In 2001 the National Cholesterol Education Program (NCEP) provided a categorical definition for metabolic syndrome (c-MetS). We studied the extent to which two ethnic groups, Blacks and Whites were affected by c-MetS. The groups were members of the Hypertension Genetic Epidemiology Network (HyperGEN), a part of the Family Blood Pressure Program, supported by the NHLBI. Although the c-MetS definition is of special interest in particular to the clinicians, the quantitative latent traits of the metabolic syndrome (MetS) are also important in order to gain further understanding of its etiology. In this study, quantitative evaluation of the MetS latent traits (q-MetS) was based on the statistical multivariate method factor analysis (FA).The prevalence of the c-MetS was 34% in Blacks and 39% in Whites. c-MetS showed predominance of obesity, hypertension, and dyslipidemia. Three and four factor domains were identified through FA, classified as "Obesity," "Blood pressure," "Lipids," and "Central obesity." They explained approximately 60% of the variance in the 11 original variables. Two factors classified as "Obesity" and "Central Obesity" overlapped when FA was performed without rotation. All four factors in FA with Varimax rotation were consistent between Blacks and Whites, between genders and also after excluding type 2 diabetes (T2D) participants. Fasting insulin (INS) associated mainly with obesity and lipids factors.MetS in the HyperGEN study has a compound phenotype with separate domains for obesity, blood pressure, and lipids. Obesity and its relationship to lipids and insulin is clearly the dominant factor in MetS. Linkage analysis on factor scores for components of MetS, in familial studies such as HyperGEN, can assist in understanding the genetic pathways for MetS and their interactions with the environment, as a first step in identifying the underlying pathophysiological causes of this syndrome. Metabolic and physiologic disorders for cardiovascular disease (CVD) and type 2 diabetes (T2D), including abdominal obesity, insulin resistance, hyperglycemia, dyslipidemia, and hypertension often cluster. This cluster is frequently identified as the "metabolic syndrome" (MetS). Reaven  related Different definitions and multivariate statistical approaches have been applied to characterize the increasing high-risk MetS premorbid state. Recently, special attention has received the categorical definition of metabolic syndrome (c-MetS) of the National Cholesterol Education Program Adult Treatment Panel III (NCEP) . The NCEa. The trait characterized as the categorical MetS (c-MetS) was studied according to the NCEP definition; b. The underlying (latent) traits or clusters of MetS (q-MetS) were evaluated by performing FA with and without Varimax rotation on 11 risk factors. All data were grouped by ethnicity and gender. Subgroups were created by excluding T2D participants, under the assumptions that T2D individuals may have a different pattern of glucose and insulin levels. Finally, our goal was to compare the expression of c-MetS and q-MetS in the Hypertension Genetic Epidemiology Network (HyperGEN) study.The objective of this study was to exemplify important facets of the MetS in the HyperGEN study. Two MetS aspects were assessed: For c-MetS the sample sizes varied from 2,025 observations for fasting triglycerides (TG) to 2,300 for high density lipoprotein (HDL) cholesterol in Blacks, and from 2,171 observations for TG to 2,471 for HDL in Whites. In the HyperGEN study, a high percentage of individuals have body waist (WAIST) and systolic blood pressure (SBP) / diastolic blood pressure (DBP) above the NCEP thresholds , fasting plasma glucose (GLUC), fasting insulin (INS), HDL, SBP, DBP, WAIST, and percent body fat (%BF), similar low density lipoprotein (LDL) cholesterol and waist to hip ratio (WHR), and lower TG. Kurtosis after adjustments varied from 0.11 for WHR to 0.85 for DBP in Blacks, and 0.17 for WHR to 1.43 for GLUC in Whites, which demonstrates normal distributions for the traits in study and also for the factors created by performing FA .Pearson correlations among the 11 adjusted and normally distributed variables are presented in Table FA with no rotation identified a factor (Factor 1) that was loaded mostly by central obesity, obesity risk factors and INS  method in S-PLUS (Insightful Corporation software), we found that the model p-values were significant, suggesting that additional factors may exist. However, although additional factors must exist to explain approximately 40 percent of the variance in the original variables, none of the remaining factors individually can explain more than about 5 percent of the variance. Therefore, we concluded that the quantitative structure of MetS can be described in terms of three to four factors when no rotation was performed, and four factors with Varimax rotation. One may even argue whether the fourth factor in the Varimax rotation is very meaningful. We chose to retain the "Central Obesity" factor particularly because it tends to reflect the well-known gender asymmetry Table . FA withWe tested the pattern of the factors between genders only for FA with Varimax rotation. This pattern was stable among ethnicities between genders Table . WHR on Another characteristic of the HyperGEN study was that at least two participants in each sibship had hypertension. A large proportion of the hypertensive participants have used anti-hypertensive and anti-cholesterol medications. Maison et al.  have comIt is a common belief that T2D participants may have a different expression of INS and GLUC, therefore it may influence also the factors pattern in the MetS analysis. In the present study, we found a consistency of the factors before and after removing type 2 diabetics. This finding is supported also by Hanson et al.  who studOther studies have provided similar results about the latent traits of MetS ,16,17. FOur study and several others have shown that FA is a useful method for studying the underlying traits of MetS. Nevertheless this methodology has not passed without been criticized. Lawlor et al.  reviewedIf c-MetS and q-MetS are explaining the same disorder in two different aspects in the HyperGEN study, can FA contribute to MetS gene finding? MetS is recognized as a precursor for cardiovascular disease and type 2 diabetes . There aThese analyses demonstrated that obesity and hypertension were the most important factors contributing to the MetS in the HyperGEN Study. Three to four distinct factor domains were identified depending on the FA rotation applied and decisions made. Results support the hypothesis that MetS is a compound phenotype, where obesity and its relationship to lipids and insulin are clearly the driving force of MetS. Insulin may play a connecting role between obesity and lipid domains.In genetic analysis, it is well known that categorical data, especially a complex trait such as MetS, encounter reduced power as compared to quantitative variables. Therefore, we suggest that genetic analysis should be performed on specific combinations of traits that belong to a factor. It is possible that some common genes may exist in the pathways for the factors identified. Linkage analysis investigating putative quantitative trait loci for MetS factor domains can be a first step which may help discover the underlying mechanisms, or generate new hypotheses, in finding the causes of MetS.The sample represents data from the HyperGEN network, part of the Family Blood Pressure Program, supported by the NHLBI as described by Williams et al.  and ProvA participant was classified as having T2D if (s)he had a fasting plasma glucose value ≥ 126 mg/dl, or is a current user of hypoglycemic medication or insulin that was documented at examination in the clinic, or if diabetes was reported in the HyperGEN questionnaire. Also, an age at diagnosis ≥ 40 years was required for T2D individuals .c-MetS according to the NCEP definition, was identified in participants by the simultaneous presence of 3 or more of the following conditions: WAIST > 102 cm in men, and > 88 cm in women; TG ≥ 150 mg/dl; high density lipoprotein (HDL) < 40 mg/dl in men, and < 50 mg/dl in women; systolic blood pressure (SBP) ≥ 130 mm Hg and/or diastolic blood pressure (DBP) ≥ 85 mm Hg or using antihypertensive medications; GLUC ≥ 110 mg/dl or on treatment for diabetes [2); WAIST measured at the level of the umbilicus in cm; WHR defined as waist circumference divided by hip circumference; GLUC in mg/dl; INS in μU/ml (where fasting time was defined as ≥ 12 hours before blood draw); LDL in mg/dl; HDL in mg/dl; TG in mg/dl; Sitting SBP in mm Hg; DBP in mm Hg ; %BF derived from the bioelectric impedance measurements based on the Lukaski formula [Factor analysis was founded on 11 variables: BMI expressed as the ratio of body weight divided by body height squared (in kg/m formula .2) and the squared transformation of %BF (%BF2) reduced the excess kurtosis considerably. The procedure transreg in SAS (version 9 for PC) was employed for finding power transformations.TG and INS had skewed distributions. A relatively skewed distribution was also present for HDL. Log transformation brought these variables