High Citrulline-to-Arginine Ratio Associated With Blood

Circulation Journal Vol.77, January 2013

Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

itric oxide (NO), a potent vasodilator, regulates sys-temic blood pressure (BP) and local blood flow. A deficiency of NO can lead to vasoconstriction and

cause diseases including hypertension and chronic kidney dis-ease (CKD).1–3 Reduced l-arginine (ARG) bioavailability and increased asymmetric dimethylarginine (ADMA) both contrib-ute to NO deficiency.3

ARG is a substrate for the synthesis of NO and this consti-tutes the rationale for manipulating ARG metabolism as a ther-apeutic approach for kidney disease and hypertension.4 Because ARG is involved in multiple metabolic pathways,5 there are discrepant findings in the literature according to the plasma level of ARG in patients with CKD and hypertension.6–8 ARG can be metabolized by arginase to generate ornithine, which can be further converted to l-citrulline (CIT). In contrast, the body can use CIT to make ARG via the argininosuccinate (AS) path-way involving AS synthetase and lyase.9 In human and experi-

mental CKD, renal CIT uptake is diminished, the amount of CIT converted to ARG in the kidney is reduced, and plasma CIT levels and turnover are elevated.6–8,10,11 We and others dem-onstrated that reduced renal ARG availability precedes hyper-tension in the young spontaneously hypertensive rat (SHR).12,13 We also found that restoration of renal ARG availability is as-sociated with lowering BP in young SHR.12 Renal ARG level, however, is not correlated with the levels of ARG and dimeth-ylarginine in the plasma.14 These findings suggest that decreased renal ARG level could develop in early CKD causing BP ab-normalities, which might be predicted by plasma CIT level and CIT-to-ARG ratio.

ADMA can compete with ARG for NO synthase (NOS) to generate NO and CIT.15,16 Therefore, the ARG-to-ADMA ratio is considered to represent NO bioavailability.17 ADMA is main-ly metabolized by dimethylarginine dimethylaminohydrolase (DDAH) in the kidney. Unlike ADMA, another structural

N

Received May 8, 2012; revised manuscript received July 26, 2012; accepted August 22, 2012; released online September 20, 2012 Time for primary review: 41 days

Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University, College of Medicine, Kaohsiung (Y.-J.L., M.-H.L., C.-F.H., S.-J.C., Y.-L.T.); Department of Pharmacy, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung (C.-N.H.); and Graduate Institute of Clinical Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung (C.-N.H.), Taiwan

Mailing address: You-Lin Tain, MD, PhD, Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, 123 Dabi Rd., Niausung, Kaohsiung 833, Taiwan. E-mail: [email protected]

ISSN-1346-9843 doi: 10.1253/circj.CJ-12-0602All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

High Citrulline-to-Arginine Ratio Associated With Blood Pressure Abnormalities in Children With

Early Chronic Kidney DiseaseYing-Jui Lin, MD; Chien-Ning Hsu, PhD; Mao-Hung Lo, MD;

Chien-Fu Huang, MD; Shao-Ju Chien, MD; You-Lin Tain, MD, PhD

Background: Nitric oxide (NO) is involved in hypertension and chronic kidney disease (CKD). NO synthase can metabolize l-arginine (ARG) to generate NO and l-citrulline (CIT). Two methylated ARG derivatives, asymmetric and symmetric dimethylarginine, are also involved in NO deficiency. Thus it was hypothesized that their combined ratios relate to blood pressure (BP) abnormalities in children with early CKD.

Methods and Results: The relationship between these ARG metabolites in plasma was examined using 24-h ambulatory BP monitoring (ABPM) profile in children and adolescents with CKD stages 1–3 (n=44). Approximately 20.4% (9/44) of children with CKD stages 1–3 were diagnosed with hypertension on clinical BP measurement, and 77.3% (33/44) had BP abnormalities on ABPM, including increased BP load, nocturnal BP non-dipping, and noctur-nal hypertension. Children with CKD stages 2–3 were more prevalent with abnormal BP on ABPM, and had a higher level of CIT and CIT-to-ARG ratio than those with stage 1. Furthermore, high CIT-to-ARG ratio was signifi-cantly correlated with abnormal ABPM profile, including nocturnal hypertension, increased diastolic BP load, and nocturnal BP non-dipping. Higher CIT level was significantly correlated with increased diastolic BP load and overall ABPM profile.

