Metabolism Unit, Shriners Burns Institute and Departments of Surgery and Anesthesiology, The University of Texas Medical Branch, Galveston, Texas 77550
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ABSTRACT |
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We have measured
skin and muscle protein kinetics and amino acid (AA) transport in
anesthetized rabbits during 1) 64-h
fast, 2) AA infusion,
3) AA plus fat emulsion infusion,
and 4) AA plus hyperinsulinemia.
L-[ring-13C6]phenylalanine
was infused as the tracer, and the ear and hindlimb were used as
arteriovenous units to reflect skin and muscle protein kinetics,
respectively. Skin protein net balance was not different from zero in
all groups, indicating a maintenance of protein mass. In contrast, the
muscle net balance differed over a range from 1.6 ± 0.6 after fasting to 0.2 ± 0.2 µmol · 100 g
1 · h
1
during hyperinsulinemia. In the skin, 59-66% of intracellular free phenylalanine came from proteolysis, and phenylalanine
availability from proteolysis was positively correlated to the protein
synthesis rate. In conclusion, normal skin maintains its constant
protein mass by efficient reutilization of AAs from proteolysis. In
contrast to muscle, skin protein is relatively insensitive to control
by nutritional and hormonal factors. Because of the metabolic
differences, when limb models are used for muscle protein metabolism,
the potential contribution by limb skin should be considered.
stable isotopes; mass spectrometry; arteriovenous balance; rabbits
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INTRODUCTION |
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NORMAL SKIN maintains its integrity under a variety of circumstances. For example, during prolonged starvation the majority of muscle mass can be depleted, yet the integrity of the skin barrier is basically preserved. On the other hand, athletes can build up their muscle mass with appropriate training and diet, without risk of overgrown skin. Protein kinetics in the skin have received little attention, and it is uncertain how the skin maintains its relatively constant protein mass under different nutritional and hormonal conditions.
To assess skin protein kinetics, it is necessary to simultaneously quantify skin protein synthesis and breakdown rates. We have previously described a tracer model to accomplish this goal in any tissue in which we can obtain arteriovenous (A-V) difference across the tissue and a tissue biopsy (5). Quantitation also requires a distinct size of tissue sampled to determine the absolute rate of protein synthesis, breakdown, and transmembrane transport of amino acids (AAs) between blood and the tissue intracellular pool (5). We recently applied this tracer model to the rabbit ear for the study of skin protein kinetics (29). The adult rabbit ear satisfies the criteria necessary to quantify skin protein kinetics because the ear exclusively represents skin protein metabolism and has measurable mass, and A-V blood sampling across the ear is possible. In our original study, skin protein kinetics were measured in the postabsorptive state (29). The present study was performed to extend our original observation to various nutritional conditions such as brief starvation, hyperaminoacidemia (with or without lipid infusion), and hyperinsulinemia. These perturbations were selected because of their well-documented anabolic or catabolic effects at the whole body level.
In addition to quantifying the response of skin protein metabolism to a variety of factors, we have simultaneously used the same balance method to quantify the response of the leg. This approach has enabled us to compare and contrast the control mechanisms in the two tissues. Furthermore, this approach enabled us to estimate the influence of limb skin metabolism on the limb A-V balance data. The limb balance technique has been widely used to represent muscle protein metabolism, and the metabolic contribution by the limb skin has not previously been quantified.
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METHODS |
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Animals. Male New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN) weighing ~4.7 kg were used for this study. The rabbits were housed in individual cages and consumed Lab Rabbit Chow HF 5326 (Purina Mills, St. Louis, MO) for weight maintenance. This study was approved by the Animal Care and Use Committee of The University of Texas Medical Branch at Galveston.
Isotopes. L-[ring-13C6]phenylalanine (99% enriched) and L-[ring-2H5]phenylalanine (98% enriched) were purchased from Cambridge Isotope Laboratories (Woburn, MA).
