Biomedical Engineering Reference
In-Depth Information
biological components, we observe no indications of altered biological processes such
as oxygen consumption rates, signaling events or enzymatic activities, or loss of viabil-
ity of isolated organelles, cells or tissues. However, if biological samples contain lipid
suspensions, the polymer membrane may accumulate a fi ne lipid fi lm layer that may
lengthen response time.
8.6 APPLICATIONS OF POLAROGRAPHIC H
2
S SENSORS IN
BIOLOGICAL SAMPLES
8.6.1 Measurement of H
2
S production
8.6.1.1 Tissue homogenates
H
2
S production in rat aorta homogenate was demonstrated by adding the substrate
L-cysteine (L-cys) and the cofactor pyridoxal phosphate (PLP) for the enzymes cys-
tathionine
β
•synthase (CBS) and cystathionine
γ
lyase (CGL) to dilute homogenate
supernatant in the respirometer chamber at
M O
2
, and recording the rise in PHSS
signal as a function of time [5] (Fig. 8.8). In all experiments, the PHSS was calibrated
prior to and after the experiment by adding either an internal Na
2
S stock standard to
check calibration or a known concentration of
Lucina pectinata
metHb I [46] to scav-
enge H
2
S. H
2
S production rate was determined at the initial steepest slope of each
trace. In all experiments, H
2
S was not produced in the absence of L-cysteine and PLP,
nor was it produced by L-cysteine and PLP in solution alone or by heat-denatured
supernatants or heat-killed cells following addition of substrate (not shown). The CGL
inhibitor
5
µ
-cyano-L-alanine (BCA) at 10 mM was effective in inhibiting aorta H
2
S
production [19, 23].
β
8.6.1.2 Cultured and isolated cells
Cultured rat vascular smooth muscle cells (VSMCs), grown and prepared for respirom-
etry as described in Doeller
et al.
, 2005 [41], were injected into the respirometer cham-
ber to a concentration of between 10
5
and 10
6
cells ml
1
. Cell viability remained at
M O
2
, H
2
S production was stimulated by the
addition of L-cysteine and PLP (Fig. 8.8). The initial H
2
S production rate was approxi-
mately 20% of the rat aorta homogenate rate. H
2
S production rate decreased after the
initial rise in H
2
S concentration, perhaps the result of product feedback inhibition. The
addition of the CGL inhibitor BCA showed an effect similar to aorta homogenate.
Isolated primary rat hepatocytes in the respirometer chamber at 10
6
cells ml
1
exhibited robust H
2
S production, achieving concentrations of
90% throughout experiments. Near 4
µ
M in 30 min as the
O
2
concentration decreased and in the presence of L-cysteine and PLP (Kraus
et al.
,
preliminary data, not shown). Inhibitors of both CBS and CGL (amino oxyacetic acid
and propargylglycine, respectively) inhibited H
2
S production, indicating the presence
of both enzymes in hepatocytes.
20
µ
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