ORIGINAL ARTICLE

Asymmetric dimethylarginine levels in children with sicklecell disease and its correlation to tricuspid regurgitant jetvelocityMohamed El-Shanshory1, Ibrahim Badraia1, Amr Donia1, Faten Abd El-hameed1, Mokhtar Mabrouk2

1Department of Pediatrics, Faculty of Medicine, University of Tanta, Tanta; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

University of Tanta, Tanta, Egypt

Abstract

Background: Pulmonary hypertension (PH) is an increasingly recognized life-threatening complication in

sickle cell disease (SCD), with associated high mortality in adults. The prevalence of PH in children with

SCD is still unknown. The etiology and pathophysiologic mechanisms are still not well understood. Aim of

the study: To assess the plasma levels of asymmetric dimethylarginine (ADMA) in children with SCD and

its correlation with elevated tricuspid regurgitant jet velocity and other hemolytic markers. Subjects &

Methods: This study was carried out on a cohort of patients (30) with SCD and 30 healthy children as a

control group. Certain investigations were carried out for all subjects: CBC, lactate dehydrogenase (LDH),

ferritin, reticulocytic count, bilirubin, AST, ALT, and plasma levels of ADMA. Doppler echocardiography

was carried out for all subjects. Results: The prevalence of high tricuspid regurgitant velocity (TRV) was

30% in SCD patients. ADMA mean plasma level was significantly higher in patients than in controls

(0.79 � 0.15 lmol/L and 0.46 � 0.11 lmol/L, respectively, P < 0.001). ADMA was significantly higher in

patients with high TRV than those with normal TRV (1.10 � 0.11 lmol/L, 0.80 � 0.06 lmol/L,

respectively, P < 0.001). There was a significant positive correlation between ADMA plasma levels and

TRV � 2.5 m/s (r = 0.475). Conclusion: High plasma ADMA levels may be implicated in the pathogenesis

of increased tricuspid regurgitant jet velocity in children with SCD.

Key words sickle cell disease; tricuspid regurgitant jet velocity; asymmetric dimethylarginine

Correspondence M. El-Shanshory, Department of Pediatrics, Faculty of Medicine, University of Tanta, Tanta, Egypt. Tel: +20

1005680834; Fax: +20 403407734; e-mail: elshanshory@hotmail.com

Accepted for publication 27 March 2013 doi:10.1111/ejh.12114

Sickle cell disease (SCD) is a hemoglobinopathy that fea-tures hemoglobin polymerization, resulting in erythrocyterigidity, hemolysis, and elements of endothelial dysfunction.Increased expression of adhesion molecules on erythrocytesand endothelial cells, interactions with leukocytes, increasedlevels of circulating inflammatory cytokines, enhanced microvascular thrombosis, and endothelial damage are all thoughtto contribute to obstruction of the arterioles by sicklederythrocytes (1). Endothelial dysfunction, defined asalterations in the normal properties of the endothelium thatare inappropriate for preservation of organ function, is char-acterized by loss of protective endothelial characteristics.Endothelial dysfunction is characterized by decreasedproduction and/or bioavailability of nitric oxide (NO) (2).

Among the mechanisms for impaired NO synthesis is theaccumulation of the endogenous nitric oxide synthaseinhibitor (NOS), asymmetric dimethylarginine (ADMA) (3).ADMA is a naturally occurring amino acid. Methylated

arginine derivatives were first isolated from human urine in1970 (4). In 1992, Vallance et al. (5) first described ADMAas an endogenous inhibitor of the arginine-NO pathway.From then, the role of this molecule in the regulation ofendothelial NO synthesis has increasingly attracted attention(6). ADMA is synthesized during the methylation of proteinarginine residues by S-adenosylmethionine: protein argi-nine methyltransferases [protein methylases, (PRMT)]. Theseenzymes transfer the methyl group from S-adenosylmethio-nine (SAM) to arginine thus forming methylated arginine