Experimental design. There were four groups. In group 1 (fasting group, n = 10), food was deprived for 64 h but rabbits had free access to water. No nutritional or hormonal supply was given during the isotope infusion. Five rabbits of this group were originally designed to match a group of rabbits that consumed very little food after thermal injury. For this reason, during the 64-h fasting period the rabbits were given an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg) at the 16th hour for sham injury. A single dose of penicillin G benzathine suspension (Bicillin, 50,000 U/kg; Wyeth Laboratories, Philadelphia, PA) was injected intramuscularly under anesthesia. When the animals awoke, buprenorphine hydrochloride (Buprenex, 0.03 mg/kg; Reckitt & Colman Pharmaceuticals, Richmond, VA) was injected intramuscularly as analgesic two times per day until 16 h before the isotope infusion study. The other five rabbits in this group were fasted for 64 h without other treatment. Because the prior anesthesia and penicillin had no discernible effects (see RESULTS), the results of all the fasted animals were grouped together.
The other three groups were studied after overnight food deprivation (16 h) with free access to water; during the isotope infusion study these animals were given a balanced mixture of AAs (10% Travasol, Baxter Healthcare, Deerfield, IL) at 1.2 ml · kg
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Sample analysis. After the collection of samples, an internal standard solution, which contained 29.7 µmol/l of L-[ring-2H5]phenylalanine, was added to the blood (0.2 ml), and the samples were deproteinized by sulfosalicylic acid (29). The supernatant was processed to make the N-acetyl,n-propyl ester (NAP) derivatives of the AAs (27). Plasma insulin concentration was determined by the microparticle enzyme immunoassay technique (1). Blood glucose concentration was measured on a glucose/L-lactate analyzer (mode 2300; Yellow Springs Instruments, Yellow Springs, OH). Blood hemoglobin (Hb) concentration was measured on an automated hematology analyzer (Coulter JT3, Hialeah, FL).
Tissue specimens, either skin or muscle, of ~60 mg were homogenized in 5% perchloric acid three times at 4°C. Phenylalanine was purified from the pooled supernatant by high-performance liquid chromatography and derivatized for the NAP derivatives (27, 29). The muscle precipitate was washed and dried at 80°C as previously described (28). The dry protein pellets were hydrolyzed in 6 N constant boiling HCl. A cation-exchange column (Dowex AG 50W-X8, Bio-Rad Laboratories, Richmond, CA) was used to isolate AAs. The samples were prepared for the N-heptafluorobutyryl-n-propyl ester (HFBPr) derivatives of AA and used to determine L-[ring-13C6]phenylalanine enrichment in the protein-bound pool (19). The isotopic enrichment in the blood and purified phenylalanine from the tissue supernatant were measured on a Hewlett-Packard 5985 gas chromatograph-mass spectrometer (GC-MS) (Hewlett-Packard, Palo Alto, CA) with chemical ionization. Ions were selectively monitored at mass-to-charge ratios of 250, 251, 255, and 256 for phenylalanine (27). L-[ring-13C6]phenylalanine enrichment in the protein hydrolysate was determined on a GC-MS (MD 800, Fisons Instrument, Beverly, MA) (19). Isotopic enrichment was expressed as tracer-tracee ratio after correction for the contribution of the abundance of isotopomers of lower weight to the apparent enrichment of isotopomers with larger weight and also a skew correction factor to calculate L-[ring-13C6]phenylalanine enrichment (22).Calculations.
The rate of appearance (Ra) of
whole body phenylalanine was calculated by the equation
Ra = (F/Ep) FPhe (27), where F is the infusion
rate of
L-[ring-13C6]phenylalanine,
Ep is the phenylalanine enrichment
(tracer:tracee ratio) in the arterial blood at isotopic plateau, and
FPhe is the infusion rate of
unlabeled phenylalanine in the Travasol solution. The muscle protein
FSR was calculated from the equation of the direct incorporation method
(16).
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Statistics. Data are expressed as means ± SD. Differences among the four groups were evaluated using a one-way analysis of variance. The Student-Newman-Keuls test (14) was used for post hoc multiple comparison testing among the groups. Linear correlation was used to test the relationship between two parameters. A P value < 0.05 was considered statistically significant.
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RESULTS |
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Table 1 lists the general characteristics of the rabbits. The body weight of the rabbits in the four groups was not significantly different. All the rabbits maintained a stable physiological state during the isotope infusion as shown by the relatively constant rectal temperature, heart rate, and mean arterial blood pressure. As described in METHODS, five rabbits in the fasting group received anesthetics, penicillin, and buprenorphine during the fasting period to match an injury group (reported elsewhere). Because these rabbits had the same physiological and metabolic responses as the other five rabbits without prior anesthesia and penicillin, the data from these two subgroups were combined.
