volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF 2 between the 2 groups.

Transcription

1 Clinical Chemistry 54: (2008) Lipids, Lipoproteins, and Cardiovascular Risk Factors Simultaneous Assessment of Endothelial Function, Nitric Oxide Synthase Activity, Nitric Oxide Mediated Signaling, and Oxidative Stress in Individuals with and without Hypercholesterolemia Renke Maas, 1* Edzard Schwedhelm, 1 Lydia Kahl, 1 Huige Li, 2 Ralf Benndorf, 1 Nicole Lüneburg, 1 Ulrich Förstermann, 2 and Rainer H. Böger 1 BACKGROUND: Endothelial function is impaired in hypercholesterolemia and atherosclerosis. Based on mostly indirect evidence, this impairment is attributed to reduced synthesis or impaired biological activity of endothelium-derived nitric oxide (NO). It was the aim of this study to directly estimate and compare wholebody NO production in normo- and hypercholesterolemia by applying a nonradioactive stable isotope dilution technique in vivo. METHODS: We enrolled 12 normocholesterolemic and 24 hypercholesterolemic volunteers who were all clinically healthy. To assess whole-body NO synthesis, we intravenously administered L-[guanidino-( 15 N 2 )]- arginine and determined the urinary excretion of 15 N-labeled nitrate, the specific end product of NO oxidation in humans, by use of gas chromatographymass spectrometry. In addition, we measured flowmediated vasodilation (FMD) of the brachial artery, expression of endothelial NOS (enos) in platelets, plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), and urinary excretion of 8-isoprostaglandin F 2 (8-iso-PGF 2 ). RESULTS: After infusion of L-[guanidino-( 15 N 2 )]- arginine, cumulative excretion of 15 N-labeled-nitrate during 48 h was 40% [95% CI 15% 66%] lower in hypercholesterolemic than normocholesterolemic volunteers [mean 9.2 (SE 0.8) mol vs 15.4 (2.3) mol/l, P 0.003]. FMD was on average 36% [4% 67%] lower in hypercholesterolemic than normocholesterolemic volunteers [6.3 (4.0)% vs 9.4 (4.6)%, P 0.027]. Normalized expression of NOS protein in platelets was also significantly lower in hypercholesterolemic volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF 2 between the 2 groups. CONCLUSIONS: This study provides direct evidence for a decreased whole body NO synthesis rate in healthy people with hypercholesterolemia American Association for Clinical Chemistry The first published studies describing impaired endothelium-dependent vasodilation due to hypercholesterolemia appeared more than 17 years ago (1). Since then, this finding has been replicated in multiple, albeit not all, studies (2). Impairment of endotheliumdependent vasodilation has largely been attributed to a reduced availability or biological activity of nitric oxide (NO) (3). Endogenous nitric oxide (NO) 3 is generated by NO synthase (NOS) from L-arginine. NO itself is a very short-living compound, which makes its direct detection and quantification very difficult and in many settings impossible (4). NO is rapidly degraded to nitrite and subsequently to nitrate, the latter being excreted into the urine. Theoretically, NOS activity in vivo is best assessed by measurement of urinary nitrate. Unfortunately, much if not most urinary nitrate originates from other sources, such as dietary nitrate intake and bacterial nitrate synthesis (5), which makes interpretation of reports that plasma nitrite/nitrate and urinary nitrate are inversely correlated with plasma LDL concentration difficult (6). In principle, this problem can be overcome by stable isotope infusion of nonradio- 1 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany; 2 Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany. * Address correspondence to this author at Institut für Experimentelle und Klinische Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, Hamburg, Germany. maas@uke.uni-hamburg.de. Received June 20, 2007; accepted November 5, Previously published online at DOI: /clinchem Nonstandard abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; FMD, flow-mediated vasodilation; 8-iso-PGF 2, 8-iso-prostaglandin F 2 ; ADMA, asymmetric dimethylarginine. 292