Conclusions: Plasma CIT-to-ARG ratio may serve as a useful marker of cardiovascular outcome in children with early CKD. (Circ J 2013; 77: 181 – 187)

Key Words: Ambulatory blood pressure monitoring; Chronic kidney disease; Citrulline; Hypertension; Nitric oxide

ORIGINAL ARTICLEPediatric Cardiology and Adult Congenital Heart Disease


182 LIN YJ et al.

isomer, symmetric dimethylarginine (SDMA), cannot directly inhibit NOS and is excreted by the kidney into the urine only. Thus the plasma ADMA-to-SDMA ratio has been proposed to reflect DDAH activity. Although plasma levels of ADMA and SDMA are both increased in patients with hypertension and CKD,18,19 little attention has been paid to understand the role of their levels and combined ratios in the development of hy-pertension in CKD. Because CIT, ARG, ADMA, and SDMA are all tightly linked to the NO pathway (Figure 1), we as-sume that simultaneous analysis of their combined ratios may provide more information to reflect NO homeostasis.

Pre-hypertension and hypertension are prevalent complica-tions in children with CKD.20 Even in early CKD, approxi-mately 50% of children have BP abnormalities, which are as-sociated with poor cardiovascular outcome.21 24-h ambulatory BP monitoring (ABPM) has proven to be a superior predictor of cardiovascular outcome when compared to clinic BP mea-surement. Our recent report showed that plasma ARG-to-ADMA ratio and ADMA-to-SDMA ratio are better markers than each individual parameter (ARG, ADMA, and SDMA) to predict hypertension in young SHR.14 In addition, we hy-pothesized that plasma CIT-to-ARG ratio reflects renal ARG availability, which may relate to BP abnormalities in children with CKD. Thus the aim of this study was to elucidate wheth-er the ARG-to-ADMA ratio, ADMA-to-SDMA ratio, and CIT-to-ARG ratio in the plasma are superior to each individual parameter in relating to BP abnormalities on ABPM in chil-dren with early CKD.

MethodsSubjectsParticipants were recruited from children and adolescents re-ferred to Kaohsiung Chang Gung Memorial Hospital for fur-ther CKD evaluation. Informed consent was obtained; the re-search protocol was approved by the Chang Gung Memorial Hospital Institutional Review Board, and followed the 1964 Declaration of Helsinki. In order to gain better insight on BP

abnormalities among children with early stages of CKD, 44 children aged 3–18 years confirmed with CKD stages 1–3 at the pediatric nephrology clinic were examined. All children were assigned to CKD stage 1 (ie, normal estimated glomeru-lar filtration rate [eGFR] ≥90 ml · min–1 · 1.73 m–2) or CKD stag-es 2–3 (ie, abnormal eGFR 30–90 ml · min–1 · 1.73 m–2) accord-ing to eGFR. The CKD staging was defined according to the K/DOQI guideline.22 eGFR was calculated by the Schwartz formula on the basis of body height and creatinine (Cr) level.23 Cr level in plasma was measured using the Jaffe reaction. Stage 1 CKD was defined as eGFR ≥90 ml · min–1 · 1.73 m–2 associ-ated with the following high-risk groups: solitary kidney, renal hypoplasia/dysplasia, obstructive nephropathy, bilateral grade IV–V vesico-ureteral reflux, neurogenic bladder, bladder out-let obstruction, steroid-resistant nephrotic syndrome plus pro-teinuria (>1 g/day), and systemic lupus erythematosus.24 Both groups of patients were followed according to the study pro-tocol for clinical and biomedical measures. The exclusion cri-teria included current pregnancy, renal transplantation, inabil-ity to complete major data collection procedures (eg, blood sampling), and history of congenital heart disease.

