Plasma concentrations of the cardiovascular risk factor asymmetric dimethylarginine (ADMA) are increased in patients with HIV-1 infection and correlate with immune activation markers

Pharmacological Research 60 (2009) 508–514

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Review

Plasma concentrations of the cardiovascular risk factor asymmetric dimethylarginine (ADMA) are increased in patients with HIV-1 infection and correlate with immune activation markers K. Kurz a,b,1 , T. Teerlink d,1 , M. Sarcletti c , G. Weiss b , R. Zangerle c , D. Fuchs a,∗ a

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz Pregl Strasse 3, 6020 Innsbruck, Austria Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria Department of Venerology and Dermatology, Innsbruck Medical University, Innsbruck, Austria d Metabolic Laboratory, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands b c

a r t i c l e

i n f o

Article history: Received 12 May 2009 Received in revised form 20 July 2009 Accepted 20 July 2009 Keywords: ADMA Neopterin Immune activation HIV-1 infection

a b s t r a c t Objective: Higher concentrations of inflammation and immune activation markers as well as the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) are associated with an increased cardiovascular risk. In vitro, parallel formation of ADMA and macrophage marker neopterin was found in stimulated human peripheral blood mononuclear cells. Methods: In 112 HIV-1 infected patients, concentrations of ADMA, SDMA and arginine were compared to C-reactive protein and neopterin concentrations before they were referred to antiretroviral therapy. Disease activity was determined by viral load (qPCR), CD4+ cell counts (FACS) and neopterin concentrations in plasma and urine (HPLC and ELISA). Additionally, concentrations of lipids were determined. Results: HIV-1 infected patients presented with increased neopterin, ADMA and SDMA concentrations, whereas CD4+ counts and arginine and plasma lipid concentrations were low. ADMA and SDMA concentrations significantly correlated with markers of immune activation, but not with plasma lipids. Conclusions: Results of this study indicate that increased ADMA and SDMA production may be related to an increased activity of immune activation pathways. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Study population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Inclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Specimen collection and assay methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Determination of ADMA, SDMA, and arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Determination of inflammatory markers and cholesterol levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

508 509 509 509 509 509 509 509 509 510 510 513 513

1. Introduction

∗ Corresponding author. Tel.: +43 512 9003 70350; fax: +43 512 9003 70350. E-mail address: [email protected] (D. Fuchs). 1 These authors contributed equally to this work. 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.07.009

Progressive immunodeficiency is the hallmark of human immunodeficiency virus type 1 (HIV-1) infection. Interestingly, the development of immune paralysis appears to be only partly due to direct effects of the virus, and rather over-whelming immune

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

activation cascades in response to the virus play an important additional role. In fact, immune activation status of the host is a major determinant of the outcome of patients [1]. Therefore, determination of several markers of immune activation like the pteridine neopterin [1–4] is well established to monitor the course of disease as well as the efficiency of antiretroviral therapy (ART). Neopterin is released by human monocyte-derived macrophages and dendritic cells particularly upon stimulation with Th1-type cytokine (IFN␥) in the course of the cellular immune response [5,6]. In HIV-1 infected patients, elevated neopterin concentrations coincide with higher viral load, lower CD4+ cell counts [1–4] and a reduced ability of peripheral blood mononuclear cells to respond to in vitro stimulation [7,8]. However, neopterin determination in body fluids is not only useful to monitor HIV-infection, but also allows a good assessment of Th1-type immune response in general [9]. Therefore, neopterin measurement also provides valuable information, e.g. regarding the prognosis of patients, in other diseases characterized by over-whelming Th1-type immune response like autoimmune diseases, cancer [9], and also cardiovascular disease [10–13]. Interestingly, cardiovascular disease has been recognized as frequent “complication” in HIV-infected patients [14,15]. Initially effects of antiretroviral treatment on lipid metabolism were held responsible for the increased risk of cardiovascular events in this population [16,17]. However, several short- as well as long-term studies have identified also other risk factors including traditional cardiovascular risk factors, HIV-infection itself, substance abuse and metabolic changes [18,19]. Furthermore, inflammation and immune activation cascades have been proposed to play a crucial role in atherogenesis in HIV-infected patients [20]. Immune activation and inflammation have been recognized to enforce the formation of plaques in vessels and impair endothelial function, thereby contributing importantly to the development of atherosclerosis and coronary lesions in non-HIV-1 infected individuals [21–24]. Besides inflammatory markers like C-reactive protein or neopterin [10,11,13,25], also asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction, has been found to predict the outcome of cardiovascular disease [26–29]. ADMA as well as its stereoisomer symmetric dimethylarginine (SDMA) derive from the amino acid arginine. ADMA functions as an endogenous inhibitor of nitric oxide synthases (NOS), thereby impairing endothelium-dependent vascular relaxation and possibly also host defence against infection [30]. Recent in vitro as well as in vivo studies indicate that enhanced ADMA formation coincides with inflammatory/immune activation cascades [31,32] and that immune activation might even be involved in ADMA accumulation. However, the relationship between arginine, ADMA and immune activation has so far not been investigated in HIV-1 infected patients. Therefore, the aim of our study was to assess plasma ADMA concentrations in HIV-1 infected patients. In addition, the relationship between ADMA and markers of immune activation was investigated. Additionally, we examined the association between lipid levels and ADMA, as hyperlipidemia has been associated with elevated ADMA levels [33]. 2. Materials and methods 2.1. Study population Patients in the outpatient cohort of the Innsbruck University Hospital were selected from an electronic database according to the inclusion criteria. Patients were included in the study according to criteria of the declaration of Helsinki 2002. Strict inclusion and exclusion criteria were chosen to reduce the number of confounding factors (see below). These criteria were selected before the analysis of the data.

