Ana Martínez-López De Castro

Susana Nieto-Cerón

Pons-Castillo Aurelio

Lilian Galbis-Martínez

Juan Latour-Pérez

Juan Torres-Lanzas

Isabel Tovar- Zapata

Pedro Martínez-Hernández

José Neptuno Rodríguez-López

Juan Cabezas- Herrera

Juan Cabezas-Herrera

Cancer-associated differences in the acetylcholinesterase activity in bronchial aspirates of lung cancer patients

Keywords: acetylcholinesterase, proliferation, airway surface fluid, lung, cancer

In non-neuronal contexts, acetylcholine is thought to be involved in the regulation of vital cell functions such as proliferation, differentiation, apoptosis and cell-cell interaction. In airways most cells express the non-neuronal cholinergic system each containing a specific set of components required for synthesis, signal transduction and acetylcholine hydrolysis. Our aim was determine the expression of cholinergic system components in bronchial aspirates from controls and lung cancer patients. We conducted an analysis of cholinergic components in stored soluble and cellular fraction of bronchial aspirates from non-cancerous patients and diagnosed lung cancer patients.

This study presents data proving that the fluid secreted by human lung cells contains enough acetylcholinesterase activity to control acetylcholine level. Thus, these data demonstrate that: (a) Acetylcholinesterase activity is significantly lower in aspirates from squamous cell carcinomas; (b) The molecular distribution of acetylcholinesterase in both bronchial cells and fluids consisted of amphiphilic monomers and dimers; (c) choline acetyltransferase, nicotinic receptors and cholinesterases are expressed in cultured human lung cells, as demonstrated by RT-PCR. It appears that the nonneuronal cholinergic system is involved in lung physiology and lung cancer. The physiological consequences of the presence of non-neuronal ACh will depend on the particular cholinergic signalling network of each cell type. Clarifying the pathophysiological actions of acetylcholine remains an essential task and warrants further investigation.

INTRODUCTION

As a neurotransmitter, acetylcholine (ACh) plays pivotal roles in both the central and peripheral nervous systems. ACh is also a signalling molecule in the lung (for review see refs 1;2 and also below). Thus, ACh plays two different biological roles, acting as a classical neurotransmitter (neuronal) and as a cytomolecule (non-neuronal).

The presence of cholinergic components in epithelial cells (intestine, skin, urogenital tract), mesothelial, endothelial, muscle and immune cell tissues points to the existence of a cholinergic phenotype, modulating basic cell functions such as gene expression, proliferation, differentiation, cellular adhesion, migration, secretion and absorption.

Multiple reports have established that human lung cells express the non-neuronal cholinergic system (reviewed in Ref. [START_REF] Wessler | The non-neuronal cholinergic system in humans: expression, function and pathophysiology[END_REF], i.e. the synthesizing enzyme, choline acetyltransferase (ChAT) [START_REF] Wessler | The non-neuronal cholinergic system in humans: expression, function and pathophysiology[END_REF][START_REF] Reinheimer | Acetylcholine in isolated airways of rat, guinea pig, and human: species differences in role of airway mucosa[END_REF][START_REF] Klapproth | Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man[END_REF], nicotinic (nAChR) and muscarinic (mAChR) receptors [START_REF] Song | Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma[END_REF][START_REF] Zia | Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells[END_REF][START_REF] Maus | Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors[END_REF][START_REF] Wang | Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors[END_REF][START_REF] Sekhon | Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys[END_REF][START_REF] Williams | Muscarinic signaling in carcinoma cells[END_REF], as well as enzymes for ACh hydrolysis, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [12;13].

In the lung, the role of ACh as an autocrine/paracrine signalling molecule is not yet generally accepted despite many data showing that lung cells synthesize the cholinergic components [START_REF] Proskocil | Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells[END_REF]. Importantly, the case for non-neuronal autocrine signalling has also been reported in lung cancers [6;15;16]. The fact that nicotine regulates cellular proliferation by activation of Akt [START_REF] West | Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells[END_REF], or by β-arrestin-mediated activation of Src and Rb-Raf-1 pathways [START_REF] Dasgupta | Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src and Rb-Raf-1 pathways[END_REF], at the same time as blocking apoptosis [16;19] provides strong evidence that stimulation of nicotinic and/or muscarinic receptors by endogenous acetylcholine or exogenous agonists associated to tobacco consumption could participate actively in lung cancer pathogenesis. The biochemical events between ligands and receptors leading to carcinogenesis and tumour progression are not fully understood. Another point of growing interest links the cholinergic system and angiogenic activity in tumours. Nicotine and endogenous cholinergic pathways could mediate on endothelial cell growth and angiogenesis [START_REF] Villablanca | Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro[END_REF][START_REF] Heeschen | Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis[END_REF][START_REF] Heeschen | A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors[END_REF].

Increasing evidence has shown that AChE may be involved in pivotal processes of carcinogenesis and tumour progression. Thus, in leukaemia, ovarian carcinoma and tumour cell lines, aberrations in the AChE gene are frequently found (reviews in [START_REF] Greenfield | Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease[END_REF][START_REF] Small | Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease[END_REF][START_REF] Soreq | Acetylcholinesterase--new roles for an old actor[END_REF]. A role of AChE expression during apoptosis has been demonstrated [26;27] and the mechanism underlying AChE upregulation is mediated by the JNK/c-Jun pathway [START_REF] Deng | Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis[END_REF].

Recently, we have shown that post-translational processing of AChE is altered in lung cancer in a cell type-dependent manner [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF].

Our goal in the present study was to determine the level of acetylcholinesterase activity, to study the pattern of molecular forms and to detect the expression of cholinergic components important to cholinergic signalling. We show that acetylcholinesterase activity was found in bronchial aspirates and the differences between lung diseases have potential diagnostic uses.

MATERIAL AND METHODS

Chemicals

Acetylthiocholine iodide, 5,5´-dithiobis-2-nitrobenzoic acid (DTNB), tetraisopropyl-pyrophosphoramide (iso-OMPA), polyoxyethylene 10 -oleyl ether (Brij 96), enzyme markers for sedimentation analysis (bovine liver catalase, 11.4S, S 20,w , Svedberg Units, and bovine intestine alkaline phosphatase, 6.1 S 20,w ,), anti-goat IgG monoclonal antibodies conjugated with horseradish peroxidase and recombinant human AChE were purchased from Sigma Chemical Co. (Madrid, Spain). Goat antisera against the N-terminal peptide of human AChE (N19) was purchased from Santa Cruz Biotechnology Inc (CA, USA). Molecular weight protein standards were provided by Invitrogene. For RT-PCR assays, mRNA was extracted using a Chemagic mRNA Direct Kit (Chemagen AG, Germany). The GenAmp RNA PCR kit (Applied Biosystems, Branchburg, NJ, USA) was used for reverse transcription. Other chemicals were of analytical grade.

Patients

We studied 105 patients admitted to the Virgen de la Arrixaca University Hospital in Murcia (Spain) between 2003 and 2006, in all of whom pulmonary function was affected. Haemolytic bronchial aspirates (n=20) were discarded. A total of 85 samples of bronchioalveolar aspirates from patients without (control; n=49) or with lung cancer (n=36) were studied. Of them 70 were male and 15 female and their ages ranged from 20 to 88 years. The mean age was 62.60 years.

The histological evaluation of the tumours according to WHO criteria, revealed that 10 corresponded to adenocarcinoma (AC), 15 to squamous cell carcinoma (SCC) and 11 to small-cell lung carcinoma (SCLC). Staging was assessed according to the TNM classification system (Tumor Node Metastasis Staging System). All patients gave their consent after being appropriately informed.

Cell culture

The human NSCLC cell lines NCI-H1264, NCI-H157, A549, H23, and the human SCLC DMS-79, NCI-H69, NCI-H187 and N-417 were a gift of Dr. L. Montuenga, Department of Histology and Pathology, Schools of Sciences and Medicine, University of Navarra, and M. Sanchez-Cespedes, Lung Cancer Group, Pathology Programme, Spanish National Cancer Centre (CNIO). Caco-2 colonic cancer cell line was a gift of SACE (University of Murcia). The human breast cancer cell line MCF-7 and human melanoma SK-MEL-28 were purchased from the American Type Culture Collection. Cell lines were maintained in RPMI medium supplemented with fetal bovine serum (10% v/v), L-glutamine (2mM), penicillin (100 U/ml) and streptomicine (100 ug/ml) at 37ºC in an atmosphere of 5% CO 2 .

Processing of the samples

Bronchial aspirates were performed by the staff physicians in a standard fashion using a 8.0-mm fiberoptic bronchoscope. Subjects were given local anesthesia using 4% lidocaine sprayed in the oropharynx and 1% lidocaine instilled over vocal cords, carina and bronchus. The bronchial aspirates were centrifuged for 10 minutes at 1,500 rpm to pellet the cells. All fractions were rapidly frozen at -80ºC until required. Serum from patients was obtained by drawing whole blood through standard procedures on the same day before the collection of bronchial aspirates.

Cytological examination of the cellular fraction of bronchial aspirates was performed by the pathologists and only cases with unequivocal malignant features allowing tumour typing were considered. Cellular fraction of bronchial aspirates was found to contain ciliated cells in much greater proportion than squamous cells and macrophages. Both, the number of recovered cells and the percentage of viable cells (as estimated by trypan-blue staining) varied significantly between samples. The number and percentage of cancerous cells in bronchial aspirates from lung cancer patients ranged from a few cells to several thousand.

Assay of the AChE activity in bronchial aspirates.

