Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid-liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4-300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7-3.5%, n=3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Arterial stiffness depends on serum ionized calcium levels during dialysis with regional citrate anticoagulation

Hemodynamic effects related to changes in serum ionized calcium (iCa) are difficult to determine during conventional hemodialysis (HD) using a fixed dialysate concentration of calcium. Regional citrate anticoagulation (RCA) allows the study of the effects of predefined iCa changes on arterial stiffness and blood pressure (BP) during a single dialysis session. In a crossover study, 15 patients with end-stage renal disease underwent two HD sessions with RCA. Each session was divided into two study phases in which iCa was titrated either to 0.8-1.0 mm or to 1.1-1.4 mm. The sequence of phases was randomly chosen and alternated for the second session. After reaching a stable iCa level, pulse wave velocity (PWV), arterial BP, and heart rate were measured. iCa levels were modified during sequence 1 (iCa low-high) from a predialysis baseline value of 1.15 ± 0.09 mm, first to 0.92 ± 0.05 mm (time point 1; P < 0.001 vs. baseline) and then to 1.18 ± 0.05 (time point 2; ns). During sequence 2 (iCa high-low), iCa levels were modified from 1.15 ± 0.12 mm first to 1.20 ± 0.05 mm (time point 1; ns vs. baseline) and then to 0.93 ± 0.03 (time point 2; P < 0.001). Assuming a basic linear repeated measures model, PWV was positively related to iCa levels (P < 0.03) independent of systolic or diastolic BP, heart rate, or ultrafiltration rate. PWV is closely related to acute changes in serum iCa levels in HD patients using RCA. RCA provides an interesting opportunity to study the effects of acute iCa changes during one dialysis procedure.

Effect of transcutaneous electrical muscle stimulation and passive cycling movements on blood pressure and removal of urea and phosphate during hemodialysis

BACKGROUND: Intradialytic exercise has been described to improve blood pressure stability and dialysis efficacy. However, comorbid conditions in the dialysis population often preclude the widespread use of active intradialytic exercise. Therefore, we investigated the effect of intradialytic transcutaneous muscle stimulation (TEMS) and passive cycling movements (PCMs) on blood pressure and dialysis efficacy in patients. STUDY DESIGN: Prospective, controlled, randomized, crossover investigation. SETTING ; PARTICIPANTS: Ten patients were randomly allocated to TEMS, PCMs, or no intervention (NI) for 9 consecutive dialysis sessions. INTERVENTION: Participants were studied with NI, PCMs using a motor-driven ergometer, and bilateral TEMS of the leg musculature. Individual dialysis prescriptions were unchanged during the investigation. OUTCOMES ; MEASUREMENTS: The effect of TEMS and PCMs on blood pressure and dialysis efficacy in patients was assessed. RESULTS: Mean blood pressure increased from 121/64 +/- 21/15 mm Hg with NI to 132/69 +/- 21/15 mm Hg (P < 0.001) during sessions with PCMs and 125/66 +/- 22/16 mm Hg (P < 0.05) during sessions with TEMS. Urea and phosphate removal during dialysis were significantly (P < 0.001) greater with TEMS (19.4 +/- 3.7 g/dialysis and 1,197 +/- 265 mg/dialysis) or PCMs (20.1 +/- 3.4 g/dialysis and 1,172 +/- 315 mg/dialysis) than with NI (15.1 +/- 3.9 g/dialysis and 895 +/- 202 mg/dialysis). Body weight, ultrafiltration, Kt/V, and increases in hemoglobin and albumin levels during dialysis did not differ among the NI, PCMs, and TEMS groups. LIMITATIONS: The study design does not allow extension of the findings to prolonged treatment. CONCLUSION: Future studies during longer observation periods will have to prove the persistence of these acute findings. Both TEMS and PCMs deserve future investigations in dialysis patients because they increase intradialytic blood pressure and facilitate urea and phosphate removal when applied short term.

Determination of carbohydrate-deficient transferrin in human serum with capillary zone electrophoresis. Sample preparation strategies for the removal of interferences caused by increased levels of immunoglobulins

Capillary zone electrophoresis (CZE) in fused-silica capillaries is an effective analytical approach for the separation and determination of the transferrin (Tf) isoforms and thus carbohydrate-deficient transferrin (CDT) in human serum. Sera of patients with progressed liver cirrhosis are prone to interferences in the beta region which prevent the proper determination of CDT by CZE without additional sample preparation. Efforts to identify, reduce or even eliminate these interferences have been undertaken. Data obtained by ultrafiltration, affinity subtraction procedures using protein A, protein L and antibodies against immunoglobulins or Tf, and immunopurification of Tf suggest that the interferences in the patient sera are caused by increased levels of IgA and IgM and are best eliminated by immunopurification. Avian IgY antibody spin column immunocapture of serum Tf followed by CZE analysis of the stripped and concentrated fraction is shown to provide an attractive approach for CDT monitoring in sera with beta region interferences.

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

Clinical Validation of a Peritoneal Dialysis Prescription Model in the PatientOnLine Software

Peritoneal transport characteristics and residual renal function require regular control and subsequent adjustment of the peritoneal dialysis (PD) prescription. Prescription models shall facilitate the prediction of the outcome of such adaptations for a given patient. In the present study, the prescription model implemented in the PatientOnLine software was validated in patients requiring a prescription change. This multicenter, international prospective cohort study with the aim to validate a PD prescription model included patients treated with continuous ambulatory peritoneal dialysis. Patients were examined with the peritoneal function test (PFT) to determine the outcome of their current prescription and the necessity for a prescription change. For these patients, a new prescription was modeled using the PatientOnLine software (Fresenius Medical Care, Bad Homburg, Germany). Two to four weeks after implementation of the new PD regimen, a second PFT was performed. The validation of the prescription model included 54 patients. Predicted and measured peritoneal Kt/V were 1.52 ± 0.31 and 1.66 ± 0.35, and total (peritoneal + renal) Kt/V values were 1.96 ± 0.48 and 2.06 ± 0.44, respectively. Predicted and measured peritoneal creatinine clearances were 42.9 ± 8.6 and 43.0 ± 8.8 L/1.73 m2/week and total creatinine clearances were 65.3 ± 26.0 and 63.3 ± 21.8 L/1.73 m2/week, respectively. The analysis revealed a Pearson's correlation coefficient for peritoneal Kt/V of 0.911 and Lin's concordance coefficient of 0.829. The value of both coefficients was 0.853 for peritoneal creatinine clearance. Predicted and measured daily net ultrafiltration was 0.77 ± 0.49 and 1.16 ± 0.63 L/24 h, respectively. Pearson's correlation and Lin's concordance coefficient were 0.518 and 0.402, respectively. Predicted and measured peritoneal glucose absorption was 125.8 ± 38.8 and 79.9 ± 30.7 g/24 h, respectively, and Pearson's correlation and Lin's concordance coefficient were 0.914 and 0.477, respectively. With good predictability of peritoneal Kt/V and creatinine clearance, the present model provides support for individual dialysis prescription in clinical practice. Peritoneal glucose absorption and ultrafiltration are less predictable and are likely to be influenced by additional clinical factors to be taken into consideration.