In vivo detection of intermediary metabolic products of [1-¹³C]ethanol in the brain using ¹³C magnetic resonance spectroscopy

Abstract

In the present study, in vivo ¹³C magnetic resonance spectroscopy (MRS) was used to study the labeling of brain metabolites after intravenous administration of [1-¹³C]ethanol. After [1-¹³C]ethanol was systemically administrated to the rats, ¹³C labels were detected in glutamate, glutamine and aspartate in the carboxylic and amide carbon spectral region. ¹³C-labeled bicarbonate HCO₃⁻ (161.0 ppm) was also detected. Saturating acetaldehyde C1 at 207.0 ppm was found to have no effect on the ethanol C1 (57.7 ppm) signal intensity after extensive signal averaging, providing direct in vivo evidence that direct metabolism of alcohol by brain tissue is minimal. To compare the labeling of brain metabolites by ethanol with labeling by glucose, in vivo time course data were acquired during intravenous co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose. In contrast to labeling by [¹³C₆]-D-glucose which produced doublets of carboxylic/amide carbons with a J coupling constant of 51 Hz, the simultaneously detected glutamate and glutamine singlets are labeled by [1-¹³C]ethanol. Since ¹³C labels originated from ethanol enter brain after being converted into [1-¹³C]acetate in liver and the direct metabolism of ethanol by brain tissue is negligible, it is suggested that orally or intragastrically administered ¹³C-labeled ethanol may be used to study brain metabolism and glutamatergic neurotransmission in studies involving alcohol administration. In vivo ¹³C MRS of rat brain following intragastric administration of ¹³C-labeled ethanol is demonstrated.

INTRODUCTION

When ethanol, an amphipathic molecule, enters the body, it distributes itself in total body water at equilibrium, and its extracellular and intracellular concentrations are approximately equal (1). In humans, studies have confirmed that ethanol is predominantly metabolized by alcohol dehydrogenase (ADH), catalase (Cata) and cytochrome P450 2E1 (CYP2E1) in liver (Fig. 1). Via oxidative metabolism, liver is responsible for almost the entire process (approximately 90%) of ethanol elimination (1,2). Acetaldehyde, the first-order or direct metabolite of ethanol, has been shown to be potentially toxic for the brain, a characteristic which is thought to be at least partially responsible for promoting chronic alcohol dependence (3). However, whether or not ethanol can be broken down directly in the brain by the in situ enzyme system remains unknown. To date, it remains unclear which chemicals are involved in the process of chronic alcohol dependence or addiction: ethanol, acetaldehyde, or both ethanol and acetaldehyde.

Figure 1.

Oxidative pathway of ethanol metabolism. Alcohol dehydrogenase (ADH), cytochrome P450E1 (CYP2E1), and catalase (Cata) are responsible for breaking down ethanol to acetaldehyde. Aldehyde dehydrogenase (ALDH) further converts acetaldehyde to acetate. Acetate from bloodstream enters the brain and is metabolized therein.

Many early studies of ethanol metabolism in the brain have shown that ethanol is not broken down directly by brain tissues (2,4,5). Acetaldehyde itself does not seem to be able to penetrate the blood-brain barrier (BBB) (6,7); furthermore, blood levels of acetaldehyde are kept extremely low by the powerful hepatic enzyme systems (6), and even when blood acetaldehyde concentrations are high, acetaldehyde can be converted into acetate by enzymes located on the BBB (e.g. aldehyde dehydrogenase (ALDH)) (8,9). Therefore, it appears that it is ethanol itself in the brain that induces chronic alcoholism. However, many recent studies suggest that ethanol may be metabolized by brain tissues and, furthermore, that the joint activity of acetaldehyde and ethanol in the brain has been proposed to be responsible for promoting chronic alcohol dependence (10–13).

Isotopic techniques are very useful for detecting the metabolism of different kinds of chemicals or energy substrates in the brain. ¹⁴C, ¹¹C labeled ethanol was previously used to detect the metabolism of ethanol in the body (2,14,15). However, despite the longer half-time (T_1/2 = 5,730±40 years), ¹⁴C labeled ethanol cannot be widely used to observe ethanol metabolism in the human body because of the harmful radiation effect associated with a large dose of ¹⁴C-labeled chemicals. Furthermore, ¹¹C-labeled ethanol cannot be used for continuous detection of ethanol metabolism in the body due to its short half-life (T_1/2 = 20.38min). Recently, ¹³C magnetic resonance spectroscopy (MRS), combined with the administration of ¹³C-labeled substrates, has become a powerful tool for investigating brain metabolism (16–18). This method allows the detection of ¹³C incorporation from ¹³C-labeled precursors into various carbon positions of metabolites, such as tricarboxylic acid (TCA) cycle intermediates and amino acids. It allows continuous, nondestructive monitoring of metabolic fluxes under different physiological or pathophysiological conditions.

