Rainer H B&ouml

Edzard Schwedhelma

Renke Maasa

Sabine Quispe-Bravo

Cord Skamirab

Prof. Dr med Rainer H B6ger
email: boeger@uke.uni-hamburg.de

ADMA and oxidative stress may relate to the progression of renal disease: rationale and design of the VIVALDI study

Keywords: endothelium, 8-iso-prostaglandin F2, nitric oxide, risk marker, telmisartan

The renin angiotensin system has been shown to be involved in the pathogenesis of vascular and renal sequelae of diabetes mellitus. In type 2 diabetes mel- litus, angiotensin receptor blockers have been shown to exert clinical benefit by reducing the progression of diabetic nephropathy. They also improve endothelium- mediated vascular function. The latter effect is partly due to the reduction of angiotensin II-associated oxidative stress. Moreover, small clinical studies have shown that treatment with angiotensin receptor blockers also reduces the circulating levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase.

In the VIVALDI trial, the ability of the angiotensin receptor blocker telmisartan to reduce the progression of diabetic nephropathy (associated with proteinuria) in com- parison with valsartan in more than 800 patients with type 2 diabetes during 1 year of treatment is being studied. In order to gain more detailed insight into the poten- tial pathomechanisms associated with this effect, further end-points have been defined. Among these are the circulating levels of ADMA and the urinary excretion rate of 8-iso-prostaglandin F 2α (8-iso-PGF 2α ). The former is an endogenous inhibitor of NO-mediated vascular function(s) and a prospectively determined marker of major cardiovascular events and mortality; the latter is a lipid peroxidation product resulting from the nonenzymatic peroxidation of arachidonic acid, which exerts detrimental vascular effects similar to those of thromboxane A 2 . Urinary 8-iso-PGF 2α has been shown in clinical studies to be an independent marker of cardiovascular disease.

Highlighting the effects of telmisartan on ADMA and 8-iso-PGF levels in such a large cohort of diabetic patients will enhance our understanding of the roles of dysfunctional NO metabolism and redox mechanisms in the pathogenesis of end-organ damage and its prevention by pharmacotherapy with angiotensin receptor blockers.

Introduction and background

Despite improved treatment of hypertension, urinary tract obstruction and infection, many forms of renal disease associated with permanent nephron loss still progress inexorably to chronic end-stage renal failure. Although this may be caused by a variety of kidney diseases, in the USA and western Europe this population group is dominated by patients with diabetes or hypertension. According to the United States Renal Data System registry data for the year 2002, diabetes was the attributed cause of end-stage renal failure in 35.7% of patients who started dialysis. Hypertension was responsible for 24% of cases. Glomerulonephritis accounts for about 15.6% of cases and cystic disease, hereditary causes and urological diseases together constitute another 6.4%. The cause is unknown in 18.3% of these patients.1 Since the early 1980s the endothelium has been rec- ognized as a major regulator of vascular homeostasis. Endothelial cells, as the inner lining of blood vessels, are strategically located between circulating blood and the vascular smooth muscle. In a person with a body weight of 70 kg, the endothelium covers an area of approximately 700 m2 and weighs about 1-1.5 kg.2 Functional integrity of the endothelium is crucial for the maintenance of blood flow and antithrombotic capacity because the endothelium releases humoral factors that control relaxation and contraction, throm- bogenesis and fibrinolysis, and platelet activation and inhibition. Thus, the endothelium contributes to blood pressure control, blood flow and vessel patency. It is now clear that impaired endothelial function con- tributes substantially to cardiovascular disorders such as atherosclerosis, hypertension and heart failure, which lead to hypoperfusion, vascular occlusion and end-organ damage.

Endothelial dysfunction is also a common feature in patients who are diabetic.3 It is considered a major step in atherosclerosis and acute atherothrombotic events. Futhermore, it predicts cardiovascular mortal- ity.4 Endothelial dysfunction has also been docu- mented in a large proportion of patients with type 2 diabetes mellitus.5 The most probable explanation for this observation is a disturbed oxidative balance in the vasculature, resulting in endothelial dysfunction.6 A major pathophysiological alteration of type 2 diabetes is insulin resistance. Studies carried out in people with type 2 diabetes mellitus show an inverse correlation between measures of oxidative stress and insulin resistance.7 Both, diabetes mellitus9 and renal dysfunction are now considered as major cardiovascular risk factors and are associated with greatly elevated cardiovascular morbidity and mortality.

Endothelial dysfunction: the role of ADMA and oxidative stress Endothelial dysfunction is mainly characterized by impaired production of the key endogenous vasodila- tor, nitric oxide (NO). In the endothelium, nitric oxide is formed from L-arginine by the endothelial isoform of nitric oxide synthase (eNOS). In humans NO syn- thesis may be impaired by asymmetrical dimethylarginine (ADMA), an endogenously formed compound that inhibits NOS activity by displacing L-arginine from the substrate binding site.ll Infusion of ADMA impairs endothelium-mediated vasodilation. 12,13 Elevated ADMA plasma levels are associated not only with endothelial dysfunctionl4,ls but also with increased oxidative stress,'6 thereby linking endothe- lial dysfunction and redox mechanisms in vascular disease. Elevated plasma ADMA concentrations have also been found in patients with diabetes.17,18 Functional impairment or decreased expression of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH) has been suggested as one possible mechanism for elevated ADMA concentra- tions in diabetes.19 This is brought about at least in part by redox-mediated dysregulation of DDAH activ- ity.20,21 Furthermore, several investigators recently demonstrated that an elevated plasma ADMA concen- tration is an independent risk factor for cardiovascular mortality in patients with cardiovascular disease and in patients with chronic renal failure. [START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF] . , Several groups of drugs have been studied with respect to their ability to lower ADMA plasma con- centration and to improve the related impairment in NO-mediated vasodilation. Until now, only met- formin, inhibitors of the angiotensin converting enzyme, and AT, receptor blockers have been found, in small clinical studies, to lower plasma ADMA con- centrations. 24-26 The strong relationship between ele- vated ADMA concentration, cardiovascular disease and mortality has made ADMA a goal of primary interest for pharmacotherapeutic intervention in large, controlled clinical trials.