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 55

European Journal of Haematology 91 (55–61)

and S-adenosylhomocysteine (SAH); the latter is subsequentlyhydrolyzed to homocysteine (7). Upon proteolytic cleavage ofarginine-methylated proteins, free monomethylarginine(MMA), ADMA, or symmetric dimethylarginine (SDMA) isgenerated and released into the cytoplasm. MMA and ADMA,but not SDMA, act as endogenous NOS inhibitors (8). As onlyminor amounts of MMA are found in human plasma andSDMA has no direct effect on NOS activity, ADMA is nowthought to be the major type of endogenously generatedmethylated arginine (2). ADMA is hydrolyzed by dimethy-larginine dimethyl aminohydrolase (DDAH) to dimethylamineand citrulline (9). Pulmonary hypertension (PH) is a recog-nized complication of SCD, which occurs in approximately6–11% of adult patients with sickle cell anemia. However, itsprevalence in children is still unknown (10).

Patients and methods

Enrollment of patient

This study was conducted between April 2011 and April2012 at Hematology and Oncology Unit of Pediatric Depart-ment, Tanta University Hospital. This cross-sectional studywas carried out on 30 children with sickle cell disease (17homozygous sickle cell anemia ‘Hb SS’ and 13 sickle thal-assemia ‘Hb Sb’). Thirty healthy children were recruited asa control group matched in ethnic, age, and sex distribution.Only seven cases of the patient group were taking hydroxy-urea. All subjects were subjected to complete history takingand full clinical examination. The following data were takenfrom the medical records: HB electrophoresis analysis, chestX-ray, X-ray on head of femur, and abdominal ultrasound.

Laboratory determinations

Venous blood was drawn for hemoglobin levels, leukocyteand platelet counts, reticulocyte counts, lactate dehydrogenase(LDH), ferritin, alanine aminotransferase, aspartateaminotransferase, bilirubin, blood urea, and serum creatinine.Human plasma ADMA was analyzed using a commercialADMA ELISA Kit (Immundiagnostik AG, Stubenwald-Allee8a, D 64625 Bensheim, made in Germany); citrated anti-coag-ulated plasma of all subjects was collected in complete aseptictechnique. Venous fasting blood samples were centrifuged atleast for 5 min at 10 000 g within 30 min of collection.Samples were frozen at �20°C till the measurement by ELISA.

Doppler echocardiography

Doppler echocardiography was carried out for all subjectsusing ultrasound machine, Vivid 7, G.E, at Tanta Universityhospital, Pediatric Department, Pediatric EchocardiographicLaboratory according to the standard protocol following theguidelines of the American Society of Echocardiography.

Subjects were screened for tricuspid regurgitant velocity(TRV) by echocardiography performed at steady state, definedas follows: � 2 wks from an acute illness including paincrisis, acute chest syndrome, febrile illness, or hospital admis-sion. TRV was measured by pulsed-wave and continuous-wave Doppler echocardiography where applicable. Multipleviews (apical 4-chamber, parasternal short axis, parasternallong axis) were obtained to record optimal tricuspid Dopplerflow signals, and a minimum of five sequential signals wererecorded (11, 12). Patients with pulmonary stenosis or otherstructural obstruction to pulmonary blood flow, evidence ofleft ventricular failure (defined as fraction shortening below28% and ejection fraction below 50%), atrial fibrillation orventricular tachycardia, significant mitral valve regurgitation>2/4 or mitral valve stenosis, severe pericardial effusion, andsubjects with no measurable TRV were excluded.

Statistics

The collected data were organized, tabulated, and statisti-cally analyzed using SPSS software statistical computer pack-age version 16 (SPSS Inc., Chicago, IL, USA). The numberand percent distribution of data was calculated. Chi-squarewas used as a test of significance and when found inappro-priate Fisher exact test was used. ANOVA test was used forcomparison among different times in the same group inquantitative data. Correlation between variables was evalu-ated using Spearman’s correlation coefficient. Significancewas adopted at P < 0.05 for interpretation of results of testsof significance.

