Vol. 274, Issue 5, H1698-H1704, May 1998
Tyrosine confounds oxidative electrochemical detection of
nitric oxide
R.
Stingele1,2,
D. A.
Wilson2,
R. J.
Traystman2, and
D. F.
Hanley1,2
Departments of 1 Neurology and
2 Anesthesiology/Critical Care
Medicine, The Johns Hopkins Medical Institutions, Baltimore,
Maryland 21287
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ABSTRACT |
We report evidence that a porphyrinic
microsensor for detection of nitric oxide (NO) also detects
biologically relevant concentrations of tyrosine (Tyr) in dog brain.
Tyr is oxidized by this sensor at the same potential as NO, and the
sensitivity for NO and Tyr are of the same order of magnitude. The
interference from Tyr is of importance because
1) Tyr is abundant and
2) there is a concentration gradient
of Tyr across the blood-brain barrier that can lead to unpredictable
results if disturbed by ischemia or hypoxia. The knowledge of
this interference is important for the interpretation of results
obtained with this sensor and for the design of future studies.
differential pulse voltammetry; neurohypophysis; dog; methodology
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INTRODUCTION |
THE RECENT SURGE of interest in nitric oxide (NO) has
led researchers to develop and refine techniques for its detection. Most techniques, such as chemiluminescence and arginine-citrulline conversion assay are indirect because they quantify some biological or
chemicophysical effect of NO. Electrochemical detection of NO with
microelectrodes, on the other hand, is a direct technique that yields
concentrations or concentration changes. Therefore, considerable effort
has been made to establish electrochemical methodology to detect NO.
Here we report two related factors that could potentially confound
electrochemical detection of NO: tyrosine (Tyr) and pH. The purpose of
this study is to characterize the influence that these two factors have
on electrochemical detection of NO.
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MATERIALS AND METHODS |
Electrode preparation.
Carbon fiber electrodes were prepared as described previously (11).
Briefly, a 30-µm carbon fiber (Textron Specialty Materials, Lowell,
MA) was inserted into a pulled capillary (Kimax 51, Kimbel), and the
tip was sealed by injection of a small quantity of resin (Epoxylite,
Irvine, CA) from the stem of the capillary with a 25-gauge needle. The
electrodes were baked (30 min at 180°F, 30 min at 300°F), and a
copper wire was glued into the stem of the capillary with two-component
conductive Ag-powder resin (EG-8020, AI Technology) to make electrical
connection to the carbon fiber. The Ag-powder resin was cured at room
temperature for 24 h. Subsequently, the exposed part of the carbon
fiber at the tip of the electrode was cut to 1 mm under a
stereomicroscope and dipped in acetone to remove residues from the
electrode surface. After drying, the carbon surface was
electrochemically coated by cyclic voltammetry in a
nickel-tetrakis-(3-methoxy-4-hydroxyphenyl)porphyrin (Ni-TMHPP) solution as previously described (11). After drying, an
additional coating was applied by immersing the carbon fiber into a
Nafion solution (Aldrich). This negatively charged polymer prevents
nitrite, the negatively charged metabolite of NO oxidation, from
reaching the electrode surface. Reference electrodes for measurements
in vivo were prepared as follows. A 200-µm Teflon-insulated Ag wire (AMS, Everett, WA) was cut, and the insulation was stripped at both
ends. A connector was soldered to one end, and the other end was cut to
an uninsulated length of 1 mm. After cleaning with acetone, the tip was
immersed in a solution of HCl (0.1 M) and NaCl (3 M). A 1.5-V direct
current (DC) was applied to the Ag wire for 2 min, resulting in a
grayish coating of AgCl. The voltage difference of these
pseudoreferences was checked against the Ag/AgCl reference electrode
used for in vitro calibrations (RE5, BAS, West Lafayette, IN) in a 3 M
solution of NaCl. The voltage difference never exceeded 10 mV.
Reference electrodes were stored in a 3 M NaCl solution in the
dark until used.
Differential pulse voltammetry.