Endothelial dysfunction and oxidative stress: the role of angiotensin 11

A growing body of evidence is strengthening the view that angiotensin II is a major stimulus for vascular oxidative stress. Angiotensin II induces a mitogenic response in vascular smooth muscle cells through protein kinase C-extracellular signal-regulated kinase- dependent and -independent pathways,27,28 together with increased vascular NAD(P)H oxidase activity29 and modulation of extracellular superoxide dismutase expression.3° The consequence is an aggravation of the oxidative burden associated with atherosclerosis and endothelial dysfunction.31 Not surprisingly, AT, receptor blockade attenuates low-density lipoprotein oxidation and improves endothelial function in hypercholesterolemia, hypertension and type 2 diabetes. [START_REF] Rachmani | Losartan and lercanidipine attenuate low-density lipoprotein oxidation in patients with hypertension and type 2 diabetes mellitus: a randomized, prospective crossover study[END_REF][START_REF] Von Zur MüHlen | Treatment with irbesartan or atenolol improves endothelial function in essential hypertension[END_REF][START_REF] Wassmann | Angiotensin II type 1 receptor antagonism improves hypercholesterolemia-associated endothelial dysfunction[END_REF] Isoprostanes: markers and mediators of oxidative injury in the vascular system Markers of oxidative stress are various and data are often conflicting.35 This may mainly be attributed to the diversity of markers used and the heterogeneity of methods applied. In recent years new markers of lipid peroxidation have shown very promising results: F2isoprostanes (reviewed by Morrow36). They are formed endogenously by arachidonic acid oxidation in phospholipids. Subsequently, they are liberated from phospholipids by phospholipases and eliminated via the kidney. F2-isoprostanes are reliable markers and potentially significant mediators of oxidative stress in disease associated with the cardiovascular system.37,38

They have also been validated as reliable markers of oxidative stress in type 2 diabetes 39-43 Oxidative stress is involved in the microvascular and macrovas- cular complications seen in patients with diabetes. These pathophysiologic alterations are mainly attrib- uted to atherosclerotic lesions in the vasculature. F2isoprostanes have been detected in atherosclerotic lesions and they may be regarded as footprints left by the enhanced oxidative burden. However, it seems rea- sonable to assume that F2-isoprostanes are not only an index of oxidative injury but also contribute to the progression of atherosclerosis.36 They may be quantified by gas chromatography-mass spectrometry (GC- MS), GC-tandem MS, liquid chromatography-tandem mass spectrometry (LC-tandem MS), and immunoas- says. Despite this variety of possible methods, reliable quantification of F2-isoprostanes is hindered by inap- propriately performed enzyme immunoassays; this is subject to recent controversy.44,45 There does not appear to be a consensus concerning the best method, but chromatographic methods (GC-(tandem) MS, and LC-tandem MS) should be superior to immunoas- says.46 Moreover, measurement of urinary F2-iso- prostanes seems to be advantageous over plasma F2-isoprostanes, at least in type 2 diabetes. [START_REF] Feillet-Coudray | Divergence in plasmatic and urinary isoprostane levels in type 2 diabetes[END_REF][START_REF] Helmersson | Association of type 2 diabetes with cyclooxygenase-mediated inflammation and oxidative stress in an elderly population[END_REF] Telmisartan: an effective and long-lasting

AT~ receptor blocker

The discovery of a new pharmacological class of anti- hypertensives, the angiotensin II receptor antagonists (or sartans), has led to the clinical development and marketing of these new therapeutic agents since 1995, with the marketing approval of losartan by the Food and Drug Administration. Telmisartan belongs to the second generation of angiotensin II receptor antago- nists. It was developed by Boehringer Ingelheim and was approved for use in hypertension by the Food and Drug Administration for North America in November 1998 and by the European Medicines Agency for Europe in December 1998. Telmisartan is an orally active nonpeptide type I angiotensin II receptor antag- onist that lowers blood pressure with once daily dos- ing over a whole 24 hours dosing interval. The antihypertensive effect of once-daily dosing of telmis- artan in patients with mild-to-moderate hypertension results in a significant reduction of sitting, supine and standing systolic and diastolic blood pressure, with usually little or no orthostatic change. The usual start- ing dose of telmisartan is 40 mg once daily, with the option to increase that dose to 80 mg daily. The 40 mg dosage reduces systolic and diastolic blood pressure by an average of 11.3/7.3 mmHg; with 80 mg this average is 13.7/8.1 mmHg. The antihypertensive activity occurs within 2 hours after single-dose admin- istration and is maintained for the full 24-hour dosing interval. With ambulatory blood pressure monitoring, the 24-hour trough-to-peak ratio for telmisartan was determined to be at least 80% for both systolic and diastolic blood pressure.47 AT~ blockers in the prevention of renal failure in type 2 diabetes mellitus In September 2001 the New England Journal of Medicine published the results of PRIME (Program for Irbesartan Mortality and Morbidity Evaluation). PRIME was the first clinical program to show beneficial effects in patients with hypertension and type 2 diabetes mellitus across the spectrum of early and late stage kidney disease. It consisted of two stud- ies : IRMA 2 (irbesartan microalbuminuria type 2) and IDNT (irbesartan diabetic nephropathy trial).

IRMA 2, a 2-year multicenter, randomised, double- blind, placebo-controlled study of 590 patients aged years, assessed the effects of irbesartan compared with other proven antihypertensive treatments in slowing the progression of diabetic renal disease in hypertensive patients with type 2 diabetes and persistent microalbuminuria (20-200 pLg/min).48 In this study irbesartan (high dose, 300 mg) led to a 70% reduction in progression to later stage disease, overt diabetic nephropathy. It was therefore shown that irbe- sartan is renoprotective independently of its blood- pressure-lowering effect in patients with type 2 diabetes mellitus and microalbuminuria.

IDNT, the second study, was conducted in 1715 patients with proteinuria (overt nephropathy), hypertension and type 2 diabetes mellitus.49 Irbesartan caused a 20% reduction in progression to doubling of serum creatinine, end-stage renal disease, or all-cause mortality compared with placebo, and a 23% reduc- tion compared with amlodipine. Moreover, there was a 37% reduction in hospitalizations owing to conges- tive heart failure compared with amlodipine. The con- clusion was that irbesartan is effective in protecting against the progression of nephropathy due to type 2 diabetes mellitus independently of the reduction in blood pressure.

In addition, in September 2001 the New England Journal of Medicine published the results of a similar trial to IDNT, the RENAAL study (Reduction of Endpoints in NIDDM with Angiotensin II Antagonist Losartan), examining the effects of losartan in patients with type 2 diabetes mellitus and overt nephropathy. The RENAAL study included 1513 patients from 29 countries.5° There was a 28% reduction in the risk of developing end-stage renal disease when losartan was included in the antihypertensive treatment regimen, and hospitalization for congestive heart failure decreased by 32%. In May 2001, data from the MARVAL study (MicroAlbuminuria Reduction with VALsartan) were presented at the 16th Annual Scientific Session of the American Society of Hypertension. MARVAL was a multicenter, double blind, randomized parallel study of 332 type 2 diabetic patients with microalbuminuria and adjusted blood pressure.51 The patients were randomized to receive either valsartan or amlodipine once daily over 24 weeks. The study was designed to assess the blood-pressure-independent effects of val- sartan versus amlodipine on urinary albumin excretion rates, a measure of microalbuminuria. The results