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To determine the tissue composition, we dissected 10 ears and 10 hindlimbs. The weight of the 10 ears was 18.8 ± 1.8 g, which was composed of 78% skin (14.7 ± 1.7 g). The 10 limbs were from seven rabbits weighing 4.5 ± 0.4 kg. On average, one hindlimb contained 215 ± 20 g muscle, 49 ± 5 g skin, and 71 ± 2 g bone. From the above dissection data, the weight of the ear skin was taken to be ear weight × 78% and the weight of limb muscle was taken to be 4.8% × body weight. Because the skin and muscle weights in the ear and limb were known, the measured blood flow rates in the ear artery and femoral artery were normalized to the amount of the skin and muscle in the ear and limb, respectively (see Table 1).
By the end of the tracer infusion, plasma insulin
concentrations were 12 ± 9, 58 ± 38, and 74 ± 45 pmol/l in
the fasting, AA, and AA-lipid groups, respectively; the value in the
fasting group was significantly (P < 0.05 or P < 0.01) lower than in the AA and AA-lipid groups. In the AA-insulin group, insulin concentration was raised to 1,055 ± 36 pmol/l by insulin infusion. Blood glucose concentration immediately before anesthesia was 3.8 ± 0.3 mmol/l (drawn from the ear vein of 6 overnight-fasted rabbits). During the
120-240 min of tracer infusion, arterial blood glucose
concentrations were 8.7 ± 2.6, 10.2 ± 2.1, and 10.4 ± 1.0 mmol/l in the fasting, AA, and AA-lipid groups, respectively
(P > 0.05 among the 3 groups). In
the AA-insulin group, it took ~90 min to reach a stable
euglycemia; during the 120-240 min of isotope infusion, arterial
blood glucose concentration was successfully clamped at 6.1 ± 0.3 mmol/l, which required exogenous glucose infusion at 10.1 ± 0.1 mg · kg1 · min
1.
During the 120-240 min of tracer infusion, isotopic plateaus were
maintained in the arterial blood in all rabbits (Fig.
3). The concentrations of phenylalanine in
the arterial blood during the 120-150 min of ear balance and
180-210 min of leg balance were almost identical (81.9 ± 18.6 vs. 81.8 ± 17.6 nmol/ml, n = 25).
In the AA, AA-lipid, and AA-insulin groups, the infusion of 10%
Travasol delivered unlabeled phenylalanine at 0.6788 µmol · kg1 · min
1.
The Ra values of endogenous
phenylalanine in the whole body were 1.09 ± 0.12, 1.00 ± 0.18, 0.82 ± 0.11, and 0.82 ± 0.15 µmol · kg
1 · min
1
in the fasting, AA, AA-lipid, and AA-insulin groups, respectively; the
values in the AA-lipid and AA-insulin groups were significantly (P < 0.05 or
P < 0.01) lower than in the fasting
and AA groups.
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Table 2 presents phenylalanine enrichment and concentration in the arterial and venous blood and tissue intracellular free AA pool. The concentration of Hb in the ear-venous blood was higher than that in the arterial blood by 1.8% (133 ± 6 vs. 131 ± 6 g/l, P < 0.05 by paired t-test, n = 12), suggesting a decrease in water content in the venous blood. Therefore, the measured AA concentrations in the ear-venous blood were multiplied by 98.2% to account for the water loss from the skin surface of the ear.
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Skin and muscle protein kinetics calculated from the three-compartment model are presented in Table 3. The highest rates of skin protein synthesis and breakdown were observed in the AA group, which was significantly (P < 0.01) higher than those in the other three groups. The values of net balance were not significantly (P > 0.05) different from zero in all groups, meaning that skin protein mass was maintained in all conditions.
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In contrast to the situation in skin, in muscle the net balance was responsive to treatment (see Table 3). Net protein loss in the fasting and AA groups was significantly (P < 0.05 or P < 0.01) greater than in the AA-lipid and AA-insulin groups. Differences in net balance were largely due to differences in breakdown, as synthesis was not significantly different among groups.
