Sebastiaan Marcel Cg Van De Poll
email: mcg.vandepoll@ah.unimaas.nl

Jp Hanssen

Maaike Berbée

Ep Nicolaas

Diethard Deutz

Wim A Monbaliu

Buurman

Hc Cornelis

Dejong

Marcel Cg Van De Poll

Sebastiaan Jp Hanssen

Nicolaas Ep Deutz

Diethard Monbaliu

Wim A Buurman

Cornelis Hc Dejong

Elevated plasma arginase-1 does not affect plasma arginine in patients undergoing liver resection

Keywords: arginase-1, arginine, cell injury

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Introduction

The amino acid arginine is an important substrate for protein synthesis and for the production of agmatine and creatine [START_REF] Wu | Arginine metabolism: nitric oxide and beyond[END_REF]. It is best known however as the precursor for the imunoregulatory and vasoactive molecule nitric oxide (NO), although the conversion to NO represents only 1% of arginine plasma flux [START_REF] Castillo | Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling[END_REF]. Quantitatively, arginine plasma flux is determined by arginine intake, endogenous arginine synthesis and protein breakdown on one side and protein synthesis and arginine catabolism by the enzyme arginase on the other side [START_REF] Castillo | Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets[END_REF]. Arginase converts arginine to urea and ornithine and arginase activity accounts for 10 % of total plasma arginine turnover [START_REF] Castillo | Plasma arginine, citrulline, and ornithine kinetics in adults, with observations on nitric oxide synthesis[END_REF]. Arginase exists in 2 isoforms with only 58% sequence identity [START_REF] Morris | Human type II arginase: sequence analysis and tissue-specific expression[END_REF]. Arginase-1 is a cytosolic protein, predominantly found in the liver and to a lesser extent in erythrocytes [START_REF] Kim | Expression of the liver form of arginase in erythrocytes[END_REF]. Arginase-2 is located in mitochondria and more ubiquitously present [START_REF] Morris | Human type II arginase: sequence analysis and tissue-specific expression[END_REF], amongst others in the kidneys and the spleen but not in mature erythrocytes [START_REF] Kim | Expression of the liver form of arginase in erythrocytes[END_REF][START_REF] Grody | Differential expression of the two human arginase genes in hyperargininemia. Enzymatic, pathologic, and molecular analysis[END_REF]. Hepatic arginase-1 activity serves urea synthesis and nitrogen homeostasis. In the liver ornithine is recycled to citrulline and back to arginine. Due to compartmentalization of this urea cycle, plasma arginine is not a substrate for hepatic arginase-1 [START_REF] Cheung | Channeling of urea cycle intermediates in situ in permeabilized hepatocytes[END_REF][START_REF] Van De Poll | Interorgan amino acid exchange in humans: consequences for arginine and citrulline metabolism[END_REF]. Ornithine generated outside the urea cycle can be converted to proline, an important constituent of collagen, and to the polyamines, which are important for cell proliferation [START_REF] Wu | Arginine metabolism: nitric oxide and beyond[END_REF]. Changes in protein turnover, arginine intake or arginase activity can affect arginine plasma levels [START_REF] Castillo | Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets[END_REF]. Arginine deficiency may result in microcirculatory disturbances, pulmonary and systemic hypertension [START_REF] Morris | Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease[END_REF][START_REF] Langle | L-arginine deficiency and hemodynamic changes as a result of arginase efflux following orthotopic liver transplantation[END_REF], disturbed collagen synthesis and wound healing [START_REF] Debats | Infected Chronic Wounds Show Different Local and Systemic Arginine Conversion Compared With Acute Wounds[END_REF], impaired immune function [START_REF] Luiking | Sepsis: an arginine deficiency state?[END_REF][START_REF] Makarenkova | CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress[END_REF] and in onset of postoperative infections [START_REF] Braga | Preoperative oral arginine and n-3 fatty acid supplementation improves the immunometabolic host response and outcome after colorectal resection for cancer[END_REF].

Injury to arginase-1 expressing cells such as hepatocytes and erythrocytes leads to arginase-1 release into the circulation and increased arginase-1 plasma levels (10,

Methods

Patients

Patients undergoing liver resection for secondary malignancies in an otherwise normal liver (n=16, Table 1) were studied. A routinely placed peripheral arterial catheter was used for blood sampling. Informed consent was obtained from every participant and the research protocol was approved by the Institutional Medical Ethical Committee.

Arginase-1 protein release and indices of in vivo arginase enzyme activity during liver resection

Arginase-1 release and its effect on arginine and ornithine levels were studied in vivo in 10 patients undergoing hepatectomy with intermittent hepatic inflow occlusion (Pringle manoeuvre) [START_REF] Patel | Early stress protein gene expression in a human model of ischemic preconditioning[END_REF]. Arterial blood was sampled pre-operatively, before liver transection, before and after each event of an intermittent Pringle manoeuvre (2 x 15 minutes ischemia and 5 minutes reperfusion) and 90 minutes postoperatively. Blood was collected in pre-chilled heparinized vacuum tubes (Becton, Dickinson & Co., Franklin Lakes, NJ), immediately placed on ice and processed as described below.

In vitro arginase enzyme activity in whole blood and plasma after liver manipulation

To study the effect of plasma arginase activity, separate from other processes regulating arginine levels in vivo, plasma samples with varying arginase-1 levels were incubated. In addition arginase activity was assessed in corresponding whole blood samples. Arterial blood was obtained from 6 patients undergoing liver resection, pre-operatively and after liver manipulation (before liver transection), when based upon prior experience peak arginase-1 concentrations were already expected [START_REF] Van De Poll | Liver manipulation causes hepatocyte injury and precedes systemic inflammation in patients undergoing liver resection[END_REF].

Pre-incubation blood processing

Blood was collected in two 4 mL heparin tubes (BD). One tube was centrifuged (4,000 x G, room temperature, 5 min) to obtain plasma. The plasma was removed and transferred to clean tubes for incubation. The whole blood samples were incubated without any further processing.

Incubation

Incubations were performed immediately after sampling. Plasma and whole blood samples were divided over three aliquots per fraction that were incubated for 0, 20 or 40 minutes at 37°C. Thereafter, samples were placed on ice and processed immediately as described below. Arginase-1, arginine, ornithine and other amino acid concentrations were measured as described below. Arginase activity was expressed as the increment of ornithine concentration per minute. Since kinetic assays like these do not discriminate between the activities of different iso-enzymes the term arginase activity will be used without further specification when referring to the data of this assay.

Arginase-1 protein release and plasma arginase enzyme activity after liver transplantation

Roth et al. described a decrease of plasma arginine levels from ±100 µmol/l to ±4 µmol/l within 30 minutes following cadaveric liver transplantation [START_REF] Roth | L-arginine deficiency after liver transplantation as an effect of arginase efflux from the graft[END_REF], which was ascribed to arginase release from the graft and overwhelming plasma arginase activity. Ethical permission was granted to include 4 patients undergoing cadaveric liver transplantation at the University Hospital, Leuven, Belgium (

Relation between arginase activity and arginase-1 concentration in plasma

Plasma arginase activity at physiological arginine concentration

Aliquots of a plasma sample from one liver transplant recipient, obtained immediately following reperfusion (arginase-1 concentration 25 µg/mL) were merged with aliquots of a plasma sample obtained from a healthy subject (arginase-1 concentration below detection limit (10 ng/ml). By this means an arginase-1 dilution curve ranging from 25 µg/ml to 195 ng/ml was created. Before merging, phosphate buffered saline (PBS, pH 7.4) containing 85 mmol/l arginine was added to the "healthy" plasma aliquots.

The volume of arginine-enriched PBS added to each aliquot was adjusted so that the final arginine concentration after merging equalled 85 µmol/l in each sample.

Accordingly, plasma samples were created containing varying arginase-1 and equal arginine concentrations, the maximum concentration of PBS in these samples was <0.1%. All samples were immediately incubated at 37ºC for 0, 5 or 30 minutes.

