As protein binding of uremic toxins is not well understood, neither in chronic kidney disease (CKD) progression, nor during a hemodialysis (HD) session, we studied protein binding in two cross-sectional studies. Ninety-five CKD 2 to 5 patients and ten stable hemodialysis patients were included. Blood samples were taken either during the routine ambulatory visit (CKD patients) or from blood inlet and outlet line during dialysis (HD patients). Total (CT) and free concentrations were determined of p-cresylglucuronide (pCG), hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS) and p-cresylsulfate (pCS), and their percentage protein binding (%PB) was calculated. In CKD patients, %PB/CT resulted in a positive correlation (all p < 0.001) with renal function for all five uremic toxins. In HD patients, %PB was increased after 120 min of dialysis for HA and at the dialysis end for the stronger (IAA) and the highly-bound (IS and pCS) solutes. During one passage through the dialyzer at 120 min, %PB was increased for HA (borderline), IAA, IS and pCS. These findings explain why protein-bound solutes are difficult to remove by dialysis: a combination of the fact that (i) only the free fraction can pass the filter and (ii) the equilibrium, as it was pre-dialysis, cannot be restored during the dialysis session, as it is continuously disturbed.

Since there is a significant difference in the isotopic composition of Cu and Fe in whole blood between men and women, it was hypothesized that menstruation and the associated Cu and Fe loss affect the isotopic composition of these metabolically relevant transition metals. To assess this hypothesis, whole blood from two groups of non-menstruating women was analyzed for its Cu, Fe and Zn isotopic composition using multi-collector ICP-mass spectrometry (MC-ICP-MS) and the results were compared to the values for a male and a female reference population. The first group of non-menstruating women consisted of women in their menopause, while the second group consisted of women that were not menstruating because of an intra-uterine device (IUD). Also the effect of age on the isotopic composition of Cu, Fe and Zn in whole blood was investigated. The Cu and Fe isotopic composition of whole blood indeed seems to be influenced by menstruation. Non-menstruating women show an isotopic signature indistinguishable from that of the male reference population. The results for both groups of non-menstruating women do not differ significantly from one another and there does not seem to be an "age effect" for these two elements. Also for the isotopic composition of Zn in whole blood, no age effect could be demonstrated. No influence of menstruation was found either. However, a significant difference in the Zn isotopic composition was found between women in their menopause and women with an IUD. Possibly, this can be explained by a hormonal effect on the isotopic signature of Zn

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To better understand the kinetics of protein-bound uremic toxins (PBUTs) during hemodialysis (HD), we investigated the distribution of hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS), and p-cresyl sulfate (pCS) in erythrocytes of HD patients. Their transport across the erythrocyte membrane was explored in the absence of plasma proteins in vitro in a series of loading and unloading experiments of erythrocytes from healthy subjects and HD patients, respectively. Furthermore, the impact of three inhibitors of active transport proteins in erythrocytes was studied. The four PBUTs accumulated in erythrocytes from HD patients. From loading and unloading experiments, it was found that (i) the rate of transport was dependent on the studied PBUT and increased in the following sequence: HA < IS < pCS < IAA and (ii) the solute partition of intra- to extra-cellular concentrations was uneven at equilibrium. Finally, inhibiting especially Band 3 proteins affected the transport of HA (both in loading and unloading), and of IS and pCS (loading). By exploring erythrocyte transmembrane transport of PBUTs, their kinetics can be better understood, and new strategies to improve their dialytic removal can be developed.

While studying and trying to optimise dialysis clearances of protein-bound uraemic toxins (PBUTs), the percentage protein binding (% PB) may be an important parameter and can be calculated from measured free and total concentrations. Since different parameters may alter this % PB, we investigated whether the ultrafiltration temperature, sample pH, and sample matrix (i. e. serum or plasma) affects the % PB of PBUTs. Pre-dialysis serum and plasma samples were obtained from 10 stable haemodialysis patients. Ultrafiltration was performed at 37 degrees C for fresh samples and at 4 degrees C, room temperature, and 37 degrees C for thawed samples (all n = 10). Total and free serum/ plasma concentrations of hippuric acid, indole-3-acetic acid, indoxyl sulphate, and p-cresylsulphate were simultaneously measured by high-performance liquid chromatography with ultraviolet and fluorescence detection. No differences in % PB were found between fresh and thawed samples at 37 degrees C or between serum and plasma samples prepared at the same temperatures. However, in both serum and plasma samples, the free concentration increased with increasing ultrafiltration temperatures and resulted in a decrease in % PB from 4 degrees C to 37 degrees C. In conclusion, the % PB of PBUTs can be determined in both thawed serum and plasma samples and ultrafiltration should be performed at 37 degrees C