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CLINICAL STUDY

Plasma asymmetric dimethylarginine and hyperemic myocardial blood flow in young subjects with borderline hypertension or familial hypercholesterolemia

Hannu Päivä, MD^*, Juha Laakso, MSc, Hanna Laine, MD, PhD, Reijo Laaksonen, MD, PhD^*, Juhani Knuuti, MD, PhD and Olli T. Raitakari, MD, PhD^||,*

^* Department of Medicine, University of Tampere, Tampere, Finland
Department of Clinical Pharmacology, University of Helsinki, Helsinki, Finland
Department of Medicine, University of Turku, Turku, Finland
Turku PET Centre, Turku University Central Hospital, Turku, Finland
^|| Department of Clinical Physiology, University of Turku, Turku, Finland

Manuscript received June 20, 2000; revised manuscript received May 20, 2002, accepted June 27, 2002.

^* Reprint requests and correspondence: Dr. Olli T. Raitakari, Turku PET Centre, Turku University Central Hospital, FIN-20520 Turku, Finland.
olli.raitakari{at}utu.fi

	Abstract

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OBJECTIVES: The goal of this study was to examine the relationship between plasma asymmetric dimethylarginine (ADMA) level and hyperemic myocardial blood flow (MBF) in subjects with borderline hypertension (BHT) and familial hypercholesterolemia (FH).

BACKGROUND: Asymmetric dimethylarginine is an endogenous competitive inhibitor of nitric oxide synthase that may modulate vascular function.

METHODS: We measured plasma ADMA levels and myocardial flow in 77 young men (mean age 35 ± 5 years), including 47 healthy controls, 16 men with BHT, and 14 men with FH. Basal and dipyridamole-induced myocardial flow was measured using positron emission tomography. Plasma ADMA levels were measured using high-pressure liquid chromatography.

RESULTS: Asymmetric dimethylarginine levels were significantly elevated in the BHT group compared with controls (0.59 ± 0.13 µmol/l vs. 0.43 ± 0.12 µmol/l, p < 0.001), and they had significantly lower dipyridamole flow (2.85 ± 1.20 ml/min/g vs. 3.69 ± 1.68 ml/min/g, p < 0.05). In a multivariate regression model adjusted for the study group, dipyridamole flow was inversely associated with ADMA (p < 0.05), age (p < 0.05), and apolipoprotein B concentration (p < 0.05).

CONCLUSIONS: We conclude that plasma ADMA concentration is related to dipyridamole-induced vasodilatory function in young men, independently of blood pressure elevation and hypercholesterolemia. Subjects with BHT have significantly increased plasma ADMA levels, which may partly explain the impaired hyperemic MBF in this condition.

Abbreviations and Acronyms   ADMA   asymmetric dimethylarginine   BHT   borderline hypertension   BP   blood pressure   FH   familial hypercholesterolemia   HDL   high-density lipoprotein   HPLC   high-performance liquid chromatography   IMT   intima-media thickness   LDL   low-density lipoprotein   LV   left ventricle/ventricular   MBF   myocardial blood flow   NO   nitric oxide   OPA   o-pthalaldehyde   PET   positron emission tomography   ROI   region of interest   SDMA   symmetrical dimethylarginine

Nitric oxide is released by vascular endothelial cells and maintains low arterial tone at rest (12). Diminished bioavailability of NO impairs endothelium-dependent vasodilation and activates other mechanisms that may play an important role in the pathogenesis of atherosclerosis (13). In the L-arginine-NO pathway, NO synthase converts L-arginine to NO and L-citrulline (14). Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase that can modulate NO production (15). A number of cells elaborate ADMA, and it is thought to be derived from proteins that have been post-translationally methylated and subsequently hydrolyzed (15). Plasma levels of ADMA and its biologically inactive structural isomer symmetrical dimethylarginine (SDMA) have been shown to be elevated in hypercholesterolemic animals (16,17) and humans (18). Clinical studies have demonstrated an association between increased plasma ADMA concentrations and hypertension (19,20). Miyazaki et al. (21) reported a direct relationship between plasma ADMA levels and carotid IMT in healthy humans. Böger et al. (18) showed that elevated plasma ADMA levels were associated with impaired endothelium-dependent brachial artery vasodilation in young hypercholesterolemic individuals. Together, these findings indicate an association of an elevated plasma ADMA level with hypertension and hypercholesterolemia, and suggest that ADMA represents a circulating marker of subclinical atherosclerosis.