Plasma arginase activity at above-Km arginine concentration

In a similar fashion as described above, plasma aliquots containing various concentrations of arginase-1 and 20 mmol/l arginine were created. However, in this case arginine was directly dissolved in the "healthy" plasma aliquots to avoid dilution of the plasma with PBS. Eventually plasma samples were created containing 15,000, 1,500 or 150 ng/ml arginase-1 with 20 mmol/l arginine. These samples were immediately incubated at 37ºC for 0, 5 or 30 minutes.

Arginase-1 concentration in erythrocytes

Blood was obtained from five healthy volunteers in vacuum tubes containing EDTA (Becton, Dickinson & Co.). To remove plasma and mononuclear cells a previously described protocol was applied using a separation fluid (Lymphoprep 1.077, Axis-Shield, PoC AS, Oslo, Norway) [START_REF] Boyum | Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality[END_REF]. One ml of packed erythrocytes was added to 74 ml distilled water and gently shaken for 10 min to induce 100% haemolysis [START_REF] Krzyzaniak | Lysis of human red blood cells 1: Effect of contact time on water induced hemolysis[END_REF]. The homogenate was centrifuged (4,000 rpm, RT, 15 min) and the supernatant was assayed for arginase-1.

Post-incubation sample processing and laboratory analysis

Whole blood samples were centrifuged (4,000 rpm, 4°C, 5 min) to obtain plasma.

One hundred µL plasma was deproteinized with 8 mg sulphosalicylic acid and stored at -80°C, the remainder was stored untreated (-80°C). Amino acid levels were analyzed in SSA deproteinized samples by high performance liquid chromatography as described before [START_REF] Van Eijk | Rapid routine determination of amino acids in plasma by high-performance liquid chromatography with a 2-3 microns Spherisorb ODS II column[END_REF]. Arginase-1 concentrations were measured in untreated samples using enzyme-linked immunosorbent assay (ELISA) [START_REF] Ikemoto | A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases[END_REF] (kindly provided by Hycult biotechnology, Uden, the Netherlands). The detection limit of this assay is 10 ng/mL arginase-1.

Statistics

Changes in amino acid and arginase-1 concentrations in vivo and in vitro were tested using a paired t-test or 1-way ANOVA for repeated measures when more than two serial observations were available. Statistical calculations were made using

Results

Effects of liver manipulation and warm ischemia

In vitro arginase activity

Blood was sampled from 6 patients undergoing liver resection pre-operatively and after liver manipulation. Pre-operative arterial arginase-1 plasma levels were 18.1 (8.0) ng/ml). In line with abovementioned data, arginase-1 plasma levels increased significantly during liver manipulation (10-fold to 184 (54) ng/mL), without affecting 3).

Whole blood

Arginase-1 plasma levels in incubated whole blood remained stable (data not shown), ruling out a potential increase of arginase activity due to haemolysis. After 40 minutes of incubation a significant decrease of arginine concentrations with a concomitant increase of ornithine concentrations was found in plasma of all whole blood samples, irrespective of the amount of arginase-1 in the plasma (Fig 2c,d).

These highly significant changes were not found for other amino acids (Table 4).

Plasma arginase activity after liver transplantation

In vivo arginase-1 levels and indices of arginase activity

In vitro arginase activity after liver transplantation

Incubation of plasma sampled 3 minutes following liver transplantation (arginase-1 13.2 (4.2 µg/ml) at 37ºC for 30 minutes resulted in a significant decrease of plasma arginine concentration (from 61.1 (7.4) to 28.4 (6.7) µmol/l). This was accompanied by a similar increase of ornithine concentration (from 125.2 (6.6) to 163.7 (10.4) µmol/l) (p=0.002) (Fig 3c). Other amino acids remained unaffected (Table 3).

Relation between arginase enzyme activity and arginase-1 protein concentration in plasma

Plasma arginase activity at physiological arginine concentration

Plasma samples containing different arginase-1 levels were incubated for 5 and 30 minutes at 37ºC in the presence of 85 µmol/l arginine. At this arginine concentration the relation between arginase-1 concentration and arginase activity appeared to be non-linear, with a lower specific activity (activity per unit of enzyme) at higher arginase-1 concentrations. Below an arginase-1 concentration of 1.6 µg/ml however, there was in fact a linear relationship between arginase-1 concentration and arginase activity (r 2 =0.99) (Figure 4a). After 30 minutes of incubation with an initial arginine concentration of 85 µmol/l, arginine plasma levels decreased dependent on the arginase-1 level. At arginase-1 levels below 2 µg/ml and an initial arginine concentration of 85 µmol/l, arginine levels decreased less than 10% within 30 minutes (Figure 4b).

Plasma arginase activity at above-Km arginine concentration

Plasma with varying amounts of arginase-1 was incubated in the presence of 20 mmol/l arginine, which is well above the Km for arginase-1. This resulted in an

Discussion

Increased plasma arginase-1 activity is frequently named as a cause of low arginine plasma levels in patients with hepatocellular or erythrocyte injury [START_REF] Morris | Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease[END_REF][START_REF] Langle | L-arginine deficiency and hemodynamic changes as a result of arginase efflux following orthotopic liver transplantation[END_REF][START_REF] Roth | L-arginine deficiency after liver transplantation as an effect of arginase efflux from the graft[END_REF][START_REF] Cheng | Remission of hepatocellular carcinoma with arginine depletion induced by systemic release of endogenous hepatic arginase due to transhepatic arterial embolisation, augmented by highdose insulin: arginase as a potential drug candidate for hepatocellular carcinoma[END_REF][START_REF] Prins | Arginase release from red blood cells: possible link in transfusion induced immune suppression?[END_REF][START_REF] Morris | Decreased arginine bioavailability and increased serum arginase activity in asthma[END_REF][START_REF] Rother | The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease[END_REF]. This could be clinically relevant since low arginine levels may induce immunological and microcirculatory dysfunction [START_REF] Makarenkova | CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress[END_REF][START_REF] De Jonge | Arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice[END_REF][START_REF] Luiking | The role of arginine in infection and sepsis[END_REF]. The results from the present study however, show that a potentially relevant effect of plasma arginase-1 on arginine metabolism only occurs when very high arginase-1 concentrations are reached. We were able to study the relation between plasma arginase-1 levels and plasma arginase activity by the application of a recently developed ELISA ( 27) that allows exact quantification of the arginase-1 concentration in plasma.

Arginase-1 was released from the liver into the plasma in patients undergoing liver resection, leading to an 8-fold increase of circulating arginase-1, without affecting arginine and ornithine plasma levels. These data show no evidence for increased arginase activity despite increased arginase-1 plasma levels. From these data alone however, it cannot be ruled out that actual arginase activity was compensated by other mechanisms regulating plasma arginine and ornithine levels. Plasma arginine levels are regulated in vivo by protein breakdown and endogenous arginine synthesis. The latter occurs in the kidney where citrulline, derived from intestinal glutamine metabolism becomes converted to arginine [START_REF] Van De Poll | Interorgan amino acid exchange in humans: consequences for arginine and citrulline metabolism[END_REF][START_REF] Van De Poll | Intestinal and hepatic metabolism of glutamine and citrulline in humans[END_REF]. We have demonstrated recently that endogenous arginine synthesis remains unchanged during liver surgery [START_REF] Van De Poll | Intestinal and hepatic metabolism of glutamine and citrulline in humans[END_REF]. Quantitatively, arginine clearance is primarily determined by protein synthesis. This is illustrated by the increased plasma levels of arginine and most other amino acids during hepatic inflow occlusion, reflecting abolishment of hepatic protein synthesis. Ornithine concentrations remained unchanged, which is in line with the absence of physiological hepatic ornithine uptake [START_REF] Van De Poll | Interorgan amino acid exchange in humans: consequences for arginine and citrulline metabolism[END_REF]. To study plasma arginase activity isolated from in vivo regulatory mechanisms, plasma and whole blood samples were incubated in vitro. In agreement with the in vivo data no significant changes in arginine and ornithine concentrations were found during incubation of plasma samples with a mean arginase-1 concentration of 185 ng/ml. To explore whether plasma arginase activity was dependent on factors present in whole blood but not in plasma, whole blood samples were incubated at 37°C for 40 minutes. A significant increase of ornithine was found, accompanied by a similar decrease of arginine. No changes were found in the plasma concentrations of other amino acids such as glutamate, an ornithine precursor or citrulline, an arginine product. This strongly suggests that the observed changes actually specifically reflect arginase activity. Whole blood arginase activity however was not related to the arginase-1 plasma concentration, which underlines that plasma arginase-1 does not affect plasma arginine levels. More likely whole blood arginase activity occurs in the cellular fraction (erythrocytes and/or leucocytes).

