2418 Mauro Forteschi1 ∗ Salvatore Sotgia1 Gianfranco Pintus1 Angelo Zinellu1 Ciriaco Carru1,2 1 Department
of Biomedical Sciences, University of Sassari, Italy 2 Quality Control Unit, Hospital University of Sassari (AOU), Sassari, Italy
Received March 1, 2014 Revised June 3, 2014 Accepted June 3, 2014
J. Sep. Sci. 2014, 37, 2418–2423
Research Article
Simultaneous determination of citrulline and arginine in human blood plasma by capillary electrophoresis with ultraviolet absorption detection† A new capillary electrophoresis method to measure human blood plasma arginine and citrulline levels in a single run without derivatization was established. After adding homoarginine as internal standard, plasma proteins were removed by a 90:10 v/v acetonitrile/ammonia mixture. Arginine and citrulline were detected by an ultraviolet detector at 190 nm and separated in 11.65 and 20.43 min, respectively, by using a 75 mmol/L Tris phosphate solution at pH 1.2 as a background electrolyte. Limits of detection were 0.8 and 5 mol/L for arginine and citrulline, respectively. Precision tests indicated a good repeatability of migration times and of peak area both for citrulline (CV% = 0.82 and 3.19) and arginine (CV% = 0.65 and 2.79). The CV% for intra- and interassay tests were, respectively, 1.84 and 3.23 for citrulline and 1.25 and 1.50 for arginine. Mean recovery was 101.5 and 98.5% for citrulline and arginine, respectively. The performance of the developed method was assessed by measuring plasma arginine levels in 52 subjects and the data were compared with those obtained by our previous assay. The new method was then applied to assess plasma citrulline and arginine in ten chronic kidney disease patients under hypolipidemic therapy with statin. Keywords: Arginine / Capillary electrophoresis / Chronic kidney disease / Citrulline DOI 10.1002/jssc.201400177
1 Introduction L-Citrulline (CIT) and L-arginine (ARG) are naturally occurring nonessential amino acids [1, 2], which play a key role in ammonia detoxification within the urea cycle [3, 4]. CIT is synthesized from ARG and glutamine in the enterocytes and once produced it is readily reconverted by the kidney’s vascular endothelium to ARG passing for argininosuccinate [5, 6]. Conversion of ARG to CIT results in nitric oxide (NO) production [7, 8], which becomes available for the target tissues within the whole body [9]. Variations in human blood plasma CIT and ARG concentrations are indicative for many diseases sharing the same consequences, i.e. an impairment of urea cycle that leads to an increase of ammonia concentration [10]. ARG is the substrate of NO synthase, whose aberrant function leads to an altered NO metabolism causing endothelial dysfunction, endothelium-dependent vasodilatation, and ultimately triggering the atherosclerosis process [11, 12]. CIT is an efficient marker for the active small bowel mass dis-
Correspondence: Dr. Ciriaco Carru, Faculty of Medicine, Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B-07100 Sassari, Sardinia, Italy E-mail: [email protected] Fax: +39-079228275
Abbreviations: ACN, acetonitrile; ARG, arginine; CIT, citrulline; CKD, chronic kidney disease; HARG, homoarginine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ease [13, 14] and for the renal failure, where hypercitrullinaemia [15] appears to be even a more sensitive marker of kidney dysfunction than the classical creatininaemia [16]. CIT also plays a role in neurological conditions such as in the Alzheimer’s disease and in multi-infarct dementia [17, 18]. Because of their implication in the above-mentioned diseases, several methods have been proposed for the quantification of CIT and ARG in biological samples. Usually based on HPLC, many methods require pre- or postcolumn derivatization techniques, such as ninhydrin staining [19], orthophtaldialdehyde [20, 21], or phenylthiocarbamyl derivatization [22] for the target compounds detection. With regard to CE, even if several assays have been proposed for amino acids analysis [23–25] only few methods are available for CIT and ARG quantification [26] and all of them require labor intensive and expensive derivatization techniques [27,28], which makes the methods more susceptible to human errors. Although a mass spectrometer detector can be a useful tool to quantify CIT and ARG in biological samples avoiding derivatization [29–35], this kind of instrumentation is still expensive and it is not yet widely available for routine screening.