Results

Patients characteristics and clinical and laboratory data areshown in Tables 1, 2, and 3. The prevalence ofTRV � 2.5 m/s in the studied cases was 30% (Table 4);nine patients with TRV 2.5–2.9 m/s and only four patientshad TRV � 3 m/s. Plasma ADMA levels were significantlyhigher in patient than in control group (Table 3) with asignificantly higher ADMA levels in patients withTRV � 2.5 m/s than in patients with TRV less than 2.5 m/s (Table 4). LDH, reticulocytic count, serum ferritin, andbilirubin levels were significantly higher in patients withTRV � 2.5 m/s when compared to patients withTRV < 2.5 m/s (Table 4). There were significant positivecorrelations between the plasma level of ADMA and LDH,reticulocytic count, ferritin, bilirubin, and tricuspid regurgit-ant jet velocity, while there was a negative correlation withHb level (Fig. 1).

Discussion

In the current study, the plasma ADMA levels were signifi-cantly higher in patient than in control group with a mean

56 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

ADMA levels in children with SCD El-Shanshory et al.

value 0.79 � 0.15 lmol, 0.46 � 0.11 lmol, respectively.Similar results were found by Schnog et al. (13), Kato et al.(14), and Landburg et al. 2010 (15).Elevated plasma ADMA levels lead to a reduced NO bio-

availability. Potential causes of elevated ADMA concentra-tions occur in SCD explained firstly due to increased releaseof free ADMA by increased proteolysis associated with theincreased erythrocyte turnover (13), which turn over at a rateup to twenty times normal (15). Furthermore, SCD is char-acterized by chronically elevated vascular wall shear stress(16), which is known to induce expression of endothelial

type-I protein arginine methyltransferase, a catalyst of argi-nine methylation (17). In addition, Billecke et al., (18)reported for the first time the presence of a large store ofprotein-incorporated ADMA in close proximity to the vascu-lar endothelium. This store could be released under certainpathological conditions. These findings may have clinicalimplications for those diseases displaying increased redblood cell (RBC) lysis and/or blood protein turnover. Davidset al., (19) also reported that intact erythrocytes play animportant role in storage of ADMA, whereas upon erythro-cyte lysis, large amounts of free ADMA are generated byproteolysis of methylated proteins, which may affect plasmalevels in hemolysis-associated diseases. The second causeoccurs due to impaired metabolism of ADMA followinginhibition of the DDAH, by oxidative stress triggered by

Table 1 Demographics of studied groups

Patients(n = 30)

Controls(n = 30) P-value

Age in years

(mean � SD)

6–18

(11.63 � 3.96)

6–18

(12.20 � 4.05)

0.586

M/F (%) 16 : 14

(53% : 47%)

17 : 13

(56.7% : 43.3%)

0.795

Table 2 Clinical characteristics of patient group

Medical history

Patients (n = 30)

Mean � SD Range

Interval between blood transfusion (wks) 4.2 � 1.9 2–7

% N

Cholelithiasis 10 3/30

Acute chest syndrome 3.3 1/30

A vascular necrosis 3.3 1/30

Stroke 3.3 1/30

Crises � 2/yr 26.7 8/30

ACS, acute chest syndrome; AVN, a vascular necrosis of femur.

