Ascorbic acid lowers central blood pressure and asymmetric dimethylarginine in chronic kidney disease

Ascorbic acid lowers central blood pressure and asymmetric dimethylarginine in chronic kidney... Background: Premature cardiovascular disease in patients with chronic kidney disease (CKD) is not explained by traditional risk factors and oxidative stress may contribute via endothelial and vascular dysfunction. We investigated the effect of ascorbic acid on oxidative stress and vascular function in CKD patients compared with controls with hypertension (HTN). Methods: A crossover study of intravenous saline and ascorbic acid was conducted. Biomarkers of oxidative stress were measured, while pulse wave analysis and brachial flow-mediated dilatation were performed to assess large artery and endothelial function. Results: Twenty HTN and 30 CKD patients Stages 3–5 were recruited. Serum ascorbic acid was significantly lower in patients with CKD. In both groups, ascorbic acid significantly increased total antioxidant potential and superoxide. Asymmetric dimethylarginine (ADMA) was reduced significantly by ascorbic acid in the CKD group and on multivariate regression analysis, age and the presence of CKD were predictors of ADMA response to ascorbic acid. Although no effect on FMD was observed, central blood pressure and augmentation index were reduced significantly in both groups. Conclusions: Ascorbic acid has pro- and antioxidant effects, reducing central blood pressure and augmentation index in HTN and CKD. Ascorbic acid reduces serum ADMA in CKD, which may have longer-term benefits. Key words: antioxidants, arterial stiffness, chronic kidney disease, endothelial dysfunction, oxidative stress partly by shared risk factors, but these do not fully account for Introduction the degree of observed CVD and its prevalence remains higher after adjustment for age and comorbidities [2]. The risk of cardiovascular disease (CVD) is significantly elevated in patients with chronic kidney disease (CKD) [1], with most Some suggest that non-conventional risk factors specific to CKD may explain this cardiovascular risk, including hyperphos- patients dying of a cardiovascular cause prior to requiring renal replacement therapy. The prevalence of CVD can be explained phataemia, anaemia and inflammation. Oxidative stress is Received: July 15, 2017. Editorial decision: December 12, 2017 V C The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 533 another risk factor characterized by an excess of reactive oxy- was calculated from serum creatinine using the Chronic Kidney gen species (ROS) and other oxidants, which cause derange- Disease Epidemiology Collaboration (CKD-EPI) formula [16]. ment in redox signalling, activating pathways that lead to deleterious changes to vascular biology [3]. Biomarkers of oxidative stress In vivo, this is demonstrated by evidence of endothelial dys- Blood samples were taken via an intravenous cannula contrala- function and by an increase in oxidative modification of macro- teral to the infusion arm after the control infusion of saline, molecules. For example, advanced oxidative protein products after ascorbic acid administration, and 1 h after ascorbic acid (AOPPs), 8-hydroxydeoxyguanosine and F2-isoprostanes are all administration. Samples were analysed for a panel of bio- elevated in CKD, while restoration of renal function by kidney markers of oxidative stress as described below. transplantation results in a significant reduction in these bio- A colorimetric assay was used to measure 8-iso-prostaglan- markers [4, 5]. Furthermore, plasma concentration of the nitric din F2a (F2-isoprostane) as a marker of lipid peroxidation (Direct oxide synthase (NOS) inhibitor asymmetric dimethylarginine 8-iso-Prostaglandin F2a Enzyme Immunoassay Kit, Assay (ADMA) are increased in CKD, which causes increased ROS pro- Designs, Ann Arbor, MI, USA). The assay has an intra- and inter- duction via uncoupling of endothelial NOS [6–8]. assay coefficient of variation (CV) of 5–10%, with a cross-reactiv- Previous studies have investigated the use of exogenous ity to other eicosanoids of <5%. The ratio of glutathione to antioxidants as therapy or as manipulators of redox homeosta- glutathione disulphide (GSH/GSSG) was taken as a measure of sis to explore the mechanisms behind oxidative stress in CKD. reduced glutathione availability, and this was measured using a For example, antioxidant supplementation has been shown to colorimetric assay (Bioxytech GSH/GSSG-412, OxisResearch, improve markers of oxidative stress and blood pressure (BP) in Portland, OR, USA). Intra- and interassay CVs for GSH were 0.96 animal models of CKD and hypertension (HTN) [9, 10]. In and 3.11% and for GSSG were 6.45 and 7.61%, respectively. Total humans, however, the data are conflicting, with some studies antioxidant potential (TAP) was measured using a quantitative showing certain benefits of antioxidant therapy in patients with colorimetric assay (Bioxytech AOP-450 Quantitative Assay for CKD [11, 12], and other studies finding no effect [13, 14]. Total Antioxidant Potential, OxisResearch). The intra- and inter- In order to explore the mechanisms underlying vascular dys- assay CVs were 0.7 and 1.5%, respectively [17]. High-perform- function in CKD and test the hypothesis that antioxidant ance liquid chromatography (HPLC) was used to measure both administration ameliorates oxidative stress and endothelial ascorbic acid and ADMA in plasma. Intra- and interassay CVs dysfunction, we carried out a crossover study of intravenous were 1.9 and 2.5% for ADMA [18] and 1.0 and 3.7% for ascorbic ascorbic acid and normal saline in a population of patients with acid. Whole blood samples were also taken and underwent elec- CKD Stages 3–5 [15] in comparison with a cohort with HTN and tron paramagnetic resonance spectrometry (EPR) to measure high cardiovascular risk. the rate of superoxide (O ) production (E-scan EPR system, Bruker, Billerica, MA, USA). Materials and methods Assessment of endothelial function Subjects Endothelial function was assessed by measuring the dilator Subjects with CKD were recruited from general nephrology and response of the brachial artery to increased blood flow induced peritoneal dialysis (PD) clinics; subjects with HTN and normal by reactive hyperaemia, termed FMD. Longitudinal recordings renal function were recruited from an HTN clinic. Participants of the brachial artery were taken using a 7-MHz ultrasound with diabetes, renovascular disease, liver disease or active transducer and a Siemens Accuson Sequoia ultrasound system infection were excluded, or if there was a history of vitamin C (Siemens, Berlin, Germany). Recordings