In vivo ¹³C MRS combined with the infusion of ¹³C-labeled substrates can be used to measure or observe the TCA cycle as well as the glutamate-glutamine and γ-amminobutyric acid (GABA)-glutamine cycles between neuronal-glial compartments (19–23). Acetate is a known glia-specific substrate that has been used to study cerebral metabolism, neurotransmission and, in particular, the two-compartment neuronal-glial interactions in both humans and animals (24–27). In the body, ethanol is predominantly metabolized into acetate by various powerful hepatic enzyme systems (1,28). However, acetate in the liver is oxidized at a much lower rate than the oxidation of ethanol itself (28). Acetate from the liver will mainly be broken down by extrahepatic tissues, or other organs (29) such as the brain. Therefore, it may be possible to use ¹³C-labeled ethanol to study brain metabolism and neurotransmission in experiments involving alcohol administration (30).

Given the uncertainty surrounding whether ethanol is directly metabolized in brain, the present study attempted to analyze the chemical reaction between ethanol and acetaldehyde using intravenous [1-¹³C]ethanol administration combined with ¹³C magnetization transfer (CMT) in the rat brain. Previously we have demonstrated CMT can detect specific enzymatic reactions and coupled processes in vivo (31–33). We found no significant conversion of ethanol into acetaldehyde in brain. We simultaneously attempted to observe ethanol and/or its multiple byproducts in the central nervous system (CNS) by using ¹³C MRS time course study. Subsequently, we acquired in vivo ¹³C MRS spectra following intravenous co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose, and compared them with those acquired during intragastric co-administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose. We simultaneously measured and distinguished multiple peaks from different isotopomer patterns originated from both ¹³C-enriched substrates in the chemical shift region of carboxylic/amide carbons. We found that ¹³C labeling of glutamate and glutamine by [1-¹³C]ethanol is characteristic of that by the glia-specific substrate [1-¹³C]acetate, therefore opening the possibility of using ¹³C-labeled ethanol to study brain metabolism and neurotransmission.

METHOD

MR Hardware

All MRS experiments were performed on a Bruker microimaging spectrometer (Bruker Biospin, Billerica, MA, USA) interfaced to an 11.7 Tesla 89-mm bore vertical magnet (Magnex Scientific, Abingdon, UK). This magnet is equipped with a 57-mm i.d. gradient (Mini 0.5; Bruker Biospin, Billerica, MA, USA) with a maximum gradient strength of 3.0 G/mm and a rise time of 100 μs for in vivo studies of adult rats. The ¹H and ¹³C radio frequency (RF) coils were coplanar and made of single-sided printed circuit board. The inner loop is the ¹³C coil, with an inner diameter and conductor width of 10.8 and 4.3 mm, respectively. The outer loop is the ¹H coil with an inner diameter and conductor width of 23.6 and 5.4 mm, respectively. No noise injection was found in the ¹³C channel due to proton decoupling. The lower end of the system was an aluminum interface box through which RF cables, ventilation tubes, rectal thermal probe, and catheters were connected. A similar design of the RF probe/animal handling system for proton and Proton-Observed, Carbon-13-Edited spectroscopy experiments of rat brain using the vertical 89-mm bore magnet was previously described (34,35). The loaded isolation between ¹H and ¹³C coils (S21), which have a large frequency separation of 375 MHz, is −30.6 dB at 125 MHz and −31.2 dB at 500 MHz, respectively, and the standard Bruker filters were used. A broadband low-pass filter was used in the ¹³C channel whose insertion loss at 125 MHz was less than 0.5 dB. Its rejection at 500 MHz was greater than 80 dB. In the proton channel, a broadband high-pass filter was used with an insertion loss at 500 MHz less than 0.5 dB and rejection at 125 MHz greater than 60 dB. The integrated RF coils/head-holder system is capable of rat head fixation (with ear pins and a bite bar), body support and RF shielding. The pulse-acquire sequence (18) and that of saturation transfer (31) were applied in the study. ¹H decoupled ¹³C MRS experiments were performed to measure the ¹³C signals in the animal brain. Anatomical images were acquired using the three-slice (coronal, horizontal, and sagittal) scout rapid acquisition with relaxation enhancement (RARE) imaging method (field of view = 2.5 × 2.5 cm², slice thickness = 1 mm, TR/TE = 200/15 ms, rare factor = 8, data matrix = 128×128).

In vivo MRS procedures

After experimental animals were put into the scanner, RARE magnetic resonance imaging (MRI) images were acquired to ensure the animal brain and the RF coils were in the proper position. The gradient isocenter of the RF probe/animal handling system in a Mini 0.5 gradient was about 0–1 mm posterior to bregma. The rat brain was shimmed as described previously using the FASTMAP/FLATNESS method (36).