509

2.2. Inclusion criteria 112 patients (71 men and 41 women; median age: 40 years) were included, 62 of whom had AIDS. Patients were either therapynaive or, if nucleoside monotherapy had been given in the past, this therapy had to be interrupted before introduction of antiretroviral combination therapy (for asymptomatic patients for the duration of 1 year and for symptomatic patients for the duration of 6 months). Patients were included in the study regardless of their HIV RNA levels and CD4+ cell count and before they were referred to combination therapy with combinations of nucleosides plus protease inhibitors. 2.3. Exclusion criteria • Acute HIV-1 infection. • Severe concomitant diseases (opportunistic infectious diseases except esophageal candidiasis, decompensated liver cirrhosis, malignant neoplasia) within 2 months of the start of or during the study period. 2.4. Specimen collection and assay methods HIV-1 RNA and CD4+ T-lymphocyte were obtained immediately after the visits of the patients at the outpatient unit. All other parameters were measured in one batch from plasma frozen at −80 ◦ C. Concentrations of HIV-1 RNA were determined with a commercially available reverse transcription-polymerase chain reaction assay (Amplicor HIV-1 Monitor Test, version 1.5, Roche Diagnostic Systems, Branchburg, NJ, USA) which quantifies HIV-1 Group M subtypes A–G. Lymphocyte numbers and subsets were determined by flow cytometry using a panel of FITC-, PE-, PerCP-, and APC-conjugated monoclonal antibodies (BD MultitestTM) from BD Biosciences, San Jose, CA, USA. CD4+ T-cell numbers were measured in the same tubes by means of calibrated beads (BD TruecountTM). Analyses were done immediately after sampling. 2.5. Determination of ADMA, SDMA, and arginine ADMA, SDMA and arginine were measured by highperformance liquid chromatography (HPLC) with fluorescence detection as previously described [34], with modified chromatographic separation conditions [35]. The intra-assay and inter-assay coefficients of variation were <2% and <4%, respectively. In earlier experiments it was found that ADMA, SDMA and arginine are stable even for years when kept frozen. 2.6. Determination of inflammatory markers and cholesterol levels Plasma neopterin was measured by ELISA (BRAHMS, Hennigsdorf, Germany) and urinary neopterin was determined by HPLC [9]. Determination of C-reactive protein (CRP), blood counts and lipid levels in routinely drawn blood samples was performed by routine automated tests. Specifically, CRP levels were measured with an ELISA with a detection limit of 0.07 mg/dl (Merck Diagnostica, Zurich, Switzerland), cholesterol, HDL-cholesterol and LDL-cholesterol as well as triglycerides were determined with enzymatic methods (DADE Behring, Vienna, Austria). 2.7. Statistical analysis Because not all the data sets showed normal distribution, nonparametric tests were applied. Group comparisons were performed