distributions to approximately normal. GLUC and %BF were highly kurtotic. Using Box-Cox transformation, it was found that the inverse of the squared transformation of GLUC . Any variables with outliers beyond ± 4 standard deviations (SD) were also adjusted for heteroscedasticity of the variance. After the adjustments for each variable, outliers beyond ± 4 SD were eliminated. Each final adjusted variable was standardized to a mean 0 and variance 1.There were two field centers recruiting Blacks and four field centers recruiting Whites. Accordingly, dummy  field center variables, one for Blacks and three for Whites, were created. All 11 risk factors were adjusted within ethnicity and gender for age, agea priori information about the structure underlying the variables. FA can be used also for a confirmatory analysis, when validation (or refutation) of a postulated structure is sought. In either case, FA seeks parsimony by summarizing a large group of interrelated variables (risk factors for a complex disease such as MetS) in terms of a small number of latent factors, thereby reducing the dimensionality. Theoretical statistical descriptions of FA can be found in the literature [factanal function, in which the MLE was employed. FA evaluated latent factors underlying the 11 original variables.Prevalence of c-MetS was estimated with the FREQ procedure of SAS. The multivariate method of factor analysis was employed for reducing a group of risk factors to a subset of latent factors. The primary goal of FA is to identify the interrelationships among a set of variables. In this study FA was used for exploratory analysis, because there was no terature -26. FA wFA was performed with and without the Varimax rotation. "No rotation" achieves the simplest latent factor structure, in the extreme case loading any variable in one of the factors and almost negligible loadings in the rest of the factors. That is the reason why some studies (extracting factors with no rotation) find a concentration of the major variables' loading on the first factor. This is also the reason why some investigators named the first factor in their studies as the "Metabolic syndrome" factor ,27. ConvMetS, metabolic syndrome; c-MetS, categorical MetS; q-MetS, latent traits of MetS; FA, Factor analysis; MLE, maximum likelihood estimate; T2D, type 2 diabetes; CVD, cardiovascular disease; BMI, body mass index; INS, fasting insulin; GLUC, fasting glucose; WHR, waist to hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure; TG, fasting triglycerides; LDL, low density lipoprotein cholesterol; HDL, high density lipoprotein cholesterol; %BF, percent body fat.The author(s) declare that they have no competing interests.All authors were equally involved in designing the MetS study, evaluating statistics, interpreting the data, writing the manuscript, and organizing the figure and tables.Table 4. This table contains information on factor loadings result of FA with and without rotation performed on 11 risk factorsClick here for file


The lipoprotein lipase (LPL) hydrolyses circulating triacylglycerol-rich lipoproteins. Thereby, LPL acts as a metabolic gate-keeper for fatty acids partitioning between adipose tissue for storage and skeletal muscle primarily for energy use. Transgenic mice that markedly over-express LPL exclusively in muscle, show increases not only in LPL activity, but also in oxidative enzyme activities and in number of mitochondria, together with an impaired glucose tolerance. However, the role of LPL in intracellular nutrient pathways remains uncertain. To examine differences in muscle nutrient uptake and fatty acid oxidative pattern, transgenic rabbits harboring a DNA fragment of the human LPL gene (hLPL) and their wild-type littermates were compared for two muscles of different metabolic type, and for perirenal fat.ex vivo using [1-14C]oleate as substrate, was lower in hLPL rabbits than in wild-type rabbits for the two muscles under study. Both insulin-sensitive glucose transporter GLUT4 and muscle fatty acid binding protein (H-FABP) contents were higher in hLPL rabbits than in wild-type littermates for the pure oxidative semimembranosus proprius muscle, but differences between groups did not reach significance when considering the fast-twitch glycolytic longissimus muscle. Variations in both glucose uptake potential, intra-cytoplasmic binding of fatty acids, and lipid oxidation rate observed in hLPL rabbits compared with their wild-type littermates, were not followed by any modifications in tissue lipid content, body fat, and plasma levels in energy-yielding metabolites.Analyses of skeletal muscles and adipose tissue showed the expression of the hLPL DNA fragment in tissues of the hLPL group only. Unexpectedly, the activity level of LPL in both tissues was similar in the two groups. Nevertheless, mitochondrial fatty acid oxidation rate, measured Expression of intracellular binding proteins for both fatty acids and glucose, and their following oxidation rates in skeletal muscles of hLPL rabbits were not fully consistent with the physiology rules. The modifications observed in muscle metabolic properties might not be directly associated with any LPL-linked pathways, but resulted likely of transgene random insertion into rabbit organism close to any regulatory genes. Our findings enlighten the risks for undesirable phenotypic modifications in micro-injected animals and difficulties of biotechnology in mammals larger than mice. The endothelial cell-associated lipoprotein lipase (LPL) works to break down triacylglycerol-rich dietary fats absorbed after a meal, thus generating free fatty acids transported in the blood. Earlier works suggested that LPL acts as a metabolic gate-keeper for fatty acid partitioning between adipose tissue for storage and muscle primarily for energy use . Then, vAltogether, metabolic studies in muscles of transgenic animals help to understand the biological links between fatty acid uptake, intracellular lipid metabolism, and some metabolic disorders such as diabetes in human beings. However, most data have been established in mice. Potential advantages of rabbit compared with mouse as human disease model ,16 relatsemimembranosus proprius and longissimus muscles of hLPL group, proving the expression of the transgene in tissues of the rabbit organism. On the contrary, no signal was detected in the same tissues of wild-type animals  was 36% higher (P = 0.07) in hLPL rabbits than in wild-type animals for semimembranosus proprius muscle, but it did not vary in other sites under study (table P < 0.05) in semimembranosus proprius and longissimus muscles, respectively (figure Muscle content in H-FABP (responsible for cytoplasmic binding of fatty acids in muscle cells), was 30% higher in y figure , in hLPLDespite a clear evidence for expression of the hLPL transgene in the tissues under study in the hLPL rabbits only, total LPL (human +native) activity in skeletal muscles or perirenal adipose tissue was similar in hLPL rabbits and in their wild-type littermates. This situation contrasts with the moderately enhanced LPL activity in post-heparin plasma reported in another line of hLPL transgenic rabbits , and witsemimembranosus proprius (a muscle composed exclusively of slow-twitch type I fibers), and to a lesser extent in the fast-twitch glycoytic longissimus muscle. However, unlike results in mice, our findings reported a lower muscle fatty acid oxidation rates in hLPL rabbits. Others have observed that over-expression of hLPL gene specifically in skeletal muscle of transgenic mice rather led to a dose-dependent increase in oxidative enzymes and to a proliferation of the oxidative specialized organelles  triolein has been incorporated, was used as the substrate. Liberated [3H]-free fatty acids were quantified by liquid scintillation.Lipoprotein lipase (LPL) activity was assessed after homogenization of the tissues in a buffer composed of ammonia-HCl (25 mM) pH 8.2, containing EDTA (5 mM), Triton-X-100 (8 g/l), sodium dodecyl sulfate (0.10 g/l), heparin (5000 IU/l) and peptidase inhibitors. Insoluble material was discarded by centrifugation at 20000 × escribed , rat serTissue lipid content was determined after chloroform/methanol extraction . Muscle 14C]oleic acid bound to defatted albumin in a 5:1 molar ratio was used as substrate. Oleate oxidation was measured using L-carnitine and other cofactors, in the absence  or presence  of mitochondrial inhibitors of the respiratory chain . The difference between total oxidation and peroxisomal oxidation was considered to be mitochondrial oxidation. All assays were performed in triplicates.Oxidation rate of oleic acid was determined in freshly excised muscles as described earlier for rat and bovine muscles , with miThe Kruskal-Wallis non-parametric test was used to analyze differences between groups . All data are presented as mean ± SEM.FG, JFH and PH conceived of the study, participated in its design and co-ordination. FG, SBJ and JFH carried out biochemical analyses. MD carried out pup genotyping. CV and LMH carried out micro-injection and provide transgenic breeder animals. FG, JFH and MD drafted the manuscript. All authors read and approved the final manuscript.


