Vernon R. Young

Relationship of plasma leucine and α-ketoisocaproate during a L-[1-13C]leucine infusion in man: A method for measuring human intracellular leucine tracer enrichment☆

The keto analog of leucine, alpha-ketoisocaproate (KIC), is formed intracellularly from leucine and is released, in part, into the systemic circulation. Therefore. KIC can be used to estimate intracellular leucine tracer enrichment in man during labeled-leucine tracer experiments without requiring tissue biopsy samples. This approach was studied in young, healthy, male adults maintained on different dietary protein intakes from generous (1.5 g kg-1d-1) to deficient (0.0 g kg-1d-1) for 5-7 day periods. At the end of each dietary period, the volunteers were given a primed, continuous infusion of L-[1-13C]leucine either after an overnight fast (postabsorptive state) or while being fed hourly aliquots of the same diet. The plasma concentrations of all 3 branched-chain amino and keto acid pairs were measured from early morning blood samples taken from 4 subjects at 4 different levels of protein intake. Leucine concentration showed a weak correlation, and valine concentration showed a strong correlation with protein intake; isoleucine and the 3 keto acids did not. However, each branched-chain amino acid concentration was strongly correlated with its corresponding keto acid concentration. In plasma samples obtained during the L-[1-13C]leucine infusions, the ratio of [1-13C]KIC to [1-13C]leucine enrichment ratio remained relatively constant (77 +/- 1% over the wide range of dietary protein intakes and for both the fed and postabsorptive states. For the tissues from which the plasma KIC originates, the rate of plasma leucine into cells will account for approximately 77% of the intracellular leucine flux with the remaining 23% coming primarily from leucine release via protein breakdown. The constant nature of the plasma KIC to leucine 13C enrichment ratio implies that relative changes in leucine kinetics will appear the same under many dietary circumstances regardless of whether plasma leucine or KIC enrichments are used for the calculations.

Suction applied to a muscle biopsy maximizes sample size

A method for increasing the size of a percutaneous needle biopsy specimen of skeletal muscle is described. Suction (700 TORR) is applied to the inner bore of the biopsy needle after the needle has been inserted into the subjects muscle. The suction pulls the surrounding muscle tissue into the needle, thus insuring the taking of a larger piece (X = 78.5 mg). In most cases, this technique will eliminate the need for repeated biopsies because of inadequate muscle sample size and enhance the validity of subsequent analysis procedures.

Protein and amino acids.

The usual source of amino acids the body cannot make (the nutritionally indispensable, or essential, amino acids) and of the nitrogen required for the synthesis of other amino acids (the nutritionally dispensable or nonessential amino acids) and numerous physiologically important nitrogen-containing compounds is from the protein-containing component of the diet. In the case of special nutritional therapies, the required amino acids and nitrogen can be supplied by formulations that are given via enteral or parenteral administration. Inadequate protein or amino acid intakes cause diminished content of protein in cells and organs and deterioration in the capacity of cells to carry out their normal function. This then results in growth faltering in the young and, in all individuals, increased morbidity and eventually death if the poor diet continues. Furthermore, intakes in considerable excess of physiologic needs also might be disadvantageous. Thus, an adequate diet, whether consisting of normal foods or specially formulated medical/nutritional products, must contain an appropriate level of protein (nitrogen) and balance of amino acids one to another, so that adequate growth, development, and/or long-term health can be achieved and sustained.

Metabolism of 3-methylhistidine in man

The metabolism of L-3-methylhistidine was studied in man using intravenously administered ((14)C)3-methylhistidine. Analysis for expired (14)CO2 for periods up to 2 hr following a single intravenous injection revealed no radioactivity, indicating that this compound is not oxidized in man. Analysis of urine samples for total radioactivity showed that 75% of the administered dose was excreted in 24 hr and 95% in 48 hr. Ion-exchange chromatography of urine samples with monitoring of the column eluated by a flow liquid-scintillation technique showed the presence of only two radioactive peaks. The time taken to elute these peaks was compatible with the major excretory component (95.5%) being ((14)C)3-methylhistidine, accompanied by a small amount (4.5%) in the form of N-acetyl-((14)C)3-methylhistidine. The plasma disappearance curves of ((14)C)3-methylhistidine suggested a half-life of approximately 130 min. The inability ot oxidize 3-methylhistidine and its quantitative excretion as the original compound as well as its N-acetyl derivative is similar to its metabolic fate in the rat and therefore suggests that 3-methylhistidine excretion may provide a reliable measure of actin and myosin turnover in the whole animal or in human subjects.