Table 3 Laboratory data of studied groups

Parameter

Patients(n = 30)mean � SD

Controls(n = 30)mean � SD P-value

HB (gm/dL) 8.36 � 0.99 12 � 1.36 0.030*

MCV (fL) 77.3 � 11.4 80.1 � 3.5 0.663

MCH (pg) 25.9 � 4.2 29.8 � 2.3 0.057

Reticulocyte (%) 9.37 + 3.89 1 � 1.99 0.036*

White blood cell counts (thousands/lL) 10.395 � 1.56 7.036 � 1.85 0.042*

Platelet counts (thousands/lL) 458.2 � 75.6 355.5 � 68.9 0.006*

Lactic dehydrogenase (u/L) 370.6 � 128.49 185 � 58.5 0.044*

Ferritin (ng/mL) 2189.73 � 548.58 122.6 � 5.4 0.020*

Total bilirubin (mg/dL) 2 � 0.67 0.5 � 0.02 0.008*

Aspartate aminotransferase (U/L) 33.3 � 9.36 22.4 � 7.53 0.009*

Alanine aminotransferase (U/L) 23.5 � 4.7 17.2 � 2.6 0.362

Creatinine (mg/dL) 0.65 � 0.18 0.60 � 0.14 0.147

Urea (mg/dL) 26.4 � 13.5 28.4 � 8.2 0.089

ADMA (lmol/L) 0.79 � 0.15 0.46 � 0.11 0.007*

TRV (m/s) 2.387 � 0.551 2.14 � 0.2343 0.032*

ADMA, asymmetric dimethylarginine; TRV, tricuspid regurgitant velocity.

*Significant.

Table 4 Clinical and laboratory associations with TRV in SCD patients

TRV � 2.5 m/s(No. = 9/30) = 30%mean � SD

TRV < 2.5 m/s(No. = 21/30) = 70%mean � SD P-value

Age (yrs) 12.5 � 1.15 11.2 � 0.91 0.414

HB (gm/dL) 7.42 � 0.30 7.54 � 0.22 0.667

LDH (u/L) 935.7 � 83.79 406.3 � 45.19 0.001*

RETICS (%) 12.9 � 0.751 7.7 � 0.735 0.001*

Ferritin

(ng/mL)

3749 � 307.4 1521.4 � 273.77 0.001*

Bilirubin

(mg/dL)

2.48 � 0.185 1.79 � 0.133 0.008*

ADMA

(lmol/L)

1.10 � 0.11 0.80 � 0.06 0.001*

LDH, lactate dehydrogenase; SCD, sickle cell disease; ADMA,

asymmetric dimethylarginine.

*Significant.

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 57

El-Shanshory et al. ADMA levels in children with SCD

several cardiovascular risk factors (20, 21). Also, hypoxiaand elevated levels of pro-inflammatory cytokines mayinhibit or down-regulate DDAH (22). Decreased DDAHexpression/activity is evident in disease states associatedwith endothelial dysfunction (23). It is notable that normalarginine metabolism is impaired in SCD through the loss ofde novo arginine synthesis from citrulline, which occurs pri-marily in the kidney. Renal dysfunction, a common occur-rence in SCD, will impair the major route for endogenousarginine biosynthesis (24). Plasma arginase activity is ele-vated in SCD as a consequence of inflammation, liver dys-function, and, most significantly, by the release oferythrocyte arginase during intravascular hemolysis, whichhas been demonstrated by the strong correlation betweenplasma arginase levels and cell-free hemoglobin levels andother markers of increased hemolytic rate (25). Low arginine

bioavailability may be exacerbated further by the presenceof elevated ADMA, which is a competitive inhibitor of argi-nine transport and all nitric oxide synthase isoenzymes (26).The state of activation or inhibition of NOS will depend onthe local intracellular concentrations of substrate and inhibi-tor, or the L-arginine/ADMA concentration ratio (27). Thus,even modest reductions in plasma arginine concentration cansignificantly impact cellular arginine uptake and bioavailabil-ity (25). On the other hand, other diseases without hemolysiscan induce ADMA elevation such as idiopathic PH (28),chronic renal failure, and moderately increased in patientswith many other diseases including hyperlipidemia, diabetesmellitus, arterial hypertension, hyperhomocysteinemia, andheart failure (7).Our results show that the prevalence of high

TRV (� 2.5 m/s) represents 30% of SCD children.