Lipoprotein lipase (LPL) is anchored at the vascular endothelium through interaction with heparan sulfate. It is not known how this enzyme is turned over but it has been suggested that it is slowly released into blood and then taken up and degraded in the liver. Heparin releases the enzyme into the circulating blood. Several lines of evidence indicate that this leads to accelerated flux of LPL to the liver and a temporary depletion of the enzyme in peripheral tissues.125I and injected. Already two min after injection about 33 % of the injected lipase was in the liver where it initially located along sinusoids. With time the immunostaining shifted to the hepatocytes, became granular and then faded, indicating internalization and degradation. When heparin was injected before the lipase, the initial immunostaining along sinusoids was weaker, whereas staining over Kupffer cells was enhanced. When the lipase was converted to inactive before injection, the fraction taken up in the liver increased and the lipase located mainly to the Kupffer cells.Rat livers were found to contain substantial amounts of LPL, most of which was catalytically inactive. After injection of heparin, LPL mass in liver increased for at least an hour. LPL activity also increased, but not in proportion to mass, indicating that the lipase soon lost its activity after being bound/taken up in the liver. To further study the uptake, bovine LPL was labeled with This study shows that there are heparin-insensitive binding sites for LPL on both hepatocytes and Kupffer cells. The latter may be the same sites as those that mediate uptake of inactive LPL. The results support the hypothesis that turnover of endothelial LPL occurs in part by transport to and degradation in the liver, and that this transport is accelerated after injection of heparin. Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and VLDL and thereby makes fatty acids available for cellular uptake and use in metabolic processes ,2. RelatHeparin releases LPL from its endothelial binding sites into the circulating blood. The uptake in the liver is retarded, but not abolished ,14. ThisLPL activity in rat liver was 26 ± 1 mU/g (Table To study the distribution of endogenous LPL in rat liver we used affinity-purified chicken antibodies raised against bovine LPL. These antibodies have previously been used for ELISA of LPL in rat tissues . There wLPL activity and mass in plasma increased many-fold after heparin injection Table . The higHepatic lipase (HL) was also measured in the livers Table . As expeThe pattern of immunofluorescence for LPL after heparin was similar to that before heparin, with faint staining over most cells and more intense staining over scattered cells, some of which were ED2 positive (not shown).To explore the origin of the LPL released into plasma and taken up by the liver we measured LPL activity in heart and adipose tissue before and 20 min after injection of heparin . Double staining with the ED2 antibody showed that the intensively LPL-positive cells were Kupffer cells. After 15 min the immunofluorescence had changed to a more punctuate pattern that still co-localized with Kupffer cells Figure . This inHeparin markedly changed the pattern of localization for the active lipase. The initial (two min after injection) staining along the sinusoids was much weaker than in sections from rats that had not received heparin. The staining was generally more intense at 15 min compared to at two min after injection of the lipase Figure . This isElectron microscopic autoradiography showed that when heparin was injected ten min after active LPL there was a strong reduction in the amount of LPL in the spaces of Disse and on endothelial cells, while the radioactivity found in hepatocytes and Kupffer cells remained (data not shown).Heparin had no marked effect on the distribution of the inactive LPL Figure . Most ofThis study shows that after injection of heparin, LPL activity and mass in liver increases several-fold, in concert with the hypothesis that heparin causes accelerated transport of LPL to the liver. In other parts of the body LPL is attached to HSPG ,2. Hepar125I-tyramine cellobiose and isolate the different cell types.Heparin markedly decreased the binding of LPL along the sinusoids. This presumably reflects that binding to HSPG was competed by heparin. Staining associated with Kupffer-like cells increased. This may be the same sites as those that bind inactive LPL. Another possibility is that the LPL-heparin complexes were recognized and taken up as such. There is evidence for binding and uptake of heparin by Kupffer cells . Most ofIt has been suggested that LPL and HL bind, at least in part, to the same sites in the liver ,28. It i125I-LPL to rats and found that about 10% of the lipase that had bound in the liver could be released [125I-LPL in a recirculating system for 10 min. After wash the perfusion was then continued in single pass mode with a heparin-containing medium. About 50% of the LPL that had bound in the liver reappeared in the perfusion medium within four min [125I-radioactivity in the liver observed in the present study.Earlier studies have shown that there are also heparin-sensitive sites that bind LPL in liver . Wallindreleased . Vilaró four min ,29. At lfour min ,31. Vilafour min . The enzfour min ,34. Immufour min ,35,36 anfour min ,25,35,37four min . The lipfour min ,40. The in vivo [Inactive LPL, as prepared here, was taken up in Kupffer cells. Most of the LPL in plasma is inactive ,10 and tin vivo  and on pin vivo . In thesin vivo ,42. Our 125I-labeled LPL in liver to that for asialofetuin, which is taken up by the galactose receptor [Before heparin, the ratio of LPL activity to LPL mass was about 0.2 mU/ng in liver and 0.3 mU/ng in blood Table . After hreceptor . The halSoleus muscle. Kuwajima et al perfused some of the rat hindlimb muscles  with heparin in situ and observed a large release in fasted but not in fed rats [To explore the source of LPL released into plasma and taken up by the liver we measured LPL activity in adipose tissue and heart before and after injection of heparin. In fed rats there was a large decrease of LPL activity in adipose tissue, in accord with a previous study . From thfed rats . These dLPL in plasma is bound to lipoproteins and it has been suggested that the lipase serves as a ligand for binding and uptake of lipoproteins in the liver. Chevreuil et al injected doubly labelled chylomicrons to rats shortly after heparin . The res• In the liver, the active form of LPL initially binds to sinusoidal surfaces but then transfers to and is taken up mainly in hepatocytes• An inactive form of LPL, presumably monomers, was mainly taken up in Kupffer cells• Heparin retards the uptake of active LPL in liver, but there are heparin insensitive binding sites for LPL both on hepatocytes and on Kupffer cells• Release of LPL into blood by heparin results in accelerated transport of the lipase to the liver• The observation that rat liver contained substantial amounts of LPL, most of which was inactive, is in accord with the hypothesis that one route for turnover for endothelial LPL is transport to and degradation in the liver• The observations that most of the LPL in blood is inactive, that injected inactive bovine LPL located to Kupffer cells, and that the immunostaining for endogenous LPL was more intense over Kupffer cells than over hepatocytes suggest that a substantial fraction of the transport from extrahepatic tissues occurs with LPL that has lost its activity.• The main source of LPL released into plasma and taken up by the liver in fed rats is the adipose tissue, whereas in fasted rats the main sources are heart and skeletal muscles.125I-albumin and Cr51-labeled red blood cells [Male Sprague-Dawley rats  weighing 180–220 g were used. They were kept on a standard pellet diet in a 12-hour light cycle. In order not to disturb blood circulation or the metabolic functions of the liver, we performed all experiments on unanaesthetized rats. They were killed through decapitation at the time of tissue removal. Injections were made in the tail vein. Mean liver weight for the rats was about 9 g. In some of the rats we dissected out all visible adipose tissue. The mean total weight was 14 g, including fibrous tissue removed with the subcutaneous adipose tissue. To correct values for LPL mass/activity in the liver we used an estimated figure of 3% for the amount of blood plasma remaining in the liver after exsanguination. This was based on earlier experiments with od cells ,46. The Vectashield mounting medium was from Vector Laboratories, Burlingame, CA. Tissue-Tec OCT compound was purchased from Sakura Finetek Europe BV, Zoeterwoude, The Netherlands. Microscope slides and cover slips were from Menzel – Gläser, Germany. Plasma, used in the preparation of catalytically inactive LPL, was taken from fasted rats with EDTA as