Dear All,

Thanks very much to all who replied to my previous question about transferrin saturation, here is the summary of the replies, I apologise for the delay.



Question:

Dear all,



I’d very much appreciate help regarding how to calculate transferrin saturation (%) using measured molar concentrations of serum iron and transferrin.  I have found an equation, but only in ‘lecture notes’ and the like on the internet, and I would like to check this out…

We are considering adding this in to our iron studies report with serum iron, transferrin and ferritin.  Is this in keeping with others out there?



Assuming each transferrin molecule can bind two Fe3+ ions transferrin saturation can be calculated  ~

Transferrin saturation % = (iron mol/L x 100)/(transferrin mol/L x 2)



Thank you very much, best wishes, Lou



Replies:

1.  Obviously not facing demands to make testing appropriate - saturation not required in most ?Fe deficiency. Guidance here [from haematologists] is that ferritin should be used as frontline test for Fe Deficiency



2.  I agree that ferritin should be used for iron deficiency, however Transferrin saturation is useful for the investigation of possible haemochromatosis. The difficulty with the calculation is that the molecular weight of transferrin is not clearly defined, therefore the conversion from g/L to mol/L can be variable depending on what figure you use.



3.  I agree with your equation for transferrin saturation but the crux of the matter is what molecular weight to use to convert gravimetric transferrin concentration to molar units.  There seems to be quite a wide range of figures used – and some labs “fiddle” their equation so that the calculated TIBC agrees better with EQA.



4.  Here in Wales we’re having to grapple with this problem as the introduction of a national LIMS requires us to all work in the same way as far as is possible.  We are fairly certain that transferrin from North Wales has the same molecular weight as that in the South!



I would suggest using a molecular weight for transferrin of 79,570 (MacGillvray RTA et al, The Primary Structure of Human Serum Transferrin. J Biol Chem 1983; 258: 3543-53) which is in close agreement with a later measurement (79,680 by  Zhao YY et al, Intact human holo-transferrin interaction with oxaloplatin. Rapid Commun Mass Spectrom 2005; 19:1956-1962).







The calculation is:



TF saturation = (serum iron (umol/L)/total iron binding capacity (umol/L)) x 100



Total iron binding capacity (umol/L) = Transferrin (umol/L) x 2        (assumes transferrin binds two ions of iron per molecule)



Transferrin (umol/L) = (Transferrin (g/L) / Mol wt transferrin) x 10^6



Final calculation is



            TF saturation (%) = serum iron [umol/L] x 100 / (Transferrin [g/L] x10^6 x2/79,570)



Which simplifies down to



            TF saturation (%) = serum iron [umol/L] x 3.98 / Transferrin [g/L]



(i.e. factor (3.98) = mol wt transferrin  x 100 / 2 x 10^6)



We tend not to measure it if ferritin is <15 ug/L even if it has been requested.  If requested then we will assay it if ferritin is between 15 & 100, or above 300 – the logic being that below 100 it is possible for an acute phase response/malignancy/chronic illness to increase ferritin even in the face of iron deficiency (I also accept the logic that transferrin is a negative acute phase protein and therefore transferrin saturation is not likely to be very helpful – so we’re not completely logical!) and if ferritin is above 300 we need to exclude the possibility of iron overload.



5.  Transferrin in µmol/L looks very unfamiliar to me.

Do you report transferrin as molar concentration to requesting physicians ? Does it also apply for ferritin (this would confuse me even more) ?

What about other proteins (albumin, antitrypsin or similar) ?



6.  I agree with you. Maybe I am a bit naïve, but surely molar quantities of any protein is technically meaningless? As most circulating proteins are not chemically homogenous, but have varying degress of modification, they must have an indefinite molar mass - converting mass to moles is not sensible.



Or have I missed something?



7.  µmol/L transferrin equals µmol/L of transferrin iron-binding sites divided by 2



transferrin (g/L) x 12.6 ==> transferrin (µmol/L)



(source: Ref ranges for adults and Children 8th edition 2004, Heil W., Koberstein R. and Zawta B.)



