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From the Clinical Research Centers |
Department of Internal Medicine and Diagnostic Radiology and General Clinical Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Linda A. Jahn, Department of Internal Medicine, MR-4 Box 5116, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Seventy-six healthy (36 females and 40 males), normal weight (body mass index, 22.04 ± 0.4 in females and 23 ± 0.3 in males), young adult (23 ± 1 yr), volunteers were admitted to the University of Virginia General Clinical Research Center the evening before the study. No subject was taking any medication, and all female participants had a negative serum pregnancy test 1–2 days before the study. The study protocol was approved by the University of Virginia human investigation committee, and each subject gave written consent.
Experimental protocol
After an overnight 12-h fast, a brachial artery and an
ipsilateral, retrograde, median cubital (deep) vein catheter were
placed percutaneously. Each subject received a primed (
33 µCi),
continuous (0.43 µCi/min) infusion of L-(ring 2, 6)-3H
phenylalanine through a catheter placed in the lower extremity. After a
90-min tracer equilibration period, quadruplicate, paired arterial and
venous samples were taken over 30 min for measurement of phenylalanine
concentration and specific activity and of glucose, lactate, and oxygen
concentrations. Forearm blood flow was measured after each set of
arterial and venous samples by capacitance plethysmography.
In a subset of 17 subjects (8 males and 9 females), magnetic resonance imaging (MRI) of the forearm was performed using a 1.5 T Magneton 63SP (Siemens, Erlangen, Germany). The image time was 6.52 min, and the resolution time was 1.2 mm in plane with a 4.7-mm effective slice thickness. The sequence was repeated with an overlapping slice to cover the full distance between the olecranon and the styloid processes. Images were evaluated using Sigma Scan (version 1.20, Jandel Scientific, Chicago, IL). A total of 16 slices/subject were used to reconstruct a 3-dimensional image of the forearm. Tissue volumes between slices were estimated as a truncated cone and then summed.
Analytic methods
Whole blood glucose and lactate concentrations were measured by a combined glucose/lactate analyzer (Yellow Springs Instruments, Yellow Springs, OH). Blood oxygen content was measured spectrophotometrically using an OSM2 hemoximeter (Radiometer, Copenhagen, Denmark). Phenylalanine concentration and specific activity were measured as previously described (7).
Calculations of forearm phenylalanine kinetics. The net forearm balances for glucose, lactate, oxygen, and phenylalanine were calculated using the Fick principle: net forearm balance = ([A] - [V]) x F (Eq I), where [A] and [V] are arterial and venous substrate concentrations, and F is forearm blood flow in milliliters per min/100 mL forearm volume. The rates of protein synthesis and degradation were estimated from the kinetics of exchange of labeled phenylalanine across the forearm using the specific activity of phenylalanine in venous plasma to reflect the precursor pool used for protein synthesis as previously described (8): synthesis = ([dpmart - dpmvein] x flow)/SAvein (Eq II), and muscle protein breakdown (B) as: breakdown = S - net balance (Eq III).
As the deep forearm venous catheter used in our forearm balance study drains nearly exclusively muscle (1) we used two approaches to calculate muscle balance and protein kinetics using Eq I–III. In the first approach, in each of the subjects whose forearm composition was measured we simply divided the balance or flux by the fractional contribution of muscle to forearm volume. This result, expressed as mass per 100 mL forearm muscle, assumes that blood flow to muscle, skin, bone, and adipose distributes in proportion to the volume that they each contribute to the forearm. The second approach to correcting forearm balances and fluxes relies on the results of recent positron emission tomographic studies (9), indicating that human leg total blood flow can be related to muscle blood flow by the equation: blood flow/100 mL leg muscle = [1.41 x total leg blood flow (mL/100 mL limb volume)] - 0.43. This estimate of flow distribution, together with the areterio-venous difference (across muscle) and the estimated fraction of the limb occupied by muscle, can be used to calculate the total substrate exchange by muscle.
For the entire study group, the extraction ratios ({[X]artery - [X]vein }/[X]artery) for glucose, oxygen, lactate, phenylalanine, and [3H]phenylalanine were also calculated to assess the handling of these substrates by muscle without blood flow as a multiplier.
Data presentation and statistical analysis. All data are presented as the mean ± SEM. Comparisons between males and females, before as well as after correcting for muscle mass, were made using Student’s t test.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Forearm phenylalanine balance was negative in these postabsorptive
subjects. Moreover, net phenylalanine release was significantly greater
in males than in females (-22 ± 2 vs. -17 ± 1;
P < 0.05). In addition, in males, the rates of both
protein degradation (Eq III; 71 ± 6 vs. 54 ± 3;
P < 0.005) and protein synthesis (49 ± 4
vs. 37 ± 3; P < 0.005) were greater
than those in females, suggesting a higher rate of muscle protein
turnover and a greater net catabolism in men than in women (Table 1
). There were no significant gender
differences in glucose uptake, lactate release, or blood flow per 100
mL forearm (Table 1). Oxygen uptake was greater in male than in female
subjects (Table 1).