Recovered bronchial aspirates are variable mixtures of sterile saline solution, airway surface fluid (ASF) and ASF components. Therefore, the concentration of recovered molecules in the ASF is usually very low because of the saline solution added. Measurement of AChE activity in such samples required a very sensitive methodology. The Ellman Method [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF] is not sensitive enough to measure AChE activity in such diluted samples. AChE activity was determined using the Acetylcholine/Acetylcholinesterase Assay kit (A12217, Molecular Probes) according to the manufacturer`s instructions. AChE activity bronchial aspirate fraction was measured by adding 0.1 ml of the serially diluted samples in duplicate onto 96-well microplates.

After that, 0.1 ml of the mixture reaction containing 50 mM tris-ClH, pH 7.5, 0.2M Amplex Red reagent, 2 U/ml horseradish peroxidase, 0.2 U/ml choline oxidase was added. Reactions were measured using a 530 nm excitation wavelength and a 590 nm emission wavelength, for 30 minutes at multiple time points to follow the kinetic. A standard curve ranging from 0 to 20 mU/mL of AChE from electric eel (included with the assay kit) was used in each experiment. One unit of AChE activity represents the hydrolysis of 1 μmol of substrate per minute at 37ºC.

Standardization of AChE activity in bronchial aspirates with Urea

It is difficult to estimate the actual concentration of recovered molecules in ASF in situ because when the saline solution is recovered by aspiration, ASF and its components are recovered along with it. The degree to which the process of collecting bronchial aspirates dilutes the airway surface fluid was determined using urea as a marker of dilution [START_REF] Rennard | Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution[END_REF]. Serum and ASF urea concentrations were determined using the kinetic UV assay for urea (Roche Diagnostic) according to the manufacturer's instructions in a microplate spectrophotometer (Biotek Instrument Inc.) and using a standard curve ranging from 0 to 20 mg/dL urea. Concentration in ASF = (Concentration in samples x serum urea concentration) / (sample urea concentration).

Solubilization of AChE from lung epithelial and cultured cells

AChE was extracted from the airway cells by homogenization in 0.5 ml Trissaline buffer (TSB; 1M NaCl, 50mM MgCl 2 and 10mM Tris, pH 7.5) supplemented with Brij 96 (0.25% w/v). After centrifugation at 30,000 rpm in a TLA Beckman rotor, 30 min at 4ºC, the supernatant containing the soluble and membrane-bound AChE molecules was recovered and stored at -80ºC until use. AChE activity in supernatants was measured by the Ellman method [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. AChE was assayed with 1mM acetylthiocholine and 100µM iso-OMPA (an inhibitor of BChE). One unit (U) of AChE activity represents the hydrolysis of 1 µmol of substrate per minute at 37ºC. The hydrolysis of substrate due to unspecific esterases was neligible.

The amount of protein was determined by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with bovine serum albumin (BSA) as the standard.

Sedimentation analysis

The molecular distribution of AChE in both bronchial cells and fluids was studied by sedimentation analysis in sucrose continuous gradients as reported before [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF]. Briefly, samples and sedimentation markers (bovine liver catalase and intestine alkaline phosphatase) were layered on the top of centrifuged tubes containing 5-20% sucrose gradients, in the presence of 0.5%w/v Brij 96 detergent. The gradient tubes were centrifuged at 35,000 r.p.m. in a SW41Ti Beckman rotor (Fullerton, CA, USA), 18h at 4ºC. After centrifugation, fractions of 250 µl were collected from the bottom of the tube and assayed for AChE activity and enzyme markers to obtain the sedimentation profiles.

Reverse trancriptase -polymerase chain reaction

The expression of the components of the cholinergic system, such as the enzyme that synthetizes ACh, choline acetyl-transferase (ChAT), the enzyme that hydrolyzes it (AChE) and nicotinic receptor subunits (nAChR) was studied by RT-PCR. For this purpose, mRNA was extracted from cultured human tumour cells using the Chemagic mRNA Direct Kit (Chemagen), and reversed transcribed into cDNA by random priming (GeneAmp RNA PCR Kit, Applied Biosystems). Subsequently, 2-5 μL of cDNA was amplified by 40 cycles of PCR (10 seconds to 95ºC, 10 seconds at 55-60ºC, 15 seconds at 72ºC) at a LightCycler Instrument (Roche) using the primer pairs listed in Table 1.

Negative controls (without reverse transcriptase) for each primer pair were assayed. The primer pairs employed to detect the mRNA of the cholinergic components are listed in Table I, where the strategy (intron-spanning/intron-flanking) employed is indicated. βactin-mRNA was used as an internal control. The PCR products were visualized by electrophoresis on 1.5% agarose gels.

Western blotting

AChE subunits from both ASF and bronchial cells were resolved by reductive SDS-polyacrylamide gel electrophoresis [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF], carried out in 12.5% polyacrilamide-gel slabs. Proteins were electro-transferred to PVDF membranes, blocked with 5% non-fat dried milk and incubated with the N19 anti-AChE antisera (Santa Cruz). Since N19 antibodies are produced against the N-terminal peptide of human AChE, it should react with the full set of AChE variants. Negative control samples (without primary antibody N-19) lacked of staining bands and verified that protein bands correspond with proteins specifically recognized by the primary antibody. Labelled proteins were revealed with anti-goat IgG horseradish peroxidase -conjugated antibodies and the ECL Western detection system (GE Healthcare UK). Blots were exposed to Hyperfilm-ECL (Amersham Biosciences UK Ltd) for various periods of time. The size of the AChE subunits was estimated in reference to appropriate protein standards (ECL DualVue markers from GE Healthcare), and the intensity of the protein bands was quantified using GelPro Analyzer Software (version 3.1; Media Cybernetic). β-actin was used as a loading control and positive control was performed loading 1, 5 or 10 μg of recombinant AChE (Figure 2).

Statistical analysis

The results are given as a mean ± SEM. The statistical significance of the difference in ACh content and AChE activity between cancerous and non-cancerous groups was evaluated by Student's t-test. The non-parametric Mann-Witney test (signed rank test at P<0.05) was used to compare AChE between the several histological types of lung cancer. Statistical analysis was performed using the SPSS software program (version 10 for Windows; SPSS, Chicago, IL).

RESULTS

AChE activity in ASF from control and cancerous patients

Airway surface fluid contained AChE activity. The AChE activity in noncancerous samples was 1.34 ± 0.17 mU/mL and decreased to 1.18 ± 0.18 mU/mL in tumoral bronchial aspirates (Table II). Bronchial aspirates from patients diagnosed with SCLC, AC and SCC showed statistically significant differences in AChE activity (Table II). Bronchial aspirates from AC patients expressed 2.17 ± 044 mU/mL AChE activity (n=7), SCLC samples expressed 0.91 ± 0.24 μU/mL (n=10) and SCC samples dropped to about 0.51 ± 0.24 μU/mL (n=10). We have previously reported that AChE activity varied according to the histological classification of lung tumours [START_REF] Martinez-Moreno | Acetylcholinesterase biogenesis is impaired in lung cancer tissues[END_REF]. In accordance with these previous data, AChE activity in ASF from SCC dropped greatly. No significant differences were observed in the AChE activity of ASF from patients with a smoking habit, COPD, bronchitis or pneumonia.

AChE molecules in bronchial aspirates

The pattern of molecular forms of AChE in both soluble and cellular fractions of bronchial aspirates was investigated by sedimentation analysis (Figure 1). Abundant 4.4 ± 0.2 S (Svedberg units) and less 5.9 ± 0.2 S and 2.8 ± 0.1 S AChE forms were identified (Figure 1). These molecular forms corresponded to dimeric (G 2 A ; 4.5 S) and monomeric (G 1 A ; 2.8 S) amphiphilic AChE molecules, while 5.9 S molecules were assigned to hydrophilic dimers of AChE (G 2 H ). All the samples included in the study gave a similar pattern of AChE forms, indicating that whatever the pulmonary diseases are they do not affect the biosynthesis and/or secretion of AChE molecules.

Sedimentation analysis of cellular extracts from cultured cancer cells of epithelial origin

such as lung (A549, H157, N417 and H69), breast (MCF-7) or colon (Caco-2) showed almost identical AChE molecular composition. Tetrameric globular forms (G4) were only detected in small cell lung cancer cells and their contribution was always scarce (Figure 1B).

Detection of AChE proteins in bronchial aspirates by western blotting

We have previously reported that lung cancer tissues express AChE molecules capable of hydrolysing ACh and also molecules with the same apparent molecular weight but that are not catalytically active [START_REF] Williams | Muscarinic signaling in carcinoma cells[END_REF]. To gain an insight into the differences in the size as well as the content of inactive AChE molecules, we decided to assay an immunoblot of both soluble and cellular fractions of bronchial aspirates. For this, samples were subjected to reductive SDS-PAGE, transferred to PVDF membranes and then incubated with antibodies able to detect all AChE molecules. Abundant 50 kDa and less 70 kDa subunits were recognized (Figure 2). As in lung tissues [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF], no correlation was found between the AChE units loaded and the labelling intensity of bands demonstrating the presence of inactive AChE molecules in ASF. The potential use of the following ratio: "AChE units/intensity of labelling" (i. e., the relative content of inactive AChE) as a diagnostic tool is under investigation.

mRNA expression of cholinergic components in the lung.

The role of the autocrine growth factor in lung cancer growth has been wellestablished [START_REF] Song | Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma[END_REF]. Analysis of human lung cancer cell lines by real time PCR shows the expression of mRNA of the enzyme responsible for (a) the synthesis of ACh (CHAT);

(b) nicotinic receptor subunits (nAChR); and (c) hydrolysing enzymes (AChE and BChE) (Figure 3). All cell lines analysed expressed ChAT, AChE and BChE needed for the synthesis and degradation of ACh. The Beta4 nicotinic subunit was present in all lung cell lines, except in H157, H187, N417. SK-MEL-28 but not MCF7 expressed beta4. Meanwhile, alpha7 and alpha3 nicotinic subunit mRNA was detected in all cell lines. The expression of mRNA coding for the cholinergic components did not indicate any specific pattern either associated with the tissue origin or to the histology of the tumours from which the cell lines are derived.