were made at baseline, supplementation, renal stone disease or systemic oxalosis. after 5 min inflation of a BP cuff and after 25 mg sublingual glyc- Haemodialysis patients were excluded, given the need for fore- eryl trinitrate (GTN). FMD was performed according to the 2002 arm cannulation. The study was carried out in accordance with guidelines [19] and recent update [20]. Image analysis was per- the Declaration of Helsinki, approved by the West of Scotland formed offline using bespoke software (Brachial Analyzer 5, Medical Imaging Applications, Coralville, IA, USA). Absolute Research Ethics Committee, with informed consent received FMD was calculated as the baseline diameter subtracted from from all participants. The study is registered with a clinical tri- the peak diameter following cuff inflation, while the percentage als registry (ISRCTN 31272864). of FMD was calculated as the absolute FMD divided by the base- line diameter. The intra-operator CV of FMD was 14.68%. Study protocol Participants attended in the morning, refraining from caffeine and Assessment of arterial function nicotine and after an overnight fast. Control infusions (100 mL Arterial properties were evaluated using the SphygmoCor Vx sys- 0.9% saline) and ascorbic acid (2000 mg in 100 mL 0.9% saline) were tem (Atcor Medical, West Ryde, NSW, Australia) of applanation given intravenously over 10 min and phlebotomy and vascular tonometry. The augmentation index (Aix) was measured at the function studies performed as described below. Phlebotomy was radial artery over 15 s and corrected to a heart rate of 75 bpm to carried out on a third occasion 1 h after ascorbic acid. Vascular derive the adjusted Aix (adjAix). Central BP was derived from the function studies and analyses were performedbyasingleopera- central pulse wave by means of a Fourier transformation of the tor. Participant details were removed from both flow-mediated measured radial pulse wave. Pulse wave velocity (PWV) was dilatation (FMD) and biomarker data to facilitate blinding. measured by measuring the distance between the right carotid and femoral pulses (via the umbilicus) and then the time between Biochemical measurements the R wave of the electrocardiogram and the upstroke of the arte- Baseline biochemistry was performed in an accredited biochem- rial waveform was measured at each pulse. The carotid to femo- istry department. The estimated glomerular filtration rate (eGFR) ral PWV is equal to the distance between the two pulses divided Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 534 | K. Gillis et al. by the transit time. The intra-operator CVs for adjAix and PWV with inclusion of variables at P < 0.05. Given the small sample were 5.25 and 4.83%, respectively. size, covariates were entered into the multivariate model based on published literature on ADMA regulation [21–24]. All statisti- cal testing was performed using SPSS version 22.0 (IBM, Statistics Armonk, NY, USA). In a sample size calculation, based on a previous study where FMD was improved by ascorbic acid in renal transplant recipi- Results ents [12], 26 patients with CKD would need to be examined to demonstrate a clinically relevant improvement in FMD from the Thirty subjects with CKD (22 CKD Stages 3–5; 8 PD) and 20 saline infusion to the ascorbic acid infusion of >2% with a subjects with HTN were recruited, with eGFRs of 22.46 12.6 mL/ standard deviation of 3%, power of 90%, a¼ 0.05 and with a 2 2 min/1.73 m and 94.46 11.7 mL/min/1.73 m , respectively paired comparison. (P< 0.001). The two groups were well matched for age, body Between-group data were compared using a two-tailed mass index (BMI), smoking status and medication history, but Student’s t-test or Mann–Whitney U-test. For related data, BP was higher in the HTN group (Table 1). a paired samples Student’s t-test or Wilcoxon’s test was used. P< 0.05 was considered significant. Biomarkers A comparison of response to ascorbic acid between the CKD and HTN groups was made using a difference in differences Table 2 and Figure 1 show the baseline and change in bio- analysis (DID): a regression analysis was performed with the markers of oxidative stress after ascorbic acid in each group. mean of the measured biomarker as the dependent variable Baseline ascorbic acid was lower in patients with CKD than and the treatment, time point and treatment  time point as in HTN (22.56 27.5 versus 44.06 14.1 mmol/L; P¼ 0.023), while predictor variables. ADMA (0.616 0.14 versus 0.486 0.07 mmol/L; P< 0.001) and TAP The relationship between the change in ADMA following (0.666 0.15 mM Trolox versus 0.586 0.10 mM Trolox; P¼ 0.027) ascorbic acid and the presence of CKD was examined via regres- were higher. At baseline there were no between-group differen- sion analysis initially with a univariate analysis (simple correla- ces in F2-isoprostanes (1036.96 1048.6 versus 899.06 643.0 pg/ tion) and then stepwise multiple linear regression analysis, mL; P¼ 0.61), GSH:GSSG (91.16 251.9 versus 140.06 448.8; P¼ 0.75) or O production (0.506 0.29 versus 0.376 0.95 arbitrary Table 1. Baseline parameters in both cohorts units; P¼ 0.08). Serum ascorbic acid levels rose immediately after its admin- Parameter HTN CKD P-value istration, before falling, albeit to higher than baseline, at Age (years) 566 10 596 14 0.34 the third time point (22.5, 705.7 and 483.2 versus 44.0, 552.9 and BMI (kg/m ) 30.96 5.4 28.16 6.7 0.13 297.5 mmol/L; P< 0.001). In CKD, TAP changed from 0.666 0.15 to Peripheral BP (mmHg) 150/956 17/10 141/826 15/10 0.04* 1.246 0.20 (P< 0.001) to 1.116 0.22 mM Trolox (P< 0.001), while Mean arterial BP (mmHg) 1126 12 101.66 10.2 0.002* O production changed from 0.506 0.29 to 0.636 0.22 (P¼ 0.038) Creatinine (mmol/L) 716 11 3596 274 <0.001* to 0.556 0.18 arbitrary units (P¼ 0.42). In HTN, TAP changed eGFR (mL/min/1.73m ) 94.46 11.7 22.46 12.6 <0.001* from 0.586 0.10 to 1.156 0.22 (P< 0.001) to 0.856 0.14 mM Trolox (P< 0.001) and O changed from 0.376 0.10 to 0.506 0.14 Angiotensin-converting 9 (45) 15 (50) 0.62 2 (P¼ 0.005) to 0.436 0.10 arbitrary units (P¼ 0.007). enzyme inhibitor, n (%) Angiotensin receptor 6 (30) 7 (23) 0.60 There was no change in F2-isoprostanes observed in blocker, n (%) CKD (1036.96 1048.6, 1015.36 994.3, 855.96 383.5 pg/mL; P¼ 0.93) Beta-blocker, n (%) 3 (15) 10 (33) 0.15 or HTN (899.06 643.0, 834.96 651.0, 728.66 463.8 pg/mL; P¼ 0.63), Calcium channel 10 (50) 15 (50) 1.00 nor was there a change in GSH:GSSG in CKD (91.16 251.9, 51.96 blocker, n (%) 92.6, 45.26 82.0; P¼ 0.30) or HTN (140.06 448.8, 111.36 390.4, Statin, n (%) 6 (30) 16 (53) 0.10 41.66 74.7; P¼ 0.84). Allopurinol, n (%) 2 (10) 10 (33) 0.06 After administration of ascorbic acid there