Differential pulse voltammetry (DPV) is an alternating-current (AC)
electrochemical technique that has previously been used in vivo for
detection of catechols and metabolites of catecholamines (13). For one
measurement, a voltage profile is applied to the working electrode that
consists of a linear sweep voltage ramp on which is superimposed a
periodic series of rectangular pulses. The current resulting from this
voltage profile is sampled in a short time period just before each
pulse (i1) and
just before the end of each pulse
(i2). The
differential current
(i2 
i1) is plotted
against the DC voltage applied (16). The current-voltage plot
(voltammogram) shows a peak if an electrochemically active substance is
present. The voltage around which the peak is centered (peak voltage)
is characteristic for the substance, and the peak current is
proportional to the concentration of the substance. We used a
polarographic analyzer (PAR-264A, Princeton Applied Research,
Princeton, NJ) for DPV. The voltammograms were recorded using an analog
X-Y plotter (PAR-RE1050, Princeton Applied Research) and digitized
(Jandel Scientific digitizer board, Sausalito, CA). Electrodes were
calibrated in an electrochemical cell filled with 0.1 M PBS that was
degassed by purging with 100% argon for at least 10 min. The
electrochemical cell was warmed to 37°C with a water-jacketed
beaker. Calibrations were obtained with a three-electrode system
consisting of a working electrode, a Ag/AgCl reference electrode (RE5,
BAS) and a Pt wire counterelectrode. For each calibration procedure, a
voltammogram was obtained without test substance and for at least three
concentrations by adding small volumes of concentrated solutions of the
test substance.
NO standard.
NO standard solutions were prepared by bubbling 98.5% NO gas (Aldrich)
through 0.1 M PBS as follows. Under a hood, a system consisting of the
NO gas tank, reduction valves, a gas washing bottle containing 0.1 M
NaOH, and a septum-sealed container for the standard solution was
thoroughly deoxygenated by purging with 100% argon for at least 2 h.
During the degassing procedure, the sample vial was heated three times
to temperatures close to the boiling point using a heat lamp.
Subsequently, the sample vial was cooled to 0°C in an ice-water
bath. Flow of argon was stopped, and NO gas was bubbled through the
system for 10 min. To prevent excess NO gas from escaping into the
atmosphere of the hood, the gas leaving the sample vial through the
outlet cannula was bubbled through a concentrated solution of potassium
permanganate, in which NO was oxidized instantly. Titration experiments
with freshly prepared solutions of oxyhemoglobin showed the NO
concentration in the standard to be 1.8 mM (data not shown). Only
freshly prepared standard solutions (<24 h) were used for
calibrations.
Experimental groups.
Responses of the NO-sensitive microelectrode were studied in two
anatomic regions of dog brain (neurohypophysis and parietal cortex)
after two treatments [nitric oxide synthase (NOS) inhibition with
40 mg NG-nitro-L-arginine methyl
ester (L-NAME)/kg iv and
bilateral intracarotid infusion of
L-Tyr]. This
resulted in four experimental groups: cortex + L-NAME
(n = 5 dogs), cortex + Tyr
(n = 3), neurohypophysis + L-NAME
(n = 5), and neurohypophysis + Tyr
(n = 3). In addition, the response of
the sensor to anoxic anoxia was studied in neurohypophysis (n = 4) and parietal cortex
(n = 4).
Surgery.
Beagle dogs (body wt 9.4 ± 0.3 kg) were anesthetized (pentobarbital
sodium 35 mg/kg iv for induction), intubated, and
ventilated. Level of anesthesia was checked regularly by
assessing the pedal withdrawal reflex, and anesthesia was maintained as
deemed necessary with pentobarbital. Lines were inserted into a femoral
artery and the left cardiac ventricle for reference withdrawal and
injection of radiolabeled microspheres, respectively, for measurement
of cerebral blood flow (CBF). A line inserted into the right
omocervical artery was used for blood pressure monitoring. In those
animals undergoing intracarotid infusion of Tyr, both thyroid arteries were cannulated in a retrograde fashion with the tip of the catheter lodging at the level of the internal carotid artery. A femoral vein
catheter was used for infusion of fluids and muscle relaxant (pancuronium bromide, ~1 mg/h). Bilateral chest tubes
were inserted to reduce oscillations of intrathoracic pressure. This
reduced movement of the brain at the tip of the electrode and hence
motion artifacts. To prevent development of negative intrathoracic
pressure and atelectasis, we ventilated the animals with a positive
end-expiratory pressure of 1-2
cmH2O after insertion of chest
tubes. The head was placed into a frame for craniotomy. In those
animals undergoing cortical implantation of the electrode, a craniotomy
(diameter ~2 cm) was performed over the left parietal cortex after
the skin, galea capitis, and temporal muscle had been
retracted. In dogs undergoing electrode implantation into
neurohypophysis, the skin, galea capitis, and temporal muscle over the
left side of the head were completely removed. An extensive craniotomy
was performed, exposing the temporal lobe down to the base of the
skull. The dura was removed over the exposed brain after coagulation of
meningeal vessels with a bipolar forceps. The temporal lobe was
carefully lifted upwards with a spatula protected by wet cotton pads to expose the lateral aspect of the pituitary gland, the sixth cranial nerve, and the internal carotid artery. Electrodes were implanted under
stereomicroscopic control with micromanipulators. The distance between
reference and working electrode was ~5 mm in parietal cortex and ~3
mm in the pituitary.