anticoagulant. Heparin was obtained from Leo Pharma AB, Malmö, Sweden. The dose given was 500 IU/kg body weight.125I using the lactoperoxidase/glucose oxidase method [in vivo experiments and incubated this at different temperatures. On incubation at 37°C the LPL activity remained essentially stable for one hour. At higher temperatures the lipase became unstable. Based on these results we decided to use 45°C for gentle inactivation of LPL aimed to prevent aggregation of the enzyme. Chromatography on heparin-Sepharose of active and inactivated LPL showed, as expected [125I-labeled LPL was included in the preparation, to enable us to follow the distribution and metabolism of the injected material. Each rat received about 40 μg lipase protein, except in the electron microscopy studies where only a trace amount of the labeled lipase was injected.LPL was purified from bovine milk as previously described  and was e method . The labe method . To findexpected , that thAntibodies against bovine LPL were raised in a chicken (chicken no 225) and IgG were isolated from egg yolks as previously described . AntibodSmall pieces of liver were mounted in Tissue – Tec OCT and snap frozen in propane chilled with liquid nitrogen. The tissue pieces were then stored at -70°C until sectioning. Cryosections were fixed for 10 min in 4 % paraformaldehyde. After rinsing, the sections were blocked in 5 % goat serum for 10 min and then incubated overnight with the primary antibody. All these procedures were made at room temperature. Incubation with the secondary antibody was then for 30 min at 37°C. The sections were rinsed in 0.01 M phosphate 0.15 M NaCl at pH 7.4 and mounted in Vectashield medium . The immunostained samples were analyzed by confocal laser scanning microscopy (Leica SP2 or Nikon Eclipse E 800). To avoid potential signal crossover the two fluorophores were sequentially scanned. Data were collected with sequential laser excitation to eliminate bleed through and with confocal parameters such as pinhole size set to minimize the thickness of the optical sections. The images were digitally optimized using the Adobe Photoshop software.125I-labeled LPL was injected to rats and 10 min later, the livers were perfused with 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 10 min. After fixation the livers were washed in 0.1 M phosphate buffer, cut in small pieces, dehydrated through graded acetone solutions and embedded in Spurr resin. Postfixation with 1% osmium tetroxide was not performed due to its known effect of fading latent images in autoradiography. Ultrathin sections, 70 nm thick, were collected over Formvar-carbonated copper grids. These sections were coated with a monolayer of Ilford L4 nuclear emulsion, diluted 1:4 with distilled water, by means of a tungsten wire loop following the "loop interference" technique. After an exposure of 8 months, the silver grains were revealed with Phenidon development. Sections were stained with uranyl acetate and lead citrate and examined in a Hitachi MT 800 electron microscope at 75 kV. Quantitative analysis was performed by counting the number of silver grains per area in each experimental condition. The area analyzed was about 25 × 104 μm2.For autoradiographic studies, 3H oleic acid-labeled triolein in egg yolk phospholipids. Hepatic lipase (HL) was inhibited by incubation of the samples on ice for two h with rabbit anti-HL IgG. In the HL assay, LPL is inactivated by 1 M NaCl. Both assays were run at 25°C for 30 min. All determinations were carried out in triplicate. The activities are expressed in mU/ml plasma. One mU corresponds to 1 nmol of fatty acid released / min. All determinations were carried out in triplicates.The activities of LPL and of hepatic lipase were determined as described . In the Plasma samples were stored frozen at -70°C before the analysis. Tissue samples were rinsed in cold 0.9 % NaCl, blotted dry, weighed and then immediately frozen in liquid nitrogen in 9 volumes of buffer at pH 8.2 containing per ml: 1 mg BSA, 10 mg Triton X-100, 1 mg SDS, 5 IE heparin, and protease inhibitor Complete Mini (Roche) 1 tablet / 50 ml buffer. They were stored at -70°C and later thawed and homogenized with a Polytron homogenizer . The homogenates were centrifuged for 15 min at 3000 rpm in a Beckman Microfuge and the supernatants were used for the assays. For assay of LPL in liver and post-heparin plasma, the activity of hepatic lipase was suppressed by incubating the extract with an excess of anti-HL immunoglobulins before assay.Detergent containing extraction buffers are needed to solubilize and stabilize active and inactive forms of LPL efficiently ,50, but LPL protein mass was measured by an ELISA, using chicken antibodies for capture and the monoclonal 5D2 antibody coupled to peroxidase for detection . Bovine LRP – low density lipoprotein receptor-related protein, LPL – lipoprotein lipase, HSPG – heparan sulphate proteoglycan, HL – hepatic lipase, ELISA – enzyme-linked immunoassay, BSA – bovine serum albumin, SDS – sodium dodecyl sulphate, VLDL – very low density lipoproteinLN carried out the immunolocalization studies, C L-I carried out the electron microscopic studies, SV participated in the design of the study and supervised the electron microscopy, JG carried out the studies on wash-out of LPL from tissues after heparin, TO conceived of the study, participated in its design and drafted the manuscript. GO participated in the design of the study and coordinated the work. All authors read and approved the final manuscript.


Increases in urokinase-like plasminogen activator (uPA) activity are reported to be amongst the earliest events occurring in remnant liver following partial hepatectomy in rats, and have been proposed as a key component of the regenerative response. Remodelling of the extracellular matrix, conversion of single chain hepatocyte growth factor to the active two-chain form and a possible activation of a mitogenic signalling pathway have all been ascribed to the increased uPA activity. The present study aimed to determine whether similar early increases in uPA activity could be detected in the remnant liver following resection of metastatic tumours in surgical patients.Eighteen patients undergoing partial hepatectomy for the removal of hepatic metastases secondary to primary colonic tumours were studied. Increased plasminogen activator activity was found in the final liver samples for the group of patients in whom the resection size was at least 50%. For smaller resections, the increased activity was not observed. The increased activity did not correlate with the age of the patient or with the time between the start of resection and the end of the operation. There was, however, a negative correlation between plasminogen activator activity and the time for which blood supply to the liver was clamped.Our findings are in accordance with those from experimental animal models and show, for the first time, that rapid increases in plasminogen activator activity can occur following similarly large liver resection in humans. Thus, increases in plasminogen activator activity are an early event in the remnant liver following major liver resection in man. Our observations provide support for the contention that increases in plasminogen activators play a key role in the initiation of hepatic regeneration in man. In vitro uPA and tissue-like plasminogen activator (tPA) have been shown to convert single chain inactive HGF into the active two chain form -thymidine into DNA and mitotic index were reduced by almost half at 44 h post-hepatectomy (the peak time for control mice), suggesting a slower hepatocyte growth response  are alsHowever, clear differences between species do exist; a notable example being the differences in the time at which DNA synthesis peaks in the remnant liver. In rats, this is at about 24 h; in mice, at about 40 h; and in man, at 180–200 h following hepatectomy. In the case of the human studies, this may partially reflect the relatively greater age of the patients since the rate of regeneration slows with age. Such age related effects are less likely in the rodent studies where the timing of hepatocyte entry into DNA synthesis following partial hepatectomy has been shown to be an intrinsic, cell-autonomous, feature . Thus, aThe unique sensitivity of the human hepatocyte to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)  likewiseThe vast majority of the literature concerns regeneration in rats and mice and much less information is available from human studies since the opportunity to study liver regeneration in humans is generally limited to units specialising in liver surgery and is necessarily constrained by ethical considerations. Surgical removal of liver metastases affords the opportunity to obtain small samples of liver at the start, time of resection and time of wound closure approximating to the early sampling times in the animal