∗ Additional corresponding author: Mauro Forteschi, E-mail: [email protected] † This paper is included in the virtual special issue on Amino acids proteins and peptides available at the Journal of Separation Science website.
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 2418–2423
CE shows several features including high separation efficiency and speed, minimal solvent consumption, and the low sample volume required, that, taken together, make it a low-cost high-throughput chemical analysis. In this study, we describe a simple and cheap CE method to quantify CIT and ARG simultaneously in human plasma in a single run. The proposed method does not require derivatization and both compounds are detected by UV detection at 190 nm. The developed method has been used to assess CIT, ARG, and CIT/ARG ratio in healthy subjects and in patients group affected by chronic kidney disease (CKD), during hypolipemic treatment with statin.
2 Materials and methods 2.1 Chemicals CIT, ARG, homoarginine (HARG), monomethyl arginine (MMARG), acetylarginine (AARG), acetonitrile (ACN), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma–Aldrich Italia (Milan, Italy). Ammonia was purchased from Merck (Darmstadt, Germany) while phosphoric acid from Carlo Erba Italia (Milan, Italy). The pH of the BGE was adjusted using 85% w/w phosphoric acid, and the standard solutions were prepared by dissolving the appropriate amount in MilliQ water. 2.2 Sample preparation Blood was collected after an overnight fast by venipuncture into evacuated tubes containing EDTA and immediately centrifuged at 3000 × g for 5 min at 4⬚C. To 200 L of plasma was added 30 L of a 383 mol/L HARG (internal standard) solution, then proteins were precipitated by adding 1 mL of a 90:10 v/v ACN/ammonia solution. After centrifugation at 17 000 × g for 5 min, 1 mL of supernatant was withdrawn in a clean tube and evaporated to dryness in a vacuum rotator at 80⬚C and resuspended in 50 L of milliQ water. 2.3 Capillary electrophoresis A CE system equipped with a diode array detector (Agilent 7100, Palo Alto, CA, USA) was used for the experiments. Analyses were performed in an uncoated fused-silica capillary 50 m internal diameter and 90 cm total length (80 cm to the detection window). Injection consisted of a water plug of 10 s at 50 mbar followed by a hydrodynamic injection of the sample for 10 s at 50 mbar. Separation was carried out at 25⬚C by using as a BGE, a 75 mmol/L Tris phosphate solution titrated at pH 1.2 with 85% w/w phosphoric acid. The voltage used was 30 kV (135 A), and the UV absorption detector was set at 190 nm for detection. The BGE was refreshed after each run and, before each run, the capillary was equilibrated with 1 M NaOH for 1 min, 0.5 M HCl for 1 min then with BGE for 2 min. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2419
2.4 Participants to study Ten CKD patients (mean age, 63.1 ± 11 years) were selected at the Department of Clinical and Experimental Medicine of University of Sassari (Sassari, Italy) with the following inclusion criteria: age over 18; LDL cholesterol >100 mg/dL (without concomitant hypolipidemic drugs); presence of proteinuric chronic nephropathy defined as creatinine clearance >20 mL/min/1.73 m2 combined with a urinary protein excretion rate >0.3 g/24 h, without evidence of urinary tract infection or overt heart failure (New York Heart Association class III or more). Patients were classified as CKD at stage 3 and 4, and were not on dialysis. Exclusion criteria were represented by previous or concomitant treatment with steroids, anti-inflammatory and immunosuppressive agents, vitamin B6, B12, folate or statin, evidence or suspicion of renovascular disease, obstructive uropathy, type I diabetes mellitus, and vasculitis. All patients were in stable treatment for at least six months with benazepril and valsartan, an inhibitor of the angiotensin-converting enzyme and an angiotensin II receptor antagonist, respectively. Enrolled patients were randomized to receive 40 mg/day of simvastatin. Patients were treated for 12 months and evaluated at baseline and after 4, 8, and 12 months of therapy. The healthy controls group included 12 subjects (mean age, 65.8 ± 5 years), recruited from accompanying relatives or friends of patients or from hospital personnel. Exclusion criteria for the control subjects were a history of the diabetes, systemic hypertension, cardiovascular or cerebrovascular disease, renal failure, blood dyscrasias, tumors, retinal vascular disorders, age under 18 years old, and current medication with vitamin B6, B12, or folic acid. The control subjects were recruited concurrently during the patients’ recruitment period. An informed consent was obtained from each patient and control, and the study was approved by local Institution’s Ethics Committee.