510

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

by Mann–Whitney test. Spearman’s rank correlations were determined to measure associations among the various variables. All tests were two-sided and p < 0.05 was considered to indicate statistical significance. All statistical analyses and tests were run using the SPSS statistical package (SPSS 11.5 for Windows, Chicago, IL). 3. Results Concentrations of all variables investigated in patients and corresponding reference values are listed in Table 1. HIV-1 infected patients had median ADMA and SDMA concentrations above reference ranges [34], whereas arginine concentrations were decreased compared to healthy individuals. However, as these reference values were determined in a group of healthy laboratory personnel and students (age range 20–40 years) that was not age-matched with the HIV-1 infected patients) and because ADMA concentrations increase with age in adults, we additionally used data from a large cohort study in the general population (n = 2311; age range 50–75 years), with mean plasma concentrations of 0.50 ± 0.06 ␮M ADMA, 0.53 ± 0.10 ␮M SDMA, and 101 ± 20 ␮M arginine [36]. ADMA concentrations in the HIV-1 patients were increased and arginine concentrations were decreased (both p < 0.001) compared to these subjects, whereas SDMA concentrations were not significantly different between both groups. Urinary and plasma neopterin concentrations were highly elevated and CD4+ counts were strongly reduced, while lipid levels were rather on the lower side of reference ranges (Table 1). Gender had no influence on ADMA, SDMA and arginine concentrations. The same was true for almost all other variables in this study, only CD4+ counts were lower in women than in men (U = 2.62, p < 0.01), whereas CRP (U = 2.78, p < 0.01), cholesterol (U = 2.07, p < 0.05), LDL-cholesterol (U = 2.04, p < 0.05) and urinary neopterin concentrations (U = 2.99, p < 0.01) were higher in females. ADMA, SDMA and arginine concentrations did not significantly differ between HIV-1 infected patients with and without AIDS, whereas CD4+ counts were lower and neopterin concentrations as well as viral load were higher in patients with AIDS (all p < 0.05). ADMA and SDMA levels were correlated with each other (r’s = 0.442, p < 0.001), whereas no significant correlation existed between dimethylarginine concentrations and arginine (Fig. 1). Elevated dimethylarginine concentrations only weakly correlated with HIV-1 load and CD4+ cell counts (Fig. 2) but

coincided with cellular immune activation: significant associations existed between ADMA and neopterin concentrations (urinary neopterin: r’s = 0.296, p < 0.01, plasma neopterin: r’s = 0.242, p = 0.01 (Fig 2). SDMA levels were also positively associated with neopterin concentrations (urinary neopterin: r’s = 0.385, p < 0.001, plasma neopterin: r’s = 0.473, p < 0.001; Fig. 2), whereas arginine and neopterin were inversely associated (urinary neopterin: r’s = −0.193, p < 0.05, plasma neopterin: r’s = −0.310, p < 0.01; Fig. 2). Neopterin concentrations were correlated with plasma HIVload (urinary neopterin: r’s = 0.354, p < 0.001; plasma neopterin: r’s = 0.389, p < 0.001; Fig. 3) and inversely with CD4+ cell counts (urinary neopterin: r’s = −0.271, p < 0.01, plasma neopterin: r’s = −0.231, p = 0.01). Also CRP concentrations correlated with HIVload (r’s = 0.219, p < 0.05) and inversely with CD4+ cell counts (r’s = −0.321, p = 0.01). Patients with higher ADMA concentrations tended to have lower HDL-cholesterol levels (r’s = −0.188, p = 0.051), ADMA and total- or LDL-cholesterol concentrations were not significantly related with each other. Higher neopterin concentrations were observed in patients with lower HDL-cholesterol levels (urinary neopterin: r’s = −0.275, p < 0.01, plasma neopterin: r’s = −0.303, p = 0.001) and CRP concentrations correlated with LDL-cholesterol (r’s = 0.228, p < 0.05) but there existed no other significant relationships among all the variables. 4. Discussion The main findings of this study are that plasma levels of ADMA and SDMA are increased in HIV-infected patients. In addition, this study shows that in patients with HIV-infection immune activation coincides with elevated levels of methylated arginine species. This finding confirms and extends earlier in vitro data, which showed that formation of ADMA and SDMA paralleled neopterin formation in stimulated peripheral blood mononuclear cells [31]. Also in patients with coronary artery disease associations between enhanced ADMA and neopterin formation have been described [32], indicating a relationship between over-whelming immune response and enhanced arginine methylation. ADMA and SDMA concentrations were not influenced by gender. The higher urinary neopterin to creatinine concentrations in females can be explained by the lower creatinine concentrations. The reason of lower CD4+ cell counts in women is unclear, but it could relate in some part to different cytokine patterns and blood loss via menstrual bleedings.