was a reduction Spironolactone, n (%) 2 (10) 0 (0) 0.07 in ADMA in the CKD group (0.616 0.14 to 0.586 0.14 mmol/L; Current smoker, n (%) 4 (20) 6 (20) 0.62 P¼ 0.039), but not the HTN group (0.486 0.07 to 0.516 0.07 mmol/ L; P¼ 0.36), which reached statistical significance in the second *Indicate a significant difference. but not the third phlebotomy time point. The degree of change Table 2. Results of biomarker assays before and after administration of ascorbic acid HTN CKD Parameter Before After P-value Before After P-value a b a b Ascorbic acid (mmol/L) 44.06 14.1 552.96 127.9 <0.001 22.56 27.5 705.76 347.5 <0.001 ADMA (mmol/L) 0.486 0.07 0.516 0.07 0.36 0.616 0.14 0.586 0.14 0.039 F2-isoprostanes (pg/mL) 899.06 643.0 834.96 651.0 0.63 1036.96 1048.6 1015.36 994.3 0.93 a b a b TAP (mM Trolox) 0.586 0.10 1.156 0.22 <0.001 0.666 0.15 1.246 0.20 <0.001 GSH:GSSG ratio 140.06 448.8 111.36 390.4 0.84 91.16 251.9 51.96 92.6 0.30 b b O production (arbitrary units) 0.376 0.10 0.506 0.14 0.005 0.506 0.29 0.636 0.22 0.038 Indicates parameters where there was a significant difference in baseline values between the two groups in an independent samples t-test. This, along with P-values, refers to a paired samples t-test comparing values measured before and after ascorbic acid. Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 535 AB CD Fig. 1. Change in (A) ascorbic acid, (B)O production, (C) ADMA and (D) TAP in both groups after administration of ascorbic acid. Measurements of biomarkers of oxida- tive stress made in both groups are shown at (1) baseline, (2) after ascorbic acid and (3) at 1 h after ascorbic acid. *Indicates a significant difference (P< 0.05) in comparison with baseline. Table 3. Simple and multiple linear regression analysis of the predictors of the change in ADMA in nanomoles in response to ascorbic acid administration. Simple regression Multiple regression Predictor Increment B 95% CI P-value B 95% CI P-value CKD 60.81 108.08 to 13.54 0.01 70.89 119.91 to 21.86 0.006 Age (years) 10 14.70 4.94–34.35 0.14 19.96 0.72–39.19 0.042 Central systolic BP (mmHg) 10 0.69 16.45–15.07 0.93 BMI (kg/m ) 1 2.05 1.80–5.90 0.29 Male 1.51 49.64–52.67 0.95 CI, confidence interval. in ADMA was also significantly different between the two a change in ADMA in response to ascorbic acid and the presence groups (–0.046 0.09 versus 0.026 0.06 mmol/L; P¼ 0.013). of CKD was negatively associated, while BMI, BP and gender In a DID analysis, there was no between-group differences in were not significant predictors (R ¼ 0.21, P¼ 0.008). the response to ascorbic acid of ascorbic acid (174.3 mmol/L; P¼ 0.12), ADMA (0.06 mmol/L; P¼ 0.22), F2-isoprostanes (42.4 pg/ Arterial stiffness mL; P¼ 0.91), TAP (0.01 mM Trolox; P¼ 0.91), GSH:GSSH ratio (26.2; P¼ 0.84) or O (0.00 arbitrary units; P¼ 0.99). Figure 2 and Table 4 demonstrate measurements of vascular A regression analysis was carried out on the determinants of function studies before and after ascorbic acid. the response of ADMA to ascorbic acid with age, presence of Baseline central BP was higher in the HTN group (132/ CKD, BMI, BP and gender entered into the model (Table 3). Due 856 15/11 versus 142/976 18/10 mmHg; P¼ 0.042) but there was to the small sample size, only a limited number of variables no difference in adjAix (25.66 8.0 versus 24.46 12.3%; P¼ 0.70). were entered. These variables were included because of pub- PWV was higher in the CKD group (10.26 2.5 versus 8.66 1.7 m/ lished data suggesting their involvement in ADMA regulation s; P¼ 0.022). [21–24] and first entered into a simple regression analysis. In a Following administration of ascorbic acid, central BP fell in multiple regression analysis, age was positively associated with both CKD (132/856 15/11 to 120/756 21/11 mmHg; P¼ 0.005) and Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 536 | K. Gillis et al. Fig. 2. Change in (A) central BP and (B) Aix in both HTN and CKD. *Indicate a significant difference (P< 0.05) occurring after ascorbic acid. SAL, normal saline; AA, ascorbic acid; NS, not significant. Table 4. Results of vascular function tests before and after administration of ascorbic acid HTN CKD Parameter Before After P-value Before After P-value a b a b Central BP (mmHg) 142/976 18/10 130/816 25/15 0.002 132/856 15/12 120/756 21/11 0.005 a b a b adjAix (%) 24.46 12.3 17.66 11.3 <0.001 25.66 8.0 15.86 10.4 <0.001 a a PWV (m/s) 8.66 1.7 8.46 1.5 0.28 10.26 2.5 9.76 3.0 0.11 b b Brachial artery diameter (mm) 4.716 0.81 5.156 0.84 <0.001 4.406 0.67 4.766 0.87 <0.001 FMD (mm) 0.196 0.07 0.256 0.14 0.12 0.206 0.11 0.276 0.15 0.10 FMD (%) 4.136 1.99 5.086 3.00 0.31 4.896 3.16 5.776 3.62 0.35 b b GTN-mediated dilatation (mm) 0.556 0.25 0.326 0.23 <0.001 0.616 0.16 0.336 0.22 <0.001 b b GTN-mediated dilatation (%) 12.306 5.91 6.636 4.91 <0.001 14.176 4.66 7.606 5.39 <0.001 Indicates parameters where there was a significant difference in baseline values between the two groups in an independent samples t-test. This, along with P-values, refers to a paired samples t-test comparing values measured before and after ascorbic acid. Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 537 HTN (142/976 18/10 to 130/816 25/15 mmHg; P¼ 0.002). A reduc- antioxidants exert a beneficial cardiovascular effect, which tion in adjAix was seen in CKD (25.66 8.0 to 15.86 10.4%; should be explored in a larger randomized placebo-controlled P< 0.001) and HTN (24.46 12.3 to 17.66 11.3%; P< 0.001), but no study. Conversely, ascorbic acid reduced central BP in both the HTN change in PWV was observed in either group. In a DID analysis, there was no between-group difference in and CKD groups by 12/15 and 12/10 mmHg, respectively, and this was associated with a reduction in Aix. This was also response to ascorbic acid of central systolic BP (0.62 mmHg P¼ 0.94), adjAix (3.0%, P¼ 0.49) or PWV (0.29 m/s, P¼ 0.77). reflected in an increase in the diameter of the brachial artery at the baseline stage of FMD, suggesting that this might have occurred due to systemic vasodilatation. Juraschek et al. [28] Endothelial dysfunction previously conducted a meta-analysis of the effect of longitudi- At baseline, no significant difference was observed in the diam- nal ascorbic acid supplementation on BP, finding that systolic eter of the brachial artery between the HTN and CKD groups and diastolic BPs were reduced by 4.85 and 1.67 mmHg, (4.716 0.81 versus 4.406 0.67 mm; P¼ 0.18) (Table 4). Neither respectively. was there a between-group difference in FMD in either absolute It is interesting that the vascular effects of ascorbic acid are (0.196 0.07 versus 0.196 0.12 mm; P¼ 0.27) or proportional little different in HTN compared with CKD and