Biomedical subjects
Publications and source records attributed to D Halliday.
Whole-body protein turnover measurements using the single dose 15N-glycine technique have been made in a patient completely paralysed by the Guillaine-Barré syndrome. Variation (difference x 100/mean) between the results of the studies was better than 0.75 per cent for protein flux, better than 0.85 per cent for protein breakdown, and better than 5 per cent for protein synthesis.
In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.
Quadriceps muscle protein turnover was assessed in the post-absorptive state in six men immediately after the end of unilateral leg immobilization (37 +/- 4 days) in a plaster cast after tibial fracture. A primed-constant intravenous infusion of L-[1-13C]leucine was administered over 7 h. Quadriceps needle biopsies, taken bilaterally at the end of the infusion, were analysed for muscle protein leucine enrichment with 13C. Quadriceps muscle protein synthetic rate, calculated from the fractional incorporation of [13C]leucine into protein compared with the average enrichment of blood alpha-ketoisocaproate, was 0.046 +/- 0.012%/h in the uninjured leg, but was only 0.034 +/- 0.007%/h in the quadriceps of the previously fractured leg (P less than 0.05, means +/- SD). Muscle RNA activity (i.e. protein synthetic rate per RNA) fell from 0.27 +/- 0.08 microgram of protein synthesized h-1 microgram-1 of RNA in the control leg to 0.14 +/- 0.03 microgram of protein synthesized h-1 microgram-1 of RNA in the immobilized leg (P less than 0.02). Immobilization was associated with a significant atrophy of type I muscle fibres (mean diameter 69.5 +/- 21 microns immobilized, 81.1 +/- 18 microns control, P less than 0.05), but no significant change occurred in type II fibre diameter. Mean quadriceps fibre volume calculated from the values for fibre diameter and percentage of each fibre type, was smaller in the injured leg by 10.6%; this value was near to the calculated difference in muscle thigh volume (calculated from thigh circumference and skin-fold thickness) which was less by 8.3%.(ABSTRACT TRUNCATED AT 250 WORDS)
1. We have used L-[1-13C,15N]leucine as the substrate tracer to study leucine and muscle protein metabolism across the forearm of eight normal fasting adults. 2. The rates of protein synthesis and breakdown, de- and re-amination of leucine, and the oxidative decarboxylation of its keto acid were calculated directly from the arteriovenous metabolite balances and isotope dilutions as described by the metabolic model. 3. The results were compared with those obtained previously when subjects were fed. The effects of fasting on protein and leucine metabolism were a significant decrease in protein synthesis from 127 (SEM 11; n = 6) to 70 (SEM 6; n = 12) nmol of leucine min-1 100 ml-1 of forearm tissue (P less than 0.001) and a marked decrease in leucine catabolism in the forearm muscle. 4. This model has demonstrated that each subject was in negative protein balance across the forearm during fasting while positive during feeding, the mean values being -29(SEM 5; n = 12) and +39(SEM 9; n = 6) nmol of leucine min-1 100 ml-1 of forearm tissue respectively. 5. These results are sufficiently encouraging to suggest a role for this model in future studies on muscle protein metabolism.
The effect of biosynthetic human growth hormone (BSHGH) on postoperative protein and energy metabolism has been studied in patients who had major gastrointestinal surgery. Seven patients received placebo and seven patients received BSHGH, 0.1 mg/kg/24 h, for the first six postoperative days. Mean total nitrogen excretion was significantly lower with BSHGH (31.5 +/- 2.4 g N) (2287 +/- 160 mmol) than with placebo (42.7 +/- 3.1 g N) (3049 +/- 219 mmol) over the 6-day study period. The mean daily measured energy expenditure over days 3-6 was higher with BSHGH (31.3 +/- 1.8 kcal/kg LBM/24 h) (131 +/- 7 kJ/kg LBM/24 h) than with placebo (27.6 +/- 0.8 kcal/kg LBM/24 h) (114 +/- 2 kJ/kg LBM/24 h). Fat oxidation with BSHGH (2.05 +/- 0.26 mg/kg LBM/24 h) was greater than with placebo (1.5 +/- 0.17 mg/kg LBM/24 h) and protein oxidation was less with BSHGH (0.68 +/- 0.07 g/kg LBM/24 h) than with placebo (0.9 +/- 0.09 g/kg LBM/24 h) on days 1-6. Postoperative nitrogen turnover (BSHGH 943 +/- 174 mg N/kg LBM/24 h, placebo 557 +/- 50 mg N/kg LBM/24 h) (BSHGH 67 +/- 13 mmol/kg LBM/24 h, placebo 40 +/- 4 mmol/kg LBM/24 h), protein synthesis (BSHGH 5.31 +/- 1.09 g prot/kg LBM/24 h, placebo 2.54 +/- 0.33 g prot/kg LBM/24 h) and protein breakdown (BSHGH 5.90 +/- 1.09 g prot/kg LBM/24 h, placebo 3.48 +/- 0.31 g prot/kg LBM/24 h) were greater with BSHGH. On the first postoperative day serum insulin and blood glucose levels were higher with BSHGH than with placebo, and on days 4 and 7 serum somatomedin-C levels were significantly elevated. This study shows that BSHGH alters postoperative protein and energy metabolism by reducing protein oxidation and increasing fat oxidation with raised rates of whole body nitrogen turnover.