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). The gender
differences in phenylalanine and oxygen balance seen in the subgroup
studied using MRI were similar to those seen in the entire study
population; although they were not statistically significant in this
subgroup, presumably due to the sample size. All these data were
expressed per 100 mL forearm volume, as has been done in virtually all
studies of forearm metabolism (1, 2, 14, 18). Forearm tissue composition
Table 2 indicates the forearm
composition determined by the reconstruction of the 16 cross-sectional
images in each of the 17 subjects. The relative contribution of adipose
tissue to total forearm volume was greater (42% vs. 30%)
and the contribution of muscle was less (46% vs. 58%) in
the female subjects (P < 0.001 for each).
Interestingly, the fractional content of bone was similar in both sexes
(12% vs. 12%). Moreover, the variances in the total
volumes and percent composition within each sex were narrow.
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Given the narrow variance of forearm percent composition within
genders, we used the forearm tissue composition given in Table 2 to
recalculate the balances for phenylalanine, glucose, lactate, and
oxygen (calculated as (arterio - venous) x F) for the
entire study population, now normalized per 100 mL forearm muscle
(Table 3). Calculating balances or fluxes
in this manner is equivalent to assuming that all blood flow to the
forearm is directed to tissue in proportion to its contribution to
forearm volume. In an effort to more precisely estimate muscle’s
contribution to forearm metabolism, fluxes were estimated assuming that
blood flow in the forearm of the present study’s subjects is
partitioned between tissues as described by recent positron emission
tomographic measurements (see Materials and Methods) (9).
These results are also shown in Table 3. Regardless of which approach
was used, the balances for each of the metabolites were closely matched
between genders (Table 3).
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The forearm extraction fraction for each substrate was calculated
as previously described. These results are given in Table 4. Again, there was no significant
difference noted between genders, with the exception of a greater
fractional lactate release in females.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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These considerations underscore the importance of recognizing some inherent assumptions of the forearm technique and how "correcting" for muscle’s contribution to forearm composition is affected by these assumptions. In forearm studies, the venous catheter is placed retrograde in the antecubital fossa and advanced into the deep forearm veins. Therefore, the arterio-venous difference measures metabolites or tracer differences between the systemic arterial inflow and the venous effluent that drains almost exclusively muscle and bone. Thus, the metabolic activity measured with the forearm technique is largely confined to those tissues. However, the forearm volume (measured by volume displacement) and forearm blood flow (measured by plethysmographic or dye dilution methods) includes contributions from all tissues in the forearm. The MRI measurements provide accurate estimates of forearm composition. However, there is no generally accepted method available to estimate the distribution of blood flow between muscle and sc adipose tissue and skin. The common practice of virtually all laboratories that use the forearm model (11, 12, 13, 14, 15, 16) involves calculating the balance by multiplying the arterio-venous difference across muscle by the total forearm flow. This tacitly assumes that muscle, bone, skin and fat behave in a quantitatively similar fashion. For many substrates this seems unlikely.
By contrast, in leg balance studies, the venous catheter (placed in the femoral vein) drains venous effluent from skin, subcutaneous adipose, bone, and muscle. Therefore, for the leg, venous sampling, blood flow, and volume (measured by volume displacement) reflect the contributions of leg tissues. This has obvious advantages, vis-á-vis the above discussion. However, it does not allow dissection of the metabolic activity of muscle (or muscle and bone) per se as can be performed in the forearm.
Several studies have demonstrated that replacement of androgens in
hypoandrogenic men increases lean body and muscle mass (4, 17, 18).
Likewise, androgen excess in women increases lean body mass and
decreases fat mass. As this androgen effect must result from an
increased rate of protein synthesis relative to breakdown (17, 19), it
is at first surprising that there was not a higher rate of protein
synthesis or more positive protein balance in the male subjects.
However, two caveats must be kept in mind. First, these were
postabsorptive subjects, and in the postabsorptive state there is a net
loss of muscle protein in all subjects. Whether males would show a
greater postprandial response to amino acid supply, insulin, or other
anabolic factors cannot be ascertained from these studies. Second, the
methods used have approximately 80% power (
< 0.05) to detect a
decrease in protein synthesis or a balance of 20% in the women. Lesser
decrements could contribute to a lower mass but not be evident by these
kinetic measures. These caveats aside, the current results suggest that
under the conditions studied, the greater concentration of androgens in
these healthy males does not have a major influence on postabsorptive
muscle oxygen, glucose, or protein metabolism.
In summary, despite proportionately greater muscle mass in males, the rates of glucose uptake, oxygen consumption, and protein metabolism per unit mass of muscle are similar in male and female subjects. These findings suggest that sex hormones are not major regulators, in postabsorptive humans, of skeletal muscle metabolism of glucose and protein or of the overall metabolic rate.
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Footnotes |
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Received July 23, 1998.
Revised November 24, 1998.
Accepted December 4, 1998.
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