2 Nitric Oxide Synthesis in Hypercholesterolemia active L-[guanidino- 15 N 2 ]-arginine ( 15 N 2 -L-arginine), which is converted by the NOS to 15 N-labeled NO, oxidized, and excreted as [ 15 N]-labeled nitrate ( 15 N- nitrate) (7, 8). 15 N-labeled nitrate can be discriminated from the naturally abundant [ 14 N]-nitrate by GC-MS. After infusion of 15 N 2 -L-arginine, only NOSderived 15 N-NO may contribute to enrichment of the 15 N-nitrate to 14 N-nitrate ratio. Nitrate from other sources primarily does not alter the natural 1:270 ratio of the 15 N- to 14 N-isotopes. This technique has not been applied in hypercholesterolemic humans, so far. Moreover, while numerous mechanisms possibly involved in hypercholesterolemia-related alterations of NO signaling have been identified in vitro and in animal models, it is still very difficult to extrapolate which of these mechanisms are relevant in humans in vivo. Major factors that may alter NO signaling in vivo are a reduced substrate availability (9), presence of endogenous NOS inhibitors (10), alterations in NOS expression (11) or localization (12), oxidative stress leading to uncoupling of NOS, and scavenging of NO by free radicals (13). In this study, we aimed to apply the stable isotope technique in humans to more specifically investigate alterations of whole-body NO synthesis in hypercholesterolemia and to explore underlying mechanisms. Materials and Methods STUDY PROTOCOL The study protocol was written in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Hamburg Board of Physicians. This work was carried out at the Institute of Experimental and Clinical Pharmacology and Toxicology of the University Hospital Hamburg-Eppendorf. All patients and volunteers provided written informed consent. Major inclusion criteria for this study were age years (all participants), LDL cholesterol 4.1 mmol/l [160 mg/dl] (patients) or LDL 4.1 mmol/l (controls). Patients and controls with history of or clinical evidence for the presence of coronary heart disease, diabetes, or renal failure were excluded. Any lipidlowering medication (e.g., statins or ezetimibe) was stopped at least 3 weeks before the study. No participant had taken nonsteroidal antiinflammatory (NSAIDs), phosphodiesterase inhibitors, antioxidant supplements, or psychotropic drugs 5 half-lives before the study. On the first day of the study, the participants arrived in the morning after an overnight fast. All patients and controls were hospitalized for the first 10 h of the study and during this period received a low-nitrate diet. Blood samples were drawn at 2, 0, 1, 2, 4, 8, 24, and 48 h before and after the infusion of 15 N 2 -Larginine (as described below). Urine was collected in special containers containing 25 mg EDTA and 25 mg butylated hydroxytoluene (BHT) as antioxidants for 2 h before infusion of 15 N 2 -L-arginine (baseline) and 0 8, 8 24, and h after the infusion. After sampling of the 2 h fasting blood sample, flow-mediated vasodilation (FMD) was measured as described below. The infusion of 15 N 2 -L-arginine was started at time 0 as detailed below. Two hours after the infusion, participants received a standardized meal low in nitrate. VASCULAR FUNCTION TESTING We measured endothelium-dependent vasodilation according to the principles set by the International Brachial Artery Reactivity Task Force (14) as described and validated by us in detail (15). In brief, we assessed endothelial function in each volunteer s right arm in a quiet, temperature-controlled room (22 C) by use of high-resolution ultrasound (12-MHz linear array transducer; Siena, Siemens). Longitudinal scans of the brachial artery were obtained approximately 5 cm proximal of the antecubital fossa. The transmit focus zone was set at the depth of the anterior wall. Anatomical landmarks and snapshot images were used to assess FMD in the same vessel section on each study day and at each time point. A view of a 5-cm longitudinal section of the brachial artery was recorded for periods 30 s at baseline and before and during peak (1 min) reactive hyperemia (after deflation of a blood pressure cuff previously inflated to 50 mmhg above the volunteer s systolic blood pressure around the forearm for 5 min, 1 min after cuff release). Each 30-s recording was digitalized (vascular imager 4.1.3; Medical Imaging Applications LLC) at a rate of 10 high-resolution frames per second (300 frames per recording), using specialized software (Brachial Analyzer 4.1.3; Medical Imaging