In vivo ¹³C saturation transfer experiments were performed as previously described (31–33). The saturation pulse was placed at 207.0 ppm, the resonant frequency of carbonyl carbon of the keto form of acetaldehyde (37–40). The chemical shift of the carbonyl carbon of acetaldehyde was measured in phantom at neutral pH.

Broadband ¹H-¹³C nuclear Overhauser enhancement (NOE) was generated using a train of nonselective hard pulse with a nominal flip angle of 180° space 100ms apart. The decoupler frequency was placed at 3.6 ppm, the resonance frequency of ethanol H2. ¹³C continuous-wave (CW) RF saturation of the carbonyl carbon (C1) of acetaldehyde at 207.0 ppm was performed with a nominal RF saturation field (B_1sat) of 1.8 × 10⁻² Gauss. When the control spectra were acquired, the saturating pulse was placed at an equal spectral distance from ethanol C1 but on the opposite side of acetaldehyde C1. To minimize motion artifacts and interference from slowly varying ethanol signal intensity, the saturation and control spectra were interleaved. A 1-ms adiabatic half passage (AHP) pulse was used for 90° excitation. The ¹³C carrier frequency was centered near ethanol C1 at 57.7 ppm. WALTZ-4 with a 400 μs nominal 90° rectangular pulse was used for ¹H decoupling. A natural-abundance baseline spectrum was also acquired before infusion. No significant overlapping resonances were found at the ethanol C1 resonance.

Because carbon in the carboxylic/amide carbon is only coupled to protons via very weak long-range ¹H-¹³C scalar couplings, ¹³C-labeled chemicals appearing in this chemical shift region can be effectively decoupled at a very low RF power (41, 42). As a result, decoupling of large one-bond ¹H-¹³C scalar couplings can be avoided by using [1-¹³C]ethanol because the resonances of its intermediary metabolic products including [1-¹³C]acetaldehyde (AcHC1, 207.0 ppm), [1-¹³C]acetate (182.6 ppm), [5-¹³C]glutamate (GluC5, 182.0 ppm), [5-¹³C]glutamine (GlnC5, 178.5 ppm), [1-¹³C] γ-amminobutyric acid (GABAC1, 182.3 ppm), [4-¹³C]aspartate (AspC4, 178.3 ppm), [1-¹³C]aspartate (AspC1, 175.0 ppm) appear in the carboxylic/amide spectral region. Furthermore, another advantage of this strategy is the lack of contamination from subcutaneous lipids, because there are no overlapping fat signals in the vicinity of the GluC5 and GlnC5 peaks. For direct detection of ¹³C labeling of brain metabolites no RF saturation pulses were applied.

Experimental animals

All experimental animals were studied according to procedures approved by the National Institute of Mental Health (NIMH) Intramural Research Program Animal Care and Use Committee, National Institutes of Health (NIH). During the experimental process, all efforts were made to minimize animal suffering, to reduce the number of animals used, and to use alternatives to in vivo techniques, if available. Animals (adult Sprague-Dawley rats, Taconic Laboratories, Germantown, NY, USA) were kept under conditions of constant temperature (21 °C) and humidity (50%), with a 12-hour light/dark cycle and free access to food and water. ¹³C-enriched [1-¹³C]ethanol and [¹³C₆]-D-glucose were purchased from Cambridge Isotope Laboratories, Inc (Andover, MA, USA). The enrichment of both ¹³C-labeled chemicals was 99%. The animals (body weight = 178–255 g) were evenly divided into four groups. Five non-fasting animals (Group A) were given intravenous infusion of [1-¹³C]ethanol [33% (vol/vol)] at a dose of 5 g/kg (body weight (BW)), including an initial bolus of 2 g/kg BW in the first 10 minutes followed by the rest of the chemical for the remaining 270 minutes (3 g/kg BW). Animals in Group B (non-fasting, n = 5) received intravenous infusion of [1-¹³C]ethanol [33% (vol/vol)] at a dose of 4 g/kg BW including a 10 minute initial bolus of 2 g/kg BW followed by the rest of the chemical (2 g/kg BW) for the remaining 170 minutes. Animals in Group C (n = 6) were fasted overnight and received intravenous co-infusion of [1-¹³C]ethanol [33% (vol/vol)] and [¹³C₆]-D-glucose (0.75 M). The [1-¹³C]ethanol infusion protocol consisted of an initial bolus of 2 g/kg BW for the first 10 minutes followed by infusion of 4 g/kg BW over a maximum period of 350 minutes; the [¹³C₆]-D-glucose infusion protocol consisted of an initial bolus of 75.5 mg/min/kg BW for the first 10 minutes followed by a maximum of 350 minute continuous infusion at 28.5 mg/min/kg BW. Group D animals (n = 3, fasted for 24 hours) were used to acquire in vivo ¹³C MRS spectra after intragastric co-administration of both [1-¹³C] ethanol [33% (vol/vol)] and [¹³C₆]-D-glucose (0.75 M).

