University of Vermont

Matthews' Group Research

Matthews' analytical chemistry program

Description of Research in Analytical Chemistry:

My research has used stable isotopically labeled tracers and kinetic models to answer questions of human physiology and biochemistry.  The research program of my group in chemistry focuses on developing new methods and techniques of mass spectrometry to measure stable isotopes in biological molecules and mathematical and computer models to interpret the stable isotope tracer kinetic data obtained from biological samples.

Photo of DE Matthews
Photo from 2001

Much of our research is focused upon amino acid and protein metabolism in humans.  Students are encouraged as part of their thesis work to apply methods developed to clinical studies of metabolism in humans.  Many simple metabolic questions have never been answered in humans.  We do not know which pathways regulate the metabolism of several important amino acids in humans.

We do not know how protein and amino acid metabolism is regulated in the body to maintain protein stores or why the body accelerates oxidation of amino acids in states of stress, trauma, or sepsis using protein stores to the point of being life threatening.

Mass spectrometry has been widely applied for compound identification, but precise measurement of isotopes in biological compounds has received less attention.  Gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and isotope ratio mass spectrometry instrumentation are all available and are used for research in our group to measure stable isotope ratio tracers in biological samples.

Work in Analytical Chemistry:

MJ MacCoss, NK Fukagawa & DE Matthews: Measurement of homocysteine concentrations and stable isotope tracer enrichments in human plasma. Anal. Chem. 71: 4527-4533, 1999.

Scheme to Reduce and Modify Protein-Bound Homocysteine for Measurement by GCMS

Hcy derivatization scheme
Elevated levels of plasma homocysteine have been established as an independent risk factor for cardiovascular disease.  Homocysteine is in low concentration in plasma (5-15 µM) and is bound to other thiols (e.g. cysteine) in plasma proteins via disulfide bonds.  Methods to measure homocysteine have difficulty in reducing and maintaining the reduction of homocysteine for measurement.  This paper describe a GCMS method that uses the reducing agent, N,N'-dimethyl-N,N'-bis(mercaptoacetyl)hydrazine, for the reduction of disulfides to thiols.  The free thiols are then alkylated with 4-vinyl pyridine to prevent the reformation of the disulfide bonds.  We use the deuterated internal standard, [3,3,3',3',4,4,4',4',-2H8]homocystine, to account for losses associated with the isolation, derivatization, and measurement of the natural homocysteine.  The amino acids are separated and derivatized to form the t-butyldimethylsilyl derivatives.  The method can detect homocysteine to <5 pmol per single injection and requires only 50 µl of plasma to measure a 5 µM sample. Total homocysteine concentrations in plasma is measured routinely from 0.5-ml samples with relative intra- and inter-day precisions of 1.3 and 4.0% respectively.  This method is sensitive enough to determine tracer enrichments of [1-13C]homocysteine with a detection limit of <0.3 mol % excess and an average tracer precision of 0.6% and has been used by us in subsequent metabolic studies.

MJ MacCoss, MJ Toth & DE Matthews: Evaluation and optimization of ion-current ratio measurements by selected-ion-monitoring mass spectrometry. Anal. Chem. 73: 2976-2984, 2001.

Stable isotopically labeled compounds are regularly used as internal standards in quantitation and as tracers of in vivo metabolism.  In both applications, the ratio of unlabeled to labeled analogs is determined from an ion-current ratio measured by a mass spectrometer.  The precision of the ion-current ratio measurement defines the detection limit for quantitation and for tracer enrichment measurement.

Ion current RSD

We have developed a method that evaluates ion-current ratio noise (i) that varies with the signal intensity and (ii) that is signal independent.  This model produces a simple equation that defines the ion-current ratio precision using constants that can be evaluated empirically from the measurement of two ion-current ratios from a single standard measured multiple times.  This approach predicts the effect of signal intensity, ion-current ratio magnitude, and both internal standard or tracer choice on the measurement precision.  The standard deviations predicted by our method are shown to equal standard deviations of samples measured experimentally.  This method allows a simple evaluation of a mass spectrometry system and can define the precision of new quantitation and tracer methods.

The above figure shows the effect of ion-current ratio (R) on the ion-current ratio precision (Y-axis).  Standards of leucine with varying amounts of [1-13C]leucine and [5,5,5-2H3]leucine were measured by EI-GCMS.  The ratios of ions ([M+1]/M (circles), [M+2]/M (triangles), [M+3]/M (diamonds), and [M+4]/M (inverted triangles)) were measured in replicate for each sample.

The Y-axis plots the relative deviation of each measurement from the mean value of each sample.  The method was used to predict the error characteristics of the GCMS system.  Predicted contour lines for 1 SD and 2 SD are shown in the figure as broken and solid lines, respectively.  If our method accurately models the noise of the ion-current ratio measurement then 68% of the points in the figure should fall within 1 SD and 95% within 2 SD.  Experimentally, 125 of 175 observations (71.4%) lie within the 1SD contours, and 167 of the 175 points (95.4%) lie within the 2 SD contours.

TS McIntosh, HM Davis & DE Matthews: A liquid chromatography-mass spectrometry method to measure stable isotopic tracer enrichments of glycerol and glucose in human serum. Anal. Biochem. 300: 163-169, 2002

LCMS of glucose & glycerol

Stable isotopes are commonly used as tracers for the measurement of glycerol and glucose kinetics in metabolic studies.  Traditionally, the analysis of these isotopes has been performed using gas chromatography-mass spectrometry (GCMS), which requires that the analytes first be derivatized.  The derivatization process adds considerable complexity to the method.  Liquid chromatography-mass spectrometry (LCMS) can measure many metabolites directly with limited sample preparation.  We present a novel analytical method for the measurement of [1,1,2,3,3-2H5]glycerol (d5-glycerol) and [6,6-2H2]glucose (d2-glucose) isotopic tracer enrichments in human serum in a single run by LCMS using electrospray ionization (ESI).  After a simple extraction step, the sample is separated isocratically by HPLC, and the isotopes measured using positive electrospray ionization with selected ion monitoring of the sodium-adduct ions.  The method is linear over a wide range of d2-glucose and d5-glycerol enrichments.  The within day standard deviation of measurement of serum samples was 0.05 mole % excess (MPE) for d2-glucose and 0.25 MPE for d5-glycerol.  The variation of tracer enrichment among days was about double that measured within a day.

The figure above shows the selected ion monitoring LCMS chromatogram of a human serum sample for glycerol & its 2H-labeled tracer and glucose and its 2H-labeled tracer.  The mass/charge (m/z) of the ions measured are indicated in the figure.  Only the 2 ions for glycerol and its label were measured during the early portion of the chromatogram when glycerol elutes and only the 2 ions for glucose and its label were measured during the second half of the run.

Last modified May 12 2009 02:31 PM

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