Table 1 Concentrations of parameters investigated in 112 HIV-infected patients and reference ranges. Males

Females

Reference ranges

Mean

S.E.M.

Median

25th–75th perc.

Mean

S.E.M.

Median

25th–75th perc.

Age (year)

41.4

1.30

41.1

34.8–46.1

38.4

2.03

36.8

30.6–41.4*

Plasma lg HIV-load (cm−3 ) CD4 (mm−3 ) ADMA (␮M) SDMA (␮M) Arginine (␮M) Neopterin (nM) CRP (mg/dl) Cholesterol (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl) Triglycerides (mg/dl)

5.2 204 0.53 0.54 67.7 25.0 0.90 155 35.7 90.8 171

0.074 15.2 0.010 0.017 2.52 2.74 0.39 4.60 1.65 4.00 15.3

5.22 185 0.53 0.52 64.4 18.7 0.38 150 35.0 88.0 134

5.88–5.06 104–252 0.46–0.55 0.38–0.48 52.8–76.5 17.5–4.8 0.15–0.09 159–189 38.0–51.0 106–130 145–111

5.23 142 0.54 0.54 66.1 26.1 1.87 170 39.4 103 155

0.10 15 0.01 0.03 3.76 3.77 0.75 6.26 1.97 5.62 12.6

5.30 180 0.53 0.52 65.1 18.7 0.47 155 35.0 92.0 139

5.70–5.38 75–186** 0.52–0.48 0.58–0.44 48.3–88.4 5.44–18.6 0.15–0.69** 156–180* 39.0–70.0 108–105* 129–53.0

>400 0.42 ± 0.06 0.47 ± 0.08 94 ± 26 <8.7 <0.7 <200 >40 <160 <150

Urine Neopterin (␮M/M creatinine)

694

76.5

524

696–262

859

87.4

524

491–456**

<250

Kyn/trp = kynurenine/tryptophan; HDL = high density lipoprotein; LDL = low density lipoprotein; n.s. = not significant. * p < 0.05 (Mann–Whitney U-test). ** p < 0.01 (Mann–Whitney U-test).

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

The mechanism responsible for the elevated levels of ADMA was not specifically investigated, but our data allow drawing some tentative conclusions. As noted above, production of ADMA by stimulated immune cells in the circulation would provide an explanation for our observations, but disturbances in the metabolism of ADMA in host tissues may also play a role. Involvement of the ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH) is not very likely, because levels of SDMA, which is not metabolized by DDAH, were elevated as well. Also reduced renal excretion is probably not involved, because this would be expected to affect SDMA levels to a larger extent than ADMA levels. Therefore, it is more likely that enhanced generation of ADMA and SDMA by increased proteolysis of methylated proteins is involved. This mechanism is consistent with the catabolic state associated with HIV-1 infections. An alternative explanation might be that HIV-1 itself is a source of ADMA. Protein methylation has been shown to play a role in maintaining optimal HIV-1 infectivity [37] and both the HIV-1 encoded

511

transactivator protein Tat and the HIV-1 nucleocapsid protein are targets of protein arginine methyltransferase 6 (PRMT6) that asymmetrically dimethylates arginine [38,39]. All in all, our results are probably best explained by changes in the production rate of ADMA (i.e. increased protein methylation and proteolysis), whereas involvement of disposal of ADMA by DDAH or renal excretion is less likely. Interestingly, in contrast to ADMA and SDMA, arginine concentrations were rather low in our patients. These data are in contrast with an earlier study [40], however, in that study a ratio of plasma arginine/total amino acid concentration was used. Possibly, the low arginine levels were caused by increased consumption of arginine by inducible isoforms of nitric oxide synthase and arginase, which are known to be upregulated by inflammatory cytokines. In accord with this notion, arginine levels were inversely correlated with neopterin concentrations. This hypothesis would fit well with data from recent cross-sectional studies [41,42], which found higher concentrations of endothelial activation markers in HIV-