studies. In this vein, the aim of the present study was to determine whether very early increases in uPA activity occur in the remnant liver following resection in man.p = 0.7), nor there was any difference between the values for male patients  and female patients  with a non-significant unpaired Student's t test (p = 0.61).The basal uPA activity associated with the membrane preparations showed a wide patient to patient variation ranging from 4 to 24 nmol/min/mg protein with a mean of 9.94 nmol/min/mg protein . This variation in basal activity correlated neither with the age of the patient – linear regression analysis gave a slope of -0.03 and correlation analysis gave a Spearman coefficient of -0.097 .Standard operative procedures were followed. The liver was mobilised and the resection delineated with diathermy. The portal inflow was clamped while resection with an ultrasonic dissector was carried out. Typically, the portal inflow was released every 15 minutes for 5 minutes intervals to prevent ischaemic damage and the total clamping time was recorded. Resection margins were sent separately for histopathology. The magnitude of the resection was estimated as percentage of the total liver volume, by the surgeon.The following samples were taken from tumour-free regions of the liver during the operation. A sample was obtained before the resection was started (this was labelled 'Start'). Immediately following resection samples were taken from the remnant liver (labelled 'Rem 0' for remnant liver at time 0) and from the resected liver as far away from the tumour as possible (labelled 'Res 0' for resected liver at time 0). The samples were placed in cryovials and immediately frozen in liquid nitrogen in the operating theatre. A second sample of the resected liver (labelled 'Res end') was kept at room temperature until the end of the operative procedure and was only transferred to liquid nitrogen when the final sample from the remnant liver was taken. The final sample ('Rem end' for remnant end) was taken from the remnant liver as late into the operation as possible and frozen immediately. The 'Res end' sample was also frozen at this time. The interval between the time of sampling 'Rem 0' and 'Rem end' ranged from 7 to 90 minutes. The median was 20.5 minutes and 15 of the 18 intervals were between 10 and 33 minutes. Samples were stored in liquid nitrogen in the laboratory and only thawed immediately prior to analysis.Casein, plasminogen, uPA (high and low molecular weight forms) and tPA were purchased from Calbiochem . The fluorometric substrates 7-amino-4-methylcoumarin (AMC), Z-Gly-Gly-Arg-AMC and EGR-CMK (Glu-Gly-Arg-Chloromethylketone) were from Bachem Ltd. (UK). Electrophoresis reagents were from BioRad and Geneflow Ltd. Other reagents were from Sigma-Aldrich Co Ltd. .Liver samples were homogenised in a ten-fold volume of homogenisation buffer: 250 mM sucrose / 10 mM MOPS pH 7.4 containing the protease inhibitors E-64 (20 μM), Pepstatin A (20 μM), and EDTA (0.2 mM). Inhibitors against the serine proteases, which include uPA, were not included.A membrane preparation was made by differential centrifugation of the homogenate in a TLS 55 swinging bucket rotor in a Beckman TL-100 bench top ultracentrifuge . The homogenate was initially centrifuged at 40,000 g, for 20 minutes, to pellet large cell organelles such as nuclei and mitochondria. After centrifugation the fat at the top of each tube was removed with a piece of tissue and the supernatants transferred to clean tubes and recentrifuged at 105,000 g, for 1 hour. The membranous pellets were then washed twice by resuspending in homogenisation buffer and recentrifuged at 105,000 g, for 1 hour. All centrifugations were carried out at 4°C.The protein content of the homogenates and membrane preparations was determined by the BCA (bicinchoninic acid) method  using a uPA activity was determined by a fluorimetric continuous rate assay of Z-Gly-Gly-Arg-AMC hydrolysis using a Perkin Elmer LS50B fluorimeter linked to an IBM compatible computer running the FLUSYS software . CleavagAt the end of the assay, EGR-CMK (Glu-Gly-Arg-Chloromethylketone), a chemical inhibitor of uPA, was added to check that this compound inhibited the measured activity. Any activity still persisting was taken as not uPA-mediated and subtracted from the rate measured in the absence of EGR-CMK.Since the biologically relevant fraction of uPA is generally considered to be associated with its receptor uPAR and therefore localised to the cell membrane, uPA activity measurements were performed with washed membrane preparations rather than with total liver homogenates. Preliminary experiments demonstrated the necessary linear response between the measured activity and the volume of membrane preparation assayed (data not shown).All samples were assayed in triplicate at two sample volumes to ensure linearity of activity with amount of extract. The measured rates were then adjusted for the protein concentration of each sample to give a rate in nmoles/min/mg of protein.et al. [Zymography was carried out with 7.5% SDS PAGE two-substrate gels essentially as described by Bryson et al. . The cont tests, simple linear regression, Spearman correlations) were performed using the software in the cited package.All Figures were generated and analysed with the GraphPad Prism package (version 3.0). Statistical analyses (Student's DM initiated the study, carried out the zymography experiments, prepared tissue extracts and drafted the manuscript. KS prepared tissue extracts and carried out the fluorometric assays. AWM and NCB participated in the design and coordination of the study. All authors have read and approved the final manuscript.


Lipoprotein lipase (LPL) has a central role in the catabolism of triglyceride-rich lipoproteins. The enzyme is anchored to the vascular endothelium through interaction with heparan sulphate proteoglycans and is displaced from this interaction by heparin. When heparin is infused, there is a peak of LPL activity accompanied by a reduction in triglycerides (TG) during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increase towards and beyond baseline. This suggests that tissue stores of LPL become depleted. It has been argued that low molecular weight (LMW) heparins cause less disturbance of the LPL system than conventional heparin does.We have followed LPL activity and TG during a dialysis-session with a LMW heparin  using the same patients and regime as in a previous study with conventional heparin, i.e. a primed infusion.The shape of the curve for LPL activity resembled that during the earlier dialyses with conventional heparin, but the values were lower during dialysis with dalteparin. The area under the curve for LPL activity during the peak period (0–180 minutes) was only 27% and for the plateau period (180–240 minutes) it was only 36% of that observed with conventional heparin (p < 0.01). These remarkably low plasma LPL activities prompted us to re-analyze LPL activity and to measure LPL mass in frozen samples from our earlier studies. There was excellent correlation between the new and old values which rules out the possibility of assay variations as a confounding factor. TG increased from 2.14 mmol/L before, to 2.59 mmol/L after the dialysis (p < 0.01). From 30 minutes on, the TG values were significantly higher after dalteparin compared to conventional heparin (p < 0.05).These results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does. Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) in circulating lipoproteins ,2. This et al. found that on a weight basis, decasaccharides released more LPL from perfused rat hearts than conventional heparin did -oleic acid-labelled triolein, 100 mg soybean TG and 10 mg egg yolk phospholipids per mL, prepared by Fresenius-Kabi, Uppsala, Sweden. Hepatic lipase was inhibited by pre-incubation of the plasma samples with immunoglobulins from a rabbit antiserum to human hepatic lipase. The assay medium contained a relatively high concentration of heparin, and possible differences in the heparin concentration or type in the sample would not affect the activity. All assays were made in triplicate and the mean value was used. A standard sample of human post-heparin plasma was run on each assay day and the value was used to calibrate for between-assay-variations. LPL protein mass was determined with an enzyme-linked immunosorbent assay, as previously described [®, Chromogenix AB, Mölndal, Sweden).Blood samples for measurement of LPL activity were collected in heparinized tubes. They were immediately chilled in ice water and centrifuged in a cooling centrifuge within 15 minutes. The plasma was frozen at -20°C and then stored at -70°C until analyses. LPL activity was measured as described  using anescribed , using iescribed . AntifacThe values are expressed in terms of median and range and were examined for significant differences by paired