The phenylalanine transport data are presented in Table 4. The rate of AA entering the ear (Fin, see Fig. 2) was lowest in the fasting group (P < 0.01 vs. AA and AA-lipid groups, P > 0.05 vs. AA-insulin group). Because of the smaller A-V shunting (Fv,A) in the fasting group (P < 0.01 vs. AA and AA-lipid groups, P < 0.05 vs. AA-insulin group), the rate of inward transport (FT,A) in the fasting group was significantly (P < 0.05) lower than only the AA group (P < 0.01) and not significantly different from those in the AA-lipid and AA-insulin groups. Similarly, whereas the rate of AA entering the limb (Fin) was lower in the fasting group (P < 0.01 vs. AA, AA-lipid, and AA-insulin groups) because of significantly (P < 0.01) smaller A-V shunting (FV,A) in the fasting group than in the other groups, the rate of inward transport (FT,A) was significantly lower than only the AA group and not different from the AA-lipid and AA-insulin groups.
When phenylalanine transport data were compared between skin and
muscle, the %shunting
(FV,A/Fin)
in the skin (84 ± 6%, n = 25) was
significantly (P < 1011) greater than in the
muscle (57 ± 12%, n = 25). The
ratio of inward transport to total
Ra, an indicator of fractional
contribution of inward transport to total phenylalanine availability in
the tissue intracellular free amino acid pool, was significantly
(P < 10
12) greater in the
muscle (61 ± 9%, n = 25) than in
the skin (36 ± 8%, n = 25). The
ratio of protein synthesis to total intracellular appearance of AAs, an
indicator of the efficiency of protein synthesis, was significantly
(P < 10
13) greater in the skin
(64 ± 8%, n = 25) than in the
muscle (32 ± 8%, n = 25).
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When the values of protein synthesis and breakdown were pooled from all the groups, significant (P < 0.01) linear correlations were found in both skin (Fig. 4A) and muscle (Fig. 4B).
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The phenylalanine enrichments in the muscle intracellular free and protein-bound pool are presented in Table 5. The muscle protein FSRs were not significantly different (P > 0.05) among groups, which is consistent with the synthesis data from the limb A-V model (see Table 3).
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DISCUSSION |
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The present study demonstrated that, in contrast to muscle, skin maintains its protein mass under a variety of conditions. Thus skin protein mass is insensitive to control by nutritional and hormonal factors, which are generally important in other tissues.
We have previously reported the maintenance of skin protein balance in the postabsorptive state using either the ear skin model (29) or a parallel tissue model (8). In the present study, the observation of a maintenance of protein mass in the absence of nutritional intake was extended to 64 h. In contrast, the muscle mass was in strikingly negative balance. This result is in agreement with the data reported by Cherel et al. (12). By determining total skin nitrogen content they demonstrated the maintenance of skin protein mass in rats fasting up to 5 days. Our data indicate that the efficient reutilization of AAs from protein breakdown was the primary mechanism responsible for the maintenance of protein mass. Not only was balance maintained in skin when nutrient intake was stopped, but the infusion of AA solution with or without lipid or glucose-insulin did not cause a significant improvement in the skin phenylalanine net balance (see Table 3).
Despite the absence of an effect on net balance, the AA infusion stimulated skin protein synthesis and breakdown (see Table 3). Cherel et al. (12) also reported that, despite unchanged skin protein mass in rats fasted for up to 5 days, skin protein FSR decreased from 9.3 to 5.8%/day over this time span. We further demonstrated that the same metabolic feature remained effective in the AA-lipid and AA-insulin groups, in which either fat emulsion or hyperinsulinemia inhibited both skin protein synthesis and breakdown rates (in comparison with AA group) without affecting net balance (see Table 3). These changes could be explained by a dominant role of protein breakdown in determining the rate of protein synthesis in the skin. Because the skin depends on its own proteolysis for the majority of AA availability in the intracellular free AA pool (see Table 4), an inhibition of breakdown would be expected to slow synthesis by decreasing the intracellular availability of AAs. This notion is supported by the almost perfect correlation between the rates of breakdown and synthesis in the skin (Fig. 4A). The inward transport of AA from blood played a lesser role in the intracellular AA availability, as only 34-41% of the intracellular AA pool in skin came from blood (see Table 4). Furthermore, the rate of inward transport was counteracted by the equal rate of outward transport (see Table 4), resulting in no net change in AA availability.