Fig. 1. Correlations of plasma HIV-1 load (left graphs, note log-scale) and CD4+ cell counts (right graphs) with concentrations of ADMA (upper; HIV-1 load: r’s = 0.024, n.s., CD4+ counts: r’s = 0.048, n.s.), SDMA (middle; HIV-1 load: r’s = 0.184, p = 0.05, CD4+ counts: r’s = 0.023, n.s.) and arginine (lower; HIV-1 load: r’s = 0.129, n.s., CD4+ counts: r’s = 0.029, n.s.). n.s. = not significant.

512

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

1 infected patients without antiretroviral therapy. Additionally, one of these studies could also demonstrate a strong relationship between inflammatory cytokines and endothelial activation [42], which is in line with our data that higher concentrations of ADMA – a marker of endothelial dysfunction – coincide with a higher degree of cellular immune activation (as reflected by higher neopterin concentrations). Thus, our ADMA-data of HIV-1 infected patients point towards the same direction: ADMA accumulation due to activation of the cellular immune system. Increased ADMA concentrations are associated with an increased cardiovascular risk in non-HIV-1 infected patients, but whether this is also true for HIV-1 infected patients needs to be examined in further longitudinal studies, which also investigate traditional cardiovascular risk factors in more detail. In our study, blood lipid concentrations were determined as a traditional cardiovascular risk factor, as ADMA accumulation has been associated with elevated cholesterol levels earlier [33]. How-

ever, ADMA was not significantly associated with either totalor LDL-cholesterol, and only a borderline significant inverse association with HDL-cholesterol was observed. Cholesterol levels were rather on the lower side of reference ranges in our study [42,43]. Low HDL-cholesterol levels were associated with higher neopterin concentrations, indicating that inflammatory cascades, which are proposed to contribute to the catabolic state of HIV-1 infected patients [44,45], also influence lipid levels. In conclusion, our study shows that dimethylarginines accumulate in the blood of patients with HIV-1 infection and the accumulation is related with Th1-type immune response. The activated T-cell/macrophage system of HIV-1 infected patients may thus contribute to the increased production of ADMA and SDMA. Whether the observed increase of ADMA is related to any increase of cardiovascular risk in HIV-1 infected patients remains to be elucidated in further, longitudinal studies.

Fig. 2. Correlations of plasma concentrations of C-reactive protein (CRP, left graphs) and neopterin (right graphs) with ADMA (upper; CRP: r’s = 0.176, n.s.; neopterin: r’s = 0.242, p = 0.01), SDMA (middle; CRP: r’s = 0.292, p = 0.01; neopterin: r’s = 0.473, p <0.001) and arginine (lower; CRP: r’s = 0.288, p < 0.01; neopterin: r’s = 0.310, p < 0.001) concentrations. n.s. = not significant.

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

513

Fig. 3. Correlations of plasma (left graphs) and urine (right graphs) concentrations of neopterin with plasma HIV-1 load (upper, note log-scale; urine: r’s = 0.354, p < 0.001, plasma: r’s = 0.389, p < 0.001) and CD4+ cell counts (lower; urine: r’s = −0.271, p < 0.01, plasma: r’s = −0.231, p = 0.01).