Several studies have reported arginase activity in plasma in clinical settings, which seemingly conflicts with our data. Roth and co-workers showed that liver transplantation leads to an immediate decrease of plasma arginine to virtually zero with a stoichiometric increase of plasma ornithine, which is highly indicative for arginase activity [START_REF] Langle | L-arginine deficiency and hemodynamic changes as a result of arginase efflux following orthotopic liver transplantation[END_REF][START_REF] Roth | L-arginine deficiency after liver transplantation as an effect of arginase efflux from the graft[END_REF]. We were able to reproduce these data, although the decrease of arginine levels was not as outspoken as described by Roth et al.

Moreover plasma concentrations of other amino acids such as methionine and alanine decreased as well, reflecting restoration of liver function. Nonetheless, the specific steep decline of arginine in the first three minutes following reperfusion and the simultaneous increase of ornithine levels is in fact highly indicative for arginase activity. Arginase-1 plasma levels were 100-fold higher after liver transplantation than Currently, there is growing interest in the role of arginine and arginase in haemolytic anaemia [START_REF] Morris | Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease[END_REF]34). In a recent publication it was reported that haemolytic patients with sickle cell anaemia had reduced arginine levels and a 5-fold increase in plasma arginase activity, measured by a semiquantitative assay [START_REF] Morris | Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease[END_REF]. In the same study it was found that erythrocyte arginase activity was similarly increased. Another recent study reported a 14-fold increase in erythrocyte arginase activity in sickle cell disease, measured semiquantitatively (34). In the light of our current data, which show that a considerable amount of plasma arginine catabolism is performed by arginase-1 within the intact erythrocyte, it can be speculated that increased arginase activity in the blood of sickle cell patients should be ascribed to increased arginase activity in the cellular fraction rather than to increased arginase levels in plasma.

Finally, it has been suggested that increased circulating arginase concentrations disturb arginine metabolism following blood transfusion due to haemolysis of stored erythrocytes [START_REF] Prins | Arginase release from red blood cells: possible link in transfusion induced immune suppression?[END_REF]. We found that normal erythrocytes contain 17 µg arginase-1 per ml cells. Therefore one unit of red blood cell concentrate (300 ml) contains 5.1 mg arginase-1. Considering a distribution volume of 3 L (plasma volume) for these erythrocytes after transfusion, one unit of packed red blood cells can increase arginase-1 plasma levels with 1.7 µg/mL in the extreme situation of 100% haemolysis of the transfused erythrocytes. This theoretical example shows that plasma arginase- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society positive control to the present study. Arterial blood was drawn in heparin tubes (BD) pre-operatively, at the end of the anhepatic phase and 3, 20 and 60 minutes following reperfusion of the graft. Whole blood was centrifuged at 4,000 x G for 5 minutes. Argininase-1, arginine, ornithine and other amino acid concentrations were measured in all samples. Plasma obtained three minutes following reperfusion was incubated at 37°C for 25-40 (mean 30) minutes to assess plasma arginase activity as described above.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Prism 4.0 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society for Windows (GraphPad Software Inc. San Diego, CA). Results are expressed as mean (SEM), patient characteristics as median (range). A p-value <0.05 was considered to indicate statistical significance. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  As expected, mean (SEM) arginase-1 plasma levels increased significantly during liver manipulation (8-fold), before hepatic inflow occlusion (Fig 1a). Despite this, plasma concentrations of arginine and ornithine remained unchanged during the same period (Fig 1b). During inflow occlusion no further significant changes of arginase-1 plasma levels were observed (Fig 1a). At the same time plasma levels of arginine (and most other amino acids, data not shown) increased. Ornithine concentrations remained unchanged (Fig 1b). Ninety minutes postoperatively, mean arginase-1 plasma concentration was declined to 17% of the last measured intraoperative value. From this, an arginase-1 plasma half life of less than 1 hour was calculated.

  arginine and ornithine levels in vivo (p=0.72 and p=0.16 respectively) (Fig 2). Plasma Incubation of pre-operatively collected plasma samples for 40 minutes at 37°C did not lead to changes in arginine and ornithine concentration (p=0.26; Fig 2a). In accordance with in vivo findings, no significant changes in arginine and ornithine concentrations were observed during incubation of plasma samples containing a 10-S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society fold increased arginase-1 concentration (p=0.33; Fig 2b). Plasma concentrations of other amino acids were not affected by in vitro incubation (Table

  After reperfusion, plasma arginase-1 levels increased steeply to 1000-fold normal values (Fig 3a). In addition we observed a rapid decline of plasma arginine levels with a concomitant increase in the plasma levels of ornithine (Fig 3a). A one-phase exponential decay curve fitted along the mean arginase-1 concentrations 3, 20 and 60 minutes post reperfusion, resulted in a calculated plasma half life of approximately 40 minutes. Within 3 minutes following reperfusion in vivo arginine plasma levels were relatively stabilized (Fig 3b). Plasma concentrations of other amino acids that depend on the liver for their plasma clearance such as alanine and methionine also declined following reperfusion of the liver, although not as steeply as arginine concentrations (Fig 3b). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society enduring linear ornithine formation. (Figure 4c), showing that the intrinsic activity of the enzyme in isolated plasma was preserved during in vitro incubation. Arginase-1 concentration in erythrocytes Erythrocyte arginase-1 concentration, measured by ELISA was 17.0 (1.1) µg per ml red blood cells. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society during liver resection and 1000-fold higher than normal reference values. In vitro assessment of plasma obtained 3 minutes following transplantation revealed specific and stoichiometric changes in arginine and ornithine concentration that were related to arginase-1 concentration. Arginase activity per µg arginase-1 (specific activity) appeared to decline at increasing arginase-1 concentrations. This is probably due to the high initial reaction velocity at high arginase-1 concentrations, leading to rapid decline of arginine concentration and arginase activity. In samples with an arginase-1 concentration <1.5 µg/ml arginine depletion did not occur. At these low enzyme concentrations there was a linear relation between plasma arginase-1 concentration and arginase activity. Moreover incubation of plasma samples with 20 mmol/l arginine (above Km) showed that the intrinsic activity of the enzyme was preserved during incubation. Theoretically, the activity of arginase-1 at low enzyme and/or substrate concentrations may be limited by endogenous arginase inhibition. The most potent endogenous arginase inhibitor known is N-ω-hydroxyarginine (NOHA), an intermediate of NO synthesis. However, regarding the high NOHA concentrations required to cause arginase inhibition (IC 50 ≈400µM) (33) it appears unlikely that any of the presently known arginase inhibitors affected the present results. Due to limited availability of methods to quantify arginase (-1 and -2) protein content in biological samples, various semiquantitative arginase activity assays, relying on supraphysiological pH and arginine concentrations have been developed (20, 34). The wide-spread use of such biochemical assays has led to the interchangeable use of the terms arginase activity and arginase concentration. The in vitro experiments in the current study were designed to study arginase activity under physiological conditions rather than to quantify plasma levels of arginase. The data show that detection of arginase activity in a plasma sample under optimal biochemical S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society conditions does not necessarily imply that arginase is active in plasma under physiological conditions.