In comparison with the skin, the limb protein metabolism was more responsive to nutritional and hormonal treatment (see Table 3). These results support the notion that skeletal muscle is a mobilizable source of AAs in the body. In the AA-lipid and AA-insulin groups the net limb balance was significantly improved because of the inhibition of breakdown rate. The inhibitory effect of lipid and insulin on protein breakdown is in agreement with previous reports (13, 18, 24, 26) and the whole body phenylalanine Ra in this study. Whereas the correlation between the rates of muscle protein synthesis and breakdown was significant (Fig. 4B), the r value was less than for skin (0.75 vs. 0.99, respectively). This reflects the fact that muscle relied to a smaller extent on protein breakdown as a source of precursors than did skin.
Some data from the limb model are not consistent with the results we previously obtained in normal subjects. Thus the infusion of AA solution did not improve net balance in the limb and hyperinsulinemia did not stimulate muscle protein synthesis in the rabbits, as it did in human subjects (4, 7). These discrepancies could have been due to the surgery in these rabbits, which apparently induced catabolic stress and modified vascular and metabolic responses. The catabolic stress was reflected by hyperglycemia during the isotope infusion period, even when no glucose was infused. We have previously found that nutrients, either alone or with short-term hyperinsulinemia, are less effective in eliminating net protein catabolism under surgical or burn stress (15, 23). In the current study, infusion of insulin and AA did not stimulate either the inward transport or the muscle synthesis. In the AA group, although the rate of inward transport was greater than in the fasting group, because of the concomitant increase in the rate of outward transport, neither the total phenylalanine Ra in the muscle intracellular pool nor the synthesis rate was significantly increased (see Tables 3 and 4). These results support the notion that the inward AA transport and/or protein synthesis did not respond normally to anabolic stimuli (9, 23). Also the limb blood flow was not increased by either AAs or insulin in the current experiment. In normal humans, we have found an increase in blood flow due to insulin infusion (7) and a significant correlation between leg blood flow and inward AA transport in a variety of circumstances, such as during local insulin infusion (7) and after exercise recovery (10). Therefore, it is likely that the vascular and metabolic responses in these rabbits were modified so that the synthesis rate of muscle protein was not normally responsive to nutrient or hormone treatments. The possible role of stress in modifying the normal responses of muscle does not reduce the significance of the difference between the responses of skin and muscle to nutritional treatments. Rather, it underscores the ability of skin protein mass to be maintained in what is likely to be a catabolic circumstance at the whole body level.
The metabolic differences between skin and muscle were also reflected by the %shunting (see Table 4). The large %shunting (84%) in the skin (in comparison to 57% in muscle) indicated that only a small portion (16%) of AAs in blood were exchanged with the AAs in the intracellular compartment. This may reflect physical shunting of blood in the skin due to its role in thermoregulation, as well as the efficiency of reutilization of intracellular AAs in skin decreasing the demand for blood-borne AAs.
In this study, we used tissue free phenylalanine enrichment as an approximation of labeling of the aminoacyl-tRNA. In muscle tissue, the aminoacyl-tRNA enrichment measured either in pigs (3) or in humans (25) is not different from the muscle free fluid enrichment, which is mainly the intracellular AA enrichment. In the case of skin, no data of aminoacyl-tRNA enrichment have been published. The available data are derived from the use of skin free AA enrichment as the precursor enrichment (8, 12, 20, 21, 29). The skin free fluid is a mixture of intracellular and interstitial fluid. Because the interstitial pool is located between blood vessels and the intracellular pool, the enrichment of interstitial AA should be lower than blood enrichment but higher than the intracellular enrichment. Thus the inclusion of interstitial fluid in the skin samples could only overestimate the true precursor enrichment. Thus the values of skin protein synthesis, breakdown, and intracellular recycling presented in this paper should be considered as minimal estimates of the true values. In any case, conclusions regarding net protein synthesis or breakdown are independent of this issue. Therefore, our conclusions about the differences between muscle and skin have a sound basis. Even though we used a two-pool model based entirely on blood values (2), the responses of skin and muscle protein synthesis, breakdown, and net balance are similar to those calculated from the three-pool model, although the rates of synthesis and breakdown from the two-pool model are much lower.
