OXIDATIVE CAPACITY AND AGEING IN HUMAN MUSCLE

Aulia Siti Nurhayati - 260110100151

INTRODUCTION

Muscle and whole body maximal aerobic performance decline with age in humans. The loss of muscle mass is an important contributor to this decline but the role of oxidative capacity per muscle mass is less clear. Age related changes in volume specific oxidative capacity have been inferred from both in vitro and in vivo measurements of muscle oxidative properties.

Magnetic resonance (MR) methods that make possible noninvasive assessment of the change in muscle energetics in vivo.
Two tools have the potential for allowing a determination of muscle oxidative capacity in vivo.

The second tool allowing us to determine oxidative capacity in muscle is the model of the control of oxidative phosphorylation

PURPOSE

The purpose of this study was to determine the oxidative capacity of muscle and how it differs between adult and elderly groups.

METHODS

Subject

An adult group consisted of nine subjects (6 males, 3 females) ranging in age from 25 to 48 years (388 79 years, mean s.d.). An elderly group consisted of 40 subjects (18 male, 22 female) ranging in age from 65 to 80 years (688 59 years). Body mass was not significantly different between the two groups (adult 698 28 kg, elderly 721 22 kg, means s.e.m.).

Subjects were not involved in a formal exercise training programme, were in good health and had no significant cardiac, neurological or musculoskeletal disease.
Nine of the elderly female subjects were receiving hormone replacement therapy.

Subjects activity profilesranged from housework, yardwork, and occasional walks to aerobic activities several times per week.
All subjects voluntarily gave informed, written consent.

Experimental Protocol

Using the dynamics of [PCr] and pH during stimulation to estimate glycolytic + production and during aerobic recovery from exercise to measure ATP supply and estimate oxidative capacity. Spectral data were collected at 6 s intervals over an 8 min period using the following protocol. Control period (60 s, 10 spectra): baseline data were obtained during resting conditions to establish initial metabolite peak areas and pH under partially saturating nuclear MR data acquisition conditions. Stimulation period (120 s, 20 spectra): a 3 Hz electrical stimulation period was used to decrease [PCr]. Glycolytic + production was determined from the [PCr] and pH changes as previously reported Aerobic recovery (300 s, 50 spectra): upon cessation of stimulation, the extent and time course of the aerobic PCr recovery was followed and used as the basis of the oxidative phosphorylation determinations.

Muscle Stimulation
activated the quadriceps muscles by transcutaneous electrical stimulation of the femoral nerve determine the intensity that evoked the maximum EMGresponse for each subject stimulations of supramaximal intensity (13 15 times maximal) were delivered for 2 min at 3 Hz.

used EMG to monitor muscle activation and establish the maximal stimulating voltage.

The active electrode was placed over the belly of the vastus lateralis muscle

Magnetic Resonance Determinations

A 9 cm diameter surface coil tuned to the phosphorus frequency (259 MHz) was placed over the vastus lateralis muscle of the thigh. The B1 field homogeneity was optimized by offresonance proton shimming on the muscle water peak. The unfiltered PCr linewidth (full width at halfmaximal height) was typically 48 Hz. Each subject had a high resolution control 31P MR spectrum of the resting muscle taken under conditions of fully relaxed nuclear spins (16 freeinduction decays (FID) with a 16 s interpulse delay) using a spectral width of 1250 Hz and 2048 data points. Measurement of changes in [PCr], [ATP], [P] and pH during and following stimulation were made using a standard 1 pulse experiment with partially saturated nuclear spins (15 s interpulse delay).

Calculations

The linear model of oxidative phosphorylation was used. The first step in using this model is to fit the recovery of [PCr] from exercise to the resting level using a monoexponential equation taken from a resistancecapacitance (RC) circuit: [PCr]t = [PCr]0 + [PCr] (1 exp(t kPCr)), (1) where the levels at time t and the beginning of recovery are [PCr]t and [PCr], respectively; kPCr is the rate constant of PCr recovery (kPCr = 1time constant ()) and [PCr] = [PCr]rest [PCr].