Applications LLC). This allowed us to average maximal, minimal, and mean vessel diameter over 25 to 40 heartbeats. Results obtained for maximal, minimal, and mean vessel diameter were very similar; data based on mean vessel diameters gave the best reproducibility and are presented here. Flow-mediated endotheliumdependent dilation (FMD) was calculated as the percent change in diameter 1 min after cuff release relative to the baseline diameter before cuff release. Ultrasound studies and image analyses were performed separately in an observer-blinded fashion. WHOLE-BODY NO SYNTHESIS Sterile, pyrogen-free infusions of L-[guanidino- 15 N 2 ]- arginine ( 15 N 2 -L-arginine, 98.5% isotope purity) dissolved in 20 ml 0.9% NaCl were prepared, tested and certified for safe use in humans in accordance with Good Laboratory Practice, Good Manufacturing Practice, and the German and European Pharmacopeia at Clinical Chemistry 54:2 (2008) 293

3 the pharmacy of the University Hospital Hamburg- Eppendorf. At 0 h (baseline), all patients and volunteers received 500 mg 15 N 2 -L-arginine in 10 min via an intravenous infusion pump. Urinary 14 N- and 15 N-nitrate and nitrite were determined before and 8, 24, and 48 h after infusion by use of GC-MS as described in detail (7, 8); all CVs were 3.8%. In brief, 100- L aliquots of urine were incubated for 30 min at 50 C in 500 L acetone containing 10 L pentafluorobenzyl (PFB)- bromide. Then samples were dried under nitrogen and resuspended in 100 L toluol for analysis of the PFB derivatives of nitrate on a gas-chromatograph (CP-3800; Varian) coupled to a quadrupole mass spectrometer (Quadrupole 1200; Varian) with single-ion monitoring at m/z 62 for 14 N-nitrate and m/z 63 for 15 N-nitrate. Concentrations 14 N- and 15 N-nitrate were calculated as follows. We measured the natural abundance of 14 N- and 15 N-nitrate in urine collected 2 h before infusion of 15 N 2 -L-arginine, then relative enrichment of 15 N-nitrate after infusion of 15 N-L-arginine. To quantify endogenous 14 N-nitrate, a separate aliquot from each urine sample was supplemented with 800 mol 15 N-nitrate (which is in vast excess of endogenous 15 N-nitrate, making the contribution of endogenous 15 N-nitrate negligible (7)). Based on the ratio of 14 N-nitrate/ 15 N-nitrate in these supplemented aliquots, we calculated the molar amount of 14 N-nitrate. We then calculated the concentration of 15 N-nitrate in the other aliquot by multiplying the molar amount of 14 N-nitrate by the relative enrichment of 15 N-nitrate. Urinary excretion of 14 N- and 15 N-nitrate was corrected for creatinine to account for differences in renal excretion. EXPRESSION OF ENDOTHELIAL NOS To obtain platelet proteins, 10 ml heparinized whole blood was centrifuged for 15 min (200g, 20 C). The resulting platelet-rich plasma was centrifuged at 2000g for 20 min, and the resulting pellet was resuspended in PBS buffer and centrifuged at 2000g for 20 min. The washed platelet pellet was lysed on ice in a lysis buffer (containing 1 mmol/l Tris, ph 7.5, 5 mmol/l KCl, 0.1 mol/l NaCl, 0.3 mmol/l -glycerophosphate, 0.5 mmol/l NaF, 0.1 mmol/l Na 3 VO 4, 1% (vol/vol) Triton X-100 and Nonidet-P40, and a standard protease inhibitor mixture (Bio-Rad) and stored at 80 C until analysis. Western blotting was performed using 50 g protein and a monoclonal anti-enos antibody that detects total enos (BD Biosciences PharMingen), as described (16). Immunocomplexes were developed using an enhanced horseradish peroxidase/luminol chemiluminescence reagent (Perkin-Elmer Life and Analytical Sciences) according to the manufacturer s instructions. enos expression was quantified as expression relative to expression of -tubulin using Gene Tools 3.06 (Synoptics Ltd). OTHER LABORATORY MEASUREMENTS We analyzed 8-iso-PGF 2 in urine samples by use of GC-MS as described (17). We measured cgmp in urine using a commercially available kit (GE) and asymmetric dimethylarginine (ADMA) and 14 N 2 - and 15 N 2 -L-arginine by use of GC-MS as described (18). All other laboratory variables such as plasma lipids, lipoproteins, glucose, uric acid, creatinine, and blood cells were obtained from the local clinical chemistry laboratory by certified routine procedures. All biochemical analyses were carried out in an observerblinded fashion. STATISTICS