Wilcoxon signed-rank test. Simple linear regression and the Spearman rank correlation test were used to evaluate relationships between variables. Two-tailed P values below 0.05 were considered to be statistically significant.Table During the dialysis with dalteparin, the anti-Factor Xa activity was between 0.52 and 0.87 IU/mL. The target value recommended by the manufacturer is 0.5–1.0 IU/mL. Hence, the plasma dalteparin concentration remained well within the range for effective anticoagulation throughout the dialysis-session.The LPL-activity rose rapidly when dalteparin was administered. The highest value was at 15 minutes, median 15 mU/mL (range 9–32). The activity remained high at 30 minutes, but then decreased so that at 120 minutes the median was only 9 mU/mL (range 5–15)  at the start, to 2.59 mmol/L (range 1.49–5.04) at the end of the dialysis represents a 21 % increase (p < 0.01). Compared to the values during the earlier dialysis with conventional heparin, there was no statistically significant difference at the start of dialysis, but from 30 minutes and through the remaining session TG values were significantly higher during the dialysis with dalteparin (p < 0.05)  at start, to 6.1 mmol/L (range 3.8–8.4) at the end . This differs from the earlier dialysis with conventional heparin, when total cholesterol did not change from baseline .HDL-cholesterol did not change from baseline. Again, this differs from the dialysis with conventional heparin when HDL-cholesterol increased from 1.09 mmol/L (range 0.67–2.06) at start to 1.19 mmol/L (range 0.67–2.31) at the end (p < 0.05). No correlation was found between LPL activity and total or HDL-cholesterol changes during any of the dialysis-sessions.The median LDL-cholesterol, calculated by the Friedewald formula, was 2.9 mmol/L (range 2.1–4.9) before the dialysis and increased to 3.75 (2.1–5.6) at 240 min. This corresponds to an increase of 29% (p < 0.05).This study shows the same pattern of plasma LPL activity in HD patients given dalteparin as observed in previous studies with control subjects given dalteparin  or conveet al. found that the early LPL activity was only about half as high compared to values observed after conventional heparin [There are several earlier studies that show that administration of LMW heparin results in lower plasma levels of LPL than conventional heparin -30. LMW  heparin ,29. This heparin .et al. have, however, found that the amount of LPL released by a bolus of heparin is restored within 24 hours [The LPL activities were lower in HD patients than observed in controls given dalteparin . A simil24 hours . In rats24 hours  and chyl24 hours . A more 24 hours . Yet ano24 hours ,35.et al. found that the clearance of injected radioactively labeled chylomicron TG was dramatically increased five minutes after rats had been given conventional heparin or LMW heparin [Administration of heparin causes a temporary derangement of lipoprotein metabolism. In our studies with control subjects given conventional heparin or dalteparin the TG concentration decreased after heparin and then gradually increased again so that at the end of the study period TG exceeded the baseline level  declare that they have no competing interests.BN participated in the design of the study, carried out the patient studies, assembled the data, did the statistical analyses, and participated in writing of the manuscript; BS and GO conceived of the study and coordinated the work. TO participated in the design of the study and in writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process.Recent studies 125I-labeled LPL added to the medium was degraded to acid soluble products, indicating that the degradation occurred extracellularly. Fragmentation of the labelled lipase occurred in conditioned medium and this process was virtually abolished by two MMP inhibitors.When explants of rat epididymal adipose tissue were incubated, LPL mass and activity decreased rapidly. Mass and activity within adipocytes remained constant for at least six hours, demonstrating that it was the extracellular portion of the enzyme that decreased. Adipocytes isolated from the explants after three or six hours of incubation retained their ability to secrete LPL to the medium. Addition of a cocktail of protease inhibitors to the incubation medium slowed down the decrease of LPL mass. Chloroquine was without effect, indicating that the degradation was not lysosomal. The decrease of LPL mass and activity that occurs when explants of rat adipose tissue are incubated is due to proteolysis of extracellular LPL. The adipocytes continue to produce and secrete the enzyme. The effect of inhibitors indicates, but does not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue. To study the underlying mechanism an in vitro model is urgently needed. Experiments with isolated adipocytes do not seem to bring out the mechanism and the in vivo experiments indicate that it is the extracellular LPL that is the target for the regulation [Lipoprotein lipase (LPL) hydrolyzes triglycerides in very low-density lipoproteins and chylomicrons . Tissue-gulation . We haveIn the first set of experiments we incubated explants of rat adipose tissue and followed LPL mass and activity Figure . LPL masWe tried variations in technique and several different incubation media in experiments such as those in Figure One possible explanation could be that LPL was released from the tissue into the medium. The amounts of LPL mass or activity that appeared in the medium were, however, small Table . To testAnother possibility was that the cells lost their ability to produce LPL. Isolated adipocytes, incubated under the same conditions as the tissue explants, released LPL activity Figure  and massHeparin release is often used to assess the LPL activity of tissues and releases mainly extracellular LPL . Figure These results indicated that the decline of LPL mass occurred through proteolytic cleavage of the extracellular enzyme. To test this hypothesis, we included a cocktail of protease inhibitors in the medium used for incubation of tissue explants. This slowed down the decrease of LPL mass Figure . Chloroq125I-LPL was added to the incubations. TCA soluble material appeared in the medium demonstrating that proteolytic degradation took place . Unless otherwise stated, the rats were 23 days old and weighed around 60 g. After transport to Umeå they were allowed to acclimatize for seven to ten days by which time they had reached a weight of approximately 120 g. The rats were kept in a well ventilated, temperature (21°C) and humidity (40–45%) controlled room with free access to a standard laboratory chow  and tap water. The light in the room was on between 6 a.m. and 6 p.m. In experiments where the rats were to be fasted, food was withdrawn from the cages at 6 a.m. and a grid was placed at the bottom of the cages to prevent coprophagia. The adipose depot used in all experiments was the periepididymal one. The rats were killed by decapitation. Animal experiments were approved by the animal ethics committee in Umeå.3H-labelled triolein in Intralipid (10%) kindly prepared by Pharmacia-UpJohn . Parker medium (Parker 199) was from SBL . 125 I-LPL was prepared as before [Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001  and Captopril, chloroquine and collagenase were from SIGMA . Protease inhibitor cocktail tablets "Complete Mini" were from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens . Substrate for the LPL activity assay was m Parker 9 was froLPL was extracted from tissues by homogenization in a Tris-HCL buffer (pH 8.2) containing detergents and protease inhibitors as described . The homLPL activity was measured as described previously . BrieflyLPL mass was measured with an ELISA as described . The chiDNA content was assayed using Labarca's method .2: 95 % O2 with continuous gentle shaking motion in a Cellstar Incubator . After incubation, the tissues were prepared for measurement of LPL activity and mass as described above. In some experiments samples of the medium were taken for assay of LPL activity and/or mass.Epididymal adipose tissue was dissected out from fed or 24 h fasted rats. The tissue was cut into small pieces (5 mg or less). A total of about maximal 100 mg tissue pieces were immediately put into culture plates. Each well contained 15 ml of Parker Medium 199 supplemented with 2% BSA, 0.5% casein hydrolysate, 10 mM glucose and adjusted to pH 7.4. Incubations were at 37°C and 5 % COIn some experiments adipocytes were prepared after collagenase treatment of the tissue pieces as described . To measStudent's t-test was used for analysis of the data.GW carried out the experiments and participated in their design and in writing of the manuscript, TO participated in the design of the study and drafted the manuscript. GO conceived of the study and coordinated the work. All authors read and approved the final manuscript.


