Nitrogen metabolism in the rat

The intermediary metabolism of 6 isotopic amino acids, 15N-aspartic acid, 15N-glutamic acid, 15N-alanine, 15N-glutamine (amide-15N), 15N-glycine and 15N-lysine (O-15N), and 15N-ammonium chloride were investigated. The aim of this study was to establish the precursor-product relationships existing between these amino acids, ammonia and urea in the amino-N pools of the major organs and tissue beds of the normal postprandial rat with the specific objective of following the movement of nitrogen to urea synthesis. It was hoped to ascertain whether glutamic acid played a central role in providing nitrogen for urea synthesis and whether there existed any relationship between 15N distribution patterns of the different isotopes and WBTP rates calculated from hepatic and renal urea-N enrichments. The method employed involved the administration of tracer quantities of the isotopes by the constant infusion technique and measuring the 15N excess of ammonia-N, glutamine amide-N, alanine-N, glutamate-N, aspartate-N and urea-N. It was found that nitrogen from 15N-alanine, 15N-aspartic acid and 15N-glutamic acid was distributed evenly in most of the amino-N pools studied. Nitrogen from the other four isotopes was distributed unevenly, preferentially to ammonia, glutamine amide and urea. 15N-glycine and 15N-lysine were only sparingly metabolised. WBPT rates obtained from urea-N enrichments were not affected by the nitrogen distribution patterns of the isotopes but by the extent to which they metabolised. WBPT rates calculated from ammonia-N enrichments were unduly affected by the extent to which each isotope contributed nitrogen to ammoniagenesis. Glutamic acid does not seem to be the precursor of both nitrogens used for urea synthesis. It supplies only one nitrogen. It is possible that urea is synthesised from an amino-N received via the glutamate to aspartate pathway and an amide-N received via the glutamine to ammonia to carbamyl phosphate pathway. Free ammonia entering the liver is first fixed as glutamine amide before being used for urea synthesis. (AU)

Protein metabolism in skeletal muscle

Two different approaches towards the study of protein turnover in skeletal muscle were made. The first approach involved the injection of 75Se Selenomethionine into rats and the subsequent measurement of the whole body decay rate of the 75Se activity in a 4 pi liquid scintillation counter. By this means it was hoped that the whole body decay curve could be analysed into exponentials representative of 'fast' (visceral protein) and 'slow' (muscle protein) pools. This proved not to be feasible. The special difficulties resulting from the use of 75Se-labelled amino acids are discussed. As a second approach a search was made for a technique for labelling muscle proteins so that radioactivity decay rates could be used directly to calculate rates of synthesis and catabolism without the usual errors arising from isotope reutilisation. 75Se selenomethionine, 14C-6 arginine, 14C-Na2 CO3 and 14C-1 glutamate were investigated. 14C-Na2 CO3 proved to be suitable especially if the decay rates of separate and glutamate isolated from muscle proteins labelled with 14C Na2 CO3 are measured. The lack of reutilisation of label is discussed in terms of the metabolic activity of the carboxyl groups of these dicarboxylic amino acids. The effects of acute deprivation of calories and protein on synthesis and catabolism of muscle and liver protein was measured in rats, using the 14C-Na2 CO3 labelling method. The synthesis rates for muscle proteins, 0.25 and 0.097 days-1 for sarcoplasmic and myofibrillar proteins respectively are the fastest reported in the literature. The total protein synthesised and catabolised in muscle sarcoplasmic and myofibrillar fractions was calculated and compared with liver. The protein free diet caused a reduction in synthesis rates in liver and muscle protein with no change in the distribution pattern between the tissues. The starved rats showed a shift in the distribution pattern of synthesis towards liver and a concomitant shift towards muscle in catabolism. The results are discussed in terms of the mobility and therefore importance of muscle protein metabolism in the economy of the whole animal (AU)

Protein turnover in skeletal muscle. I. The measurement of rates of synthesis and catabolism of skeletal muscle protein using [14C]Na2CO3 to label proteins

The turnover of rat skeletal muscle protein was studied using [75Se]selenomethionine, [6-14C]arginine and [14C]Na2CO3 to label protein. In rats labelled with both [75Se]selonomethionine and [14C]Na2CO3 the 14C activity of mixed skeletal muscle protein fell rapidly with a half-life of 6.0 days for the specific activity and 10.5 days for the total activity. There was no loss of 75Se activity from muscle protein during the 12 days of the experiment. Following the injection of [6-14C]arginine both sarcoplasmic and myofibrillar proteins continued to incorporate label for 6 days after which time the label was lost fairly rapidly. Following injection of [14C]Na2CO3 muscle protein was maximally labelled by 6h, at which time specific activity of the free amino acids had fallen to a very low level. Aspartate and glutamate in particular had lost over 99 percent of their maximum activity by this time in comparison to arginine which was still highly labelled after 24h. 14C activity was lost more rapidly from aspartate and glutamate isolated from sarcoplasmic and myofibrillar protein than from other labelled amino acids. The half-lives of the two protein fractions were 3.9 and 7.2 days from the specific activity curves and 6.0 and 19.0 days from the total activity curves. The differences between the half lives of muscle proteins labelled with amino acids are discussed in terms of the effects of reutilization of the labelled amino acids used. It is postulated that aspartate and glutamate labelled by the injection [14C]CO3= are only reutilized to a very small extent and therefore afford the means by which the rates of protein synthesis and catabolism in skeletal muscle can be measured with reasonable accuracy (Summary)