that there was (4.136 1.99 versus 4.896 3.16%; P¼ 0.51) terms. little difference in either biomarkers of oxidative stress or In the HTN group, after administration of ascorbic acid, FMD endothelial dysfunction at baseline. While baseline FMD in the changed from 0.196 0.07 to 0.256 0.14 (P¼ 0.12) and the per- CKD group was similar to other studies in this cohort [29–31], centage FMD changed from 4.136 1.99 to 5.086 3.00 (P¼ 0.31). In in the HTN group, FMD was 4.13%, which is lower than in the CKD group FMD changed from 0.206 0.11 to 0.276 0.15 mm many other studies in similar populations. Having been (P¼ 0.10) and proportional FMD changed from 4.896 3.16 to recruited from a tertiary referral BP clinic, it is possible that 5.776 3.62 (P¼ 0.35) after ascorbic acid. There was a significant this represents a population with higher cardiovascular risk reduction in endothelial independent dilatation after ascorbic than in previously studied populations with HTN and normal acid administration, in both the HTN group (12.306 5.91 versus renal function [32]. Similarly, although the two study groups 6.636 4.91%; P< 0.001) and the CKD group (14.176 4.66 versus were well matched for age, BMI, smoking status and medica- 7.606 5.39%; P< 0.001). tion history, BP was higher in the HTN group. In the absence In a DID analysis there was no between-group difference in of any confounding factors, such as diabetes or pre-existing the change in FMD after ascorbic acid (0.43 mm, P¼ 0.75) coronary artery disease, it is possible that such higher BP might offset the differences in vascular disease occurring due to renal impairment. Discussion There are limitations to this study that should be acknowl- Oxidative stress is a state of disturbed redox signalling due to edged. Patients with diabetes were excluded to examine the an excess of ROS and derivatives and a consequent depletion of specific effect of uraemia on vascular function and oxidative cardioprotective signalling molecules such as nitric oxide (NO). stress, but it could be argued that this limits the applicability of Understanding the mechanisms underlying these processes these data to those with diabetes. Bias may also have been may lead to the development of novel therapeutics that will introduced by the need for participants to undergo a prolonged reduce the burden of CVD in patients with CKD. study visit that may select out healthier patients. Vascular In both cohorts there was a numerical increase in FMD follow- function studies and biomarkers are evaluated on a single day ing administration of ascorbic acid, which did not reach statisti- and repeated arterial occlusion and exposure to GTN may con- cal significance. Results of previous studies into the vascular found the interpretation of brachial artery ultrasound measure- effects of ascorbic acid are conflicting: Cross et al. [13]found no ments. Nevertheless, performing the study on a single day improvement in FMD after parenteral administration of ascorbic removes the confounding effect of day-to-day variability from acid, but Williamsetal. [12] found improvement in endothelium- the results. Serum levels of ascorbic acid rose to very high lev- dependent vasodilatation after ascorbic acid supplementation in els during the study, higher than would be predicted in healthy a cohort of transplant patients with mild impairment of kidney subjects [33] or during oral supplementation [34], such that the function. In an earlier study by Taddei et al.[25], ascorbic acid relevance of the effects of such levels of antioxidant could be augmented the increase in forearm blood flow caused by acetyl- questioned, although it has previously been shown that supra- choline but not sodium nitroprusside, and this effect was physiological doses of ascorbic acid are required to manipulate reversed by NOS inhibition, suggesting that ascorbic acid vascular function [35]. Furthermore, the sample size calculation increases endothelium-dependent vasodilatation in an NO- was based on between-group differences in FMD, so differences dependent manner. That no effect on FMD was observed in this in other measured parameters could be due to chance rather study could be attributed to the method of administration, given than true between-group differences. Indeed, the study is lim- as an intravenous pulse rather than an intra-arterial infusion or ited in large part by its small sample size and should be viewed oral supplement. It is also possible that the studied population as a proof-of-concept study. Nevertheless, our findings demon- was of inherently high cardiovascular risk compared with other strate that more robust studies with well-characterized cohorts studies, as demonstrated by the lower FMD and high PWV at are required to more precisely measure the oxidative stress baseline, which may result in more resistant vascular associated specifically with uraemia, separate from other risk dysfunction. factors. The presence of CKD was associated with a decrease in In summary, in comparison with matched hypertensive con- ADMA after administration of ascorbic acid, echoing the results trols, CKD patients have ascorbic acid deficiency but otherwise of earlier studies that have shown a reduction in ADMA levels similar levels of oxidative stress and endothelial dysfunction. In in patients with CKD supplemented with vitamin E [26]. Given both CKD and in a high-risk hypertensive population, parenteral that ADMA is a significant predictor of cardiovascular outcome ascorbic acid reduces central BP and Aix in a manner independ- [27], this may represent an important mechanism by which ent of endothelial function. Further studies are required to Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 538 | K. Gillis et al. 15. 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Sherman DL, Keaney JF Jr, Biegelsen ES et al. rosclerosis. Hypertension 2010; 55: 1210–1216 Pharmacological concentrations of ascorbic acid are 33. Khaw K-T, Bingham S, Welch A et al. Relation between required for the beneficial effect on endothelial vasomo- plasma ascorbic acid and mortality in men and women in tor function in hypertension. Hypertension 2000; 35: EPIC-Norfolk prospective study: a prospective population 936–941 study. Lancet 2001; 357: 657–663 Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Kidney Journal Oxford University Press

Ascorbic acid lowers central blood pressure and asymmetric dimethylarginine in chronic kidney disease

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European Renal Association - European Dialysis and Transplant Association
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© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA.
