Vitamin A The first vitamin
Abstract / Clinica Chimica Acta 411 (2010) 896–914
of tryptophan due to cytokine induction of GTP-cyclohydrolase-1, nitric
oxide synthetase and indolamine dioxygenase activities.
HPLC profiling of neurotransmitters, vitamins, cofactors, and inflammatory 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: The first 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, cystic fibrosis. The first 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 at λmax325nm.
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 standardized. 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
907
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 D3 and D2 have 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 D3 was b2 nmol/l and linearity extended to 1500 nmol/l. The
intra-assay imprecision was less than 3% and the inter-assay imprecision 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,
r2N0.994).
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
of tryptophan due to cytokine induction of GTP-cyclohydrolase-1, nitric
oxide synthetase and indolamine dioxygenase activities.
HPLC profiling of neurotransmitters, vitamins, cofactors, and inflammatory 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: The first 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, cystic fibrosis. The first 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 at λmax325nm.
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 standardized. 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
907
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 D3 and D2 have 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 D3 was b2 nmol/l and linearity extended to 1500 nmol/l. The
intra-assay imprecision was less than 3% and the inter-assay imprecision 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,
r2N0.994).
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