Kinetic study of benzyl [1-¹⁴C]acetate as a potential probe for astrocytic energy metabolism in the rat brain: Comparison with benzyl [2-¹⁴C]acetate

Abstract

Brain uptake of [¹⁴C]acetate has been reported to be a useful marker of astrocytic energy metabolism. In addition to uptake values, the rate of radiolabeled acetate washout from the brain appears to reflect CO₂ exhaustion and oxygen consumption in astrocytes. We measured the time–radioactivity curves of benzyl [1-¹⁴C]acetate ([1-¹⁴C]BA), a lipophilic probe of [1-¹⁴C]acetate, and compared it with that of benzyl [2-¹⁴C]acetate ([2-¹⁴C]BA) in rat brains. The highest brain uptake was observed immediately after injecting either [1-¹⁴C]BA or [2-¹⁴C]BA, and both subsequently disappeared from the brain in a single-exponential manner. Estimated [1-¹⁴C]BA washout rates in the cerebral cortex and cerebellum were higher than those of [2-¹⁴C]BA. These results suggested that [1-¹⁴C]BA could be a useful probe for estimating the astrocytic oxidative metabolism. The [1-¹⁴C]BA washout rate in the cerebral cortex of immature rats was lower than that of mature rats. An autoradiographic study showed that the washout rates of [1-¹⁴C]BA from the rat brains of a lithium–pilocarpine-induced status epilepticus model were not significantly different from the values in control rat brains except for the medial septal nucleus. These results implied that the enhancement of amino acid turnover rate rather than astrocytic oxidative metabolism was increased in status epilepticus.

Introduction

Labeled acetate has been widely used in radiometric^1–3 and magnetic resonance spectroscopy (MRS)^4–8 studies of astrocytic energy metabolism. For example, ¹³C-labeled acetate has been used in studies of metabolic trafficking between neurons and astrocytes (including the glutamate–glutamine cycle).^4–8 The MRS technique has many advantages for determining chemical forms of various intermediates produced during oxidative metabolism in astrocytes. In addition, MRS can provide a high potential boost in analysis of detailed mapping of the metabolic pathways when used with gas chromatography–mass spectrometry. However, MRS itself requires the infusion of large amounts of [¹³C]acetate, which may affect normal physiological conditions.^9,10

On the other hand, ¹⁴C-labeled acetate is used to measure astrocytic energy metabolism in rats and mice with trace amounts of substrates,¹¹ although chemical analysis of labeled products in the brain is limited.

Clarke et al. have reported that fluorocitrate and fluoroacetate inhibit astrocytic metabolism.¹² We previously reported that fluorocitrate significantly inhibited the accumulation of [¹⁴C]acetate in the rat brain in a dose-dependent manner.¹³ We also showed that the brain uptake of [¹⁴C]acetate significantly decreased in a very short-term (e.g. 5 min) ischemia–reperfusion injury rat model.¹⁴ In contrast, the brain uptake of [¹⁴C]acetate significantly increased in a lithium–pilocarpine-induced status epilepticus (SE) rat model, and this phenomenon was not dependent on increased regional cerebral blood flow or glucose utilization.¹⁵ These results indicated that the brain uptake of [¹⁴C]acetate could be a useful index for reflecting astrocytic oxidative metabolism.

Wyss et al. have determined the kinetics of [1-¹¹C]acetate in rat and human brains using β-scintillator and positron emission tomography, respectively, and found that the [1-¹¹C]acetate washout rate in the somatosensory cortex is significantly increased by infraorbital nerve stimulation.¹⁶ The authors suggested that the [1-¹¹C]acetate washout rate from the brain reflects astrocytic [¹¹C]CO₂ production. Therefore, the ¹¹C- or ¹⁴C-labeled acetate washout rate is a potentially useful indicator for estimating astrocytic energy metabolism. Recently, we improved the brain uptake of [¹⁴C]acetate using the highly lipophilic probe benzyl [1-¹⁴C]acetate ([1-¹⁴C]BA).¹⁷ The brain uptake of [1-¹⁴C]BA depends on the cerebral blood flow and hydrolytic process. Because [1-¹⁴C]BA is rapidly hydrolyzed to [1-¹⁴C]acetate in the brain and [1-¹⁴C]acetate selectively enters astrocytes, the uptake of [1-¹⁴C]BA in rat brain was significantly decreased by fluorocitrate.¹⁷