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  activity during blood transfusion will not likely affect arginine metabolism substantially. This assumption is further confirmed by clinical observations in patients receiving massive blood transfusion during thoracoabdominal aorta surgery, showing S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society moderate changes in plasma arginase-1 concentrations but no changes in plasma arginine concentrations (Hanssen et al., unpublished data). In conclusion, increased plasma levels of arginase-1 occurring during liver resection do not lead to arginine breakdown. The threshold beyond which the plasma level of arginase-1 significantly affects plasma arginine concentration is probably rarely reached in clinical practice, with the exceptions of liver transplantation. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .Figure 3

 23 Figure 2. Blood was drawn from 6 patients undergoing liver resection immediately

Figure 4 .

 4 Figure 4. Plasma samples with varying arginase-1 concentrations were created by

  . The sum of arginine and ornithine concentrations remained unchanged during all experiments. Data are presented as mean (SEM), absolute and relative changes were tested vs. zero by a one-sample t-test. Σ AA = sum of all amino acids, Σ Arginine & Ornithine = sum of arginine and ornithine concentrations. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  concentrations before incubation are given as well as absolute (µmol/l) and relative (%) increases at the end of the incubation (mean 37.5 minutes). Results were similar for both experiments. Data are presented as mean (SEM), absolute and relative changes were tested vs. zero by a one-sample ttest. Σ AA = sum of all amino acids, Σ Arginine & Ornithine = sum of arginine and ornithine concentrations. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Cecil R, Parkin L, Woods G, Ohene-Frempong K, Asakura T. Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy. Br J Haematol 2005;131:389-94. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

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Figure 2 S

 2 Figure 2 S t a g e 2 ( a ) P O S T -P R I N T

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Table 2 )

 2 as optimal

Table 1 . patient characteristics -liver resection

 1 In vivo study (n=10) In vitro study (n=6) Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	Surgical trauma	Liver resection with	Liver
		intermittent Pringle	mobilization/manipulation
		manoeuvre	prior to liver resection
	Gender (M/F)	5/5	3/3
	Age (years)	50 (33-75)	54 (36-74)
	Colorectal liver	8/2	6/0
	metastases/Other liver
	metastases
	AST (IU/L)	23 (7-37)	34 (8-59)
	LDH (IU/L)	360 (295-490)	387 (351-422)
	Creatinine (µmol/l)	103 (67-118)	93 (56-106)

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 . patient characteristics -liver transplantation

 2 Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

	M/F Age F 23 M 55 M 61 M 73 Gender, Age (years), Indication for liver transplantation, Cold ischemia time (hours:min) and AST (IU/L), LDH (IU/L) and indication Cold ischemia AST Pre AST 3' rep LDH Pre LDH 3' rep Creat Pre Creat 3' rep 1 Acute Budd-Chiari 10:51 1956 1786 2417 4680 410 318 2 Alcoholic cirrhosis + HCC 8:57 40 479 274 1544 62 61 3 Alcoholic cirrhosis 11:26 71 458 572 1582 62 68 4 Hepatitis C cirrhosis + HCC 12:29 68 290 329 855 21 98 creatinine (µmol/l) levels preoperative and 3 minutes after reperfusion. HCC=hepatocellular carcinoma S t a g e 2 ( a ) P O S T -P R I N T

Table 3 . Changes in plasma amino acid concentrations during incubation of plasma

 3

		concentration	absolute increase	relative increase
		(µmol/l)	(µmol/l)		(%)
				p-value		p-value
	Glutamate	114 (9.3)	2.3 (2.9)	0.451	3.0 (3.1)	0.350
	Asparagine	68 (11.7)	4.3 (2.2)	0.083	4.6 (2.8)	0.130
	Serine	137 (14.3)	2.6 (2.7)	0.355	1.3 (1.8)	0.481
	Glutamine	626 (52.9)	11.6 (12.5)	0.372	1.7 (2.3)	0.481
	Histidine	188 (105.1)	0.6 (5.3)	0.915	-5.7 (9.8)	0.573
	Glycine	329 (40.3)	9.8 (8.7)	0.287	8.8 (12.5)	0.496
	Threonine	155 (18.9)	0.5 (4.4)	0.911	-1.3 (2.8)	0.653
	Citrulline	40 (2.7)	0.7 (1.0)	0.526	1.3 (2.3)	0.582
	Alanine	404 (38.5)	12.8 (8.3)	0.148	3.2 (2.2)	0.187
	Taurine	61 (7.5)	2.1 (1.2)	0.113	10.2 (8.2)	0.237
	α-amino butyric acid	17 (1.3)	0.7 (0.4)	0.136	3.7 (2.3)	0.140
	Tyrosine	84 (15.6)	6.6 (5.4)	0.246	5.1 (4.4)	0.272
	Valine	156 (12.0)	7.5 (5.1)	0.173	3.2 (2.7)	0.273
	Methionine	38 (11.1)	1.5 (0.8)	0.098	2.8 (2.3)	0.265
	Isoleucine	52 (3.5)	2.3 (1.5)	0.157	3.5 (2.6)	0.207
	Phenylalanine	68 (11.5	2.0 (1.2)	0.122	2.2 (1.7)	0.213
	Tryptophane	51 (7.5)	1.3 (1.3)	0.370	3.7 (3.1)	0.251
	Leucine	101 (8.5)	4.3 (2.2)	0.075	3.2 (2.0)	0.134
	Lysine	212 (19.0)	8.0 (5.8)	0.193	2.8 (2.6)	0.313
	Σ AA	3073 (291.4)	90.3 (61.7)	0.171	2.3 (2.0)	0.283
	Σ Arginine & Ornithine	161 (10.0)	4.8 (3.7)	0.219	1.7 (2.1)	0.424
	Pooled data of all plasma incubation experiments. Plasma was obtained from patients undergoing
	liver resection before liver manipulation (n=6), after liver manipulation (n=6) and after liver
	transplantation (n=4). Arterial concentrations before incubation are given as well as absolute
	(µmol/l) and relative (%) increases at the end of the incubation (mean 37.5 minutes). Results were
	similar for all three experiments except for changes in arginine and ornithine concentrations in
	plasma obtained after liver transplantation. Changes in arginine and ornithine concentrations during
	the various experiments are presented in Figures 2 and 3c

Table 4 . Changes in plasma amino acid concentrations during incubation of whole blood

 4 Pooled data of all whole blood incubation experiments. Whole blood was obtained from patient undergoing liver resection before liver manipulation (n=6) and after liver manipulation (n=6). Arterial

		concentration	absolute increase	relative increase
		(µmol/l)	(µmol/l)		(%)
				p-value		p-value
	Glutamate	109 (6.8)	9.6 (4.7)	0.078	8.3 (4.0)	0.077
	Asparagine	43 (2.0)	3.8 (1.7)	0.066	7.7 (3.8)	0.079
	Serine	118 (10.7)	3.6 (5.3)	0.514	1.7 (4.4)	0.711
	Glutamine	527 (9.3)	3.1 (23.6)	0.898	0.5 (4.5)	0.924
	Histidine	61 (13.7)	6.3 (4.4)	0.196	6.6 (5.2)	0.247
	Glycine	305 (53.0)	45.9 (41.8)	0.308	55.4 (41.3)	0.221
	Threonine	117 (9.5)	4.1 (5.0)	0.433	2.0 (4.8)	0.692
	Citrulline	39 (3.0)	1.0 (1.1)	0.394	2.2 (2.7)	0.437
	Arginine	68 (3.1)	-18.3 (2.2) <0.001	-26.7 (3.1) <0.001
	Alanine	321 (26.0)	28.4 (14.6)	0.094	9.3 (4.2)	0.062
	Taurine	58 (5.3)	-1.4 (10.9)	0.903	3.2 (16.3)	0.850
	α-amino butyric acid	14 (0.8)	1.3 (0.5)	0.049	8.4 (3.4)	0.043
	Tyrosine	49 (2.9)	2.1 (1.7)	0.239	3.5 (3.3)	0.327
	Valine	148 (11.0)	9.3 (5.3)	0.127	5.0 (3.2)	0.161
	Methionine	16 (0.9)	0.5 (0.5)	0.351	2.3 (3.0)	0.473
	Isoleucine	46 (3.6)	2.9 (1.6)	0.111	4.6 (3.3)	0.209
	Phenylalanine	43 (2.6)	1.9 (1.3)	0.201	4.0 (3.0)	0.214
	Tryptophane	34 (4.0)	2.1 (2.3)	0.391	7.8 (8.7)	0.401
	Leucine	85 (7.4)	7.3 (2.9)	0.042	7.1 (2.9)	0.045
	Ornithine	73 (5.9)	37.6 (7.5) <0.001	45.1 (5.8) <0.001
	Lysine	167 (14.1)	19.0 (11.2)	0.132	10.0 (5.2)	0.094
	Σ AA	2445 (111.6)	172.1 (105.1)	0.145	6.9 (4.3)	0.153
	Σ Arginine & Ornithine	145 (8.5)	19.3 (7.4)	0.024	12.0 (4.1)	0.015