To further examine the role of ADMA in early atherosclerosis, we measured plasma ADMA levels in young subjects with borderline hypertension (BHT) and familial hypercholesterolemia who participated in a noninvasive positron emission tomography (PET) study for the assessment resting and dipyridamole-induced MBF. We tested the hypothesis that these conditions are associated with elevated plasma ADMA levels compared with healthy control subjects, and examined whether high ADMA level is associated with hyperemic MBF responses.

	Methods

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Subjects.   The study population included 47 healthy controls (control group), 16 men with BHT (BHT group), and 14 men with familial hypercholesterolemia (FH group). The control group and BHT group were recruited from an ongoing intervention study for children, the Special Turku Coronary Risk Factor Intervention Project (STRIP)-baby study (22). Families in the STRIP-baby intervention study were recruited in the Turku area (a city of approximately 160,000 inhabitants in Finland). Those families with newborn babies born between March 1991 and May 1992 were contacted and recruited for the study. A total of 1,054 families with 1,062 infants agreed to participate (56.5% of the 1,880 eligible infants in the cohort). The subjects in the present study were fathers of these children. The inclusion criteria for healthy controls were: age <45 years, body mass index <27 kg/m², BP <150/90 mm Hg, nonsmoking, no history of diabetes, and no history of atherosclerotic disease. The men were recruited by a letter of invitation. Those fulfilling the criteria and living in the Turku area were recruited by a letter of invitation; on average 65% of those invited participated in PET studies.

The BHT group included healthy fathers with three to four BP measurement values during the previous two to three years (average follow-up 2.7 years) defined according to the Sixth Joint National Committee classification as high-normal (systolic BP 130 to 140 mm Hg or diastolic BP 85 to 89 mm Hg in all previous measurements). Otherwise, the inclusion criteria for the BHT group were the same as for controls. None of the subjects in the BHT group was receiving antihypertensive medication.

All eligible FH patients meeting the inclusion criteria were selected from the patient registry of Turku University Central Hospital and asked to participate by a letter of invitation. Subjects with a clinical history or evidence of coronary artery disease or other cardiac diseases, diabetes, systemic hypertension, or current smoking were excluded. Fourteen men (of 19 eligible men) with heterozygous FH volunteered for the study. All FH patients had at least one first-degree relative with hypercholesterolemia. The diagnosis of familial hypercholesterolemia was confirmed by deoxyribonucleic acid test in five men by identifying a known Finnish mutation using enzymatic amplification of genomic deoxyribonucleic acid by the polymerase chain reaction. Seven men had either clinical signs of tendon xanthomas or a positive finding in ultrasound scanning of the Achilles tendon (23) (distinctly thickened tendons, more than 10 mm). Three men had at least one first-degree relative with hypercholesterolemia and tendon xanthomata. Twelve patients had been receiving cholesterol-lowering medication for several years. Eight patients were receiving lovastatin, one was receiving lovastatin in combination with colestipol, one was receiving lovastatin in combination with acipimox, one was receiving bezafibrate, and one patient was receiving simvastatin in combination with cholestyramine. Two men were only undergoing diet therapy.

To rule out significant coronary heart disease, stress echocardiographic examinations were performed on all subjects with relatively low myocardial perfusion reserve values (the ratio of dipyridamole flow to basal flow <3). All these men had normal exercise capacity, were asymptomatic during the test, had no diagnostic ST-segment changes in electrocardiogram, and no wall motion disturbances either at rest or after the exercise test.

The study protocol was approved by the Joint Commission on Ethics of the Turku University and the Turku University Central Hospital. Each subject gave a written informed consent.

PET study protocol
The study subjects were instructed to fast (including coffee, tea, and caffeine-containing soft-drinks) 12 h before the PET scan. Heart rate and electrocardiogram were monitored continuously during the studies. Blood pressure was monitored with an automatic oscillometric BP monitor (OMRON HEM-705C, Omron Corporation, Kyoto, Japan) during the PET study. Myocardial perfusion was measured twice, once at rest and once after administration of dipyridamole using PET and [¹⁵O]-labelled tracers. To calculate myocardial perfusion noninvasively without arterial blood sampling, a mathematical model is used that corrects for the spillover of radioactivity both from the myocardium into the left ventricular (LV) region of interest (ROI) and the blood into the myocardial ROI. The method requires the measurement of a time-activity curve in the LV chamber during the dynamic [¹⁵O]H₂O study and the measurement of the recovery coefficient of the LV ROI using a [¹⁵O]-carbon monoxide scan and venous blood sampling (24).

