Susanne Møller Pedersen

Soluble serum Klotho levels in healthy subjects: Comparison of two different immunoassays

OBJECTIVE Soluble serum Klotho is a new biomarker linked to chronic kidney disease, cardiovascular disease and diabetes. This study describes the evaluation and comparison of two different immunoassays and establishment of assay specific reference intervals in adults. DESIGN AND METHODS Serum Klotho concentrations were determined in 120 healthy adults aged 19-66 years. Blood samples were collected, and stored sera were assayed for Klotho according to age and gender. In addition several other clinical and laboratory characteristics were determined in the cohort and compared to the levels of serum Klotho. RESULTS Serum Klotho levels were significantly higher in time-resolved fluorescence immunoassay (TRF) compared to an ELISA (IBL) and no correlation was found between the assays. No signal was obtained in either assay when the standard curve was switched between the two different immunoassays. The median serum Klotho concentration using TRF was 61 ng/mL (2.5-97.5% reference limits; 11-181 ng/mL) for males and 99 ng/mL (2.5-97.5% reference limits; 19-316 ng/mL) for females while the ELISA gave a mean value of 472 pg/mL (2.5-97.5% reference limits; 204-741 pg/mL) with no difference between genders. Concentrations of serum Klotho were independently associated with estimated glomerular filtration rate (eGFR) and body weight using TRF whereas serum Klotho concentrations were associated with age using the ELISA. CONCLUSION Comparison of two different immunoassays for serum Klotho indicate, that the protein exists in human beings in different forms which may function as independent factors and whose role and potential value as biomarkers needs to be evaluated separately. Reference intervals specific for the different forms recognized by the different assays were calculated in this study.

Changes in fibroblast growth factor 23 during treatment of secondary hyperparathyroidism with alfacalcidol or paricalcitol

BACKGROUND Fibroblast growth factor 23 (FGF23) increases renal phosphate excretion and decreases the formation of 1,25 dihydroxyvitamin D. In patients with chronic kidney disease, plasma FGF23 levels are markedly elevated by unknown mechanisms. We explored the changes in FGF23 during treatment of secondary hyperparathyroidism (SHPT) with alfacalcidol or paricalcitol in haemodialysis patients. METHODS Intravenous alfacalcidol and paricalcitol were compared in haemodialysis patients with SHPT in a randomized 2 × 16-week cross-over study, with 6 weeks washout period preceding treatment and between treatment periods. In 57 of the enrolled patients, blood samples were frozen before and after each treatment period and available for measurement of FGF23. RESULTS Treatment with both analogues increased FGF23 (P < 0.05 in all treatment periods). The magnitude of increase was similar for alfacalcidol and paricalcitol (Period 1: 223 versus 314%; P = 0.384 and Period 2: 174 versus 227%; P = 0.510) and the levels returned to pre-treatment levels during the washout period. Independent predictors of rise in FGF23 were baseline levels of FGF23 (P < 0.01), changes in ionized calcium (P < 0.01) and phosphate (P < 0.01) and cumulative dose of vitamin D analogues (P = 0.024). CONCLUSION Alfacalcidol and paricalcitol increase the plasma levels of FGF23 equally and substantially in haemodialysis patients.

Acute hyperinsulinemia is followed by increased serum concentrations of fibroblast growth factor 23 in type 2 diabetes patients.