showed that more patients returned to a normal albu- minuric status after 24 weeks with valsartan (29.9%) compared with amlodipine (14.5%) (p = 0.001). These two groups experienced similar rates of blood pressure reduction. The conclusion was that the angiotensin receptor blocker valsartan is more effec- tive than the calcium channel blocker amlodipine in reducing microalbuminuria in type 2 diabetic patients with adjusted blood pressure. The effects of angiotensin converting enzyme inhibitors and angiotensin II ATj -receptor antagonists on mortality and renal outcomes in diabetic nephropathy have recently been reviewed.52 Available data sug- gest that AT1 receptor antagonists are beneficial for renal outcomes, with about a 22% reduction in risk of end stage renal disease and doubling of the serum creatinine concentration, around a 51 % reduction in progression rates from microalbuminuria to macroal- buminuria, and about a 42% increase in regression from microalbuminuria to normoalbuminuria.52 Design of the VIVALDI trial ... The VIVALDI trial has been designed as a prospective, multicenter, randomized, double-blind, double- dummy, parallel group trial to investigate the efficacy of telmlsartan versus VALsartan in hypertensive type 2 Dlabetic patients with overt nephropathy. The primary goal is to show non-inferiority of telmisartan 80 mg with valsartan 160 mg in reducing proteinuria after 1 year of treatment. Furthermore, the treatment effects for telmisartan 80 mg versus valsartan 160 mg with regard to several renal function parameters, clin- ical end-points and parameters of endothelial function and oxidative stress after 1 year of treatment will be evaluated.

Between April 2003 and November 2004, 884 patients aged 30-80 years with type 2 diabetes and hypertension and overt nephropathy (defined by pro- teinuria in 24 h urine >-900 mg and serum creatinine <265 J..Lmol/1 or :!~;3.0 mg/dl) were randomized in the study. Study medication is given while other antihy- pertensive therapy may be maintained, with the excep- tion of angiotensin converting enzyme inhibitors and other angiotensin II receptor antagonists. If the goal blood pressure of 130/80 mmHg cannot be reached with the high dose of study medication, other antihypertensive medication may be given at any time during the study, starting 4 weeks after randomization. The sample size of the per protocol analysis population is set to include a total of 680 patients, 340 administered telmisartan 40 mg daily with mandatory titration after 2 weeks to 80 mg daily, and 340 patients taking valsartan 80 mg daily, with mandatory titration after 2 weeks to 160 mg daily.

The primary efficacy variable is the change from baseline in 24-hour proteinuria, after 1 year of treatment with telmisartan 80 mg versus valsartan 160 mg. Secondary end-points are the following: renal parameters (24-hour urinary albumin excretion rate, creatinine clearance, glomerular filtration rate, serum creatinine, 24-hour sodium excretion in urine); clinical end-points (composite of doubling of the serum crea- tinine concentration, end-stage renal disease or all- cause death; composite of morbidity and mortality from cardiovascular causes); parameters characteriz- ing endothelial dysfunction and oxidative stress (change from baseline in C-reactive protein, ADMA and 8-iso-PG F2a levels); parameters characterizing insulin sensitivity (change from baseline in home- ostasis model assessment index in patients not receiving insulin therapy; change from baseline in adiponectin).

Taken together, the VIVALDI trial will evaluate different aspects of the effects of the angiotensin II receptor blockers telmisartan and valsartan on renal function and the processes underlying the pathogene- sis and facilitating the prevention of diabetic nephropathy.

ADMA and urinary 8-iso-prostaglandin F2a as secondary end-points in the VIVALDI trial ~ ~ :

As detailed above, serum levels of ADMA and urinary excretion rates of 8-iso-PGF2a are novel markers of endothelial dysfunction and oxidative stress, respec- tively. For both, elevated levels have been reported in patients with type 2 diabetes. Moreover, both are asso- ciated with cardiovascular disease, although this association was more difficult to assess for 8-iso-PGF2a because only in a few studies were sufficiently reliable urine collections carried out to allow the assessment of this marker in urine. However, in one study in which we collected urine samples carefully, a significant relationship between urinary excretion of 8-iso-PGF2a and coronary heart disease was found. 37 In the case of ADMA, several prospective as well as numerous cross-sectional clinical studies have confirmed the predictive value of this marker for major cardiovascular adverse events and death. These data have been extensively reviewed elsewhere in this issue. 53 The analysis of ADMA in a large cohort of type 2 diabetic patients in the VIVALDI trial will allow us to establish the relationship between ADMA levels and cardiovascular end-points in a prospective manner in the diabetic population. Moreover, it will also allow firm conclusions on the efficacy of the AT1 receptor blocker telmisartan to lower ADMA levels. Only a few previous studies have been reported in which the effects of AT1 blockers on ADMA were tested; these included very small numbers of participants (see the overview on therapeutic interventions by Maas in this issue54). Moreover, they may have been flawed by the method of assessing ADMA, which has only recently been improved (see the overview by Schwedhelm, also in this issue55).

Although angiotensin II has long been viewed as a major determinant of oxidative stress in the vascular system, the effects of AT1 receptor blockade on uri- nary F2-isoprostane excretion in type 2 diabetes melli- tus have not yet been studied.

Thus, the VIVALDI study will give us detailed and high-quality information on the roles of these novel markers of endothelial dysfunction and oxidative stress to allow us to predict outcomes in diabetic patients. It will also allow us to study the inter- relationship between ADMA, lipid peroxidation and angiotensin receptor blockade with telmisartan, and thus to find potential new clues to the mechanisms of action of this class of drugs in cardiovascular disease and diabetes mellitus.


Markus Reiter
email: markus.reiter@meduniwien.ac.at

Susan Wirth

Ali Pourazim

Stefan Puchner

Mehrdad Baghestanian

Erich Minar

Robert A Bucek

Skin tissue cholesterol is not related to vascular occlusive disease

Keywords: atherosclerosis, cholesterol, Framingham, PREVU POC Skin Sterol Test, PROCAM, skin tissue cholesterol