In all experimental groups, a stabilization period of at least 60 min
after implantation was allowed before the respective treatment was
administered. The treatments were intravenous infusion of 40 mg
L-NAME/kg or a 5-min bilateral
infusion of a 20 mM solution of
L-Tyr at 7 ml · min
1 · carotid1.
At the end of eight experiments (n = 4 for cortex and n = 4 for
neurohypophysis), anoxic anoxia was started by turning off the
ventilator. Care was taken that anesthesia and muscle relaxation were
sufficient before terminal anoxia was started. DPV was performed repeatedly up to 20 min after onset of anoxia.
Mean arterial blood pressure (MAP) was recorded continuously (Gould,
Cleveland, OH). Arterial blood gases were sampled regularly and
processed by a blood gas analyzer (ABL3, Radiometer, Copenhagen, Denmark). CBF was measured by cardiac injection of radiolabeled microspheres (time points: control and 30 and 60 min after
L-NAME or Tyr administration).
For Tyr administration, an additional microsphere injection was made at
5 min (diameter 15 ± 1.5 µm, DuPont NEN, Boston, MA). After the
experiments, brains were removed and stored in a 10% buffered Formalin
solution for 3 days before processing of the trapped microspheres. The
reference sample technique was used for quantification of blood flow
(4).
In vitro studies.
All electrodes used were calibrated for NO and Tyr by the
method of additions of standards. Three of the electrodes underwent an
extensive characterization with calibrations for NO, Tyr, and nitrite
at four concentrations and four pH values each. The resulting 144 voltammograms were analyzed for peak current and peak potential, and
the sensitivities for the three substrates were calculated at different
pH values. The shift of the peak potential with pH was analyzed for the
three substances.
Statistics.
One-way analysis of variance for repeated measurements was used to test
for significantly different treatments. A
P < 0.05 was considered significant.
To identify significantly different treatments, we used a post hoc
analysis (Bonferroni's test for multiple comparisons vs. a single
control level).
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RESULTS |
In vitro studies.
Table 1 shows the results of the
calibrations of three electrodes with nitrite, NO, and Tyr in PBS at
four pH values. Sensitivities for NO and Tyr were of the same order of
magnitude (2.00 ± 0.20 nA/µM NO and 1.79 ± 0.09 nA/µM Tyr)
at pH = 7.4. The sensitivities for both substances did not change
significantly with pH over the range under investigation (6-7.8).
Sensitivity for nitrite, the metabolite of NO oxidation at the tip of
the electrode, was lower, with 0.43 ± 0.10 nA/µM at pH = 7.4, compared with the sensitivity for NO and Tyr. Sensitivity to nitrite
did not change with pH.
At pH = 7.4, NO and Tyr were oxidized at similar potentials (670 ± 10 mV for NO and 677 ± 8 mV for Tyr; see Fig.
1 for DPV). Nitrite was oxidized at a
higher potential (810 ± 0 mV at pH = 7.4). The peak potentials for
NO and nitrite did not change significantly with pH (see Table 1),
whereas there was a considerable shift of the oxidation potential for
Tyr from 650 ± 0 mV at pH = 7.4 to 760 ± 0 mV at pH = 6.0. To illustrate this shift, the voltammograms for 30 µM Tyr at
four pH values are shown in Fig. 2.
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Fig. 1.
Differential pulse voltammograms (DPV) showing calibration of an
electrode by addition of nitric oxide (NO; 0, 9, 18, and 27 µM;
A) and tyrosine (Tyr; 0, 10, 20, and
30 µM; B). Vertical offsets are
added to subsequent voltammograms to allow display without
overlap. Tyr and NO oxidize at ~680 mV vs. Ag/AgCl. Sensitivities are
comparable.
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Fig. 2.
DPV of a 30 µM solution of Tyr in PBS at 4 pH values. Peaks from
left to
right correspond to pH = 7.8, 7.4, 7.0, and 6.0. Vertical dashed line at +680 mV marks peak potential at
pH = 7.4. Acidic pH leads to oxidation of Tyr at higher voltages. For
explanation of arrow, see
DISCUSSION.