ABPMIn this study 3 consecutive seated BP readings were recorded and clinical BP was the mean of the last 2 readings. The 24-h ABPM data were collected using an Oscar II monitoring de-vice (SunTech Medical, Morrisville, NC, USA). A single ex-perienced nurse specialist applied the monitor. The ABPM was set to record the BP and pulse rate at 20-min intervals over 24 h. Patient diary cards were used to document each sub-ject’s daily activities and times of sleep and waking. BP read-ings >25% of any individual recordings were likely erroneous and excluded from analysis. Abnormal ABPM profile was de-termined based on (1) average daytime, average night-time, or average 24-h BP exceeding 95th percentile stratified by gen-der and height using ABPM reference data;25 (2) 24-h systolic or diastolic BP load ≥25%; and (3) nocturnal decrease of BP by <10% compared with average daytime BP.25,26 Because

Figure 1. Scheme of synthesis and metabolism of CIT, ARG, ADMA, and SDMA. ARG has multiple metabolic fates, including metabolism by NOS and arginase. CIT can be generated by NOS, DDAH, and OCT. The only route of CIT metabolism is via the ASS/ASL pathway. Both ADMA and SDMA come from methylated ARG by PRMT, but only ADMA can be metabolized by DDAH. ADMA, asymmetric dimeth-ylarginine; ARG, l-arginine; ASL, ar-gininosuccinate lyase; ASS, arginino-succinate synthetase; CIT, l-citrulline; DDAH, dimethylarginine dimethylami-nohydrolase; DMA, dimethylamine; NOS, nitric oxide synthase; OCT, orni-thine carbamoyltransferase; PRMT, protein arginine methyltransferase; SDMA, symmetric dimethylarginine.


183CAR and BP Abnormalities in Pediatric CKD

ARG, nitrate, and nitrite levels can be affected by food intake, we collected blood samples after overnight fast. We also sug-gested that families avoid giving their children foods rich in ARG (eg, peanut and gelatin), CIT (eg, watermelon), nitrates (eg, sausage), and nitrites (eg, green leafy vegetables) for 1 week before blood sampling, but complete food restriction was not practical in this age group.

Biochemistry and High-Performance Liquid ChromatographyFasting plasma specimens were aliquoted and stored at –80°C until analysis. Uric acid, glucose, total cholesterol, triglyceride, calcium, phosphate, and hematocrit were measured by stan-dard laboratory assays. Plasma levels of nitrite (NO2

–) and NOx (NO2

–+NO3–) were measured by the Griess reaction as previ-

ously described.27

CIT, ARG, ADMA, and SDMA in plasma were measured using high-performance liquid chromatography (HP series 1100, Agilent Technologies) with the o-phthaldialdehyde 3-mercaptopropionic acid (OPA-3MPA) derivatization reagent as described previously.12 The standards contained ARG, CIT, ADMA, and SDMA at 1–100 μmol/L, 1–100 μmol/L, 0.5–5 μmol/L, and 0.5–5 μmol/L, respectively. The recovery rate was approximately 85–105%. The inter-assay coefficient of variation (CV) for CIT and ADMA was 7.5% and 12.5%, whereas the intra-assay CV was 4.6% and 4.9%, respectively.

Statistical AnalysisContinuous data are presented as mean ± SD. The independent t-test or the Mann-Whitney U-test was used to test differences in variables between children with CKD stage 1 and those with CKD stages 2–3. The association between variables was ex-amined using the Pearson correlation coefficient. Chi-squared analysis was used to compare frequency (proportion, %) of abnormal ABPM parameters between groups with biomarkers

above and below the median. P<0.05 was considered statisti-cal significant. All analysis was done using SPSS 14.0 (SPSS, Chicago, IL, USA).

ResultsWe enrolled a total of 44 patients (M=28, F=16) with stages 1–3 CKD, which included 21 cases of congenital anomaly of genitourinary tract (48%), 14 cases of renal parenchymal dis-ease (32%), 7 cases of systemic disease (16%), 1 case of ge-netic disorder (2%), and 1 case of renal vascular disease (2%). As shown in Table 1, children with CKD stage 2–3 had high-er Cr but lower eGFR compared to those with CKD stage 1. Approximately 20.4% (9/44) of patients were diagnosed as hav-ing hypertension on clinical BP measurement. There was no statistical difference in patients with hypertension between the 2 groups. Except for uric acid level, there were no significant differences between the groups in terms of clinical character-istics, BP, or CKD-related biochemistry.