In brief, the subjects were positioned supine in a 15-slice ECAT 931/08-12 tomograph (Siemens/CTI, Knoxville, Tennessee) with a measured axial resolution of 6.7 mm and in-plane resolution of 6.5 mm. The subjects inhaled [¹⁵O]-carbon monoxide for 2 min (mean dose 3,400 ± 410 Mbq). After the inhalation, 2 min were allowed for carbon monoxide to combine with hemoglobin in red blood cells before a static scan for 4 min was started. During the scan period, three blood samples were drawn at 2-min intervals for blood radioactivity. A 10-min period was allowed for the radioactive decay of [¹⁵O]-carbon monoxide before the flow measurements. Flow was measured at the baseline and 2 min after the end of intravenous administration of dipyridamole (0.56 mg per kg of body weight over a period of 4 min). A total of 1,650 ± 110 MBq of [¹⁵O]H₂O was injected intravenously during 2 min, and a dynamic scan was started for 6 min. Previous data have shown that resting and hyperemic MBF can be measured reproducibly with PET (25).

Calculation of regional blood flow
Regions of interest were placed on representative transaxial ventricular slices in each study covering anterior, septal, and lateral free wall of the LV. The ROIs were drawn on the images obtained at rest and copied to the images obtained after dipyridamole administration. Values of regional MBF (expressed in milliliters per gram of tissue per minute) were calculated according to the previously published method employing the single compartment model (26,27).

Qualitative analysis of the PET data did not reveal any regional differences in the distribution of blood flow. Therefore, in order to enhance accuracy and statistics of flow measurements, the average flow of the global left ventricular myocardium was calculated, and no detailed regional analysis was carried out.

Ultrasound imaging of carotid artery IMT
All measurements were performed by Acuson 128XP/10 (Acuson Inc., Mountain View, California) ultrasonography, using 7 MHz scanning frequency linear array transducer.

The common carotid artery far-wall IMT were measured from both sides in three different cardiac cycles approximately 10 mm distally from carotid bulb, and the values of 12 measurements were averaged, as described previously (28). In our laboratory the interobserver and intraobserver coefficients of variation of carotid IMT measurements were 5.2 ± 4.1% and 4.0 ± 3.2%, respectively (28). Intima-media thickness measurements were available from the majority of the study subjects: control subjects (43/47), BHT group (16/16), and FH group (7/14).

Serum lipoproteins
Venous blood samples were taken after a 12-h overnight fast during the same week as the PET study was performed. Serum total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride concentrations were measured using standard enzymatic methods with a fully automated analyzer (Hitachi 704, Hitachi Ltd., Tokyo, Japan). High-density lipoprotein cholesterol was measured after polyethyleneglycol precipitation. The low-density lipoprotein (LDL) cholesterol concentration was calculated using the Friedewald formula (29). Apolipoprotein-B concentrations were measured by immunonephelometric method Behringen BNA (Behringswerke AG, Marburg, Germany).

Plasma ADMA, SDMA, and arginine
Plasma arginine and its dimethylated endogenous derivatives ADMA and SDMA were determined with a novel high-performance liquid chromatography (HPLC) method. Briefly, 200 µl serum was diluted with 160 µl of distilled water, and solution was mixed with 40 µl of internal standard (homo-arginine, 400 µmol/l). Arginine, ADMA, and SDMA were then absorbed on a 100-mg Bond Elut silica solid phase extraction column (Varian, Harbor City, California), pre-treated with 800 µl of methanol and with 800 µl of distilled water. After washing, arginine, ADMA, and SDMA were eluted from the SPE column with o-pthalaldehyde (OPA) derivatizing reagent. The OPA reagent was modified by adding acetonitrile and tetrahydrofuran (2:1:1). The stable OPA derivatives of arginine, ADMA, and SDMA were then separated with HPLC on an Waters Bondapak phenyl column (5 µm, 150 x 4.6 mm) with 10 mmol/l phosphate buffer containing 870 mg/l of hexanesulphonic acid as a mobile phase. Fluorescence was monitored at 328 nm (exitation) and 445 nm (emission). The HPLC equipment consisted of an LKB Pharmacia 2248 pump, a Hewlett-Packard 1050 autosampler, and a Shimadzu RF-551 fluorescence detector. Total imprecision (coefficient of variation %) for ADMA, SDMA, and arginine were not more than 12% as studied by repeated analyses of a pooled sample during the analyses of the actual samples.