Potential use of 3-methylhistidine excretion as an index of progressive reduction in muscle protein catabolism during starvation

Abstract Based on animal studies, the amino acid 3-methylhistidine, a component of actin and myosin, is not reutilized for protein synthesis following the breakdown of muscle protein but is quantitatively excreted in the urine and should thus provide an index of the rate of muscle protein breakdown. The urinary outputs of total N, urea N, 3-methylhistidine, 1-methylhistidine, and creatinine are reported for three obese subjects during a 20-day starvation period. The excretion of 3-methylhistidine decreased progressively and on day 20 was 34% less than on day 3. This reduction was proportionally similar to the reduction in urinary total N output but was much greater than the 15% decrease in creatinine excretion and in calculated loss of muscle mass. Assuming that tissues other than muscle are not significant sources of urinary 3-methylhistidine, these findings suggest that the reduced output of urinary N is due to an adaptive decrease in the rate of catabolism of muscle proteins as starvation progressed.

Increased rates of whole body protein synthesis and breakdown in children recovering from burns.

The rates of whole body protein synthesis and breakdown were determined, with the aid of a constant administration of [15N]glycine, during recovery in 11 acutely burned children, involving a total of 24 studies. Eleven studies were also conducted in seven healthy children before and after reconstructive surgery. Rates of whole body protein synthesis and breakdown, expressed as g protein/kg body weight/day, were significantly (p less than 0.05) and positiviely correlated with per cent body surface area total burn, per cent third-degree burn, and per cent open wound. These rates (synthesis, 7.1 +/- 2.1 g protein/kg/day; breakdown, 6.3 +/- 1.8 g protein/kg/day) were 80 to 100% greater (p less than 0.05) in patients with total burns greater than or equal to 60%, as compared to patients with less than 25% total burns or to the surgical patients. Because of the high energy cost of protein synthesis, it is proposed that an increased whole body protein turnover is partly responsible for the reported elevations in rates of heat production occurring in patients recovering from thermal injury.

Total human body protein synthesis in relation to protein requirements at various ages

THE intensity of body and tissue protein metabolism per kg declines with increased adult body size in mammals1. This fall parallels a similar progressive decline in the intensity of energy metabolism2–4. It has also been concluded that protein metabolism per unit of body weight is about four to five times faster in young rats than in adult man1; this pattern of change extends to cellular and subcellular aspects of protein metabolism, such as plasma albumin synthesis, liver RNA content and enzyme activity1,5. Similarly, the rate of protein synthesis per kg total body weight declines during growth and development within a species, such as the rat6. This parameter again parallels the reduction in the intensity of energy metabolism which occurs during the growth period3.

Albumin synthesis in young and elderly subjects using a new stable isotope methodology: Response to level of protein intake

Albumin synthesis was evaluated in 5 young adult males (19-25 yr) and 6 elderly males (64-78 yr) by a procedure involving oral administration of 15N-glycine every 3 hr over a 60-hr period. From about 40 hr onwards, urinary urea achieved a plateau of 15N-enrichment, which was estimated from the average of the last five (low protein) or seven (adequate protein) consecutive three-hourly urinary samples of the 60-hr period. This enrichment plateau was used as an index of the 15N-enrichment of the guanidine N of hepatic free arginine. The 15N-enrichment of the guanidine N of arginine in serum albumin was determined and albumin synthesis was estimated by comparing this value with the estimated enrichment of precursor hepatic arginine. Using this methodology, serum albumin concentration, synthesis, rate and plasma volume were measured when the young and elderly subjects had received an adequate protein intake (1.5 g x kg-1 for 7 days) or a low protein intake (0.4 g x kg-1 for 14 days). Serum albumin concentration was lower in the elderly at both levels of protein intake; protein intake did not affect this parameter in either age-group. Plasma volume (per kg body weight) did not differ between young and old, but increased in both groups when they were given the low-protein diet, so that the total intravascular albumin mass increased in both age groups significantly in the case of the young, and was probably due to net transfer of albumin from the extravascular pool. The fractional synthesis rate of the whole body albumin pool with adequate intake of protein was 4.0%/day in the young and 3.4%/day in the elderly. This fractional rate was reduced significantly by giving the low-protein diet to the young subjects, but was not reduced in the elderly. Absolute synthesis rates, calculated per kg body weight and per kg body cell mass, led to a similar conclusion. Whole body protein synthesis was also estimated from urinary 15N-urea enrichment using the Picou and Taylor-Roberts model. Albumin synthesis as a percentage of whole body protein synthesis (5%-6%) was reduced in the young adults by giving the low-protein diet, but was unchanged in the elderly. In conclusion, the rate of albumin synthesis in the young, but not in the elderly, is sensitive to changes in protein intake. It is suggested that albumin synthesis in the elderly is controlled at a lower set point, which prevents its response to higher protein intakes.