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2048-8505
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2048-8513
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10.1093/ckj/sfx158
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Abstract

Background: Premature cardiovascular disease in patients with chronic kidney disease (CKD) is not explained by traditional risk factors and oxidative stress may contribute via endothelial and vascular dysfunction. We investigated the effect of ascorbic acid on oxidative stress and vascular function in CKD patients compared with controls with hypertension (HTN). Methods: A crossover study of intravenous saline and ascorbic acid was conducted. Biomarkers of oxidative stress were measured, while pulse wave analysis and brachial flow-mediated dilatation were performed to assess large artery and endothelial function. Results: Twenty HTN and 30 CKD patients Stages 3–5 were recruited. Serum ascorbic acid was significantly lower in patients with CKD. In both groups, ascorbic acid significantly increased total antioxidant potential and superoxide. Asymmetric dimethylarginine (ADMA) was reduced significantly by ascorbic acid in the CKD group and on multivariate regression analysis, age and the presence of CKD were predictors of ADMA response to ascorbic acid. Although no effect on FMD was observed, central blood pressure and augmentation index were reduced significantly in both groups. Conclusions: Ascorbic acid has pro- and antioxidant effects, reducing central blood pressure and augmentation index in HTN and CKD. Ascorbic acid reduces serum ADMA in CKD, which may have longer-term benefits. Key words: antioxidants, arterial stiffness, chronic kidney disease, endothelial dysfunction, oxidative stress partly by shared risk factors, but these do not fully account for Introduction the degree of observed CVD and its prevalence remains higher after adjustment for age and comorbidities [2]. The risk of cardiovascular disease (CVD) is significantly elevated in patients with chronic kidney disease (CKD) [1], with most Some suggest that non-conventional risk factors specific to CKD may explain this cardiovascular risk, including hyperphos- patients dying of a cardiovascular cause prior to requiring renal replacement therapy. The prevalence of CVD can be explained phataemia, anaemia and inflammation. Oxidative stress is Received: July 15, 2017. Editorial decision: December 12, 2017 V C The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 533 another risk factor characterized by an excess of reactive oxy- was calculated from serum creatinine using the Chronic Kidney gen species (ROS) and other oxidants, which cause derange- Disease Epidemiology Collaboration (CKD-EPI) formula [16]. ment in redox signalling, activating pathways that lead to deleterious changes to vascular biology [3]. Biomarkers of oxidative stress In vivo, this is demonstrated by evidence of endothelial dys- Blood samples were taken via an intravenous cannula contrala- function and by an increase in oxidative modification of macro- teral to the infusion arm after the control infusion of saline, molecules. For example, advanced oxidative protein products after ascorbic acid administration, and 1 h after ascorbic acid (AOPPs), 8-hydroxydeoxyguanosine and F2-isoprostanes are all administration. Samples were analysed for a panel of bio- elevated in CKD, while restoration of renal function by kidney markers of oxidative stress as described below. transplantation results in a significant reduction in these bio- A colorimetric assay was used to measure 8-iso-prostaglan- markers [4, 5]. Furthermore, plasma concentration of the nitric din F2a (F2-isoprostane) as a marker of lipid peroxidation (Direct oxide synthase (NOS) inhibitor asymmetric dimethylarginine 8-iso-Prostaglandin F2a Enzyme Immunoassay Kit, Assay (ADMA) are increased in CKD, which causes increased ROS pro- Designs, Ann Arbor, MI, USA). The assay has an intra- and inter- duction via uncoupling of endothelial NOS [6–8]. assay coefficient of variation (CV) of 5–10%, with a cross-reactiv- Previous studies have investigated the use of exogenous ity to other eicosanoids of <5%. The ratio of glutathione to antioxidants as therapy or as manipulators of redox homeosta- glutathione disulphide (GSH/GSSG) was taken as a measure of sis to explore the mechanisms behind oxidative stress in CKD. reduced glutathione availability, and this was measured using a For example, antioxidant supplementation has been shown to colorimetric assay (Bioxytech GSH/GSSG-412, OxisResearch, improve markers of oxidative stress and blood pressure (BP) in Portland, OR, USA). Intra- and interassay CVs for GSH were 0.96 animal models of CKD and hypertension (HTN) [9, 10]. In and 3.11% and for GSSG were 6.45 and 7.61%, respectively. Total humans, however, the data are conflicting, with some studies antioxidant potential (TAP) was measured using a quantitative showing certain benefits of antioxidant therapy in patients with colorimetric assay (Bioxytech AOP-450 Quantitative Assay for CKD [11, 12], and other studies finding no effect [13, 14]. Total Antioxidant Potential, OxisResearch). The intra- and inter- In order to explore the mechanisms underlying vascular dys- assay CVs were 0.7 and 1.5%, respectively [17]. High-perform- function in CKD and test the hypothesis that antioxidant ance liquid chromatography (HPLC) was used to measure both administration ameliorates oxidative stress and endothelial ascorbic acid and ADMA in plasma. Intra- and interassay CVs dysfunction, we carried out a crossover study of intravenous were 1.9 and 2.5% for ADMA [18] and 1.0 and 3.7% for ascorbic ascorbic acid and normal saline in a population of patients with acid. Whole blood samples were also taken and underwent elec- CKD Stages 3–5 [15] in comparison with a cohort with HTN and tron paramagnetic resonance spectrometry (EPR) to measure high cardiovascular risk. the rate of superoxide (O ) production (E-scan EPR system, Bruker, Billerica, MA, USA). Materials and methods Assessment of endothelial function Subjects Endothelial function was assessed by measuring the dilator Subjects with CKD were recruited from general nephrology and response of the brachial artery to increased blood flow induced peritoneal dialysis (PD) clinics; subjects with HTN and normal by reactive hyperaemia, termed FMD. Longitudinal recordings renal function were recruited from an HTN clinic. Participants of the brachial artery were taken using a 7-MHz ultrasound with diabetes, renovascular disease, liver disease or active transducer and a Siemens Accuson Sequoia ultrasound system infection were excluded, or if there was a history of vitamin C (Siemens, Berlin, Germany). Recordings were made