Last Month's Most Accessed Feature: Ischemic amino acids: Rapid mass spectrometric estimation in the brain

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  • Published: Feb 6, 2015
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Ischemic amino acids

The levels of the neurotransmitters glutamic acid and aspartic acid have been measured in the brains of diabetic rats following reperfusion by a rapid LC/MS procedure.

When the blood supply to the brain becomes limited in an ischemic episode, like during a stroke, it initiates a sequence of events that results in brain cell death. The initial step in this cascade is the stimulation of two amino acid neurotransmitters, glutamic acid (Glu) and aspartic acid (Asp), which are produced in increasing amounts.

The degree of brain injury can be estimated by changes in the levels of these two compounds, while treatments to reduce their concentrations have been proposed as potential therapies for stroke. Clearly, a rapid and accurate method is needed to measure them and a team of Chinese researchers has explored the use of LC/MS with encouraging results.

Yi Fang and Libo Zhao from Peking University People's Hospital, Jia Xu from Mawangdui Hospital, Changsha, and Zhonglian Qian from The First Hospital, Huhhot, devised a procedure which is better than several published HPLC and LC/MS methods in terms of sensitivity, selectivity and speed.

Rapid tandem method

Using standard solutions of the two amino acids, the best LC/MS conditions were derived for Asp and Glu. They were separated on C18 column with a fixed mobile phase of acidified aqueous acetonitrile and both eluted within three minutes.

For the mass spectrometric measurement in multiple reaction monitoring mode, positive and negative ionisation were compared. The most abundant product ions were seen in positive mode for Glu and negative mode for Asp, so these were adopted. With a modern mass spectrometer like the hybrid triple quadrupole-linear ion trap that was used, switching polarity is a routine operation and does not affect the instrument performance.

Pentadeuterated Glu and trideuterated Asp were tested as internal standards but the researchers were confounded by very low ionisation efficiency for the latter so it was dropped with Glu-d5 being adopted for both amino acids.

Using rat brain homogenates, no interferences from the tissue were observed in the chromatograms and the amino acids were measured with a lower limit of quantification of 10 ng/mL. With good accuracy, precision and recoveries, the method was extended to live diabetic rats because diabetes is a known risk factor for stroke.

Accurate amino acids levels in brain tissue

The animals were fed a high-fat diet and treated with streptozotocin to make them diabetic before their brains were sutured internally to induce the classical ischemia/reperfusion mode via the middle cerebral artery. After 120 minutes the rats were injected with saline or with nimodipine, a drug which protects the brain against ischemia nerve injury following ischemia/reperfusion. A sham-operated group was also involved as controls.

Reperfusion was continued for 48 hours, then the rats were sacrificed and the brain tissue was removed and homogenised for analysis, using Glu-d5 as the internal standard.

In the sham group, there was almost no brain damage and the levels of Glu and Asp were equivalent to the normal levels observed. After the 48-hour perfusion period in the ischemic rats, the concentrations increased initially then fell back near to those of the control group. However, those treated with nimodipine decreased the Glu level compared with the controls while the levels of Asp in all of the groups were the same.

The novel LC/MS method allowed the amounts of Glu and Asp to be measured with high accuracy and specificity. Compared with published LC/MS procedures, it reduced the total run time to 5 minutes and has the advantage over many HPLC methods of requiring no derivatisation. It holds great promise for the study of ischemia/perfusion as well as other conditions in which Glu and Asp concentrations are modified.

Related Links

Biomedical Chromatography 2014, 28, 1822-1827: "An optimized HPLC/MS/MS method for quantification of excitatory amino acids in rat hippocampus and its application in brain ischemia/reperfusion research"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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