Cysteine metabolism and whole blood glutathione synthesis in septic pediatric patients.

ObjectiveTo investigate whole body in vivo cysteine kinetics and its relationship to whole blood glutathione (GSH) synthesis rates in septic, critically ill pediatric patients and controls. DesignProspective cohort study. SettingMultidisciplinary intensive care unit and pediatric inpatient units at a children’s hospital. PatientsTen septic pediatric patients and ten controls (children admitted to the hospital for elective surgery). InterventionsSeptic patients (age, 31 months to 17 yrs) and controls (age, 24 months to 21 yrs) received a 6-hr primed, constant, intravenous tracer infusion of l-[1-13C]cysteine. Blood samples were obtained to determine isotopic enrichment of plasma cysteine and whole blood [1-13C]cysteinyl-glutathione by gas-chromatography mass spectrometric techniques. The plasma flux and oxidation rate of cysteine and the fractional and absolute synthesis rates of GSH were determined. Septic patients received variable protein and energy intake, as per routine clinical management, and controls were studied in the early postabsorptive state. Measurements and Main Results Plasma cysteine fluxes were increased in the septic patients when compared with the controls (68.2 ± 17.5 [sd] vs. 48.7 ± 8.8 &mgr;mol·kg−1·hr−1;p < .01), and the fraction of plasma cysteine flux associated with oxidative disposal was similar among the groups. The absolute rates of GSH synthesis in whole blood were decreased (p < .01) in the septic patients (368 ± 156 vs. 909 ± 272 &mgr;mol·L−1·day−1). The concentration of whole blood GSH also was decreased in the septic group (665.4 ± 194 vs. 1059 ± 334 &mgr;M;p < .01) ConclusionsWhole blood glutathione synthesis rates are decreased, by about 60%, in critically ill septic children receiving limited nutritional support. Plasma cysteine fluxes and concentration of cysteine were increased in the septic patients, suggesting a hypermetabolic state with increased protein breakdown. The mechanisms whereby GSH synthesis rates are decreased in these patients are probably multifactorial, presumably involving an inflammatory response in the presence of limited nutritional support. The role of nutritional modulation and the use of cysteine prodrugs in maintaining GSH concentration and synthesis remain to be established.

Nτ-Methylhistidine content of mixed proteins in various rat tissues

Abstract In order to use N τ -methylhistidine (3-methylhistidine) excretion in the urine as a measure of muscle protein breakdown, it is necessary to demonstrate that other tissues are not important sources of this protein constituent. Accordingly, the concentration of N τ -methylhistidine in blood serum and in the mixed proteins of heart, brain, lung, kidney, diaphragm, spleen, testis, stomach, liver and hind leg skeletal muscle was measured in male rats of approx. 400 g body weight. The free N τ -methylhistidine concentration of rat serum was less than 2 nmol per ml. In contrast, measurable amounts of N τ -methylhistidine were found in the mixed proteins of all tissues and organs examined. The highest concentration was found in skeletal muscle (658 nmol/g tissue). Assuming muscle mass to be 45% of body weight, it has been estimated that the muscle contains more than ten times the total amount of this amino acid present in all of the other organs analyzed, which together account for about 20% of total body weight. These findings indicate that skeletal muscle is likely to be the major source of urinary N τ -methylhistidine and the latter is, in consequence, a reflection of myofibrillar protein breakdown in skeletal muscle.