DISCUSSION

This study presents data showing that human lung epithelium expresses the components for establishing a physiologically active cholinergic system, possibly needed for a correct pulmonary function and involved in cancer growth. This study shows that: (1) ASF contains enough AChE activity to control the effective levels of the neurotransmitter and/or its degradation product choline; (2) AChE activity varies according to the histological type of lung tumours being the highest in ASF from lung adenocarcinomas and the lowest in squamous cell lung carcinomas; (3) The pattern of molecular forms of AChE do not change with cancer; (4) As in lung tissues, inactive AChE molecules are present in bronchial aspirates, increasing their content in cancerous samples and showing histological-type differences; and (5) both small-and non-smallcell lung cancer cells express genes needed for a cholinergic autocrine or paracrine loop.

It has been clearly demonstrated that nicotine and other tobacco components

increased the proliferation of non-cancerous and cancerous [6;18;33;34] human lung cell lines, at the same time as acetylcholine or carbachol also modulated the proliferation of lung cells [6;35]. By using agonist or antagonist of nicotinic and muscarinic receptors the growth of lung cells, and other cell lines, is affected (for review see refs [15;36;37]. It becomes evident that cholinergic signalling can modulate cellular phenotype and function. Further, it appears likely that nicotine plays its pathological role through its direct interaction with cholinergic receptors [18;20;37].

The recent studies elucidating the events implicated in these receptor-dependent responses [6;17;36-39] are important not only for tobacco consumption-associated cancer but also for lung tumours, such as adenocarcinomas that have a weaker correlation with smoking than SCC and SCLC.

According to RT-PCR, our results are consistent with others (revised in refs 1, 2) showing that lung cells express mRNAs needed for establishing functional cholinergic systems in both small-cell and non-small lung cancer cells. Nevertheless, as far as we know there is not an association between particular cholinergic gene expression patterns and lung cancer pathogenesis. A very interesting study presented by Minna et al. [START_REF] Lam | Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non-Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers[END_REF] shows that tobacco consumption could change the composition of nicotinic receptors of bronchial epithelial cells. It remains to be seen if these changes are important for the pathogenesis of lung cancers but they clearly indicate the existence of particular nAChR subunit gene expression patterns which change upon several physiological conditions.

As ACh levels could also change with other pathologies, for example in response to inflammation processes [1;41], the same pathways that are involved in nicotineassociated lung cancer progression could also play a role in the biology of lung cancer not linked to tobacco consumption.

Our results show that airway surface fluid contains an important level of AChE activity which varies depending on the histology of the lung carcinoma (Table II). AChE activity in ASF was higher than the control in AC, and lower in SCLC and SCC.

We have previously shown that surgery pieces from SCC express very less AChE activity than non-cancerous and AC samples [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF]. It is interesting that ASF from smoking-associated lung cancers such as SCC and SCLC contain less AChE activity than ASF from controls or AC. The actual role of AChE activity in cancer is controversial. Cancer promotes changes affecting both the AChE gene structure and expression [42;43] and the protein properties [13;44-47]. More recently, it has been proposed that AChE plays a role in apoptosis [START_REF] Zhang | Induction of acetylcholinesterase expression during apoptosis in various cell types[END_REF][START_REF] Park | Acetylcholinesterase plays a pivotal role in apoptosome formation[END_REF][START_REF] Deng | Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis[END_REF] while immunohistochemistry staining is inversely associated with survival in ovary cancer [START_REF] Motamed-Khorasani | Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression[END_REF]. AChE biology is a very complex process and the precise physiological role of this enzyme in non-neuronal contexts is poorly understood. AChE can be found as soluble or as membrane-bound subunits and little is known about the regulation and its physiological significance. Whether secretion of AChE molecules to epithelial lining fluid of the bronchi is a controlled process needed for the normal pulmonary function need to be further investigated. Thus, it cannot be assumed, but also it cannot be excluded, a correlation between the AChE activity in the fluid of aspirates and in the malignant epithelium cells. A correlation between AChE activity in both airway surface fluid and surgical pieces in squamous cell carcinomas (SCC) with a great reduction of the AChE activity was found, whereas no correlation between AChE activity in ASF and surgical pieces drived from adenocarcinoma (AC) was observed. We discussed in our previous work [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF] that the heterogeneous cellular origin of lung cancers included in the AC group may mask possible differences, and the same can happen with the results of AChE activity in the airway surface fluid. It is tempting to think that in AC the secretion of active AChE is up-regulated and that this phenomenon affects the tumour biology. By other hand, the differences between the AChE activity in airway surface fluid from AC and SCLC could be of diagnostic interest.

With regard to the molecular composition of AChE, sedimentation analysis revealed that ASF contains abundant amphiphilic dimers (G 2 A ) and fewer monomers (G 1 A ) (Figure 1), the same pattern as in the cellular fraction of bronchial aspirates. The similarity between the ASF AChE pattern and the expression by tumours of epithelial origin such as lung [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF], breast and metastatic lymph nodes [44;45] and colon [49] reinforces the belief that human epithelia only express light molecules of AChE as enzymatic active forms. As human serum contains a very small amount of AChE activity and great butyrylcholinesterase activity, and the opposite is found in ASF, it is also probable that ASF AChE comes from the bronchial epithelium. The pattern of AChE molecular forms from cultured cancer lung cells also shows that only small-cell lung cancer cells express AChE tetramers and this seems to be related to adhesionindependent growth but not to the histological type.

Immunoblotting results indicate that the same labelled protein bands are found in ASF (Figure 2) and cellular fraction of bronchial aspirates (not shown). It is remarkable that ASF contains inactive AChE molecules as occurs in lung tissue (50 kDa protein band) and active AChE subunits (about 70 kDa). In agreement with previous work [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF], squamous cell carcinoma showed the lowest content of active subunits (lane S, Figure 2). We have previously assigned the 50 kDa protein band to the stress-induced AChE variant (AChR-R), which is synthesized more abundantly in lung cancer tissues and especially in SCC. Interestingly, Soreq´s Group found that CREB´s basal levels were insufficient to block the AChE-R proliferative effect in AChE-R transfected glial cells and hypothesized that this could increase the risk of tumour growth in individuals exposed to anti-cholinergic agents [50]. The potential diagnostic use derived from this study and the physiological significance of cholinesterases in lung cancer warrants further investigation. According to previous data [START_REF] Martinez-Moreno | Cholinesterase activity of human lung tumours varies according to their histological classification[END_REF], the labelled bands were assigned to the synaptic AChE

Legend to Figures

Figure 1 .

 1 Figure 1. Sedimentation profiles with AChE forms in bronchial aspirates and cultured lung cells. A, AChE forms in the soluble (ASF) and the cellular (Cells) fraction of bronchial aspirates were identified by sedimentation analysis in sucrose gradients (5-20%) containing Brij-96. Sedimentation coefficients were calculated by taking internal sedimentation markers (C, catalase 11.4S; P, alkaline phosphatase 6.1S) as the reference. Figure shows a profile corresponding to a cancerous ASF and cellular fraction. No differences between non-cancerous and cancerous samples were observed.B, Enzymatic extracts from NSCLC (A549, H157), SCLC (N417, H69), breast cancer cells (MCF7) and colonic cancer cells (Caco-2) were fractionated by sucrose gradients centrifugation as before. Amphiphilic globular AChE dimers and monomers appear in all samples but globular tetramers are restricted to cells that grew unattached to the culture plate. AChE activity is expressed in arbitrary units.

Figure 2 .

 2 Figure 2. Immunoblotting of AChE protein in airway surface fluid. Proteins (50 μg) in non-tumoral (NT), adenocarcinoma (A), squamous cell carcinoma (S) and small-cell lung carcinoma (SCLC) ASF were resolved by reductive SDS-PAGE as described in material and methods. N19 antibodies recognize the complete set of AChE variants.

(

  variant T; 68 kDa) and to the read-through AChE-R (stress-associated; 50 kDa). Note the weaker labelling of the catalitically active AChE subunit (68 kDa) in squamous cell cancer (S) and the stronger labelling of the 50 kDa protein band in tumoral ASF. Right panel shows the correlation between AChE activity loaded and intensity of labelling.

Figure 3 .

 3 Figure 3. Expression of cholinergic components in cultured cancer cells. mRNA was prepared and RT-PCR performed for the given cell lines with primers shown in Table 1. "AChE T"= acetylcholinesterase variant T. "AChE H"= acetylcholinesterase variant H. "AChE R"= acetylcholinesterase variant R. "E1d"= N-terminus extended acetylcholinesterase. "BChE" = butyrylcholinesterase. "ChAT" = cholineacetyltranferase. "Alpha3" = nicotinic acetylcholine receptor α3 subunit. "alpha7" = nicotinic acetylcholine receptor α7 subunit. "Beta4" = nicotinic acetylcholine receptor β4 subunit. No products were visualized when RT-PCR was performed in the absence of reverse transcriptase (non shown)

( 1 )

 1 Intron-spanning, (2) Intron-flanking S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070393 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070393 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 1 . RT-PCR primers Gene Forward

 1

		Reverse	Lenght (bp)
	AChE variant T	5´ AACTTTGCCCGCACAGGGGA 5´ GCCTCGTCGAGCGTGTCGGT	203 (1)
	AChE variant H	5´ AACTTTGCCCGCACAGGGGA 5´ GGGAGCCTCCGAGGCGGT	201 (1)
	AChE variant R	5´ CCCCTGGACCCCTCTCGAAAC 5´ ACCTGGCGGGCTCCCACTC	315 (2)
	AChE E1d	5´ CCTGGTGACGAAAGTCCGA 5´ TCCTCCACCCAGGAGCCAGAG	247 (2)
	BChE	5´ TGTCTTTGGTTTACCTCTGGAA 5´ CACTCCCATTCTGCTTCATC	297 (2)
	Chat	5´ TAGCCAGGTGCCCACAAC 5´ ATGAGGCTTTCTTCCACAGC	177 (1)
	Alfa 7	5´ CAGTACTTCGCCAGCACCAT 5´ AGCCGATGTACAGCAGGTTC

Acknowledgments

This work was supported in part by grants from FIS (Projects 01/3025 and 02/1564), from SEPAR (Sociedad Española de Neumología y Cirugia Toracica) and MEC (Projects SAF2006-070040-C02-01 and Projects SAF2006-070040-C02-02). S. N-C has a fellowship from Ministerio de Sanidad y Consumo and FFIS (Murcia). S. N-C, J. T-L, I. T-Z, P. M-H and J. C-H belong to RTIC Cancer (Instituto de Salud Carlos III). This paper is in memoriam of Dr. Sanchez Gascón, a great man, friend and physician.

