Malcolm F. Fuller

The optimum dietary amino acid pattern for growing pigs

A series of four nitrogen-balance experiments was carried out with growing pigs to determine the optimum balance amongst the amino acids in the diet. The reduction in N retention when 20% of a single amino acid was removed from the diet was used to calculate a dietary amino acid pattern in which each amino acid would be equally limiting. A mixture of amino acids simulating the amino acid pattern of casein was used with the same efficiency as casein. From two successive deletion experiments an optimum balance amongst the essential amino acids was derived. Expressed relative to lysine = 100 this had threonine 72, valine 75, methionine + cystine 63, isoleucine 60, leucine 110, phenylalanine + tyrosine 120, tryptophan 18. No estimate was made for histidine. Essential amino acids in this pattern were mixed with non-essential amino acids in ratios of 36:64 up to 57:43. The highest efficiency of N retention was achieved with diets having a ratio of at least 45:55. This included (g/16 g N) lysine 6.5, threonine 4.7, valine 4.9, methionine + cystine 4.1, isoleucine 3.9, leucine 7.2, phenylalanine + tyrosine 7.8, tryptophan 12. The N of diets with this amino acid pattern was utilized significantly better than when the pattern proposed by the Agricultural Research Council (1981) was used. The flow of amino acids past the terminal ileum of pigs given the semi-synthetic diet with this amino acid pattern was no greater than that observed with protein-free diets. The proposed pattern thus describes the intrinsic requirements of the growing pig for absorbed amino acids.

The optimum dietary amino acid pattern for growing pigs: 2. Requirements for maintenance and for tissue protein accretion

Experiments were made to estimate separately the amino acid requirements of growing pigs for maintenance and for protein accretion. The relationship between nitrogen retention and amino acid intake was estimated for each essential amino acid (except histidine) by giving, at rates of N intake of 0.25 and 2.0 g/kg body-weight (W)0.75 per d, diets in which one amino acid was made specifically deficient. From the regression coefficients it was calculated that, for the accretion of 1 g body protein, the dietary amino acid requirements were (mg) threonine 47, valine 53, methionine + cystine 36, methionine 19, isoleucine 43, leucine 78, phenylalanine + tyrosine 84, phenylalanine 41, lysine 68 and tryptophan 12. The daily amino acid requirements for N equilibrium were also estimated. From the relationship between N retention and amino acid intake the daily amino acid requirements for N equilibrium were estimated to be (mg/kg W0.75 per d) threonine 53, valine 20, methionine + cystine 49, methionine 9, isoleucine 16, leucine 23, phenylalanine + tyrosine 37, phenylalanine 18, lysine 36 and tryptophan 11. It was estimated that both for maintenance and for protein accretion tyrosine could provide close to half the total phenylalanine + tyrosine needs. Cystine could supply close to half the total sulphur amino acid needs for protein accretion but 0.8 of the needs for maintenance.

Protein turnover in growing pigs. Effects of age and food intake

1. Measurements were made of the nitrogen and energy balances of pigs of 30, 60 and 90 kg given a conventional diet at various daily rates. 2. Body protein synthesis was estimated from the irreversible loss of leucine from the blood following the infusion of [1-14C]leucine, and from the oxidation of the labelled amino acid. 3. Protein synthesis (g/d) increased by 2.17 for each 1 g increase in daily protein accretion and by 1.55 for each 1 g increase in daily protein intake. 4. At 30 kg, pigs close to energy equilibrium synthesized 270 g protein daily compared with 406 g and 512 g when their ration supplied twice and three times their maintenance requirement. 5. There was a close correlation between the daily urinary excretion of urea + ammonia and total amino acid catabolism estimated from the catabolism of leucine, but the latter underestimated the observed excretion by 2.5 g N/d. 6. The results imply that protein turnover accounts for only a proportion of the heat production associated with protein deposition.

Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid concentrations and branched-chain keto-acid dehydrogenase activity.

