What (?) is the source/scientific justification for the myth enunciated by:
Steve Goodall.
>One possible explanation (for a higher tryptophan concentration with
>increasing time >in an ultrafiltrate) (not scientifically proved) was
>that, as the ultrafiltrate passed >through the membrane, the equilibrium
>between bound and free in the remaining serum
>altered, and more tryptophan was released from protein, hence increasing
>the apparent free tryptophan.
and
Sten Öhman
>Yes, this effect should be seen in any method using ultrafiltration. There
>is always a dynamic equilibrium between free and bound substance and
>removal of free component e g by ultrafiltration will disturb this
>equilibrium and hence affect the result.
>
>Therefore only the very first portion of ultrafiltrate reflects the true
>free concentration. Hence you should use the largest possible volume of
>sample and use a very sensitive method needing smallest possible volume of
>ultrafiltrate.
These propositions are nonsense!
Imagine a vessel containing free and bound drug/hormone andserum binding
protein(s) in equilibrium. Imagine also dragging a net (i.e. a
semi-permeable membrane) through the vessel (thereby progressively
confining the protein(s) to a smaller volume) but allowing the free
drug/hormone to pass unhindered through the net. The equilbrium state DOES
NOT CHANGE. The free drug/hormone concentration will remain exactly the
same on both sides of the membrane, and also exactly the same as existed
before the net was dragged through the vessel. Thus dialysing 1 ml serum
against 10 mls buffer has exactly the same effect on the ambient free
drug/hormone concentration as diluting 1ml serum with 10 mls buffer.
(See, e.g . Oppenheimer JH and Surks M. Determination of free thyroxine in
serum: a theoretical and experimental analysis. J clin Endocrinol. Metab.
42, 98, 1964. See also Ekins, R. Measurement of free hormones in blood.
Endocrine Reviews: 11; 5-46, 1990. and Ekins, R. The science of free
hormone measurement. Proc UK NEQAS meeting 1998; 3; 35-59. )
In other words, the location of binding proteins within the overall
compartment is irrelevant.
One important implication is that attaching antibodies to a solid support
has no effect on the final equilibrium attained in an immunoassay system
(second order effects - such as a modification of antibody structure -
aside), albeit on and off reaction rates will be (equally) affected.
Ultrafiltration can be likened to passing a semi-permeable membrane
through serum, and has no effect on the equilibrium state.
These simple propositions can readily be mathematically proved on the back
of an envelope.
When, oh when, will biochemists finally come to understand the basic
implications of the mass action laws?
Roger Ekins
Molecular Endocrinology
UCL Medical School.
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