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In vivo physiological parameters.
A summary of physiological parameters is given in Table
2 for the four groups. MAP,
arterial pH, arterial PO2
(PaO2), and arterial
PCO2
(PaCO2) remained stable throughout the
experiment. Because of the relatively long time of exposure of the
brain in the neurohypophysis + L-NAME group, we
performed hyperventilation resulting in mild respiratory alkalosis in
this group. MAP underwent a transient rise after intravenous infusion of L-NAME and was back to
control levels after 30 min in both groups with
L-NAME infusion. Global CBF was
unchanged 30, 60, and 120 min after
L-NAME infusion for all brain
regions investigated (brain stem, cerebellum, right and left
hemisphere, right and left white matter, and right and left cortex)
with the exception of the neurohypophysis. When the electrode was
implanted in cortical tissue, blood flow to the neurohypophysis (NHBF)
was lowered from 278 ± 58 ml · 100 g1 · min1
at control to 59 ± 10 ml · 100 g1 · min1
30 min after L-NAME and remained
at this level 60 and 120 min after
L-NAME
(P < 0.05). For experiments with the
electrode implanted into the neurohypophysis, NHBF was low at control
compared with control values of the groups with cortical implantation
(P < 0.05). Neither
L-NAME nor Tyr infusion led to a
significant decrease of NHBF with the electrode positioned in the
neurohypophysis. Furthermore, right-left differences of blood flow to
white matter and cortex were close to zero with cortical implantation
of the electrode but positive for experiments with a large craniotomy for neurohypophysial implantation (P < 0.05, Mann-Whitney rank-sum test on right-left differences of blood
flow). This indicates a lower blood flow on the side of the craniotomy
with neurohypophysial implantation.
Electrode recordings.
After implantation of the electrode, a stable baseline was reached in
15 min in almost all cases, both for implantation into the cortex and
neurohypophysis. In all animals, the signal was stable during a 30-min
period preceding the administration of L-NAME or Tyr. A typical example
of DPV recorded during the stabilization period is shown in Fig.
3. Neither peak current nor peak potential changed for the last 30 min of the stabilization period (Fig. 3, top
two scans).
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Fig. 3.
Stabilization period after implantation of sensor into parietal cortex.
DPV (from bottom to
top) were obtained 5, 15, 30, and 60 min after implantation.
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In all voltammograms obtained after administration of
L-NAME, including early
measurements just after administration of
L-NAME (not shown), peak current
and peak potential did not change significantly for both
recordings in cerebral cortex and neurohypophysis (see Table 3). A typical set of voltammograms is
shown in Fig. 4 for an electrode implanted
in cortex.
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Fig. 4.
DPV obtained in parietal cortex in a 3-h time period after infusion of
NG-nitro-L-arginine methyl ester
(L-NAME; 40 mg/kg iv) show a
stable peak at +680 mV.
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In the cortex, a bilateral intracarotid infusion of Tyr did not change
peak current and potential significantly (see Table 4). In neurohypophysis, a large increase of
the peak current resulted directly after the start of the
infusion. The peak current was back to preinfusion levels 30 min after
start of infusion. The potential at which the peaks occurred was
between 627 and 657 mV for cortex and between 707 and 743 mV for
neurohypophysis. A typical set of voltammograms for intracarotid
infusion of Tyr is shown in Fig. 5 for
neurohypophysis.
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Fig. 5.
DPV obtained in neurohypophysis before, during, and after a 5-min
intracarotid infusion of a 20 mM Tyr solution. Immediately after start
of infusion, a large peak at +680 mV becomes apparent that slowly
disappears after infusion is stopped. Arrow marks end of infusion. Note
small peak at +680 mV before Tyr infusion is started
(bottom).
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Anoxic anoxia did not change peak current significantly, but peak
voltage shifted from 670 ± 12 mV to 724 ± 10 mV after 20 min
(see Table 5) in four dogs with the
electrode implanted in cortex. DPV obtained in neurohypophysis showed
both a shift of the oxidation potential and an increase of the peak
current with anoxia. The continuous shift of the oxidation potential
over a 15-min period after start of anoxia is shown for neurohypophysis in Fig. 6.
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Fig. 6.
DPV before and during terminal anoxia obtained with an electrode
implanted in neurohypophysis. Peak current and peak potential increase
with anoxia.