2.5 Statistical analysis All results are expressed as mean values (mean ± SD) or median values (median and range). The distribution of variables in the study group was assessed by the Kolmogorov– Simirnov test. The differences between cases and controls for quantitative variables were analyzed by Student’s t test or the Mann–Whitney test, when appropriate. The effect of drug treatment was evaluated by one-way repeated measures of analysis of variance. Calculations were performed using the software packages MedCalc for Windows, version 12.5 64 bit (MedCalc Software, Ostend, Belgium).
3 Results and discussion 3.1 Electrophoretic conditions In order to optimize the electrophoretic performances of the new method, we tested a range of BGE concentrations from www.jss-journal.com
2420
M. Forteschi et al.
J. Sep. Sci. 2014, 37, 2418–2423
Figure 2. Effect of preinjection water plug length on CIT resolution, area, and height.
Figure 1. Effect of BGE pH and concentration on CIT/ARG migration time, peak area and resolution.
50 to 100 mmol/L. A pH range from 1.1 to 3 was also tested. However, satisfactory results were obtained when pH was comprised between 1.1 and 1.3, whereby this interval was investigated in a detailed way. All the experiments were performed in triplicate on plasma samples. The best separation condition was obtained with a 75 mmol/L Tris phosphate buffer at pH 1.2 as a BGE. As can be seen in Fig. 1, by using such a BGE, the migration times were, respectively, 11.65 and 20.43 min for ARG and CIT, and the sensitivity was enough to detect CIT, which is the analyte at the lowest concentration.
3.2 Injection optimization Since ARG concentration in human plasma does not cause any sensitivity problem for CE quantification, injection was optimized principally to improve the CIT measurement. We tested a set of injection times ranging from 10 and 40 s at 50 mbar. Efficiency and resolution were shown to decrease as the injection times increases until the overlapping of the peaks at 30 s of injection. Based on the above consideration, an injection of 10 s at 50 mbar was chosen and used for the following experiments. The influence of a 0, 2, 5, 7, 10, and 15 s preinjection water plug and its effects on the electrophoretic performances were also evaluated. With a plug length
of Biomedical Sciences, University of Sassari, Italy 2 Quality Control Unit, Hospital University of Sassari (AOU), Sassari, Italy
Received March 1, 2014 Revised June 3, 2014 Accepted June 3, 2014
J. Sep. Sci. 2014, 37, 2418–2423
Research Article
Simultaneous determination of citrulline and arginine in human blood plasma by capillary electrophoresis with ultraviolet absorption detection† A new capillary electrophoresis method to measure human blood plasma arginine and citrulline levels in a single run without derivatization was established. After adding homoarginine as internal standard, plasma proteins were removed by a 90:10 v/v acetonitrile/ammonia mixture. Arginine and citrulline were detected by an ultraviolet detector at 190 nm and separated in 11.65 and 20.43 min, respectively, by using a 75 mmol/L Tris phosphate solution at pH 1.2 as a background electrolyte. Limits of detection were 0.8 and 5 mol/L for arginine and citrulline, respectively. Precision tests indicated a good repeatability of migration times and of peak area both for citrulline (CV% = 0.82 and 3.19) and arginine (CV% = 0.65 and 2.79). The CV% for intra- and interassay tests were, respectively, 1.84 and 3.23 for citrulline and 1.25 and 1.50 for arginine. Mean recovery was 101.5 and 98.5% for citrulline and arginine, respectively. The performance of the developed method was assessed by measuring plasma arginine levels in 52 subjects and the data were compared with those obtained by our previous assay. The new method was then applied to assess plasma citrulline and arginine in ten chronic kidney disease patients under hypolipidemic therapy with statin. Keywords: Arginine / Capillary electrophoresis / Chronic kidney disease / Citrulline DOI 10.1002/jssc.201400177
1 Introduction L-Citrulline (CIT) and L-arginine (ARG) are naturally occurring nonessential amino acids [1, 2], which play a key role in ammonia detoxification within the urea cycle [3, 4]. CIT is synthesized from ARG and glutamine in the enterocytes and once produced it is readily reconverted by the kidney’s vascular endothelium to ARG passing for argininosuccinate [5, 6]. Conversion of ARG to CIT results in nitric oxide (NO) production [7, 8], which becomes available for the target tissues within the whole body [9]. Variations in human blood plasma CIT and ARG concentrations are indicative for many diseases sharing the same consequences, i.e. an impairment of urea cycle that leads to an increase of ammonia concentration [10]. ARG is the substrate of NO synthase, whose aberrant function leads to an altered NO metabolism causing endothelial dysfunction, endothelium-dependent vasodilatation, and ultimately triggering the atherosclerosis process [11, 12]. CIT is an efficient marker for the active small bowel mass dis-