Acetate labeled at carbon position 1 or 2 has been used to measure astrocytic energy metabolism.^18–23 Both [1-¹⁴C]acetate and [2-¹⁴C]acetate are metabolized to [¹⁴C]CO₂ and also ¹⁴C-labeled amino acids, such as glutamine, glutamate, and gamma-aminobutyric acid (GABA). Almost all C-1-labeled carbon were released from the brain within the second turn of the tricarboxylic acid (TCA) cycle, whereas C-2-labeled carbon was eliminated gradually, beginning during the third turn of the TCA cycle.^24,25 This finding indicates that C-1-labeled acetate seems to be a more suitable probe than C-2-labeled acetate for measuring washout rates from the brain. In this study, we measured and compared the kinetics and washout rates of [1-¹⁴C]BA and [2-¹⁴C]BA in rat brain. In addition, we compared the washout rates of [1-¹⁴C]BA in mature and immature rats. A simplified two-time-point quantitative autoradiography method was used to estimate the radioactivity washout rate of [1-¹⁴C]BA and compare it in SE model rats with that in control rats.

Materials and methods

Radioprobes

We obtained [1-¹⁴C]acetic acid, sodium salt ([1-¹⁴C]acetate) and [2-¹⁴C]acetic acid, sodium salt ([2-¹⁴C]acetate) from PerkinElmer Life Science Inc. (Boston, MA). [1-¹⁴C]BA was prepared as described previously.¹⁷ Briefly, benzyl alcohol (Nacalai Tesque, Inc.; Kyoto, Japan) and [1-¹⁴C]acetate were mixed with dicyclohexylcarbodiimide (Nacalai Tesque, Inc.) for 24 h at room temperature, followed by thin-layer chromatographic purification. [2-¹⁴C]BA was obtained using the same method with [2-¹⁴C]acetate instead of [1-¹⁴C]acetate.

Animals

All animal experiments were performed according to the local regulations of the Graduate School of Medicine of Osaka University. The local Experimental Animal Committees approved the animal experimental protocols. All studies involving animals were performed in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

We purchased male Wistar rats from Japan SLC (Shizuoka, Japan). The rats were housed in a temperature- and humidity-controlled room (about 23℃ and 50%, respectively) under a 12-h light cycle and allowed free access to food and water. All rats were randomly assigned to groups. Sample sizes were calculated according to previous reports,^15,24 with α and β errors at 0.05 and 0.2, respectively, via a priori power analysis performed using G Power.²⁶

Animal model of SE

SE was induced using lithium and pilocarpine.^15,27 Rats (8-week-old, 195–205 g, n = 13) were administered lithium chloride (127 mg/kg, dissolved in distilled water; Wako Pure Chemical Industries, Ltd., Osaka, Japan) via intraperitoneal injection, followed by an intraperitoneal injection of pilocarpine hydrochloride (30 mg/kg, dissolved in distilled water; Nacalai Tesque, Inc.) 21 h later. We monitored the behavioral changes of these experimental rats for 90 min and classified them according to Racine stage.²⁸ This classification includes six stages: Stage 0, normal activity; Stage 1, mouth and facial movements and hyperactivity; Stage 2, head nodding; Stage 3, forelimb clonus; Stage 4, forelimb clonus and rearing; and Stage 5, rearing and falling. We used rats (n = 8) classified higher than Stage 4 for further studies. Control rats were (n = 8) injected with lithium chloride and the same volume of distilled water instead of pilocarpine hydrochloride.

Kinetics of [1-¹⁴C]BA and [2-¹⁴C]BA in rat brain

Rats (8-week-old, 185–220 g) were intravenously administered [1-¹⁴C]BA (approximately 39.4 kBq, n = 20) or [2-¹⁴C]BA (approximately 72.1 kBq, n = 22) and were decapitated under isoflurane at various time intervals (1, 5, 10, 20, and 30 min) after radioprobe administration. The brains were quickly removed, dissected into three parts (cortex, cerebellum, and pons), and weighed. The extracted brain tissues were mixed with a solubilizer (Soluene-350; Packard Instruments Co. Inc./PerkinElmer, Inc., Waltham, MA) and counted using a liquid scintillation counter (LS6500; Beckman Coulter, Inc., Brea, CA). Fifty microliters of blood obtained from each [1-¹⁴C]- or [2-¹⁴C]BA-injected rat were mixed with 2 mL of ethyl acetate. The organic fraction was counted using the liquid scintillation counter to measure unmetabolized radioprobes.