Unlike clinical BP measurement, we found up to 77.3% (34/44) of children with CKD had at least 1 of the BP abnor-malities on ABPM in this study. ABPM identified 6 patients (13.6%) with 24-h hypertension, 6 patients (13.6%) with day-time hypertension, 13 patients (29.5%) with night-time hyper-tension, 23 patients (52.3%) with increased systolic BP load, 7 patients (15.9%) with increased diastolic BP load, and 22 pa-tients (50%) with nocturnal BP non-dipping.

As shown in Table 2, plasma CIT and CIT-to-ARG ratio were higher in children with CKD stages 2–3 than in those with CKD stage 1. Plasma levels of ARG, ADMA, and SDMA and their combined ratios, however, were not different between children with different stages of CKD. Similarly, the plasma nitrite levels were not different between children with differ-ent stages of CKD (CKD stage 1, 0.52±0.65 μmol/L vs. CKD stages 2–3, 0.92±0.68 μmol/L). There were also no differences in plasma NOx (NO2

–+NO3–) level between the 2 groups

(49.12±57.98 μmol/L vs. 75.4±48.28 μmol/L). Table 3 lists the correlations between NOx and CIT/ARG/ADMA/SDMA pro-files. Both nitrite and NOx were positively correlated with CIT and CIT-to-ARG ratio in the plasma. There was a mild posi-tive correlation between nitrite level and plasma ARG-to-ADMA ratio.

Next, plasma CIT level was significantly higher in children with nocturnal BP non-dipping and abnormal ABPM profile than those with normal profile (Table 4). Plasma CIT-to-ARG ratio was significantly increased in children with abnormal

Table 1. Subject Characteristics vs. CKD Stage

CKD

Stage 1 Stage 2–3

n 19 25

Gender (M/F) 10/9 18/7

Hypertension 21% (4/19) 20% (5/25)

Age (years) 12.7±4.3　　 13.9±4.2　　Body height (cm) 149±18　　 153±23　　Body weight (kg) 46±19 47±20

Systolic BP (mmHg) 123±19　　 118±18　　Diastolic BP (mmHg) 74±14 74±11

Systolic BP load (%) 36±29 27±26

Diastolic BP load (%) 17±23 12±11

Creatinine (mg/dl) 0.55±0.13 　1.1±0.4*eGFR (ml · min–1 · 1.73 m–2) 116±26　　　65±16*Uric acid (mg/dl) 5.6±2.2 　7.4±1.9*Glucose (mg/dl) 87.5±7.2　　 86.2±8.9　　Cholesterol (mg/dl) 174±64　　 168±29　　Triglyceride (mg/dl) 80±59 133±100

Calcium (mg/dl) 9.3±0.3 9.2±0.4

Phosphate (mg/dl) 4.4±0.6 4.4±0.9

Hematocrit (%) 40.6±2.8　　 41.5±2.8　　

Data given as mean ± SD. *P<0.05 (t-test).BP, blood pressure; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate.

Table 2. Plasma CIT, ARG, ADMA, and SDMA vs. CKD Stage

CKD

Stage 1 Stage 2–3

CIT (μmol/L) 59±43 　89±44*ARG (μmol/L) 116±25　　 109±29　　ADMA (μmol/L) 0.68±0.39 0.75±0.43

SDMA (μmol/L) 1.12±0.5　　 1.21±0.48

AAR 211±120 213±152

ASR 0.6±0.18 0.64±0.25

CAR 0.57±0.45 　0.88±0.49*

Data given as mean ± SD. *P<0.05 (t-test).AAR, ARG-to-ADMA ratio; ADMA, asymmetric dimethylarginine; ARG, l-arginine; ASR, ADMA-to-SDMA ratio; CAR, CIT-to-ARG ratio; CIT, l-citrulline; CKD, chronic kidney disease; SDMA, symmet-ric dimethylarginine.


