Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects

Rosenblatt, Judah; Chinkes, David L.; Wolfe, M. H.; Wolfe, Robert R.

doi:10.1152/ajpendo.1992.263.3.e584

Cited by 88 publications

(126 citation statements)

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“…The isotopic enrichment in the blood and in the skin protein hydrolysate for AA content was measured on a Hewlett-Packard 5985 gas chromatograph-mass spectrome- The isotopic enrichment was expressed as mole percent excess (MPE) 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-13 C6]Phe enrichment (21).…”

Section: Measurement Of Isotopic Enrichmentmentioning

confidence: 99%

Measurement of protein metabolism in epidermis and dermis

Zhang

Chinkes

Wolfe

2003

American Journal of Physiology-Endocrinology and Metabolism

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; 10.1152/ajpendo. 00460.2002.-We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 Ϯ 37.6 mg ⅐ 100 g Ϫ1 ⅐ h Ϫ1 , which was 10-fold greater than that of the dermal protein (9.3 Ϯ 5.8 mg ⅐ 100 g Ϫ1 ⅐ h Ϫ1 ). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state. stable isotopes; gas chromatograph and mass spectrometer; arteriovenous balance; fractional synthesis rate; rabbits THE SKIN IS ONE OF THE LARGEST TISSUES in the body. In normal circumstances, the skin maintains a constant protein mass, whereas skeletal muscle may have a significantly positive or negative protein balance (28). The homeostasis of skin protein mass plays a crucial role in preserving an intact barrier on the body surface. Once the integrity of the skin barrier is destroyed, several problems ensue immediately, such as loss of water and electrolytes, invasion of microorganisms, and impairment of immune functions (11,19,20). Thus knowledge of skin protein metabolism is important in understanding the maintenance and repair process of the skin barrier.In our previous experiments (28-30), we investigated protein metabolism in normal and scalded skin by use of a rabbit ear model. This model has been the only approach to simultaneously quantify the in vivo rates of protein synthesis and breakdown as well as amino acid (AA) transport in the skin. However, this model is limited by the consideration of the skin as a single pool. It is well known that the epidermis and dermis have different structure, function, and turnover rates (12,17,25). The dermis serves as a basis for supporting and nourishing the epidermis, whereas the epidermis exerts the barrier function of the skin. It is therefore important to distinguish between the metabolic regulation of the epidermis and that of the dermis under physiological and pathological conditions.The goal of this study was to establish an approach for quantitation of protein kinetics and AA transport in the epidermis and dermis. This...

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Section: Measurement Of Isotopic Enrichmentmentioning

confidence: 99%

Measurement of protein metabolism in epidermis and dermis

Zhang

Chinkes

Wolfe

2003

American Journal of Physiology-Endocrinology and Metabolism

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“…13 C6]Phe enrichment was corrected for the contribution of the abundance of isotopomers of lower weight to the apparent enrichment of isotopomers with larger weight, and a skew correction factor was applied (15). Isotope enrichment was expressed as the tracerto-tracee ratio for the internal standard method and as mole percent excess for calculation of protein kinetics and AA transport.…”

Section: Animalsmentioning

confidence: 99%

Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids

Zhang

Chinkes

Irtun

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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Zhang, Xiao-Jun, David L. Chinkes, Øivind Irtun, and Robert R. Wolfe. Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids. Am J Physiol Endocrinol Metab 282: E1308-E1315, 2002; 10.1152/ ajpendo.00361.2001.-To investigate the metabolic basis of skin wound healing, we measured in anesthetized rabbits the responses of protein kinetics in scalded skin to insulin and amino acids. L-[ring-13 C6]Phe was infused on the 7th day after the ear was scalded, and the scalded ear was used as an arteriovenous unit to reflect protein kinetics in skin wound. The ipsilateral carotid artery was clamped to control the wound blood flow within four-to fivefold the normal skin rate to measure the enrichment difference in the scalded ear during hyperaminoacidemia. Neither insulin (2.5 mU ⅐ kg Ϫ1 ⅐ min Ϫ1 ) nor amino acid (2.5 mg ⅐ kg Ϫ1 ⅐ min Ϫ1 ) infusion alone improved net protein balance in the skin wound. In contrast, combined infusion of insulin and amino acids increased the net protein balance in skin wound from Ϫ6.5 Ϯ 4.5 to 1.4 Ϯ 5.2 mol ⅐ 100 g Ϫ1 ⅐ h Ϫ1 (P Ͻ 0.01, control vs. insulin plus amino acids). We conclude that there is an interactive effect of insulin and sufficient amino acid supply on protein metabolism in skin wound, meaning that their combined anabolic effect is greater than the sum of their individual effects. stable isotopes; gas chromatograph-mass spectrometer; rabbit ear; arteriovenous balance INSULIN IS A KEY ANABOLIC HORMONE and not only plays an important role in substrate metabolism but also is a regulator of protein synthesis and breakdown. The effect of insulin on muscle protein has been investigated extensively in the normal state (2, 6, 7, 9, 11-13, 18, 21) and also in catabolic states such as after severe burns (5, 17). However, although the general role of insulin in wound healing is well known (10, 14, 16), the effect of insulin on protein metabolism in skin wound has not been assessed sufficiently.The lack of information regarding the effect of insulin on protein metabolism in skin wound is largely because of the lack of a method to quantify both protein synthesis and breakdown in vivo. To this end, we developed a rabbit ear model for measurement of protein metabolism in skin wound (24,26). Using this animal model, we demonstrated in our previous experiment (24) that insulin had an anabolic effect on wound protein resulting from the inhibition of protein breakdown. It is possible that the failure of insulin to stimulate protein synthesis was because of an insufficient supply of amino acids (AAs). Although AAs were infused during the insulin infusion, the plasma AA concentrations were only maintained at levels that were either comparable to the postabsorptive state (during insulin infusion at 0.6 mU ⅐ kg Ϫ1 ⅐ min Ϫ1) or even lower (during insulin infusion at 2.3-3.4 mU ⅐ kg Ϫ1 ⅐ min Ϫ1). Consequently, the potential role of sufficient AA supply in conjunction with insulin on wound protein metabolism is not clear.The present study was designed to investigate the p...