Statistical methods
The results are expressed as mean ± SD. The distribution for triglycerides was skewed; therefore, the values were log₁₀-transformed before analysis. Data comparison among three groups was based on analysis of variance with respective all pairwise multiple comparison post-hoc analysis using the Bonferroni method. Student t tests were used for data comparison between two groups. The univariate association between dipyridamole flow and plasma ADMA concentration was assessed by correlation analysis. Multivariate determinants of flow variables (baseline and dipyridamole flow) were assessed by stepwise linear regression technique including ADMA, age, study group, serum lipids, and BP as covariates, with p < 0.15 as model entry criteria. All statistical tests were performed using SAS software (SAS Institute, Cary, North Carolina).

	Results

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The characteristics of study subjects are shown in Table 1. The FH group had higher serum LDL, apolipoprotein B, triglycerides, and carotid IMT compared with healthy controls but comparable ADMA, SDMA, and arginine levels. The BHT group had higher BP values, apolipoprotein B, triglycerides, and carotid IMT compared with control subjects and significantly higher ADMA levels. The levels of SDMA and arginine were similar between the three groups.

There was a weak inverse correlation between ADMA and dipyridamole flow (r = –0.22, p < 0.05) (Fig. 1). To study the association of very high ADMA levels on flow variables, we compared those subjects in the highest quintale of ADMA distribution (n = 15, ADMA >0.58 µmol/l) to others (n = 62). Subjects with high ADMA levels had significantly higher carotid IMT (0.60 ± 0.10 mm vs. 0.67 ± 0.14 mm, p = 0.03), serum triglycerides (1.79 ± 1.02 mmol/l vs. 1.17 ± 0.56 mmol/l, p = 0.04), and lower dipyridamole flow (2.60 ± 1.12 ml/min/g vs. 3.63 ± 1.65 ml/min/g, p = 0.03). The association between high ADMA levels and low dipyridamole flow was also observed when the analysis was restricted to healthy control subjects (high ADMA: n = 5 vs. others, n = 42; dipyridamole flow: 2.35 ± 1.24 vs. 3.85 ± 1.66, p = 0.05). A nonsignificant trend toward lower dipyridamole flow with high ADMA levels was also seen in the FH group (high ADMA, n = 4 vs. others, n = 10; dipyridamole flow 2.51 ± 2.37 vs. 3.50 ± 1.3, p = 0.33).

View larger version (16K): [in this window] [in a new window]   Figure 1 Scatterplot showing the correlation between plasma asymmetric dimethylarginine (ADMA) and hyperemic myocardial blood flow in pooled data.

	Discussion

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ADMA and hypertension.   Because methylarginines are, in part, eliminated by renal excretion, they accumulate in the plasma of patients with end-stage renal failure (30–32), and it has been suggested that the accumulation of ADMA contributes to the development of hypertension in these patients (33). Elevated ADMA levels may also play a role in the development of essential hypertension. Surdacki et al. (19) observed that men with newly diagnosed and untreated hypertension had increased plasma ADMA levels and depressed systemic NO formation. Observations showing increased plasma levels of ADMA in young hypertensive children (20) and in women with pregnancy-induced hypertension (34,35) also indicate that elevations in ADMA levels may play a role in the early stages of hypertension. Borderline hypertension is a strong risk factor for established hypertension, and this condition offers a useful model for "prehypertension" to examine the early metabolic manifestations related to elevated BP levels without the confounding effects mediated by other risk factors commonly clustering with advanced stages of hypertension (28). The finding of the present study showing high ADMA levels in subjects with BHT is in line with previous observations and supports the idea that high ADMA level may be a risk factor for BP elevation.