Because both skin and muscle protein kinetics were measured in the same
animals, this study enables us to estimate the contribution of limb
skin to the limb balance data. The use of limb models to reflect muscle
protein kinetics is based on the assumption that the metabolic
contribution from nonmuscle tissue is negligible. Nonmuscle tissue
(mainly skin) is estimated to account for 10-15% of total leg
protein kinetics in humans (6) and dogs (8). In some species, this
source of error could be greater. Preedy and Garlick (20) demonstrate
that the rat hemicorpus contains 39% by weight of nonmuscle tissue.
Because skin and bone have faster protein synthesis rates than muscle,
Lobley et al. (17) and Preedy and colleagues (20, 21) state that the
metabolic contribution by the nonmuscle tissue could be a considerable
portion of the measured protein kinetics in the hemicorpus. Our
dissection data showed that the rabbit hindlimb contains 36% by weight
of nonmuscle tissue (skin and bone). In particular, the weight of skin
was equal to 23% of muscle weight, and the protein synthesis rate in
the ear skin was 1.8- to 3.5-fold that in the limb muscle (see Table
3). The real contribution of limb skin to limb A-V data should be
smaller than that estimated from the protein mass and synthetic rate
because the skin had a large intracellular AA cycle. Using the data in
Table 2 and blood flow rates in Table 1, we can calculate that the
rates of skin tracer uptake were 0.44 ± 0.15, 0.43 ± 0.09, 0.27 ± 0.08, and 0.27 ± 0.06 µmol · 100 g1 · h
1
in the fasting, AA, AA-lipid, and AA-insulin groups, respectively. If
the limb skin had the same tracer uptake as the ear skin and 100% of
limb skin was drained by the femoral vein, the limb skin accounted for
35 ± 16, 42 ± 11, 25 ± 7, and 26 ± 10% of the total limb tracer uptake. Statistical analysis shows that the rates of skin
tracer uptake were significantly (P < 0.05) higher in the fasting than in the AA-lipid and AA-insulin
groups, indicating a variable contribution of skin to the limb balance.
Consequently, attention should be paid to the nonmuscle tissue when
limb models are used to reflect muscle protein metabolism, especially
when the skin and muscle have opposite responses. In this study, this source of error would not affect the validity of the comparison between
skin and muscle because the inclusion of skin protein kinetics in the
limb balance data could only reduce the metabolic differences between
skin and muscle. Even if we simultaneously calculate protein kinetics
in the limb muscle and limb skin using the parallel tissue model (8),
we will reach the same conclusion regarding the different regulation of
protein metabolism in these two tissues (data not presented here).
In summary, normal skin maintains a relatively constant protein mass under both catabolic and anabolic conditions. This is because the majority of AAs for synthesis come from protein breakdown and the inward AA transport from blood is counteracted by the outward transport. The skin has a large A-V functional shunting and a large intracellular AA cycle, both of which ensure protein homeostasis and insensitivity to the changes of nutrients and hormones in blood circulation. Whereas the protein mass in the skin is well maintained, the absolute rate of turnover can change in response to nutritional and hormonal conditions. Because the skin may have substantial and variable contribution to the limb balance data, when limb models are used to reflect muscle protein kinetics, potential contribution from the limb skin should be considered.
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ACKNOWLEDGEMENTS |
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The authors thank John Guy, Yunxia Lin, and Zhiping Dong for technical assistance. We also thank the staff in the Clinical Laboratory of Shriners Hospital for measurement of blood hemoglobin concentration and the staff in the Animal Resource Center of University of Texas Medical Branch for professional care of the animals.
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FOOTNOTES |
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This work was sponsored by Grants 8490 and 8630 from Shriners Hospital.
This work was presented in abstract form at Experimental Biology '96, Washington, DC, April 14-17, 1996.
Address for reprint requests: R. R. Wolfe, Shriners Burns Institute, 815 Market St., Galveston, TX 77555-1220.
Received 2 June 1997; accepted in final form 9 December 1997.
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