The relation between hepatic glucose production rate (HGPR) and plasma concentrations of insulin and glucagon was investigated in four term neonates who had severe hypoglycaemia. The hepatic glucose production rate was less than 20% of normal for fasting term neonates in all four babies and yet insulin concentrations were never greater than 12 microU/ml; two babies had very low glucagon concentrations (less than 60 ng/l). Two further neonates with similar histories also had plasma glucagon concentrations that were also extremely low (less than 20 ng/l). A single intravenous bolus of glucagon caused a rapid rise in hepatic glucose production rate towards the normal range, which was sustained for many hours after the bolus had been given. Diazoxide given to one baby suppressed previously 'normal' insulin concentrations still further (4.2 to less than 1.6 microU/ml) and thereby restored the hepatic glucose production rate to normal. In view of the normal plasma insulin concentrations at a time when the hepatic glucose production rate was reduced, we feel that the absolute concentration of insulin may be less important than the insulin/glucagon molar ratio in the control of glucose homeostasis in this group of infants. The changing of this ratio by means of boluses of glucagon may be useful in preventing rebound hypoglycaemia, which so often occurs when dextrose infusions are reduced either accidentally or in an attempt to restart oral feeds.
Hyperglucagonemia coexists with insulin deficiency or insulin resistance in many conditions where urinary nitrogen excretion is increased, but the precise role of glucagon in these conditions is controversial. The purpose of this study was to evaluate the effect of hyperglucagonemia on protein metabolism in insulin-deficient subjects. We used the stable isotope of an essential amino acid (L-[1-13C]leucine) as a tracer of in vivo protein metabolism. A combined deficiency of insulin and glucagon was induced by intravenous infusion of somatostatin. Hyperglucagonemia and hypoinsulinemia were induced by infusions of somatostatin and glucagon. When somatostatin alone was infused leucine flux increased, indicating a 6-17% increase in proteolysis. When somatostatin and glucagon were infused, leucine flux increased, indicating a 12-32% increase in proteolysis. The increase in leucine flux during the infusion of somatostatin and glucagon was higher than the increase during infusion of somatostatin alone. Somatostatin alone did not change leucine oxidation, whereas the somatostatin plus glucagon increased leucine oxidation 100%. We conclude that hyperglucagonemia accelerates proteolysis and leucine oxidation in insulin-deficient humans.
The influence of anaerobic and aerobic exercise, promoted by computer regulated functional electrical stimulation (FES) was evaluated in four paraplegic males. Quadriceps muscle bulk was monitored by serial computerised axial tomography (CT) scanning and histology by muscle biopsies from the vastus intermedius. Anaerobic exercise consisted of 60 degree straight leg raising against increasing weights (range 1.4-11.4 kg) over a period of ten weeks. Aerobic exercise consisted of pedalling a modified Monark bicycle ergometer at 50 rpm against a fixed load ranging from 0-3/8 kilopond (0-18.75 watts) over a period of eight months. In both exercise studies the same work was not achieved by each paraplegic. FES was regulated by a closed loop system which is not presently commercially available, the frequency of the sequential muscle stimulator was 40 Hz with a pulse width of 300 microseconds. Quadriceps muscle area of both legs increased 62.7% (p less than 0.01) after anaerobic exercise; similar but less pronounced effects followed aerobic exercise. Histologically two distinct patterns were noted from the outset, one had normal fibre type distribution the remainder had marked Type 1 loss. Both exercise regimens failed to change these although the number of internal nuclei per 100 fibres steadily increased (from 7.0% to 13.8% to 26.0%) as did the % of fibres with internal nuclei (5.4% to 10.5% to 25.7%) throughout the exercise periods. The significance of these observations is not immediately apparent but may signify continuing damage which may be due to the eccentric rather than the concentric nature of FES promoted muscular contraction.
