Pathophysiological Consequences of Protein N-Homocysteinylation - Homocysteine in Protein Structure/Function and Human Disease - page 106

This finding is consistent with ex vivo observations that cultured human CBS-
deficient fibroblasts synthesize much more Hcy-thiolactone than normal fibroblasts
[73].
Moreover, 5-methyltetrahydofolate deficiency, secondary to a mutation in the
MTHFR gene, leads to elevation of Hcy-thiolactone levels in humans [93]: plasma
Hcy-thiolactone in MTHFR-deficient patients (11.8 nM) is 24- or 59-fold higher
than in MTHFR heterozygous or normal individuals, respectively (Table 3.12 ). This
in vivo finding is consistent with ex vivo observations showing that limited avail-
ability of folic acid greatly enhances Hcy-thiolactone synthesis in human fibroblasts
[73] and vascular endothelial cells [74]. It should be noted that because MTHFR /
patients, like CBS / patients, were on Hcy-lowering therapy, their Hcy-
thiolactone concentrations represent minimal values. In fact, in one MTHFR /
patient for whom samples were obtained before therapy, the therapy resulted in
lowering plasma Hcy-thiolactone from 47.3 to 16.6 nM (tHcy was lowered from
208 M after therapy) [93].

A high-Met diet causes elevation of Hcy levels in humans and animals and is
often used as a model of experimental hyperhomocysteinemia [44, 48]. Feeding a
high methionine diet to mice for 6 weeks also increases plasma and urinary Hcy-
thiolactone levels, 3.7- and 25-fold, respectively, compared to mice fed a normal
chow diet. Normal plasma and urinary Hcy-thiolactone levels in mice are 3.7 nM
and 140 nM [93].
Taken together, these findings show that genetic or dietary hyperhomocys-
teinemia increases Hcy-thiolactone levels in human and mice, as predicted by the
Hcy-thiolactone hypothesis (Fig. 6.1 ). A clinical study showing that plasma Hcy-
thiolactone levels are associated with the development and progression of vascular
complications in diabetic patients [362] provides additional support for the Hcy-
thiolactone hypothesis.

μ M before therapy to 66.2 μ

6.1.2 Protein N -Linked Hcy in Hyperhomocysteinemia

The Hcy-thiolactone hypothesis [93] predicts that protein N-homocysteinylation
will be elevated under conditions conducive to atherosclerothrombotic diseases,
such as hyperhomocysteinemia. Indeed, as predicted by the Hcy-thiolactone
hypothesis, protein N-linked Hcy [76, 79, 96, 139] is elevated in human subjects
with hyperhomocysteinemia due to mutations in CBS or MTHFR gene (Tables 3.12
and 5.1 ) [115].
To explain the link between hyperhomocysteinemia and higher cardiovascular
risk and mortality observed in uremic patients, protein N-homocysteinylation was
examined [372]. Significantly higher protein N-linked Hcy levels are found in
uremic patients on hemodialysis than in control subjects [300, 301]. Interestingly,
the ratio of protein N-linked Hcy to tHcy is lower in hemodialysis patients than in
control subjects [300, 301]. Similarly, the ratio of protein N-linked Hcy to tHcy in
patients with high plasma tHcy (50-120

μ M) is lower than in patients with low