Kunihiko Shibata

An improved method for the detection of changes in brain extracellular glutamate levels

We developed a method for in vivo real-time monitoring of the concentration of extracellular glutamate ([Glu]e) in the brain under anoxic conditions. A dialysis electrode (Sycopel Int., UK) was employed as a sensing device to measure the concentration of glutamate by enzyme amperometry, and an electron mediator, ferrocene, was introduced into the electrode together with glutamate oxidase. The ferrocene was covalently conjugated with a high molecular weight molecule, bovine serum albumin, to avoid outward diffusion through the dialysis membrane. With this set-up, the amperometric response was independent of the pO2 around the electrode in vitro up to 400 microM glutamate. Using this method, we investigated the dynamics of [Glu]e in the rat striatum during anoxia. [Glu]e increased rapidly at 102+/-5.4s (n = 6) after the start of nitrogen inhalation. The increase continued for about 30 s, and then [Glu]e decreased. The peak value of delta[Glu]e was 141+/-37 micro M. [Glu]e subsequently underwent another gradual increase, reaching 213+/-69 microM at 15 min after the start of nitrogen inhalation. This distinct biphasic profile was reproducible. We conclude that this method is very useful for monitoring [Glu]e in the brain under low pO2 conditions.

High Correlation between Results of the [1-13C]-Phenylalanine Breath Test and Phenylalanine Hydroxylase (EC 1.14.16.1) Activity of the Liver in Rats

Background:¹³CO₂ is decreased in patients with end-stage liver disease by the [1-¹³C]-phenylalanine breath test. Decreased ¹³CO₂ is supposed to be caused by the decreased ability of the liver to oxidize phenylalanine. However, no direct evidence has been reported. Methods: The [1-¹³C]-phenylalanine breath test was performed in galactosamine hepatitis rats (n = 14) and control rats (n = 8). Plasma phenylalanine concentration before intravenous administration of [1-¹³C]-phenylalanine, the elimination rate of phenylalanine and the phenylalanine hydroxylase (PAH; EC 1.14.16.1) activity of the whole liver were examined. Results: Increase of ¹³CO₂ in the breath [Δ¹³CO₂ (‰)] of galactosamine hepatitis rats 2 min after administration of [1-¹³C]-phenylalanine was only 1/5 of that in control rats. The concentration of plasma phenylalanine and the elimination rate of plasma phenylalanine in hepatitis rats did not show significant differences compared to control rats. On the other hand, a clear difference in the activity of PAH was observed between hepatitis rats and control rats. Δ¹³CO₂ (‰) 2 min after administration of [1-¹³C]-phenylalanine was highly correlated to the PAH activity of the whole liver (r = 0.917). Conclusion: It was strongly indicated that decreased Δ¹³CO₂ in hepatitis rats was the result of decreased activity of PAH.

Synthetic 13C-dipeptide breath test for the rapid assessment of pancreatic exocrine insufficiency in rats

Objective. 13C-breath tests have been investigated in order to assess pancreatic exocrine function using various 13C-compounds, but they have not been accepted for routine clinical use. One of the barriers to their acceptance is that these tests are time-consuming and require up to several hours for breath collection. The purpose of this study was to design a novel 13C-compound that would make a rapid 13C-breath test for assessing exocrine pancreatic function possible. Material and methods. N-benzoyl-L-tyrosyl-1-13C-L-alanine was synthesized, and the characteristics of its cleavage in duodenal juice and in the duodenum of rats were examined. Thereafter, a 13C-breath test was carried out in which N-benzoyl-L-tyrosyl-1-13C-L-alanine was given orally to pancreatic exocrine-insufficient and normal control rats. Results. N-benzoyl-L-tyrosyl-1-13C-L-alanine was readily cleaved and liberated 1-13C-L-alanine in the duodenal juice. Carboxypeptidase was a major contributor to the cleavage. When N-benzoyl-L-tyrosyl-1-13C-L-alanine was injected into the duodenum and orally administered to the rats, the 13C atom% of CO2 in breath increased rapidly. This indicated that N-benzoyl-L-tyrosyl-1-13C-L-alanine in the duodenum liberated 13C-Ala on cleavage. 13C-Ala is absorbed and metabolized to liberate 13CO2, which is exhaled. It was shown that the Δ13CO2 (‰) in the N-benzoyl-L-tyrosyl-1-13C-L-alanine breath test in the pancreatic exocrine-insufficient rats, in whom the absorption and metabolism of 13C-Ala was unimpaired, was significantly lower than that in the control rats. Conclusions. The rate of increase in the Δ13CO2 (‰) in the N-benzoyl-L-tyrosyl-1-13C-L-alanine breath test is expected to be proportional to the rate of N-benzoyl-L-tyrosyl-1-13C-L-alanine cleavage by pancreatic proteases in the duodenum. We propose the N-benzoyl-L-tyrosyl-1-13C-L-alanine breath test as a rapid test for assessing pancreatic exocrine function.