All chemicals were purchased from Sigma-Aldrich (Shanghai, China). The procedures of the animal experiments performed in this study were approved by the Animal Care Committee of Ningxia Medical University (Ningxia, China).

Blood was collected retroorbitally from specific pathogen-free Sprague Dawley (SD) rats weighing 200–250 g, which were obtained from the Animal Center of Ningxia Medical University (Ningxia, China). The animals were incubated at 37°C in the presence of injected Pyr, Oxa or other chemicals at different concentrations. Blood aliquots were then taken at different time-points in order to measure the glutamate levels. In order to study the depletion of glutamate from the blood cell pool, the blood was centrifuged at 1,300 × g for 7 min at 4°C and the cell pellet was resuspended in Ringer-HEPES buffer containing 2.75 mM glucose, 5 mM HEPES, 0.15 M NaCl, 2 mM CaCl₂, 0.2 mM MgCl₂·6H₂O, 5 mM KCl and 6 mM NaHCO₃ (pH 7.4). It was then washed three times by centrifugation and resuspension. Glutamate depletion was achieved by supplementing the blood cell pool, maintained at 37°C, with 1 mM Pyr/Oxa or other chemicals added every 15 min, and the glutamate value was measured at different time-points.

Blood samples were deproteinated by adding an equal volume of ice-cold 1 M perchloric acid (PCA) and then centrifuging the samples at 16,000 × g for 10 min at 4°C. The pellet was discarded and the supernatant collected, adjusted to pH 7.2 with 2 M K₂CO₃, and, when required, stored at −20°C for later analysis. The glutamate concentration was measured in the supernatant using the fluorometric method by Graham and Aprison (6). A 20 μl aliquot of the PCA supernatant was added to 480 μl reaction buffer containing 15 units of glutamate dehydrogenase in 0.2 mM nicotinamide adenine dinucleotide (NAD), 0.3 M glycine and 0.25 M hydrazine hydrate adjusted to pH 8.6 with 1 N H₂SO₄. Following incubation for 30–45 min at room temperature, fluorescence was measured at 460 nm with excitation at 350 nm. A glutamate standard curve was established with concentrations ranging between 0 and 6 μM. All determinations were performed at least in duplicate. The results are expressed as the mean ± standard deviation.

In accordance with a previously described method (4), female SD rats (250–300 g) were anesthesized by intraperitoneal injection of urethane (0.125 g/0.2 ml per 100 g body weight). Catheterization of the tail vein (for drug injections) and of the femoral vein (for blood aliquot withdrawals) were performed using PE10 polyethylene tubing linked to PE50 polyethylene tubing (Smiths Medical, Barking, UK). All catheters were secured with 5-0 silk thread (Smiths Medical) and flushed with heparin (3–5 μl of 182 U/ml). The body temperature was maintained using a lamp and the rectal temperature was monitored. Intravenous injections of Pyr/Oxa and lipoamide diluted in phosphate-buffered saline (PBS) were performed at a rate of 0.05 ml/min for 30 min with a Pharmacia pump P-1 (Pharmacia, Uppsala, Sweden). During and following the injections, aliquots of 150 μl blood were removed from the femoral vein every 15 min.

A steel cannula made from a 27G needle was implanted into the right lateral ventricle of the rat using the following stereotactic coordinates: 0.8 mm posterior to the bregma; 1.4 mm lateral from the midline; 4 mm below the skull surface and 3.5 mm from the dura. A total volume of 11 μl [³H]glutamate solution in PBS was injected into the lateral ventricle in ~3 min through the implanted cannula using a Hamilton syringe (25 μl) connected to a PE20 tube filled with the solution. For radioactivity determination, 50-μl blood samples, collected from the femoral vein, were diluted in 500 μl H₂O and added to a 16-ml scintillator (PerkinElmer Lifesciences, Waltham, MA, USA). The measured counts per minute (cpm) values were corrected for quenching as determined by comparing the measured cpm of a set volume of [³H]glutamate added to water or to diluted blood.

Results are presented as the mean ± standard error of the mean. Data were analyzed by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The optimal concentration and time-point for minimizing blood glutamate levels with Pyr/Oxa were evaluated. Fig. 1 illustrates the changes in blood glutamate levels upon addition of various concentrations of the mixture of Pyr/Oxa, at t = 0, 20, 30, 40, and 60 min. The results show that the blood glutamate levels were reduced by ≤50% by basal levels of the blood resident transaminases GPT and GOT with addition of a mixture of 1 mM Pyr/Oxa for 60 min. Pyr/Oxa act in synergy to decrease glutamate levels but their effects are limited by the build-up of α-ketoglutarate, which facilitates the back reaction of glutamate formation.

Blood contains resident enzymes, such as GPT (substrate, Pyr), GOT (substrate, Oxa) and glutamate dehydrogenase (substrate, GDH). In the presence of their substrates, glutamate is transformed into α-ketoglutarate by these transaminases (Fig. 2). The products and cofactors of these reactions may affect the efficiency of the glutamate conversion.

In the presence of Pyr, blood glutamate is converted to α-ketoglutarate by GPT, and Pyr to alanine (Fig. 2). However, this process is reversible as the enzyme operates equally well in both directions. As shown in Fig. 3, the addition of alanine diminished the ability of Pyr to decrease the glutamate concentration. As the concentration of alanine was increased, the glutamate conversion into α-ketoglutarate was reduced.