6

7

8

9

10 A

B

C

D

E

F

ADMA (umol/L)

HB

(gm

/dL)

1.251.151.050.950.850.750.65

1.251.151.050.950.850.750.65

1.251.151.050.950.850.750.65

1500

1000

500

0

ADMA (umol/L)

LDH

(u/l)

r = –0.352

P = 0.021*

r = 0.749

P = 0.001

1.251.151.050.950.850.750.650

1000

2000

3000

4000

ADMA (umol/L)

ferri

tin (n

g/m

l)

r = 0.707

P = 0.001

1.251.151.050.950.850.750.652

7

12

17

ADMA (umol/L)

Ret

icul

ocyt

ic %

r = 0.615

P = 0.001

2

3

4

ADMA (umol/L)

TJV

(m/s

)

1.251.151.050.950.850.750.65

1

2

3

ADMA (umol/L)

bilir

ubin

(mg/

dL)

r = 0.554

P = 0.001

r = 0.481, P = 0.007

Figure 1 Correlations between asymmetric dimethylarginine (ADMA) levels and Hb level (A), lactate dehydrogenase (LDH) (B), ferritin (C), reticul-

ocytic count (D), bilirubin (E), and tricuspid regurgitant velocity (TRV) (F) in patient group. The reference cutoff value for ADMA levels was

0.58 lmol/L.

58 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

ADMA levels in children with SCD El-Shanshory et al.

TRV � 2.5 m/s represents a threshold that is associatedwith increased hemolytic rate and oxygen desaturationduring 6-min walk test (29). However, it is still unclearwhether this measurement in SCD reflects true pulmonaryhypertension per se or is a biomarker of disease severity andsystemic vasculopathy (30). Also, Voskaridou et al., (31)found that the prevalence of high TRV in their study was33%. This is similar to prevalence in previous pediatric stud-ies (32, 33, 34, 35). Prevalence of elevated TRV in the pedi-atric population of SCD is almost comparable to occurrencesin the adult population, although similar effects on mortalityare not observed (36).However, the prevalence and prognosis of PH in children

with SCD remain largely unknown despite retrospectivestudies that suggest a similar prevalence to adults (34, 35).As the pathophysiological changes that lead to developmentof PH may be reversible in children, it may be necessaryto do effective screening of PH using TRV measurementsto select candidates for RHC. On the other hand, Qureshiet al., (37) reported only 16% prevalence of high TRV inSCD; however, their study was carried out on subjects asyoung as 6 months, too early for development of pulmo-nary hypertension. Minniti C et al., (38) showed that theprevalence of elevated tricuspid regurgitant velocity was11%, which is lower than the prevalence in most of theprevious studies of pediatric sickle cell disease patients(32). This could be due to using a threshold of 2.6 m/s ormore in their study.This study demonstrates that age and HB levels were not

significantly associated with high TRV, while reticulocytecount, LDH, ferritin, and bilirubin levels were significantlycorrelated to high TRV. A similar observation was made byQureshi et al., (37) and Pashankar et al., (32). The dataemerging from the PUSH study and others provide addi-tional evidence to suggest that children with a high TRVrepresent a sicker subpopulation of pediatric SCD patients(39). These children will likely grow up to be adults with anelevated TRV (11).Minniti et al., (38) generated the ‘hemolytic index’ by

principal component analysis of the levels of lactate dehy-drogenase, aspartate aminotransferase, bilirubin, and reticulo-cyte count. This hemolytic index (and not Hb levels) wascorrelated with high TRV in their children subjects withSCD. These children may still be at greater risk of complica-tions in young adulthood and warrant close observation (40).Ataga et al., (41) found that high TRV was correlated withthe degree of hemolysis as manifested by significantly higherlactate dehydrogenase and bilirubin, lower hemoglobin, andhematocrit levels. However, after statistical adjustment forage and sex, increased serum LDH was not associated withincreased TRV. Voskaridou et al., (31) found that patientswith high TRV had elevated values of reticulocyte countsand serum ferritin compared with patients with normal TRV.This is consistent with Ambrusko et al., (34) who found that