All radioactivities were expressed as a percentage of the injected dose per gram of tissue (%ID/g), and means ± standard deviations were plotted on a semi-logarithmic plot. The radioactivities were fitted by a monoexponential curve, A_t = A₀e^−kt, where A_t is radioactivity in the brain at time t, A₀ is extrapolated initial concentration (intercept), and k is washout rate constant. The washout rate values were determined from the slopes (k) of the lines. Immature rats (3-week-old, 43.4–53.9 g, n = 20) were also used in the [1-¹⁴C]BA study (approximate injected dose: 28.5 kBq), and radioactivity was expressed as a differential absorption ratio [DAR: %ID/g × body weight (g)/100] to allow comparison with mature (8-week-old) rats.

Simplified two-time-point autoradiography

Control (8-week-old, 195–216 g, n = 8) and SE model (8-week-old, 200–205 g, n = 8) rats were intravenously administered [1-¹⁴C]BA (139–207 kBq) and sacrificed under isoflurane 3 or 30 min later. The brains were quickly removed and frozen to obtain coronal brain slices (thickness: 20 µm). The brain slices were exposed to an imaging plate with a ¹⁴C-standard scale (RPA 511; Amersham Biosciences/GE Healthcare) for one week. Autoradiograms were obtained using a bioimaging analyzer system (FLA-7000; Fujifilm/GE Healthcare Japan, Tokyo, Japan). Regions of interest were manually placed on the images, and the obtained photo-stimulated luminescence values per area were calibrated to %ID/g according to the standard scale.

Statistical analysis

All of the data were analyzed by at least two people (one of whom did not acquire the raw data). The analyzed data obtained from the investigators were similar. Statistical differences in brain uptake between different radioprobes ([1-¹⁴C]BA versus [2-¹⁴C]BA) and different rat groups (mature versus immature rats and control versus SE model rats) were detected using Student’s or Welch’s t-test at each time point. Regional differences in the brain were detected using a one-way ANOVA and post-hoc Tukey–Kramer test at each time point. Comparisons of washout rates (mature versus immature rats and control versus SE model rats) were assessed by performing analysis of covariance.

Results

Kinetics of [1-¹⁴C] and [2-¹⁴C]BA radioactivity in mature rat brain

Time–radioactivity curves of [1-¹⁴C] and [2-¹⁴C]BA in mature rat brain are shown in Figure 1. The maximum [1-¹⁴C]BA and [2-¹⁴C]BA radioactivity levels in the cortex, cerebellum, and pons were observed 1 min post injection; these levels decreased in a single-exponential manner (R²> 0.81), and the slopes of the lines appeared to indicate the washout rate. The radioactivity level of [2-¹⁴C]BA was significantly higher than that of [1-¹⁴C]BA in all three regions beginning 10 min after administration (p < 0.05 in each region except for the cortex at 20 min). We did not consider the radioactivity in the cerebral blood; therefore, the actual radioactivity in the brain tissue was thought to be slightly lower than the values measured in this study. However, the measurement values did not affect the estimation of the washout rates, because of only 3% of blood volume in the brain as well as lower level of radioactivity in blood. Because it is difficult to identify the chemical forms of ¹⁴C, we measured only unmetabolized [1- or 2-¹⁴C]BA in blood using an organic solvent extraction method. The radioactivity level of unmetabolized [2-¹⁴C]BA was also higher than [1-¹⁴C]BA in the blood (Figure 1d). The estimated washout rates of [1-¹⁴C] and [2-¹⁴C]BA in the various brain regions are shown in Table 1. The washout rate of [1-¹⁴C]BA in the cerebral cortex was significantly higher than that of [2-¹⁴C]BA (p < 0.01); however, the washout rates in the cerebellum and pons were similar.

Figure 1.

The time–radioactivity curves of [1-¹⁴C]BA(•) and [2-¹⁴C]BA(▴) in the cortex (a), cerebellum (b), and pons (c) and of the parent compound in the blood (d) (means ± standard deviations; n = 4–5/group) %ID/g: % of injected dose per g of tissue. The brain radioactivity was derived from brain tissue included cerebral blood. *:p < 0.05, **p < 0.01 between [1-¹⁴C]BA and [2-¹⁴C]BA using Student’s t test at each time point.

Table 1.

Comparison of the washout rate of [1-¹⁴C]BA and [2-¹⁴C]BA (min⁻¹).

	Cortex	Cerebellum	Pons
[1-14C]BA	0.043 ± 0.006	0.031 ± 0.005	0.028 ± 0.005
[2-14C]BA	0.013 ± 0.003	0.022 ± 0.002	0.027 ± 0.003
p*	4.5 × 10−5	0.100	0.886

Data are calculated from time–radioactivity curves (%ID/g), and are expressed as the mean ± standard error.