Acknowledgments

The authors would like to thank Dr J Rozing (Dept. of Biochemistry, Maastricht University), Dr WH Lamers (Dept. of Anatomy and Embryology, Maastricht University) and Dr AJ Meijer (Dept. of Biochemistry, University of Amsterdam, the Netherlands) for fruitful discussions. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Aug 2007 as manuscript CS20070143 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070143 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

This study was supported by grants from The Netherlands Organization for Health Research and Development to MCGvdP (920-03-317 AGIKO) and CHCD (907-00-033 Clinical Fellowship).


Whittall Ra

Cooper Ja

HAW Neil

Humphries Se

The PCSK9 gene R46L variant is associated with lower plasma lipid levels and cardiovascular risk in UK healthy men

Keywords: PCSK9, total cholesterol, LDL-C, Familial Hypercholesterolaemia

We have determined the relative frequency of the R46L, I474V and E670G variants in the protein convertase subtilisin/kexin type 9 (PCSK9) gene and its association with plasma lipid levels and coronary heart disease (CHD) in UK healthy men and patients with clinically-defined definite familial hypercholesterolaemia (FH). Genotypes were determined using PCR and Restriction Enzyme digestion in 2444 healthy middle-aged (50-61 years) men from the prospective NPHSII Study, with 275 CHD events (15 years followup) and in 597 UK FH patients from the Simon Broome Register. In the NPHSII healthy men the R46L genotype distribution was in Hardy-Weinberg equilibrium and the frequency of 46L was 0.009 (95% CI 0.007-0.012), with one man homozygous for the 46L allele.

There was significant association of the 46L allele with lower mean (+SD) total cholesterol (RR 5.74 ± 1.01 vs RL 5.22 ± 1.05; p=0.001), apo B (RR 0.87 ± 0.24 vs RL 0.73 ± 0.27; p<0.0001) and LDL-C (RR 4.01 ± 0.95 vs RL 3.55 ± 1.01;p=0.02) levels, after adjustment for age, GP practice, smoking, BMI and SBP. As expected, 46L carriers had a low risk of definite or possible CHD HR=0.46 (0.11-1.84)) but this was not statistically significant (p=0.27). Two other common PCSK9 variants I474V (frequency V = 0.179, CI 0.17-0.19) and E670G (frequency G = 0.034 CI 0.03-0.04) were not associated with any significant effects on lipid levels or CHD risk. In FH patients the frequency of 46L was 0.003 (95% CI 0.00-0.01) which was significantly lower (p=0.037) than the healthy subjects. In the four FH patients carrying 46L mean untreated total cholesterol levels were not different in carriers and non-carriers (median.10.3 mmol/l vs 10.2mmol/l p=0.91 after adjustment for age, gender and mutation type).

Conclusions.

The PCSK9 46L allele is more frequent in UK healthy men than in FH patients and is strongly associated with a protective plasma lipid profile risk to CHD. Its low frequency (~2% carriers) means that it is not making a major contribution to determining population CHD risk in the UK.

INTRODUCTION

The protein convertase subtilisin/kexin type 9 (PCSK9) gene, codes for an enzyme which has been shown to be involved in degrading the LDL-receptor protein in the lysosome of the cell and prevents it recycling to the cell surface [START_REF] Horton | Molecular biology of PCSK9: its role in LDL metabolism[END_REF][START_REF] Maxwell | Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment[END_REF]. Gain of function mutations in PCSK9 that increase activity therefore cause increased degradation of LDL-receptors, reduced numbers of receptors on the surface of the cell [START_REF] Horton | Molecular biology of PCSK9: its role in LDL metabolism[END_REF][START_REF] Cameron | Effect of mutations in the PCSK9 gene on the cell surface LDL receptors[END_REF] and hence monogenic familial hypercholesterolaemia (FH) [START_REF] Abifadel | Mutations in PCSK9 cause autosomal dominant hypercholesterolemia[END_REF] However, loss-of function mutations that inactivate PCSK9, result in less degradation of the LDL-receptor and thus higher levels of on the cell surface [START_REF] Zhao | Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote[END_REF]. This results in faster clearance of LDL from the plasma and has been reported to be associated with lower LDL-C levels and lower risk of Coronary Heart Disease CHD [START_REF] Cohen | Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9[END_REF][START_REF] Cohen | Sequence variations in PCSK9, low LDL, and protection against coronary heart disease[END_REF][START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF].

PCSK9 is a highly polymorphic gene [START_REF] Shioji | Genetic variants in PCSK9 affect the cholesterol level in Japanese[END_REF][START_REF] Chen | A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis[END_REF] and one of the DNA changes 137G>T in exon 1 results in the replacement of Arginine 46 by Leucine (R46L). The reported prevalence for 46L heterozygosity was 0.7% in black subjects (N=3363), and 3.2% in European-Americans (N=9524), 8 of whom were homozygous for the 46L allele [START_REF] Cohen | Sequence variations in PCSK9, low LDL, and protection against coronary heart disease[END_REF]. Carriage of the 46L allele was associated with significantly lower plasma levels of total cholesterol and LDL-C, 9% and 15% respectively, and a 47% reduction in the rate of coronary events in those carrying the 46L allele. These results have been confirmed by others [START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF]. A recent study from Norway confirmed the association of R46L PCSK9 with hypocholesterolemia, and suggested that in FH subjects, 46L carriers also showed a better lipid-lowering response to statin therapy [START_REF] Berge | Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy[END_REF].

The aims of this work are to determine the relative frequency of the PCSK9 46L allele and its association with plasma lipid levels and coronary heart disease (CHD) in healthy UK men [START_REF] Miller | The effects of quality and timing of venepuncture on markers of blood coagulation in healthy middle-aged men[END_REF]13] and patients with clinically-defined definite familial hypercholesterolaemia (FH), of whom 35% had evidence of premature CHD [START_REF] Neil | Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia[END_REF]. We also determined the frequency and examined the impact on lipid levels of two other reported common PCSK9 variants, I474V and E670G. Previous studies from Japan [START_REF] Shioji | Genetic variants in PCSK9 affect the cholesterol level in Japanese[END_REF] and the US [START_REF] Chen | A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis[END_REF] have also suggested that carriers of the rare alleles of these variants and haplotypes containing them have lower and higher plasma lipid levels respectively.

MATERIALS AND METHODS

Subjects

The Second Northwick Park Heart Study (NPHS-II) recruited 3012 healthy European Caucasian men aged 50 to 61 years from nine general medical practices in the UK and has followed them for the development of CHD from 1989 [START_REF] Miller | The effects of quality and timing of venepuncture on markers of blood coagulation in healthy middle-aged men[END_REF]13]. In brief, at baseline all of the participants were free of a history of unstable angina, myocardial infarction, coronary surgery, anticoagulant therapy (including aspirin), malignancy and cardiovascular disease or any other condition precluding informed consent. Ethical approval was granted by the local institutional review committee and patients gave written, informed consent.

