Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

OBJECTIVES: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples. METHODS: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. RESULTS: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. CONCLUSIONS: Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Free cortisol and free 21-deoxycortisol in the clinical evaluation of congenital adrenal hyperplasia.

CONTEXT: Some patients with classic congenital adrenal hyperplasia (CAH) survive without glucocorticoid treatment. Increased precursor concentrations in these patients might lead to higher free (biological active) cortisol concentrations by influencing the cortisol-protein binding. In 21-hydroxylase deficiency (21OHD), the most common CAH form, accumulated 21-deoxycortisol (21DF) may further increase glucocorticoid activity. Both mechanisms could explain the low occurrence of symptoms in some untreated classic CAH patients. OBJECTIVE: Develop and validate an LC-MS/MS method for free cortisol and free 21DF to quantify these steroids in untreated patients with classic CAH (n=29), non-classic CAH (NCCAH, n=5), other forms of adrenal insufficiency (AI, n=3), and controls (n=11) before and 60 minutes after Synacthen® administration. RESULTS: Unstimulated total cortisol concentrations of untreated classic CAH patients (median 109 nmol/L) were lower than in untreated NCCAH patients (249 nmol/L, p=0.010) and controls (202 nmol/L, p=0.016), but free cortisol concentrations were similar. Basal free 21DF concentrations were high in 21OHD patients (median 5.32 nmol/L) and undetectable in AI patients and controls (<0.19 nmol/L). After Synacthen® administration, free 21DF concentrations increased in 21OHD patients, while free cortisol concentrations did not change. CONCLUSIONS: Free cortisol concentrations in classic CAH patients were similar to those in controls and NCCAH patients, indicating comparable cortisol availability. Additionally, 21OHD patients produce high concentrations of 21DF, possibly explaining the low occurrence of symptoms in some classic 21OHD patients. Free cortisol and 21DF levels should be considered in evaluating adrenal insufficiency in patients with CAH.

Pre-evacuation hCG glycoforms in uneventful complete hydatidiform mole and persistent trophoblastic disease.

OBJECTIVE: To investigate whether the glycoform distribution patterns of human chorionic gonadotropin (hCG) obtained by chromatofocusing in pre-evacuation serum are different for patients who will eventually develop into persistent trophoblastic disease in case of complete hydatidiform mole pregnancy as compared to those patients for whom trophoblastic tissue will regress uneventfully. METHODS: Pre-evacuation blood samples were collected from women with complete hydatidiform mole with uneventful spontaneous regression after molar evacuation (n=32), from women with complete hydatidiform mole who developed persistent trophoblastic disease after evacuation of their mole (n=28) and, as a control group, from women during the first trimester of normal pregnancy (n=22). The serum specimens were subjected to chromatofocusing, and hCG was determined in the fractions collected in the pH range 7.0-3.0. RESULTS: Receiver operating characteristics (ROC) analysis revealed that 36% of complete hydatidiform mole patients with post-molar persistent trophoblastic disease development had different hCG glycoform profiles at 97% specificity (pH interval 6.3-5.1, hCG cutoff 9.9%). There was a significant difference between complete hydatidiform mole with and without persistent trophoblastic disease for the cumulative percent amounts of hCG in the pH interval 6.3-5.1 (p<0.0003). CONCLUSION: In 36% of the patients with complete hydatidiform mole with subsequent development of persistent trophoblastic disease, typical glycoform profiles for hCG are observed in pre-evacuation serum samples. This result suggests that hCG glycoform profiles are of potential use in the prediction of persistent trophoblastic disease.

Prognostic significance of VEGF and components of the plasminogen activator system in endometrial cancer.

OBJECTIVE: The plasminogen activator system (PAS) and vascular endothelial growth factor (VEGF) are important in the carcinogenesis and play a key role in cancer invasion and mediating metastasis of carcinomas. The aim of the study was to evaluate the correlation of serum levels of VEGF and components of the PAS with clinicopathological risk factors and outcome in patients with endometrial cancer (EC). METHODS: Preoperative blood was collected from 173 patients treated for EC between 1999 and 2009. Serum concentrations of VEGF, urokinase plasminogen activator (uPA) tissue plasminogen activator (tPA), plasminogen activator inhibitor type-1 (PAI-1) and -2 (PAI-2) were assessed by enzyme-linked immunosorbent assays (ELISA). RESULTS: Serum levels of VEGF and components of the PAS were significantly associated with stage of the disease, tumor histology, tumor grade, myometrial invasion (MI), presence of lymphovascular space invasion (LVSI) and lymph node metastases (LNM). Preoperative serum levels of PAI-1 and -2 and tPA were higher in patients who experienced a recurrence than in patients who remained disease free (p < 0.01). PAI-1 and -2 and tPA were significantly independent prognostic factors for DFS with a HR of 3.85 (95% CI 1.84-8.07), 3.90 (95% CI 1.75-8.66) and 2.53 (95% CI 1.16-5.55), respectively. PAI-1 and tPA turned out to be independent prognostic factors for OS, with a HR of 2.09 (95% CI 1.08-4.05) and 2.16 (95% CI 1.06-4.44), respectively. CONCLUSION: Serum levels of VEGF and components of the PAS at primary diagnosis were associated with well-known clinicopathological risk factors such as; FIGO stage, tumor histology, tumor grade, MI, LVSI and LNM. High concentrations of PAI-1 and-2 and tPA are independent factors for poor prognosis in patients with endometrial cancer.

Insights into the formation of carlactone from in-depth analysis of the CCD8-catalyzed reactions.

Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis-configured ß-carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used 13 C and 18 O-labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC-MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.