Biomedical subjects

M J MacCoss

Publications and source records attributed to M J MacCoss.

7 recordsLinked to original sources

PubMed2001

Evaluation and optimization of ion-current ratio measurements by selected-ion-monitoring mass spectrometry.

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 analogues 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. We have used standard models of noise to develop 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. We demonstrate that our approach can predict the effect of signal intensity, ion-current ratio magnitude, and 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.

Ions↗

PubMed2001

Proteomics: analytical tools and techniques.

Scientists have long been interested in measuring the effects of different stimuli on protein expression and metabolism. Analytical methods are being developed for the automated separation, identification, and quantitation of all of the proteins within the cell. Soon, investigators will be able to observe the effects of an experiment on every protein (as opposed to a selected few). This review presents a discussion of recent technological advances in proteomics in addition to exploring current methodological limitations.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

Effects of estradiol and progesterone on body composition, protein synthesis, and lipoprotein lipase in rats.

Prior studies suggest that estradiol and progesterone regulate body composition in growing female rats. Because these studies did not consider the confounding effect of changes in food intake, it remains unclear whether ovarian hormones regulate body composition independently of their effects on food intake. We utilized a pair-feeding paradigm to examine the effects of these hormones on body composition. In addition, skeletal muscle protein fractional synthesis rate and adipose tissue lipoprotein lipase activity were measured to examine pathways of substrate deposition into fat and fat-free tissue. Female Sprague-Dawley rats [pubertal: 7-8 wk old; 190 +/- 0.5 (SE) g] were separated into four groups: 1) sham-operated (S; n = 8), 2) ovariectomized plus placebo (OVX; n = 8), 3) ovariectomized plus estradiol (OVX+E; n = 8), and 4) ovariectomized plus progesterone (OVX+P; n = 8). All ovariectomized groups were pair-fed to the S group. Body composition was measured using total body electrical conductivity. The relative increase in fat-free mass was greater (P < 0.01) in the OVX group (31 +/- 2%) than in the S (17 +/- 2%), OVX+E (18 +/- 2%), and OVX+P (22 +/- 2%) groups. The fractional synthetic rates of gastrocnemius muscle protein paralleled changes in fat-free mass: OVX had a higher (P < 0.05) synthesis rate (21 +/- 3%/day) than S (12 +/- 2%/day), OVX+E (11 +/- 2%/day), and OVX+P (8 +/- 1%/day) groups. Body fat increased in the S group (31 +/- 7%; P < 0.01), whereas the OVX groups lost fat (OVX: -10 +/- 7%; OVX+E: -15 +/- 7%; OVX+P: -13 +/- 7%). No differences in lipoprotein lipase were found. Our results suggest that estradiol and progesterone may regulate the growth of fat and fat-free tissues in female rats. Moreover, ovarian hormones may influence skeletal muscle growth through their effects on skeletal muscle protein synthesis.

Adipose Tissue↗

PubMed2001

Measurement of intracellular sulfur amino acid metabolism in humans.

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.

Adult↗

PubMed2001

Recovery of (13)CO(2) from infused [1-(13)C]leucine and [1,2-(13)C(2)]leucine in healthy humans.

Carbon (C) in the 1-position of leucine is released as CO(2) with the decarboxylation of alpha-ketoisocaproate (KIC). Carbon in the 2-position of leucine undergoes several additional metabolic steps before entering the tricarboxylic acid (TCA) cycle in the 1-position of acetyl-CoA, where it can be released as CO(2) or be incorporated into other compounds. This study examined the metabolic fate of C in the 2-position of leucine. We infused 11 healthy subjects with [1-(13)C]leucine and [1,2-(13)C(2)]leucine for 3.5--4 h to measure leucine kinetics and the oxidation of the tracers from enrichments of (13)C in blood and expired CO(2). The fraction of leucine infused that was oxidized (f(ox)) was used to define the degree of recovery of the (13)C label(s) for each tracer. As expected, leucine appearance (means +/- SE) did not differ between tracers ((13)C(1): 92.1 +/- 3.1 vs. (13)C(2): 89.2 +/- 3.2 micromol x kg(-1) x h(-1)) when calculated using plasma leucine enrichments as an index of intracellular enrichment. A small (3%) but significant (P = 0.048) difference between tracers was found when KIC was used to calculate leucine appearance ((13)C(1): 118.0 +/- 4.1 vs. (13)C(2): 114.4 +/- 4.5 micromol x kg(-1) x h(-1)). The value of f(ox) was 14 +/- 1% for [1,2-(13)C(2)]leucine and was lower than the f(ox) for [1-(13)C]leucine (19 +/- 1%). From the f(ox) data, we calculated that the recovery of the 2-(13)C label in breath CO(2) was 58 +/- 6% relative to the 1-(13)C label. These findings show that, although a majority of the 2-(13)C label of leucine is recovered in breath CO(2), a significant percentage (approximately 42%) is retained in the body, presumably by transfer to other compounds, via TCA exchange reactions.

Adult↗

PubMed1999

Measurement of homocysteine concentrations and stable isotope tracer enrichments in human plasma.

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 microM) and is bound to other thiols (e.g., cysteine in plasma proteins) via disulfide bonds. Existing methods for measuring homocysteine have difficulty in reducing and maintaining the reduction of homocysteine for measurement. We describe a GC/MS method that first reduces the disulfides in the physiological sample matrix and then immediately alkylates the free thiols with 4-vinylpyridine to prevent the reformation of the disulfide bonds. We use a 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 tert-butyldimethylsilyl derivatives. This method requires only 50 microL of plasma to measure homocysteine concentrations to 5 microM. Total homocysteine concentrations in plasma can be measured routinely from 0.5-mL samples with relative intra- and interday 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%.

Algorithms↗

PubMed1995

Direct database searching with MALDI-PSD spectra of peptides.

The analysis of matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) mass spectra of peptides by using the cross-correlation method for database searching is illustrated. MALDI-PSD mass spectra are shown to contain sufficient fragmentation information to uniquely identify the correct amino acid sequence from large protein databases (approximately 160,000 entries). A search employing the MALDI-PSD mass spectrum of a phosphorylated peptide that correctly identifies the amino acid sequence and the site of phosphorylation is also illustrated.

Amino Acid Sequence↗