Participants attended for examination in a non-fasting state, having avoided smoking, vigorous exercise, or heavy meals from midnight beforehand. A resting 12-lead electrocardiogram (ECG) was recorded at baseline and during the sixth year of surveillance with results coded by Minnesota criteria [START_REF] Prineas | The Minnesota Code Manual of Electrocardiographic Findings: standards and procedures for measurement and classification[END_REF]. Weight was measured on a balance scale and height on a stadiometer to calculate the body mass index (BMI, kg/m 2 ). Smoking habits were assessed by questionnaires [START_REF]Medical Research Council's Committee on Chronic Bronchitis and Questionaire on respiratory symptoms[END_REF] completed at baseline and annually thereafter for five years. Blood pressure was recorded with a random zero mercury sphygmomanometer after the subject had been seated for five minutes. Details of possible events were obtained through primary care medical practices, hospitals and coroners' offices. The clinical history, ECGs, cardiac enzymes and pathology were assessed by independent review to diagnose myocardial infarction according to World Health Organization criteria [START_REF]Myocardial Infarction Community Registers[END_REF].

Normal limits for cardiac enzymes were those for the reporting laboratory. Other terminating events were a new major Q wave on the ECG after 5 years of follow-up (Minnesota codes 1 1, 1 2.1 to 1 2.7 , 1 2.8 plus 5 1 or 5 2 ) [START_REF] Prineas | The Minnesota Code Manual of Electrocardiographic Findings: standards and procedures for measurement and classification[END_REF] and surgery for angina pectoris with CHD angiographically demonstrated. In the 2444 men with genotyping there had been 275 terminating events comprising 161 acute CHD events, 67 coronary artery revascularisation procedures and 22 silent myocardial infarctions and 25 possible MIs.

Patients with clinically-defined definite familial hypercholesterolaemia [START_REF] Simon | Scientific Steering Committee on behalf of The Simon Broome Register Group: (1991) The risk of fatal coronary heart disease in familial hypercholesterolaemia[END_REF] were recruited for the Simon Broome British Heart Foundation study, which is a cross-sectional comparison of white Caucasian patients aged 18 years or more with treated heterozygous FH with and without clinically documented CHD [START_REF] Neil | Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia[END_REF][START_REF] Simon | Scientific Steering Committee on behalf of The Simon Broome Register Group: (1991) The risk of fatal coronary heart disease in familial hypercholesterolaemia[END_REF]. Recruitment methods, inclusion and exclusion and diagnostic criteria were as defined in [START_REF] Neil | Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia[END_REF][START_REF] Simon | Scientific Steering Committee on behalf of The Simon Broome Register Group: (1991) The risk of fatal coronary heart disease in familial hypercholesterolaemia[END_REF]. Pre-treatment cholesterol levels were obtained from patient records.

Molecular Genetic analysis

Genomic DNA was isolated from whole blood samples using standard methods [START_REF] Humphries | Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk[END_REF][START_REF] Heath | A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom[END_REF]. PCR primers were designed to introduce a recognition site for the restriction enzyme RsaI.

Genotyping for the PCSK9, exon 1 c137G>T (R46L) mutation was carried out using primers and conditions as follows: Forward, 5'-CAC GGC CTC TAG GTC TTC TC -3'; Reverse, 5'-AGG CCG TCC TCC TCG GTA -3' where the forced base is underlined. The 20µl PCR reaction contained [START_REF]Medical Research Council's Committee on Chronic Bronchitis and Questionaire on respiratory symptoms[END_REF] sizes of 287bp, 69bp, 51bp, 33bp, 30bp, 18bp and 6bp; the G allele produces band sizes of 215bp, 72bp, 69bp, 51bp, 33bp, 30bp, 18bp and 6bp. Products were separated on 7.5% polyacrylamide gel stained with Ethidium Bromide using the microarray diagonal gel electrophoresis (MADGE) system [START_REF] Day | Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose[END_REF].

Statistical analysis

All statistical analyses were carried out using STATA (Intercooled Stata 9.2, Stata Corporation, Texas). Differences in proportions were determined by a χ² test or Fisher's exact test. Lipid levels were compared by genotype after adjustment using analysis of covariance. R-squared values were used to estimate the percentage of the sample variance explained by each genotype. CHD risk was assessed by Cox proportional hazards models (which allowed for varying lengths of follow-up, producing hazard ratios (HR) with 95% confidence intervals (CI). For each variant the common homozygotes served as the reference category. Adjustments for possible confounding factors (age, BMI, smoking, cholesterol, triglyceride and systolic blood pressure (SBP)) were made by including them as covariates in the model, and differences in the baseline hazard by general practice (recruitment site) were permitted (using the strata option in STATA). Haplotype analysis was conducted using THESIAS. Pre-treatment cholesterol levels were not available for all FH patients. Throughout, a p-value of <0.05 was taken as statistically significant.

RESULTS

Healthy NPHSII men

The R46L genotype distribution was in Hardy-Weinberg equilibrium and there was one healthy man homozygous for the 46L allele, the rare allele frequency was 0.009 (95% CI 0.007-0.012). Table 2a shows the mean (+ SD) baseline characteristics of lipids levels of NPHSII men by PCSK9 R46L genotype. There was significant association of the 46L allele with lower mean (+SD) total cholesterol (RR 5.74 ± 1.01 vs RL 5.22 ± 1.05; p=0.001), apo B (RR 0.87 ± 0.24 vs RL 0.73 ± 0.27; p<0.0001) and LDL-C (RR 4.01 ± 0.95 vs RL 3.55 ± 1.01;p=0.02) levels in NPHSII men, after adjustment for age, practice, smoking, BMI and SBP. The 46L carriers had a low risk of definite or possible CHD with a Hazard Ratio (HR) of 0.46 (0.11-1.84) but this was not statistically significant (p=0.27).

The majority of the men had lipid levels available at baseline and for five annual visits and these are presented in Figure 1. As can be seen, the mean levels of the 46L carriers was approximately 0.5mmol/l lower at baseline and the one LL man 2.3mmol/l lower. These levels were maintained over the next five years. The 46L carriers were at the 33 rd percentile of the total cholesterol distribution of the whole sample, while the LL man was at the 0.4 th percentile. Over the 5 year period there was a progressive drop-out of subjects with recorded lipid levels (10 46L carriers and 659 non-carriers). During this time, there was a small but non-significant 3.8% increase in the mean levels in 46L carriers and a statistically significant 3% decrease in levels in non-carriers, the result of which was that the difference between the two groups lost statistical significance by year 4.

To put the impact of this variant in context, the proportion of sample variance for each of the lipid traits was determined and compared to the impact of the well known common APOE gene E2, E3 and E4 alleles. While APOE explained 5.1% of the variance in LDL-C, 3.5% for ApoB and 2.9% for Total cholesterol in this sample, the PCSK9 R46L genotype explained 0.6%, 0.9% and 0.6% respectively. There was no evidence for interaction with APOE genotype in NPHSII men, with effects on cholesterol levels of variation in the two genes not being different from additive (p interaction = 0.39). The genotype was also determined for two other common variants in PCSK9, the I474V and E670G. For both genotype distribution was in Hardy-Weinberg equilibrium and as shown in Table 1 in healthy men the 474V frequency was 0.18 (95% CI 0.17-0.19) and for 670G the frequency was 0.034 (95% CI 0.028-0.040). Table 2b and c shows that there was no significant association with any measured plasma lipid trait. There was also no significant association with CHD risk (for I474V V allele carriers vs II men the HR= 1.16 (0.89-1.50) and for E670G G allele carriers vs EE men HR = 1.20 (0.73-1.98). These estimates were not substantially altered by adjustment for age, practice, smoking, BMI and SBP and remained non-significant (not shown). When data from these two variants was combined into haplotypes three common haplotypes were seen (V474-G670 frequency 0.79, V474-E670 frequency 2,6%, V474-E670 frequency 0.18) with significant evidence for linkage disequilibrium (D' = 0.93). There were no significant associations between any haplotypes and plasma levels of Total-or LDL-cholesterol or with CHD risk (not shown).

