High resolution magic angle spinning (hr-MAS) NMR where a sample spinning rate of a few kHz or more is used has become a powerful tool for metabolic profiling of intact biological tissues. However, there are a few critical issues that nee be addressed in order for MAS NMR to be used widely in biomedical, clinical, and translational researches. First, hr-MAS technique is destructive due to the large centrifugal force associated with fast sample spinning. Second, the sample volume in an hr-MAS experiment is restricted to ~15 to 60 ml also due to a variety of technical challenges associated with fast spinning. The goal of our research is to develop a non-destructive, high resolution and high sensitivity MAS-NMR method that complements hr-MAS for metabolomics investigations. To reach our goal, we have formulated two specific Aims.
Aim 1 : Development of a non-destructive MAS NMR metabolomics technique by using slow sample spinning, including a slow-MAS probe and rotor position synchronized slow-MAS pulse sequences on a 500 MHz NMR spectrometer. The slow-MAS technology will be capable of high resolution and high sensitivity metabolic profiling on biological tissue samples with volume variable from as small as 200 nanoliters (nL) to as large as 1000 microliters (1.0 cm3) or more using a single probe. The nL capability will make it possible to follow the metabolic changes through a continued investigation on a single small laboratory animal, and ultimately on a patient, over a long period of time using minimally invasive tissue biopsy and blood samples. The micro-liter to cm3 capability will serve the wide spread need of metabolic profiling on intact biological tissues of variable sizes, thus enabling large scale metabolic profiling on intact tissues. We have successfully performed concept- proven experiment on a 300 MHz NMR spectrometer using a concept-proven slow-MAS NMR probe to justify our proposed research.
Aim 2 : Application of the nL slow-MAS method. We will apply the nL feature of the slow-MAS probe to continuously follow the metabolic changes using minimal invasive biopsy skeletal muscle and blood samples of 200 to 500 nL in volume on 8 obese C57BL/6 mice, and 8 normal C57BL/6 mice (controls) over ages 8 to 16 weeks to identify possible metabolite biomarkers that are related to obesity. At the end of the in life sampling, the mice will be sacrificed and whole organs will be studied using the cm3 feature of the probe. We will also carry out slow-MAS metabolomics studies on artery excised from an obese- accelerated atherosclerosis mouse model. Obesity has become a recognized risk factor for a variety of metabolic disorders, including in particular atherosclerotic cardiovascular diseases. However, the disordered metabolic pathways that contribute to obesity-accelerated atherosclerosis are not well established. This is mainly due to the difficulties of direct metaboli profiling on very small amount of tissue samples. Our nL slow- MAS capability will make the metabolic profiling on the intact artery excised from a diet-induced obese + atherosclerosis mouse model possible.
We propose to develop a non-destructive magic angle spinning metabolomics technique that is capable of high resolution and high sensitivity metabolic profiling on biological samples, in particular, on tissue samples with sample volume from as small as 200 nanoliters (nL) to as large as a milliliter or more using a single probe and using only a few minutes. If successful, this technique will enable large scale metabolic profiling on intact biological tissues of various sizes that will have wide application in biomedical, clinicl and translational researches.
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