The initial rate of change of PCr is given by the following derivative of eqn (1):
d[PCr]/dt = kPCr [PCr] exp(t kPCr). (2) (3) The instantaneous rate at t = 0 reduces the equation to: d[PCr]/dt = kPCr [PCr]. This equation predicts the oxidative phosphorylation rate (d[PCr]dt) at any given [PCr] based on the characteristic kPCr of each muscle. To estimate oxidative capacity, we assumed that [PCr]rest reflects the maximum range of change in [PCr] (i.e. [PCr]max = [PCr]rest 0). Thus, an estimate of the oxidative capacity of the muscle comes from the characteristic kPCr of that muscle and [PCr]rest in eqn (3).

Oxidative capacity per mitochondrial volume

The average oxidative capacity of mitochondria has been measured as the whole body maximum oxygen consumption divided by the total mitochondrial volume of the musculature. This method yielded a oxidative capacity of between 4 and 5 ml 2 1 (ml mitochondria)1 , which averages 27 mol ATP 1 (ml mitochondria )1 for a P/ 2 (phosphorylation to oxidation ratio of mitochondrial respiration) of 6.

Conts

An independent estimate of the maximal rate of mitochondrial respiration comes from isolated mitochondrial studies . Since muscle oxidizes >85% pyruvate at the maximal oxygen uptake rate, the substrate pair best suited for comparison with in vivo mitochondria is pyruvate and malate. This substrate pair yielded a maximum oxidative rate of 17 mol ATP 1 (ml mitochondria)1 (2.7 ml 2 1 ( ml mitochondria )1 ) at 30 C, when corrected for a P/2 of 6 and 0.7 ml 2 O per ml tissue. Correcting the rate to 37 C (assuming a doubling of rate with each 10 C, i.e. 10 = 2) yields 27 mol ATP 1 (ml mitochondria)1 Thus isolated mitochondria and in vivo mitochondria have the same maximum oxidative rate per mitochondrial volume. This value is used to estimate muscle oxidative capacity based on mitochondrial volume density.

Glycolisis

The glycolytic H generated was quantified from the change in PCr and pH through stimulation: + = pHtot + (-)PCr, (4) where pH and PCr are the changes in pH and PCr during exercise, tot is the buffering capacity of the individuals muscle, and is the proton stoichiometric coefficient of the coupled Lohman reaction Glycolytic ATP synthesis is related to H production by the ATP/ + stoichiometry of glycogenolysis and glycolysis

Tissue Analysis
used the Bergstrom needle biopsy technique to acquire tissue from the midthigh level of the right vastus lateralis muscle.

A 25 mg piece was immersion fixed in glutaraldehyde, processed for electron microscopy, and morphometrically analysed

Any remaining tissue was freezeclamped immediately after collection and stored at 80C prior to HPLC metabolite analysis

Metabolite concentrations were expressed per volume of cell water by assuming 07 ml intracellular water (g muscle mass))1 as found for human muscle biopsy samples

Statistics

Using Students twotailed paired and unpaired t tests to determine differences between groups and standard linear regression methods for analysis of correlations. Statistical significance was defined at the 0.05 level.

RESULTS
MR spectroscopy was used to quantify the metabolite levels in resting muscle, during stimulation and through recovery.

three steps to achieve the goal of evaluating the change in oxidative capacity with age.

the PCr recovery from exercise to estimate the muscle oxidative capacity

evaluation the contribution of a loss of mitochondrial volume density and mitochondrial function to the change in oxidative capacity with age.

Metabolite levels and dynamics

The metabolite levels quantify using a combination of HPLC analysis of muscle biopsies and the peak areas in the MR spectra

Resting levels

Figure 1. Stack plot of every third 31P NMR spectrum during rest, stimulation and recovery in an elderly individual The abscissal scale references PCr to a chemical shift () of 254 p.p.m. P, inorganic phosphate.

Table 1. Metabolite levels determined by HPLC from muscle biopsies of the vastus lateralis and by MR of the same muscle Metabolite Adult Elderly Literature [ATP] (mM) 68 06 (3) 59 02 (32) 82 Total [Cr] (mM) 434 52 (2) 467 11 (31) 42 [PCr] (Mm) 267 35 (3) 260 10 (32) 32 [PCr]/[ATP] 430 02 (32) 39 MR [PCr]/[ATP] 400 011 (9) 453 010 (38) * MR [P]/[ATP] 052 003 (9) 062 003 (38) MR [PDE]/[ATP] 075 014 (9) 076 005 (38)

Values are means s.e.m. with the sample size given in parentheses. Literature values are from Harris et al. (1974) for subjects 1830 years old. PDE, phosphodiester. * Significant difference between adult and elderly groups.

