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Letters to the Editor |
1 Department of Clinical Biochemistry, Southend Hospital, Westcliff-on-Sea, Essex, UK
2 Department of Clinical Biochemistry, Kent and Canterbury Hospital, East Kent Hospitals NHS Trust, Canterbury, Kent UK
aAddress correspondence to this author at: Department of Clinical Biochemistry, Kent and Canterbury Hospital, East Kent Hospitals NHS Trust, Canterbury, Kent CT1 3NG United Kingdom. Fax 32-1-227-783077; e-mail: Edmund.lamb{at}ekht.nhs.uk.
To the Editor:
In contrast to Van Dieijen-Visser’s group, who concluded that in patients with kidney failure the presence of fragments of circulating cardiac troponin T (cTnT) led to increased cTnT concentrations (1)(2), our studies led us to conclude that the increased concentrations were attributable to free, intact cTnT(3). Michielsen et al. suggest that our conclusion is erroneous because we used a partially purified cardiac troponin (cTn) standard preparation that was not properly characterized and because the analysis of our gel filtration chromatography (GFC) results was incorrect.
With respect to the first point of Michielsen et al., we used a commercially available extract of human heart tissue containing cTn at high concentration (SCIPAC Ltd.; product code 184–4). The suppliers had made no claims regarding the relative abundance of troponin forms within this material. However, native–polyacrylamide gel electrophoresis and Western blot analysis of the batch of material we used indicated that the majority of the cTn present was of a molecular mass consistent with the ternary cardiac troponin T-I-C (cTnT-I-C) complex (personal communication, Simon Packer, July 10, 2006).
With regard to the analysis of our GFC results, Michielsen et al. reanalyze our data and relate elution to their Stokes radius (Rs), because they consider elution of nonglobular proteins on GFC to be more closely related to Rs than to relative molecular mass (Mr). Certainly, within the cTnT-I-C complex cTnT is thought to have both a globular domain and an elongated tail region (4)(5), but the 3-dimensional shape adopted when the protein is present in isolation is unknown. GFC is, like any analytical tool, imperfect, and there are many other variables that influence the elution of proteins in this technique, including the gel, the mobile phase, the protein surface charge, and the hydrophobic domain distribution. The report Michielsen et al. cite in support of their argument concluded that although GFC is not a satisfactory basis for the precise determination of molecular mass, it can be used to obtain an approximation of the relative magnitude of molecular masses(6). In practice, Wu et al.(7) have clearly shown that on GFC with Sephacryl S-200 gel (Pharmacia), the cTnT-I-C complex and free cTnT behave as globular proteins and their elution is closely related to Mr. Our results with Sephacryl S-100 gel and the SCIPAC cTn preparation are consistent with those of Wu et al.(7). The first peak eluting in Fig. 2D of our article (peak 1), showing GFC of the SCIPAC material(3), elutes in a position consistent with the Mr of the cTnT-I-C complex. We have shown that fractions from this peak react in both the Roche cTnT assay and the Beckman Access cardiac troponin I (cTnI) assay (Fig. 1
), supporting our conclusion that this peak represents the cTnT-I-C complex. cTnT reactivity elutes as a shoulder on the leading edge of the peak of cTnI reactivity, suggesting that this cTnT peak represents the cTnT-I-C complex. The cTnI peak contains cTnT-I-C complex poorly resolved from the cTnI-C complex. Wu et al.(7) also found the cTnT-I-C and cTnI-C complexes to be poorly resolved. A minor higher–molecular mass form of cTnI, the nature of which we are investigating, is also seen at the void volume. When SCIPAC cTn is subjected to GFC, as in Fig. 2D of our article(3) and in the Fig. shown here, added cTnT immunoreactivity is quantitatively recovered and none is detected in the void volume, as predicted by Michielsen et al.
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On the basis of reanalysis of our GFC results by use of Rs rather than Mr, Michielsen et al. speculate that the second peak in Fig. 2D may correspond to immunoreactive fragment(s) of cTnT, a circumstance that seems unlikely because Wu et al. (7) and ourselves(3) have shown that the increased circulating cTnT immunoreactivity in patients with acute coronary syndrome results largely in elution in the same position, with an Mr consistent with that of free, intact cTnT.
In their reanalysis of our data, Michielsen et al. calculated Rs for peaks 1 and 2 to be 37 Å and 26 Å, respectively. Using data from the publication of Wu et al. (7), we calculated Rs of 36.9 Å for the cTnT-I-C complex and 27.9 Å for cTnT, in excellent agreement with Michielsen et al. The disagreement over the identity of the peaks in Fig. 2D arises from Michielsen et al.’s interpretation of the relationship between elution and Rs on GFC. Because primary data are not available, Michielsen et al. have speculated on the Rs for cTnT and refer to an Rs value for the cTnT-I-C complex, a value that was determined by use of recombinant, bovine proteins in a different chromatography system and therefore may not be appropriate in the context of our study.
Further work is underway to confirm the identity of the peaks in our GFC system, but practical experience indicates that the elution of the cTnT-I-C complex and free, intact cTnT on GFC using Sephacryl S-100 and S-200 gels is related to Mr, as with globular proteins. We would be happy for Michielsen et al. to subject fractions from our GFC system to their Western blotting analysis. However, we feel that Michielsen et al.’s use of Rs rather than Mr in their reanalysis of our data is incorrect, and the conclusions of our study (3) are valid; cTnT circulates in the free, intact form in patients with kidney failure. Such a conclusion would appear to be more in keeping with the outcomes data that are now emerging, which show increased cTnT concentrations to be powerful predictors of death in both predialysis(8) and end-stage renal disease(9) patients.
References
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) symbols, respectively. Albumin and prolactin are shown as short- and long-dashed lines respectively.