The present experiment was designed to elucidate the mechanism of the methionine-sparing effect of excess branched-chain amino acids (BCAA) reported in the previous paper (Langer & Fuller, 2000). Twelve growing gilts (30-35 kg) were prepared with arterial catheters. After recovery, they received for 7 d a semipurified diet with a balanced amino acid pattern. On the 7th day blood samples were taken before (16 h postabsorptive) and after the morning meal (4 h postprandial). The animals were then divided into three groups and received for a further 7 d a methionine-limiting diet (80% of requirement) (1) without any amino acid excess; (2) with excess leucine (50% over requirement); or (3) with excesses of all three BCAA (leucine, isoleucine, valine, each 50% over the requirement). On the 7th day blood samples were taken as in the first period, after which the animals were killed and liver and muscle samples taken. Plasma amino acid and branched-chain keto acid (BCKA) concentrations in the blood and branched-chain keto-acid dehydrogenase (BCKDH; EC 1.2.4.4) activity in liver and muscle homogenates were determined. Compared with those on the balanced diet, pigs fed on methionine-limiting diets had significantly lower (P < 0.05) plasma methionine concentrations in the postprandial but not in the postabsorptive state. There was no effect of either leucine or a mixture of all three BCAA fed in excess on plasma methionine concentrations. Excess dietary leucine reduced (P < 0.05) the plasma concentrations of isoleucine and valine in both the postprandial and postabsorptive states. Plasma concentrations of the BCKA reflected the changes in the corresponding amino acids. Basal BCKDH activity in the liver and total BCKDH activity in the biceps femoris muscle were significantly (P < 0.05) increased by excesses of leucine or all BCAA.

Effects of changes in the intakes of protein and non-protein energy on whole-body protein turnover in growing pigs.

1. The relationships between the intakes of protein and of non-protein energy (NPE), nitrogcn retention and body protein synthesis have been studied in female pigs weighing 30 and 35 kg. 2. Four animals were assigned to three regimens and given a conventional (basal) diet supplemented with fat, carbohydrate or protein. After 1 week, measurements of N excretion in urine and faeces (7 d collection) and gaseous exchange (3–4 d) were made. At the end of the balance period a solution of [l- 14 C]leucine was infused at a constant rate. Body protein synthesis was then calculated as the difference between the apparent irreversible loss of blood leucine and the loss of 14 C in expired air. The animals were then offered the basal diet without supplement for 10 d and the measurements of N retention, energy retention and protein synthesis were repeated. 3. The intakes of metabolizable energy (ME; MJ/kg body-weight (W) 0.75 per d) were 1.75 for fat, 1.58 for carbohydrate, 1–25for protein and 1.18 for the basal diet; corresponding intakes of apparently digestible N (ADN; g N/kgW 0.75 per d) were 2.30,2.31,4.35 and 2–17. Daily N retention, which during the period of basal feeding was 13.6 g was increased by between 3.4 and 7.2 g by the supplements. Daily fat deposition was also increased in the animals that received the diets supplemented with carbohydrate and fat. 4. The rate of leucine catabolism was significantly reduced in the animals receiving the diets that were supplemented with W Eand increased by the addition of protein to the diet. 5. When based on the specfic radioactivity of blood leucine both the synthesis and breakdown of body protein (per unit metabolic body-weight) were increased by 30% in the animals receiving the high-protein diet but the increases in protein synthesis associated with the addition of carbohydrate (+14%) and fat (+12%) were much less marked. Consideration of these results together with previous observations (Reeds et al. 1980) suggested that body protein synthesis(g N/d) increased by 0.88 for each g increase in daily ADN and by 0.93 for each MJ increase in daily ME intake. 6. Comparison of the results obtained with the animals given high-carbohydrate diets and those given high-protein diets suggested an increase in heat production of 14 KJ/g of additional fat deposition. A similar comparison of animals receiving the high-protein and basal diets suggested a heat increment of 23.5KJ/g additional protein deposition. The changes in heat production and protein synthesis in the animals given the protein supplement were compatible with a heat increment of 5.3 KJ/g additional protein synthesized. Because of the large proportion of heat production associated with the deposition of fat this could not be confirmed with either of the other supplements, but it is possible that the energy cost of protein accretion varies with the relative proportions of protein and NPE in the diet.

Body fat in lean and overweight women estimated by six methods

Body fat content of seven lean women (body mass index (BMI) 20.6 (SD 1.8) kg/m2) and seven overweight women (BMI 31.1 (SD 3.3) kg/m2) was estimated by six different methods: underwater weighing (UWW), body-water dilution (BWD), whole-body counting (40K), skinfold thickness (SFT), bioelectrical impedance (BEI) and magnetic resonance imaging (MRI). Using UWW as the reference method, the differences between percentage fat by each other method and the percentage fat by UWW were calculated for each subject. The mean difference was lowest for SFT and highest for BWD. MRI showed the lowest variability in individual results, and 40K the highest. 40K and BWD methods used in combination gave better agreement with UWW results than either 40K or BWD methods alone. There was a weak negative correlation between the difference from the UWW results and percentage fat in the SFT measurements, but not in the BWD, 40K, BEI or MRI measurements, suggesting that for these methods the assumptions involved produced no greater inaccuracy in the overweight women than in the lean women. In all subjects the BEI offered little improvement over the traditional SFT measurements. The agreement between MRI and UWW estimates in both lean and overweight women suggests that MRI may be a satisfactory substitute for the more established methods of body fat estimation in adult women.

