Abstract
BackgroundIt has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest.ResultsTTR levels as determined by ELISA in plasma and urine of healthy individuals were 489 ± 155 μg/ml plasma and 46 ± 24 ng/g creatinine, respectively. Average levels in urine of pregnant women were 45 ± 65 μg/g creatinine. The molecular heterogeneity of TTR was analyzed using a high-throughput mass spectrometric immunoassay system. TTR was extracted from plasma or urine onto an antibody-coated (via protein A) affinity chip surface (PS20) using the surface-enhanced laser desorption/ionization (SELDI) technique. Subsequently samples were subjected to time-of-flight mass spectrometry (TOF-MS). In healthy individuals, TTR in plasma occurred rather consistently in two variants of 13732 ± 12 and 13851 ± 9 Da for the native and S-cysteinylated forms and at a smaller signal of 14043 ± 17 Da for the S-glutathionylated form. In urine of pregnant women, various signals were observed with a dominant signal at 13736 ± 10 Da and a varying number of smaller immunoreactive fragments. These fragments are possibly the consequence of metabolism in plasma or kidney.ConclusionThis chip-based approach represents a rapid and accurate method to characterize the molecular variants of TTR including protein or peptide fragments which are either related to TTR or have resulted from its catabolism. These molecular variants may be of diagnostic importance as alternative or novel biomarkers due to their predominant relation to the TTR metabolism both in healthy and diseased individuals.
Highlights
It has been shown that transthyretin (TTR) exists in different molecular variants
Any TTR or retinol-binding protein (RBP) molecules that are filtered are rapidly bound to megalin, the multiligand receptor expressed on the luminal surface of the renal proximal tubules and internalized
TTR levels in plasma and urine TTR levels in plasma determined by enzyme-linked immunoassay (ELISA) in 10 healthy individuals were 489 ± 155 μg/ml
Summary
It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest. Transthyretin (TTR, formerly called prealbumin) belongs to a group of proteins including thyroxine-binding globulin and albumin which bind and transport thyroid hormones in the blood. It is a single polypeptide chain of 127 amino acids (14 kDa) and is present in the plasma as a tetramer of non-covalently bound monomers. The macromolecular complex plays an important physiological role in vitamin A homeostasis because it binds the specific transport protein for retinol, the lipocalin retinol-binding protein (RBP) [4,5] This reduces the glomerular filtration of the low molecular weight transport protein (21 kDa) in the (page number not for citation purposes). Under physiological conditions, TTR and RBP are present in urine if at all, only in trace amounts [6]
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