80 J
determined by ultrafiltration at 388C using Centrifree The operating assumption is that the fraction of the marker
ultrafiltration devices Amicon, Beverley, MA. The con- molecule lost from the dialysate, as it is perfused through
centration of VPA in plasma and brain samples was the probe, is equivalent to the fraction of the analyte
analyzed by a capillary gas chromatographic assay de- recovered from the tissue surrounding the probe. The
scribed by Semmes and Shen [27]. A more sensitive assay, concentration of VPA in the ECF was calculated using the
utilizing mass spectrometry, was performed for the mea- following equation:
surement of subnanogram per ml VPA concentrations in DC
dialysate samples. The dialysate assay used was a modified ]
ECF 5 1
FL version of the assay previously described by Rettenmeier
et al. [25]. Briefly, 10 ml of 1-methyl-1-cyclohexane where DC is the concentration of unlabeled VPA found in
3
carboxylic acid MCCA were added as the internal the dialysate sample and FL is the fraction of [ H]VPA lost
standard to each sample. Extraction of VPA and MCCA from the dialysate. The ICC VPA concentration was
from the dialysate was accomplished by acidifying the estimated using Eq. 2:
sample with 500 ml of 1 N HCl, adding 3 ml of Br 2 fe 3 ECF
chloroform, and shaking for 15 min. The organic phase ]]]]
ICC 5 2
containing the VPA and MCCA was then dried over 1 2 fe
anhydrous magnesium sulfate powder and evaporated to where Br is the concentration of VPA found in the brain
|50 ml under dry nitrogen. The VPA and MCCA were tissue samples and fe is the fractional volume of ECF in
converted to trimethylsilyl TMS derivatives by adding 40 cortical brain tissue, which has been determined to be
ml of bistrimethylsilyltrifluoroacetamide BSTFA and |0.18 [5].
heating to 658C for |40 min. The derivatized samples were Brain tissue-to-plasma concentration ratios of VPA were
then diluted to |100 ml with chloroform and analyzed by computed using the average total or free plasma con-
gas chromatography mass spectrometry GC MS. centration of unlabeled VPA at steady state. The steady-
Quantitative GC MS
analyses of
VPA-TMS and
state plasma concentration in each rabbit was computed by MCCA-TMS derivatives were performed on a Carlo Erba
averaging concentrations of samples taken from 90 min 8000 gas chromatograph Fisons Instruments, Manchester,
through 210 min. The mean steady-state dialysate and ECF UK interfaced to a VG MD 800 Quadrupole mass
values were computed by averaging dialysate samples and spectrometer VG BioTech, Cheshire, UK using a fused
the corresponding ECF values from 90 min through 240 silica capillary column 60 m30.32 mm ID30.25 mm film
min, respectively. The steady-state ICC concentration of thickness coated with a DB-1 stationary phase JW
VPA for each rabbit was calculated using the mean ECF Scientific, Folsom, CA. The temperature of the ion source
concentration and concentration of the brain tissue sur- was 2008C. The temperatures at the injector and the GC
rounding the dialysis probe sampled at the termination of interface were held at 2508C. Helium was used as the
the experiment. carrier gas, at a head pressure of 15 psi. The initial oven
All data are expressed as means6S.E. Data at serial temperature was 408C. After 1 min, the temperature was
time points from the control and probenecid treatment raised rapidly to 808C. The temperature was then raised
groups were analyzed by repeated measure ANOVA, from 80 to 1208C at 28 min, followed by a 408 min
followed by post-hoc paired-wise comparisons. Statistical gradient to 2508C. Molecular ions with mass-to-charge
significance was defined as P 0.05. ratios of 201 and 199 were monitored that corresponded,
respectively, to VPA and MCCA. Under these chromato- graphic conditions, VPA-TMS and MCCA-TMS eluted at
|15.7 min and 16.7 min, respectively. The concentration
3. Results
of VPA in samples was determined by comparing VPA-to- internal standard peak area ratios with standard curves,
3.1. Physiologic data which were generated from the extracts of VPA standards.
3
Radioactivity of [ H]VPA in dialysate samples was Table 1 compares the results of the physiologic data
assayed by liquid scintillation counting using a TriCarb collected from the control and probenecid-treatment
2000CA Packard Instrument, Downers Grove, IL. groups. Between-group comparisons indicated that there
were no significant differences in any of the physiological 2.7. Data analysis
parameters monitored. Within-group comparisons indicated a time-related decrease of blood pH and bicarbonate
The method of retrodialysis was used to determine concentration in the probenecid group. Post hoc paired-
relative recovery of each probe. This method typically wise comparisons of the data within the probenecid group
includes, in the perfusing dialysate, either a marker mole- indicated that only the mean pH at 220 min decreased
cule of similar molecular weight and structure to that of significantly from baseline value.