In regard to the fact that glutamate is converted into α-ketoglutarate and that the latter is able to drive the GPT- and GOT-catalyzed back reactions, thereby limiting the conversion of glutamate, the present study investigated whether α-ketoglutarate, the common coproduct of GPT and GOT, was able to serve as a substrate of α-ketoglutarate dehydrogenase, which converts α-ketoglutarate into succinyl coenzyme A (CoA) in the presence of CoA and NAD (Fig. 2). Therefore, the consequences of the addition of CoA and NAD on glutamate conversion were assessed. In addition, the effects of the addition of pyridoxal phosphate, which is the cofactor of GPT/GOT, and of cocarboxylase and lipoamide, which are cofactors of α-ketoglutarate dehydrogenase (Fig. 2), were examined. As shown in Fig. 4, the extent of glutamate scavenging in the blood increases with increasing concentration of Pyr/Oxa. Pyridoxal phosphate exerted a marginal effect, while cocarboxylase decreased the glutamate conversion. However, lipoamide increased the conversion from a value of 55% in the control (1 mM Pyr/Oxa) to 83%.

Since, as observed in Fig. 4, lipoamide appeared to contribute to the scavenging of blood glutamate, the dose-efficiency correlation was investigated by applying various concentrations of lipoamide to blood. Fig. 5 shows that a maximal extent of glutamate conversion was obtained with 100 μM lipoamide.

As blood contains, in addition to GPT and GOT, other enzymes, such as GDH, the possible contribution of GDH to the scavenging of glutamate was investigated. GDH is a multimeric enzyme that uses NAD or nicotinamide adenine dinucleotide phosphate (NADP) as cofactors to transform glutamate into α-ketoglutarate and ammonia. It is allosterically activated by leucine and ADP (Fig. 2). Therefore, the glutamate scavenging effects of NAD, ADP and leucine (all at 1 mM) were assessed. Fig. 6 illustrates the effects of the repeated addition of 1 mM NAD to blood on glutamate levels in the cell and plasma compartments: While a decrease in glutamate levels was found to occur in the blood cells, an increase in glutamate levels occured in the plasma. These results may be interpreted as NAD possibly activating GDH in the cellular compartment, with the α-ketoglutarate produced being released from the cells and converted back into glutamate via the GPT or GOT present in the plasma. In all instances of blood being incubated with NAD, leucine or ADP either separately or in combination, increases in plasma glutamate levels occurred (Fig. 7). However, systemic decreases in the cellular compartment were observed when NAD was present alone or in combination with leucine and ADP, while the latter did not significantly affect glutamate levels alone (data not shown). When blood was incubated with a mixture of NAD, Pyr and Oxa, glutamate levels decreased to a marginally greater extent than with Pyr or Oxa alone (Fig. 8).

Previously, we reported that upon continuous intravenous administration of Pyr/Oxa, blood, as well as CSF/interstitial fluid glutamate levels were reduced to ≤50% (4). Since the present in vitro study demonstrated that by administration of lipoamide together with Pyr/Oxa, it was possible to further reduce blood glutamate levels by 83% compared with 50% for Pyr/Oxa alone, an in vivo study was performed to examine whether Pyr/Oxa together with lipoamide was able to further reduce blood glutamate levels compared with the effect of Pyr/Oxa alone. Fig. 9 illustrates the effect of Pyr/Oxa alone or together with lipoamide administered continuously through an intravenous catheter at a rate of 50 μmol/min for Pyr/Oxa and 100 μmol/min for lipoamide for a duration of 30 min. A marked decrease (55% for Pyr/Oxa and 70% for Pyr/Oxa/lipoamide) in blood glutamate occurred after 15 min. This is consistent with the in vitro results. However, as soon as the administration of Pyr/Oxa and lipoamide was ceased, the blood glutamate levels increased. When the levels of glutamate were monitored for ~200 min following the completion of the injection of Pyr/Oxa and lipoamide, glutamate levels clearly exceeded basal blood levels, suggesting that the increase in glutamate levels is not only due to the back reactions as a result of the build-up of the enzymatic products, α-ketoglutarate, alanine and aspartate, but possibly to additional compensatory processes causing various organs to release their glutamate content into the plasma to adjust to the novel conditions.

To monitor the fate of excess glutamate in the CSF, a bolus injection of 10 μCi [³H]Glu (230 pmol glutamate/10 μl) was administered to the lateral ventricles of rats and the appearance of radioactivity in the blood was monitored in relation to time. Fig. 10 shows the changes in radioactivity levels in the blood upon intracerebroventricular injection of 10 μCi [³H]glutamate followed by blood glutamate scavenging by Pyr/Oxa and lipoamide. As soon as a constant level of radioactivity was reached in the blood, blood glutamate levels were transiently decreased by the intravenous administration of Pyr/Oxa and lipoamide. The changes in blood radioactivity levels originating from the brain mirrored those of blood glutamate. While the latter decreased by ~50% during the administration of Pyr/Oxa with lipoamide and then increased, the blood radioactivity increased by ~40% and then decreased.