184 LIN YJ et al.

ABPM profile, including nocturnal hypertension, increased dia-stolic BP load, and non-night dipping. Both plasma CIT level and CIT-to-ARG ratio, however, were not different in children with early CKD stratified by abnormal vs. normal clinical BP. We also found that plasma NOx was significantly increased in children with increased diastolic BP load. In addition, the fre-quency of each abnormal ABPM parameter was not different between children with different stages of CKD (Figure 2A). Nevertheless, children with CKD with an elevated plasma CIT-to-ARG ratio (above the median) were more likely to have in-creased systolic and diastolic BP loads, nocturnal BP non-dip-ping, and abnormal ABPM profile (Figure 2B). This tendency, however, was not observed for CIT or ARG individually.

Using data pooled from all subjects (CKD stages 1–3), cor-relations of eGFR with NOx, CIT, ARG, ADMA, SDMA and their combined ratios (ARG-to-ADMA ratio, ADMA-to-SDMA ratio, and CIT-to-ARG ratio) were not significant. Similarly,

systolic and diastolic BP loads were not correlated with these parameters except for CIT-to-ARG ratio, which had a slight correlation with diastolic BP load (r=0.335, P=0.026).

DiscussionIn this study of children with early CKD, we found a remark-ably high prevalence of increased BP load, non-dipping, and nocturnal hypertension. We found that approximately 77.3% of children with early CKD had BP abnormalities on ABPM, which is consistent with previous studies that indicated that hypertension is a prevalent comorbidity in children with CKD but which may be often under-diagnosed.20,21 Importantly, high CIT level and CIT-to-ARG ratio in the plasma were associated with BP abnormalities on ABPM in children with early CKD.

Elevated plasma CIT was found in patients and rats with advanced CKD, indicating reduced CIT uptake and ARG

Table 3. NOx and CIT/ARG/ADMA/SDMA Profiles

Factor CIT ARG ADMA SDMA AAR ASR CAR

NO2–

r-value 0.496 –0.221　　 –0.128　　 0.032 0.338 –0.215　　 0.496

P-value 0.001 0.149 0.406 0.836 0.025 0.16　　 0.001

NOx

r-value 0.409 –0.174　　 –0.018　　 0.069 0.092 –0.069　　 0.456

P-value 0.006 0.258 0.907 0.656 0.552 0.658 0.002

NOx, NO2– + NO3

–. Other abbreviations as in Table 2.

Table 4. Plasma Biochemistry vs. BP and ABPM Profile in Pediatric CKD Patients

Plasma n CIT (μmol/L)

ARG (μmol/L)

ADMA (μmol/L)

SDMA (μmol/L) AAR ASR CAR NOx

(μmol/L)

24-h BP

Abnormal 6 72±57 103±25 0.55±0.28 1.03±0.48 254±192 0.61±0.28 0.73±0.56 70.8±89.6

Normal 38 76±44 111±27 0.75±0.43 1.19±0.48 206±130 0.62±0.21 0.75±0.49 63±47.5

Daytime BP

Abnormal 6 72±57 103±25 0.55±0.28 1.03±0.48 254±192 0.61±0.28 0.73±0.56 70.8±89.6

Normal 38 76±44 111±27 0.75±0.43 1.19±0.48 206±130 0.62±0.21 0.75±0.49 63±47.5

Night-time BP

Abnormal 13 94±61 100±21 0.64±0.39 1.24±0.52 220±149 0.57±0.3　　　0.97±0.61* 77.3±71.2

Normal 31 68±36 114±28 0.75±0.43 1.14±0.47 209±135 0.64±0.18 0.65±0.4　　 58.5±44.7

Systolic BP load

Abnormal 23 83±50 106±22 0.77±0.43 1.28±0.53 189±128 0.63±0.24 0.82±0.51 72±59.4

Normal 21 68±41 115±31 0.67±0.4　　 1.05±0.41 238±146 0.62±0.2　　 0.66±0.47 55.4±46.5

Diastolic BP load

Abnormal 7 　111±43*　　 106±28 0.71±0.31 1.25±0.37 206±188 0.62±0.27 　1.15±0.55* 　114.2±69.3*　　 Normal 37 70±44 111±27 0.72±0.43 1.16±0.5　　 213±130 0.62±0.21 0.67±0.45 54.6±45.4