The metabolic syndrome (MetS), as well as postprandial hypertriglyceridemia, is associated with coronary heart disease. This study aimed to evaluate the postprandial lipemia after oral fat tolerance test (OFTT) in subjects with MetS and compare them to hypertensive (HTN) and healthy subjects.OFTT was given to 33 men with MetS (defined by the Adult Treatment Panel III), 17 HTN and 14 healthy men. The MetS group was further divided according to fasting triglycerides (TG) into TG ≥ 150  or <150 mg/dl [MetS-TG (n = 11)], and into those with or without hypertension . TG concentrations were measured before and at 4, 6 and 8 h after OFTT and the postprandial response was quantified using the area under the curve (AUC) for TG.The postprandial response was significantly higher in MetS compared to HTN and healthy men . The TG levels were increased significantly in MetS+TG compared to MetS-TG subjects at 4 (p = 0.022), 6 (p < 0.001) and 8 hours (p < 0.001). The TG were increased significantly in MetS-TG compared to healthy subjects at 4 (p = 0.011), 6 (p = 0.001) and 8 hours (p = 0.015). In linear regression analysis only fasting TG levels were a significant predictor of the AUC .Fasting TG concentration is the main determinant of postprandial lipemia. However, an exaggeration of TG postprandialy was found in normotriglyceridemic MetS and HTN compared to healthy subjects. This suggests that intervention to lower fasting TG levels should be recommended in MetS subjects. In 1988, Reaven et al. proposed insulin resistance as the underlying metabolic aberration linking essential hypertension (HTN), dyslipidemia, type 2 diabetes and other abnormalities associated with an increased risk of atherosclerotic cardiovascular disease in adults . MetS isPostprandial hypertriglyceridemia is also associated with cardiovascular disease . During All participants ingested their fatty meal and tolerated it well. The amount of fatty meal ingested by the MetS group was 362(28) g, by the HTN was 333(23) g, and by the Healthy was 347(16) g.Clinical characteristics of main groups  are shown in Table Plasma total and HDL cholesterol, apolipoproteins A and B, and lipoprotein (a) were measured postprandialy in a 60% of the study population and no significant difference was found between groups.The following changes were noticed pre and post OFTT:A) Comparison of the TG concentrations at 0, 4, 6, 8 h in MetS, HTN and Healthy subject groups are shown in Figure B) In MetS+HTN and HTN groups: The TG levels were significantly increased in MetS+HTN vs. HTN subjects at 4 hours p = 0.022), 6 p < 0.001) and 8 hours (p < 0.001). .All participants gave their informed consent and the Ethics Committee of the Onassis Cardiac Surgery Centre, Athens, Greece, approved the study protocol.2).All patients were studied in the outpatient clinic between 8.00–9.00 am after 12 h overnight fast. The fatty meal was consumed within 20 min and plasma TG concentrations were measured before and 4, 6 and 8 h after the fat load. During this 8 h period, the participants did not eat and did not smoke. They were only allowed to drink water. Blood samples were drawn at 8:00 , at 12:30 am , at 2:30 pm , and at 4:30 pm . In all four samples total cholesterol, TG, HDL cholesterol, apolipoproteins A and B, and lipoprotein (a) were measured. The content of the fatty meal has been described in a previous study . BrieflyPlasma total cholesterol, TG and HDL cholesterol were measured using enzymatic colorimetric methods on a Roche Integra Biochemical analyzer with commercially available kits . The serum LDL cholesterol levels were calculated using the Friedewald formula  only in The body mass index was calculated as weight divided by height expressed in kg/m2. We assessed the whole-body insulin resistance with the following formula: HOMA-IR = (fast glucose × fast insulin)/22.5 .Categorical variables are presented as percentages. Values of numerical characteristics were tested for normality and are presented as mean value (± SD), if normally distributed. An ANOVA or Kruskal-Wallis H test with a Bonferroni correction (whichever appropriate) was performed for three group comparisons. The t-test for independent samples or the Mann Whitney U test was used for the comparison of numerical values between two groups. Areas under the curve (AUC) for serial measurements of TG levels at baseline and after the fatty meal were calculated using the trapezoid rule. Linear regression analysis, where the AUC was the dependent variable and age, BMI, HDL cholesterol, fasting TG, fasting glucose and HOMA-IR were the independent variables was performed in order to uncover the significant predictors of postprandial lipemia . The level of significance was set at p < 0.05.GDK conceived the study and participated in the development of the hypothesis, the study design and drafting of the manuscript. KKA is a research associate who participated in the development of the hypothesis, the study design and drafting of the manuscript. ANP is a physician who participated in the study design, recruitment of subjects and clinical evaluation. KDS is a research associate who participated in data analysis and interpretation of the findings. SAI, KT and DSD are physicians who participated in the study design, recruitment of subjects and clinical evaluation. AM is a physician who participated in the study design and drafting of the manuscript. DVC is a physician who participated in the design of the study and its coordination.


Clinical performance targets are intended to improve patient outcomes in chronic disease through quality improvement, but evidence of an association between multiple target attainment and patient outcomes in routine clinical practice is often lacking.2/dl2), dialysis dose (Kt/V≥1.2), and vascular access type (fistula). Outcomes included mortality, hospital admissions, hospital days, and hospital costs.In a national prospective cohort study , we examined whether attainment of multiple targets in 668 incident hemodialysis patients from 74 U.S. not-for-profit dialysis clinics was associated with better outcomes. We measured whether the following accepted clinical performance targets were met at 6 months after study enrollment: albumin (≥4.0 g/dl), hemoglobin (≥11 g/dl), calcium-phosphate product  = 0.55, 95% confidence interval (CI), 0.41–0.75], hospital admissions , hospital days , and hospital costs , relative to those who did not. Increasing numbers of targets attained were also associated, in a graded fashion, with decreased mortality (P = 0.030), fewer hospital admissions and days (P < 0.001 for both), and lower costs (P = 0.029); these trends remained statistically significant for all outcomes after adjustment (P < 0.001), except cost, which was marginally significant (P = 0.052).Attainment of each of the five targets was associated individually with better outcomes; Attainment of more clinical performance targets, regardless of which targets, was strongly associated with decreased mortality, hospital admissions, and resource use in hemodialysis patients. Both improvement in the quality of delivered care and its measurement are of considerable interest to healthcare providers and researchers -3. In paHigher serum albumin, a marker of better nutrition and lower inflammatory burden, has been well-established as a predictor of better survival in dialysis patients -10. LoweThese guidelines have led to improvement in the process of kidney disease care, as evidenced by improvement in clinical performance measures, and have even led to calls for financial incentives for further improvement . HoweverThe ESRD Quality (EQUAL) study was designed to measure processes of care and outcomes in the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE)  cohort. 2/dl2; dialysis dose, Kt/V≥1.2; and vascular access type, presence of functioning arteriovenous fistula. In addition to individual targets, the total number of targets attained was also used as an independent variable. For total number of targets attained, data were collapsed into four categories . The numbers of patients attaining no (n = 3) or all five targets (n = 17) were small. We also examined a subset of targets  that are regularly measured as part of the usual laboratory panels.Measures of whether patients attained clinical performance targets at 6 months after enrollment in the study served as the independent variables for this study. The clinical performance measures examined included 6-month values for albumin, hemoglobin, Ca-P product, and dialysis dose and ascertainment of the type of vascular access in use at 6 months. Albumin, hemoglobin, calcium, and phosphate levels were obtained from routine laboratory data. All serum albumin levels were measured at a central laboratory using the Bromocresol Green method . Calcium levels were corrected for albumin [corrected calcium = calcium level + 0.8*] before Ca-P products were calculated. Vascular access information was obtained on a subset of individuals through review of discharge summaries, dialysis flow sheets, and dialysis clinic progress notes . DialysiOutcome variables included all-cause mortality, number of hospitalizations, days hospitalized, and hospitalization costs. Mortality information was ascertained from clinic report, medical records, and Centers for Medicare & Medicaid Services . Follow-up continued until death, transplantation, loss to follow-up, or the last follow-up date of November 2003. Hospitalization data were obtained through the United States Renal Data System (USRDS)  and wereWe also collected data on demographic, laboratory, and clinical characteristics. Data regarding patient demographics  and socioeconomic status (education and employment) were collected from a baseline self-report questionnaire. Laboratory values and height and weight [used to calculate body mass index (BMI)] were obtained from clinic records and from the CMS Medical Evidence report (CMS Form 2728). Comorbidity was assessed at baseline using the Index of Coexistent Disease (ICED), a validated measure whose composite integer score ranges from 0 to 3 (with 3 as the highest severity level) and is a measure of both the presence and severity of comorbid conditions -32. The 2 tests for categorical variables and two-sided t tests or ANOVA for continuous variables. Observed survival time was assessed starting at time of patient enrollment and ending