Protein turnover in skeletal muscle. I. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver

Rates of synthesis and catabolism of liver and muscle sarcoplasmic and myofibrillar protein have been measured in young control, starved and protein (deprived) rats using [14C]Na2CO3 to label protein. Half-lives for synthesis of 1.35, 2.8 and 7.2 days for liver, sarcoplasmic and myofibrillar proteins, respectively were obtained, whilst half-lives for catabolism were 1.55, 3.6 and 15.6 days in each case in the control animals. The protein free diet for 3 days caused a small decrease in the rate of synthesis of liver and muscle proteins. The catabolic rate of liver protein was increased by 20 percent whilst there was a smaller increase in the catabolic rate of myofibrillar proteins. Starvation for 3 days caused a 20 percent reduction in the rate of liver protein synthesis whilst there were greater reductions in muscle protein synthesis. The catabolic rate of liver protein was only slightly increased whereas there was a 75 percent increase in the rate of myofibrillar protein breakdown. The total amount of protein synthesis and catabolism in liver and the 2 muscle protein fractions over the first 3 days of the 3 regimes were calculated. Muscle protein turnover, in particular myofibrillar, was shown to be very sensitive to dietary protein and/or calorie deficiency. These results are discussed in terms of the mobility and therefore importance of muscle protein metabolism in the economy of the whole animal. (AU)

The turnover of [75 Se] selenomethionine in infants and rats measured in a whole body counter

The gamma-emitting amino acid [75Se]selenomethionine was given to rats and to human infants, and the rate and route of excertion of 75Se was followed for several weeks by daily measurements in a 4 pi whole body counter. These data were used to calculate the turnover rate of total body protein, and the results were checked against other, technically more difficult, methods.(AU)

Observations on the mechanisms of adaptation to the low protein intakes

Experiments are described which attempt to throw light on the mechanism by which animals and man can adapt to low protein intakes. In the rat, studies by constant infusion of labelled amino acid have shown that in the protein depleted animal there is a small reduction in the total protein turnover: this, however, is not enough to account for the great reduction in urinary nitrogen output. Constant infusion and single injection experiments agree in showing that in rats on a low protein diet there is a change in the pattern of protein turnover: synthesis of carcass protein (muscle and skin) is reduced, while that of liver protein is well maintained. The preservation of synthesis in liver seems to depend partly on increased re-utilization of amino acids liberated by the catabolism of tissue protein. This economy may be brought about by adaptive enzyme changes -decreased activity of the urea cycle enzymes and increased activity of amino acid activating enzymes in the liver. These changes, previously described by others in the rat have been shown to occur in the human liver also. Studies in human infant with 75selenium-labelled methionine provide some support for the concept that when the protein intake is limited, turnover is preferentially maintained in the liver. However, not all liver-produced proteins behave in the same way; studies of albumin kinetics in infants show that when the protein intake is altered, there is a rapid change in the rate of albumine synthesis, together with a redistribution of albumin between intra and extravascular spaces. Later and more slowly occurs a change, presumably compensatory, in the rate of albumin catabolism. Hormonal changes may play a part in these adjustments. Increased cortisone and decreased insulin activity would have the effect of promoting amini acid uptake at the expense of muscle. It is concluded that the net nitrogen loss which occurs when the protein intake is reduced results simply from the time-lag before the adaptive mechanisms come into play, and therefore cannot logically be regarded as the loss of reserve protein. The practical implications of this concept are discussed (Summary)

The protein content of liver and muscle as a measure of protein deficiency in human subjects

The major nutritional problem in underdeveloped countries is deficiency of protein. At the present time there is no way of assessing with any accuracy the degree to which a human being is depleted of protein.Until this gap in scientific and medical knowledge is filled, it will be difficult to plan preventive measures in the most effective way. In clinical work the problem becomes one of finding some objective measure that will indicate the severity of malnutrition. With this aim analyses have been made of biopsy and autopsy specimens of the liver from malnourished children. The degree of fatty infiltration of the liver seems to be of little importance. There is a large loss of liver protein, which however, shows no clear relation to the general condition of the patient. It is suggested that in chronic malnutrition the main loss of protein is from muscle, rather than from liver. This is confirmed by the results of preliminary measurements on fatal cases. Measurement of the protein depletion of the tissues may provide a baseline for assessing the value of simpler tests, of the kind that can be applied in the field. (AU)