Homocysteine is important because of its association with elevated levels of plasma homocysteine as an independent risk factor of heart disease. Because existing gas chromatography-mass spectrometry (GCMS) and HPLC methods were difficult to apply and assure complete and continued reduction of thiol-species in plasma and homocysteine concentration was difficult to measure precisely and sensitively enough to be able to obtain measurements of stable isotopic enrichments at tracer amounts, we have spent effort defining a new and novel method for measuring homocysteine by GCMS. The ultimate application is measurement of tracer enrichments in homocysteine with sufficient precision and sensitivity to be able to determine its rates of synthesis and disposal. The internal standard, [3,3,3',3',4,4,4',4',-2H8]homocystine is added to plasma samples to account for losses associated with the isolation, derivatization, and measurement of the natural homocysteine as previously used by other. We have modified this method in two ways: First, we use a novel reducing agent N,N'-dimethyl-N,N'-bis(mercaptoacetyl)hydrazine (DMH) for the reduction of disulfide bonds to reduced thiols. Secondly, the free thiols are then immediately alkylated with 4-vinyl pyridine to prevent permanently any reformation of the disulfide bridges. The plasma amino acids are separated and derivatized to form the t-butyldimethylsilyl derivatives as per our usual protocol. Using this new preparation scheme, we can measure amounts of homocysteine to <5 pmol and can determine plasma total homocysteine concentrations with an inter-day precision of 3.4% using 0.5-ml plasma samples. This method is also sensitive enough to determine enrichments of stable isotopic tracers (e.g. [1-13C]) with precisions typical of other amino acids (10.1 mole % excess tracer). This method will be used in our studies of sulfur amino acid metabolism in humans this year.

Project Start
1998-12-01
Project End
1999-11-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
35
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Scagnelli, Connor N; Howard, Diantha B; Bromberg, Mark B et al. (2018) Hydration measured by doubly labeled water in ALS and its effects on survival. Amyotroph Lateral Scler Frontotemporal Degener 19:220-231
Horne, Hisani N; Sherman, Mark E; Pfeiffer, Ruth M et al. (2016) Circulating insulin-like growth factor-I, insulin-like growth factor binding protein-3 and terminal duct lobular unit involution of the breast: a cross-sectional study of women with benign breast disease. Breast Cancer Res 18:24
Kien, C Lawrence; Matthews, Dwight E; Poynter, Matthew E et al. (2015) Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of ?-oxidation. J Lipid Res 56:1795-807
Gierach, Gretchen L; Patel, Deesha A; Falk, Roni T et al. (2015) Relationship of serum estrogens and metabolites with area and volume mammographic densities. Horm Cancer 6:107-19
Albert, Kimberly; Pruessner, Jens; Newhouse, Paul (2015) Estradiol levels modulate brain activity and negative responses to psychosocial stress across the menstrual cycle. Psychoneuroendocrinology 59:14-24
Bodelon, Clara; Heaphy, Christopher M; Meeker, Alan K et al. (2015) Leukocyte telomere length and its association with mammographic density and proliferative diagnosis among women undergoing diagnostic image-guided breast biopsy. BMC Cancer 15:823
Morris, Erin A; Hale, Sarah A; Badger, Gary J et al. (2015) Pregnancy induces persistent changes in vascular compliance in primiparous women. Am J Obstet Gynecol 212:633.e1-6
Miller, Mark S; Bedrin, Nicholas G; Ades, Philip A et al. (2015) Molecular determinants of force production in human skeletal muscle fibers: effects of myosin isoform expression and cross-sectional area. Am J Physiol Cell Physiol 308:C473-84
Kien, C Lawrence; Bunn, Janice Y; Fukagawa, Naomi K et al. (2015) Lipidomic evidence that lowering the typical dietary palmitate to oleate ratio in humans decreases the leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes. J Nutr Biochem 26:1599-606
Fox, James R; Gray, Weili; Koptiuch, Cathryn et al. (2014) Anisotropic tissue motion induced by acupuncture needling along intermuscular connective tissue planes. J Altern Complement Med 20:290-4

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