On the day of in vivo ¹³C MRS study, the animals were orally intubated and mechanically ventilated (SAR-830/AP, CWE, Inc., Ardmore, PA, USA) with a mixture of 70% N₂O, 30% O₂, and 1.5% isoflurane. One artery was cannulated for periodic sampling of arterial blood in order to monitor blood gases (pO2, pCO2), pH, and blood glucose concentrations using a blood analyzer (Bayer Rapidlab 860, East Walpole, MA, USA), as well as to monitor and regulate arterial blood pressure levels. For Groups A and B animals, one femoral vein was cannulated for infusion of ¹³C-labeled ethanol; for Groups C and D animals, bilateral femoral veins were cannulated, one for intravenous infusion of [1-¹³C]ethanol, the other for intravenous infusion of [¹³C₆]-D-glucose. For the rats administered with [¹³C₆]-D-glucose (groups C and D), total plasma glucose levels were maintained at 12.0~16.0 mM during in vivo ¹³C MRS data acquisition. Throughout the experiment, normal physiological conditions were maintained according to results obtained from the blood analyzer (pH ~7.4, pCO₂ ~35 mmHg and pO₂ > 100 mmHg), respiration monitor (SurgicalVet, SIMS BCI, Inc. Wisconsin, USA), and by real-time regulation of ventilation. Blood pressure, heart rate, and respiration changes were also monitored simultaneously (BIOPAC System, Inc. GenuineIntel, Goleta, CA). An external pump for heat exchange by circulating warm water (BayVoltex, Modesto, CA, USA) was used to maintain body temperature at 37.8 ± 0.5 °C.

RESULTS

¹³C labeling of brain metabolites following intravenous infusion of [1-¹³C]ethanol

Figure 2 shows in vivo ¹³C MRS spectra acquired from one Group A animal after intravenous infusion of [1-¹³C]ethanol. The results showed that ¹³C labels carried by infused [1-¹³C]ethanol were incorporated into carbonyl carbons of multiple metabolites including GluC5 (182.0 ppm), GlnC5 (178.5 ppm), and GluC1 (175.4 ppm). The time domain data were zero-filled to 16 K. Exponential line broadening (lb = 10) was applied before Fourier transform. The ¹³C-labeled bicarbonate HCO₃- (161.0 ppm) was also detected. Although ethanol itself can be easily detected as a singlet at 57.7 ppm (not shown in Fig. 2), its direct metabolite AcHC1 (207.0 ppm) was not observed. Similar results were obtained for all Group A animals. The chemical shift position of AcHC1 (207.0 ppm) was marked in Fig. 2.

Figure 2.

Accumulated in vivo ¹³C MRS spectrum (150~210 ppm) from the rat brain after 280 minutes intravenous infusion of [1-¹³C]ethanol. TR = 18.75s, NA = 448. The spectrum was summed from one rat in Group A over 280 minutes. Data processing: lb = 10, si = 16k. Glu-C5 = glutamate C5, Gln-C5 = glutamine C5, Glu-C1 = glutamate C1, Gln-C1 = glutamine C1, AcH-C1 = acetaldehyde C1.

¹³C saturation transfer

Figure 3(A) shows the results of ¹³C saturation transfer experiments performed on one Group B animal targeting the [1-¹³C]ethanol↔[1-¹³C]acetaldehyde reaction. The difference spectrum (bottom trace) represents signal averaging for 180 min. The difference spectrum (bottom trace) was obtained by subtracting the subspectrum with the saturation of AcHC1 at 207.0 ppm (middle trace) from the control spectrum with the saturation pulse placed at −149.3 ppm (top trace). The results indicate that no significant saturation transfer effect was found for the [1-¹³C]ethanol↔[1-¹³C]acetaldehyde reaction in brain. We also added up the results from all Group B animals (n = 5) in Figure 3(B) and no signal above the noise level was detected in the summed difference spectrum. The noise level in Fig. 3B was measured by first integrating the ethanol C1 signal in the control spectrum. Then the same integration interval length was used to divide the difference spectrum into equal segments and to integrate the noise in each segment. The concentration of ethanol was estimated to be 15.4 mM based on separate proton MRS measurements using N-acetylaspartate (NAA) as a concentration reference standard ([NAA] = 10.8 mM, 27). In the proton MRS experiments the infusion protocol used unlabeled ethanol. The noise level (σ) of the difference spectrum was found to be 0.089 mM. Since the detection threshold for 95% confidence interval is 1.65σ, the detection limit using our procedure is 1.65 × 0.089 = 0.15 mM for ethanol C1, which corresponds to a ¹³C saturation transfer pseudo first order rate constant in the range of 3.2 × 10⁻³ sec⁻¹ ~ 9.7 × 10⁻³ sec⁻¹ (for T₁ = 3~1 sec). That is, the detection threshold of our method for the pseudo first order rate constant of the ethanol acetaldehyde exchange reaction in rat brain is ca. 0.003~0.01 sec⁻¹. In contrast to the brain, when rat liver homogenate was measured using the same ¹³C saturation transfer MRS protocol, a prominent drop in the ethanol C1 signal was observed upon irradiating the AcHC1 resonance at 207.0 ppm. The amplitude of the difference ethanol C1 signal was determined to be 72.5% of that in the control spectrum (data not shown), indicating a very fast exchange between acetaldehyde and ethanol in liver.