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DISCUSSION |
The experiments presented here were originally designed to test the
hypothesis that the unusually high blood flow in the neurohypophysis of
the dog is caused by high NO concentrations in this organ. We had
reason to believe that NO is present in the neurohypophysis from
immunohistochemical stains of rat hypothalamus and pituitary, which
showed abundant NOS (2). In addition, previous experiments in our
laboratory showed a dramatic decrease of neurohypophysial blood flow
after inhibition of NOS (20). Therefore, we chose to use a porphyrinic
microsensor that had previously been shown to detect NO (11).
CBF was lowered 30, 60, or 120 min after
L-NAME in the neurohypophysis
only. This finding can be explained by the abundance of NOS in this
organ. Presumably, high concentrations of NO lead to a sustained
vasodilation in neurohypophysis, and this allows for an efficient
washout of hormones. In experiments with implantation of the electrode
into the neurohypophysis, a large craniotomy was necessary to expose
the site of implantation. The temporal lobe was lifted rostrally with a
padded copper spatula, resulting in some degree of compression of the
exposed left hemisphere and possibly also compression of midline
structures. We therefore believe that the surgical procedure in
experiments with hypophysial implantation is the reason for the low
control NHBF. This argument is also supported by a significantly higher
right-to-left difference of blood flow in experiments with
neurohypophysial implantation compared with experiments with cortical
implantation. In addition, control NHBF was significantly lower in
experiments with large craniotomy compared with cortical implantation
with small craniotomy.
The results show that an electrochemically active substance is detected
at ~680 mV (vs. Ag/AgCl) in the cortex and neurohypophysis. This is
consistent with the potentials of oxidation of NO previously described
for this sensor (11). If the detected peak currents are translated into
concentrations of NO ([NO]) using the sensitivity values
obtained by in vitro calibrations, baseline [NO] of 3.4 ± 1.2 and 1.4 ± 0.1 µM would result in the cortex and
neurohypophysis, respectively.
Inhibition of NOS with 40 mg
L-NAME/kg did not lead to a
decrease of the concentration of the detected substance in the cortex and neurohypophysis. It has been previously shown in our laboratory that this dosage of L-NAME
blocks NOS in dogs to ~25% of the control level within 30 min (17).
We therefore had to question the identity of the detected species. In
the in vivo electrochemical literature, Tyr is reported to be oxidized
between 0.6 and 0.8 mV (vs. Ag/AgCl) (5), which led us to characterize
the response of the porphyrinic microsensor to Tyr.
Tyr is a nutritional amino acid with three dissociable protons. At pH = 7.4, Tyr is present in its neutral and negative forms in a ratio of
100/1 (pKa2 = 9.1) (15). Therefore, diffusion of Tyr to the electroactive surface of
the electrode through the negatively charged Nafion layer is
theoretically possible. Tyr is abundant throughout the body. Typical
concentrations for dogs are 100 µM in blood and 6 µM for cerebral
extracellular space and cerebrospinal fluid (14). This
gradient is maintained by the presence of the blood-brain barrier (BBB)
with specialized transporters for amino acids.
The peak potential of the DPV obtained in the cortex and
neurohypophysis is consistent with detection of both NO and Tyr. Sensitivity of the microsensor for NO and for Tyr are comparable, and
the typical concentrations of Tyr are above the detection limit for
Tyr. Intracarotid infusion of Tyr led to an increase of the peak
current in neurohypophysis but not in cortex. This can be explained by
the different anatomy of the BBB in these regions. In cortex, the BBB
is complete, with tight junctions along the entire perimeter of
endothelial cells. In neurohypophysis, on the other hand, the BBB is
fenestrated (14), leaving gaps for diffusion of Tyr. Because the Tyr
carrier of the BBB is operating close to saturation (14), even a
considerable increase of the Tyr concentration in blood will not lead
to a large increase of the Tyr concentration in the extracellular space
in the presence of a tight BBB as in the case of cortex.
Anoxic anoxia led to a shift of the peak potential to higher voltages
in cortex and neurohypophysis. In addition, in neurohypophysis the
concentration of the detected species also increased with anoxia. Such
a shift towards higher voltages occurs in vitro with decreasing pH for
detection of Tyr but not for NO (in acid environments, the base Tyr
acquires positive charges and more energy is therefore required for
oxidation). A plausible explanation for the potential shift encountered
with anoxia in vivo is that anoxic tissue acidosis leads to oxidation
of Tyr at a higher potential. This is a finding inconsistent with NO
being the substance detected in these experiments. If baseline peak
currents 60 min after implantation of the electrode are translated into
Tyr concentrations, 5.9 ± 1.2 and 5.7 ± 2.0 µM result for
cortex and neurohypophysis, respectively. This is in good agreement
with Tyr concentrations reported for canine cerebral extracellular
space (6 µM; Ref. 14).