the analyte, or radiolabeled analyte, as in the present study. The EEG data are displayed in Table 2. Baseline mean
J .L. Scism et al. Brain Research 884 2000 77 –86
81 Table 1
a
Comparison of physiologic parameters between the control and probenecid treatment groups n 55 in each Group time
Mean arterial Heart rate
PaO PaCO
pH Arterial
Rectal
2 2
blood pressure beats min
mmHg mmHg
bicarbonate conc. temperature
mmHg mEq l
Control Baseline
7465 280615
18063 3161
7.3960.02 18.761.0
38.160.2 60 min
7765 267610
18165 3362
7.3760.02 19.061.4
38.160.1 110 min
7364 27169
18064 3161
7.3860.02 17.960.7
38.060.1 170 min
7763 26267
18165 3261
7.3660.01 17.861.0
38.160.1 220 min
7863 27466
18165 2961
7.3760.01 16.760.6
38.160.1 Probenecid
b b
Baseline 7762
287616 17964
3262 7.4360.01
20.861.4 37.460.4
b b
60 min 8465
294612 18162
3262 7.3860.02
18.360.9 37.560.3
b b
110 min 8064
300611 18463
3361 7.3260.02
16.861.0 37.760.3
b b
170 min 7763
290616 18163
3162 7.3060.02
15.160.9 37.660.3
b,c b
220 min 6763
31265 18162
2962 7.2660.03
13.060.7 37.760.3
a
Values represent mean6S.E.
b
Significant variation with respect to time, P ,0.001.
c
Significantly different from baseline mean value, P ,0.05.
value in the control group was not different from that in representative control rabbit. Plasma VPA concentration
the PBD group; therefore, they were combined for the reached steady state within the first 25 min of the study
purpose of statistical analysis. Administration of VPA or and remained constant through 240 min. Cerebral dialysate
VPA plus probenecid decreased power in the delta and VPA concentration reached steady state by 50 min, and
theta frequency bins at the end of the study as compared to remained relatively constant throughout the study.
baseline values. Moreover, probenecid co-infusion in- creased power and number of delta waveforms, and
3.3. Microdialysis probes decreased number of theta waveforms in comparison to
3
values in the control group. The mean fractional loss of [ H]VPA during re-
trodialysis in vivo was 0.2860.014 for the control group 3.2. VPA pharmacokinetics
and 0.2660.014 for the probenecid treatment group data not shown. The two mean values do not differ sig-
Fig. 1 illustrates the concentration–time profile of VPA nificantly, indicating that the probes used in each group
in the plasma and dialysate during VPA infusion for one had comparable dialytic characteristics.
Table 2
a
Electroencephalographic activity in the control and probenecid treatment groups Frequency bin
Combined baseline Values at the end of the
values n 510 experimental period
Control Probenecid
n 55 n 55
Delta
3 b
c c
Power, mV310 min 22.467.1
6.161.9 11.261.3
c c
Waveform, number min 4769
4168 7269
Theta
3 b
Power, mV310 min 49.4614.1
19.964.9 10.363.7
c c
Waveform, number min 150623
169625 84621
Alpha
3
Power, mV310 min 9.363.1
3.561.1 3.060.7
Waveform, number min 9363.1
90612 89616
Beta
3
Power, mV310 min 4.96.2.1
1.760.7 2.060.7
Waveform, number min 88632
91623 152648
3
Total power, mV310 min 87.2624.3
31.267.9 26.662.9
Activity edge, Hz 1263
1261 1362
a
Values represent mean6S.E.
b
Significant difference from experimental period, P ,0.05.
c
Significant difference between groups, P ,0.05.
82 J
Fig. 1. Typical time course of VPA concentration in the plasma and cerebral dialysate during a cerebral cortex microdialysis experiment in a control rabbit. At time zero, an i.v. priming dose 7.5 mg kg was administered and immediately followed by a constant rate infusion 125 mg kg min that was
maintained throughout the experiment. The respective mean6S.E. of steady-state plasma and dialysate concentration from 90 min to the end of the study was 52.862.9 mg ml and 0.5960.06 mg ml.
