Structural modifications of serum transthyretin in rats during protein-energy malnutrition.

Transthyretin (TTR) is a sensitive marker of protein-energy malnutrition and changes in serum and expression levels during protein and energy deficiency are well described. However, little is known about structural modifications of TTR during protein and/or energy deprivation. Therefore, the aim of this study was to determine the effects of protein inadequacies on post-translational modifications of TTR. For this purpose, male Wistar rats were fed a diet with either casein or gelatine as sole protein source subsequent to a protein wash-out period. Changes in TTR serum levels as well as other markers of nutritional status as body weight, food consumption, total serum protein and serum RBP4 levels as well as antioxidative capacity were determined. Post-translational modifications of TTR were examined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) analysis. The rats from the gelatine group revealed a marked change in the post-translational modification pattern of TTR which was reflected by a significant elevation of sulfonated TTR and which was inversely correlated to the antioxidative capacity. Additionally, the elevation of sulfonated TTR was accompanied by a decrease in body weight and food consumption, low antioxidative capacity as well as a deprivation of serum TTR, RBP4 and total serum protein levels in the animals of the gelatine group. Protein-energy malnutrition leads therefore next to changes in TTR serum concentration, also to changes in the post-translational modification pattern of TTR. Such changes are probably induced by protein-energy malnutrition-driven oxidative stress and might be linked to alterations in protein function and stability.

Alteration of transthyretin microheterogeneity in serum of multiple trauma patients.

Transthyretin (TTR) which exists in various isoforms, is a valid marker for acute phase response and subclinical malnutrition. The aim of the study was to investigate the relationship between inflammation, oxidative stress and the occurrence of changes in microheterogeneity of TTR.A prospective, observational study at a level-I trauma center of a large urban medical university was performed. Patients were severely injured (n = 18; injury severity score (ISS): 34-66), and were observed within the first 24 hours of admittance and over the following days until day 20 after injury. 20 healthy subjects, matched by age and sex, were used as controls.TTR was enriched by immunoprecipitation. Microheterogeneity of TTR was determined by linear matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Four major mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated and S-glutathionylated TTR. In the course of their ICU stay, 14 of the 18 patients showed a transient change in microheterogeneity in favour of the S-cysteinglycinylated form of TTR (p < 0.05 vs. controls). The occurrence of this variant was not associated with the severity of trauma or the intensity of the acute-phase response, but was associated with oxidative stress as evidenced by Trolox.Our results demonstrate that changes in microheterogeneity of TTR occur in a substantial number of ICU trauma patients. The diagnostic values of these changes remains to be elucidated. It is speculated that TTR modification may well be the mechanism underlying the morphological manifestation of amyloidose or Alzheimer's diseases in patients surviving multiple trauma.

Peptide and protein profiles in serum and follicular fluid of women undergoing IVF.

BACKGROUND: Proteins and peptides in human follicular fluid originate from plasma or are produced by follicular structures. Compositional changes reflect oocyte maturation and can be used as diagnostic markers. The aim of the study was to determine protein and peptide profiles in paired serum and follicular fluid samples from women undergoing IVF. METHODS: Surface-enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS) was used to obtain characteristic protein pattern. RESULTS: One hundred and eighty-six individual MS signals were obtained from a combination of enrichment on strong anion exchanger (110), weak cation exchanger (52) and normal phase surfaces (24). On the basis of molecular masses, isoelectric points and immunoreactivety, four signals were identified as haptoglobin (alpha(1)- and alpha(2)-chain), haptoglobin 1 and transthyretin (TTR). Immunological and MS characteristics of the TTR : retinol-binding protein (RBP) transport complex revealed no microheterogeneity differences between serum and follicular fluid. Discriminatory patterns arising from decision-tree-based classification and regression analysis distinguished between serum and follicular fluid with a sensitivity and specificity of 100%. CONCLUSIONS: Quantitative and qualitative differences indicate selective transport processes rather than mere filtration across the blood-follicle barrier. Identified proteins as well as characteristic peptide and/or protein signatures might emerge as potential candidates for diagnostic markers of follicle and/or oocyte maturation and thus oocyte quality.

Microheterogeneity of transthyretin in serum and ascitic fluid of ovarian cancer patients.

BACKGROUND: Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR. METHODS: Subjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay (ELISA) and C-reactive protein (CRP) levels by a high-sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining. RESULTS: TTR and RBP (microg/ml) levels in serum were 148.5 +/- 96.7 and 22.5 +/- 14.8 in affected women compared to 363.3 +/- 105.5 and 55.8 +/- 9.3 in healthy postmenopausal women (p < 0.01). In ascitic fluid, levels were 1.02 +/- 0.24 and 4.63 +/- 1.57 microg/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml (p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 +/- 7, 13876 +/- 13 (greatest intensity), 13924 +/- 21 and 14062 +/- 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR (12828 +/- 11 Da). No immunoreactive TTR was observed in the tumor sites. CONCLUSION: The severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid.

Influence of hormone replacement therapy on proteomic pattern in serum of postmenopausal women.

OBJECTIVES: Proteomics approaches to cardiovascular biology and disease hold the promise of identifying specific proteins and peptides or modification thereof to assist in the identification of novel biomarkers. METHOD: By using surface-enhanced laser desorption and ionization time of flight mass spectroscopy (SELDI-TOF-MS) serum peptide and protein patterns were detected enabling to discriminate between postmenopausal women with and without hormone replacement therapy (HRT). RESULTS: Serum of 13 HRT and 27 control subjects was analyzed and 42 peptides and proteins could be tentatively identified based on their molecular weight and binding characteristics on the chip surface. By using decision tree-based Biomarker Patternstrade mark Software classification and regression analysis a discriminatory function was developed allowing to distinguish between HRT women and controls correctly and, thus, yielding a sensitivity of 100% and a specificity of 100%. The results show that peptide and protein patterns have the potential to deliver novel biomarkers as well as pinpointing targets for improved treatment. The biomarkers obtained represent a promising tool to discriminate between HRT users and non-users. CONCLUSION: According to a tentative identification of the markers by their molecular weight and binding characteristics, most of them appear to be part of the inflammation induced acute-phase response.