Confict of interest statement

None declared


Makrina D Savvidou
email: msavvidou@dsla.ndo.co.uk

Alexandros Sotiriadis

Christine Kaihura

Kypros H Nicolaides

Naveed Sattar

Circulating levels of adiponectin and leptin at 23-25 weeks of pregnancy in women with impaired placentation and in those with established fetal growth restriction

Keywords: adiponectin, leptin, Doppler, pre-eclampsia, fetal growth restriction, maternal

come

Introduction

Pre-eclampsia (PE) is a major cause of maternal and perinatal mortality and morbidity [START_REF] Meis | The preterm prediction study: risk factors for indicated preterm births. Maternal-Fetal Medicine Units Network of the National Institute of Child Health and Human Development[END_REF]. Although its exact pathophysiology remains elusive, the widely accepted model is that of a hypoxic placenta, which releases factor(s) in the maternal circulation that cause endothelial dysfunction, which results in hypertension and proteinuria [START_REF] Roberts | Pre-eclampsia: more than pregnancyinduced hypertension[END_REF].

Inflammatory upregulation and adipocyte lipolysis are also probably involved [START_REF] Redman | Preeclampsia: an excessive maternal inflammatory response to pregnancy[END_REF][START_REF] Pinkney | Adipose tissue as an endocrine and paracrine organ[END_REF][START_REF] Greer | Increased concentrations of cytokines interleukin-6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: a mechanism for endothelial dysfunction?[END_REF][START_REF] Chappell | A longitudinal study of biochemical variables in women at risk of preeclampsia[END_REF][START_REF] Lorentzen | Fasting serum free fatty acids and triglycerides are increased before 20 weeks of gestation in women who later develop pre-eclampsia[END_REF].

Fetal growth restriction (FGR), another major cause of perinatal morbidity and mortality, seems to share the same pathophysiology with PE. In both conditions there is histological evidence of impaired trophoblastic invasion of the uterine spiral arteries, and Doppler evidence of increased impedance to flow in the uterine arteries [START_REF] Lyall | The human placental bed revisited[END_REF][START_REF] Yu | An integrated model for the prediction of preeclampsia using maternal factors and uterine artery Doppler velocimetry in unselected low-risk women[END_REF].

Adiponectin and leptin are two adipose tissue-derived proteins with important metabolic effects. The plasma concentration of adiponectin, which is derived mainly from adipose tissue and possibly from placenta, is inversely correlated with insulin resistance and is consequently reduced in obesity and in type 2 diabetes but it is paradoxically increased in PE [START_REF] Hu | AdipoQ is a novel adiposespecific gene dysregulated in obesity[END_REF][START_REF] Lindsay | Adiponectin and development of type 2 diabetes in the Pima Indian population[END_REF][START_REF] Ramsay | Paradoxical elevation in adiponectin concentrations in women with preeclampsia[END_REF][START_REF] Naruse | Peripheral blood concentrations of adiponectin, an adipocyte-specific plasma protein, in normal pregnancy and preeclampsia[END_REF][START_REF] Haugen | Increased plasma levels of adipokines in preeclampsia: relationship to placenta and adipose tissue gene expression[END_REF][START_REF] D'anna | Adiponectin and insulin resistance in early-and late-onset pre-eclampsia[END_REF][START_REF] Caminos | Expression and regulation of adiponectin and receptor in human and rat placenta[END_REF].

Leptin, a protein regulating body weight, is produced in large amounts by adipocytes and human placental trophoblasts [START_REF] Ashworth | Placental leptin[END_REF]. Leptin gene expression in the placenta is augmented in severe PE and it has been suggested that placental hypoxia may play a role in this augmentation [START_REF] Mise | Augmented placental production of leptin in preeclampsia: possible involvement of placental hypoxia[END_REF]. Maternal leptin levels have also been reported to be elevated in women with established severe PE, and in women destined to develop the condition but not in women who subsequently delivered FGR infants [START_REF] Chappell | A longitudinal study of biochemical variables in women at risk of preeclampsia[END_REF][START_REF] Anim-Nyame | Longitudinal analysis of maternal plasma leptin concentrations during normal pregnancy and pre-eclampsia[END_REF].

The aim of the current study was to investigate whether alterations of adiponection and leptin levels predate the development of PE and fetal growth restriction (FGR) in women at increased risk of these complications, as assessed by Doppler examination of the uterine arteries during the second trimester of pregnancy.

We also sought to investigate the circulating levels of adiponection and leptin in women with established severe, early onset FGR.

Methods

Study Participants

This was a retrospective study, which included three groups of healthy women with singleton pregnancies at 23-25 weeks of gestation. They were all recruited consecutively from the Ultrasound Department of King's College Hospital, where color Doppler examination of the uterine arteries is routinely performed, as previously described [START_REF] Yu | An integrated model for the prediction of preeclampsia using maternal factors and uterine artery Doppler velocimetry in unselected low-risk women[END_REF]. Group A included 44 women with normal uterine artery Doppler waveforms and normally grown fetuses. Group B, included 49 with abnormal uterine artery Doppler waveforms (presence of early diastolic notch bilaterally) and normally grown fetuses at the time of the examination, and Group C included 15 women with estimated fetal weight <5 th centile for the gestation at the time of the examination; all of them had abnormal uterine artery Doppler waveforms, 11 of these fetuses had absent or reversed end-diastolic flow in the umbilical artery and 8 of these fetuses had reduced amniotic fluid. The fetal weight was estimated from the sonographic measurements of head circumference, abdominal circumference and femur length [START_REF] Hadlock | Sonographic estimation of fetal weight. The value of femur length in addition to head and abdomen measurements[END_REF].

Maternal age, ethnic group, smoking status, parity, body mass index (BMI) and blood pressure (BP) were recorded. BP was measured in the right arm with the subject seated using an ambulatory blood pressure monitor (SpaceLabs Medical 90207, WA, US). Three measurements were taken and averaged. The study was approved by the Local Research Ethics Committee and all women gave written informed consent.

Analysis of adiponectin and leptin

All women had a venous blood sample taken and plasma was separated by centrifugation, frozen and stored at -80°C. Plasma adiponectin concentration was determined using ELISA (R&D systems, Minneapolis, US). The intra-assay and the interassay coefficients of variability were each 7.5%. The minimal detectable concentration was 0.01µg/ml. Leptin was measured by an in-house radioimmunoassay that was validated thoroughly against the commercially available Linco assay [START_REF] Mcconway | Differences in circulating concentrations of total, free and bound leptin relate to gender and body composition in adult humans[END_REF]. In this case the intra-assay and inter-assay coefficients of variation were <7% and <10%, respectively, over the sample concentration range and the detection limit of the assay was 0.5 ng/ml.

Definition of Clinical Outcome

Information on pregnancy outcome, including gestation at delivery, birth weight (BW) and sex were obtained from examination of individual patient hospital records.

The diagnosis of PE was made according to the criteria of the International Society for the Study of Hypertension in Pregnancy. Under this classification, PE is defined as diastolic BP of at least 110 mm Hg on one occasion or diastolic BP of at least 90 mmHg on two consecutive occasions more than four hours apart, in combination with proteinuria (≥300 mg total protein in a 24-hour urine collection or, if this was not available, ≥ +2 proteinuria by dipstick on two consecutive occasions at least four hours apart) developing after 20 weeks of gestation in previously normotensive women [START_REF] Davey | The classification and definition of the hypertensive disorders of pregnancy[END_REF]. The diagnosis of FGR was based on the delivery of an infant with BW below the 5 th percentile for gestation and sex [START_REF] Gardosi | Customised antenatal growth charts[END_REF].

Statistical analysis

Normality of the distribution of continuous data was examined with the Shapiro-Wilk test. Logarithmic transformation was performed for non-normally-distributed data.

Descriptive data were expressed as mean±SD or as median (interquartile range) for normally and non-normally distributed data respectively. Comparisons between groups were performed using one-way analysis of variance followed by the Bonferroni post-hoc test, Kruskal-Wallis or Mann-Whitney as appropriate. The chisquare (χ 2 ) test was used to compare categorical variables among groups. In order to compare the levels of adiponectin and leptin in the different groups of women, adjusting for BMI, smoking and ethnic group, variables that are known determinants of both adiponectin and leptin [START_REF] Hu | AdipoQ is a novel adiposespecific gene dysregulated in obesity[END_REF][START_REF] Mcconway | Differences in circulating concentrations of total, free and bound leptin relate to gender and body composition in adult humans[END_REF][START_REF] Abbasi | The relationship between plasma adiponectin concentration and insulin resistance is altered in smokers[END_REF][START_REF] Abate | Adipose tissue metabolites and insulin resistance in nondiabetic Asian Indian men[END_REF][START_REF] Wannamethee | Plasma leptin: associations with metabolic, inflammatory and haemostatic risk factors for cardiovascular disease[END_REF], the following steps were taken: Power analysis indicated that a sample of 13 cases of PE and 44 controls would have a power over 80%, with an alpha .05 (2-tails), for the detection of a mean difference of 6.9µg/ml in the plasma adiponectin concentration and a mean difference of 11.6 ng/ml in the plasma leptin concentration between the two groups. The effect size was estimated from previous publications [START_REF] Ramsay | Paradoxical elevation in adiponectin concentrations in women with preeclampsia[END_REF][START_REF] Anim-Nyame | Longitudinal analysis of maternal plasma leptin concentrations during normal pregnancy and pre-eclampsia[END_REF].