^*Statistical significance was determined by performing analysis of covariance.

Kinetics of [1-¹⁴C]BA in mature and immature rats

The cerebral radioactivity of [1-¹⁴C]BA in immature rats decreased in a single-exponential manner (Figure 2), similar to that observed in mature rats (Figure 1). The cortical and cerebellar radioactivity levels were lower in immature rats than in mature rats. The [1-¹⁴C]BA washout rates in mature and immature rats are shown in Table 2; these values were lower in immature rats than in mature rats in all regions, despite the lack of statistically significant differences.

Figure 2.

The time–radioactivity curves of [1-¹⁴C]BA in the cortex (a), cerebellum (b), and pons (c) and of the parent compound in the blood (d) of mature (8-week-old: •) and immature (3-week-old: ▴) rats (means ± standard deviations; n = 4/group). DAR: % of injected dose/g tissue × body weight (g)/100. The brain radioactivity was derived from brain tissue included cerebral blood. *p < 0.05, **p < 0.01 between mature and immature rats using Student’s t test at each time point.

Table 2.

Comparison of the washout rates of [1-¹⁴C]BA in mature and immature rats (min⁻¹).

	Cortex	Cerebellum	Pons
8 weeks old	0.042 ± 0.006	0.030 ± 0.005	0.027 ± 0.005
3 weeks old	0.029 ± 0.004	0.019 ± 0.002	0.022 ± 0.003
p*	0.056	0.068	0.372

Data are calculated from time–radioactivity curves (DAR), and expressed as the mean ± standard error.

^*Statistical significance was determined by performing analysis of covariance.

Simplified two-time-point autoradiography

The kinetics data showed that the radioactivity of [1-¹⁴C]BA in the normal rat brain decreased in a single-exponential manner. To reduce the number of experimental animals required, a simplified two-time-point quantitative autoradiographic method was used to estimate the radioactivity washout rate following the injection of [1-¹⁴C]BA into SE model and control rats. Typical [1-¹⁴C]BA autoradiograms obtained from control and SE model rats are shown in Figure 3. To account for regional differences in radioactivity distribution, we placed regions of interest in the cingulate cortex, sensory cortex, motor cortex, retrosplenial cortex, cortex (entire region of cortex, including the above-mentioned segments), striatum, medial septal nucleus, hippocampus, thalamus, cerebellum, and pons. The radioactivity levels in the retrosplenial cortex, medial septal nucleus, and thalamus were higher in SE model rats than in control rats. The washout rates in these regions are shown in Table 3. There were no significant differences of the values between SE model and control rats in all regions studied except for the medial septal nucleus.

Figure 3.

Typical quantitative autoradiograms of [1-¹⁴C]BA at early (3 min) and late (30 min) phases in control and SE model rats.

Table 3.

The washout rates of [1-¹⁴C]BA in control and status epilepticus model rats (min⁻¹).

	Control	SE	p*
Cingulate cortex	0.041 ± 0.005	0.046 ± 0.008	0.562
Sensory cortex	0.042 ± 0.003	0.056 ± 0.007	0.084
Motor cortex	0.037 ± 0.006	0.050 ± 0.007	0.167
Retrosplenial cortex	0.045 ± 0.005	0.048 ± 0.006	0.637
Whole cortex	0.038 ± 0.003	0.047 ± 0.006	0.186
Striatum	0.029 ± 0.004	0.031 ± 0.004	0.781
Medial septal Nucleus	0.028 ± 0.005	0.048 ± 0.005	0.010
Hippocampus	0.026 ± 0.004	0.034 ± 0.006	0.266
Thalamus	0.034 ± 0.004	0.043 ± 0.005	0.198
Cerebellum	0.028 ± 0.003	0.035 ± 0.005	0.223
Pons	0.028 ± 0.004	0.030 ± 0.004	0.663

Data are expressed as the mean ± standard error.

^*Statistical significance was determined by performing analysis of covariance.