The relative recovery of VPA in vitro was 0.4160.04 for steady-state VPA concentration was 63.666.1 mg ml,
probes used in control animals, and was not significantly which was not significantly different from that in the
different from the relative recovery for probes used in control group. Determination of the plasma protein binding
probenecid-treated animals 0.3860.03. Our fractions of of VPA for both groups showed that the unbound fraction
VPA recovery in vitro are comparable to the in vitro VPA of VPA in the plasma was not significantly different
value reported by Golden et al. [9] using microdialysis between
control and
probenecid treatment
groups, probes of similar construction to those used in this study.
0.1960.03 versus 0.2360.02. The mean steady-state con-
3
The loss of [ H]VPA in vitro was 0.4660.04 for probes centration of unbound VPA in the plasma was calculated to
used in control animals, which was not significantly be 11.362.2 mg ml for control animals and 14.962.3
different from the value for probes used in probenecid- mg ml for probenecid-treated animals. The difference
treated animals 0.4260.01. The in vitro relative recovery between the group means was not statistically significant.
value for VPA was not statistically different from the in The mean steady-state tissue concentration of VPA at the
3
vitro loss of [ H]VPA. This was in keeping with the probe site was lower than in plasma in both groups of
operating assumption of retrodialysis, i.e. the fractional animals. Co-treatment with probenecid resulted in a greater
loss of tracer molecule is equivalent to the fractional than twofold increase in brain tissue VPA concentration,
3
recovery of analyte. The relative loss of [ H]VPA in vivo from 4.1560.24 mg g to 9.3461.37 mg g. Steady-state
was |60 of the in vitro loss, most likely due to restricted
Table 3
diffusion of VPA in tissue as compared to water; this is
Comparison of steady-state VPA concentrations in plasma, dialysate, and
a
commonly observed in microdialysis [23].
brain tissue between control and probenecid treatment groups Control
Probenecid
3.4. Plasma, brain, and dialysate distribution of VPA
n 55 n 55
Plasma mg ml 60.463.5
63.666.1
A comparison of the mean steady-state concentrations of
Free fraction 0.1960.03
0.2360.02
VPA in plasma, dialysate, and brain tissue between the
Free plasma mg ml 11.362.2
14.962.3
b
control and probenecid treatment groups is presented in
Brain tissue mg g 4.1560.24
9.3461.37
b
Dialysate mg ml 0.4960.04
0.7260.07
Table 3. An average steady-state plasma VPA concen-
a
tration of 60.463.5 mg ml was reached in the control
Values represent mean6S.E.
b
animals. With concurrent i.v. infusion of probenecid,
Significantly different from control, P ,0.05.
J .L. Scism et al. Brain Research 884 2000 77 –86
83
VPA concentration in the dialysate was much lower calculated and was found to be somewhat higher for
compared to either plasma or brain tissue in control probenecid-treated animals 0.04460.0048 than for con-
animals 0.4960.04 mg ml, and increased significantly by trol animals 0.02960.0026. However, when plasma
probenecid treatment 0.7260.07 mg ml. protein binding was taken into account, difference in the
mean ECF-to-free plasma VPA concentration ratio between 3.5. Estimated ECF and ICC VPA concentrations
the control group 0.1760.034 and the probenecid group 0.1960.015 was no longer statistically significant.
The estimated mean steady-state concentrations of VPA The ICC-to-ECF VPA concentration ratio was calculated
in the ECF and the ICC in the control and probenecid to examine the VPA concentration gradient between the
groups are shown in Table 4. The ECF VPA concentration, brain ECF and ICC. On average, the control animals had
calculated directly from the dialysate VPA concentration, an ICC VPA concentration that was 2.8160.28 times
was 1.7260.16 mg ml for control animals, and was higher than the extracellular VPA concentration. Treatment
significantly higher in the probenecid-treated animals, at with probenecid increased this ratio to 4.2460.44.
2.7860.36 mg ml. The mean estimated ICC VPA con- The ICC-to-free plasma VPA concentration ratio, which
centration for control animals was 4.6960.27 mg ml, serves as an index of intracellular delivery, was computed
which was almost threefold higher relative to the ECF to gain an appreciation of the overall effect of sequential
VPA concentration. Treatment with probenecid signifi- exchange processes at both the parenchymal cell mem-
cantly increased the mean ICC VPA concentration to brane and the BBB. The ICC-to-free plasma VPA con-
11.661.6 mg ml. centration ratio was 0.4660.068 for control animals and
Mean concentration ratios of brain tissue-to-plasma, was significantly increased by probenecid treatment to
brain tissue-to-ECF, ECF-to-plasma, ICC-to-plasma, and 0.8160.10.
ICC-to-ECF are also presented in Table 4. The mean ratio of VPA concentration in the cerebral cortical to that in total
plasma was well below unity for both the control group
4. Discussion