Correspondence: Dr. Ciriaco Carru, Faculty of Medicine, Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B-07100 Sassari, Sardinia, Italy E-mail: [email protected] Fax: +39-079228275
Abbreviations: ACN, acetonitrile; ARG, arginine; CIT, citrulline; CKD, chronic kidney disease; HARG, homoarginine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ease [13, 14] and for the renal failure, where hypercitrullinaemia [15] appears to be even a more sensitive marker of kidney dysfunction than the classical creatininaemia [16]. CIT also plays a role in neurological conditions such as in the Alzheimer’s disease and in multi-infarct dementia [17, 18]. Because of their implication in the above-mentioned diseases, several methods have been proposed for the quantification of CIT and ARG in biological samples. Usually based on HPLC, many methods require pre- or postcolumn derivatization techniques, such as ninhydrin staining [19], orthophtaldialdehyde [20, 21], or phenylthiocarbamyl derivatization [22] for the target compounds detection. With regard to CE, even if several assays have been proposed for amino acids analysis [23–25] only few methods are available for CIT and ARG quantification [26] and all of them require labor intensive and expensive derivatization techniques [27,28], which makes the methods more susceptible to human errors. Although a mass spectrometer detector can be a useful tool to quantify CIT and ARG in biological samples avoiding derivatization [29–35], this kind of instrumentation is still expensive and it is not yet widely available for routine screening.
∗ Additional corresponding author: Mauro Forteschi, E-mail: [email protected] † This paper is included in the virtual special issue on Amino acids proteins and peptides available at the Journal of Separation Science website.
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 2418–2423
CE shows several features including high separation efficiency and speed, minimal solvent consumption, and the low sample volume required, that, taken together, make it a low-cost high-throughput chemical analysis. In this study, we describe a simple and cheap CE method to quantify CIT and ARG simultaneously in human plasma in a single run. The proposed method does not require derivatization and both compounds are detected by UV detection at 190 nm. The developed method has been used to assess CIT, ARG, and CIT/ARG ratio in healthy subjects and in patients group affected by chronic kidney disease (CKD), during hypolipemic treatment with statin.
2 Materials and methods 2.1 Chemicals CIT, ARG, homoarginine (HARG), monomethyl arginine (MMARG), acetylarginine (AARG), acetonitrile (ACN), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma–Aldrich Italia (Milan, Italy). Ammonia was purchased from Merck (Darmstadt, Germany) while phosphoric acid from Carlo Erba Italia (Milan, Italy). The pH of the BGE was adjusted using 85% w/w phosphoric acid, and the standard solutions were prepared by dissolving the appropriate amount in MilliQ water. 2.2 Sample preparation Blood was collected after an overnight fast by venipuncture into evacuated tubes containing EDTA and immediately centrifuged at 3000 × g for 5 min at 4⬚C. To 200 L of plasma was added 30 L of a 383 mol/L HARG (internal standard) solution, then proteins were precipitated by adding 1 mL of a 90:10 v/v ACN/ammonia solution. After centrifugation at 17 000 × g for 5 min, 1 mL of supernatant was withdrawn in a clean tube and evaporated to dryness in a vacuum rotator at 80⬚C and resuspended in 50 L of milliQ water. 2.3 Capillary electrophoresis A CE system equipped with a diode array detector (Agilent 7100, Palo Alto, CA, USA) was used for the experiments. Analyses were performed in an uncoated fused-silica capillary 50 m internal diameter and 90 cm total length (80 cm to the detection window). Injection consisted of a water plug of 10 s at 50 mbar followed by a hydrodynamic injection of the sample for 10 s at 50 mbar. Separation was carried out at 25⬚C by using as a BGE, a 75 mmol/L Tris phosphate solution titrated at pH 1.2 with 85% w/w phosphoric acid. The voltage used was 30 kV (135 A), and the UV absorption detector was set at 190 nm for detection. The BGE was refreshed after each run and, before each run, the capillary was equilibrated with 1 M NaOH for 1 min, 0.5 M HCl for 1 min then with BGE for 2 min. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2419
