Pathophysiological Consequences of Protein N-Homocysteinylation - Homocysteine in Protein Structure/Function and Human Disease - page 107

plasma tHcy (5-40 M) [79]. The lower protein N-linked Hcy/tHcy ratios suggest

that the Hcy-thiolactone clearance is more effective at high plasma tHcy levels.
This suggestion is supported by a finding that in mice fed a hyperhomocysteinemic
high-Met or high-Hcy diet, the ratio of urinary to plasma Hcy-thiolactone is
sevenfold or fourfold higher, respectively, compared to mice fed a normal diet [93].
Hyperhomocysteinemia is linked to increased mortality in CAD patients [37]. In
a clinical study that examined a relationship between Hcy and CAD, plasma protein
N-linked Hcy levels are significantly higher in coronary heart disease patients than
in controls and are associated with the risk of CAD [168]. Furthermore, there is a
weak but significant positive correlation between protein N-linked Hcy level and
the number of diseased coronary arteries: the higher protein N-linked Hcy level, the
greater the number of afflicted arteries.
Studies using polyclonal rabbit anti-N-Hcy-protein IgG antibodies [310] dem-
onstrate that N-Hcy-protein is present in diseased human cardiac tissues [357, 373].
A positive immunohistochemical staining of myocardium and aorta samples from
cardiac surgery patients is observed. Control experiments demonstrate that the
staining is specific for N-Hcy-protein. No immunostaining is observed with rabbit
preimmune IgG, with iodoacetamide-treated tissues (which destroys the Nε

μ

-Hcy-
Lys-protein epitope), or with the antibody pre-adsorbed by preincubation of IgGs
with N-Hcy-albumin. That immunostaining is specific for Hcy-thiolactone-
modified protein is shown by increased intensity of the staining in tissues pretreated
with Hcy-thiolactone [357, 373].
Immunohistochemical studies also demonstrate that N-Hcy-protein accumulates
within atherosclerotic lesions in ApoE / mice [357]. ApoE / mice develop
atherosclerosis spontaneously on a normal chow, but the process is accelerated by
hyperhomocysteinemia caused by a high-Met diet [45]. Chemical assays establish
that high-Met diet causes significant elevation also in Hcy-thiolactone and N-Hcy-
protein levels [93, 115]. Positive staining for N-Hcy-protein is observed in aortas of
ApoE / mice fed with a normal chow diet, and the staining increases in animals
fed with a hyperhomocysteinemic high-Met diet [357].
These findings show that hyperhomocysteinemia increases levels in human and
mice and that elevated N-Hcy-protein levels are linked to CAD in humans and to
atherosclerosis in mice, as predicted by the Hcy-thiolactone hypothesis (Fig. 6.1 ).

6.2 N -Hcy-Protein and Adaptive Autoimmune Responses

6.2.1 Atherosclerosis Is an Inflammatory Disease

Atherosclerosis is now widely recognized as a chronic inflammatory disease [43]
that involves innate and adaptive immunity [374-376]. Lipid peroxidation is
thought to play a central role in the initiation of both cellular and humeral