Acknowledgements The authors thank Miss Astrid Haara for excellent technical assistance and Sigrid de Jong for the skillful analysis of ADMA, SDMA, and arginine. References [1] Mildvan D, Spritzler J, Grossberg SE, Fahey JL, Johnston DM, Schock BR, et al. Serum neopterin, an immune activation marker, independently predicts disease progression in advanced HIV-1 infection. Clin Infect Dis 2005;40: 853–8. [2] Fuchs D, Spira TJ, Hausen A, Reibnegger G, Werner ER, Werner-Felmayer G, et al. Neopterin as a predictive marker for disease progression in human immunodeficiency virus type 1 infection. Clin Chem 1989;35:1746–9. [3] Fahey JL, Taylor JM, Detels R, Hofmann B, Melmed R, Nishanian P, et al. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1. N Engl J Med 1990;322:166–72. [4] Krämer A, Biggar RJ, Hampl H, Friedman RM, Fuchs D, Wachter H, et al. Immunologic markers of progression to acquired immunodeficiency syndrome are time-dependent and illness-specific. Am J Epidemiol 1992;136:71–80. [5] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response-associated production of neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med 1984;160:310–6. [6] Wirleitner B, Reider D, Ebner S, Böck G, Widner B, Jaeger M, et al. Monocytederived dendritic cells release neopterin. J Leukocyte Biol 2002;72:1148–53. [7] Fuchs D, Shearer GM, Boswell RN, Clerici M, Reibnegger G, Werner ER, et al. Increased serum neopterin in patients with HIV-1 infection is correlated with reduced in vitro interleukin-2 production. Clin Exp Immunol 1990;80:44–8. [8] Wirleitner B, Schroecksnadel K, Winkler C, Fuchs D. Neopterin in HIV-1 infection. Mol Immunol 2005;42:183–94. [9] Murr C, Widner B, Wirleitner B, Fuchs D. Neopterin as a marker for immune system activation. Curr Drug Metabol 2002;3:175–87. [10] van Haelst PL, Liem A, van Boven AJ, Veeger NJ, van Veldhuisen DJ, Tervaert JW, et al. Usefulness of elevated neopterin and C-reactive protein levels in predicting cardiovascular events in patients with non-Q-wave myocardial infarction. Am J Cardiol 2003;92:1201–3. [11] Ray KK, Morrow DA, Sabatine MS, Shui A, Rifai N, Cannon CP, et al. Longterm prognostic value of neopterin: a novel marker of monocyte activation in patients with acute coronary syndrome. Circulation 2007;115:3071–8.

[12] Avanzas P, Arroyo-Espliguero R, Cosín-Sales J, Aldama G, Pizzi C, Quiles J, et al. Markers of inflammation and multiple complex stenoses (pancoronary plaque vulnerability) in patients with non-ST segment elevation acute coronary syndromes. Heart 2004;90:847–52. [13] Grammer TB, Fuchs D, Böhm BO, Winkelmann BR, Maerz W. Neopterin as a predictor of total and cardiovascular mortality in individuals undergoing angiography (The Ludwigshafen Risk and Cardiovascular Health Study). Clin Chem 2009;55:115–46. [14] Friis-Moller N, Sabin CA, Weber R, d’Arminio Monforte A, El-Sadr WM, Reiss P, et al. Data Collection on Adverse Events of Anti-HIV Drugs (DAD) Study Group. Combination antiretroviral therapy and the risk of myocardial infarction. N Engl J Med 2003;349:1993–2003. [15] Lohse N, Hansen AB, Pedersen G, Kronborg G, Gerstoft J, Sørensen HT, et al. Survival of persons with and without HIV infection in Denmark, 1995–2005. Ann Intern Med 2007;146:87–95. [16] Henry K, Melroe H, Huebsch J, Hermundson J, Levine C, Swensen L, et al. Severe premature coronary artery disease with protease inhibitors. Lancet 1998;351:1328. [17] Vittecoq D, Escaut L, Monsuez JJ. Vascular complications associated with use of HIV protease inhibitors. Lancet 1998;351:1959. [18] Dube MP, Lipshultz SE, Fichtenbaum CJ, Greenberg R, Schecter AD, Fisher SD. Effects of HIV infection and antiretroviral therapy on the heart and vasculature. Circulation 2008;118:e36–40. [19] Grinspoon SK, Grunfeld C, Kotler DP, Currier JS, Lundgren JD, Dubé MP, et al. State of the science conference: Initiative to decrease cardiovascular risk and increase quality of care for patients living with HIV/AIDS: executive summary. Circulation 2008;118:198–210. [20] Zangerle R, Fuchs D. Increased cardiovascular risk in patients with human immunodeficiency virus infection under highly active antiretroviral therapy. Am J Cardiol 2008;102:373–4. [21] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340: 115–26. [22] Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. [23] Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res 2002;53:31–47. [24] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95. [25] Shah SH, Newby LK. C-reactive protein: a novel marker of cardiovascular risk. Cardiol Rev 2003;11:169–79. [26] Böger RH, Sullivan LM, Schwedhelm E, Wang TJ, Maas R, Benjamin EJ, et al. Plasma asymmetric dimethylarginine and incidence of cardiovascular disease and death in the community. Circulation 2009;119:1592–600.

