With regards to EFA analysis, thanks for posting that information Bart.

It goes to show that there is a large difference of opinion as to how
EFA's should be processed and quantitated!

1) I have tried both methanol/HCl and boron trifluoride over the years
and my conclusion is that BF3 is a better way of doing the
methylation, both in terms of yield and reliability

2) I would be cautious of any method using BHT as an antioxidant. Many
papers suggest that this produces artifactual peaks which interfere
with FAME analysis

3) As with any analysis, quantitative results should be based on
dose-response curves using the analyte being estimated. In the case of
EFA's, the approach outlined (calcuating from peak areas and assuming
the same response factor for each peak) can produce serious errors for
the low abundance fatty acids,

4) I understand the value of looking at relative percentages of fatty
acids in the final report - but this should be derived from the
quantitative analysis, and not the other way around

5) What Prof Muskiet says about the value of red cell membrane
analysis as opposed to plasma analysis is quite correct. However, we
sometimes do plasma analysis to assess compliance and absorption,
especially in children.

Any comments?

Nick Miller
London

> From: Clinical biochemistry discussion list
> [mailto:[log in to unmask]] On Behalf Of Bart Ballieux
> Sent: 07 December 2010 20:12
> To: [log in to unmask]
> Subject: Re: Essential Fatty Acids
>
>
>
> Dear Dr Wright,
>
>
>
> Please find enclosed below a reaction from prof Muskiet of Groningen
> University.
>
> You have already been in contact with Dr Vaz of AMC.
>
>
>
> Best regards,
>
>
>
> Bart Ballieux
>
> Dr.Ir. B.E.P.B. Ballieux
> Klinisch Chemicus Endocrinoloog
> CKCL, E2-P
> Leids Universitair Medisch Centrum
> Postbus 9600
> 2300 RC  Leiden
> Tel: 071-5262165/2278
> Fax: 071-5266753
> email: [log in to unmask]
>
> Dear Dr. Wright,
>
>
>
> Plasma is not the preferred compartment for establishment of essential fatty
> acid (EFA) status; in plasma one deals with fatty acids in various lipids
> (TG, PL and CE), located in various lipoproteins, each with different half
> lives. The RBC fatty acid profile provides you with the intermediate term
> EFA status and the isolated omega-3 status. RBC fatty acids on the other
> hand are convined to membrane PL and the RBC half live is usually 35 days
> (cave hemoglobinopathies!).
>
>
>
> The protocol for RBC fatty acid profiling with capillary GC-FID that we
> follow in Groningen is briefly as follows: Take EDTA blood. Wash the RBC
> three times with 0.9% saline. Centrifuge each time 10 min at 800
> g. Resuspend the washed RBC to a 50% Ht in 0.9% saline. Transfer 200 uL of
> the 50% hematocrit suspension to a Sovirel tube (Tevlon cap!). The Sovirel
> tubes contains 2 mL methanol/ HCl (5:1,v/v) and 5 mg butylated
> hydroxytoluene (antioxidant). This mixture is stable for years at room
> temperature; store preferably in the dark. You can now send to a laboratory
> that performs the GC profiling like this: In the laboratory: heat for 4 h at
> 90 C in a heating block. After cooling extract with 2*2 mL hexane, evaporate
> hexane to dryness, add  200 uL hexane and inject 2 uL into the GC. For RBC
> fatty acid composition in g%: measure peak areas and quantify by assuming
> that weight amounts give rise to equal peak areas.
>
>
>
> For interpretation the laboratory needs cut-off values for establishment of
> EFA deficiency, isolated omega-3 deficiency and omega 3/DHA deficiency. We
> use: 0.46 mol% 20:3w9 for EFAD, 0.068 mol/mol 22:5w6/20:4w6 for
> w3-deficiency, 0.22 mol/mol 22:5w6/22:6w3 for w3/DHA-marginality and 0.48
> mol/mol 22:5w6/22:6w3 for w3/DHA-deficiency. See Fokkema-MR et al. PLEFA
> 2002 for reference values. See Muskiet-FA et al. J Chromatogr Biomed Appl
> 1983 for original analytical method.
>
>
>
> Hope that the above is of some value to your question.
>
>
>
> Best wishes,
>
> Frits Muskiet
>
> Dr. Frits A.J. Muskiet
> Professor of Pathophysiology and Clinical Chemical Analysis
> Laboratory Medicine, CMC-V, Room Y1.147, EA22
> University Medical Center Groningen (UMCG)
> P.O. Box 30.001, 9700 RB Groningen
> The Netherlands
> Tel. +31 50 361 2733 or 9228
> Fax. +31 50 361 2290
> Email. [log in to unmask]
>
>
>
>
>
> ________________________________
>
> From: Clinical biochemistry discussion list
> [mailto:[log in to unmask]] On Behalf Of Wright Katherine
> Sent: donderdag 2 december 2010 11:55
> To: [log in to unmask]
> Subject: Essential Fatty Acids
>
> I was wondering whether anyone has any experience of sending samples to
> Amsterdam Medical Centre for erythrocyte essential fatty acids ? Do you have
> protocols for washing the cells prior to sending the sample, or do you
> courier the whole blood sample prior to any sample prep ? Is there a
> particular person to contact in Amsterdam ?
>
>
>
> As far as I am aware nowhere in the UK offers the assay anymore although
> plasma essential fatty acids are measured by the lab at West Park Hospital,
> Epsom. If anyone knows any different please let me know.
>
>
>
> Thanks,
>
>
>
> Katherine Wright
>
>
>
> Principal Biochemist
>
> Alder Hey Childrens Hospital
>
> Liverpool, UK
>
>
>
> Tel : 0151 2525486
>
>
>
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