The optimal amino acid supplementation of barley for the growing pig. 1. Response of nitrogen metabolism to progressive supplementation.

1. In five experiments, involving 142 female pigs weighing on average 33 kg, estimates were made of the amounts of essential amino acids which minimized urinary N excretion when diets with barley as the only source of protein were given at the rate of 120 g/kg0.75 per d. 2. With additions of lysine (4.0 g/kg diet) and threonine, (1.2 g/kg diet) to barley urinary N excretion decreased from 0.91 to 0.36 g/kg0.75 per d, corresponding to an increase in biological value (bv) from 0.51 to 0.86. 3. With these additions of lysine and threonine, there were no responses to additions of tryptophan, methionine or isoleucine, or to further additions of lysine or threonine, but addition of histidine significantly reduced N excretion. 4. No optimal addition of histidine could be determined; the mean rate of N excretion after addition of histidine (not less than 0.3 g/kg diet) was 0.27 g/kg0.75 per d, corresponding to a BV of 0.93. 5. There was a variation between pigs from different litters in their responses to added histidine. Those with low rates of N excretion on the unsupplemented diet did not respond to additions of histidine, but those with high rates did. 6. It is concluded that additions of only three amino acids can greatly improve the nutritive value of barley protein for the growing pig and that the amino acid composition of the supplemented protein closely approaches the ideal; it is also similar to whole-body tissue protein.

The protein-sparing effect of carbohydrate. 1. Nitrogen retention of growing pigs in relation to diet.

1. Measurements were made of the losses of nitrogen in the faeces and urine of sixty pigs of approximately 33 kg, given dietary regimens comprising twenty-nine combinations of fish flour (0-800 g/d) and maize starch (0-1200 g/d). 2. The results were used to develop a generalized equation describing N retention as a joint function of N intake and starch intake. 3. The protein-sparing effect of starch was exerted in all circumstances. It was greatest when protein intake exceeded 220 g/d but some effect persisted with protein-free diets. With a high protein supply, the increase in N retention per unit increase in dietary starch decreased from 36 mg/g with the first increment to 3 mg/g with the highest attainable starch intake. 4. From the generalized equation the relationships between net protein utilization and protein concentration and food intake could be described as continuous functions. The equations may be of use in comparing the protein values of diets measured under non-standardized conditions.

Effects of the amount and quality of dietary protein on nitrogen metabolism and protein turnover of pigs

1. The interrelation between protein accretion and whole-body protein turnover were studied by varying the quantity and quality of protein given to growing pigs. 2. Diets with 150 or 290 g lysine-deficient protein/kg were given in hourly meals, with or without lysine supplementation, to female pigs (mean weight 47 kg). 3. After the animals were adapted to the diets, a constant infusion of [14C]urea was given intra-arterially for 30 h, during the last 6 h of which an infusion of [4,5-3H]leucine was also infused at a constant rate. At the same time, yeast-protein labelled with 15N was given in the diet for 50 h. 4. The rate of urea synthesis was estimated from the specific radioactivity (SR) of plasma urea. The rate of leucine flux was estimated from the SR of plasma leucine. The irrevocable breakdown of leucine was estimated from the 3H-labelling of body water. Total N flux was estimated from the 15N-labelling of urinary urea. 5. Addition of lysine to the low-protein diet significantly increased N retention, with a substantial reduction in leucine breakdown, but there was no significant change in the flux of leucine or of total N. 6. Increasing the quantity of the unsupplemented protein also increased N retention significantly, with concomitant increases in leucine breakdown and in the fluxes of leucine and of total N. 7. It is concluded that a doubling of protein accretion brought about by the improvement of dietary protein quality is not necessarily associated with an increased rate of whole-body protein turnover.

Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-(13)C]lysine and L-[6,6-(2)H(2)]lysine, respectively. [(15)N(2)]urea or (15)NH(4)Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with (15)NH(4)Cl. [(15)N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [(13)C]- and [(2)H(2)]lysine enrichments during the 12-h fed period were observed between the two (15)N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after (15)NH(4)Cl and [(15)N(2)]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg. kg(-1). day(-1), depending on the [(15)N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration ofl-[1-13C]lysine andl-[6,6-2H2]lysine, respectively. [15N2]urea or15NH4Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with15NH4Cl. [15N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [13C]- and [2H2]lysine enrichments during the 12-h fed period were observed between the two15N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after15NH4Cl and [15N2]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg ⋅ kg-1 ⋅ day-1, depending on the [15N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.