8.  I understand that you report transferrin in umol/L and that your manufacturer uses a molecular mass of 79,570 to convert from mass to molar units.  The conversion factor depends on what you believe the molecular mass to be.  Recently I’ve looked at the equations used within Wales for the calculation of transferrin saturation and the apparent molecular mass for transferrin used in those calculations ranged from 75,188 to 90,909 which goes some way to explaining variability in transferrin saturation on EQA returns!  With the pending introduction of a national pathology LIMS in Wales we have decided to standardise on a figure of 79,570 (MacGillvray RTA et al, The Primary Structure of Human Serum Transferrin. J Biol Chem 1983; 258: 3543-53 ) which is in close agreement with a later measurement of 79,680 by Zhao YY et al, Intact human holo-transferrin interaction with oxaloplatin. Rapid Commun Mass Spectrom 2005; 19:1956-1962.



Slightly off the original thread – but could we as a community agree on an equation for the calculation of transferrin saturation? i.e. all use the same molecular mass in the calculation.



9.  This is very topical.



We (DH, CfH, Prof Bodies incl ACB)  are currently trying to agree standard units for pathology reporting across the NHS as there are considerable nmbers of errors visible in current electronic reporting messages.



If you wish to see current "draft" recommendations or to contribute to the work please take a look at



www.ychi.leeds.ac.uk/pmipunits<http://www.ychi.leeds.ac.uk/pmipunits> (cut and paste if the link doesn't click through)



10.  We use Transferrin saturation (%)= (Iron umol/L /Transferrin g/L ) x 3.98



11.  If there are two eggs in an eggbox which has a capacity to hold six eggs then the box is 30%full; you don't multiply the box by it's capacity. Hence, % saturation is ([iron]/[transferrin]*100. The reference range will depend on the ratio between transferrin expressed as 'g' and 'mols'.



12.  Except...



If you've measured both eggs and eggboxes in molar units. You'd then have 1 eggbox, 2 eggs and ([eggs]/[eggbox] x100) would give you 200%.



13.  IRON STUDIES



Iron:

8.1 - 28.6 µmol/L (male)

5.4 - 28.6 µmol/L (female)

Transferrin: 2.0 - 3.6 g/L

Transferrin binds 2 atoms of iron when fully saturated.

Transferrin % saturation = (2 x iron x 0.07957)/transferrin

Transferrin index >1 compatible with iron overload - ?haemochromatosis

I hope this is helpful,



14.  There is little point in measuring iron (poisoning excluded and there is no need to measure TIBC in a case of poisoning), transferrin is an acute phase reactant which complicates the situation, then to make matters worse there is anaemia of chronic disorder.



If it's necessary to assess an individual's iron status then we should measure' hepcidin'; those of us old enough will remember reading the elctrolyte section in books referring to the 'third factor' before natriuretic hormones were discovered.



15.  So why do we not all measure UIBC/TIBC and iron to produce a real saturation ?



16.  When we assay serum iron concentration, usually most of the iron is bound to transferrin, but some is not (some may be bound to other proteins, some may be free or unbound, and the magnitude of these components is unknown).



When we determine UIBC or TIBC, we are determining the uptake of iron by proteins in the serum, mostly by transferrin, but some by other proteins (and the magnitude of this component is unknown).





So a transferrin saturation calculated from these measurements is no more “real” than one calculated from serum iron and transferrin calculations.



17.  Derived TIBC

We report:

•       iron

•       dTIBc (from the measured transferrin)

•       % saturation.

We measure transferrin in g/L and then convert it to micromol/L.  For that you need the m.w.  we use 87,900 but it would appear Wales use 79,570



Thus

1 g/L Transferrin = 11.4 micromoles/L using 87,900 or

1 g/L Transferrin = 12.6 micromol/L using 79,570.



As there are 2 iron binding sites, then the TIBC is twice the concentration in micromoles.



Thus 1

1 g/L Transferrin = 11.4 micromol/L = dTIBC of 22.8 micromoles/L using 87,900  or

1 g/L Transferrin = 12.6 micromol/L  = dTIBC of 25.2 micromoles/L using 79,570.



% saturation is (iron/dTIBC)*100% with both iron and dTIBC in micromol/L, which is the same as your calculation (except your calculation uses transferrin and not dTIBC)



Thus the key is what molecular weight to use.  The Welsh are a good bunch and it may be that I will re look at the m.w. that we use. However we look good in NEQAS albeit a bit high (even though they do not score it).  If we were to use a higher conversion factor, a la Wales, then our results will be even higher.  So perhaps we are using the better m.w.







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