Results

Characteristics of the study participants

In Group A (normal Doppler findings, n=44), none of the women developed PE and all of them delivered babies of appropriate size. The women in Group B (abnormal Doppler findings and normal fetal growth at presentation, n=49) were sub-classified into three groups according to the outcome of pregnancy; those with no complications (n=19, 39%), those who delivered FGR infants (n=17, 35%) and those who developed PE (n=13, 26%). In Group C (abnormal Doppler findings and FGR at presentation, n=15), three women had PE and in nine cases there was intra-uterine or early neonatal death. The demographic and clinical characteristics of the groups, obtained at study entry, are presented in Table I. Systolic and diastolic BP's were significantly higher in women who eventually developed PE and in women with established FGR. This was the case even after exclusion of the three women in Group C that developed PE (data not shown).

Levels of adiponectin and leptin

The multiple regression equations for adiponectin and leptin levels in Group A are: Adiponectin= For each patient and using the formulas above, we calculated the expected adiponectin and leptin level and the ratio of the observed to expected value. The concentrations of adiponectin and leptin (expressed as raw values and as MoM's of the unaffected Group A) in the different groups of women are shown in Table 2. All groups of women studied had similar adiponectin levels, expressed as MoM's of the unaffected Group A (overall p=0.3). Leptin concentrations, expressed as MoM's of the unaffected Group A, were higher in women with established, early onset FGR (Group C; overall p<0.001, Figure 1). Post-hoc analyses revealed that women of Group C had higher levels of leptin compared to all the other groups and subgroups. This was the case, even after exclusion of the three women in this group that developed PE.

Discussion

The study has demonstrated that maternal levels of adiponectin during the second trimester of pregnancy do not seem to predate the development of PE and/or FGR in women at risk of these complications, as assessed by Doppler examination of the uterine arteries. Furthermore, maternal adiponectin levels are not different between women with established, early onset, severe FGR and women with uncomplicated pregnancies.

We have previously shown that women who subsequently develop PE have evidence of endothelial dysfunction at 23-25 weeks, as assessed by flow-mediated dilatation of their brachial artery [START_REF] Savvidou | Endothelial dysfunction and raised plasma concentrations of asymmetric dimethylarginine in pregnant women who subsequently develop preeclampsia[END_REF]. Adiponectin may be considered as a useful marker of endothelial function as it attenuates the excessive inflammatory response in the vascular wall [START_REF] Ouchi | Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin[END_REF] and it also increases nitric oxide production by activating and increasing the expression of the endothelial nitric oxide synthase enzyme [START_REF] Hattori | Globular adiponectin upregulates nitric oxide production in vascular endothelial cells[END_REF].

In the current study, the lack of difference in the adiponectin levels between women who subsequently develop PE and/or FGR compared to those with uncomplicated pregnancies may suggest that either adiponectin does not play a fundamental role in the development of PE or that the second trimester of pregnancy is not a good time to use adiponectin as a marker of PE. Studies have demonstrated that adiponectin concentration decreases throughout normal pregnancy [START_REF] Nien | Plasma adiponectin concentrations in non-pregnant, normal and overweight pregnant women[END_REF] and that its level, in women who subsequently develop PE, is lower in the first and higher in the third trimester compared to women with uncomplicated pregnancies [START_REF] Ramsay | Paradoxical elevation in adiponectin concentrations in women with preeclampsia[END_REF][START_REF] D'anna | Plasma adiponectin concentration in early pregnancy and subsequent risk of hypertensive disorders[END_REF].

Taken together these findings indicate that in women who develop PE later on in pregnancy, the adiponectin levels in mid-pregnancy may overlap with those of the women with uncomplicated pregnancies. Consequently, despite the fact that adiponectin may play a role in the development of PE, it is unlikely to be a useful biomarker for it in mid-pregnancy. In line with this, we were not able to demonstrate a significant difference in the adiponectin concentration between women with established FGR and women with normal pregnancies. This was the case even after exclusion of the three women in this group that had PE developed.

Another finding of the current study is that maternal leptin concentrations do not seem to predate the development of PE and/or FGR. However, its levels were increased in women with established FGR.

Leptin is a peptide hormone, which was introduced as an adipocyte-derived messenger of energy metabolism [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF]. Maternal leptin concentration has been reported to be elevated in women with established, severe PE and especially in those who delivered FGR infants as well [START_REF] Mise | Augmented placental production of leptin in preeclampsia: possible involvement of placental hypoxia[END_REF][START_REF] Sagawa | Role of leptin in pregnancy--a review[END_REF]. Longitudinal studies have demonstrated that elevations of maternal leptin levels occur weeks prior to the clinical manifestation of PE [START_REF] Chappell | A longitudinal study of biochemical variables in women at risk of preeclampsia[END_REF][START_REF] Anim-Nyame | Longitudinal analysis of maternal plasma leptin concentrations during normal pregnancy and pre-eclampsia[END_REF]. However, these studies did not comprehensively adjust for potential differences in maternal weight, smoking and ethnic group, factors that are strongly linked to leptin levels [START_REF] Mcconway | Differences in circulating concentrations of total, free and bound leptin relate to gender and body composition in adult humans[END_REF][START_REF] Abate | Adipose tissue metabolites and insulin resistance in nondiabetic Asian Indian men[END_REF][START_REF] Wannamethee | Plasma leptin: associations with metabolic, inflammatory and haemostatic risk factors for cardiovascular disease[END_REF]. In the current study, we expressed the levels of adiponectin and leptin as MoM's of the unaffected groups and consequently we were able to adjust for all the potential confounders. We were unable to detect any difference in maternal leptin levels between women that subsequently developed PE and those with uncomplicated pregnancies and this is in accordance with other studies [START_REF] Tommaselli | Serum leptin levels and uterine Doppler flow velocimetry at 20 weeks' gestation as markers for the development of pre-eclampsia[END_REF].

Maternal levels of leptin in women with established, early onset FGR were twice as high as in the other groups. Usually FGR is defined as a fetus below a certain weight percentile. This definition includes fetuses with a low genetic growth potential, that do not have a placental problem, and fetuses that do not reach their growth potential due to placental insufficiency. All the FGR pregnancies (Group C) included in our study were characterised by impaired placentation. However, it has to be mentioned that others did not find elevated maternal levels of leptin in FGR pregnancies [START_REF] Laivuori | Relationships between maternal plasma leptin, placental leptin mRNA and protein in normal pregnancy, preeclampsia and intrauterine growth restriction without pre-eclampsia[END_REF]36]. The reason for this inconsistency could be the above mentioned difficulties to define FGR. Certainly, we cannot exclude that some of the FGR pregnancies would have had PE developed if they did not have their pregnancies terminated. The tendency of elevated maternal BP in FGR pregnancies when compared to controls, even after exclusion of those women who definitely developed PE, indicate that some of the FGR pregnancies were possibly close to the threshold of PE and this has to be considered in the interpretation of the data. Therefore, it could be stated that although leptin may play a role in the clinical manifestation of estsblished FGR, it is unlikely that this adipokine plays a fundamental role in the initiation of this condition and therefore it is unlikely that leptin alone would be a useful biochemical marker in screening for the development of either PE or FGR.

In summary, maternal levels of adiponectin and leptin during the second trimester of pregnancy do not seem to predate the development of PE and/or FGR and consequently it is unlikely that these biochemical markers will be useful to allow improved prediction of the above pregnancy disorders. However, elevated levels of maternal leptin, but not adiponectin, seem to characterise pregnancies complicated by severe, early onset FGR. Adiponectin (MoM's of Group A) 0.9 (0.7-1.2) 0.8 (0.5-1) 0.9 (0.7-1.1) 0.9 (0.6-0.9) 0.9 (0.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409

1 .

 1 The patients were subdivided into five groups according to the outcome of pregnancy: Group A: normal Doppler-normal outcome, Group B: abnormal Dopplernormal outcome, abnormal Doppler-development of FGR, abnormal Dopplerdevelopment of PE and Group C: abnormal Doppler-established FGR. 2. Linear regression analysis was used to determine which factors among the maternal demographic and clinical characteristics were significant predictors of the adiponectin and leptin levels in Group A.3. The distribution of adiponectin and leptin levels, expressed as multiples of the median (MoM) of Group A, were determined for all the subgroups of Group B and Group C.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409 4. Due to the small number of patients, a non-parametric test, followed by post-hoc tests, was used to compare the MoM's of adiponectin and leptin in the different groups.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409Licenced 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 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409Licenced 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 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409

Figure 1 .