Abstract Background. Phosphate homeostasis is connected to glucose metabolism and is influenced by insulin, but the role of fibroblast growth factor 23 (FGF23) is unknown in this relation. Therefore, the levels of FGF23 and phosphate were investigated during a euglycemic-hyperinsulinemic clamp in healthy and type 2 diabetic individuals. Methods. The study population consisted of ten type 2 diabetic patients, ten weight-matched glucose-tolerant obese subjects, and ten healthy lean subjects. All subjects underwent a 4-h euglycemic-hyperinsulinemic clamp using physiological hyperinsulinemia (40 mU/min per m2) to determine glucose disposal rates. Blood samples for measurement of serum FGF23 and insulin, plasma phosphate and glucose, and HbA1c were drawn before and after insulin infusion. Results. The three groups did not differ in baseline levels of serum FGF23. Insulin infusion increased serum FGF23 in the diabetic group (p = 0.009), but not in the other groups. The increase in serum FGF23 correlated strongly with increase in insulin levels in the diabetic group (r = 0.83; p = 0.003). In the overall group insulin infusion suppressed plasma phosphate concentrations (p = 0.006), but with no differences between the groups. Conclusions. Physiological hyperinsulinemia is under euglycemic conditions followed by a significant increase in serum FGF23 concentrations in diabetic individuals, which correlated with change in insulin level. The interplay between insulin effects and FGF23 may be important for the understanding of phosphate metabolism in relation to type 2 diabetes and its vascular complications.

Influence of ionic strength on the binding of sodium aurothiosulphate to human serum albumin

The effect of ionic strength on the binding of aurothiosulphate to human serum albumin has been studied at 37 degrees and neutral pH by equilibrium dialysis in unbuffered solutions. The effect of ionic strength is more pronounced on the lower association constants K2-K4 than on the high association constant K1. Furthermore a reduction in the number of lower affinity binding sites is observed at low ionic strength. The main ionic strength dependence on the association constants agrees with the Debye-Hückel theory. The extrapolated values of K1 and the sum of K2 to K4 at zero ionic strength are 7.6 X 10(5) M-1 and 1.1 X 10(5) M-1, respectively. It is shown that the observed changes in pH of the albumin solutions during dialysis contains valuable information of the aurothiosulphate-albumin interaction. A molecular binding mechanism is discussed.

Large discrepancy in the results of sensitive measurements of thyroglobulin antibodies in the follow-up on thyroid cancer: a diagnostic dilemma.

During follow-up on patients treated for differentiated thyroid cancer, thyroglobulin (Tg) antibodies can interfere with the Tg assay, making the use of Tg less reliable as a tumor marker. Purpose: To compare Tg and Tg autoantibodies (Tg-Ab) methods used in Denmark, regarding the number of patient samples being accepted for evaluating the result of a serum thyroglobulin (s-Tg) measurement. Design: 95 consecutive blood samples drawn from patients in 2006 in one center were selected according to the following criteria: s-Tg <1µg/l and accepted BRAHMS Tg+ recovery test using 50 ng of Tg. Samples were retested with: (1) DPC IMMULITE 2000 Tg and Tg-Ab, (2) BRAHMS Tg and Tg-Ab on Kryptor, (3) BRAHMS Tg+ and Dynotest anti-Tg, (4) DELFIA hTg and recovery test using 25 ng of Tg, and (5) BRAHMS Tg+ with recovery test using 1 and 50 ng of Tg. Results: The number of patient samples that was not accepted for Tg evaluation varied from 2 to 26% when the reference values suggested by the manufacturers of the assay were used. When using the detection limit to the cutoff seen in epidemiological studies the number increased to 40%. Conclusion: We found large discrepancies in acceptance of patient samples for s-Tg evaluation, thus illustrating a diagnostic dilemma.

The effect of pH on the binding of sodium aurothiosulphate to human serum albumin: A possible binding mechanism

The effect of pH on the binding of aurothiosulphate to human serum albumin was studied in unbuffered solutions at 37 degrees and ionic strength 0.15-0.16 M. In the investigated pH range, 6.3-8.4, the effect of pH on the high affinity association constant K1 was very different from that on the lower affinity constants K2-K4. K1 was virtually constant except for a two-fold decrease in the narrow pH range 7.5-7.9, which was explained as a H+ induced local conformation change in the environment of site 1. Contrary to this, K2-K4 decreased monotonically with increasing pH, which could be entirely accounted for by a change in electrostatic interaction. A conceivable binding mechanism consistent with the results might be: that gold binds as Au+ to the high affinity binding site by exchanging a H+ and that this site might be the free sulphydryl group in cysteine or the terminal alpha-amino group; and that gold binds as Au(S2O3)3-(2) to the lower affinity binding sites which might be the protonated basic side chain group, i.e. epsilon-amino groups.

