The authors declare that they have no conflict of interest.
In this work we estimated the contribution of the fluorescence of 4-pyridoxic acid (4-PA) to the total fluorescence of spent dialysate with the aim of evaluating the on-line monitoring of removal of this vitamin B-6 metabolite from the blood of patients with end-stage renal disease (ESRD).
Spectrofluorometric analysis of spent dialysate, collected from hemodialysis and hemodiafiltration sessions of 10 patients receiving regularly pyridoxine injections after dialysis treatment, was performed in the range of Ex/Em 220–500 nm. 4-PA in dialysate samples was identified and quantified using HPLC with fluorescent and MS/MS detection.
Averaged HPLC chromatogram of spent dialysate had many peaks in the wavelength region of Ex320/Em430 nm where 4-PA was the highest peak with contribution of 42.2±17.0% at the beginning and 47.7±18.0% in the end of the dialysis. High correlation (R = 0.88–0.95) between 4-PA concentration and fluorescence intensity of spent dialysate was found in the region of Ex310-330/Em415-500 nm, respectively.
4-PA elimination from the blood of ESRD patients can be potentially followed using monitoring of the fluorescence of the spent dialysate during dialysis treatments.
Vitamin deficiency is common in chronic kidney disease (CKD) patients. One vitamin that CKD patients are lacking is vitamin B-6 (B6) which is the term for a group of interconvertible molecules containing pyridoxine, pyridoxal, pyridoxamine and their phosphates. The deficiency of B6 has been linked to many pathologies including impaired gluconeogenesis and glucose tolerance [
The main active form of B6 is pyridoxal-5’-phosphate (PLP). PLP acts as a cofactor for 147 EC-classified enzymes, 64 of which are known to be present in multicellular animals [
Our previous studies have shown that UV and fluorescence spectra data, measured directly at the outflow of the spent dialysate from dialysis machine, can be used to calculate contents of different characteristic uremic solutes [
The aim of this study was to investigate the potential of measurement of fluorescence in spent dialysate for monitoring of the elimination of 4-PA from the blood of dialysis patients receiving regular B6 treatment. The set aim was achieved.
The study was approved by the Tallinn Medical Research Ethics Committee at the National Institute for Health Development, Estonia decision no. 2349. A written informed consent was obtained from all participating patients.
39 dialysis sessions of 10 patients (age 59 ± 15 years) were followed. 100 mg B6 was routinely injected to patients after each dialysis session. The dialysis machine used was Fresenius 5008H (Fresenius Medical Care, Germany), dialyzers were FX8 or FX1000, the dialysate and blood flow varied from 500–800 mL/min and 300–350 mL/min, respectively. The dialysate samples were collected 7–10, 60, 120, 180, 240 minutes after the start of the dialysis session from the outlet dialysate line and from tank (145 HD and 50 HDF samples in total). All dialysate samples were acidified down to pH 4.25 with formic acid before the HPLC analysis for the best chromatographic separation and stable retention times. Full fluorescence spectra of spent dialysates in the range of excitation/emission 220–500 nm and emission with excitation increment 10 nm were recorded with the spectrofluorophotometer RF-5301 by Shimadzu (Kyoto, Japan). The cell with optical path 4 mm was used for measurement and the Panorama Fluorescence 1.2 software by Shimadzu for spectral data processing.
The HPLC system consisted of a gradient pump unit, a thermostated auto sampler, a column oven, a diode array spectrophotometric detector (DAD) and a fluorescence detector (FLD), all Ultimate 3000 Series instruments from Dionex (Sunnyvale, CA, USA), column of Kinetex C18 100A column (Phenomenex, USA) with a security guard KJO-4282 from Phenomenex (Torrance, CA, USA). The fluorescence was recorded at the wavelength of Ex320/Em430 nm and measurement interval of 0.5 s. Chromatographic data was processed with Chromeleon 7.1 software by Dionex Thermo Scientific (Waltham, USA).
The micrOTOF-Q II instrument by Bruker Daltonik GmbH (Bremen, Germany) with ESI source was used for mass-spectrometric analyses. For the identification of 4-PA both positive and negative ion mode were used. Sample analysis were done with the following parameters: mass range of 60–1700 m/z, ion source temperature of 200°C, ESI voltage of 4.5 kV, ESI nebulization gas flow of 8.0 L/min, drying gas flow of 1.2 bar, detector voltage of 2.03 kV and acquisition rate of 1 Hz. Mass calibration was performed with sodium formate solutions from m/z 60 to 1700. For data acquisition software Compass HyStar version 3.2 and for processing Compass DataAnalysis version 4.0 SP1 was used (both Bruker, Billerica, USA).
The two-component eluent was used as mixture of A: 0.05 M formic acid adjusted to pH 4.25 with ammonium hydroxide and B: the mixture of methanol and acetonitrile in the volume ratio of 9:1, both HPLC-grade from Rathburn (Walkerburn, Scotland). The five-step linear gradient elution program was used, as specified in
Step | Time (min) | Buffer (A) % | Organic solvent (B) % | Curve type |
---|---|---|---|---|
0 | 0 | 100 | 0 | |
1 | 0 | 100 | 0 | linear |
2 | 30 | 90 | 10 | linear |
3 | 60 | 5 | 95 | concave |
4 | 80 | 5 | 95 | linear |
5 | 82 | 100 | 0 | linear |
The total flow rate of 0.8 mL/min was used with the column temperature of 40°C. The sample volume injected was 20–50 μL.