All data were tested for normal distribution with the Kolmogorov-Smirnov test. Continuous variables were expressed as arithmetic mean (SE) if normally distributed or otherwise by median (25% and 75% percentiles). Parametric and nonparametric tests (Mann- Whitney U 2-sided) and Kruskal-Wallis H were used for comparisons, as appropriate. Categorical variables were compared by 2 test or exact Fisher test as appropriate. Correlations were assessed by Pearson or Spearman test, as appropriate. P 0.05 was considered significant. SPSS 13.0 was used for statistical analysis. Results Baseline characteristics of the 24 volunteers with hypercholesterolemia and 12 volunteers with normal cholesterol values are shown in Table 1. The 2 groups differed only in lipid-related markers. FLOW-MEDIATED VASODILATION The FMD of patients with hypercholesterolemia was on average 36% (95%CI 4% 67%) lower in patients with hypercholesterolemia than in healthy controls [6.30% (0.85%) vs 9.81% (1.33%), P 0.027] (Fig. 1A). URINARY EXCRETION OF NITRATE After infusion of 15 N 2 -L-arginine, the mean urinary excretion rate of 15 N-nitrate calculated for the respective 8, 16, and 24 h collection intervals was significantly lower in hypercholesterolemic patients than in healthy controls (Table 2). Correspondingly, cumulative excretion of 15 N-nitrate during the whole 48-h collection period was 40% (15% 66%) lower in patients with hypercholesterolemia than in normocholesterolemic 294 Clinical Chemistry 54:2 (2008)

4 Nitric Oxide Synthesis in Hypercholesterolemia Table 1. Baseline characteristics of normo- and hypercholesterolemic individuals. a Hypercholesterolemic LDL Normocholesterolemic LDL P value n Age, years 59 (1) 58 (3) Sex (M/F), n 12/12 7/ Height, cm 171 (2) 173 (3) Weight, kg 73 (2) 70 (3) Body mass index, kg/m 2 25 (1) 24 (1) Systolic blood pressure, mmhg 126 (3) 127 (3) Diastolic blood pressure, mmhg 77 (2) 80 (2) Serum creatinine, mol/l 76.9 (1.8) 77.8 (4.4) Creatinine clearance, ml/min 105 (4) 106 (8) High-sensitivity C-reactive protein, mg/dl a 1.15 ( ) 0.75 ( ) Fasting glucose, mmol/l 4.9 (0.1) 4.8 (0.2) Parent with premature cardiovasular event b 9 (38) 1 (8) History of smoking b 2 (8) 1 (8) Total cholesterol, mmol/l 7.8 (0.3) 5.3 (0.2) LDL cholesterol, mmol/l 5.6 (0.2) 3.2 (0.2) HDL cholesterol, mmol/l 1.4 (0.1) 1.6 (0.1) Triglycerides, mmol/l 1.7 (0.1) 1.1 (0.1) Regular intake of cardiovascular drugs b ASS 0 (0) 0 (0) Statins 0 (0) 0 (0) blockers 1 (4) 1 (8) ACE inhibitors 1 (4) 0 (0) Brachial artery baseline diameter, mm 4.6 (0.1) 4.5 (0.2) Data are arithmetic mean (SE) and P value from t test, unless noted otherwise. Hypercholesterolemia was defined as LDL 4.1 mmol/l ( 160 mg/dl). a Median (25th 75th percentile), P value from Mann-Whitney U test. b n (%), P value from 2 test or Fisher exact test as appropriate. volunteers [9.2 (0.8) mol vs 15.4 (2.3) mol in 48 h, P 0.003] (Fig. 1B). In contrast, as shown in Table 2, patients with hypercholesterolemia and normocholesterolemic volunteers did not significantly differ in the urinary excretion rate of 14 N-nitrate (reflecting not only NOderived nitrate but also nitrate from dietary and other sources). There was also no significant difference in the cumulative excretion of 14 N-nitrate during the whole 48-h collection period [2730 (188) mol in hypercholesterolemic patients vs 2698 (246) mol in normocholesterolemic volunteers, P 0.923] (Fig. 1C). Urinary excretion of 15 N-nitrate (r 0.481, P 0.003) but not 14 N-nitrate (r 0.085, P 0.620) was inversely correlated with LDL. The correlation coefficients of 15 N-nitrate and 14 N-nitrate excretion and urinary cgmp excretion (indexed to creatinine) were r (P 0.098) and (P 0.645), respectively. There was no significant correlation of 15 N- nitrate and 14 N-nitrate excretion with FMD or isoprostane excretion (all r 0.2 and all P 0.05). cgmp The difference mean urinary excretion of cgmp between normo- and hypercholesterolemic subjects failed to reach statistical significance [71.4 (10.6) nmol cgmp/ mmol creatinine vs 64.2 (5.5) nmol cgmp/mmol creatinine, P 0.504] (Fig. 1D). enos PROTEIN IN PLATELETS As shown in Fig. 1E, expression of enos protein in platelets of patients with hypercholesterolemia was 38% (15% 60%) lower than in normocholesterolemic volunteers [ratio enos/ -tubulin expression 0.37 (0.04) vs 0.59 (0.04), P 0.002]. Clinical Chemistry 54:2 (2008) 295

