PPi synthesis remains unaltered post-hemodialysis.

<p>Thin layer chromatography. (<b>A</b>) Plasma hydrolysis of 1 μmol/L ATP showed PPi and Pi production at the indicated times. (<b>B</b>) PPi quantification at the indicated conditions and sample types following 1 hr of incubation with 1 μmol/L ATP and [γ³²P]ATP as a radiotracer. Pre-hemodialysis plasmas without levamisole were used as controls. Results are represented as mean ± SEM for all plasma samples (n = 45). There was a significant difference in post-hemodialysis (postHD) without interaction (two-way ANOVA; <i>P</i> = 0.017). The Bonferroni post-test was used for statistical analysis. PreHD, pre-hemodialysis. PPi, pyrophosphate; Pi, phosphate. <b>CPM</b>: counts per minute. <b>* <i>P</i> < 0.05</b>.</p

Plasma phosphate levels inhibit ALP activity.

<p>(<b>A</b>) PreHD (left) and postHD (right) levels of urea and phosphate in plasma. Results are represented as mean ± SEM (n = 45). Wilcoxon’s matched pairs test was used for statistical analysis. (<b>B</b>) ALP activities of post-hemodialysis samples (with the exception of the controls, which were pre-hemodialysis samples) without or with potential inhibitors: (<b>1</b>) 120 mg/mL (20 mmol/L) urea; (<b>2</b>) no addition; (<b>3</b>) 4.5 mg/dL (1.5 mmol/L) inorganic phosphate; (<b>4</b>) 9 mg/dL inorganic phosphate; (<b>5</b>) 20 mmol/L EDTA; and (<b>6</b>) 100 μmol/L levamisole. Results are represented as mean ± SEM (n = 45). For statistical analysis, Friedman’s one-way ANOVA test and Dunn’s post-test were used. (<b>C</b>) The scattergraph demonstrates a correlation between ΔPi (the difference in post- and pre-hemodialysis phosphate levels) and ΔALP (the difference in post- and pre-hemodialysis ALP levels). PreHD, pre-hemodialysis; PostHD, post-hemodialysis. <b>* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001</b>.</p

Influence of plasma p<i>H</i> on alkaline phosphatase activity.

<p>(<b>A</b>) A p<i>H</i>-dependent curve using ten pools comprising four different post-hemodialysis plasma samples per pool at the p<i>H</i> indicated. Friedman’s one-way ANOVA test and Dunn’s post-test were used for statistical analysis. (<b>B</b>) Box and whiskers graph demonstrating pre- and post-hemodialysis plasma p<i>H</i> values (preHD and postHD, respectively; n = 10). (<b>C</b>) ALP activity in post-hemodialysis plasmas was quantified at the p<i>H</i> indicated. (<b>B, C</b>) Plasma samples were collected from all patients (n = 45), and results were analyzed using the Wilcoxon matched pairs test. (<b>D</b>) The scattergraph demonstrates a correlation between Δp<i>H</i> (the difference in post- and pre-hemodialysis p<i>H</i>) and Δ Relative ALP (the difference in post- and pre-hemodialysis relative ALP levels). <b>* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001</b>.</p

Physiological ALP activity increases post-hemodialysis.

<p>Maximal (<b>A</b>) and physiological (<b>C</b>) alkaline phosphatase (ALP) activities of pre-hemodialysis (preHD) and post-hemodialysis (postHD) plasma samples were quantified. For relative ALP activity (<b>E</b>), physiological ALP activities were divided by maximal ALP activities for each sample. (<b>A, C, E</b>) The Wilcoxon matched pairs test was used for statistical analysis. (<b>B, D, F</b>) The scattergraph demonstrated a correlation between pre- and post-hemodialysis ALP activities in maximal (<b>B</b>) and physiological (<b>D</b>) conditions, and relative ALP activity (<b>F</b>). Lineal regression demonstrated a significant deviation in all cases. <b>*** <i>P</i> < 0.001</b>.</p

PPi availability is reduced post-hemodialysis.

<p>(<b>A</b>) Plasma PPi quantification. (<b>B</b>) The scattergraph shows a correlation between plasma Pi and PPi. (<b>C</b>) Plasma (Ca)/PPi ratio. (<b>D</b>) Plasma ATP quantification and PPi/ATP ratio. (<b>E</b>) The scattergraph shows a correlation between PPi/ATP ratio and relative ALP activity in PostHD plasma. (<b>B, E</b>) A significant deviation was observed using linear regression. (<b>A, C, D</b>) Results are represented as mean ± SEM for all paired samples (n = 45). Wilcoxon’s matched pairs test was used for statistical analysis. PPi, pyrophosphate; Pi, phosphate; Ca, calcium; ALP, alkaline phosphatase; PreHD, pre-hemodialysis; PostHD, post-hemodialysis. <b>* <i>P</i> < 0.05; *** <i>P</i> < 0.001</b>.</p

Calcium deposition in the rat aortic wall <i>ex vivo</i> under calcium and phosphate concentrations matching pre- and post-haemodialysis levels in haemodialysis patients.

<p>Rat aortic rings were incubated <i>ex vivo</i> in MEM medium containing the indicated concentrations of calcium and phosphate. The medium was replaced every 2 days and contained ⁴⁵calcium as a radiotracer. After 6 days of incubation, aortic rings were dried and radioactivity was measured by liquid scintillation counting. Results are represented as mean ± SD from three independent experiments, with 15 or 16 rings per condition (five or six rings per condition per experiment). One-way ANOVA and Tukey’s multicomparison test were used for statistical analysis. Calcium and phosphate concentrations are in mmol/L. *<b><i>p</i> < 0.05; ***<i>p</i> < 0.001</b>. Pre-dialysis conditions were used as a reference. <b>PreHD</b>, pre-haemodialysis; <b>PostHD</b>, post-haemodialysis.</p

Demographic and clinical characteristics of the study population.

<p>Data expressed as mean±SD or %.</p

Inorganic phosphate and ionized calcium concentration in haemodialysis patients.

<p>(<b>A</b>) Plasma phosphate concentration in pre- and post-haemodialysis samples (n = 92). (<b>B</b>) Plasma ionized calcium concentration in pre- and post-haemodialysis samples (n = 45). The Wilcoxon matched pairs test (pre- versus post-haemodialysis) was used for statistical analysis. ***<b><i>p</i> < 0.001</b>. <b>PreHD</b>, pre-haemodialysis; <b>PostHD</b>, post-haemodialysis.</p

Study design: Timing of plasma sampling for BPA assessment.

<p>Baseline pre-dialysis plasma sampling took place after >6 months OL-HDF and prior to the first session with the switch dialyzer. Baseline post-dialysis sampling was obtained immediately after the first session with the switch dialyzer. Sampling was repeated pre- and post-dialysis after 3 months on the switch dialyzer.</p

Summary of clinical and biochemical variables.

<p>Summary of clinical and biochemical variables.</p