of tryptophan due to cytokine induction of GTP-cyclohydrolase-1, nitric oxide synthetase and indolamine dioxygenase activities.
HPLC profiling of neurotransmitters, vitamins, cofactors, and infl am-matory markers in CSF, elucidating the clinical biochemistry of all these analytes within the brain and understanding the many interactions between them, allows identification of genetic diseases, helps to distinguish between metabolic and neurodegenerative brain diseases, can be more sensitive than brain MRI in detecting specific neuronal losses and is extremely useful for detecting and monitoring inflammatory brain diseases.
doi:10.1016/j.cca.2010.02.040
Symposium VIII
Vitamin A: Thefirst vitamin!
R.F.Greaves, L.M. Jolly, G.A. Woollard, K.E. Hoad, T.A. Walmsley, S. Briscoe, L.A. Johnson, C. Salonikas, J.P. Gill
The AACB Vitamins Working Party, The Australasian Association of Clinical Biochemists, Mt Lawley, WA, 6929, Australia
http://www.aacb.asn.au/web/Scientific_&_Regulatory_Affairs/ Working_Parties/Vitamins/
Email:Ronda.greaves@rch.org.au
Historically, vitamin A deficiency is associated with the oldest genetic disease known to man, cysticfibrosis. Thefirst description of an essential growth factor present in food occurred in the early 20th century as the“vital amine”. Quickly it became clear that there was more than one growth factor and the term“vitamin A”was coined for the fat soluble vital amine and“vitamin B”for the water soluble form. In 1960 vitamin A was recognized to exist in 3 parent forms retinal, retinol and retinoic acid. Now the term vitamin A is used as“the generic description for retinoids exhibiting qualitatively the biological activity of retinol”.
Today vitamin A deficiency is the leading vitamin deficiency worldwide and listed by the WHO as a major health issue of epidemic proportions in some developing countries. Typically chronic deficiency of vitamin A is described in association with preventable childhood night blindness. Deficiency is also linked to changes in epithelial cell differentiation and immune function which is attributed to causing the increased infectivity and mortality rates seen in malnourished children. Worldwide 190 million pre-school children are thought to be vitamin A deficient.
Serum or plasma retinol is routinely measured by HPLC to assess vitamin A status in medical testing laboratories. Sample preparation usually involves precipitation of proteins with methanol or ethanol (with retinol acetate as the internal standard included) followed by extraction of the aqueous phase by hexane. The organic layer is evaporated under nitrogen and the residue redissolved for subsequent chromatographic analysis. The separation is usually performed with an alkane bonded silica (typically, octadecylsilane) column. Vitamin A is quantitated spectrophotometrically atλmax325nm. Clinical deficiency of vitamin A is deemed to be≤0.35 µmol/l. To provide consistent and accurate results between laboratories it is essential that the assay methodology is robust and uniformly standard-ized. To this end the AACB Vitamins Working Party is developing a consensus method to guide laboratories in the best practice for analysis of this important vitamin.
doi:10.1016/j.cca.2010.02.041
Symposium VIII
LC/MS/MS measurement of vitamin D3 and D2: Present and future
B.C.McWhinney
Chemical Pathology, Pathology Queensland, Brisbane, QLD 4029, Australia Email:brett_mcwhinney@health.qld.gov.au
Introduction
The demand for serum 25-hydroxyvitamin D3 analysis has increased dramatically in recent years. A number of studies have linked Vitamin D3 deficiency to a number of disease states, including heart disease, multiple sclerosis and some cancers. Liquid chromatography and tandem mass spectrometry (LC MS/MS) methods for Vitamin D3and D2have now been adopted by many clinical laboratories. The advantages of LC MS/MS methods for Vitamin D3 and D2 over immunological based methods include greater analytical specificity and sensitivity and the ability to determine both compounds of interest in a single analytical run. Several different extraction procedures including liquid–liquid extraction and solid phase extraction have been described for LC–MS/MS.
Methods
Serum (100 µl) was extracted using liquid–liquid extraction and the dried extract dissolved in mobile phase prior to analysis on the UPLC MS/MS. The analytes were separated on an Acquity BEH C8 column (2.1 × 50 mm) with a water/methanol/ammonium acetate gradient. A Waters Quattro Premier tandem mass spectrometer was used to quantitate both analytes by monitoring two transitions for each.
Results
The analytical run time was 5 min. The limit of quantitation for Vitamin D3wasb2 nmol/l and linearity extended to 1500 nmol/l. The intra-assay imprecision was less than 3% and the inter-assay impreci-sion less than 5.0%. Comparison with an automated immunoassay method produced the following correlation y= 0.749x + 19.07 (r2= 0.903). External proficiency program samples were also assayed with very good agreement with target values (y= 1.046x−1.1, r2
N0.994).
Conclusions
LC MS/MS offers significant advantages over other methods due to reduced sample volume requirements and increased sensitivity and specificity. Automating the sample extraction is the next challenge to be tackled.
doi:10.1016/j.cca.2010.02.042
Symposium VIII
Coenzyme Q10 in health and disease
P.M.George, R.J. Mackay, S.L. Molyneux, J.M. Young, M. Lever, C.M. Florkowski
Clinical Biochemistry, Canterbury Health Laboratories, Christchurch, New Zealand
Email:Peter.george@cdhb.govt.nz