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The Framingham Heart Study defined the traditional cardiovascular risk factors such as heredity, sex, age, smoking, arterial hypertension, glucose intolerance, obesity, physical inactivity and high levels of serum lipids. [START_REF] Grundy | Primary prevention of coronary heart disease: guidance from Framingham: a statement for healthcare professionals from the AHA Task Force on Risk Reduction[END_REF] The National Cholesterol Education Program has made extensive use of Framingham data for preventing coronary heart disease (CHD), especially by controlling high serum cholesterol levels, which has been demonstrated to profoundly reduce cardiovascular events and mortality. [START_REF]Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report[END_REF][START_REF] Prabhakaran | The metabolic syndrome: an emerging risk state for cardiovascular disease[END_REF][START_REF] Macmahon | Effects of lowering average and below-average cholesterol levels on the progression of carotid atherosclerosis: results of the LIPID Atherosclerosis Substudy[END_REF][START_REF]MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial[END_REF] However, of the 1.5 million heart attacks and 600 000 strokes that occur in the USA each year, almost half will affect apparently healthy men and women with normal or even low serum cholesterol levels. [START_REF] Ridker | C-reactive protein: a simple test to help predict risk of heart attack and stroke[END_REF] In an effort to better predict cardiovascular events, the determination of skin tissue cholesterol (SkinTc) as a marker for deposited extracellular cholesterol has been suggested to be a further independent cardiovascular risk factor. [START_REF] De Graeve | Is cutaneous apoprotein B a better discriminator than serum lipoproteins for atherosclerosis?[END_REF][START_REF] Bouissou | Skin cholesterol and skin apoprotein B in atherosclerosis[END_REF][START_REF] Bouissou | Cutaneous cholesterol in the young and aged coronary patient[END_REF][START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] Previous data indicated a relation between higher concentrations of SkinTc and the presence of CHD, but the reported SkinTc concentrations differed significantly between the studies and suffered from high standard deviations and high interquartile ranges. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] Additionally, a further study reported a missing correlation between SkinTc and high-sensitive CRP (hs-CRP) levels and the history of CHD, cerebrovascular disease (CVD) and peripheral arterial disease (PAD). [START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF] It also has to be pointed out that the differences of SkinTc concentrations between patients with CHD and the corresponding controls were in the range of the reported with-in day and day-to-day precision and the reported natural fluctuation. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF][START_REF] Reiter | Statin therapy has no significant effect on skin tissue cholesterol: results from a prospective randomized trial[END_REF] Consequently, the value of SkinTc as a cardiovascular risk marker may be overrated and created by the natural variation of this parameter. A further limitation of the previous trials is that the authors provided no information about the general vascular status of the patients, which may have a major impact on SkinTc concentrations. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] Therefore, we performed a prospective trial to clarify the role of SkinTc in cardiovascular risk assessment and evaluated the correlation of SkinTc with documented CVD and PAD as well as with the calculated cardiovascular risk based on the Framingham and PROCAM risk scores. [START_REF] Pearson | New tools for Coronary Risk Assessment. What are their advantages and limitations[END_REF][START_REF] Wilson | Prediction of coronary heart disease using risk factor categories[END_REF][START_REF] Assmann | Simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow-up of the prospective cardiovascular Munster (PROCAM) study[END_REF]

Methods

Consecutive outpatients, referred to the department of angiology of a university clinic for evaluation of CVD or PAD were eligible for the present prospective trial. Patients were admitted for the assessment of their general vascular status (i.e. owing to recently diagnosed arterial hypertension or diabetes mellitus, carotid bruit or carotid pulse asymmetry or weakness, leg pain to exclude or confirm intermittent claudication, skin ulceration or peripheral pulse deficit). Exclusion criteria were (1) current lipid-lowering therapy or lipidlowering therapy within the last year; (2) age Ͻ 18 years; (3) pregnancy; (4) psoriasis or eczema on either hand; and (5) recent use (within 24 hours prior to testing) of a topical medication or cream or lotion on either hand. The study protocol was approved by the local ethics committee and written informed consent was obtained from all patients.

At admission, a standardized questionnaire was used to record the patient's medical history with a special focus on symptomatic cardiovascular disease, drug intake, arterial blood pressure (Riva-Rocci) and body mass index (BMI).

Blood samples

Antecubital venous blood samples were obtained for the determination of total plasma cholesterol (TC), serum low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose and glycosylated hemoglobin A1c (HbA1c) levels. An Olympus AU5400 analyzer with standardized Olympus System Reagents (Olympus Diagnostica GmBH, Hamburg, Germany) was used for the determination of lipid levels, a Menarini-Arkray HA 8140 hemoglobin (Hb) A1c analyzer (Menarini Diagnostics, Florence, Italy) was used for the measurement of HbA1c levels.

PREVU POC Skin Sterol Test

The PREVU POC Skin Sterol Test (PreMD Inc., Toronto, Canada) was performed on the palmar surface of each hand as described recently. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF][START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF][START_REF] Reiter | Statin therapy has no significant effect on skin tissue cholesterol: results from a prospective randomized trial[END_REF] The subsequent statistical analysis used the mean value obtained from both hands. The test required no prior fasting and after vigorously cleaning the palm of the hand with an alcohol swab, the photometric reaction of a detector (Digitonin horseradish peroxidase)/indicator (tetramethylbenzidine hydrogen peroxide solution) system was analyzed by a MD22 Spectrophotometer (X-Rite, Inc., Michigan, USA) interfaced with a computer (Sony Computer, Tokyo, Japan) generating a numeric absorber units value for SkinTc. Negative and positive controls applied to the palm in parallel with the detector solution served to monitor test performance. In the pre-study phase, we observed a within-day imprecision of 3.8% and a between-day imprecision of 8.6% for the right hand and 4.3% for the left hand. [START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF][START_REF] Reiter | Statin therapy has no significant effect on skin tissue cholesterol: results from a prospective randomized trial[END_REF] Cardiovascular screening tests and definition of vascular disease groups Carotid duplex ultrasound was performed by experienced vascular technicians on an Acuson XP 10 scanner (Acuson, Mountain View, CA, USA) with a 5-MHz linear probe according to the principles of the Society of Radiologists in Ultrasound concerning the quantification of stenosis of the extracranial carotid artery and the proposals of Nicolaides et al. [START_REF] Nicolaides | Angiographic and duplex grading of internal carotid stenosis: can we overcome the confusion?[END_REF] According to previously published data, our duplex laboratory achieves an excellent agreement of 96% ( ϭ 0.85) with angiography and was therefore used to reliably classify the macrovascular CVD in three groups: group 1 (no plaques visible); group 2 (Ͻ 50% stenosis, hemodynamically not significant); and group 3 (Ͼ 50% stenosis, hemodynamically significant). [START_REF] Sabeti | Quantification of internal carotid artery stenosis with duplex US: comparative analysis of different flow velocity criteria[END_REF] Patients with a prior history of carotid surgery or carotid artery stenting were classified in group 3 irrespective of the results of duplex ultrasound.

Complete peripheral pulse status and determination of the ankle-brachial index (ABI) (calculated as ratio of the systolic arterial ankle pressure to brachial artery pressure) were obtained in all patients. PAD was classified in three groups: group 1 (ABI index Ͼ 0.9 at rest, regular pulse status); group 2 (ABI index Ͻ 0.9 at rest; asymptomatic patients); and group 3 (ABI index Ͻ 0.9 at rest; patients suffer from intermittent claudication or ischemic rest pain). [START_REF] Dormandy | Management of peripheral arterial disease (PAD). TASC Working Group. TransAtlantic Inter-Society Concensus (TASC)[END_REF] Patients with a history of peripheral vascular surgery or percutaneous transluminal angioplasty and/or stenting were classified in group 3. Diabetic patients with incompressible arteries due to mediasclerosis, which precludes accurate pressure measurements, were classified in group 2 if asymptomatic, symptomatic diabetic patients were classified in group 3.

