Biomedical Engineering Reference
In-Depth Information
and this reactivity limits the usefulness of most standard analytical methods for real-
time H
2
S measurements. Several analytical methods are suffi ciently sensitive to detect
the micromolar to submicromolar H
2
S levels found in biological samples, but they
often require multiple chemical steps and/or chromatographic procedures, and rely
on the conversion of all sulfi de species to H
2
S or S
2
under acidic or alkaline condi-
tions, respectively [37]. Sulfur can exist in several different oxidation states, and in
animal tissues is more commonly found as reduced divalent or fully oxidized hexav-
alent states. Sulfur not liberated by acid or reducing agents such as dithiothreitol is
considered stable. Reduced organic thiols and sulfane sulfur atoms, existing as diva-
lent anions covalently bound to other sulfur atoms, are more easily liberated as inor-
ganic hydrosulfi de anion or hydrogen sulfi de by enzymatic cleavage or mild reducing
conditions [37]. Determination of free H
2
S in biological samples may be infl uenced
by these sulfur sources especially if measurement conditions are not similar to the
physiological conditions of the sample. Many of the standard methods used to meas-
ure free H
2
S rely on sample manipulations to generate products for detection by spec-
trophotometry, fl uorescence, ion-specifi c electrodes or titration. For the most part it is
unclear if these methods also measure H
2
S that existed as a persulfi de or other labile
species.
8.1.2.2 Methods for H
2
S measurements
Measurements of H
2
S production rate, reported for a variety of homogenized tissues
[6, 20, 23, 31, 38], are often performed by placing the homogenized tissue in the outer
well of a fl ask that is fl ushed with N
2
and sealed to limit spontaneous H
2
S oxidation.
A separate center well contains H
2
S-trapping agents such as alkaline zinc acetate. After
a specifi ed time, enzymatic H
2
S production is halted by the addition of trichloroace-
tic acid to precipitate protein and convert any remaining S
2
and HS
to H
2
S but per-
haps also resulting in the uncontrolled liberation of acid-labile H
2
S. The amount of
H
2
S trapped as ZnS at a single time point is determined with an assay that produces a
proportional amount of methylene blue dye, and the H
2
S production rate is calculated
assuming zero H
2
S at time zero. Alternative methods used to determine H
2
S produc-
tion rate or tissue H
2
S levels include gas chromatography following acid or alkaline
extraction, and the silver/silver-sulfi de (Ag
2
S) electrode to measure the concentration
of S
2
in tissue samples placed in a pH 14 sulfi de anti-oxidant buffer [20, 31]. The bare
Ag
2
S electrode in combination with a reference electrode was developed to detect S
2
in solution at alkaline pH, and recent work suggests that it may also report HS
down
to submicromolar concentrations [39]. However, the bare Ag
2
S coating requires daily
reconditioning to remove interfering deposits that accumulate from constituents present
in biologically relevant solutions [39].
With both the acidic and alkaline single time point assays, it remains unclear if acid
labile H
2
S or desulfuration of proteins [40], respectively, creates artifactual overesti-
mates of free H
2
S levels, as described earlier. Tissue H
2
S concentrations determined by
thiol derivatization using bromobimane near neutral pH followed by HPLC also indicate
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