Leucine kinetics were studied in six obese subjects (W/H2 = 39 +/- 4) and six normal subjects (W/H2 = 21 +/- 3) before and after an oral load of 150 g glucose. An intravenous infusion of 1(-13)C leucine was given to the fasting subjects for 450 min: a steady state of plasma leucine enrichment was established 90 min after the start of the infusion, and the glucose load was given 220 min after the start of the infusion. Compared with the lean controls the obese subjects showed a greater area under the curve of blood glucose after the glucose load (P less than 0.025) and higher insulin and glucagon levels both before and after the meal (P less than 0.05), thus indicating the well-known insulin insensitivity of obese (but not diabetic) subjects with respect to glucose metabolism. After the glucose load the lean subjects showed a significant and sustained decrease in leucine oxidation (from 20.0 +/- 2.2 to 13.3 +/- 1.5 mumol/kg LBM/h: P less than 0.01). This response is similar to that observed when insulin-dependent diabetic subjects are given insulin. However the obese subjects showed no decrease in leucine oxidation after the glucose meal (20.3 +/- 1.9 before, and 21.2 +/- 3.6 after). This indicates that obese subjects show insensitivity to the action of insulin with respect to protein metabolism as well as carbohydrate metabolism.
Muscle wasting in myotonic dystrophy may result from decreased muscle anabolic processes rather than from increased catabolism. Male patients with myotonic dystrophy often have low levels of circulating androgens, and androgen administration has been shown to increase their muscle mass. We have studied the effect of testosterone enanthate administration (3 mg/kg weekly for 3 months) on muscle and whole body protein synthesis in 6 male patients with myotonic dystrophy. Muscle protein synthesis was estimated from the rate of isotope incorporation into muscle protein obtained by quadriceps muscle biopsy during a primed continuous infusion of L-[1-13C]leucine. Testosterone administration resulted in a significant increase in muscle protein synthesis in all patients. Whole body protein synthesis did not increase, indicating that protein synthesis in other tissues may have declined. Muscle ribonucleic acid content rose significantly in response to testosterone administration, suggesting that testosterone initiated its effect by hormone receptor interaction with muscle nuclei.
Explore the source record for details and available documents.
The rate of muscle protein synthesis in patients with myotonic dystrophy has been studied, and results correlated with total muscle mass. Whole body and skeletal muscle protein synthesis were estimated by stable isotope methodology with a primed, continuous infusion of 1-[13C]leucine with measurement of incorporation of [13C]leucine into muscle protein in biopsy samples. Whole body leucine flux, protein synthesis, and protein breakdown were only slightly depressed, but muscle protein synthesis was markedly decreased, in myotonic dystrophy. This depression of muscle protein synthesis in myotonic dystrophy correlates with previous observations of impaired insulin-induced muscle uptake of amino acids and supports the suggestion that muscle wasting in this disease is the consequence of defective anabolism in muscle.
Protein metabolism after surgery in the presence of disseminated malignancy has been investigated and compared with results obtained from patients undergoing similar surgical procedures for benign disease and localized malignancy. Whole body nitrogen turnover, measured by primed continuous infusion of 15N glycine, was highest with disseminated malignancy. Similarly rates of whole body protein synthesis and breakdown, calculated from turnover and nitrogen excretion (nitrogen intake being zero), were elevated in the presence of disseminated malignant disease. The increased rates of protein metabolism may represent adaptation to the demands of inevitably growing malignant tissue.