 1 Figure 1. Box and whisker plot comparing leptin levels, expressed a Multiples of the Median (MoM's) of the unaffected Group A, at 23-25 weeks of gestation between the different patient groups: Group A (n=44), women with normal Doppler and normal outcome, Group B (n=49), with abnormal Doppler and either normal outcome (n=19), or subsequent development of fetal growth restriction (FGR) (n=17), or subsequent development of pre-eclampsia (PE) (n=13), and Group C (n=15) with abnormal Doppler and established FGR. Boxes represent interquartile range, where the line represents the median. Whiskers at top and bottom of the box represent the highest and lowest values. Group C had the highest leptin levels compared to all the other groups (*P<0.001). S t a g e 2 ( a ) P O S T -P R I N T

Table 1

 1 Demographic, clinical maternal and neonatal characteristics in each group of women, according to the outcome of pregnancy. Group A: women with normal Doppler examination and normal outcome, Group B: women with abnormal Doppler examination, with subgroups according to the pregnancy outcome, Group C: women with established fetal growth restriction at 23-25 weeks of gestation. All statistical comparisons were performed with Group A.

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Table 2 Levels of adiponectin and leptin, expressed as raw values and as MoM's of the unaffected Group A, in each group of women according to the outcome of pregnancy. Group A: women with normal Doppler examination and normal outcome, Group B: women with abnormal Doppler examination and three subgroups according to outcome, Group C: women with abnormal Doppler examination and established FGR at 23-25 weeks of gestation.

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  17.008-0.264 X BMI (kg/m 2 ) + (3.434 if smokers, 0 if non-smokers) +

	(1.599 if Caucasian, -0.910 if Afro-Caribbean, 0 if any other ethnic group); R 2
	=0.305, p=0.006.
	Leptin= -27.915 + 2.744 X BMI (kg/m 2 ) -(4.626 if smokers, 0 if non-smokers) -
	(15.699 if Caucasian, 18.472 if Afro-Caribbean, 0 if any other group); R 2 =0.665,
	p<0.001.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements:

The study was supported by The Fetal Medicine Foundation (UK Registered Charity number: 1037116). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 23 Jan 2008 as manuscript CS20070409 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070409 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society


Carolyn M Webb
email: c.webb@imperial.ac.uk

Christopher S Hayward

Mark J Mason

Charles D Ilsley

Peter Collins

Coronary vasomotor and blood flow responses to isoflavone-intact soy protein in subjects with coronary heart disease or risk factors for coronary heart disease

Keywords: Soy, isoflavones, coronary circulation, cardiovascular disease, humans

Coronary vasomotor and blood flow responses to isoflavone-intact soy protein in subjects with coronary heart disease or risk factors for coronary heart disease

INTRODUCTION

Soy beans, the source of most soy protein consumed in the diet, contain soy protein and other constituents including isoflavones (sometimes termed 'phytoestrogens' due to their similar chemical structure to estradiol and weak binding affinity for estrogen receptor alpha and beta [1;2]). Evidence suggests that isoflavones have health benefits in humans against hormone dependent cancers of the breast and prostate, bone sparing effects, and some favourable effects on cardiovascular disease risk factors such as cholesterol concentrations in hypercholesterolemic subjects [START_REF] Adlercreutz | Phyto-oestrogens and cancer[END_REF][START_REF] Anderson | Meta-analysis of the effects of soy protein intake on serum lipids[END_REF][START_REF] Arjmandi | The role of phytoestrogens in the prevention and treatment of osteoporosis in ovarian hormone deficiency[END_REF][START_REF] Lissin | Phytoestrogens and cardiovascular health[END_REF]. The American Heart Association dietary guidelines recommended 'the consumption of soy protein containing isoflavones, along with other hearthealthy diet modifications' in 'high-risk populations with elevated total and low-density lipoprotein (LDL) cholesterol' [START_REF] Krauss | AHA Dietary Guidelines: revision 2000: A statement for healthcare professionals from the Nutrition Committee of the American Heart Association[END_REF], and the Food and Drug Administration approved labels on foods containing 25g/day soy protein claiming a reduction in heart disease risk [START_REF]Food labeling, health claims, soy protein, and coronary heart disease[END_REF]. However, a recent American Heart Association dietary advisory committee counsels caution with regard to these recommendations [START_REF] Sacks | Soy protein, isoflavones, and cardiovascular health. An American Heart Association science advisory for professionals from the nutritional committee[END_REF].

Our group has demonstrated a relaxing effect of the isoflavone genistein in precontracted coronary arteries [START_REF] Figtree | Plant derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. S t a g e 2 ( a ) P O S T -P R I N T J[END_REF]. In vivo animal studies indicate a beneficial effect of soy protein with intact isoflavones on coronary reactivity in female but not male atherosclerotic monkeys, and indicate an endothelium-mediated effect [START_REF] Adams | Soy protein containing isoflavones reduces the size of atherosclerotic plaques without affecting coronary artery reactivity in adult male monkeys[END_REF][START_REF] Honore | Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques[END_REF][START_REF] Williams | Dietary soy isoflavones inhibit in-vivo constrictor responses of coronary arteries to collagen-induced platelet activation[END_REF]. Investigation of the vascular effects of soy protein and isoflavones in humans have largely been confined to the peripheral circulation, mostly in healthy postmenopausal women [START_REF] Squadrito | The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women[END_REF][START_REF] Squadrito | Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study[END_REF][START_REF] Teede | Dietary soy has both beneficial and potentially adverse cardiovascular effects: a placebocontrolled study in men and postmenopausal women[END_REF][START_REF] Teede | Isoflavones reduce arterial stiffness: a placebo-controlled study in men and postmenopausal women[END_REF]. These studies show both positive and negative results using non-standard soy protein or isoflavone preparations and doses. In humans with risk factors for coronary heart disease (CHD) the data are also unclear. One uncontrolled study in hypercholesterolemic non-smoking men showed increases in baseline brachial artery diameter and reactive hyperaemia after 6 weeks of low-fat diet and soy protein substitution [START_REF] Yildirir | Soy protein diet significantly S t a g e 2 ( a ) P O S T -P R I N T improves endothelial function and lipid parameters[END_REF]. In contrast, a cross-over study in postmenopausal hypercholesterolemic women showed no effect of 25 g soy protein compared with placebo for 6 weeks on lipid profile or brachial artery reactivity [START_REF] Blum | Effects of soy protein on endotheliumdependent vasodilatation and lipid profile in postmenopausal women with mild hypercholesterolemia[END_REF], and a recent study in hypertensive subjects showed no effect of isoflavone-intact soy protein on arterial function compared with placebo [START_REF] Teede | Randomised, controlled, cross-over trial of soy protein with isoflavones on blood pressure and arterial function in hypertensive subjects[END_REF].

There are very few published studies investigating the effects of soy protein, with or without intact isoflavones, on the coronary circulation of humans [START_REF] Pepine | Phytoestrogens and coronary microvascular function in women with suspected myocardial ischemia: a report from the Women's Ischemia Syndrome Evaluation (WISE) Study[END_REF]. We therefore investigated the effects of short-term isoflavone-intact soy protein ingestion on basal coronary artery tone and stimulated coronary vasoreactivity and blood flow in patients with CHD or risk factors for CHD undergoing clinically-indicated coronary angiography.

Study design

Subjects were randomised in a double-blind parallel design to twice daily isoflavone-intact soy protein drinks (SUPRO ® SOY, The Solae Company, PO Box 88940, St Louis, Missouri, USA)

or isoflavone-free placebo drinks in addition to an unrestricted diet for 5 days before coronary angiography with the last drink consumed approximately 5 hours before cardiac catheterisation.

Blinding was achieved by having the drinks flavoured the same, labelled blindly, and randomised and dispensed by our pharmacy department. Cardiac medication and caffeinecontaining beverages were withheld for at least 24 hours prior to cardiac catheterisation.

Firstly, a 10 ml blood sample was taken from the in situ femoral artery sheath for evaluation of plasma isoflavone and lipid concentrations. All subjects then underwent clinicallyindicated coronary angiography and these images were used to measure basal coronary artery mean luminal diameter (method described later). Subjects with risk factors for CHD and with at least one non-obstructed major coronary artery proceeded immediately to the intracoronary substudy.

Intracoronary protocol. Following full heparinisation, a 0.014" Doppler wire (Cardiometrics ® Inc, Mountain View, California) was positioned in the proximal portion of an unobstructed coronary artery from which continuous traces of average peak blood flow velocity were recorded. Arterial pressure, heart rate (HR) and electrocardiogram were displayed continuously.

Baseline measurements were made then adenosine (30 µg) was given as a bolus intracoronary infusion. This was followed by infusions of increasing concentrations of intracoronary acetylcholine (ACh; 10 -7 -10 -5 M) for 2 minutes each or until peak velocity response. The study protocol was completed with an intracoronary bolus of 300 µg isosorbide dinitrate (ISDN).

Coronary angiograms were performed at baseline, then at peak response to each vasoactive substance. A rest period of at least 1 minute was observed between each infusion to allow all S t a g e 2 ( a ) P O S T -P R I N T measured parameters to return to baseline.

Quantitative coronary angiography and calculation of flow

Coronary angiograms were acquired digitally using a real-time digital image acquisition system (Siemens AG, Berlin and Munich, Germany) and analysed off-line using quantitative coronary angiography (QCA; MEDIS, Leiden, The Netherlands). Basal luminal diameter of the entire coronary artery (mean luminal diameter) was measured for all subjects. For the intracoronary protocol angiograms mean luminal diameter and luminal diameter approximately 4 mm distal to the tip of the Doppler wire were measured. The latter measurements were used to quantify volume flow as described previously [START_REF] Webb | Effects of testosterone on coronary vasomotor regulation in men with coronary artery disease[END_REF].

Calculation of CFR and coronary resistance

In the intracoronary protocol, adenosine was infused to induce maximal hyperaemic response.

Coronary flow reserve (CFR) was then calculated as the quotient of maximal coronary blood flow/baseline coronary blood flow. Coronary resistance was calculated as the quotient of mean arterial pressure (MAP, mmHg) and coronary blood flow (ml/min)).