Dynamic

Figure 2. Metabolite levels as a function of time during the stimulation and recovery experiment Symbols indicate means and error bars indicate s.e.m. Doubleheaded arrows indicate the duration of stimulation. adult subjects; elderly subject

 Table 2. Metabolite levels determined by MR at rest and at the end of stimulation Quantity Group Rest Stimulation end Stimulation Rest [PCr] (mM) Adult 2924 160 2044 132 880 137 * Elderly 2711 088 1824 079 887 038 * [P1] (mM) Adult 432 069 1162 178 730 132 * Elderly 387 022 1112 074 724 056 * Ph Adult 7063 0011 7040 0016 0022 0022 Elderly 7058 0006 7020 0009 0036 0011 * [ADP] (mM) Adult 0030 0004 0062 0007 0032 0005 * Elderly 0031 00015 0065 0002 0033 0002 * Values are means s.e.m. The sample size was 9 for the adult group and 40 for the elderly group. Significant difference between conditions.

Oxidative phosphorylation rate

Next step was to determine the oxidative phosphorylation rate and estimate the oxidative capacity. Which compared the initial rate of oxidative phosphorylation estimated from the monoexponential fit of the PCr recovery to that measured directly.

Oxidative phosphorylation rate

Figure 3. [PCr] recovery following stimulation Continuous lines are monoexponential fits to the data for an adult and an elderly subject. The vertical dashed lines denote the time constant for each recovery.

Oxidative phosphorylation rate

Figure 4. [PCr] recovery rate constant (kPCr) as a function of age The line represents the regression equation: y = 00005x + 0057 ( 2 = 035)

Oxidative phosphorylation rate

Figure 5. Oxidative capacity as a function of age The line represents the regression equation: y = 0017x + 1777 ( 2 = 034)

Oxidative and mitochondrial properties vs. age

The final step was to evaluate the structural basis for the decline in oxidative capacity with age by determining the mitochondrial volume density (VV(mt,f)) from biopsies taken at the same site as the MR determinations.
These results confirm the lower oxidative capacity per mitochondrial volume for the elderly vs. adult muscle shown in Fig. 6.

Oxidative and mitochondrial properties vs. age

Figure 6. Oxidative capacity, mitochondrial volume density (VV(mt,f)) and oxidative capacity/VV(mt,f) in the adult and elderly groups Values are means s.e.m. and asterisks denote significant difference from the

DISCUSSION

Oxidative recovery Muscle vs. mitochondrial oxidative capacity Adult vs. elderly oxidative capacity

Oxidative recovery

A clear indication of the change in oxidative properties between adult and elderly muscle is the twofold difference in the time course of PCr recovery from exercise shown in Fig. 3 Two results support the notion that kPCr is a characteristic of the oxidative properties of mitochondria in muscle. First, kPCr is independent of [PCr] to 50% PCr depletion in a number of rodent and human muscles . Only at low pH levels does kPCr vary in a given muscle, because [H] significantly affects the creatine kinase equilibrium thereby altering the link between [PCr] and oxidative phosphorylation.

Second, kPCr is proportional to oxidative enzyme activity in both human and animal muscle, which indicates that kPCr is determined by the mitochondrial properties of he muscle.

Muscle vs. mitochondrial oxidative capacity

These results indicate a lower oxidative capacity of elderly compared with adult mitochondria and suggest that the loss of muscle oxidative capacity per muscle volume reflects not only mitochondrial volume loss but also the reduced capacity of the mitochondria themselves.

Adult vs. elderly oxidative capacity

The lower oxidative capacity per mitochondrial volume of the elderly vastus lateralis compared with the adult group shown in Fig. 6 is consistent with a lower mitochondrial oxidative enzyme activity with age. The reduction in mitochondrial function with age may be caused by a number of factors, such as mitochondrial DNA mutatiions, oxidative damage by reactive oxygen species, or reduced synthesis of mitochondrial proteins Brierly et al. (1997a,b) suggest that the reduction in mitochondrial function seen in elderly subjects is not directly caused by an ageing process per se. The end result is decline of nearly half of the muscle oxidative capacity between adults and elderly subjects due to reduced mitochondrial content as well as a significantly lower oxidative capacity per mitochondrial volume.