5 Fig. 1. Comparison of clinically healthy volunteers with normal (<4.1 mmol/l) or increased (>4.1 mmol/l) LDL cholesterol in serum. *P (A), Flow-mediated vasodilation in % of baseline diameter. (B), Urinary excretion of 15 N-nitrate during 48 h after infusion of 500 mg 15 N-L-arginine. (C), Urinary excretion of 14 N-nitrate during 48 h after infusion of 500 mg 15 N-L-arginine. (D), Urinary excretion of cgmp in nmol/mmol creatinine. (E), Expression enos protein relative to -tubulin as determined by immunoblotting of whole protein from lysed platelets. (F), Plasma concentration of the endogenous NOS inhibitor ADMA L-ARGININE, ADMA There was no significant difference in the plasma concentration of L-arginine [68.3 (3.0) mol/l HC vs 66.3 (4.4) mol/l, P 0.709] or the endogenous NOS inhibitor ADMA [0.61 (0.02) mol/l vs 0.64 (0.03) mol/l, P 0.190) (Fig. 1F). ISOPROSTANES We determined urinary excretion of isoprostanes as an indicator of oxidative stress. Excretion of isoprostanes in hyper- and normocholesterolemic participants was not significantly different [8.8 ( ) ng/h vs 8.0 ( ) ng/h, P 0.746]. 296 Clinical Chemistry 54:2 (2008)

6 Nitric Oxide Synthesis in Hypercholesterolemia Table 2. Urinary excretion of nitrate. Hypercholesterolemic LDL Normocholesterolemic LDL P value 14 N-nitrate excretion, mol/h 0 8 h 61.6 (3.3) 64.3 (9.4) h 47.8 (4.8) 50.4 (5.2) h 61.3 (5.5) 57.4 (6.0) N-nitrate excretion, mol/h 0 8 h (0.038) (0.075) h (0.019) (0.063) h (0.021) (0.046) Urinary excretion of 14 N-nitrate, pmol/mmol creatinine 0 8 h 127 (16) 168 (33) h 112 (14) 123 (15) h 126 (14) 116 (15) Urinary excretion of 15 N-nitrate, pmol/mmol creatinine 0 8 h (0.091) (0.173) h (0.051) (0.142) h (0.034) (0.108) Data are arithmetic mean (SE), P value from t test. Hypercholesterolemia was defined as LDL 4.1 mmol/l (160 mg/dl). SEX-RELATED DIFFERENCES There were no sex-related differences in the data with regard to the major variables, FMD, 15 Nor 14 N-nitrate excretion, and enos expression (data not shown). Discussion This study is the first to provide direct evidence for a reduction in whole-body NO synthesis rate in clinically healthy persons with hypercholesterolemia by demonstrating a reduced urinary excretion of NOS-derived 15 N-nitrate in vivo. NITRIC OXIDE SYNTHESIS IN VIVO Infusion of 15 N 2 -labeled L-arginine and subsequent measurement of labeled metabolites (nitrite/nitrate) is currently considered to be the most accurate method to study whole body NO production in vivo (4). Anupto-date review covering the application of isotopes in the L-arginine/NO pathway, including a discussion of interferences and possible limitations of the method, was recently provided by van Eijk et al. (19). A major advantage of the stable isotope method is that it allows to distinguish NO-derived nitrate from nitrate from other (dietary) sources. Unlabeled NO-derived 14 N- nitrate (generated from endogenous 14 N 2 -L-arginine) may simply be diluted in the vast endogenous nitrate pool (4), making it difficult to detect any impact of hypercholesterolemia on NO synthesis. And indeed the urinary excretion of unlabeled 14 N-nitrate was not significantly different in the 2 groups. The latter finding is in line with data from a large population-based study in which only a weak and rather inconsistent inverse relationship of unlabeled plasma NOx and serum cholesterol was observed. Most data available, so far, concerning the impact of hypercholesterolemia on NO synthesis were obtained in cell culture or isolated tissues predominantly containing enos. When interpreting the present data, it is important to note that urinary excretion of 15 N- nitrate represents stimulated and unstimulated wholebody NO synthesis. It does not provide any information regarding the site of NO synthesis or the relative contribution of any of the 3 NOS isoforms (20). In principle, the conversion of 15 N 2 -L-arginine to 15 N-NO may have occurred at sites other than the endothelium, as well as by other NOS isoforms such as inos (20). In this study, a significant contribution of inos is unlikely, though, because C-reactive protein levels were low and all participants were free from infection or trauma. It should also be kept in mind that NO is finally degraded to nitrate whether or not it has activated guanylyl cyclase, which is responsible for the formation of cgmp. The latter acts as the key second messenger for the physiological effects of NO. Especially under conditions of oxidative stress, a substantial proportion of the NO formed may be scavenged by reactive Clinical Chemistry 54:2 (2008) 297