Influence of temperature on the binding of sodium aurothiosulphate to human serum albumin.

The influence of temperature on the binding of aurothiosulphate by human serum albumin was studied in unbuffered solutions at pH 7.4 and ionic strength 0.15 M by means of equilibrium dialysis. It was found that the high affinity association constant was temperature dependent. The thermodynamic characteristics of binding delta G1 degrees less than 0, delta H1 degrees greater than 0 and delta S1 degrees greater than 0 indicated that the binding process was endothermic and entropically driven. It was concluded that electrostatic interaction was predominantly involved in the binding of aurothiosulphate to the high affinity binding site on albumin. This is consistent with the molecular mechanism that the ligand binds as Au+ to a sulfhydryl group of albumin by replacing a hydrogen ion.

Mediastinal microdialysis: early diagnosis of anastomotic leakage after resection for esophageal cancer

The aim of the present study was to evaluate the safety of mediastinal microdialysis and its efficacy regarding the early diagnosis of anastomotic leakage after gastroesophageal resection for esophageal cancer. Eight consecutive patients were included; one patient was excluded for reasons of catheter malfunction. The tip of the mediastinal microdialysis catheter was placed close to the anastomosis and held by a 4-0 absorbable suture. A subcutaneous microdialysis catheter placed in the pectoral region served as a reference. Samples collected every 4 h in the first 8 postoperative days were analyzed for lactate, glucose, pyruvate, and glycerol and the lactate/pyruvate ratio (L/P ratio) was calculated. There were no procedure-related complications. Six patients had an uncomplicated postoperative course. In one patient, the L/P ratio was within normal range during the first 20 h postoperatively, but a steady and significant increase in L/P ratio then occurred, reaching a maximum of 105 after 124 h. The patient developed leakage symptoms on day 3, but endoscopy was unable to demonstrate any leakage. On the sixth postoperative day, blue dye administered through the nasogastric tube was recovered in the pleural drain. The leakage was treated with a covered selfexpanding metal stent; an immediate and significant drop in the L/P ratio occurred. Mediastinal microdialysis seems to be a safe and promising tool in the early diagnosis of anastomotic leakage in patients undergoing gastroesophageal resection for cancer.

Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

Background The clinical utility of serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma may be compromised by the presence of endogenous antithyroglobulin antibodies. To prevent interference by antithyroglobulin antibodies several groups have developed real-time PCR-based assays for quantification of blood thyroglobulin mRNA levels. For accurate quantification of thyroglobulin mRNA in blood preanalytical factors must be recognized and controlled. In this study, we evaluate the effect of different blood RNA stabilizing systems – the Tempus Blood RNA system and the PAXgene Blood RNA system – and storage time on RNA yield and quality, and thyroglobulin mRNA stability. Methods Blood samples from 11 patients previously treated for differentiated thyroid carcinoma were collected in K2-EDTA, Tempus and PAXgene tubes and maintained at room temperature. RNA was isolated following storage for 0 and 72 h, and RNA yield, integrity and purity was determined. Thyroglobulin, GAPDH and ACTB mRNA levels were quantified by semi-quantitative real-time PCR. Results The RNA yield was significantly higher for blood collected in Tempus tubes compared with PAXgene tubes following storage for 72 h at room temperature (P = 0.0011). High-quality RNA could be extracted from blood collected in PAXgene and Tempus tubes. Blood collected in K2-EDTA tubes, but not in PAXgene and Tempus tubes, showed significant changes in thyroglobulin mRNA levels following storage for 72 h at room temperature (P = 0.0263). Conclusions Stabilization of blood in PAXgene and Tempus tubes enables storage at room temperature for up to 72 h, without compromising thyroglobulin mRNA levels.