514

K. Kurz et al. / Pharmacological Research 60 (2009) 508–514

[27] Valkonen VP, Päivä H, Salonen JT, Lakka TA, Lehtimäki T, Laakso J, et al. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127–8. [28] Schnabel R, Blankenberg S, Lubos E, Lackner KJ, Rupprecht HJ, Espinola-Klein C, et al. Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the AtheroGene Study. Circ Res 2005;97:e53–9. [29] Siroen MP, Teerlink T, Nijveldt RJ, Prins HA, Richir MC, van Leeuwen PA. The clinical significance of asymmetric dimethylarginine. Annu Rev Nutr 2006;26:203–28. [30] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–5. [31] Schroecksnadel K, Weiss G, Stanger O, Teerlink T, Fuchs D. Increased asymmetric dimethylarginine concentrations in stimulated peripheral blood mononuclear cells. Scand J Immunol 2007;65:525–9. [32] Murr C, Meinitzer A, Grammer T, Schroecksnadel K, Böhm BO, März W, et al. Association between asymmetric dimethylarginine and neopterin in patients with and without angiographic coronary artery disease. Scand J Immunol 2009;70:63–7. [33] Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98: 1842–7. [34] Teerlink T, Nijveldt RJ, de Jong S, van Leeuwen PA. Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography. Anal Biochem 2002;303:131–7. [35] de Jong S, Teerlink T. Analysis of asymmetric dimethylarginine in plasma by HPLC using a monolithic column. Anal Biochem 2006;353:287–9.

[36] Teerlink J. HPLC analysis of ADMA and other methylated l-arginine analogs in biological fluids. Chromatogr B 2007;851:21–9. [37] Willemsen NM, Hitchen EM, Bodetti TJ, Apolloni A, Warrilow D, Piller SC, et al. Protein methylation is required to maintain optimal HIV-1 infectivity. Retrovirology 2006;3:92. [38] Boulanger MC, Liang C, Russell RS, Lin R, Bedford MT, Wainberg MA, et al. Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression. J Virol 2005;79:124–31. [39] Invernizzi CF, Xie B, Frankel FA, Feldhammer M, Roy BB, Richard S, et al. Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function. AIDS 2007;21:795–805. [40] Ollenschläger G, Jansen S, Schindler J, Rasokat H, Schrappe-Bächer M, Roth E. Plasma amino acid pattern of patients with HIV infection. Clin Chem 1988;34:1787–9. ˜ [41] de Larranaga GF, Bocassi AR, Puga LM, Alonso BS, Benetucci JA. Endothelial markers and HIV infection in the era of highly active antiretroviral treatment. Thromb Res 2003;110:93–8. [42] Ross AC, Armentrout R, O’Riordan MA, Storer N, Rizk N, Harrill D, et al. Endothelial activation markers are linked to HIV status and are independent of antiretroviral therapy and lipoatrophy. J Acquir Immune Defic Syndr 2008;49:499–506. [43] Zangerle R, Sarcletti M, Gallati H, Reibnegger G, Wachter H, Fuchs D. Decreased plasma concentrations of HDL cholesterol in HIV-infected individuals are associated with immune activation. J Acquir Immune Defic Syndr 1994;7:1149–56. [44] Rodríguez-Carranza SI, Aguilar-Salinas CA. Metabolic abnormalities in patients with HIV infection. Rev Invest Clin 2004;56:193–208. [45] Schroecksnadel K, Zangerle R, Bellmann-Weiler R, Garimorth K, Weiss G, Fuchs D. Indoleamine-2, 3-dioxygenase and other interferon-gamma-mediated pathways in patients with human immunodeficiency virus infection. Curr Drug Metab 2007;8:225–36.