The total percent risk of heart disease in 10 years for the Framingham risk score and the total PROCAM points were calculated as described previously. [START_REF] Wilson | Prediction of coronary heart disease using risk factor categories[END_REF][START_REF] Assmann | Simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow-up of the prospective cardiovascular Munster (PROCAM) study[END_REF] All patients were classified in three groups separated for each score: group 1 (low, Ͻ 10% estimated risk); group 2 (average, 10-20% estimated risk); and group 3 (high, Ͼ 20% estimated risk). [START_REF] Wilson | Prediction of coronary heart disease using risk factor categories[END_REF][START_REF] Assmann | Simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow-up of the prospective cardiovascular Munster (PROCAM) study[END_REF]

Definition of cardiovascular risk factors

Individuals who smoked regularly during the previous 12 months were classified as current smokers. Arterial hypertension was classified in patients receiving antihypertensive medication or in patients with systolic blood pressure values Ն 140 mmHg or diastolic values Ն 90 mmHg according to the guidelines of the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. [START_REF] Chobanian | The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report[END_REF] Diabetes mellitus was classified in patients with a HbA1c level Ͼ 6% accompanied by a fasting blood glucose level greater than 110 mg/dl or in patients with a pathologic oral glucose tolerance test and was assumed to be present in patients with a history of treatment with insulin or oral hypoglycemic agents. [START_REF] Anand | Diagnostic strategies to detect glucose intolerance in a multiethnic population[END_REF][START_REF] Little | The national glycohemoglobin standardization program: a five-year progress report[END_REF] Hyperlipidemia was classified in patients with a fasting total serum cholesterol Ͼ 200 mg/dl, a serum LDL-C value greater than 130 mg/dl or a serum TG value of greater than 172 mg/dl according to the local standard values.

Statistical analysis

A sample size calculation (based on an estimated power of 0.8) assumed a standard deviation (SD) of 30 U concerning SkinTc and revealed a minimal sample size of 289 patients; we assumed a safety margin of an additional 10%. Statistical analysis was performed by using SPSS 13.0 (SPSS Inc, Chicago, IL, USA). Numeric values were tested for normal distribution and are stated as mean Ϯ standard deviation. Student's t-test was used to evaluate differences between groups and correlation analysis used Pearson's correlation coefficient (r). Univariate ANOVA analysis was used to assess differences of SkinTc concentrations between the three stages of CVD, PAD and the Framingham and PROCAM calculated cardiovascular risk. Regression analyses were used to assess influence factors on the presence of atherosclerosis and to evaluate the influence of the presence of atherosclerotic disease on SkinTc. A general linear regression analysis included traditional cardiovascular risk fac-tors (age, sex, diabetes mellitus, arterial hypertension, smoking, TC, LDL-C) and the presence of PAD and CVD with SkinTc as the dependent variable. A logistic regression analysis was adapted in a forward manner, including traditional cardiovascular risk factors with the presence of atherosclerosis as the dependent variable. A p-value Ͻ 0.05 was considered significant.

Results

In total, 318 consecutive patients were included in the present trial. Their baseline demographic data and laboratory results distributed to the three stages of CVD are shown in Table 1. The mean SkinTc concentration of the total study population was 107.7 Ϯ 23.8 U. Univariate analyses revealed no significant correlation of SkinTc with traditional cardiovascular risk factors such as age, BMI, TC, HDL-C, LDL-C and TG and no significant differences for sex, diabetes mellitus, arterial hypertension, hyperlipidemia and smoking concerning SkinTc concentrations (all p Ͼ 0.05).

The t-test analyses demonstrated no significant differences in SkinTc concentrations between the patients with (groups 2 and 3) and without (group 1) CVD or PAD ( p Ͼ 0.05). In addition to that, we failed to detect significant higher SkinTc concentrations in patients with a cardiovascular risk Ͼ 10% in 10 years compared with patients with a risk Ͻ 10% in 10 years according to the Framingham and PROCAM scoring systems (all p Ͼ 0.05); detailed results of the ANOVA analyses are stated in Table 2.

A comparison was made of the 245 patients with at least one documentation of atherosclerotic disease (PAD or CVD 2 and 3) and/or a cardiovascular risk Ͼ 10% in 10 years and the remaining 73 patients, who were classified in group 1 for PAD and CVD and had a cardiovascular risk Ͻ 10% in 10 years and therefore exhibited no evidence or increased risk of atherosclerotic disease and served as the control group. SkinTc was not significantly higher in patients with atherosclerotic disease (108.0 Ϯ 24.7 U vs 106.9 Ϯ 20.5 U; p Ͼ 0.05). Our results were confirmed by a general linear regression analysis, which detected no significant influence factor on SkinTc concentrations, based on the inclusion of the following parameters: sex, age, BMI, TC, LDL-C, HDL-C, TG, arterial hypertension, diabetes mellitus, smoking, the presence of PAD and the presence of CVD (all p Ͼ 0.05). In addition to that, our analyses showed that the 23 patients with a history of myocardial infarction exhibited no significantly higher SkinTc concentrations compared with the remaining individuals (116.5 Ϯ 28.7 U vs 107.1 Ϯ 23.3 U; p Ͼ 0.05).

In order to rule out a systematic failure of our study cohort we performed additional analyses, which demonstrated that our data are in accordance with the Framingham risk profile because the patients in the atherosclerosis group were significantly older and had significantly higher levels of TC, TG and LDL-C (all p Ͻ 0.005) and a higher incidence of arterial hypertension (176 vs 15 patients), diabetes mellitus (97 vs three patients) and smoking history (91 vs 14). A logistic regression analysis, which included sex, age, diabetes mellitus, arterial hypertension, smoking, TC and LDL-C confirmed this finding and demonstrated a significant influence of all included parameters (with the exception of LDL-C) on the presence of atherosclerosis (all p Ͻ 0.05).

Discussion

The major risk factors included in the Framingham risk score account for Ͼ 80% of the excess risk for premature CHD. [START_REF] Stampfer | Primary prevention of coronary heart disease in women through diet and lifestyle[END_REF] An abnormal ABI and the visualization of carotid artery plaques by ultrasound add to the information provided by traditional risk assessment. [START_REF] Pearson | New tools for Coronary Risk Assessment. What are their advantages and limitations[END_REF][START_REF] Wyman | Ultrasounddetected carotid plaque as a predictor of cardiovascular events[END_REF] The determination of SkinTc has been suggested as an adjunct to the traditional cardiovascular risk factor testing. [START_REF] Grundy | Primary prevention of coronary heart disease: guidance from Framingham: a statement for healthcare professionals from the AHA Task Force on Risk Reduction[END_REF][START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF][START_REF] Wilson | Prediction of coronary heart disease using risk factor categories[END_REF] Previous trials suggested a correlation between SkinTc and the presence of CHD, but these trials provided no further information about the general vascular status of their patients. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] Atherosclerosis is of course not limited to the coronary arteries and a cardiovascular risk predictor additionally includes the atherosclerotic status of the remaining arterial system.