2.4 Participants to study Ten CKD patients (mean age, 63.1 ± 11 years) were selected at the Department of Clinical and Experimental Medicine of University of Sassari (Sassari, Italy) with the following inclusion criteria: age over 18; LDL cholesterol >100 mg/dL (without concomitant hypolipidemic drugs); presence of proteinuric chronic nephropathy defined as creatinine clearance >20 mL/min/1.73 m2 combined with a urinary protein excretion rate >0.3 g/24 h, without evidence of urinary tract infection or overt heart failure (New York Heart Association class III or more). Patients were classified as CKD at stage 3 and 4, and were not on dialysis. Exclusion criteria were represented by previous or concomitant treatment with steroids, anti-inflammatory and immunosuppressive agents, vitamin B6, B12, folate or statin, evidence or suspicion of renovascular disease, obstructive uropathy, type I diabetes mellitus, and vasculitis. All patients were in stable treatment for at least six months with benazepril and valsartan, an inhibitor of the angiotensin-converting enzyme and an angiotensin II receptor antagonist, respectively. Enrolled patients were randomized to receive 40 mg/day of simvastatin. Patients were treated for 12 months and evaluated at baseline and after 4, 8, and 12 months of therapy. The healthy controls group included 12 subjects (mean age, 65.8 ± 5 years), recruited from accompanying relatives or friends of patients or from hospital personnel. Exclusion criteria for the control subjects were a history of the diabetes, systemic hypertension, cardiovascular or cerebrovascular disease, renal failure, blood dyscrasias, tumors, retinal vascular disorders, age under 18 years old, and current medication with vitamin B6, B12, or folic acid. The control subjects were recruited concurrently during the patients’ recruitment period. An informed consent was obtained from each patient and control, and the study was approved by local Institution’s Ethics Committee.
2.5 Statistical analysis All results are expressed as mean values (mean ± SD) or median values (median and range). The distribution of variables in the study group was assessed by the Kolmogorov– Simirnov test. The differences between cases and controls for quantitative variables were analyzed by Student’s t test or the Mann–Whitney test, when appropriate. The effect of drug treatment was evaluated by one-way repeated measures of analysis of variance. Calculations were performed using the software packages MedCalc for Windows, version 12.5 64 bit (MedCalc Software, Ostend, Belgium).
3 Results and discussion 3.1 Electrophoretic conditions In order to optimize the electrophoretic performances of the new method, we tested a range of BGE concentrations from www.jss-journal.com
2420
M. Forteschi et al.
J. Sep. Sci. 2014, 37, 2418–2423
Figure 2. Effect of preinjection water plug length on CIT resolution, area, and height.
Figure 1. Effect of BGE pH and concentration on CIT/ARG migration time, peak area and resolution.
50 to 100 mmol/L. A pH range from 1.1 to 3 was also tested. However, satisfactory results were obtained when pH was comprised between 1.1 and 1.3, whereby this interval was investigated in a detailed way. All the experiments were performed in triplicate on plasma samples. The best separation condition was obtained with a 75 mmol/L Tris phosphate buffer at pH 1.2 as a BGE. As can be seen in Fig. 1, by using such a BGE, the migration times were, respectively, 11.65 and 20.43 min for ARG and CIT, and the sensitivity was enough to detect CIT, which is the analyte at the lowest concentration.
3.2 Injection optimization Since ARG concentration in human plasma does not cause any sensitivity problem for CE quantification, injection was optimized principally to improve the CIT measurement. We tested a set of injection times ranging from 10 and 40 s at 50 mbar. Efficiency and resolution were shown to decrease as the injection times increases until the overlapping of the peaks at 30 s of injection. Based on the above consideration, an injection of 10 s at 50 mbar was chosen and used for the following experiments. The influence of a 0, 2, 5, 7, 10, and 15 s preinjection water plug and its effects on the electrophoretic performances were also evaluated. With a plug length