at baseline, supplementation, renal stone disease or systemic oxalosis. after 5 min inflation of a BP cuff and after 25 mg sublingual glyc- Haemodialysis patients were excluded, given the need for fore- eryl trinitrate (GTN). FMD was performed according to the 2002 arm cannulation. The study was carried out in accordance with guidelines [19] and recent update [20]. Image analysis was per- the Declaration of Helsinki, approved by the West of Scotland formed offline using bespoke software (Brachial Analyzer 5, Medical Imaging Applications, Coralville, IA, USA). Absolute Research Ethics Committee, with informed consent received FMD was calculated as the baseline diameter subtracted from from all participants. The study is registered with a clinical tri- the peak diameter following cuff inflation, while the percentage als registry (ISRCTN 31272864). of FMD was calculated as the absolute FMD divided by the base- line diameter. The intra-operator CV of FMD was 14.68%. Study protocol Participants attended in the morning, refraining from caffeine and Assessment of arterial function nicotine and after an overnight fast. Control infusions (100 mL Arterial properties were evaluated using the SphygmoCor Vx sys- 0.9% saline) and ascorbic acid (2000 mg in 100 mL 0.9% saline) were tem (Atcor Medical, West Ryde, NSW, Australia) of applanation given intravenously over 10 min and phlebotomy and vascular tonometry. The augmentation index (Aix) was measured at the function studies performed as described below. Phlebotomy was radial artery over 15 s and corrected to a heart rate of 75 bpm to carried out on a third occasion 1 h after ascorbic acid. Vascular derive the adjusted Aix (adjAix). Central BP was derived from the function studies and analyses were performedbyasingleopera- central pulse wave by means of a Fourier transformation of the tor. Participant details were removed from both flow-mediated measured radial pulse wave. Pulse wave velocity (PWV) was dilatation (FMD) and biomarker data to facilitate blinding. measured by measuring the distance between the right carotid and femoral pulses (via the umbilicus) and then the time between Biochemical measurements the R wave of the electrocardiogram and the upstroke of the arte- Baseline biochemistry was performed in an accredited biochem- rial waveform was measured at each pulse. The carotid to femo- istry department. The estimated glomerular filtration rate (eGFR) ral PWV is equal to the distance between the two pulses divided Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 534 | K. Gillis et al. by the transit time. The intra-operator CVs for adjAix and PWV with inclusion of variables at P < 0.05. Given the small sample were 5.25 and 4.83%, respectively. size, covariates were entered into the multivariate model based on published literature on ADMA regulation [21–24]. All statisti- cal testing was performed using SPSS version 22.0 (IBM, Statistics Armonk, NY, USA). In a sample size calculation, based on a previous study where FMD was improved by ascorbic acid in renal transplant recipi- Results ents [12], 26 patients with CKD would need to be examined to demonstrate a clinically relevant improvement in FMD from the Thirty subjects with CKD (22 CKD Stages 3–5; 8 PD) and 20 saline infusion to the ascorbic acid infusion of >2% with a subjects with HTN were recruited, with eGFRs of 22.46 12.6 mL/ standard deviation of 3%, power of 90%, a¼ 0.05 and with a 2 2 min/1.73 m and 94.46 11.7 mL/min/1.73 m , respectively paired comparison. (P< 0.001). The two groups were well matched for age, body Between-group data were compared using a two-tailed mass index (BMI), smoking status and medication history, but Student’s t-test or Mann–Whitney U-test. For related data, BP was higher in the HTN group (Table 1). a paired samples Student’s t-test or Wilcoxon’s test was used. P< 0.05 was considered significant. Biomarkers A comparison of response to ascorbic acid between the CKD and HTN groups was made using a difference in differences Table 2 and Figure 1 show the baseline and change in bio- analysis (DID): a regression analysis was performed with the markers of oxidative stress after ascorbic acid in each group. mean of the measured biomarker as the dependent variable Baseline ascorbic acid was lower in patients with CKD than and the treatment, time point and treatment  time point as in HTN (22.56 27.5 versus 44.06 14.1 mmol/L; P¼ 0.023), while predictor variables. ADMA (0.616 0.14 versus 0.486 0.07 mmol/L; P< 0.001) and TAP The relationship between the change in ADMA following (0.666 0.15 mM Trolox versus 0.586 0.10 mM Trolox; P¼ 0.027) ascorbic acid and the presence of CKD was examined via regres- were higher. At baseline there were no between-group differen- sion analysis initially with a univariate analysis (simple correla- ces in F2-isoprostanes (1036.96 1048.6 versus 899.06 643.0 pg/ tion) and then stepwise multiple linear regression analysis, mL; P¼ 0.61), GSH:GSSG (91.16 251.9 versus 140.06 448.8; P¼ 0.75) or O production (0.506 0.29 versus 0.376 0.95 arbitrary Table 1. Baseline parameters in both cohorts units; P¼ 0.08). Serum ascorbic acid levels rose immediately after its admin- Parameter HTN CKD P-value istration, before falling, albeit to higher than baseline, at Age (years) 566 10 596 14 0.34 the third time point (22.5, 705.7 and 483.2 versus 44.0, 552.9 and BMI (kg/m ) 30.96 5.4 28.16 6.7 0.13 297.5 mmol/L; P< 0.001). In CKD, TAP changed from 0.666 0.15 to Peripheral BP (mmHg) 150/956 17/10 141/826 15/10 0.04* 1.246 0.20 (P< 0.001) to 1.116 0.22 mM Trolox (P< 0.001), while Mean arterial BP (mmHg) 1126 12 101.66 10.2 0.002* O production changed from 0.506 0.29 to 0.636 0.22 (P¼ 0.038) Creatinine (mmol/L) 716 11 3596 274 <0.001* to 0.556 0.18 arbitrary units (P¼ 0.42). In HTN, TAP changed eGFR (mL/min/1.73m ) 94.46 11.7 22.46 12.6 <0.001* from 0.586 0.10 to 1.156 0.22 (P< 0.001) to 0.856 0.14 mM Trolox (P< 0.001) and O changed from 0.376 0.10 to 0.506 0.14 Angiotensin-converting 9 (45) 15 (50) 0.62 2 (P¼ 0.005) to 0.436 0.10 arbitrary units (P¼ 0.007). enzyme inhibitor, n (%) Angiotensin receptor 6 (30) 7 (23) 0.60 There was no change in F2-isoprostanes observed in blocker, n (%) CKD (1036.96 1048.6, 1015.36 994.3, 855.96 383.5 pg/mL; P¼ 0.93) Beta-blocker, n (%) 3 (15) 10 (33) 0.15 or HTN (899.06 643.0, 834.96 651.0, 728.66 463.8 pg/mL; P¼ 0.63), Calcium channel 10 (50) 15 (50) 1.00 nor was there a change in GSH:GSSG in CKD (91.16 251.9, 51.96 blocker, n (%) 92.6, 45.26 82.0; P¼ 0.30) or HTN (140.06 448.8, 111.36 390.4, Statin, n (%) 6 (30) 16 (53) 0.10 41.66 74.7; P¼ 0.84). Allopurinol, n (%) 2 (10) 10 (33) 0.06 After administration of ascorbic acid there was a reduction Spironolactone, n (%) 2 (10) 0 (0) 0.07 in ADMA in the CKD