The methods used for the determination of the concentration and isotope enrichment of (1-13C)leucine and its metabolite (13C) alpha-ketoisocaproic acid (KIC) in plasma for the study of whole-body protein turnover are described. Leucine was analysed as its N-heptafluorobutyryl isobutyl ester and KIC as its quinoxalinol-TMS derivative, both by chemical ionization selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The sensitivity of the leucine assay was improved 30 times by monitoring the negative ions under the conditions described. The coefficient of variation for enrichment and concentration measurements were 0.5% and 2%, respectively, with a minimum detectable enrichment of 0.1 at% excess for both assays.
Tserng and Kalhan have raised the question of the appropriate equation to use for the measurement of turnover rate in a stable-isotope study. In a comparison of glucose turnover measured with 2H-glucose and with 13C-glucose they used five apparently different equations and obtained conflicting answers. There is, however, no difference in principle between the use of a stable isotope as a tracer and the use of a radioactive isotope, and the rate of appearance of tracee in a steady-state system (the turnover) can therefore be shown to be proportional to the equilibrium dilution of the infused tracer. Because the sensitivity of measurement of this dilution made using a gas chromatograph-mass spectrometer is lower than that made by radioactivity measurement, the contribution to the measured turnover rate due to the infusate cannot be neglected, as it usually is in radioisotope work. A convenient calibration curve to establish this dilution is the mole ratio of the pure infusate against the area ratio for the relevant ions. Tserng and Kalhan's apparently conflicting results for glucose turnover using 13C-glucose as the tracer can all be shown to amount to approximately 11.6 mumol min-1 kg-1. This value is only slightly lower (0.05 less than P less than 0.1) than that obtained using 2H-glucose as the tracer and supports the use of 13C-glucose as an alternative.
We have used the forearm model to study protein metabolism in six normal healthy subjects in the fed state using L-[1-13C, 15N]-leucine as the substrate tracer. Deep venous and arterialized venous blood samples from the forearm were collected at 10-min intervals 2.5 h into a primed-continuous infusion of the dilabelled tracer. Arterialized venous blood was obtained using a 'hot-box' technique and forearm blood flow was measured by mercury strain-gauge plethysmography. The concentration and isotope enrichment of leucine and its metabolites, alpha-ketoisocaproic acid and CO2, in deep venous and arterialized venous blood were measured by gas chromatography-mass spectrometry and isotope ratio-mass spectrometry. The rates of leucine deamination and reamination were 388 +/- 24 (mean +/- SEM) and 330 +/- 23 nmol (100 ml)-1 min-1 respectively, whilst protein synthesis and breakdown rates were 127 +/- 11 and 87 +/- 10 nmol (100 ml)-1 min-1 respectively across the forearm in the fed state. We have demonstrated that the use of doubly labelled leucine as tracer and application of the mathematical model developed in this study, permits the comprehensive quantification of leucine kinetics including protein breakdown.
Rates of synthesis of protein were measured in vivo in skeletal muscle and in the whole body of cachectic patients with cancer and in normal healthy men, using a tracer infusion of leucine labelled with a stable isotope. Synthesis of protein in muscle was significantly reduced in the patients with cancer (0.030 v 0.198%/hour; p less than 0.01), whereas whole body rates of protein synthesis and degradation did not differ significantly between the two groups. Thus depression of synthesis of protein in muscle appeared to be the immediate cause of muscle wasting in cancerous cachexia. Any therapeutic intervention that aims at preventing the onset of cachexia should be designed to stimulate the synthesis of protein in muscle, and measurement of turnover of protein may be used to evaluate such treatment provided that rates of protein synthesis are measured directly in specific tissues.
Measurements of protein and amino acid metabolism in man using stable isotopes and selected ion monitoring gas chromatographic/mass spectrometric techniques are limited by the requirement of relatively high levels of labelling for adequate precision (greater than 0.05 at % excess). We describe here a means of extending the scope of such studies by measurement of lower levels of enrichment achieved in gaseous CO2 derived from whole blood or protein-bound amino acids following the administration of tracer amounts of appropriately labelled substrates. Construction and operation of a novel glass vacuum line required for this work are described in detail and specific applications relevant to clinical investigations are outlined. Measurements of both the total amount of CO2 and its 13C enrichment are performed in an isotope-ratio mass spectrometer which provides acceptable levels of accuracy and reproducibility for both measurements (+/- 0.1% and +/- 0.0001 at % excess respectively).