UVM Theses and Dissertations
Format:
Print
Author:
Luo, Yue
Dept./Program:
Chemistry
Degree:
MS
Abstract:
The goal of this thesis research was to develop and optimize the Europa 20-20 gas chromatograph-pyrolysis-isotope ratio mass spectrometry (GC-P-IRMS) to measure deuterium enrichment in amino acids. The modified system is expected to be applied to slow turnover protein metabolism studies that use deuterium labeled tracers. Isotopically labeled amino acids (tracers) are often used in combination with kinetic models to study human protein metabolism. The basic concept of tracer study is simple: after being infused into the human body, tracers mix with circulating free amino acids, and over time are incorporated into body proteins. The protein synthesis rate then can be determined by measuring the rate of the tracer incorporation into the protein. The ¹³C, ²H or ¹⁵N labeled tracers are commonly used in protein tracer studies. The key factor that discourages wider use of ²H labeled amino acid tracers is the large tissue or blood sample size required for high precision isotope ratio measurement by mass spectrometry.
The instrument modifications of the Europa Hydra 20-20 GC-P-IRMS served two purposes. One is to simplify the system and make it more specific for deuterium enrichment analysis, and the modifications jnclude (I) removal of the redundant second GC, and (II) elimination of the solvent diversion step. The other is to improve the system sensitivity in order to reduce the sample size and the modifications include (III) optimization of the pyrolysis temperature, (IV) increasing the restriction at the isolation valve to force more flow entering the IRMS, and (V) removal of the second helium makeup gas. These modifications successfully increased the system sensitivity about 3 - 4 times.
To optimize the modified GC-P-IRMS system for ²H₃-leucine enrichment of serum albumin measurement, (1) the chromatographic conditions were optimized for leucine deuterium enrichment analysis, and (2) multiple hydrolyzed bovine serum albumin (BSA) samples with leucine concentration ranging form 1 - 10 mM were analyzed to optimize the injecting sample size and estimate the system sensitivity and precision. The results showed that 4-5 nmol leucine injection was enough for reproducible and reliable deuterium enrichment measurements, and the modified system was able to detect deuterium enrichment as low as 5 - 10 ppm.
To validate the modified system for a slow turnover protein study, the fractional synthesis rate (FSR) of human serum albumin (HSA) was determined by measuring the rate of ²H₃-leucine incorporation into HSA using the modified GC-P-IRMS. Five patients' ²H₃-leucine enriched plasma albumin samples were measured and the synthesis rate of albumin was determined. The measured FSR of albumin was 8.38 (± 0.42) %/day, which agrees with the literature. The precision of the measured albumin FSR was 11.2%, characterized in terms of coefficient of variance (CV), and it is much better than the average precision in the literature (29%). The precision of the albumin FSR measurements on the same patient is very low (0.4%), indicating that the instrument measurements are of high precision, and the major cause of the albumin FSR variance among the subjects shall be greatly attributed to the difference between biological samples.
Patterson (1997) suggested that in a regular tracer study of slow turnover proteins, such as muscle proteins and collagen, the tracer to tracee ratio is approximately 50 - 1000 ppm. Using the test model in this dissertation (²H₃-leucine tracer and N-acetyl methyl ester derivatives), then the 50 - 1000. ppm tracer to tracee ratio equals to 9 - 176 ppm deuterium enrichment. The modified Europa Hydra 20-20 GC-P-IRMS system was demonstrated to be able to measure deuterium enrichment as low as 5 - 10 ppm. For its highprecision, sensitivity and robustness, the modified system shall be able to be applied in slow turnover protein tracer studies.
The instrument modifications of the Europa Hydra 20-20 GC-P-IRMS served two purposes. One is to simplify the system and make it more specific for deuterium enrichment analysis, and the modifications jnclude (I) removal of the redundant second GC, and (II) elimination of the solvent diversion step. The other is to improve the system sensitivity in order to reduce the sample size and the modifications include (III) optimization of the pyrolysis temperature, (IV) increasing the restriction at the isolation valve to force more flow entering the IRMS, and (V) removal of the second helium makeup gas. These modifications successfully increased the system sensitivity about 3 - 4 times.
To optimize the modified GC-P-IRMS system for ²H₃-leucine enrichment of serum albumin measurement, (1) the chromatographic conditions were optimized for leucine deuterium enrichment analysis, and (2) multiple hydrolyzed bovine serum albumin (BSA) samples with leucine concentration ranging form 1 - 10 mM were analyzed to optimize the injecting sample size and estimate the system sensitivity and precision. The results showed that 4-5 nmol leucine injection was enough for reproducible and reliable deuterium enrichment measurements, and the modified system was able to detect deuterium enrichment as low as 5 - 10 ppm.
To validate the modified system for a slow turnover protein study, the fractional synthesis rate (FSR) of human serum albumin (HSA) was determined by measuring the rate of ²H₃-leucine incorporation into HSA using the modified GC-P-IRMS. Five patients' ²H₃-leucine enriched plasma albumin samples were measured and the synthesis rate of albumin was determined. The measured FSR of albumin was 8.38 (± 0.42) %/day, which agrees with the literature. The precision of the measured albumin FSR was 11.2%, characterized in terms of coefficient of variance (CV), and it is much better than the average precision in the literature (29%). The precision of the albumin FSR measurements on the same patient is very low (0.4%), indicating that the instrument measurements are of high precision, and the major cause of the albumin FSR variance among the subjects shall be greatly attributed to the difference between biological samples.
Patterson (1997) suggested that in a regular tracer study of slow turnover proteins, such as muscle proteins and collagen, the tracer to tracee ratio is approximately 50 - 1000 ppm. Using the test model in this dissertation (²H₃-leucine tracer and N-acetyl methyl ester derivatives), then the 50 - 1000. ppm tracer to tracee ratio equals to 9 - 176 ppm deuterium enrichment. The modified Europa Hydra 20-20 GC-P-IRMS system was demonstrated to be able to measure deuterium enrichment as low as 5 - 10 ppm. For its highprecision, sensitivity and robustness, the modified system shall be able to be applied in slow turnover protein tracer studies.