Comparison of the [[sup.2][H.sub.5]]phenylalanine model with the [1-[sup.13]C]leucine method to determine whole body protein synthesis and degradation in sheep fed at two levels

Asian - Australasian Journal of Animal Sciences, Oct, 2007 by M. Al-Mamun, C. Ito, A. Sato, T. Fujita, H. Sano

ABSTRACT : The [[sup.2][H.sub.5]]phenylalanine model was compared with the [1-[sup.13]C]leucine method to determine whole body protein synthesis (WBPS) and degradation (WBPD) in sheep fed at two levels. The animals were fed either 103 (M-diet) or 151 (H-diet) kcal ME/[kg.sup.0.75]/day once daily in a crossover design for 21 days ere simultaneously conducted as a primedcontinuous infusion of [[sup.2][H.sub.5]]phenylalanine, [[sup.2][H.sub.2]]tyrosine and [1-[sup.13]C]leucine on each dietary treatment. The WBPS and WBPD calculated from the [[sup.2][H.sub.5]]phenylalanine model were lower (p = 0.009 and p = 0.003, respectively) than those calculated from the [1-[sup.13]C]leucine method. The WBPS tended to be higher (p = 0.08) and WBPD was numerically higher (p = 0.33) for H-diet than M-diet in the [[sup.2][H.sub.5]]phenylalanine model, whereas the WBPS was numerically higher (p = 0.37) for H-diet and WBPS remained similar (p = 0.79) between diets in the [1-[sup.13]C]leucine method. However, the absolute values and the directions of WBPS as well as WBPD from M-diet to H-diet were comparable between the [[sup.2][H.sub.5]]phenylalanine model and [1-[sup.13]C]leucine method. Moreover, the values vary depending on the use of the respective amino acid contents in the carcass protein when calculating WBPS and WBPD. Therefore, it is concluded that the [[sup.2][H.sub.5]]phenylalanine model could be used as an alternative to the [1-[sup.13]C]leucine method for the determination of WBPS and WBPD in sheep. (Key Words : Isotope Dilution Method, Stable Isotope, Dietary Intake, Protein Synthesis, Protein Degradation, Sheep)

INTRODUCTION

A number of well-established isotope dilution techniques using radioactive and stable isotopes have been used to estimate whole body protein metabolism in humans and animals (Wolfe, 1984; Marchini et al., 1993; Sano et al., 2004). Among the methods, isotope kinetics of [1-[sup.13]C]leucine is the most widely used method for the assessment of whole body protein synthesis (WBPS) and degradation (WBPD) through the measurement of plasma [alpha]-[1-[sup.13]C]ketoisocaproic acid ([alpha]-[1-[sup.13]C]KIC), the true precursor of intracellular leucine metabolism, enrichment in humans, sheep, and cows (Matthews et al., 1982; Krishnamurti and Janssens, 1988; Lapierre et al., 2002; Sano et al., 2004). The [[sup.2][H.sub.5]]phenylalanine model, as proposed by Clarke and Bier (1982), could also be used for the accurate measurement of WBPS and WBPD quickly and, in addition, it offers the advantage of not requiring the measurement of expired air C[O.sub.2] production or [sup.13]C[O.sub.2] enrichment, which enables the analyses to be performed using gas chromatography-mass spectrometry (GC/MS) alone.

Although, the [[sup.2][H.sub.5]]phenylalanine model has been used in humans and animals (Pacy et al., 1994; Borel et al., 1997; Clark et al., 1997; Whittaker et al., 1999; Gibson et al., 2002; Bregendahl et al., 2004; Fujita et al., 2006) for the determination of whole body protein metabolism, a few experiments have been reported in sheep (Harris et al., 1992; Connell et al., 1997; Lobley et al., 2003). Harris et al. (1992) used [[sup.3]H-2,6]phenylalanine and [U-[sup.14]C]phenylalanine and stated that WBPS increased with increased nutritional** level. However, they used previous nitrogen balance data (Harris et al., 1989) to calculate WBPD without measuring nitrogen balance simultaneously. In another study, Connell et al. (1997) used [1-[sup.13]C]glycine, [1-[sup.13]C]leucine and [[sup.2][H.sub.5]]phenylalanine and stated that irreversible loss rate (ILR) and whole body protein flux (WBPF) were higher for fed than fasted sheep. However, the authors did not measure the oxidation of leucine to C[O.sub.2] via [alpha]-KIC and phenylalanine to tyrosine. Furthermore, there is some debate about the application of the [[sup.2][H.sub.5]]phenylalanine model which has been elaborated by Fujita et al. (2006). Dietary energy intake also influences whole body protein metabolism in goats (Fujita et al., 2006).

The present experiment was conducted using isotope dilution and nitrogen balance tests simultaneously in sheep fed at two levels of dietary intake to determine whether or not the same WBPS and WBPD could be obtained from the [[sup.2][H.sub.5]]phenylalanine model and the [1-[sup.13]C]leucine method.

MATERIALS AND METHODS

Animals, diets and management

Four cross-bred (Suffolk x Corriedale) sheep, aged around two years and weighing, initially, 44[ or -]1 kg, were used. The animals were surgically prepared under anesthesia with a skin loop enclosing the left carotid artery at least three months before starting the experiment. During the adjustment period the animals were kept in individual pens in an animal barn, and were shorn closely before starting the experiments. The animals were fed mixed hay of orchardgrass and reedcanary grass (1.8 kcal metabolizable energy (ME)/g and 12% crude protein (CP)) and concentrate mixture (2.8 kcal ME/g and 12% CP) in a 1:1 ratio at two different levels, 103 kcal ME/[kg.sup.0.75]/day (M-diet) and 151 kcal ME/[kg.sup.0.75]/day (H-diet), equivalent to 107 and 157%, respectively, of the ME required for maintenance (National Research Council, 1985), with a crossover design for a period of 21 days on each diet. The sheep were fed at 14:00 h everyday with ad libitum water access. On day 15 the sheep were moved to metabolic cages in an environmentally controlled room with an air temperature of 20[ or -]1[degrees]C. A polyvinyl catheter was placed in the right jugular vein one day before the experiment. Also, a catheter for blood sampling was inserted into the skin loop of the left carotid artery about 2 h before starting the experiment. The catheters were kept flushed with 3.8% (w/v) trisodium citrate sterile solution. The sheep were moved back to individual pens after completing each dietary treatment. They were weighed at the onset, on day 15 and after finishing each dietary treatment. The handling of animals including surgery and blood sampling was carried out according to the guidelines established by the Animal Care Committee of the Iwate University.