Figure 3.

(A) Results of the ¹³C saturation transfer experiments on one Group B animal targeting the [1-¹³C]ethanol↔[1-¹³C]acetaldehyde reaction. Top trace: control spectrum; middle trace: with saturation of AcH-C1 at 207.00ppm; bottom trace: difference spectrum (×3). The difference spectrum (right bottom trace) represents signal averaged for 180 min (TR = 9.375s, NA = 1152, lb = 10). (B) Summed spectra from 5 Group B rats. The difference spectrum (bottom right trace) represents signal averaged for 900 min (TR = 9.375s, NA = 5760, lb = 10).

Simultaneous intravenous administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose

Figure 4(a) shows the in vivo proton decoupled ¹³C MRS time course spectra of rat brain in the carboxylic/amide carbon spectral region acquired from one animal in Group C during co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose. The time domain data were zero-filled to 16K. The resolution enhancing Lorentz-Gauss transformation (lb = −5, gb (Gaussian broadening factor) = 0.1) was applied before Fourier transform. ¹³C labeling of multiple metabolites was detected in the carboxylic/amide region, including GluC5 (182.0 ppm), GluC1 (175.4 ppm), GlnC5 (178.5 ppm), GlnC1 (174.9 ppm), Asp C4 (178.3 ppm) and [1-¹³C]lactate (183.3 ppm). Each spectrum corresponds to 20-minute signal averaging. The singlets from [1-¹³C]ethanol (57.7 ppm) and ¹³C-labeled HCO₃⁻ (161.00 ppm) were also observed but are not shown here. Figure 4(b) shows the spectrum from one animal accumulated over the 120–360 minute interval after the initiation of co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose. In separate control experiments using unlabeled ethanol and [¹³C₆]-D-glucose no GluC5 singlets were detected (data not shown). This is expected because the glutamate ¹³C4-¹³C5 moiety always comes from [1,2-¹³C₂]acetylCoA regardless of the number of turns of the TCA cycle. Therefore, glutamate C5 singlet cannot be formed as long as [1,2-¹³C₂]acetylCoA is the only labeled input to the TCA cycle. By inference, glutamine C5 and aspartate C4 singlets cannot be formed either under this condition.

Figure 4.

(a) Time-course in vivo ¹³C MRS spectra from an individual rat brain during co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose compared with baseline (bottom trace). Each individual spectrum was averaged for 20 minutes with NA = 32. (b) In vivo ¹³C MRS spectrum from an individual rat brain accumulated during the 120–360 minute interval after the start of co-infusion of [1-¹³C]ethanol and [¹³C₆]-D-glucose. Green: resonance lines originated from [¹³C₆]-D-glucose; Red: resonance lines originated from [1-¹³C]ethanol. lb= -5, gb = 0.1. Note the glutamate C5 resonance labeled by [¹³C₆]-D-glucose formed a doublet with a ¹³C-¹³C homonuclear J coupling constant of 51 Hz; the glutamate C5 resonance labeled by [1-¹³C]ethanol formed a singlet. From downfield to upfield, the resonance lines in the 178–179 ppm region were Gln C5 by [¹³C₆]-D-glucose, Gln C5 by [1-¹³C]ethanol + Asp C4 by [¹³C₆]-D-glucose, Gln C5 by [¹³C₆]-D-glucose + Asp C4 by [1-¹³C]ethanol, Asp C4 by [¹³C₆]-D-glucose. The two downfield Glu C1 lines originated from [¹³C₆]-D-glucose and [1-¹³C]ethanol were also marked.

