An Investigation of the Protein Metabolism in Healthy Subjects and Weight-Losing Cancer Patients

The majority of cancer patients with progressive disease lose weight and a proportion become emaciated to the point where they appear to die of starvation. This complex metabolic syndrome characterised clinically by progressive involuntary weight loss is termed cancer cachexia. The main aim of this thesis was to develop suitable methodology to allow investigation of the basis of the increased whole body protein synthesis in cancer patients. It has been estimated from work on animals that liver and skeletal muscle make up approximately 60% of daily protein synthesis in the whole body. Therefore, following a review of literature methods were developed, using stable isotope-labelled amino acids, to measure simultaneously not only whole body protein synthetic rate but also protein synthetic rates in liver and skeletal muscle in man. Protein synthetic rate measurements involve detection of small changes in isotopic enrichment requiring the use of a low resolution mass spectrometer. It is essential to purify samples for presentation to the mass spectrometer. Therefore, sample preparation protocols were developed to allow separation of leucine and glycine, in a pure form, from complex samples such as plasma, intracellular fluid and hydrolysed protein. Sample preparation methods were developed to enable measurement of enrichment in urinary ammonia, plasma glycine, homogenate free glycine and glycine from tissue protein hydrolysate. Sample preparation methods were also developed to enable measurement of enrichment in breath CO2, plasma leucine and leucine from tissue protein hydrolysate. Continuous Flow-lsotope Ratio Mass Spectrometry was used for the isotope enrichment measurements presented in this thesis, rather than the conventional approach (requiring both a Gas Chromatography-Nass Spectrometer and Isotope Ratio Mass Spectrometer). This permitted the analysis of all samples with a single mass spectrometer. To obtain normal values for whole body and tissue protein synthetic rates, a group of weight-stable normal subjects (n = 6) undergoing elective cholecystectomy were studied. Two labelled amino acids ([13C]leucine and [15N]glycine) were used to assess which in the context of the present study would give the most reliable results. The precision and accuracy of the measurement of [13C]leucine enrichment was poorer than that of [15N]glycine. Furthermore, [15N]glycine appeared to give the most reliable results and therefore was used in the study of a group of (n = 6) weight-losing cancer patients. In comparison with the control group the mean rate of whole body protein synthesis in the cancer patients was increased by 71%. However, when individual tissues were studied, non-export liver protein synthesis was reduced by 32% whereas skeletal muscle protein synthesis was increased by 86% in the cancer patients. Therefore, much of the increase in whole body protein synthesis in weight-losing cancer patients appears to be due to an increase in skeletal muscle protein synthesis. There was no significant difference in resting energy expenditure between the two groups. Furthermore, the increase in skeletal muscle protein synthesis conflicts with previous reported results and other indirect measurements in this group of patients. These results posed two questions. Firstly, does the incorporation of 15N into a metabolic end-product (ammonia) reflect the change in whole body protein synthesis in weight-losing cancer patients? Secondly, what is the basis of the increased incorporation of [15N]glycine into the muscle of the weight-losing cancer patients? One explanation, discussed in detail, is that in the cancer patients increased amounts of glycine-rich proteins are synthesised as part of the inflammatory response and bring about the apparent increase in whole body protein synthesis and increased [15N]glycine enrichment in the skeletal muscle. It is concluded, firstly, that the analytical approach developed in this thesis was satisfactory for use in [15N]glycine tracer studies. Secondly, that the data presented draws attention to the limitations of extrapolating the kinetics of a single labelled amino acid to the rate of synthesis of protein either in individual tissues or in the whole body. A variety of mechanisms and mediators are discussed which might account for the altered tracer kinetics observed in the weight-losing cancer patient. (Abstract shortened by ProQuest.)