Discussion

Acetate is a well-known potential agent for monitoring astrocytic energy metabolism because it is only taken up by astrocytes. Acetate enters the TCA cycle after conversion to acetyl-CoA by acetyl-CoA synthetase, followed by normal oxidation. Various MRS and radiometric kinetic studies of acetate in brain have been conducted.^1,5 The results of these studies demonstrated that acetate is metabolized into amino acids (e.g. glutamate, glutamine, aspartate, and GABA). In particular, glutamate and glutamine are produced during the glutamate–glutamine cycle from α-ketoglutarate, an intermediate metabolite of the TCA cycle. We previously found that [1-¹⁴C]BA is converted to [¹⁴C]glutamate and [¹⁴C]glutamine in rat brain within 10 min after injection.¹⁷ On the other hand, [¹⁴C]acetate-derived radioactivity decreased as [¹⁴C]CO₂ is expelled from the TCA cycle. Wyss et al. suggested that this decrease in brain radioactivity resulted from the release of [¹¹C]CO₂ and the washout rate was related to oxygen consumption.¹⁶ This suggestion was based on a well-established method using [1-¹¹C]acetate for measuring myocardial oxygen consumption.²⁹ In the present study, we compared the kinetics of C-1 - and C-2-labeled BA, a lipophilic probe of acetate, in rat brain. The results showed that the radioactivity in rat brain associated with [¹⁴C]BA decreased in a single-exponential manner regardless of the labeling positions. The disappearance rate of [1-¹⁴C]BA in the cerebral cortex was significantly faster than that of [2-¹⁴C]BA. The acetate carbons were oxidized through different pathways in TCA cycle. C-2 (methyl) radiolabeled acetate was retained longer in brain (in TCA cycle intermediates or amino acids) relative to C-1 (carbonyl) radiolabeled acetate. Therefore, [1-¹⁴C]BA appeared to be a better radioprobe for the estimation of the washout rate in rat brain. However, the washout rate is also influenced by the exchange rate of aminotransferase and activity of glutamine synthetase in addition to the turnover rate of TCA cycle, it is important to take into consideration these factors.

The radioactivity of unmetabolized [1-¹⁴C]BA in blood also decreased faster than that of [2-¹⁴C]BA, although these radioactivity levels were extremely low and did not affect brain uptake. We performed radiometabolite analysis in arterial plasma with [1-¹¹C]BA instead of [1-¹⁴C]BA and detected radiolabeled acetate, ${\text{HCO}}_3^{-}$, some amino acids (glutamate and glutamine), and unknown metabolites (see Supplementary material). Although this unknown metabolite was assumed to be [¹¹C]glucose, Berl and Frigyesi suggested that glucose formed in liver from [¹⁴C]acetate existed less than 5% of total radioactivity in the brain at the early time point.¹ Taken together the fact, a very high radioactivity in the brain as well as a very lower level of labeled metabolites indicated almost negligible effect of labeled metabolites in plasma on brain radioactivity.

In the present study, as calculated from Table 1, the ratios of the washout rates of C-1 to C-2 in the cortex, cerebellum, and pons were about 3.42, 1.42, and 1.03, respectively. These regional differences of relative ratios of the washout rates between C-1 and C-2 might be due to regional difference of the CO₂ elimination rate rather than the turnover of amino acids. Since in a study by Berl and Frigyesi¹ using cats, the turnover of amino acids was reported to be similar among all regions studied (caudate nucleus, thalamus, and motor cortex).

The uptake values of C-1- and C-2-radiolabeled BA in the cerebellum and pons were different at 1 min after injection (Figure 1). However, in a previous study using C-1- and C-2-labeled acetate that we conducted (unpublished data), the radioactivity levels between labeled positions in the cerebellum and pons were not different at 1 min after injection. Therefore, the differences in uptake values at 1 min were only seen for BA. The reason for the difference in the uptakes of C-1- and C-2-labeled BA is unknown, but one hypothesis is that the turnover of labeled acetate is very rapid in these regions, as expected. Further studies on the uptake of BA at earlier time points (15 and 30 s) after the injection are needed.

We compared the kinetics of [1-¹⁴C]BA in the brains of mature and immature rats. The [1-¹⁴C]BA washout rate in the cortex of immature rats was lower than that of mature rats (not statistically significant: p = 0.056). This result implies that astrocytic oxidative metabolism remains a developing process in the brains of three-week-old rats,^{4,18,19,23,30} although additional experiments are necessary to demonstrate the relationship between astrocytic oxidative metabolism and the development process. Future studies of comparing the washout rates in rats of various ages, such as neonatal and older rats, are needed to investigate age-related changes in astrocytic oxidative metabolism.