The present trial is the first providing a complete objectively determined vascular status of the study cohort by evaluating the patients by carotid duplex sonography, the determination of the ABI and standardized cardiovascular risk scores. We demonstrated that SkinTc concentrations are not higher in patients with documented vascular disease and/or an increased cardiovascular risk compared to patients without any evidence of atherosclerotic disease. Additionally, separated ANOVA analyses of each vascular disease group and both cardiovascular risk groups revealed no significant difference between patients without (group 1), with limited (group 2) and with advanced (group 3) disease. Even patients with passed myocardial infarction did not exhibit elevated SkinTc concentrations.

Our data is in accordance with a previously published trial [START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF] SkinTc concentrations and a history of cardiovascular events, but which challenges the remaining data and therefore has to be analyzed more precisely. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] A systematic technical failure in the execution of the skin test does not seem a probable explanation because the SkinTc concentrations and standard deviations of our study are comparable with previously published data. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF][START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF][START_REF] Reiter | Statin therapy has no significant effect on skin tissue cholesterol: results from a prospective randomized trial[END_REF] A misinterpretation based on our study population is also not likely because traditional cardiovascular risks were significantly associated with the presence of atherosclerosis in our patients, indicating that a representative study cohort was investigated.

Additionally, the number of patients included in our study was based on a detailed sample size calculation in order to provide analysis with high statistical power. Therefore, we reviewed the previous trials in detail. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] It has to be pointed out that the SkinTc concentrations of patients and controls varied considerably among the studies. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] While Vaidya et al reported on a median SkinTc concentration of 91 U (IQR 24) for patients exhibiting coronary artery calcium (CAC) versus 83 U (IQR 20) for controls, Sprecher et al showed mean SkinTc levels of 124 Ϯ 30 U for patients with coronary artery stenosis versus 118 Ϯ 30 U for patients without stenoses and mean SkinTc levels of 127 Ϯ 29 for patients with a history of myocardial infarction versus 120 Ϯ 20 with those without. [START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF] A further study suggested a threshold value of 84 U for an increased 10-year Framingham risk. [START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF] A potential explanation for the variation of SkinTc concentrations might be that most of these trials investigated a relatively small number of patients. Vaidya and coworkers demonstrated the correlation between SkinTc and CAC only in a small subgroup analysis of 99 white patients and failed to detect a significant correlation in the remaining 123 patients -all African-Americans -of their study cohort. [START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF] In addition to that, the significant differences reported in their study diminished after hypertension was adjusted for using logistic regression. [START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF] Tzou et al included 81 patients and demonstrated an increased IMT not until the highest quartile of SkinTc, but the low statistical power of these results has to be considered in the interpretation of this study reporting data from small groups. [START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] The findings of Mancini et al was based on the evaluation of an inhomogeneous group of 60 individuals: eight healthy volunteers, five patients with known vascular disease and 47 patients with unknown vascular status. [START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF] Zawydiwski et al investigated 111 individuals without known CHD, but only 15 patients had a positive stress test and the reported SkinTc concentrations exhibited a wide range for patients (median 0.059 U, range 0.031-0.166 U) and controls (median 0.040 U, range 0.003-0.118 U). [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF] Our literature research detected only two statistically powerful publications, both by Sprecher et al. [START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF] However, only one of these trials reports original data about a correlation between SkinTc and the presence of coronary artery stenosis. [START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF] The second article, describing a correlation between SkinTc and a history of myocardial infarction, reports findings corresponding with the first study; obviously for the same patient cohort, so no additional information has been provided. [START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF] A further major limitation of these two studies and the majority of the previous studies is the use of the SkinTc concentrations of one hand instead of the mean value of both hands for their analyses. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] This fact is of special interest because only a moderate correlation between the right and the left hand could be observed in our study (r ϭ 0.55) and in two further studies (r ϭ 0.42 and r ϭ 0.57). [START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF] Additionally, as mentioned above, most studies including the data of Sprecher et al suffered from a limited description of the study population. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] None of the trials reported on concomitant vascular occlusive disease. Only Vaidya et al and Tzou et al evaluated the patients by carotid ultrasound, but only focused on the determination of the IMT and provided no information about potential concomitant carotid stenoses even though the investigated population had a mean age of 66 years and 56 years and the presence of advanced CVD was therefore likely. [START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] Data about the potential presence of PAD are missing in both publications. Thus, the incomplete evaluation of the study populations may be a potential explanation for the inconsistent findings of the previous trials because patients without evidence of CHD potentially suffered from concomitant CVD or PAD and therefore may have been wrongly classified as 'healthy controls'. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF] The only study providing information about concomitant vascular diseases failed to detect a correlation between SkinTc and a history of cardiovascular events. [START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF] Finally, a significant natural fluctuation of SkinTc concentrations -which has been observed during a period of 6 months and constitutes about 12% -and a within-day and day-to-day precision of the skin test of 4-11% and 7-9% have to be considered in the interpretation of the previous trials and may have influenced or created their results. [START_REF] Zawydiwski | A novel test for the measurement of skin cholesterol[END_REF][START_REF] Mancini | Association of skin cholesterol content, measured by a non-invasive method, with markers of inflammation and Framingham risk prediction[END_REF][START_REF] Sprecher | Skin tissue cholesterol (SkinT(c)) is related to angiographically-defined cardiovascular disease[END_REF][START_REF] Vaidya | Skin tissue cholesterol assay correlates with presence of coronary calcium[END_REF][START_REF] Sprecher | Elevated skin tissue cholesterol levels and myocardial infarction[END_REF][START_REF] Tzou | Skin cholesterol content identifies increased carotid intimamedia thickness in asymptomatic adults[END_REF][START_REF] Reiter | Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation[END_REF][START_REF] Reiter | Statin therapy has no significant effect on skin tissue cholesterol: results from a prospective randomized trial[END_REF] A limitation of the present trial was that a direct visualization of the coronary arteries by angiography is missing. We used the 10-year risk according to the PROCAM and Framingham scoring systems instead of an invasive procedure in order to reflect cardiovascular disease by a risk calculation because these scores accurately predict coronary events with an area under the receiver-operating characteristics curve of about 80%. [START_REF] Wilson | Prediction of coronary heart disease using risk factor categories[END_REF] In conclusion, SkinTc concentrations determined by the PREVU POC Skin Sterol Test are neither related to the presence of CVD or PAD nor to an elevated cardiovascular risk, indicating that this parameter cannot be used as a reliable indicator of atherosclerosis.

SkinTc and vascular disease 131 Vascular

 131 Medicine 2007; 12: 129-134

  reporting a missing correlation between 132 M Reiter et al Vascular Medicine 2007; 12: 129-134

Table 1

 1 Baseline demographic data and laboratory results of the study distributed according to the three-group system for CVD.

	Group 1	Group 2	Group 3
	n ϭ105	n ϭ 143	n ϭ 70

BMI, body mass index; TC, total cholesterol; LDL-C, serum low-density lipoprotein cholesterol; HDL-C, serum high-density lipoprotein cholesterol; TG, serum triglycerides; SkinTc, skin tissue cholesterol; U, units.

