to the
tube. The mixture was then heated at 97 ^ C for 3 minutes.
T he S D S PA G E gel electrop h oresis system is a discontin u ous system con sistin g
o f a stacking and resolv in g gel. For the stacking gel a 4 % solution w as used in
order to align the contractile proteins prior to separation.
Resolving gel preparation
In order to produce a 7% resolvin g gel the fo llo w in g reagents w ere m ixed in
sp ecific volum es.
R eagent V o lu m e (m l) A crylam ide - B is (1 0 0 :1 ) 3.13 L ow er Tris (pH 6 .8 ) 1.34 D istilled H2O 1.05 G lycine 1.0 10% S D S 0 .4 G lycerol 3 .0 APS 100 /// T E M E D 8 ///
In order to set the resolvin g g el a set o f g la ss plates w as p osition ed in a cradle
d esigned to hold the resolvin g g el plates. A pproxim ately 4m l o f the solution w as
poured into the sp ace b etw een the g la ss plates. T he solution w as allo w ed to set for
Stacking gel preparation
In order to produce a stacking gel the following reagents were mixed in specific
volumes Reagent Volume (ml) Acrylamide - Bis (37.5:1) 0.5 Upper Tris (pH 8.8) 1.25 Distilled H2O 3.25 APS 50/// TEMED 10///
The solution was added on top of the resolving gel and allowed to set for a further 1 hour
A running buffer consisting of 50 ml buffer concentrate (Biorad Tris, Glycine)
added to 450 ml distilled H2O pre cooled to approximately 5 ® C is placed into the
running tank and the samples are loaded into the stacking gel wells (Figure 4.1). A
further 200/// of beta mercaptoethanol was added to the centre of the tank prior to
Figure 4.1 Gel electrophoresis setup. A) Loading gel plates with samples B) Loaded tank with supply leads ready to run
Sample staining
The gels were removed from the glass plates and placed into a small container.
The gels were rinsed for approximately 10 minutes in distilled water this was then
repeated. Next, the gels were covered with 20ml of Invitrogen, ‘simply blue safe
stain’ (LC 6060) and were left staining for approximately 1 hour, depending on
the required intensity and the amount of protein present. The stained gels were
then rinsed again in distilled water for approximately 2 hours, which decreased the
background staining. This improved protein band resolution during light
densitometry.
Light densitometry
Once stained the resolving gels were placed into a biorad densitometer (Biorad
Geldoc 2000). The gels were scanned and a density map produced along with a
calculated proportional percentage with respect to the separate myosin isoform
bands using the associated software (Biorad quantity 1 ver 4.2). The gel
background was subtracted from the protein band density in order to align the
baseline to zero.
Data analysis
Student t tests were used to compare mechanical variables between the two
groups. The significance level set was 0.05. In order to determine the association
between mechanical variables and the muscle MHC isoform composition, linear
4.3 RESULTS
Inertial sprint cycling
Lower limb volumes
Anthropometric measures of the lower limbs for all subjects are shown in Table
4.2
Group Total limb volume Lean limb volume
Elderly 7942± 2 lO' 5352± 160'
Young 10205 ±224 7670± 178
Table 4.2 Anthropometric measures of group mean lower limb volumes (cm^) for
the young and elderly subjects. All values are ± SE. * Significantly different from
young group (P < 0.001).
Power output and optimal velocity
Figure 4.2 shows the absolute values of average crank power plotted against the
associated crank velocity for both the elderly and young groups. It can be seen
that the older group have significantly lower levels of both power output and
velocity (P = <0.001). The mean group optimal velocity (Vopt) ( 120 ±3.6 rpm, vs.
89 ± 5.8 rpm) (P = 0.001), and maximal power being (847 ± 46.8 W vs. 406 ±
o CL 1200 1 1000 - 800 - 600 - 400 - 200 - Y o u n g I E ld erly « « Velocity (rpm)
Figure 4.2 Individual subject power output vs. crank velocity during inertial sprint cycling. Each curve represents the polynomial fit to the average power and
velocity values for each crank downstroke at all inertial loads tested. Young (•) Elderly (•).
Normalised power output and optimal velocity
Power output was normalised to the lean limb volume (muscle plus bone) in order
to allow for the greater volumes observed in the young group. Using the
normalised values for the third order polynomial fit, the values for maximal power
for the young and elderly group were 0.11 W/cm^ and 0.08 W/cm^ respectively.
Figure 4.3 shows the normalised group mean power output vs. the associated
crank velocity. In spite of the power output being normalised for lean limb
volume the younger group still produced significantly greater power output (P =
0.01).
Optimal torque
Mean torque was plotted against mean velocity during each crank down stroke
(Figure 4.4). The torque generated at maximal power (Topt) was determined for
each individual from the line fit generated from the plots in Figure 4.4. The group
mean value for Topt was 14.7 ± 0.6 and 8.9 ± 0.7 Nm for the young and old group
respectively. The young group generated significantly greater Topt than the elderly
group (P < 0.001). The torque and velocity obtained by the elderly group as a
o CL 0.16 n 0.14 - 0.12 - 0.10 - 0.08 - 0.06 - 0.04 - 0.02 - 0.00 - Y o u n g E ld erly 50 100 150 200 Velocity (rpm)
Figure 4.3 Individual normalised power output vs. mean crank velocity during inertial sprint cycling. All plotted data represents individual values at each crank downstroke during sprint cycling over the inertial range tested. Young (•) Elderly
30 1 25 ^ 20 - 0) 15 - p- 10 - 5 1 Y o u n g E ld erly 50 100 150 Velocity (rpm) 200
Figure 4 .4 Individual plots of torque vs. velocity during inertial sprint cycling. All data represent mean values of torque and velocity during each crank downstroke over the inertial range tested. Young (•) Elderly (•)
M) 5 s V s Ou 80 70 ^ 60 j 50 i 40 4 30 - 20 - 10 1 0 ■
Figure 4.5 Elderly group mean values of velocity and torque at maximal power as a percentage of the young group mean values. Velocity (•) Torque (•).
Percentage M H C II composition
The MHC-I and II composition of each subject was determined by SDS PAGE
electrophoresis and expressed as a percentage value of the total MHC content.
Plate 1 shows an example gel which contains both young and elderly myosin
samples. For the purposes of analysis the MHC-II content was pooled (HA + IIX),
this was in part due to the fact that only single young subject had a sample
containing both MHC-IIA and IIX .
Figure 4.6 shows the mean values of the relative percentage of MHC-II for the
vastus lateralis. The MHC-II content was significantly greater in the younger
group (P = 0.01). The relative percentage values for the young and elderly group
being 52.1 and 25.6 % respectively.
Muscle percentage MHC II composition and optimal velocity
In order to allow the pooling of data, individual regression fit lines were examined
to ensure no significant difference with respect to the slope. Vopt during sprint
cycling is plotted against the percentage of MHC-II determined from the muscle
biopsy for each individual (Fig 4.7). The MHC-II composition is seen to be
positively related to the Vopt. The relationship between the percentage MHC-II