Following dissatisfaction from Clinicians, with transketolase results
not mirroring clinical symptoms we had a close look at thiamin assays
by HPLC and Thiochrome (Schultz et all 1972), over 20 years ago. Both
of these methods were hampered by insensitivity and long winded pre
assay extraction which essentially precluded them when large number
of requests were received. The thiochrome method involved a heat
hydrolysis step and as thiamin is heat sensitive (particularly at
neutral or alkaline pH), we had some reservations.
We eventually settled for a microbiological assay. By creating a
chloramphenicaol resistant strain of the test organism (Lactobacillus
fermenti), this allowed chloramphenicol to be added to the assay
medium to suppress bacterial contamination. This eliminated the need
for sterile reagents or aseptic procedures and enabled the tests to
be run on automated equipment (Gilson diluting platform) with highly
reproducible results. Results can be available within 24 hours if
required.
The organism is sensitive to around 2.5 nmol/L which makes
it suitable for assaying both serum and red cell thiamin (lower limit
of ref range for serum 11.3 nmol/L as derived from almost 2,000
normal subjects). This is akin to the folate situation in which the
serum levels give an indication of recent intake whereas red cell
levels give an indication of body stores (intake over the last
several weeks) - thiamin does not readily cross the red cell
membranes.
Thiamin is predominantly in the pyrophosphate form which the organism
is well able to utilise.
We have been running this assay for over 20 years (around 100-150
tests per week), with minimal problems.
Best wishes
Graham
Graham ICKE
A/Principal Scientist
Division of Laboratory Medicine
Royal Perth Hospital
Wellington St
PERTH
Western Australia
6001
Tel: (08) 9224 2265
Fax: (08) 9224 2491
Private e.mail: [log in to unmask]
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