Table 2

 2 Distribution of CVD, PAD and the PROCAM and Framingham scores stated with the corresponding SkinTc concentrations.

		SkinTc concentration
	n	(U) Ϯ SD

Vascular Medicine 2007; 12: 129-134

Acknowledgements

We thank PreMD Inc. (formerly IMI International Medical Innovations Inc.) for providing the test kits. We thank Prof. Harald Heinzl, member of the core unit of Medical Statistics and Informatics of the University of Vienna, for statistical advice.

Conflicts of interest

There are no conflicts of interest.


Raghu Kolluri

Onsi Kamel

teaching and research institutions in France or abroad, or from public or private research centers.

A 61-year-old Caucasian female with prior history of bilateral femoro-popliteal bypass grafts, chronic venous insufficiency and long-standing rheumatoid arthritis was admitted with painful bilateral lower extremity ulcerations. Presumptive diagnosis of infected venous or vasculitic ulcers was made and vascular medicine consultation was obtained. Anterolateral aspects of bilateral lower extremities were erythematous and revealed several tender ulcers with well-defined edges. The ulcer bases were composed of predominantly pale granulation tissue and serous discharge (Panel A). Ankle-brachial index and toe pressures were normal at rest. Pertinent laboratory results included a rheumatoid factor of 392 IU/ml and a Westergren sedimentation rate of 115 mm/h. The serum cryoglobulins, C-ANCA, P-ANCA and complement levels (C3/C4) were within normal limits. An MRI did not suggest osteomyelitis. A wound biopsy was requested due to the atypical nature of the ulcers and revealed cutaneous large T-cell lymphoma. Panel B shows the microscopic appearance of the large T-cell lymphoma with angioinvasive features. Beneath an ulcerated epidermis, malignant lymphoid cells infiltrate the deep dermis and subcutis and invade small and medium-sized arterioles in the deep dermis (1: hematoxylin and eosin, ϫ 200). At higher magnification, the cells have a large cell morphology (2: hematoxylin and eosin, ϫ 400). Immunohistochemistry for the T-cell-associated antigen CD3 shows a brown colorimetric reaction decorating the cytoplasmic membranes of the neoplastic cells (3: anti-CD3, ϫ 400).

CHOP chemotherapy was started but she died several months after the diagnosis.

Malignant transformation of chronic ulcers is well documented, although uncommon. [START_REF] Mekkes | Causes, investigation and treatment of leg ulceration[END_REF] Patients with rheumatoid arthritis frequently present with leg ulceration 2 and are at increased risk for lymphoma. [START_REF] Askling | Haematopoietic malignancies in rheumatoid arthritis: lymphoma risk and characteristics after exposure to tumour necrosis factor antagonists[END_REF] This underscores the importance of tissue biopsy in chronic ulcerations with atypical appearance and in unusual locations.

Images in vascular medicine

Unusual etiology of painful leg ulcers

Raghu

  Kolluri a and Onsi Kamel b a Prairie Vascular Institute and b Department of Pathology, St John's Hospital, Springfield, IL, USA Address for correspondence: Raghu Kolluri, Prairie Vascular Institute, 401 East Carpenter St, Springfield, IL 62702, USA. Tel: ϩ1 217 788 0706; Fax: ϩ1 217 523 4520; E-mail: rkolluri@ prairieheart.com Panel A Panel B 'Images in vascular medicine' is a regular feature of Vascular Medicine. Readers may submit original, unpublished images related to clinical vascular medicine to: Mark A Creager, Editor in Chief, Vascular Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.


Alexandros Kafetzakis

Andreas Foundoulakis

Christos V Ioannou

Emilia Stavroulaki

Anastassios Koutsopoulos

Asterios N Katsamouris
email: asterios@med.uoc.gr

Acute lower

Keywords: arterial thromboembolic disease, leucostasis, leukapheresis

Introduction

Although disorders of the coagulation system are well recognized in patients with acute leukemia, these usually present with hemorrhagic complications or thrombosis of small vessels. Large vessel thrombosis is a very rare complication. To our knowledge, there are only nine cases reported in the English literature of acute leukemia presenting as acute lower limb ischemia due to large artery occlusion.

This report describes a patient who was admitted to our hospital under this clinical scenario and was discharged with complete remission without amputation at any level.

Case report

A 57-year-old Caucasian ex-smoker (45 pack-years) with a previous history of chronic obstructive pulmonary disease presented in the emergency department with a 6-hour progressive history of coolness, pallor and pain of the right (Rt) lower limb. He had no history of previous claudication. Clinical examination was unremarkable apart from the findings in the Rt leg. Electrocardiography revealed a regular sinus cardiac rhythm. The Rt leg was cool and pallid with mild sensory loss of the toes, without any motor deficits. The left leg was absolutely normal. Pulses were palpable in all arteries of his left lower limb whereas palpable pulses were found only in the Rt femoral artery. The ankle-brachial pressure index (ABPI) of the left limb was 1.0 (120 mmHg/120 mmHg) and of the right limb was 0.2 (25 mmHg/120 mmHg).

Blood analysis revealed a hematocrit of 33.9% (normal values [NV] 37-47%), platelet count of 50 ϫ 103 (NV: 150-450 ϫ 103), white blood cell count (WBC) of 120 300 cells/l (95% blasts) (NV: 3.8-10.5 ϫ 103 cells/l), INR of 1.37 (NV: 0.85-1.15), aPTT of 43.2 s (NV: 28-40 s), D-dimers Ͼ20 g/ml (NV: 0.0-0.5 g/ml) and fibrinogen of 196 mg/dl (NV: 200-400 mg/dl). In view of these results, an emergent arterial angiography was avoided. Duplex ultrasonography revealed normal flow waveforms up to the level of the popliteal artery, occlusion of the tibioperonial trunk and single-phase flow in the anterior tibial artery. Since the limb was still viable and the coagulation status precluded thombolysis, the patient was admitted to the hematology-oncology department for heparinization and leukapheresis because of possible leucostasis. After this procedure the WBC fell to 50 000 cells/l. Under the provisional morphological diagnosis of acute myeloid leukemia (AML), chemotherapy with AML protocol was initiated (idarubicin 12 mg/m 2 /24 h for 3 days followed by cytarabine 200 mg/m 2 /24 h for 7 days). The immunophenotypic analysis revealed a unique blast population compatible with AML-M5.

