Real time RT-PCR analysis of thyroglobulin mRNA in peripheral blood in patients with congenital athyreosis and with differentiated thyroid carcinoma after stimulation with recombinant human thyrotropin.

OBJECTIVE: Thyroglobulin (Tg), measured by immunometric assay, is the most sensitive and widely used clinical marker for thyroid cancer progression and relapse. However, these Tg determinations are of limited sensitivity and susceptible to interference by Tg autoantibodies. As a possible diagnostic alternative, we tested a real time RT-PCR protocol to determine Tg mRNA levels in peripheral blood. METHODS: Tg mRNA was determined by real-time RT-PCR using total RNA from peripheral blood. Tg mRNA blood levels were calibrated to the mRNA encoding the housekeeping enzyme glyceraldehyde phosphate dehydrogenase (GAPDH); pooled blood from ten healthy subjects served as a RT-PCR positive control. RESULTS: Tg mRNA and serum Tg were detected in twelve patients with differentiated thyroid cancer (DTC) after thyroidectomy and radioiodine therapy, however, there was no correlation with the clinical stage. An increase in Tg mRNA and protein was observed after application of recombinant human thyrotropin (rhTSH) in four patients with DTC stimulated with rhTSH for postoperative follow up. Tg mRNA and protein were also detected in four congenital athyreotic patients. Analysis of Tg mRNA levels using a commercial multiple tissue Northern blot revealed Tg hybridization signals in several extrathyroidal tissues (salivary gland, trachea, kidney, pancreas, adrenal gland, etc.). CONCLUSIONS: Our data suggests that RT-PCR detects Tg mRNA of extrathyroidal origin, from leukocytes or from metastasizing carcinoma cells under basal conditions or after TSH stimulation. However, considering the marked and highly variable individual Tg mRNA backgrounds, interpretation of real time PCR results requires caution. This limits the clinical use of Tg mRNA determination by real time PCR to an individual tumor progression marker in follow-up.

Technical evaluation of a new immunoradiometric and a new immunoluminometric assay for thyroglobulin.

BACKGROUND: After removal of differentiated thyroid carcinoma (DTC), serum thyroglobulin (Tg) can indicate persistent or recurrent disease. We describe two novel two-step assays designed to measure low Tg concentrations. METHODS: We evaluated prototypes of the new IRMA, DYNOtest Tg-pluS, and the new immunoluminometric assay (ILMA), LUMItest) Tg-pluS. In the first step, a high-salt incubation buffer leads to dissociation of Tg-Tg antibody complexes in serum and is intended to reduce nonspecific interference and interference of potential Tg autoantibodies in the system. We studied recovery of human Tg (from thyroid glands) added to horse serum. We also studied 58 patients with DTC in whom Tg values under thyroid-stimulating hormone (TSH) suppression and TSH stimulation (without thyroxine) were available. RESULTS: The detection limits were 0.04 microg/L Tg for the IRMA and 0.02 microg/L for the ILMA. Intraassay imprecision (CV) was <10% over the range of the calibration curve in both assays. The day-to-day CV was <20% at 0.2 microg/L for the IRMA and at 0.06 microg/L for the ILMA. No high-dose hook effect was seen with up to 200 000 microg/L added Tg or in dilutions of 12 patient sera with Tg values of 307-38 880 microg/L. Mean recovery of 50 microg Tg/L was 96% in those patients. Among 77 samples with Tg antibody values of 65.2-8150 kilounits/L, recovery by the IRMA was disturbed in 7 cases (9%) and by the ILMA in 9 cases (12%). Tg increased as measured in both assays in 50 of 58 patients after thyroxine withdrawal. CONCLUSIONS: The new assays have improved precision for Tg <1 microg/L, and even low measured Tg concentrations respond physiologically to thyroxine withdrawal. The assays are free of a high-dose hook effect up to a Tg concentration of at least 38 000 microg/L and may further reduce Tg antibody interference.

L-arginine deficiency and supplementation in experimental acute renal failure and in human kidney transplantation.

BACKGROUND: The "L-arginine paradox" refers to situations where L-arginine (L-Arg) supplementation stimulates nitric oxide (NO) synthesis, despite saturating intracellular concentrations. This paradox is frequently observed in acute renal failure (ARF). First, the effects of L-Arg on renal function of rats with ARF were studied. Based on the promising results from these initial studies, the second part of our study searched for a form of ARF in humans that could be studied easily under conditions with little variance and yet was linked with endothelial dysfunction. Thus, we investigated the effects of L-Arg supplementation immediately after kidney transplantation in 54 patients. METHODS: In uranyl nitrate-induced ARF in rats the effects of L-Arg and L-NNA (inhibitor of nitric oxide synthase; NOS) on glomerular filtration rate (GFR), renal plasma flow (RPF), blood pressure (BP) and NOx (NO2- +NO3-) excretion were examined. Tissue L-Arg levels, NOS activities, immunodetection of NOS and superoxide dismutase (SOD), activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase, and nitrotyrosine immunoreactive protein (NT-IR) were determined and compared to sham operated animals. Secondly, in a randomized, double-blind study, the effects of L-Arg on GFR and RPF were investigated in 54 kidney transplant recipients, receiving IV L-Arg for three days. GFR and RPF were measured on days 1, 3, 5 and 10 by scintigraphy. RESULTS: In experimental ARF, decreased RPF and GFR were associated with reduced tissue L-Arg levels, endothelial NOS-III expression, NO formation and NOx excretion. Reduction in GFR, RPF and NOx excretion were reversed upon administration of exogenous L-Arg. There also was a loss of Cu,Zn-SOD, a key enzyme against oxidative stress, and an elevation of NT-IR, an indicator of nitrosative stress and suggested marker for pathological actions of NO. However, NT-IR was not dependent on de novo NO synthesis and not related to the functional effects of l-Arg administration. In kidney transplant recipients receiving organs with a short cold ischemia time (CIT) and from young donors, that is, those with a higher likelihood of a functional endothelium, early administration of L-Arg improved renal function. CONCLUSION: Both experimental and clinical data show that \L-Arg deficiency and endothelial dysfunction are pathomechanistically relevant in ARF. The data suggest a therapeutic potential for the administration of L-Arg in ARF and kidney transplantation, at least in patients receiving kidneys with shorter CIT and from younger donors.