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“…The isotopic enrichment was expressed as mole percent excess for the A-V model and FSR calculations and as tracer-to-tracee ratio for FBR calculations, which were required by the methods. The L-[ring-13 C6]Phe enrichment was corrected 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 (15).…”

Section: Methodsmentioning

confidence: 99%

Acute responses of muscle protein metabolism to reduced blood flow reflect metabolic priorities for homeostasis

Zhang¹,

Irtun²,

Chinkes³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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Zhang X-j, Irtun O, Chinkes DL, Wolfe RR. Acute responses of muscle protein metabolism to reduced blood flow reflect metabolic priorities for homeostasis. Am J Physiol Endocrinol Metab 294: E551-E557, 2008. First published December 18, 2007 doi:10.1152/ajpendo.00467.2007.-The present experiment was designed to measure the synthetic and breakdown rates of muscle protein in the hindlimb of rabbits with or without clamping the femoral artery. L-[ring-13 C6]phenylalanine was infused as a tracer for measurement of muscle protein kinetics by means of an arteriovenous model, tracer incorporation, and tracee release methods. The ultrasonic flowmeter, dye dilution, and microsphere methods were used to determine the flow rates in the femoral artery, in the leg, and in muscle capillary, respectively. The femoral artery flow accounted for 65% of leg flow. A 50% reduction in the femoral artery flow reduced leg flow by 28% and nutritive flow by 26%, which did not change protein synthetic or breakdown rate in leg muscle. Full clamp of the femoral artery reduced leg flow by 42% and nutritive flow by 59%, which decreased (P Ͻ 0.05) both the fractional synthetic rate from 0.19 Ϯ 0.05 to 0.14 Ϯ 0.03%/day and fractional breakdown rate from 0.28 Ϯ 0.07 to 0.23 Ϯ 0.09%/day of muscle protein. Neither the partial nor full clamp reduced (P ϭ 0.27-0.39) the intracellular phenylalanine concentration or net protein balance in leg muscle. We conclude that the flow threshold to cause a fall of protein turnover rate in leg muscle was a reduction of 30 -40% of the leg flow. The acute responses of muscle protein kinetics to the reductions in blood flow reflected the metabolic priorities to maintain muscle homeostasis. These findings cannot be extrapolated to more chronic conditions without experimental validation. stable isotope; gas chromatograph-mass spectrometer; arteriovenous balance; fractional synthetic rate; fractional breakdown rate PROTEIN KINETICS in the skeletal muscle have been studied intensively under various conditions, including fasting, spaceflight, exercise, and catabolic illness (5,8,18). The importance of muscle protein metabolism is not only because of its contractile function but also because muscle is the largest mobilizable source of protein. Muscle catabolism provides free amino acids under catabolic states, thereby supporting the continual synthesis of essential proteins in other parts of the body (9,10,19,21). The synthesis of muscle protein, as well as breakdown, depends on blood flow, since the metabolic precursors and products have to be transported into, and removed from, the muscle through the blood stream.The muscle is proposed to have two vascular flow routes: nutritive and nonnutritive (4). Nutritive flow is able to exchange nutrients and hormones with the skeletal muscle myocytes. Although the nonnutritive flow does not have direct nutritional function, it carries a flow reserve and redistributes to nutrition routes in response to increased metabolic demand. It has been demonstrated that increased rates of m...

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Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects

Cited by 88 publications

References 1 publication

Measurement of protein metabolism in epidermis and dermis

Measurement of protein metabolism in epidermis and dermis

Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids

Acute responses of muscle protein metabolism to reduced blood flow reflect metabolic priorities for homeostasis

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