Most previous studies (1, 3, 6-12, 18, 19, 21) with the
porphyrinic sensor were done with differential pulse amperometry (DPA).
The advantage of DPA is that it yields quasicontinuous signals
(differential current vs. time), whereas DPV scans a preselected voltage range (here, 0.2-0.9 V) at a preselected scan rate (here, 10 mV/s), resulting in an acquisition time of ~70 s for one
voltammogram. DPA and DPV are closely related techniques. In both
techniques, the voltage profile applied to the working electrode has a
DC and an AC component. The AC component is a series of rectangular pulses (here, 25-mV amplitude, 50-ms duration, and 500-ms duty cycle),
and in both techniques the differential current is measured for each
pulse (see Differential pulse
voltammetry). The difference between
DPA and DPV resides in the DC component of the applied voltage profile.
In DPV, the DC voltage is a linear sweep over the preselected range; in
DPA, the DC voltage is held constant at a preselected value. Therefore,
with DPA, consecutive pulses are identical. In DPA, the DC voltage at
which the differential current is determined is set to a value at which
the substance under investigation is known to be oxidized (for NO,
typical DC voltage settings are 0.65-0.68 V vs. Ag/AgCl). A change
in the analytic signal of DPA can result from a change in the
concentration of the electrochemically active substance or from a shift
of the potential at which the substance reacts. The advantage of DPV is
that it is possible to differentiate between these two possibilities (shift of peak voltage vs. change of peak amplitude), whereas DPA does
not allow this. From Fig. 2 it can be seen that a small change of pH
can result in a considerable change of the differential current at any
voltage if Tyr is detected. For example (see Fig. 2), when the pH
changes from 7.4 to 6.0, a change of 40 nA would result with DPA if the
DC voltage is set to +680 mV (corresponding to the vertical dashed
line), without change in the concentration of Tyr (see arrow). That
such a shift of peak voltage occurs in vivo is documented in Fig. 6. In
protocols with inherent changes of pH (e.g., ischemia/hypoxia)
this could lead to unpredictable results if DPA is used. We believe
that in vivo measurements are particularly affected by this artifact
because of the large quantities of Tyr present in blood and
extracellular space. In the brain, the presence of the BBB separating
two compartments at different Tyr concentrations complicates the
situation even further if the integrity of the BBB is disturbed during
the measurement.
In summary, we propose that the porphyrinic microsensor used here not
only detects NO but also Tyr. We base this on the following observations. 1) Oxidation of Tyr by
this sensor is theoretically possible and occurs in vitro.
2) In vitro, Tyr is oxidized at the
same potential as NO for pH = 7.4. 3) Sensitivities of the sensor for
NO and Tyr are comparable. 4) The
sensor is able to detect biologically relevant concentrations of Tyr.
5) Measured concentrations of Tyr in
cerebral extracellular space are close to reported values.
6) The sensor is able to detect Tyr
infused in carotid artery in neurohypophysial extracellular space.
Observations that argue against NO being the detected species are that
1) NOS inhibition does not decrease
peak current and 2) anoxia shifts
the oxidation potential to higher values. The sensor does detect NO,
but the interference from Tyr can make interpretation of results
difficult or impossible. Especially in protocols using the sensor in
brain tissue undergoing ischemia, anoxia, or acidosis (see,
e.g., Ref. 8), this may yield erroneous data because of BBB damage and
subsequent Tyr inflow from blood and shift of the Tyr oxidation
potential with changes in pH. Appropriate design of
control groups is crucial for work with this sensor to make sure that
effects are caused by changes of [NO] and not [Tyr].
We suggest that DPV should be used instead of DPA if the sensor is
applied to systems known to contain Tyr and/or to undergo pH
changes. Furthermore, in vitro the presence of Tyr should be avoided.
Other techniques using oxidation of NO for its detection should be
tested for this interference. Further research into refinement of the
coating technique with Nafion is necessary and might lead to the
development of a sensor with better selectivity against Tyr.
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ACKNOWLEDGEMENTS |
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Sti 124/1) and the National Institutes of
Health (NS-24282 and HL-48517).
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FOOTNOTES |
Address for reprint requests: R. Stingele, Dept. of Neurology, Univ. of
Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany.
Received 18 August 1997; accepted in final form 28 January 1998.
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Copyright © 1998 the American Physiological Society
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Abstract
Introduction