Chromatographic peak of the 4-PA was identified by comparing retention time, UV absorption, fluorescence and mass spectra data of an unknown found in the sample with the corresponding characteristics of the reference compound (4-pyridoxic acid, Sigma Aldrich, USA). HPLC fluorescence data of reference 4-PA solution with different known concentrations were used to create a calibration curve. Concentration of 4-PA was calculated on the basis of HPLC fluorescence chromatograms.
The relative contribution (RC) of the 4-PA peak in the total fluorescence of the samples was calculated as a ratio of the area of 4-PA peak (APA) to the total area of all peaks appeared on the chromatogram (Atotal): RC (%) = (APA/Atotal)*100
Student’s t-test was used to compare Two-Sample dataset, Assuming Unequal Variances, while p < 0.05 was considered significant.
Excitation at 320 nm.
Samples were collected 7–10 min after the start of the dialysis. Compound 2 was identified as 4-pyridoxic acid. Raised chromatogram is of a reference 4-pyridoxic acid solution (1 μM).
The MS spectrum of the highest peak no 2 (
All 39 dialysis sessions followed were used to calculate concentration of 4-PA found chromatographically in spent dialysates. It was found that the average concentration of 4-PA in the beginning of dialysis was 4.20 ± 2.29 μmol/L and in the end 1.71 ± 0.67 μmol/L. Average contribution of fluorophores in HPLC fluorescence chromatograms of the spent dialysates were calculated using 10 dialysis sessions data. The calculation showed that 4-PA appears to be the main contributor of the fluorescence signal (
Mean contribution ± SD at the start of the dialysis | Mean contribution ± SD at the end of the dialysis | |
---|---|---|
Unknown 1 | 7.4 ± 2.1 | 7.2 ± 3.1 |
4- PA (peak 2) | 42.2 ± 17.0 | 47.7 ± 18.0 |
Unknown 3 | 22.4 ± 5.4 | 19.9 ± 3.1 |
Linear correlation was calculated between concentration of 4-PA found chromatographically in spent dialysate and directly measured fluorescence intensity of the dialysate. High correlation (R > 0.88, N = 195) was found in the wavelength region Ex310-330/Em415-500 nm (
More detailed examination of the region with the highest R values revealed that the best correlation was found at the wavelengths Ex310/Em460 nm (Rmax = 0.95, N = 195 (
The best correlation was found with Ex310/Em460 nm (Rmax value of 0.95, N = 195).
As vitamin B6 is commonly used in the treatment of patients of chronic kidney disease, the monitoring of the vitamin status of the patient is important. Our present study confirms that measurement of fluorescence in spent dialysate may be useful for assessment of elimination of 4PA, as the main metabolite of B6, from the blood of dialysis patients receiving regular B6 treatment.
PLP, the main active form of B6 is mostly used to evaluate the status of B6. However plasma PLP levels are low in dialysis patients [
To our knowledge, B6 catabolite 4-PA has not been measured in spent dialysate so far. Many 4-PA measurement methods consist of several derivatization steps to enhance fluorescence intensity [
The results of this study indicate that the majority of the fluorescence signal (Ex320/Em430 nm) derives from 4-PA, which is the biggest contributor to the fluorescence signal with 42.2±17.0% to the total fluorescence intensity at the beginning of the dialysis and 47.7±18.0% at the end (
As 4-PA may be a potentially versatile marker that may help assess multiple aspects of health status of the dialysis patient an easy measurement method would be beneficial for the patient and hospital staff. Our previous study showed that spent dialysate provides a good substitution for blood for diagnostic analyses [
Consequently, on-line fluorescence measurements in the region of Ex310-330/Em415-500 nm could potentially help for assessment of the status of the vitamin B-6 metabolism of ESRD patients during regular dialysis treatment.
The limitations of this study were relatively small data material (only 10 dialysis patients during 40 dialysis sessions were included) and the study did not cover heterogeneity of total dialysis population. The possible role of AGE-s in 4-PA-linked fluorescence as well as the possibility of not-radiative energy transfer (FRET) between fluorophores in dialysate need to be explained for final interpretation of the wavelength shift on the correlation graph (
The 4-pyridox acid (4-PA) appeared to be the highest contributing peak of the HPLC chromatogram measured in the wavelength of Ex320/Em430 nm. The intensity of the fluorescence in the region Ex310-330/Em415-500 nm has high (R>0.88) correlation with 4-PA concentration in spent dialysate. It can be concluded from these observations, that 4-PA elimination from the blood of end stage renal disease patients can be potentially followed using monitoring of the fluorescence of the spent dialysate during regular dialysis treatment.
Includes the following: Figure A: data for normalized emission spectra of spent dialysate and 4-pyridoxic acid at excitation of 320 nm. Figure B: data for an averaged Ex320/Em430 nm chromatogram of the spent dialysate. Table A: data for mean contribution calculations. Figure C: dependence of the correlation between fluorescence intensity of spent dialysate and 4-pyridoxic acid concentration. Figure D: Data for the correlation between fluorescence intensity of spent dialysate and 4-pyridoxic acid concentration at Ex310 nm and at Em460. Figure E: Data of an example of the regression line between concentration of 4-PA in spent dialysate and fluorescence intensity.
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