N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 95

determination of N-linked Hcy in ferritin is illustrated in Fig. 5.14b . Hemoglobins
from a variety of species (baboon, bovine, horse, pig, rat, mouse, and chicken)
contain more protein N-linked Hcy than did corresponding albumins
(0.0127-0.0828 vs. 0.0027-0.0086 mol/mol). Normal human plasma and milk
were found to contain sub-micromolar concentrations of protein N-linked Hcy,
whereas cow milk and whey contained micromolar concentrations of protein N-
linked Hcy. This assay has been extensively used in the studies of plasma and tissue
N-Hcy-protein in genetic deficiencies in folate and Hcy metabolism in humans
[115] and mice [113] as well as in studies of physiological roles of paraoxonase 1
and bleomycin hydrolase using Pon1-null [140] and Blmh-null [141] mice.
An alternative HPLC assay for protein N-linked Hcy uses tris(2-carboxyethyl)
phosphine (TCEP) for S-linked Hcy reduction and removal and on-column deriva-
tization with alkaline OPA and fluorescence detection [220]. The method's limit of
quantification for protein N-linked Hcy is 0.25 M. The utility of the method was

demonstrated with plasma samples from 18 subjects, in whom protein N-linked Hcy
varied from 1.6 to 3.3

μ

μ M (mean ¼ 2.52 μ M) and tHcy concentrations varied from

7.7 to 134 μ M.

5.5.2.2 Derivatization with 4-Fluoro-7-sulfamoyl-benzofurazan

Two other N-Hcy-protein assays rely on pre-column derivatization with other
fluorescent thiol reagents. In one assay, thiols in plasma protein are derivatized
with 4-fluoro-7-sulfamoyl-benzofurazan. The derivatized proteins are then acid-
hydrolyzed, and the liberated derivatized Hcy is quantified by C18 reversed-phase
HPLC and fluorescence detection [300]. The method was applied to 35 plasma
samples from 20 healthy adults and 15 hemodialysis patients. The mean
concentrations for control group were 0.51 0.11 μ M for protein N-linked Hcy

and 13.9 5.8 μ M for tHcy. In 15 hemodialysis patients, the mean concentrations

were 0.74 M for tHcy. The

peptide fraction containing protein N-linked Hcy accounts for 4.2 % of tHcy in
healthy adults and 2.0 % in hemodialysis patients.

0.20 μ M for protein N-linked Hcy and 47.2 26.1 μ

5.5.2.3 Derivatization with 7-Fluorobenzo-2-Oxa-1,3-Diazole-4-Sulfonate

In another assay [301], plasma protein N-linked Hcy is quantified by HPLC after
sample reduction with tri-n-butyl-phosphine, gel filtration, derivatization with
ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, and acid hydrolysis.
Using this assay, it is found that protein N-linked Hcy and S-linked Hcy in uremics
are significantly elevated relative to control subjects. The protein N-linked Hcy and
tHcy concentrations in uremic patients were 0.86 0.14 μ M and 69.38 15.15

M, respectively. After folate therapy, protein N-linked Hcy in uremic patients is
normalized to the levels observed in healthy subjects (0.40

μ

0.06 μ M), whereas

S-linked Hcy is lowered (to 26.01 3.80 μ M) but remains elevated relative to

control subjects (11.35 1.03 μ M).