Fig 1.
Physiological ALP activity increases post-hemodialysis.
Maximal (A) and physiological (C) alkaline phosphatase (ALP) activities of pre-hemodialysis (preHD) and post-hemodialysis (postHD) plasma samples were quantified. For relative ALP activity (E), physiological ALP activities were divided by maximal ALP activities for each sample. (A, C, E) The Wilcoxon matched pairs test was used for statistical analysis. (B, D, F) The scattergraph demonstrated a correlation between pre- and post-hemodialysis ALP activities in maximal (B) and physiological (D) conditions, and relative ALP activity (F). Lineal regression demonstrated a significant deviation in all cases. *** P < 0.001.
Fig 2.
PPi synthesis remains unaltered post-hemodialysis.
Thin layer chromatography. (A) Plasma hydrolysis of 1 μmol/L ATP showed PPi and Pi production at the indicated times. (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; P = 0.017). The Bonferroni post-test was used for statistical analysis. PreHD, pre-hemodialysis. PPi, pyrophosphate; Pi, phosphate. CPM: counts per minute. * P < 0.05.
Fig 3.
PPi availability is reduced post-hemodialysis.
(A) Plasma PPi quantification. (B) The scattergraph shows a correlation between plasma Pi and PPi. (C) Plasma (Ca)/PPi ratio. (D) Plasma ATP quantification and PPi/ATP ratio. (E) The scattergraph shows a correlation between PPi/ATP ratio and relative ALP activity in PostHD plasma. (B, E) A significant deviation was observed using linear regression. (A, C, D) 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. * P < 0.05; *** P < 0.001.
Fig 4.
Plasma phosphate levels inhibit ALP activity.
(A) 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) ALP activities of post-hemodialysis samples (with the exception of the controls, which were pre-hemodialysis samples) without or with potential inhibitors: (1) 120 mg/mL (20 mmol/L) urea; (2) no addition; (3) 4.5 mg/dL (1.5 mmol/L) inorganic phosphate; (4) 9 mg/dL inorganic phosphate; (5) 20 mmol/L EDTA; and (6) 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. (C) 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. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 5.
Influence of plasma pH on alkaline phosphatase activity.
(A) A pH-dependent curve using ten pools comprising four different post-hemodialysis plasma samples per pool at the pH indicated. Friedman’s one-way ANOVA test and Dunn’s post-test were used for statistical analysis. (B) Box and whiskers graph demonstrating pre- and post-hemodialysis plasma pH values (preHD and postHD, respectively; n = 10). (C) ALP activity in post-hemodialysis plasmas was quantified at the pH indicated. (B, C) Plasma samples were collected from all patients (n = 45), and results were analyzed using the Wilcoxon matched pairs test. (D) The scattergraph demonstrates a correlation between ΔpH (the difference in post- and pre-hemodialysis pH) and Δ Relative ALP (the difference in post- and pre-hemodialysis relative ALP levels). * P < 0.05; ** P < 0.01; *** P < 0.001.