Nocturnal dipping

Abnormal 22 89±50 113±31 0.79±0.32 1.28±0.39 176±113 0.64±0.23 　0.89±0.58* 69.6±60.1

Normal 22 63±37 108±21 0.65±0.49 1.06±0.54 249±153 0.6±0.21 0.6±0.34 58.5±47.2

Overall ABPM profile

Abnormal 34 　84±49* 111±28 0.74±0.39 1.23±0.48 200±131 0.61±0.22 　0.81±0.52* 65.3±55　　　 Normal 10 50±20 108±22 0.67±0.5　　 0.97±0.44 252±159 0.64±0.23 0.51±0.31 59.9±51.4

Clinical BP

Abnormal 9 93±44 97±23 0.74±0.48 1.23±0.45 213±180 0.62±0.29 0.99±0.49 99.1±68.1

Normal 35 72±46 113±27 0.72±0.4　　 1.16±0.49 212±128 0.62±0.2　　 0.68±0.48 55.1±46.3

Data given as mean ± SD. *P<0.05 (t-test).ABPM, ambulatory BP monitoring. Other abbreviations as in Tables 1–3.



production in the damaged kidney.6,11 Because the metabolism of CIT is primarily regulated by the intestine (synthesis) and the kidney (degradation), plasma CIT level can be used as a marker of bowel or renal dysfunction.28 We found that chil-dren with CKD stages 2–3 have higher plasma CIT level and CIT-to-ARG ratio compared to those with CKD stage 1, but plasma ARG level was not different between the 2 groups. These findings suggest that in early CKD the kidney has a com-pensatory increase in CIT synthesis and/or uptake, resulting in increased renal ARG production to maintain a steady level of

ARG in the plasma. If these compensatory mechanisms fail, renal ARG deficiency may develop, leading to hypertension. It is in line with previous observations that renal NO deficiency due to impaired CIT–ARG conversion precedes hypertension, and that restoration of renal ARG availability is associated with lowering BP in young SHR.12,13

Importantly, children with early CKD with high CIT have abnormal ABPM profile. Combined analysis of plasma CIT and ARG to generate a CIT-to-ARG ratio improved the sig-nificance for predicting BP abnormalities on ABPM in chil-

Figure 2. Frequency of blood pressure (BP) abnormalities on ambulatory BP monitoring (ABPM) according to (A) chronic kidney dis-ease (CKD) stage and (B) plasma l-citrulline-to-l-arginine (CIT-to-ARG) ratio stratified ac-cording to the median. *P<0.05.


186 LIN YJ et al.

dren with early CKD. Although children with CKD stages 2–3 have increased plasma CIT-to-ARG ratio, the high fre-quency of abnormal ABPM profile is associated with high CIT-to-ARG ratio but not high CKD stage. Similarly, data from the ESCAPE study showed that children with stages 2–4 CKD are prone to have nocturnal BP non-dipping, which is not associ-ated with GFR.29 Yet with strict BP control in these children, progression of CKD can be delayed.30 The present data high-lighted that BP abnormalities, especially at night, develop early in children with CKD, which is related to plasma CIT-to-ARG ratio. Thus early detection and aggressive management target-ing the CIT-ARG-NO pathway might be a promising strategy to treat hypertension in children with early CKD. Given that ABPM has been shown to be superior in predicting either tar-get organ damage or comorbidity,31,32 and ABPM has been used in the evaluation of the pharmacologically anti-hyperten-sive effects, we must determine whether the CIT-to-ARG ratio is beneficial in predicting progression of CKD, cardiovascular outcomes, and therapeutic response in future studies.

Although most evidence supports NO deficiency in CKD and hypertension,1–3 some reports noted that NO synthesis was increased in patients with CKD.7 In the present study, nitrite and NOx levels were not different between children with CKD stage 1 and those with CKD stages 2–3. Interestingly, we found that high NOx is related to high diastolic BP load and tends to high clinical BP. The present findings suggest that even as CKD is characterized by reduced NO synthesis in the kidney,3 a com-pensatory increase of total NO production might be expected instead. We found that both nitrite and NOx were positively related to CIT and CIT-to-ARG ratio in the plasma. Thus high CIT-to-ARG ratio in early CKD children with BP abnormali-ties might reflect high conversion rate of ARG to CIT to gen-erate NO as a compensatory response to elevation of BP.