7 species such as superoxide (13). Formation of 15 N- labeled nitrate is therefore not equivalent to biologically active NO. In line with the lower NO synthesis in vivo, one would expect lower excretion of cgmp as well. However, this was not the case, despite the fact that hyperemia-induced vasodilation, which has been suggested to be a physiological measure of endothelium-dependent, NO-mediated vascular function, was also impaired. Similar to our observation regarding the excretion of unlabeled 14 N-nitrate, differences in cgmp related to NO signaling may simply have been diluted. When using cgmp formation as an indicator for biologically active NO, it has to be taken into account that in vivo, activation of guanylyl cyclase by natriuretic peptides may be a more prominent source of urinary cgmp than NO signaling (21). Determination of cgmp in venous plasma most likely would have been subject to similar limitations (21).In principle, some of these limitations could be overcome by additional arterial cannulations to obtain the arteriovenous difference in cgmp (which may be more specific (22)), but this was deemed too invasive in our healthy volunteers. HYPERCHOLESTEROLEMIA AND NO SYNTHESIS In the literature, the impact of hypercholesterolemia and NO synthesis is discussed quite controversially. Yet several lines of evidence support a causal role of LDL cholesterol in endothelial dysfunction and impaired NO synthesis. Most notably, improvement in endothelium-dependent vasodilation in patients with hypercholesterolemia has been achieved by different methods of LDL cholesterol reduction including apheresis (23) and statins (24). Flow-mediated vasodilation, in turn, appears to be predominantly mediated by NO. A pilot study applying the stable isotope technique in cholesterol-fed rabbits found a reduced conversion of 15 N 2 -L-arginine to 15 N-nitrate (8). In clinical practice, hypercholesterolemia frequently occurs together with other factors known to affect the L-arginine NO pathway and vascular function. There is abundant evidence that the individual risk associated with increased serum LDL cholesterol is substantially modified by additional factors (25). Although elevation of LDL cholesterol is associated with premature cardiovascular disease in some families, it appears to have little impact on outcome in others (26). Hence, a possible contribution of factors frequently associated with hypercholesterolemia has to be addressed as well. With regard to prevalence in the population, especially hypertension and diabetes have to be considered. Moreover, using similar 15 N- nitrate based methods it has already been shown that hypertension (27) and diabetes (28) are both associated with reduced NOS activity and therefore constitute potential confounders. In this study, we observed no significant difference in blood pressure or fasting glucose between the hypercholesterolemic and the normocholesterolemic groups, however. The lower 15 N- nitrate excretion in the hypercholesterolemic group also could not be explained by impaired renal function or a reduced capacity for urinary excretion of nitrate in general, because neither glomerular filtration rate nor urinary excretion of unspecific 14 N-nitrate was different from the normocholesterolemic group. Thus, looking at individual risk factors, we found no significant differences between the 2 groups other than hypercholesterolemia that could explain the difference in NO synthesis we observed. MOLECULAR MECHANISMS The precise pathomechanisms through which LDL cholesterol may affect endothelial function and NO synthesis are only partly understood (3). Impaired NO synthesis in hypercholesterolemia has been linked to increased plasma concentrations of the endogenous NOS inhibitor ADMA, which have frequently but not consistently been observed in patients with hypercholesterolemia (29). In this respect, it is of interest to