The next day, the lower limb showed clinical deterioration and now had rest pain. The pulses were no longer palpable in the Rt femoral artery and Doppler signals were no longer audible at the femoral level and distally. This clinical deterioration was obviously due to a new thrombotic event. Without having any blood or blood product transfusions, laboratory results were hematocrit: 32%; platelet count: 53 ϫ 103; INR: 1.35; aPTT: 38.4 s. Despite any risk of possible hemorrhagic complications, an emergency Rt iliofemoral and femoral-popliteal arterial thromboembolectomy was performed under local anesthesia in an effort to salvage the limb. A white-clot plug was removed from the bifurcation of the Rt common iliac artery (Figure 1A). An improvement of perfusion of the lower limb was noted immediately after the procedure, with restoration of palpable pulses on the dorsalis pedis and posterior tibial arteries as well as restoration of a normal ABPI. Histological examination of the clot revealed cells with similar characteristics with those of AML-M5 cells (Figure 1B).

Complete remission was achieved after the completion of chemotherapy within the next 10 days, without recurrence of further arterial insufficiency, and the patient was discharged after 20 days of hospitalization. At 3 months the patient remains free of symptoms, without any sign of claudication and with complete recovery of initial sensory loss as confirmed after clinical evaluation. Antiplatelet therapy was not prescribed due to an increased risk of hemorrhaging because of a decrease in the platelet count following chemotherapy.

Discussion

Ischemic events in acute leukemia usually involve small vessels of the brain, spleen and lungs. Only nine cases of lower limb ischemia resulting from large arterial occlusion have been presented in the past (Table 1). In the first case, the affected artery was the aorta [START_REF] Brinson | Acute lymphoblastic leukemia manifested as aortic occlusion[END_REF] and in the second one the external iliac, the profunda femoral and all tibial arteries. [START_REF] Redmond | Acute ischaemia of the lower limb: an unusual presenting feature of acute lymphoblastic leukemia[END_REF] In both cases, leucostasis was the mechanism of ischemia. This is of interest since the most common cause of acute ischemia in acute lymphoblastic leukemia is arterial embolism in patients being actively treated with L-asparaginase or other chemotherapeutic regimens who have associated depression of antithrombin III, plasminogen, fibrinogen and coagulation factor levels. The patients in these case reports had not received any treatment previously and no evidence of coagulopathy was present.

The next three cases involved acute promyelocytic leukemia where the mechanism of ischemia was disseminated intravascular coagulation (DIC). [START_REF] Rolston | Arterial occlusions as a presenting feature of acute promyelocytic leukemia[END_REF][START_REF] Digiovanni | Peripheral arterial occlusion in acute promyelocytic leukemia[END_REF][START_REF] Kalk | Acute arterial thrombosis in acute promyelocytic leukemia[END_REF] In one case with acute promyelocytic leukemia [START_REF] Fass | Recurrent peripheral arterial occlusion by leukemic cells sedimentation in acute promyelocytic leukemia[END_REF] and in three cases [START_REF] Kootte | Acute myelocytic leukemia with acute aortic occlusion as presenting symptom[END_REF][START_REF] Campbell | Acute leg ischaemia as a manifestation of the hyperleukocytosis syndrome in acute myeloid leukemia[END_REF][START_REF] Mataix | Acute leg ischaemia as a presentation of the hyperleukocytosis syndrome in acute myeloid leukemia[END_REF] involving AML, the mechanism of lower limb ischemia was leucostasis and subsequent occlusion of an artery by leukemia cells.

The case reported here is interesting in many respects. First, acute ischemia of the lower limb was the presenting symptom of AML in this patient who had no previous history of fever, weight loss, petechia or other hematologic disorders, which is highly unusual. Moreover, the FAB type of leukemia in our patient was M5. According to the FAB classification, M5 is the monocytic variant of AML. There are two subtypes: M5a (monoblastic variant) and M5b (monocytic variant). This type of leukemia develops leukocytosis in 10-30% of patients as well as a relatively high frequency of DIC. The excessive numbers of leucocytes may seriously affect arterial flow by obstructing microchannels or by forming aggregates and white thrombi. Moreover, leukemic blasts may compete for oxygen in the microcirculation, damaging vessel walls. [START_REF] Litchman | Hyperleukocytic leukemias: rheological, clinical, and therapeutic considerations[END_REF] The above, of course, occur in small and medium size arteries, whereas the exact mechanism of clot/emboli development is poorly understood. One may hypothesize that abnormal accumulation of lymphocytes may initially cause the sedimentation of leukemic cells which may then trigger the accumulation of platelets, thus forming a white clot. This clot may form in any part of the arterial tree, though it is usually seen in the periphery (smaller vessels). If the clot is formed in a larger artery then it will of course flow downstream until it is lodged at an artery of smaller diameter or, as usually seen, at a bifurcation site. In general, the embolic material is composed of a mixture of leukemic cells and thrombus, but the amount contained of the two varies among patients.

Treatment of this patient was challenging since, unlike the previously presented cases, he exhibited both leucostasis and coagulation disorders that precluded the use of thrombolytic therapy and interventional radiology. Despite the above-mentioned difficulties, this patient was successfully treated without recurrence of ischemia or amputation at any level. Treatment was rapidly undertaken with urgent leukapheresis and prompt initiation of chemotherapy in this patient in an effort to inhibit any further sedimentation. Despite these therapies, the patient required emergency surgery the following day because of limb deterioration and the presentation of limb-threatening ischemia. Though we cannot draw a definite conclusion from just one case, it is our feeling that leukapheresis and chemotherapy may aid in avoiding thromboembolic recurrence. This is in agreement with Fass et al [START_REF] Fass | Recurrent peripheral arterial occlusion by leukemic cells sedimentation in acute promyelocytic leukemia[END_REF] who attributed the unsuccessful outcome of their patient to delayed initiation of chemotherapy and quoted: 'if remission could have been induced earlier, after initiation of chemotherapy, further propagation of the pathological sedimentation might have been obviated'. To the best of our knowledge there are no articles reporting on the proper approach for treating acute arterial occlusions due to leukemic cells because such embolic occlusions of large arteries are very rare.

Taking into account that thrombolytic therapy is usually not feasible or is unsuccessful and that heparinization is ineffective in these patients, we believe that combined leukapheresis and chemotherapy is the first therapeutic choice unless the limb is immediately threatened. Despite the possibility of coagulation disorders which often cause difficult to control hemorrhage or recurrent thromboembolic events, prompt surgical thromboembolectomy or revascularization should be attempted in such cases presenting with limb-threatening ischemia.

Figure 1 (

 1 Figure 1 (A) Section of arterial white embolus; (B) showing fibrin material in which isolated or small groups of large monoblastic cells are recognized (hematoxylin & eosin, ϫ400) that are positive in CD68 monoclonal antibody (KP1 clone ϫ400) (insert).

Table 1

 1 All reported cases of acute lower limb ischemia due to large artery occlusion: treatment and outcome.

	Acute ischemia in leukemia 201

M, male; ALL, acute lymphoblastic leukemia; APL, acute promyelocytic leukemia; F, female; AML, acute myelocytic leukemia; NR, not reported.