group (0.616 0.14 to 0.586 0.14 mmol/L; Current smoker, n (%) 4 (20) 6 (20) 0.62 P¼ 0.039), but not the HTN group (0.486 0.07 to 0.516 0.07 mmol/ L; P¼ 0.36), which reached statistical significance in the second *Indicate a significant difference. but not the third phlebotomy time point. The degree of change Table 2. Results of biomarker assays before and after administration of ascorbic acid HTN CKD Parameter Before After P-value Before After P-value a b a b Ascorbic acid (mmol/L) 44.06 14.1 552.96 127.9 <0.001 22.56 27.5 705.76 347.5 <0.001 ADMA (mmol/L) 0.486 0.07 0.516 0.07 0.36 0.616 0.14 0.586 0.14 0.039 F2-isoprostanes (pg/mL) 899.06 643.0 834.96 651.0 0.63 1036.96 1048.6 1015.36 994.3 0.93 a b a b TAP (mM Trolox) 0.586 0.10 1.156 0.22 <0.001 0.666 0.15 1.246 0.20 <0.001 GSH:GSSG ratio 140.06 448.8 111.36 390.4 0.84 91.16 251.9 51.96 92.6 0.30 b b O production (arbitrary units) 0.376 0.10 0.506 0.14 0.005 0.506 0.29 0.636 0.22 0.038 Indicates parameters where there was a significant difference in baseline values between the two groups in an independent samples t-test. This, along with P-values, refers to a paired samples t-test comparing values measured before and after ascorbic acid. Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 535 AB CD Fig. 1. Change in (A) ascorbic acid, (B)O production, (C) ADMA and (D) TAP in both groups after administration of ascorbic acid. Measurements of biomarkers of oxida- tive stress made in both groups are shown at (1) baseline, (2) after ascorbic acid and (3) at 1 h after ascorbic acid. *Indicates a significant difference (P< 0.05) in comparison with baseline. Table 3. Simple and multiple linear regression analysis of the predictors of the change in ADMA in nanomoles in response to ascorbic acid administration. Simple regression Multiple regression Predictor Increment B 95% CI P-value B 95% CI P-value CKD 60.81 108.08 to 13.54 0.01 70.89 119.91 to 21.86 0.006 Age (years) 10 14.70 4.94–34.35 0.14 19.96 0.72–39.19 0.042 Central systolic BP (mmHg) 10 0.69 16.45–15.07 0.93 BMI (kg/m ) 1 2.05 1.80–5.90 0.29 Male 1.51 49.64–52.67 0.95 CI, confidence interval. in ADMA was also significantly different between the two a change in ADMA in response to ascorbic acid and the presence groups (–0.046 0.09 versus 0.026 0.06 mmol/L; P¼ 0.013). of CKD was negatively associated, while BMI, BP and gender In a DID analysis, there was no between-group differences in were not significant predictors (R ¼ 0.21, P¼ 0.008). the response to ascorbic acid of ascorbic acid (174.3 mmol/L; P¼ 0.12), ADMA (0.06 mmol/L; P¼ 0.22), F2-isoprostanes (42.4 pg/ Arterial stiffness mL; P¼ 0.91), TAP (0.01 mM Trolox; P¼ 0.91), GSH:GSSH ratio (26.2; P¼ 0.84) or O (0.00 arbitrary units; P¼ 0.99). Figure 2 and Table 4 demonstrate measurements of vascular A regression analysis was carried out on the determinants of function studies before and after ascorbic acid. the response of ADMA to ascorbic acid with age, presence of Baseline central BP was higher in the HTN group (132/ CKD, BMI, BP and gender entered into the model (Table 3). Due 856 15/11 versus 142/976 18/10 mmHg; P¼ 0.042) but there was to the small sample size, only a limited number of variables no difference in adjAix (25.66 8.0 versus 24.46 12.3%; P¼ 0.70). were entered. These variables were included because of pub- PWV was higher in the CKD group (10.26 2.5 versus 8.66 1.7 m/ lished data suggesting their involvement in ADMA regulation s; P¼ 0.022). [21–24] and first entered into a simple regression analysis. In a Following administration of ascorbic acid, central BP fell in multiple regression analysis, age was positively associated with both CKD (132/856 15/11 to 120/756 21/11 mmHg; P¼ 0.005) and Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 536 | K. Gillis et al. Fig. 2. Change in (A) central BP and (B) Aix in both HTN and CKD. *Indicate a significant difference (P< 0.05) occurring after ascorbic acid. SAL, normal saline; AA, ascorbic acid; NS, not significant. Table 4. Results of vascular function tests before and after administration of ascorbic acid HTN CKD Parameter Before After P-value Before After P-value a b a b Central BP (mmHg) 142/976 18/10 130/816 25/15 0.002 132/856 15/12 120/756 21/11 0.005 a b a b adjAix (%) 24.46 12.3 17.66 11.3 <0.001 25.66 8.0 15.86 10.4 <0.001 a a PWV (m/s) 8.66 1.7 8.46 1.5 0.28 10.26 2.5 9.76 3.0 0.11 b b Brachial artery diameter (mm) 4.716 0.81 5.156 0.84 <0.001 4.406 0.67 4.766 0.87 <0.001 FMD (mm) 0.196 0.07 0.256 0.14 0.12 0.206 0.11 0.276 0.15 0.10 FMD (%) 4.136 1.99 5.086 3.00 0.31 4.896 3.16 5.776 3.62 0.35 b b GTN-mediated dilatation (mm) 0.556 0.25 0.326 0.23 <0.001 0.616 0.16 0.336 0.22 <0.001 b b GTN-mediated dilatation (%) 12.306 5.91 6.636 4.91 <0.001 14.176 4.66 7.606 5.39 <0.001 Indicates parameters where there was a significant difference in baseline values between the two groups in an independent samples t-test. This, along with P-values, refers to a paired samples t-test comparing values measured before and after ascorbic acid. Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 BP and ADMA in CKD | 537 HTN (142/976 18/10 to 130/816 25/15 mmHg; P¼ 0.002). A reduc- antioxidants exert a beneficial cardiovascular effect, which tion in adjAix was seen in CKD (25.66 8.0 to 15.86 10.4%; should be explored in a larger randomized placebo-controlled P< 0.001) and HTN (24.46 12.3 to 17.66 11.3%; P< 0.001), but no study. Conversely, ascorbic acid reduced central BP in both the HTN change in PWV was observed in either group. In a DID analysis, there was no between-group difference in and CKD groups by 12/15 and 12/10 mmHg, respectively, and this was associated with a reduction in Aix. This was also response to ascorbic acid of central systolic BP (0.62 mmHg P¼ 0.94), adjAix (3.0%, P¼ 0.49) or PWV (0.29 m/s, P¼ 0.77). reflected in an increase in the diameter of the brachial artery at the baseline stage of FMD, suggesting that this might have occurred due to systemic vasodilatation. Juraschek et al. [28] Endothelial dysfunction previously conducted a meta-analysis of the effect of longitudi- At baseline, no significant difference was observed in the diam- nal ascorbic acid supplementation on BP, finding that systolic eter of the brachial artery between the HTN and CKD groups and diastolic BPs were reduced by 4.85 and 1.67 mmHg, (4.716 0.81 versus 4.406 0.67 mm; P¼ 0.18) (Table 4). Neither respectively. was there a between-group difference in FMD in either absolute It is interesting that the vascular effects of ascorbic acid are (0.196 0.07 versus 0.196 0.12 mm; P¼ 0.27) or proportional little different in HTN compared with CKD and that there was (4.136 1.99 versus 4.896 3.16%; P¼ 0.51) terms. little difference in either biomarkers of oxidative stress or In