Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 31

( 20) and are negatively correlated with urinary pH. This assay

is now routinely used in studies of the role of Hcy-thiolactone in human [93], mouse
[93, 113, 140, 141], and bacterial [67] physiology.
An alternative method for urinary Hcy-thiolactone determination utilizes a solid-
phase extraction on C18 cartridges, an on-column derivatization with OPA, and
fluorescence detection [220]. The on-column derivatization results in narrower
peaks and shorter run times (3 min), compared with the post-column derivatization
method. The limit of quantification is 20 nM. Using the on-column derivatization,
Hcy-thiolactone concentrations in human urine from 15 subjects were found to vary
from 25 to 297 nM (mean 103 89 nM), similar to the values reported previously
using the post-column derivatization method [95].

0.1-22.6 nM; n ¼

<

3.3.4 Gas Chromatography/Mass Spectrometry Assay

Plasma Hcy-thiolactone can also be assayed by a gas chromatography/mass spec-
trometry (GC-MS) method with deuterated d 4 -Hcy-thiolactone as an internal
standard [225]. Hcy-thiolactone is extracted from the plasma using silica solid-
phase and then derivatized with heptafluorobutyric anhydride. The derivatized
sample is analyzed by GC-MS in NCI mode with methane as the reagent gas and
Hcy-thiolactone ion [M(-)[bond]HF] and the d 4 -Hcy-thiolactone standard are
quantified in a single-ion monitoring mode. The calibration line shows a dynamic
linear range up to 40 nM. Within-day precision (n ¼ 20, nominal concentration

5.2 nM) was 0.96 %, and between-day precision was 3.9 %, with a detection limit of
1.7 nM and quantification limit of 5.2 nM. This procedure has had a very limited
application, being used with only two human plasma samples, in which Hcy-
thiolactone concentration was found to be 18 and 25 nM.

3.3.5 Gold Nanoparticle Homocysteine-Thiolactone Sensor

Gold nanoparticles are known to interact with thiols [226, 227], including Hcy
[228], which results in appearance of new absorption maxima at 610-660 nm.
This principle has been exploited to develop a method for detection and quantifica-
tion of Hcy-thiolactone. For this purpose fluorosurfactant (FSN)-capped gold
nanoparticles (AuNPs) are used as aminothiol removers [227] and as sensors
[229]. Hcy-thiolactone does not bind to the surface of the FSN-AuNPs in the pH
range of 4.0-10.0. In contrast, under these pH conditions, the FSN-AuNPs are
aggregated by Hcy and cysteine. Thus, FSN-AuNPs are effective sorbent materials
for Hcy and cysteine, but not for Hcy-thiolactone. When added to a solution
containing thiols and Hcy-thiolactone, FSN-AuNPs bind

99 %
cysteine. Subsequent removal of FSN-AuNPs particles by centrifugation leaves
Hcy-thiolactone in the supernatant. Treatment of the supernatant with NaOH

98 % Hcy and

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