elevated TRV is associated with low hemoglobin and ele-vated reticulocyte count. Also, Gladwin et al., 2004 (11)demonstrated a TRV � 2.5 m/s is a risk factor for PH inSCD, and screening for SCD-related PH with echocardiogra-phy is generally recommended in adults with SCD. In con-trast, Pashankar et al., 2008 (32), Suell et al. 2005 (35), andKato et al. (42) did not find a significant associationbetween markers of hemolysis and high TRV.This study demonstrates that ADMA is significantly

higher in patients with high TRV than in patients with nor-mal TRV. This is consistent with Kato et al., 2009 whonoticed that ADMA levels in patients with SCD are linkedto high TRV (43). A similar observation was found byLandburg et al., (15) who reported that plasma ADMA con-centrations were significantly higher in patients with highTRV. Moreover, plasma ADMA concentrations were identi-fied as a risk factor for early death in SCD. Also, Landburget al., (16) identified an association of plasma ADMA con-centrations with high TRV and hence risk of PH in SCD,possibly identifying a novel factor of importance in itspathophysiology. They hypothesize that chronic hemolysis-induced ADMA elevations significantly contribute to endo-thelial activation and dysfunction in SCD via NOS inhibitionand that patients with higher ADMA concentrations aremore prone to develop a vasculopathy leading to complica-tions such as PH over time. Multivariate analysis was carriedout and showed that high TRV was significantly associatedwith high ferritin, high reticulocytic count, high bilirubin,and high ADMA. In addition, ADMA was associated signifi-cantly with high LDH, high reticulocytic count, high ferritin,and high serum bilirubin. Although difference in ADMAbetween patients with and without high TRV seems modest,even small increases in extracellular ADMA lead to signifi-cant intracellular NOS inhibition through preferred cellularADMA uptake over arginine (44). Although ADMA levelsare now documented by literature to be associated with bio-markers of hemolytic rate, yet this is the first study to dem-onstrate this on children.The present study has some limitations that deserve men-

tion. First of all, our study includes relatively a small numberof patients. In addition, we used ELISA method for measure-ment of ADMA. Yokoro et al. (44) reported that ADMAhas been measured by HPLC/MS; however, these methodshave disadvantages such as difficulty in getting reproducibleresults and the necessity of using expensive instrument.Although the ELISA method is characterized by simplicity,sensitivity, and specificity and can measure the concentra-tions of ADMA in large numbers of samples efficiently (45),yet comparative studies revealed that ELISA overestimatesADMA concentration as compared with LC-MS/MS and itruns varyingly in different laboratories (46). So, Pecchiniet al. have showed in their study that appropriate calibrationis needed when ADMA is measured by ELISA (47). Anotherlimitation in our study is that echocardiography was used to

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 59

El-Shanshory et al. ADMA levels in children with SCD

estimate the TRV instead of performing a right heart cathe-terization to avoid subjecting the patients to invasive mea-sures (48). Parent et al. found that true pulmonaryhypertension, using right heart catheterization, was only 6%of SCD patients while it was 27% using the TRV threshold.However, Gladwin et al., 2004 (11) demonstrated aTRV � 2.5 m/s is a risk factor for PH in SCD and screen-ing for SCD-related PH with echocardiography is generallyrecommended in adults with SCD.

Conclusion

From this study, it can be concluded that the increasedtricuspid regurgitant jet velocity is common in pediatricSCD. ADMA may be implicated in the pathogenesis ofSCD; high plasma ADMA levels are a risk factor for hightricuspid regurgitant jet velocity in children with SCD, andit is correlated with the hemolytic markers.

Recommendation

Further pediatric studies are warranted to determine the roleof ADMA in children with SCD and its association with anelevated TRV, vasculopathy, and alterations of the argininemetabolome.

Funding

The authors declare no competing financial interests.

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