Oxidative stress as cause for hyperhomocysteinemia

Homocysteine, a sulfur containing aminoacid, is an intermediate of the methionine metabolism. Hyperhomocysteinemia is established as a risk factor for cardiovascular and cerebrovascular diseases and can be treated by supplementation of the vitamin folic acid. Tetrahydrofolic acid is essential for the conversion of homocysteine to methionine thereby reducing the concentration of the former. To date, the biochemical link between hyperhomocysteinemia and vascular risk has remained unclear although there is experimental data that homocysteine might be involved in the generation of oxidative stress in turn evoking vascular events. Oxidative stress occurs during immune system activation, and clinical investigations have revealed an association between immune activation and the increase of serum homocysteine concentrations. Furthermore, tetrahydrofolic acid is very susceptible to oxidation which may become relevant under oxidative stress conditions. Therefore we assume immunologically-induced oxidative stress to be a main cause for endogenous folic acid depletion resulting in hyperhomocysteinemia. With the submitted project it is planned to substantiate this hypotheses on an (1) in vivo and an (2) in vitro level. (1) In patients with neurodegenerative diseases, homocysteine and folic acid metabolism will be investigated with respect to immune activation and oxidative stress. (2) In five independent cell culture systems, i.e., monocytic U-937, myelomonocytic THP-1, dendritic cells, peripheral blood mononuclear cells and human dermal microvascular endothelial cells, homocysteine and folate metabolism will be investigated upon cytokine stimulation and exogenous or endogenous oxidative stress generation. By the addition of antioxidants, the impact of oxidative stress on homocysteine metabolism is examined. The results of the study may provide new insights into the interaction between immune system activation and homocysteine metabolism and the pathogenesis of cardio- and cerebrovascular diseases.

Results of this project strengthen the view that immune activation and proliferation of T-cells play a major role in the development of moderate hyperhomocysteinemia. B-vitamins folic acid and B12 are essential cofactors for the remethylation of homocysteine to methionine and are prone to be oxidized irreversibly. Therefore, oxidative stress may lead to the enhanced oxidation of B-vitamins resulting in the accumulation of homocysteine in the blood. In our studies the association between immune activation, oxidative stress and hyperhomocysteinemia was examined in different patient collectives as well as by in vitro studies. First, a new method for the measurement of homocysteine was set-up. In our in vivo studies, not only coronary artery disease but also rheumatoid arthritis, neurodegenerative disease (with different forms of dementia: mild cognitive impairment, vascular dementia, Alzheimer`s disease, with Parkinson`s or Huntington`s disease) or malignant melanoma were found to be associated with increased concentrations of homocysteine, in parallel immune activation markers neopterin and sTNF-R75 were increased. The concentrations of B-vitamins were mainly in the lower range of normal, except for patients with rheumatoid arthritis substituted with B-vitamins. Also the age-dependent increase of homocysteine concentrations in healthy control persons also coincided with increased immune activation, whereas interferon- therapy only influenced immune activation, but not homocysteine accumulation in the blood. In vitro, homocysteine metabolism was studied in human peripheral blood mononuclear cells (PBMC), T- lymphocytes, dendritic cells (DC), human dermal microvascular endothelial cells (HDMEC), and in myelomonocytic (THP-1) and monocytic cells (U-937). Effects of pro-inflammatory cytokines and mitogens and the influence of supplementation of cells with folate, methionine and the combination of both on homocysteine metabolism were investigated. Freshly isolated human PBMC and T-lymphocytes and THP-1 and U-937 cells were able to release and also metabolize homocysteine, whereas HDMEC and DC did not produce relevant amounts of homocysteine. In PBMC, mitogen treatment significantly increased homocysteine production, and increase of homocysteine production was accompanied by neopterin release from macrophages. Homocysteine accumulation in stimulated PBMC could be referred to T-cell proliferation. Homocysteine production was further enhanced by methionine supplementation, folate supplementation slightly lowered homocysteine production in all cells. Various immunomodulating compounds including acetyl-salicylic acid (aspirin), atorvastatin and resveratrol were studied in the PBMC model system and they were found to dose-dependently inhibit homocysteine formation, neopterin production and cell proliferation.