Soy preparation

The isolated soy protein drink (SUPRO ® SOY) was prepared using water-washing to retain the isoflavones. One 65g powdered beverage or one 316g ready-to-drink beverage was consumed twice daily approximately 12 hours apart. Each SUPRO ® SOY drink (active) provided 25.8g soy protein containing 71mg isoflavones (aglycone weight). The placebo (control) was made with milk protein isolate and provided 25.7g milk protein with no isoflavones. The mucosal lining of the gut is necessary for the activation of the isoflavones in this form, which takes approximately 3-5 hours. Each beverage (active or control) typically contained 210-240 calories, 1-2 g fat (0g saturated fat), 24-30g carbohydrate (21-26g sugars), 26 g protein (25g from soy or milk), 170-260mg sodium, 480-800mg potassium and 900mg calcium.

Blood analysis methods

Isoflavones genistein, daidzein and equol were isolated and measured from serum using liquidchromatographic tandem mass spectrometric bioanalytical assays (HFL Ltd, Fordham, Cambridgeshire, UK). Total cholesterol, high density lipoprotein (HDL)-cholesterol and triglyceride concentrations were measured using a Beckman CX7 analyser. LDL-cholesterol was estimated with the formula of Friedewald.

Statistics

Sample size

As there were no previous studies of isoflavones in the human coronary circulation on which to base our calculations, the sample size calculation for the basal epicardial coronary artery tone endpoint was based on a previous study of 17β-estradiol published by our laboratory [START_REF] Webb | 17β-Estradiol decreases endothelin-1 levels in the coronary circulation of postmenopausal women with coronary artery disease[END_REF],

estimating a 12% difference in basal luminal diameter with SD of 15% at 80% power and 5% significance level, giving an estimate of 38 subjects in each group.

The sample size calculation for the intracoronary study was determined by the treatment response of coronary luminal diameter to ACh, using in vivo animal data to estimate the difference between groups [START_REF] Honore | Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques[END_REF]. A mean difference of 12% with SD of 7% required 13 subjects in each group, with assumptions of a 5% significance level and 80% power to detect a difference between groups.

Statistical methods

Outcome measures that were normally distributed were compared between groups using a twosample t-test and are presented as mean±SD. The Mann-Whitney test was used to compare between groups when outcomes were not normally distributed and are presented as median (interquartile range). p<0.05 was considered significant.

RESULTS

Subject characteristics

Seventy-one subjects were consented and randomised (Table 1). Thirty-three subjects did not have an coronary artery suitable for the intracoronary protocol (for example due to the extent of coronary atherosclerosis or difficulty placing the coronary catheter in a stable position), 4 did not comply with the study treatment regimen, 1 had severe chest pain on intracoronary contrast injections at diagnostic angiography, 1 had pre-procedural ST-segment changes, 1 had pre-procedural syncope, 2 had taken anti-anginal medication prior to angiography, and 4 had exclusion criteria that were unknown at randomisation.

The remaining 25 subjects proceeded to the intracoronary protocol (Table 2). Thirteen received the active drink and of these 2 subjects had no significant coronary stenosis, 9 had 1vessel disease (diameter stenosis ≥70% in a major epicardial coronary artery) and 2 had 2-vessel disease. In the active group the left anterior descending coronary artery was studied in 1 subject, the left circumflex in 2 subjects and the right coronary artery in 10 subjects. Twelve subjects received placebo and of these 2 subjects had unobstructed coronary arteries, 7 had 1-vessel disease and 3 had 2-vessel disease. In the placebo group the left circumflex coronary artery was 2).

Serum isoflavone concentrations

Genistein (median (95% CI); 340 (247,515) vs 3.9 (1.

Lipid profile

There was no difference in lipid profile after treatment in either the main study group (active vs placebo; total cholesterol 4.5±0.9 vs 5.1±1 mmol/l, p=0.06; HDL-cholesterol 1.2±0.4 vs 1.2±0.4 mmol/l, p=0.99; LDL-cholesterol 2.7±1.1 vs 3±0.9 mmol/l, p=0.17

Luminal diameter

There was no difference in basal mean luminal diameter between treatment groups (2.9±0.7 vs 2.73±0.44 mm, active vs control respectively; p=0.31).

Intracoronary protocol

Luminal diameter

Mean luminal diameter responses to adenosine, ACh (10 -7 -10 -5 M) and ISDN were significantly increased in both treatment groups but there was no difference in response between groups (Figure 1).

Velocity

Blood flow velocity responses significantly increased after each infusion in both treatment groups except after ACh 10 -7 M in the active group (Figure 1). There was no significant difference between groups, but a trend towards a significantly lower peak velocity response to ACh 10 -7 M (p=0.07) and ACh 10 -5 M (p=0.09) in subjects taking the active drinks was shown.

Volume flow

Volume blood flow increased significantly compared to baseline after adenosine, ACh 10 -6 and 10 -5 M and ISDN in both treatment groups (Figure 1), but there was no difference between groups.

CFR and resistance

There was a trend towards a lower coronary vascular resistance in response to adenosine (0.94±0.33 vs 1.24±0.

Systemic haemodynamics

Baseline systolic blood pressure and diastolic blood pressure were not different between groups (146±18 vs 158±20 mmHg, p=0.59 and 79±7 vs 76±5 mmHg, p=0.25; active vs placebo). There was no difference in MAP (100±9 vs 97±11 mmHg, p=0.37; active vs placebo) and HR (69±11 vs 67±9 bpm, p=0.73; active vs placebo) at baseline, and these measurements did not change significantly throughout the procedure.

DISCUSSION

This is the first report to describe invasive in vivo coronary vasomotor and blood flow effects of soy-derived isoflavone consumption in humans. We found that isoflavone-intact soy protein and placebo-treated subjects with CHD or risk factors for CHD had similar basal coronary luminal diameter, and comparable vasodilator and blood flow responses to adenosine, ACh and ISDN. Until now intracoronary studies of soy protein and isoflavones have been confined to animals, and studies in humans investigating vascular function after isoflavone-intact soy protein ingestion have largely been limited to the peripheral circulation, namely the brachial artery. A strength of our study is the direct investigation of coronary vascular effects of isoflavone-intact soy protein in humans. Our results complement those of van der Schouw and colleagues who found no effect of higher dietary intake of phytoestrogens on cardiovascular disease risk [START_REF] Van Der Schouw | Prospective study on usual dietary phytoestrogen intake and cardiovascular disease risk in Western women[END_REF]. Indeed recent evidence has cast uncertainty on the purported benefits of soy and isoflavone supplementation on cardiovascular health [START_REF] Sacks | Soy protein, isoflavones, and cardiovascular health. An American Heart Association science advisory for professionals from the nutritional committee[END_REF].

Effect of variations in isoflavone preparation and ingestion

Vascular responses to isoflavones vary depending on preparation used (isoflavone-intact soy protein vs isolated isoflavones for example), dose of isoflavones, duration of exposure, and existence of risk factors for CVD vs healthy subjects. SUPRO ® SOY was a commerciallyavailable preparation containing isoflavone-intact soy protein at levels recommended for lipid lowering at the time our study was conceived [7;8]. We favoured this supplement over intracoronary infusions of isoflavones as the combination of soy-protein together with isoflavones had been shown to result in enhanced lipid effects compared to isoflavones alone (summarised in Sacks and colleagues [START_REF] Sacks | Soy protein, isoflavones, and cardiovascular health. An American Heart Association science advisory for professionals from the nutritional committee[END_REF]) and in practice therefore isoflavone-intact soy protein would be the supplement of choice. We chose a relatively short treatment duration as pilot data showed that serum isoflavone levels were significantly raised after taking this supplement for 4 days, and that relaxation of precontracted isolated coronary arteries exposed to phytoestrogens occurred within a short time period [START_REF] Figtree | Plant derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. S t a g e 2 ( a ) P O S T -P R I N T J[END_REF]. It is possible that longer-term treatment may have different effects on the human coronary circulation however primate coronary data would argue against this [START_REF] Adams | Soy protein containing isoflavones reduces the size of atherosclerotic plaques without affecting coronary artery reactivity in adult male monkeys[END_REF][START_REF] Honore | Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques[END_REF][START_REF] Williams | Dietary soy isoflavones inhibit in-vivo constrictor responses of coronary arteries to collagen-induced platelet activation[END_REF]. In the peripheral circulation of healthy postmenopausal women there does appear to be an effect of treatment duration but clear interpretation of a duration effect is complicated by use of non-uniform treatments (isolated isoflavones vs red clover isoflavones vs isoflavone-intact soy protein) [START_REF] Squadrito | The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women[END_REF][START_REF] Squadrito | Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study[END_REF][START_REF] Teede | Dietary soy has both beneficial and potentially adverse cardiovascular effects: a placebocontrolled study in men and postmenopausal women[END_REF][START_REF] Teede | Isoflavones reduce arterial stiffness: a placebo-controlled study in men and postmenopausal women[END_REF]. Our study population had a clinical indication for undergoing coronary angiography, to investigate chest pain, and all subjects had at least one risk factor for CHD. Whether a similar lack of effect on coronary artery tone and stimulated reactivity would be demonstrated in healthy subjects ingesting isoflavone-intact soy protein is unknown, however data from the peripheral circulation would suggest that there may be a differing effect of isoflavone-intact soy protein on the coronary circulation of healthy subjects [START_REF] Squadrito | The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women[END_REF][START_REF] Squadrito | Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study[END_REF][START_REF] Teede | Dietary soy has both beneficial and potentially adverse cardiovascular effects: a placebocontrolled study in men and postmenopausal women[END_REF][START_REF] Teede | Isoflavones reduce arterial stiffness: a placebo-controlled study in men and postmenopausal women[END_REF].