ADMA and hypercholesterolemia
Contrary to the study by Böger et al. (18), we could not demonstrate an association between hypercholesterolemia and ADMA. Similarly, Miyazaki et al. (21) were unable to show a significant relationship between serum cholesterol concentration and ADMA in middle-aged subjects. Also, a recent study by Cardinale et al. (36) in subjects with established atherosclerosis did not observe any association between cholesterol concentration and ADMA. Differences in study populations may explain the discrepant results. For example, the majority of our FH patients in the current study were receiving lipid-lowering therapy. Furthermore, FH patients have a single-gene defect and may differ from subjects with polygenic hypercholesterolemia. These observations, however, do not necessarily imply that ADMA levels are irrelevant to vascular reactivity or atherosclerotic risk. In the study by Miyazaki et al. (21), plasma ADMA level was significantly associated with elevated BP and increased carotid artery IMT, a marker of subclinical atherosclerosis. Furthermore, in the study by Cardinale et al. (36), elevated plasma ADMA level was associated with increased atherosclerosis in the thoracic aorta. In the present study high ADMA level was associated with dipyridamole flow independently of the study group, and a trend towards lower dipyridamole flow with high ADMA level was also seen within the FH group.

Effect of dipyridamole
Dipyridamole causes accumulation of intracellular adenosine that has a direct vasodilatory effect on smooth muscle cells via purinergic vascular receptors (37). Part of the effects of dipyridamole may be mediated by vascular endothelium because its effects can be reduced by inhibiting NO synthesis (10). Furthermore, it has been suggested that an increase in shear stress due to increased flow after dipyridamole administration will induce the release of vasodilating substances from endothelial cells and elicit more prominent vasodilation in the vessels with preserved endothelial function (38). Recent study showed that approximately one-third of the adenosine-induced myocardial hyperemic blood flow response is blocked by simultaneous administration of NO synthase inhibitor N^G-nitro-L-arginine (39). Therefore, the myocardial flow response to dipyridamole may be regarded as an integrated measure of vascular smooth muscle relaxation and endothelial function.

Measurement of ADMA
We used HPLC and OPA derivatization to measure plasma ADMA. The method produced sharp HPLC peaks, and the variability of repeated measurements was low. The plasma levels in control subjects averaged 0.43 ± 0.12 µmmol/l (range 0.21 to 0.66 µmmol/l). These values are similar to those reported in healthy controls in several previous studies (21,30,31,40) but slightly higher than in some reports (41,42) and lower than in others (18,19) that have reported ADMA levels approximately 1.0 µmmol/l in healthy controls. The levels of plasma ADMA in subjects with BHT in the current study were similar than previously reported in dialysis patients (30) or patients with preeclampsia (35). The variable results regarding plasma ADMA levels are likely explained by differences in methodology. Serum ADMA levels may be determined employing a variety of methods including HPLC-ultraviolet or HPLC with fluorescence detection after a derivatization step, capillary electrophoresis with laser-induced fluorescence detection, and HPLC tandem mass spectrometry. At present no interlaboratory comparisons have been reported. Consequently, the reasons for discrepancy in ADMA levels obtained with different methods have not been clarified.

Study limitations
The present study examined the relationships between ADMA and myocardial reactivity in a cross-sectional setting. A more ideal approach would be prospective study of subjects before and after therapeutic interventions aimed at altering plasma ADMA levels. As there is currently no effective means to reduce plasma ADMA levels, such intervention study would not be possible to perform. Therefore, these data must be interpreted carefully, as they do not necessarily indicate a causal relationship between elevated ADMA levels and reduced hyperemic MBF in hypertension; ADMA may play a causal role or be simply a marker of the disorder. We have studied only those volunteers approached and willing to consent to studies on the effects of risk factors on arterial physiology, and, therefore, some selection bias may be present. Only men were investigated; therefore, it is unknown whether the same results can be extrapolated to female subjects. In the FH group, 12 patients had been receiving cholesterol-lowering medication for several years. Despite this, their cholesterol levels were elevated. It is possible, however, that the results obtained in these medically treated patients may underestimate the impact of FH on myocardial reactivity. It is also unknown whether long-term lipid-lowering therapy influences ADMA concentrations.

Conclusions
We conclude that plasma ADMA concentration correlates with dipyridamole-induced vasodilatory function in young males, independently of BP elevation and hypercholesterolemia. Subjects with BHT have significantly increased plasma ADMA levels, which may partly explain impaired myocardial reactivity in this condition.

	Acknowledgments

 
The authors thank the personnel of the Turku PET Centre for their technical assistance.

	Footnotes

 
Supported by the grants of the Academy of Finland, the Novo Nordisk Foundation, the Turku University Foundation, the Finnish Foundation for Cardiovascular Research, the Emil Aaltonen Foundation, the Sigrid Juselius Foundation, the Yrjö Jahnsson Foundation, the Leiras Scientific Foundation, the Ida Montin Foundation, and the Maud Kuistila Foundation.

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