note that the present data are in line with a subgroup analysis of a study in patients with renal failure, which revealed a nonsignificant trend toward a lower NO synthesis rate in patients with familial hypercholesterolemia (30). In patients with renal failure, impaired NO synthesis and poor outcome has also been closely linked to the endogenous NOS inhibitor ADMA (31). However, in the present study, plasma ADMA concentrations in the normal range in hyper- and normocholesterolemic participants argue against a significant impact of ADMA on NO synthesis in hypercholesterolemia. We assessed expression of enos in platelets, which have previously been shown to contain enos (32) as, for obvious reasons, vascular biopsies were not available from our clinically healthy participants. The present expression data suggest that differences in enos expression may provide a simple explanation for the differences in NO synthesis we observed. However, the literature suggests that this explanation may be too simplistic (12, 33). Possible differences in the regulation of enos expression in platelets or megakaryocytes and endothelial cells have to be considered as well (34). There is conflicting evidence available regarding the impact of hypercholesterolemia on enos expression. Although some investigators report that expression of enos is increased by native or LDL cholesterol (34), others suggest that expression may be downregulated by oxidized (35) or native LDL 160 mg/dl (36). Downregulation of enos expression by native LDL could be prevented by simvastatin through a posttranscriptional mechanism (37). Similarly, cholesterol was 298 Clinical Chemistry 54:2 (2008)

8 Nitric Oxide Synthesis in Hypercholesterolemia found to decrease enos expression in swine (11). Considering that atherosclerosis is not a static disease, these partly conflicting observations regarding the effect of LDL cholesterol on enos gene expression may not be so conflicting at all. The effect of LDL cholesterol on gene expression may strongly depend on the stage of disease. The load of oxidative stress and isoprostane excretion appears to increase with the stage of atherosclerotic disease (17). The excretion rate of isoprostanes as an indicator of oxidative stress was less pronounced in our hypercholesterolemic group than suggested by data from the literature (37) but consistent with another previous study (38), indicating that our subjects were in less advanced stages in the chain of atherosclerotic events. Although effects unrelated to oxidative stress may dominate the early stages of atherosclerosis in hypercholesterolemic patients, there may be a substantial increase in oxidative stress in later stages of the disease, leading to an upregulation of enos expression (17, 39). A further mechanism to be considered is the phosphorylation state of enos. This is very difficult References to assess in vivo, however, owing to the speed of the phosphorylation-dephosphorylation reaction. In summary, this study provides first direct evidence for reduced whole-body NO synthesis in clinically healthy persons with hypercholesterolemia. Looking at possible confounding factors, we found no significant differences between the 2 groups other than hypercholesterolemia that could explain the difference in NO synthesis we observed. Grant/funding Support: This study was supported in part by an unrestricted research grant from Pfizer GmbH Germany to Rainer H. Böger and by the Institute of Clinical and Experimental Pharmacology and Toxicology. Financial Disclosures: None declared. Acknowledgments: We thank Mariola Kastner and Anna Steenpaß for their excellent technical assistance. And Dr. Hans-Jürgen Staude from the department of pharmacy for preparing the 15 N-L-arginine for human use. 1. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest 1990;86: Benjamin EJ, Larson MG, Keyes MJ, Mitchell GF, Vasan RS, Keaney JF Jr, et al. Clinical correlates and heritability of flow-mediated dilation in the community: the Framingham Heart Study. Circulation 2004;109: Adams MR, Kinlay S, Blake GJ, Orford JL, Ganz P, Selwyn AP. Atherogenic lipids and endothelial dysfunction: mechanisms in the genesis of ischemic syndromes. Annu Rev Med 2000;51: Baylis C, Vallance P. 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