We previously reported that the radioactivity of [1-¹⁴C]acetate and [¹⁴C]2-deoxyglucose was significantly increased in SE model rats throughout most of the brain.¹⁵ The radioactivity of [1-¹⁴C]BA in brain was higher than that of [1-¹⁴C]acetate both in control and SE model rats. The uptake of [1-¹⁴C]BA was increased in nearly all regions of brain in SE model rats in the present study; in particular, statistically significant differences were observed in the retrosplenial cortex, medial septal nucleus, and thalamus. However, regional cerebral distribution of the enhancement of [¹⁴C]BA uptake in SE rat was significantly different from that of [¹⁴C]acetate or [¹⁴C]2-deoxyglucose. Acetate is transported into astrocytes via monocarboxylic acid transporter-1, the mRNA expression of which is upregulated in the acute phase of SE.³¹ In addition, monocarboxylic acid transporter-1 was deficient in microvessels, whereas it was upregulated in astrocytes in the hippocampus of temporal lobe epilepsy model rats.³² Thus, an increase in [¹⁴C]acetate uptake in SE rat brain might be partly due to upregulation of monocarboxylic acid transporter-1. However, as [1-¹⁴C]BA is transported by the diffusion process due to its high lipophilicity (Partition coefficient = 1.41),¹⁷ the increased uptake of [1-¹⁴C]BA in the brain of SE model rats appears to reflect an increase in cerebral blood flow¹⁵ rather than the upregulation of monocarboxylic acid transporter-1 in astrocytes.

As shown in Figure 1, the results of a kinetic study of [1-¹⁴C]BA in normal rat brain showed the disappearance of radioactivity in a single-exponential manner. Therefore, simplified two-time-point autoradiography could be used to estimate the washout rate of [1-¹⁴C]BA from brain. Comparisons of the estimated washout rate values in various regions of SE model and control rats are summarized in Table 3. The washout rates of [1-¹⁴C]BA in SE model rats in all regions studied except for the medial septal nucleus were not different with those in control rats. Although the washout rates in most of brain regions were not significantly different between control and SE model rats in this study, these results indicated that the apparent values obtained by this simplified autoradiographic method could be useful for the estimating the washout rates in various types of model animals, and suggested its ability to reduce the number of experimental animals.

As previously reported, [¹⁴C]BA freely passed through the blood–brain barrier because of its high lipophilicity and was then hydrolyzed to [¹⁴C]acetate in rat brain. It is very important that [¹⁴C]BA should be almost hydrolyzed quickly in extracellular space in the brain since extracellular [¹⁴C]BA can enter into neurons as well as astrocytes. If [¹⁴C]BA itself enters and is hydrolyzed in neurons, labeled acetate is also converted to acetyl-CoA via acetyl-CoA synthetase in neurons. Therefore, the hydrolysis process of [¹⁴C]BA in extracellular space seems to be a rate-limiting step that depends on localization of the activity of hydrolysis enzymes, including carboxyl esterase and acetylcholine esterase. The localization of carboxyl esterase in brain is unknown, whereas most acetylcholine esterase exists in extracellular space. However, the previous study using rats brain showed that fluorocitrate reduced the radioactivity of [¹⁴C]BA by 79.6% in rats striatum¹⁷ and that value was similar with [¹⁴C]acetate.¹³ Therefore, [¹⁴C]BA seemed to be rapidly hydrolyzed to [¹⁴C]acetate in brain extracellular space. It supported the idea that [¹⁴C]BA was a suitable protracer for [¹⁴C]acetate, which was selectively taken up and metabolized by astrocytes in rats. In future studies using other species, detailed analysis of the hydrolysis process in the brain should be carefully evaluated.

In conclusion, we measured the differences in the cerebral kinetics of [1-¹⁴C]BA and [2-¹⁴C]BA, which are lipophilic probes of [1-¹⁴C]acetate and [2-¹⁴C]acetate, to estimate astrocytic oxidative metabolism. [¹⁴C]BA showed single-exponential kinetics regardless of the labeling positions, but the washout rate of [1-¹⁴C]BA was higher than that of [2-¹⁴C]BA. Thus, [1-¹⁴C]BA appeared to be a potential radioprobe for estimating the washout rate in rat brain. However, the hydrolysis process and metabolite incorporation in various species of brain should be carefully evaluated in future studies.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: JSPS KAKENHI, Grant Number 23591767.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

MO involved in analysis and interpretation of data and drafting the article. KY, TK, KY, and RH contributed to acquisition of data and analysis and interpretation of data. KA, M-RZ, and OI contributed to substantial contributions to conception and design of data. All the authors involved in revising the article critically for important intellectual content and final approval of the version to be published.

Supplementary material

Supplementary Material for this paper can be found at http://jcbfm.sagepub.com/content/by/supplemental-data.