FH patients

Genotype was only determined for the PCSK9 R46L variant, and was obtained in 597 FH patients. The genotype distribution was in Hardy-Weinberg equilibrium, and the 46L allele frequency was 0.03 (95% CI 0.00-0.01), which was significantly lower than in the NPHSII healthy subjects (p=0.037). In the four FH patients carrying 46L, whose characteristics are shown in Table 3, there was no difference in mean untreated total cholesterol levels than in the FH patient group overall, (median 10.3 mmol/l vs 10.2mmol/l (p=0.91) geometric mean 10.4 (+1.4)mmol/l vs 10.3 (+1.8)mmol/l (p=0.94) after adjustment for age, gender and mutation type). None of the 46L carriers had evidence of premature CHD, compared to the reported [START_REF] Neil | Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia[END_REF] prevalence in the sample as a whole (38.5%) or in those with a detected mutation causing FH [START_REF] Humphries | Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk[END_REF] (38.6%) although these differences were not statistically significant (all p>0.2). Because of low numbers, no analysis of response to lipid-lowering therapy by R46L genotype was attempted.

DISCUSSION

This study strongly confirms earlier reports [START_REF] Cohen | Sequence variations in PCSK9, low LDL, and protection against coronary heart disease[END_REF] that in healthy middle-aged men, carriers of the 46L allele in PCSK9 have lower plasma levels of total-and LDL-C and apo B, and demonstrate that this effect is maintained over at least 5 years of follow-up. The one individual who was homozygous for this variant had LDL-C levels that put him at the 0.5 th percentile of the sample of healthy men from the UK general population. In this group of healthy men the 46L variant was not associated with a significant effect on any other measured lipid trait. As expected, carriers also had a 54% lower risk of early CHD, which although not statistically significant in this sample is of similar magnitude to the 47% lower risk reported previously [START_REF] Cohen | Sequence variations in PCSK9, low LDL, and protection against coronary heart disease[END_REF]. Based on data from the effect on CHD risk reduction seen with lipid lowering therapy [START_REF] Baigent | Efficacy and safety of cholesterollowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins[END_REF], the observed reduction in LDL-C would be predicted to result in only a 10% reduction in risk. This supports the view [START_REF] Cohen | Sequence variations in PCSK9, low LDL, and protection against coronary heart disease[END_REF] that the life-long lower LDL-C experienced by 46L carriers has a greater than expected CHD benefit than lowering in middle-age. In support of this, none of the FH patients who were carrying the 46L variant had evidence of CHD. It is well known that the age of onset of CHD varies widely in FH patients [START_REF] Slack | Risks of ischaemic heart-disease in familial hyperlipoproteinaemic states[END_REF][START_REF] Kotze | Phenotypic variation among familial hypercholesterolemics heterozygous for either one of two Afrikaner founder LDL receptor mutations[END_REF] and this suggests that variants in the PCSK9 gene may be explaining in part this variability.

The possible mechanism of the LDL-C lowering effect of 46L has been discussed elsewhere [START_REF] Horton | Molecular biology of PCSK9: its role in LDL metabolism[END_REF][START_REF] Zhao | Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote[END_REF]. The enzyme encoded by PCSK9 is involved in degrading the LDLreceptor protein in the lysosome of the cell and preventing it recycling. Loss of function mutations in the PCSK9 gene result in less degradation of LDL-receptors, increased numbers of receptors on the surface of the cell and hypocholesterolaemia [START_REF] Cohen | Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9[END_REF]. There are two possible sites for this effect. The first is in a post-ER compartment such as the Golgi apparatus, where PCSK9 binds to the LDL-R and targets it for degredation in the acidic lysosome. A second possiblity is that secreted PCSK9 binds to surface located LDL-R and, after internalisation, prevents LDL-R recycling from the endosome to the surface,and reroutes it to the lysosome. Currently it is still unclear if the LDL-R is itself cleaved directly by PCSK9. A definite proof of the effect of 46L in vitro has not yet been published, but a direct effect of the substitution on an aspect of PCSK9 function seems highly likely. The R46L substitution has no measurable effect on the synthesis, processing or secretion of PCSK9 in transfected HEK-293 cells [START_REF] Zhao | Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote[END_REF] but modeling indicates that R46 is in a N-terminal extension of the prodomain, which may make intra-molecular contacts with the catalytical C-domain after autocatalytical cleavage. It has been suggested [START_REF] Zhao | Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote[END_REF] that the substitution of a hydrophobic leucine for a polar arganine may alter this association and influence function in this way. The amino-acid at position 46 is hydrophobic in most species [START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF].

However, although the effect seen in the NPHSII healthy men is statistically robust, and is maintained over time, because of the relatively low frequency of 46L carriers the overall impact on LDL-C levels in the population is low, explaining roughly 12% of the effect of the well known APOE gene common variants. Although the 46L-lowering effect on LDL-C of roughly 0.5mmol/l is similar to that seen in APOE e2 carriers compared to APOE e3e3 subjects [25], the population level impact of APOE is greater because the variants are much more common (e2 carriers ~12 % of the UK population).

The two other PCSK9 variants examined here, I474V and E670G, were not associated with any significant impact on lipid levels in the NPHSII healthy men. The 474V variant was first reported to be present at low frequency (0.05) and associated with roughly 7% lower LDL-C levels in carriers in a Japanese study [START_REF] Shioji | Genetic variants in PCSK9 affect the cholesterol level in Japanese[END_REF], but although the frequency was higher (0.18-0.20) in whites and blacks from the US [START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF] no association with lipid levels were detected. The frequency in UK men was similar to that reported in the US, and also no effect on lipid levels as detected. For the E670G variant, in the original study in moderately hyperlipidaemic subjects from the US, homoygosity for the 670G allele was associated with roughly 20% higher LDL-C levels than E/E subjects [START_REF] Chen | A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis[END_REF], an effect which again was not observed in either whites or blacks in the study by Kotowsky [START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF]. In the UK men examined here, LDL-C levels in 670G homozygotes tended to be lower, although not statistically significant. The study here has the power (80%) to be able to detect a 0.14 mmol/l difference in LDL-C levels associated with carriage of the 474V and 0.6 mmol/l difference for homozygosity of the 670G variant. The original report suggested a larger effect with a PCSK9 haplotype [START_REF] Chen | A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis[END_REF], but no such effect was seen here (not shown). We were thus able to confirm the reports [START_REF] Kotowski | A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol[END_REF] of the negligible (or at the very best very modest) impact of these variants in healthy subjects and therefore did not examine their effect in FH patients.

For the FH patients examined, although the low frequency of 46L limits our ability to make strong interpretations, the lower frequency of the 46L variant and the possible protection from early CHD suggests that PCSK9 is one of the genes that is involved in modulating the expression of FH. Thus individuals with FH (commonly due to a mutation in LDLR or APOB) who also are carrying 46L may be less likely to be diagnosed as FH, resulting in the lower than expected allele frequency. Since all the patients examined had a diagnosis of definite FH (ie a personal or family history of Tendon Xanthoma, [START_REF] Simon | Scientific Steering Committee on behalf of The Simon Broome Register Group: (1991) The risk of fatal coronary heart disease in familial hypercholesterolaemia[END_REF], it may be that the an affected father with the same LDLR mutation (age 54 years) who did not carry the 46L variant, and his recorded untreated total cholesterol level was 13.4mmol/l (data not shown), compatible with a lowering effect of the PCSK9 variant. Confirmation of these observation in larger patient cohorts, and particularly studies in the relatives of these patients, to determine their plasma lipid levels, will be informative. In 169 FH patients with a detected LDLR mutation the recorded mean (+SD) untreated total cholesterol levels were 10.3 (1.9) mmol/l, and the average age of onset of CHD was 47.3 (11.3) years, while in 113 patients with no detected mutation these values were 9.85 (1.9) mmol/l and 47.7 (12.9) years respectively (data from [START_REF] Humphries | Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk[END_REF]).

