Cultured fibroblasts as a model for methionine metabolism in man : effect of growth and substrates on homocysteine and related metabolites

Abnormal homocysteine metabolism has been implicated in a number of common diseases, but mainly cardiovascular disease. Though this association is well established, it remains to be proven whether homocysteine itself is directly responsible. Consideration of the causal mechanism depends on clearly defining the interrelationships between homocysteine and the other metabolites within the transmethylation-transsulphuration pathways, as they may also play an integral role in each disease process. In considering the pathogenesis of sulphydryl compounds, the relationship between intra- and extra-cellular levels is an often underestimated and important regulatory factor. Homocysteine metabolism is regulated by complex mechanisms involving its own export, B vitamins, S-adenosyl-methionine, (AdoMet), S-adenosyl-homocysteine (AdoHcy) and amino acids, as well as the proper function of various enzymes. These mechanisms in human cells, and particularly with export studies, have so far been performed in media containing un-physiological substrate levels. Difficulties in attempting to understand these mechanisms are compounded by the use of serum supplemented media, and the need to unravel the metabolic interrelationships by piecing together information from various models.
This work therefore, involved the development and application of a model for methionine metabolism, using human skin fibroblasts cultured in medium containing similar amino acid and vitamin levels to those found in normal human plasma. This required the formulation and production of a basal physiological medium (P-med) that would sustain cell growth, the development and application of methods for metabolite determination, the modification of sample handling and preparation procedures, and improvements to the existing analytical methods. Following the successful completion of this time consuming but indispensable step, it was possible to measure (i) total, free (non-protein bound) and protein-bound homocysteine, cyst(e)ine, cysteinyl-glycine and glutathione, (ii) 5-methyltetrahydrofolate (5-MeTHF), (iii) AdoMet and AdoHcy, and (iv) free amino acids, in media and/or cells for the first time using conditions more comparable to the physiological situation than those obtained previously.
In a multi-time point study designed to examine changes in media sulphydryl compound levels with log phase and post-confluent cells in basal and supplemented media, homocysteine and glutathione export, cyst(e)ine uptake and media cysteinyl-glycine levels where reproducible in eight cell lines at each time point for each media and growth state. Differences in media sulphydryl compound levels between each media and growth state however, were only subtle. Nevertheless, folate levels accounted for similarities in homocysteine export from log phase cells in each media, and a growth resurgence in post-confluent cells on renewing media accounted for similarities between each media and the post-confluent and log phase cells.
the second part of the work, which included cystathionine -synthase (CBS)-deficient cells, answered the important question, could the model be used to reliably measure intracellular metabolites? Indeed, total homocysteine, cysteine, cysteinyl-glycine, glutathione, AdoMet and the free amino acids could be measured, 5-MeTHF and AdoHcy could not. This study also highlighted the importance of export in the control of intracellular homocysteine, folate receptors and folate in homocysteine remethylation in post-confluent CBS-deficient cells, transsulphuration in glutathione synthesis, and the unrelated nature of media amino acid concentrations in amino acid transport.
In a final study to evaluate the outcome of methionine and folate on metabolites, supplementation resulted in methionine catabolism and conservation respectively, but was less effective than anticipated and previously shown in commercial media. Although this approach has been extensively applied to a variety of cell types in various commercial media, it may still have scope for examining the effects of additional substrates, for example arginine, taurine and the antioxidant vitamins A, C and E on homocysteine, its export or its modification of low-density lipoprotein.
In conclusion, this thesis added to the existing work by obtaining more comprehensive data on the main metabolite levels in media and in human fibroblasts under similar conditions to better understand the metabolism, and gave support and validation to our knowledge of export from previous studies as applicable to the human physiological situation.