Therefore, co-infusion of differently labeled substrates and the detection of signals in the carboxylic/amide ¹³C spectral region allow clear separation of contribution to glutamate, glutamine and aspartate from the different substrates. As shown in Fig. 4, glutamate C5 originated from [¹³C₆]-D-glucose appears as a doublet with a J coupling constant of 51 Hz while glutamate C5 originated from [1-¹³C]ethanol appears as a singlet. The large homonuclear ¹³C-¹³C J coupling between an aliphatic carbon and a carboxylic or an amide carbon and the lack of interference from other one-bond couplings allow a clear separation of signals originated from singly and doubly labeled substrates. At 11.7 Tesla, the chemical shift separation between glutamine C5 and aspartate C4 is coincidentally one half of the one-bond J coupling between an aliphatic carbon and a carboxylic or amide carbon. As a result, a pseudo quartet was detected in the 178–179 ppm region, allowing easy detection of contributions to glutamine C5 and aspartate C4 from different substrates. Similarly, glutamate C1 originated from [¹³C₆]-D-glucose and singly labeled [1-¹³C]ethanol were also detected because glutamate C1 is widely separated from glutamine C1, aspartate C1 and NAA C5. Quantitative analysis revealed the expected brain’s preference for glucose as the respiration fuel. At 220 min after the initiation of co-infusion (all group C and D rats were infused for a minimum of 230 minutes), [GluC5]_Glucose/[GluC5]_Ethanol, [GlnC5]_Glucose/[GlnC5]_Ethanol and [GluC5 + GlnC5 + AspC4]_Glucose/[GluC5 + GlnC5 + AspC4]_Ethanol ratios from Group C animals were found to be (3.29 ± 0.12, 2.65 ± 0.17, 3.36 ± 0.15):1.

Intragastric administration of ¹³C labels

Figure 5(a) shows the in vivo proton decoupled ¹³C MRS time course spectra acquired from one Group D rat during intragastric co-administration of [1-¹³]ethanol and [¹³C₆]-D-glucose. Figure 5(b) shows the accumulated ¹³C MRS spectrum acquired during the 140~360 minute interval after the start of intragastric co-administration of [1-¹³]ethanol and [¹³C₆]-D-glucose. Data processing was the same as previously described for the co-infusion experiments (lb = −5, gb = 0.1). At 220 min after the initiation of co-infusion, [GluC5]_Glucose/[GluC5]_Ethanol, [GlnC5]_Glucose/[GlnC5]_Ethanol and [GluC5 + GlnC5 + AspC4]_Glucose/[GluC5 + GlnC5 + AspC4]_Ethanol ratios from Group C animals were found to be (4.04 ± 0.36, 0.92 ± 0.13, 2.83 ± 0.34):1. Compared with Group C, the Group D data showed a much slower rate of glucose transport into the brain as expected from the additional delay by the stomach (43). In contrast, ethanol is freely diffusible in the body and therefore not affected by intragastric administration. As a result, the doublet signals originated from intragastrically administered [¹³C₆]-D-glucose are lower in (Fig. 5(b) than the corresponding doublet signals in Fig. 4(b). The largest difference between intravenous and intragastric administration is seen in GlnC5. In Fig. 5(b), singly labeled GlnC5 is rapidly generated in astroglia by ¹³C labels originated from [1-¹³C]ethanol. At the same time, the synthesis of doubly labeled GlnC5 is delayed by both the gastric barrier to glucose and the glutamate-glutamine cycle. By the same mechanism, the GluC1 signal of Group C rats originated from [¹³C₆]-D-glucose is much higher than the GluC1 signal of Group D rats originated from [¹³C₆]-D-glucose.

Figure 5.

(a) Time-course ¹³C MRS spectra from an individual rat brain during intragastric co-administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose. Each individual spectrum was averaged for 20 minutes. The bottom spectrum was the baseline. (b) Accumulated in vivo ¹³C MRS spectra from an individual rat brain during intragastric co-administration of [1-¹³C] ethanol and [¹³C₆]-D-glucose. The spectrum was summed during the 140~360 minute interval after the start of the co-infusion. Green: resonance lines originated from [¹³C₆]-D-glucose; Red: resonance lines originated from [1-¹³C]ethanol. Data processing parameters were: lb = −5, gb = 0.1.

DISCUSSION

The metabolism of ethanol and its intermediary metabolic products in the brain

Previously ¹⁴C, ¹¹C-labeled ethanol combined with the techniques of radioactivity test and positron emission tomography (PET) were used to measure the metabolism of ethanol in the body (2,14,15). In one study where ¹⁴C-labeled ethanol was used to observe ethanol metabolism in monkeys (44), Mushahwar and colleagues found that some of the glutamate and glutammine in the brain tissue was originated from the ¹⁴C-labeled ethanol (14). Because they did not find ¹⁴C-labeled acetaldehyde or acetate, their results cannot be taken as evidence that ethanol is broken down directly in the brain. Using results obtained from isolated, perfused rat brain, Mukherji and colleagues concluded that ethanol itself cannot be metabolized by brain directly, although they simultaneously found that acetaldehyde can be broken down by the brain (2). The current study used ¹³C MRS, combined with the administration of ¹³C-labeled substrates to study the labeling of brain metabolites by [1-¹³C]ethanol. We found that ¹³C labels from [1-¹³C]ethanol are readily incorporated into glutamate, glutamine and aspartate in brain, suggesting that ethanol and/or its metabolites (e.g., acetaldehyde, acetate) are metabolized by brain tissues (see Fig 2). However, neither AcHC1 (207.0 ppm) nor [1-¹³C]acetate (182.6 ppm) metabolized from [1-¹³C]ethanol were detected in the brain ¹³C MRS spectra. Recent studies of ethanol metabolism reported that the concentration of ethanol and acetaldehyde could reach 40 mM and 250 μM by using an aldehyde dehydrogenase inhibitor (45). Earlier studies have also found that, under normal physiological conditions without using enzyme inhibitors, acetaldehyde levels were only 6.720 μM and 18.165 μM (or: 6.4 nM/g and 17.3 nM/g) for C57 and DBA mice, respectively (46), which were well beyond the detection threshold of in vivo ¹³C MRS.

