Study population and study design
Selected study subjects were part of a cross-over intervention study protocol investigating whether an acute intravenous sodium load, as compared to a chronic dietary sodium load, differs in its effects on blood pressure, the endothelial surface layer and microcirculation.(17) Participants included healthy men, and both male type 1 diabetes mellitus and hereditary multiple exostosis patients (i.e., patients with, respectively, acquired and genetically determined glycocalyx changes).(23) Exclusion criteria were hypertension (≥ 140/90 mmHg), obesity (body mass index (BMI) ≥ 30 kg/m2), history of primary hyperlipoproteinemia, coagulation disorders, and renal or cardiovascular diseases. All subjects were randomized to a low sodium diet (LSD, <50 mmol Na+ daily) or to a high sodium diet (HSD, >200 mmol Na+ daily) for eight days, separated by a crossover period of at least one week. The study was performed at the Amsterdam UMC, location AMC, Amsterdam, The Netherlands. All participants provided written informed consent and approval was obtained from the local ethics committee. The trial is registered in the Netherlands Trial Register (NTR4095 and NTR4788).
Transcapillary escape rate of rHSA
An intravenous (IV) bolus of saline solution with rHSA labeled with 100 kBq I-125 was administered in a cubital vein. Blood samples were drawn from the contralateral arm at baseline and between 3 and 60 minutes after injection of rHSA. Radioactivity in plasma was measured in duplicate with a Wizard2 2480 automatic gamma counter (PerkinElmer, Waltham, Massachusetts, USA) with a coefficient of variation of <3%. The routine quality controls of the gamma counter were performed according to the standard GLP features of PerkinElmer, including detector energy resolution, background, absolute - and relative detector efficiency, detector stability probability and calibration.
The TERalb was calculated with PKSolver, a free Microsoft Excel add-in for pharmacokinetic (PK) and pharmacodynamic (PD) data analysis.(24) PKSolver has been validated and has been used in different PK/PD studies.(24-29)
TERalb was expressed as percentage decline in plasma radioactivity per hour (%/h). The TERalb calculation with PKSolver was performed for an IV bolus administration. The formula used for the calculation of TERalb was:
TERalb = (A 0 min – A 60 min) / A 0 min
The predicted activity of rHSA at T0 min (A 0 min) and at T60 min (A 60 min) were calculated by PKSolver (Microsoft Excel 2016) based on a mono- and bi-exponential kinetic model. This program also calculated the correlation coefficient (R) between the observed and predicted data.
Sampling schemes
After acquiring the PK curves of rHSA, we calculated the TERalb according the following simulated blood sampling schemes:
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T3 – 60 min: 3, 4, 5, 10, 15, 20, 30, 45, and 60 minutes
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T5 – 60 min: 5, 10, 15, 20, 30, 45, and 60 minutes
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T10 – 60 in: 10, 15, 20, 30, 45, and 60 minutes
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T15 – 60 min: 15, 20, 30, 45, and 60 minutes
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T20 – 60 min: 20, 30, 45, and 60 minutes
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Tmax – 60 min: from individual A max till 60 minutes
All blood samples before A max of the PK curves were excluded for the calculation of TERalb, irrespective of the sampling scheme.
Statistics
TERalb values were excluded if the correlation coefficient (R) was below <0.80.(30) All data were log-transformed and the effect of different blood sampling schemes on the TERalb values were analyzed by fitting a mixed model as implemented in IBM SPSS Statistics (version 25, IBM, USA). This mixed model uses a compound symmetry covariance matrix and is fitted using maximum likelihood. In the absence of missing values, this method results in the same p values as multiple comparisons tests (e.g. repeated measures ANOVA) that are less able to deal with missing values. Therefore, in the presence of missing values, the results can be interpreted like repeated measures ANOVA.(31) We used Bonferroni correction as post hoc test and p values < 0.05 were considered statistically significant. Results were reported as mean ± standard error of the mean (SEM). Bland-Altman plots were used to evaluate the level of agreement between two different blood sample schemes. All presented values represent non -transformed data.