So far, NOx remains a useful index of NO generation, al-though it cannot represent biologically active NO.33 Instead, it is possible that NOx levels increase while bioactivity of NO decreases, or vice versa. Given that apparent variation in meth-odologies used to evaluate NO synthesis in humans exists,34 there is a need to develop an ideal measure of bioactive NO in vivo in order to unravel these mysteries in future studies.

CIT supplementation improves ARG availability and en-hances NO production more than ARG itself because it by-passes splanchnic extraction, it is not a substrate for arginase, and it avoids the harmful effects of an excessive ARG sup-ply.28,35 Thus supplemental CIT has therapeutic use as a treat-ment of cardiovascular disease involving NO deficiency, in-cluding hypertension.35–37 Taking into consideration that high plasma CIT is associated with hypertension in children with early CKD and that this high ratio relates to high NOx level, the question of whether CIT supplementation is beneficial to treat hypertension in children with early CKD needs further evaluation. There are potentially several mechanisms involved in the development of hypertension. In addition to NO defi-ciency, oxidative stress and inflammation may contribute to endothelial dysfunction,1,38,39 an early event of cardiovascular disease. Given that endothelial function can be protected by CIT or ARG supplementation,5,9,35,36 it is possible that the CIT-ARG pathway may modulate oxidative stress and inflamma-tion to influence BP.

Although a previous report demonstrated that plasma SDMA level correlated positively with BP load in children with early CKD,40 such findings are not supported by the present data. The present results, however, are in good agreement with that previous study in that there was no association between BP abnormalities and ARG, ADMA, ARG-to-ADMA ratio, or

ADMA-to-SDMA ratio.40 Of significant interest is that the cor-relation between BP abnormalities and ARG-to-ADMA ratio or ADMA-to-SDMA ratio was not significant, which is incon-sistent with our previous findings showing that plasma ARG-to-ADMA ratio and ADMA-to-SDMA ratio are superior mark-ers to predict hypertension in young SHR.14 Given the fact that SHR is not a CKD-induced hypertension model, additional studies are needed across all insults of hypertension regarding the relationship between these markers and BP abnormalities in children.

This study has several limitations. First, statistical compari-sons were performed in a small cohort of children and adoles-cents with early CKD. A larger sample size is required to de-tect the true differences. Next, we did not recruit non-CKD controls because we examined the difference of clinical and biomedical features between 2 levels of renal function. That is, in the present study children with CKD stage 1 served as the controls. We found that there was no difference in each citrulline-related parameter between healthy controls and chil-dren with CKD stage 1 (data not shown). In addition, we used ABPM reference from the German group.26 Ethnic differences should be considered,41 even though 1 study found no differ-ence in ABPM between Taiwanese children and German chil-dren in the age group 6–14 years of age.42 Plasma ARG, CIT, and dimethylarginine measurements in patients are not yet performed on a routine basis and thus there is still a lack of simultaneous measurements and of their combined ratios in numerous clinical studies. To date, there is no clear cut-off for ARG-to-ADMA ratio, ADMA-to-SDMA ratio, and CIT-to-ARG ratio to define the limit between healthy subjects and children with different diseases, including hypertension and CKD. Last, measurement of biologically active NO instead of plasma NOx might provide more information about in vivo NO generation in early CKD.

ConclusionHypertension was highly prevalent in children and adolescents with early CKD. BP abnormalities on ABPM including noc-turnal hypertension, increased diastolic BP load, and nocturnal BP non-dipping were associated with increased plasma CIT-to-ARG ratio, which suggested the impact of the CIT-ARG-NO pathway on CKD-related hypertension. It is imperative to detect BP abnormalities early and to translate the study find-ings into an effective therapeutic approach to improve cardio-vascular outcome in children with CKD.

AcknowledgmentsFunding: This work was supported by grant NHRI-EX101-9826SC from National Health Research Institutes, Taiwan and grants CMRPG890261 and CMRPG890262 from the Chang Gung Memorial Hospital, Taiwan.

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High Citrulline-to-Arginine Ratio Associated With Blood