Enzymatic activity for acetaldehyde production in the brain

To further explore ethanol metabolism in the CNS, the present study used intravenous [1-¹³C]ethanol administration combined with ¹³C saturation transfer to detect the conversion of ethanol to acetaldehyde in the rat brain.

Because fasting reduces ethanol metabolism (47–49), the saturation transfer experiment conducted here did not use animals that had fasted before ¹³C MRS data acquisition. Results from the saturation transfer showed that when the pulse was set on 207.0 ppm (the resonant frequency of acetaldehyde C1), no significant difference in [1-¹³C]ethanol signal (57.7 ppm) between the saturation and control spectra was observed (Fig. 3). Because the in vivo ¹³C saturation (or inversion) transfer effect of enzyme-catalyzed reactions can be used to measure enzyme-specific reactions in vivo (31–33,50), the negative saturation transfer results obtained here are consistent with previous studies that found minimal ADH, CYP2E1 and catalase activities in brain tissue. In comparison, a large ¹³C saturation transfer effect caused by the exchange between ethanol and acetaldehyde is easily observable using rat liver homogenate (data not shown).

The results of the present study echo previous work noting that powerful enzyme systems exist for metabolizing ethanol in extracerebral tissues, especially the liver, which is the biggest digestive gland in the body. When entering the body, ethanol is predominantly metabolized into free acetate in the liver, and subsequently enters the bloodstream (1,28,47–49). Thus, after the administration of ethanol, the main role of the brain is simply to use the ethanol product acetate originated from extracerebral tissues. This conclusion is further corroborated by separation proton MRS measurements performed during the course of infusing unlabeled ethanol. Using the same infusion protocol as that used for Group B rats, no change in the lactate signal at 1.32 ppm in the proton time course spectra was found (data not shown). If cerebral ethanol metabolism were significant, the increased NADH/NAD ratio would have caused an elevation of the cerebral lactate signal. The results shown in Figs. 4 and 5 therefore open the possibility that intravenous or oral administration of ethanol could be used to study brain metabolism.

Co-administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose for the study of cerebral metabolism

Co-administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose allows simultaneous detection of the labeling kinetics of brain metabolites by singly labeled [1-¹³C]ethanol and uniformly labeled glucose. Similar to a previous study using infusion of [¹³C₆]-D-glucose (without ethanol) and detection of ¹³C label incorporation into the alkyl C4 carbons of glutamate and glutamine (35), the results in Figs 4(b) and 5(b) show a temporal lag in the incorporation of ¹³C labels from [¹³C₆]-D-glucose into the glutamine C5 carbon as compared to the incorporation of ¹³C labels into glutamate C5 carbons. This is consistent with the recently validated glutamate-glutamine cycle between neurons and astroglia with ¹³C-labeled neuronal glutamate as the main metabolic precursor for ¹³C-labeled astroglial glutamine when ¹³C-labeled glucose is administered. The precursor-product relationship between ¹³C-labeled neuronal glutamate and ¹³C-labeled astroglial glutamine is reversed when ¹³C-labeled acetate is used for introduction of extragenous ¹³C labels. This is because acetate is predominantly metabolized in the glial cells. Recently, ¹³C labeled acetate and glucose have been used to study cerebral metabolism, particularly with regards to the relationship between neurons and the glial cells (20,24,51). This reversed precursor-product relationship between ¹³C-labeled neuronal glutamate and ¹³C-labeled astroglial glutamine is shown by the results given in Figs. 4 & 5 with co-administration of [1-¹³C]ethanol and [¹³C₆]-D-glucose. In particular, in Figs 4(b) and 5(b), there is a clear preference by glutamine for ¹³C labels originated from the singly labeled [1-¹³C]ethanol.

Accumulating evidence indicated that co-infusion of specific ¹³C-enriched glucose and acetate could be used to measure both neuronal and glial metabolism in the brain using ¹H-decoupled ¹³C MRS (22). This approach was based on the findings that ¹³C-labeled glucose and acetate can produce distinct isotopomers of metabolites, which could be used to track multiple intermediate products of TCA cycle in the CNS. Therefore, specific ¹³C-labeled glucose and acetate (or ethanol) combined with in vivo ¹³C MRS can be used to observe brain metabolism and glutamatergic neurotransmission. In other words, co-administration of differently ¹³C-labeled substrates combined with analysis of multiple ¹³C MRS signals in the spectral region of carboxylic/amide carbons allows investigators to cleanly separate the contributions to Glu, Gln, and Asp from different energy substrates.