Soy isoflavones and coronary endothelial function

A normally-functioning endothelium is vital for the maintenance of vascular health. The age-related decline in endothelial function predisposes to vascular diseases however evidence suggests that diet assists in the preservation of endothelial function [START_REF] Brown | Dietary modulation of endothelial function: implications for cardiovascular disease[END_REF]. We observed no effect of isoflavone-intact soy protein consumption on stimulated coronary endothelium-derived nitric oxide however. Results of previous coronary studies, using longer-term soy-isoflavone treatment in primates, suggest that endothelium-dependent responses in the coronary circulation may be gender-related [START_REF] Adams | Soy protein containing isoflavones reduces the size of atherosclerotic plaques without affecting coronary artery reactivity in adult male monkeys[END_REF][START_REF] Honore | Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques[END_REF][START_REF] Williams | Dietary soy isoflavones inhibit in-vivo constrictor responses of coronary arteries to collagen-induced platelet activation[END_REF] although inhibition of atheroma progression appears unrelated to gender [START_REF] Adams | Soy protein containing isoflavones reduces the size of atherosclerotic plaques without affecting coronary artery reactivity in adult male monkeys[END_REF]. A recent observational study found that higher genistein levels in women referred for coronary angiography are associated with reduced coronary endothelium-dependent andindependent coronary vascular function [START_REF] Pepine | Phytoestrogens and coronary microvascular function in women with suspected myocardial ischemia: a report from the Women's Ischemia Syndrome Evaluation (WISE) Study[END_REF]. Our study did not include a sufficient number of women to perform a gender-specific analysis so we cannot exclude this as a possible reason for the null result on endothelial function.

Isoflavone concentrations

Dietary isoflavones such as genistein and daidzein are metabolised by intestinal bacteria, conjugated in the liver, circulated in the plasma and excreted in the urine [START_REF] Cassidy | Potential risks and benefits of phytoestrogen-rich diets[END_REF].

Daidzein is further converted to equol in approximately one-third of adults depending on gut microflora or/and intestinal conditions [START_REF] Setchell | Bioavailability of pure isoflavones in S t a g e 2 ( a ) P O S T -P R I N T[END_REF]. Equol has higher estrogenic potency than daidzein, with higher binding affinity for estrogen receptors and lower binding to sexhormone binding globulin and albumin [28;29]. Serum concentrations of genistein measured in our study are similar to those reported in a previous pilot study using the same preparation (1.6 µM vs 2.1 µM respectively) [START_REF] Figtree | Plant derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. S t a g e 2 ( a ) P O S T -P R I N T J[END_REF]. In the present study only 2 subjects receiving the isoflavone-intact soy protein drinks produced significant amounts of serum equol, fewer than the previous report of one-third of the general population [START_REF] Setchell | Bioavailability of pure isoflavones in S t a g e 2 ( a ) P O S T -P R I N T[END_REF]. Investigation of coronary vasoreactivity and blood flow responses in active daidzein-equol metabolisers may further clarify the role of soy protein products in the coronary circulation.

Limitations

We acknowledge that pre-treatment data would have helped the interpretation of our data and this is a flaw in our study design. Our protocol was of parallel design as performing cardiac catheterisation before and after treatment raised ethical issues relating to risk/benefit of the additional procedure. We did not control the general diet of subjects enrolled in the study, however measurement of serum isoflavone concentrations confirmed that the subjects in the placebo group did not consume significant amounts of isoflavone-intact soy protein. We feel that consumption of a supplemental isoflavone-intact soy protein drink in addition to an unrestricted diet better mimics the way in which isoflavones would generally be consumed in the diet and therefore enhances the application of our results. We also felt that consuming a supplement would provide more pertinent information than giving isolated isoflavones via intracoronary infusion as we have done in previous intracoronary studies [22;23]. We enrolled 12 and not the required 13 patients into the placebo group meaning that the intracoronary study is slightly underpowered. Whether our results would be replicated in a larger and longerterm study, possibly using non-invasive measures of coronary blood flow is an area for further investigation.

Conclusions

In our study, short-term ingestion of isoflavones together with soy protein does not affect coronary vascular tone or stimulated vasoreactivity and blood flow compared to isoflavone-free placebo. There was no effect on ACh-stimulated coronary luminal diameter and blood flow indicating no effect on endothelium-derived nitric oxide. Our results suggest that consumption of isoflavone-intact soy protein is neither harmful nor beneficial to the coronary circulation of humans with risk factors for, or established CHD. A recent nutritional advisory committee has placed caution on past statements by National organisations asserting that consumption of plant sterols is recommended to combat diseases such as CHD [START_REF] Sacks | Soy protein, isoflavones, and cardiovascular health. An American Heart Association science advisory for professionals from the nutritional committee[END_REF], and our results provide further physiological evidence to support this advice.

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society METHODS Subjects Subjects aged 30-75 years with clinical indications for coronary angiography, and on the waiting list for the procedure at the Royal Brompton and Harefield hospitals were invited to participate in the study. Female subjects were postmenopausal (follicular stimulating hormone >40 IU/L) and had not taken hormone replacement therapy during the previous 6 months. Subjects with significant bradycardia, asthma, left ventricular hypertrophy or dysfunction, primary valvar heart disease or known 3-vessel coronary disease were excluded. The study complied with the Declaration of Helsinki, the protocol was approved by the local research ethics committee and all subjects gave written informed consent to the full study.

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society studied in 2 subjects and in the right coronary artery in 10 subjects. There was no significant difference between groups in extent of coronary atherosclerosis (p=0.81), artery studied (p=0.62), risk factors for CHD, anthropometric measurements or cardiovascular medications (Table

; triglycerides 1 .

 1 8±1.5 vs 1.8±1 mmol/l, p=0.98) or the intracoronary protocol group (active vs placebo; total cholesterol 5.1±0.9 vs 4.5±1.2 mmol/l, p=0.19; HDL-cholesterol 1.3±0.3 vs 1.2±0.5 mmol/l, p=0.34; LDL-cholesterol 3.2±0.8 vs 2.7±1.4 mmol/l, p=0.29; triglycerides 1.5±0.8 vs 2±2 mmol/l, p=0.44).

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Figure 1

 1 Figure 1 Coronary artery change in mean luminal diameter, blood flow velocity and volume blood flow after infusions of adenosine (AD), ACh 10 -7 -10 -5 M (A7, A6, A5) and ISDN in subjects taking isoflavone-intact soy protein (black) or placebo (grey). ** p≤0.01, ***p≤0.001 Active vs respective baseline; † p≤0.05, † † p≤0.01, † † † p≤0.001 control vs respective baseline. There was no significant difference in response between the two groups. S t a g e 2 ( a ) P O S T -P R I N T

  .03) concentrations were greater in the active vs control groups. Two subjects who consumed the isoflavone-intact soy protein drink produced significantly greater equol concentrations than the rest of the active group (282.2 and 171.3 ng/mL compared to a mean concentration of 1.2 ng/mL for the rest of the active group). There was no dietary explanation (vegetarianism, herbal supplements) for this variation.

	7, 5.8) ng/mL, p<0.001), daidzein (168
	(108, 360) vs 1.2 (0.5, 2) ng/mL, p<0.001) and equol (0.88 (0.72, 2.41) vs 0.59 (0.35, 0.76)
	ng/mL, p=0

Table 1

 1 Subject characteristicsValues are mean±SD or number. M, male; F, female; MI, myocardial infarction; BMI, body mass index. Clinical Science Immediate Publication. Published on 28 Apr 2008 as manuscript CS20070443 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070443Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	Characteristic	Active	Placebo	p
		(n=33)	(n=38)
	Age (years)	58±8	61±8	0.051
	Gender (M/F)	31/2	31/7	0.12
	Previous MI	10	10	0.69
	Hypercholesterolaemia	21	26	0.51
	Hypertension	18	18	0.7
	Diabetes mellitus	4	4	0.83
	Cigarette smoking status			0.69
	current	4	6
	ex	19	17
	never	10	6
	unknown	0	2
	CHD			0.81
	Unobstructed	8	6
	1 vessel disease	9	10
	2 vessel disease	9	13
	3 vessel disease	7	9
	Height (cm)	175±6	174±9	0.62
	Weight (kg)	90±15	85±14	0.3
	BMI (kg/m 2 )	29±4	28±3	0.46

S t a g e 2 ( a ) P O S T -P R I N T © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 2 Subject characteristics intracoronary flow protocol

 2 Values are mean±SD or number. M, male; F, female; MI, myocardial infarction; BMI, body mass index; ACE, angiotensin converting enzyme; K, potassium; ISMN, isosorbide mononitrate.

	Characteristic	Active	Placebo	p
		(n=13)	(n=12)
	Age (years)	58±6	59±7	0.66
	Gender (M/F)	13/0	9/3	0.13
	Previous MI	1	3	0.11
	Hypercholesterolaemia	6	5	0.85
	Hypertension	6	6	0.54
	Diabetes mellitus	3	1	0.32
	Cigarette smoking status			0.34
	current	3	2
	ex	8	5
	never	2	5
	Height (cm)	175±7	174±7	0.9
	Weight (kg)	90±15	84±12	0.2
	BMI (kg/m 2 )	29±4	28±4	0.4
	Cardiovascular medications
	ACE inhibitor	2	3	0.57
	Statin	6	5	0.83
	β-blocker	6	8	0.32
	Calcium antagonist	7	4	0.32
	K channel activator	1	3	0.26
	ISMN	5	4	0.8

ACKNOWLEDGEMENTS

We gratefully acknowledge the subjects who participated in this study, the staff in the day-case unit and the cardiac catheterisation laboratories of the Royal Brompton and Harefield Hospitals. Financial support was provided by The Solae Company, St. Louis, MO. Carolyn Webb was supported by the British Heart Foundation and is currently funded by the Victor Phillip Dahdaleh Charitable Foundation, UK. Christopher Hayward was supported by an Overseas Research Fellowship from the National Heart Foundation of Australia.