  while heterozygotes (GT) produce band sizes of 205bp, 188bp and 17bp. The TT (homozygote) produced one band size of 205bp. Products were separated on 2.0 % agarose gel and visualised by staining with Ethidium Bromide. For R46L, all gels included a positive heterozygous control sample (confirmed by direct sequencing), and genotypes were read by two independent observers blind to case/control status and any discrepancies confirmed by PCR and digestion. Genotyping for PCSK9, exon 9 c.1420A>G (I474V) and exon 12 c.2009A>G (E670G) variants (rs562556, rs505151 respectively) was carried out using PCR and RFLP conditions as follows: Primers used for amplifying exon 9: Forward, 5'-TCC CTT CTC CCT TGT CTG TG -3'; Reverse, 5'-CTG TGG CTC TCT CCA GCA G -3'. Primers for exon 12: Forward, 5'-GAT GTC GGA GGG AGA AAT GA -3'; Reverse, 5'-GGC ACC CAG AGT GAG TGA GT -3'. The 20µl PCR reaction contained 16mM (NH 4 ) 2 SO 4 ; 67mM Tris, pH8.3; 1.5mM MgCl 2 ; 0.01% Tween 20; 0.2mM dATP, dGTP, dTTP, dCTP; 0.032pmoles of each primer (Invitrogen, UK); 0.3U of Taq DNA Polymerase (Bioline, UK); and 15ng genomic DNA. The PCR cycling conditions consisted of 95 o C 5 mins; 95 o C 30 secs, 55 o C 30 secs, and 72 o C 30 secs for 35 cycles; and 72 o C 5 min. Restriction digest for the I474V variant was carried out using 4U/sample BccI (NEB) and digest for the E670G variant was carried out using 2U/sample of Sau96I (NEB), both overnight at 37 0 C. The A allele of the BccI digest produces band sizes of 267bp, 193bp, 96bp and 25bp; the G allele produces band sizes of 267bp, 218bp and 96bp. The A allele of the Sau96I digest produces band Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society 1

  likely lower LDL-C levels in patients carrying 46L reduces the likelihood of xanthoma development. One of the patients (SB 2825, untreated total cholesterol level 8.4mmol/l) has Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society 1

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society 7

1 Table1

 1 Baseline characteristics of NPHSII men divided on the basis of absence or presence of subsequent CHD (definite plus possible)

		No event	CHD	p-value
		(n = 2169)	(n = 275)
	Age (years)	56.1 (3.4)	56.6 (3.5)	0.02
	SBP (mmHg) a	136.6 (18.8)	141.7 (19.2)	<0.0001
	DBP (mmHg)	84.3 (11.3)	87.1 (11.1)	<0.0001
	BMI (kg/m 2 ) d	26.2 (3.4)	26.9 (3.4)	0.001
	Smoking % (N)	27.1 (588)	37.5 (103)	<0.0001
	Cholesterol (mmol/l)	5.68 (1.00)	6.08 (1.05)	<0.0001
	Triglyceride (mmol/l) a	1.76 (0.92)	2.09 (1.11)	<0.0001
	ApoA (g/l)	1.63 (0.32)	1.62 (0.30)	0.64
	ApoB (g/l) a	0.86 (0.24)	0.92 (0.23)	0.0001
	HDL(mmol/l) a	0.80 (0.24)	0.75 (0.22)	0.03
	Genotype Distribution
	RR/RL/LL	2128/40/1	273/2/0	0.31
	L allele frequency	0.010	0.004	0.23
	(95% CI)	(0.007-0.013)	(0.0-0.013)
	Genotype Distribution
	II/IV/VV	1428/618/64	175/86/11	0.44
	V allele frequency	0.18	0.20	0.21
	(95% CI)	(0.17-0.19 )	(0.17-0.23 )
	Genotype Distribution
	EE/EG/GG	1712/115/3	218/16/1	0.44
	G allele frequency	0.03	0.04	0.55
	(95% CI)	(0.028-0.039 )	(0.023-0.060 )
	a Geometric mean given for log-transformed data (approx sd)

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society 1Table 2 : Mean (+ SD) Baseline lipids levels of NPHSII men by PCSK9 genotype a) R46L, b) I474V and c) E670G genotypes

 2

	a) R46L
		GG	GT	TT	P value	Adjusted p
		N=2401	N=42	N=1	GG vs	value $
					GT+TT
	Total
	cholesterol	5.74 (1.01) 5.26 (1.03)	3.40 (0)	0.0008	0.001
	(mmol/l)
	Triglyceride*	1.80 (0.95) 1.85 (0.96)	1.33 (0)	0.77	0.32
	(mmol/l)
	ApoB*	0.87 (0.24) 0.75 (0.26)	0.37 (0)	0.0003	<0.0001
	(g/l)
	HDL-C*	0.79 (0.24) 0.85 (0.20)	1.01 (0)	0.16	0.18
	(mmol/l)
	LDL-C	4.01 (0.95) 3.62 (0.97)	1.79 (0)	0.01	0.02
	(mmol/l)
	b) I474V genotype
		AA	AG	GG	P value	Adjusted p
		N=1603	N=704	N=75		value $
	Total
	cholesterol	5.72	5.76 (1.03) 5.80 (1.05)	0.56	0.56
	(mmol/l)	(0.99)
	ApoB*	0.87	0.87 (0.24) 0.83 (0.23)	0.44	0.29
	(g/L)	(0.24)
	HDL*	0.80	0.81 (0.24) 0.82 (0.24)	0.53	0.62
	(mmol/l)	(0.24)
	LDL	3.98	4.02 (0.88) 4.02 (0.99)	0.71	0.60
	(mmol/l)	(0.98)
	c) E670G genotype
		AA	AG	GG	P value	Adjusted
		N=1930	N=131	N=4		p value $
	Total
	cholesterol	5.73	5.66 (0.97)	5.35 (0.19)	0.56	0.46
	(mmol/l)	(0.99)
	ApoB*	0.86	0.88 (0.23)	0.84 (0.06)	0.61	0.37
	(g/L)	(0.23)
	HDL*	0.81	0.83 (0.26)	0.77 (0.10)	0.72	0.87
	(mmol/l)	(0.24)
	LDL	3.98	3.93 (0.95)	3.55 (0.30)	0.73	0.76
	(mmol/l)	(0.94)
	*geometric mean (approximate sd)
	$ adjusted for age, practice, smoking, BMI, SBP

Table 3 : Characteristics of FH patients carrying the PCSK9 46L variant

 3

	SB	M/	Age at	Untreated Chol	Age onset	LDLR
	No	F	recruitment*	(mmol/l)	CHD	mutation
			(years)
	2825	F	30	8.6	No CHD	splice i9 $
	3049	F	57	9.7	No CHD	C337Y
	3154	M	62	12.6	No CHD	P664L
	8	M	63	11.3	No CHD	none
	* in 1999
	$ mutation is intron 9 c.1359-23del9/ins5 [26]

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society Figure 1

  Change in cholesterol levels over five years by PCSK9 R46L genotype in NPHSII men

				RR	RL	LL
	Total Cholesterol (mmol/l)	2.8 3.3 3.8 4.3 4.8 5.3 5.8 6.3
		2.3
		Baseline Year 1	Year 2	Year 3	Year 4	Year 5
	N = 2379/43/1 2218/38/1 2121/39/1 2048/37/1 1835/34/1 1720/33/1
	p = 0.0005,	0.005	0.0005	0.01	0.07	0.18

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150

Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We would like to thank the members of the Simon Broome BHF study for access to patients samples, Dr Rossi Naoumova, Prof Gil Thompson, Dr Mary Seed, Prof Paul Durrington, Dr Paul Miller, and Prof John Betteridge. The study was supported by a grant from the British Heart Foundation (grant RG93008). SEH and RW acknowledge BHF support (RG 2005/014) and a grant from the Department of Health to the London IDEAS Genetics Knowledge Park. CH is supported by the Pinto Foundation. Clinical Science Immediate Publication. Published on 6 Jun 2007 as manuscript CS20070150 © 2007 The Authors Journal compilation © 2007 Biochemical Society