In the present study, when [1-¹³C]ethanol and [¹³C₆]-D-glucose were intravenously administrated to the animals, GluC5, GluC1, GlnC5, GlnC1, and AspC4, originated from both [¹³C₆]-D-glucose and [1-¹³C]ethanol could be detected over the 171–186 ppm range (Fig. 4), as could the fine isotopomer structure arising from homonuclear ¹³C-¹³C J-couplings. The isotopomer patterns observed in vivo are consistent with the known ¹³C-¹³C J-couplings previously detected in both in vitro and in vivo studies (19,35).

There was a striking similarity between the steady state spectra acquired using [¹³C₆]-D-glucose and [1-¹³C] ethanol with the corresponding results of the co-infusion of [¹³C₆]-D-glucose and [1-¹³C] acetate (52,53). Obviously, in both spectra, the singly labeled substrate (acetate or ethanol) makes a predominant contribution to glial glutamine. This similarity can be explained by the conversion of ethanol into acetate in the liver and the subsequent recirculation of acetate into the brain. In addition, the features of the spectra acquired during co-infusions match the sum of the corresponding single substrate ([¹³C₆]-D-glucose, [1-¹³C]ethanol and [1-¹³C]acetate) infusion spectra (52,53). Thus, the present study demonstrate that the metabolic consequence of ethanol consumption can be detected in the brain by noninvasive ¹³C MRS. The strong similarity of in vivo ¹³C spectra acquired with ethanol administration to intravenous acetate infusion suggests that orally administered ethanol may be used as a glia-specific substrate to study cerebral metabolism.

Following the above reasoning, a mixture of [¹³C₆]-D-glucose and [1-¹³C]ethanol was intragastrically administered to the group D animals. Fig. 5 indicates that the results are similar to those experiments that used intravenous co-infusion of [¹³C₆]-D-glucose and [1-¹³C]ethanol, except that a temporal lag in the appearance of doublet peaks was observed in the intragastric administration experiments. This was due to the fact that it took more time for uniformly labeled glucose to reach blood circulation. Many early studies of acetate metabolism found that it was impossible to raise blood acetate levels by oral ingestion of acetic acid or acetate, however, when moderate amounts of ethanol were given, acetate concentrations increased several-fold over oral ingestion of acetate (47). Furthermore, for safety reasons, studies with ¹³C-labeled chemicals in humans are generally conducted using lower doses of acetate infusion than in animal studies (20). The rate at which ethanol was cleared from the blood was the same as the rate of acetate production (47), suggesting that the enzymatic and chemical processes leading from ethanol to free acetate are quite powerful and rapid.

Chronic alcohol consumption is usually associated with metabolic disturbances of sulfur-containing amino acids, leading to increased levels of glutamate, aspartate, and homocysteine in alcoholic patients, and may result in dependence, addiction, and even liver cirrhosis. When high resolution proton NMR spectroscopy, which offers superior dynamic range and detects many classes of molecules in samples of brain tissue extracts or serum, was used to observe the effects of an acute or single administration of ethanol on metabolic alterations in the CNS, no detectable changes in the brain were observed (54). Even at pharmacologically relevant concentrations, and particularly in the concentration range of 10 to 20 mM, the effect of ethanol on the “fluidity” of the membrane bulk lipids is very small, or undetectable (1). In contrast, the results of the present study suggest that oral or intragastric administration of ¹³C-labeled ethanol could be used to study brain metabolism and neurotransmission using ¹³C MRS.

Abbreviations used

ADH

dehydrogenase

Cata

catalase

CYP2E1

cytochrome P450 2E1

BBB

blood-brain barrier

ALDH

Aldehyde dehydrogenase

MRS

magnetic resonance spectroscopy

TCA

tricarboxylic acid

GABA

γ-amminobutyric acid

CMT

¹³C magnetization transfer

CNS

central nervous system

RF

radio frequency

RARE

rapid acquisition with relaxation enhancement

MRI

magnetic resonance imaging

NOE

nuclear Overhauser enhancement

CW

continuous-wave

AHP

adiabatic half passage

AcHC1

[1-¹³C]acetaldehyde

GluC5

[5-¹³C]glutamate

GlnC5

[5-¹³C]glutamine

[1-¹³C]γ-aminobutyric acid

GABAC1

[4-¹³C]aspartate

AspC4

AspC1

[1-¹³C]aspartate

BW

body weight

lb

line broadening

NAA

Nacetylaspartate

gb

Gaussian broadening

PET

positron emission tomography