the HTN group, after administration of ascorbic acid, FMD endothelial dysfunction at baseline. While baseline FMD in the changed from 0.196 0.07 to 0.256 0.14 (P¼ 0.12) and the per- CKD group was similar to other studies in this cohort [29–31], centage FMD changed from 4.136 1.99 to 5.086 3.00 (P¼ 0.31). In in the HTN group, FMD was 4.13%, which is lower than in the CKD group FMD changed from 0.206 0.11 to 0.276 0.15 mm many other studies in similar populations. Having been (P¼ 0.10) and proportional FMD changed from 4.896 3.16 to recruited from a tertiary referral BP clinic, it is possible that 5.776 3.62 (P¼ 0.35) after ascorbic acid. There was a significant this represents a population with higher cardiovascular risk reduction in endothelial independent dilatation after ascorbic than in previously studied populations with HTN and normal acid administration, in both the HTN group (12.306 5.91 versus renal function [32]. Similarly, although the two study groups 6.636 4.91%; P< 0.001) and the CKD group (14.176 4.66 versus were well matched for age, BMI, smoking status and medica- 7.606 5.39%; P< 0.001). tion history, BP was higher in the HTN group. In the absence In a DID analysis there was no between-group difference in of any confounding factors, such as diabetes or pre-existing the change in FMD after ascorbic acid (0.43 mm, P¼ 0.75) coronary artery disease, it is possible that such higher BP might offset the differences in vascular disease occurring due to renal impairment. Discussion There are limitations to this study that should be acknowl- Oxidative stress is a state of disturbed redox signalling due to edged. Patients with diabetes were excluded to examine the an excess of ROS and derivatives and a consequent depletion of specific effect of uraemia on vascular function and oxidative cardioprotective signalling molecules such as nitric oxide (NO). stress, but it could be argued that this limits the applicability of Understanding the mechanisms underlying these processes these data to those with diabetes. Bias may also have been may lead to the development of novel therapeutics that will introduced by the need for participants to undergo a prolonged reduce the burden of CVD in patients with CKD. study visit that may select out healthier patients. Vascular In both cohorts there was a numerical increase in FMD follow- function studies and biomarkers are evaluated on a single day ing administration of ascorbic acid, which did not reach statisti- and repeated arterial occlusion and exposure to GTN may con- cal significance. Results of previous studies into the vascular found the interpretation of brachial artery ultrasound measure- effects of ascorbic acid are conflicting: Cross et al. [13]found no ments. Nevertheless, performing the study on a single day improvement in FMD after parenteral administration of ascorbic removes the confounding effect of day-to-day variability from acid, but Williamsetal. [12] found improvement in endothelium- the results. Serum levels of ascorbic acid rose to very high lev- dependent vasodilatation after ascorbic acid supplementation in els during the study, higher than would be predicted in healthy a cohort of transplant patients with mild impairment of kidney subjects [33] or during oral supplementation [34], such that the function. In an earlier study by Taddei et al.[25], ascorbic acid relevance of the effects of such levels of antioxidant could be augmented the increase in forearm blood flow caused by acetyl- questioned, although it has previously been shown that supra- choline but not sodium nitroprusside, and this effect was physiological doses of ascorbic acid are required to manipulate reversed by NOS inhibition, suggesting that ascorbic acid vascular function [35]. Furthermore, the sample size calculation increases endothelium-dependent vasodilatation in an NO- was based on between-group differences in FMD, so differences dependent manner. That no effect on FMD was observed in this in other measured parameters could be due to chance rather study could be attributed to the method of administration, given than true between-group differences. Indeed, the study is lim- as an intravenous pulse rather than an intra-arterial infusion or ited in large part by its small sample size and should be viewed oral supplement. It is also possible that the studied population as a proof-of-concept study. Nevertheless, our findings demon- was of inherently high cardiovascular risk compared with other strate that more robust studies with well-characterized cohorts studies, as demonstrated by the lower FMD and high PWV at are required to more precisely measure the oxidative stress baseline, which may result in more resistant vascular associated specifically with uraemia, separate from other risk dysfunction. factors. The presence of CKD was associated with a decrease in In summary, in comparison with matched hypertensive con- ADMA after administration of ascorbic acid, echoing the results trols, CKD patients have ascorbic acid deficiency but otherwise of earlier studies that have shown a reduction in ADMA levels similar levels of oxidative stress and endothelial dysfunction. In in patients with CKD supplemented with vitamin E [26]. Given both CKD and in a high-risk hypertensive population, parenteral that ADMA is a significant predictor of cardiovascular outcome ascorbic acid reduces central BP and Aix in a manner independ- [27], this may represent an important mechanism by which ent of endothelial function. Further studies are required to Downloaded from https://academic.oup.com/ckj/article-abstract/11/4/532/4840701 by Ed 'DeepDyve' Gillespie user on 07 August 2018 538 | K. Gillis et al. 15. Chapter 2: Definition, identification, and prediction of CKD assess the effects of chronic ascorbic acid on vascular function in these populations. progression. Kidney Int Suppl 2013; 3: 63–72 16. Levey AS, Stevens LA, Schmid CH et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150: Funding 604–612 This study was funded by the Glasgow Renal and 17. Apak R, Gu ¨c ¸lu¨K, Ozyu ¨ rek M et al. Total antioxidant capacity Transplant Endowment fund. assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. Free Radic Res 2005; 39: 949–961 Conflict of interest statement 18. Blackwell S, O’Reilly DS, Talwar DK et al. HPLC analysis of None declared. asymmetric dimethylarginine (ADMA) and related arginine metabolites in human plasma using a novel non- endogenous internal standard. Clin Chim Acta 2009; 401: References 14–19 1. Culleton BF, Larson MG, Wilson PW et al. Cardiovascular dis- 19. 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Clinical Kidney JournalOxford University Press

Published: Aug 1, 2018

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