at death or censoring , and we assessed individual cumulative mortality by whether patients attained targets by calculating overall crude mortality rates at the average follow-up (2.8 years) and by using Kaplan-Meier methods. We used Cox proportional hazards models to assess the strength and independence of an association between target attainment and survival. The proportional hazards assumption was not violated for any of the targets or for the total number of targets attained . Crude hospitalization rates were calculated, and Poisson regression models were used to assess the relation between target attainment and hospitalization (incidence rate ratio). Linear regression was used to assess the association between attainment of targets and hospitalization (after logarithmic transformation) costs per year. For all regression models, attainment of individual targets, the subset of three laboratory targets, and all five targets were examined.We first compared patient characteristics by whether patients attained individual targets and by the total number of targets they attained using Pearson's χi.e., significantly associated with both target attainment and the outcome measure) or prior evidence of their association with the outcome. Adjustment for both baseline target values (the major confounders) and other confounders of the outcome was performed. We also used models incorporating both target attainment and propensity scores for target attainment. Propensity score modeling is an established technique used to address selection bias [P value of 0.05/5 = 0.01 for the individual target analyses. All analyses were performed using STATA v. 8.2 .Variables were considered for adjustment in the multivariable regression models based on either their demonstration to be confounders (ion bias . For muli) albumin target attainers were younger, more likely to be male, and less likely to have a fistula at the start of hemodialysis, and had lower comorbidity scores, higher baseline albumin and creatinine, and lower initial CRP; (ii) Ca-P target attainers were older and less likely to be employed or noncompliant with dialysis and had higher comorbidity scores, higher Kt/V, lower creatinine, higher hemoglobin, and lower initial Ca-P product; (iii) hemoglobin target attainers were older and more likely to be male and white and had less frequent physician visits, lower BMI, higher Kt/V, and higher initial hemoglobin; (iv) dialysis dose target attainers were older and less likely to be male and had more frequent physician visits lower BMI, lower creatinine, higher hemoglobin, lower Ca-P, and higher initial dose (Kt/V); and (v) access target attainers were younger and more likely to be male and white and had lower dose, higher albumin, lower CRP, and fewer fistulas (none) at start of dialysis, relative to those not attaining the target. For those who were at or above targets at 6 months, 26.1%, 81.2%, 23.2%, 61.5%, and 56.5% of patients were already at the albumin, Ca-P product, hemoglobin, dialysis dose, and vascular access targets, respectively, at baseline. Finally, those patients attaining a greater number of targets at 6 months (Table n = 344) at 6 months, 39% (n = 133) had attained 0 or 1 target at baseline; 41%, 16%, and 4% of these patients had attained 2, 3, and 4 or 5 targets at baseline, respectively.Relative to patients not attaining 6-month targets Table , (i) albThe crude mortality rates at the median follow-up of 2.8 years were lower in the patients who attained the albumin target and in those who attained multiple targets Table  suggesten = 558, 159, 517, 244, and 356 for albumin, Ca-P product, hemoglobin, dialysis dose, and access type, respectively), only albumin remained statistically significant as a predictor of decreased mortality (data not shown). By likelihood ratio tests in multivariable-adjusted models, albumin and hemoglobin (P < 0.001 for both) and Ca-P product (P = 0.039) contributed most to the variation in mortality. Dialysis dose (P = 0.052) and access (P = 0.806) did not significantly contribute to variation in mortality.In adjusted models, the attainment of all individual targets was associated with a decreased risk of mortality Table . These aAttaining one or more of the laboratory targets was associated with decreased risk of mortality Table . When alP = 0.091; n = 154). By likelihood ratio test, albumin, Ca-P, hemoglobin and access  all contributed significantly to the variation in hospital admission rate, whereas dialysis dose (P = 0.903) did not.All individual targets were associated with a decreased incidence of hospitalizations, with the exception of attaining the dialysis dose target, which was only statistically significantly associated with decreased hospitalizations with propensity score adjustment Table . Addition = 23), results were not significant (P = 0.435). Again, history of cardiovascular disease was significantly associated with hospital admissions, but adjusting for it did not change our results. Less frequent physician contact, less frequent sit-down rounds, and noncompliance were all associated with increased hospital admissions but did not affect our estimates of the association of targets with hospital admissions when they were added as adjusters.Each increase in number of targets attained among the laboratory targets was associated with a statistically significantly decreased incidence of hospitalization Table . There wP < 0.001 for all). By likelihood ratio test, all five individual targets  all contributed significantly to the variation in number of days hospitalized.All individual targets except dialysis dose were associated with a decreased hospital days Table , but pron = 23) still gave a significant trend (P = 0.030) toward decreased hospital days with more targets attained at 6 months.Each increase in number of targets attained among the laboratory targets was associated with decreased hospitalized days Table , and theP = 0.002) and access (P = 0.039) contributed to variation in hospital costs, but Ca-P product (P = 0.977), hemoglobin (P = 0.679), and dialysis dose (P = 0.535) did not.The attainment of the albumin target was associated with a reduction of approximately $3000 per patient-year in Medicare hospital payments Table  with mulP for trend = 0.129 for all five targets). Subpopulation analyses of those attaining 0 or 1 targets at baseline showed that hospital payments decreased with each additional 6-month target attained , but the trend was not significant (P = 0.470).Increases in the number of targets attained among the laboratory targets were generally associated with decreased payments Table . Also, tAttainment of clinical performance targets in patients requires considerable time and effort on the part of providers and patients. Resistance to work on quality improvement may in part be due to skepticism that attainment will lead to meaningful improvement in health and other outcomes ,3. This e.g., through provider education, competition from public reporting, and rewards, such as payment for performance) might reap large benefits in morbidity, mortality, and efficiency for both patients and payers.For example, we found that attainment of each additional target resulted in a mortality risk reduction of approximately 35%, a hospitalization risk reduction of approximately 20%, a reduction in number of days hospitalized of approximately 24%, and a decrease in annual Medicare hospital payments of approximately $762 per patient-year. The results support the idea that efforts to improve clinical performance  or outcomes .Although this study was observational, the associations we report do meet many of the criteria for causality, including plausibility, temporality, dose-response relationship, and consistency. It is certainly plausible that attaining clinical performance targets would improve patient outcomes, and it is just as plausible that increasing numbers of targets attained in a patient would result in incrementally better outcomes. The use of an incident dialysis cohort, capturing patients at the inception of their dialysis treatment, ensures temporality, as the exposure to provider efforts to meet clinical performance targets for ESRD patients begins with the initiation of kidney replacement therapy. We observed a dose-response relationship, reflected in the graded trend toward better outcomes with more targets attained. Also, our results were consistent across various modeling strategies and consistent with other studies focusing on single static measures  than others . Importantly, there may be fixed and possibly unmeasurable patient characteristics that might or might not allow target attainment with exposure to same quality of provider care. Such characteristics might also affect the amount of time needed to attain a goal, which may be more or less than 6 months for some patients and some targets. Finally, despite collection of and adjustment with extensive data on determinants of patient outcomes, selection bias and residual confounding due to lack of data on variables for which we could not account may still exist.Some possible limitations to this study deserve mention. First, this study was underpowered to determine which particular individual targets or combinations among the five performance targets examined yield the most benefit in terms of each outcome. Second, it should be remembered that this is an observational study and that only the associations between target and outcome can be ascertained, not cause and effect. Despite the fact that some clinical performance targets have been chosen based upon observational data and consensus , the value of observational data should not be overstated. Because some of the factors that we studied here were not the subject of national guidelines at the time we started our study, guideline-directed therapeutic intent cannot be shown. The hemoglobin, dialysis dose, and vascular access targets were in place in 1997 and have not changed since this time, although they were not yet widely implemented by 1998. The albumin and Ca-P targets used here were not introduced until well after the study period. Third, as with any study of clinical performance targets, the biological markers studied may be influenced by other factors than the quality of providers' care. For example, albumin and hemoglobin levels could be affected in part by the degree of ultrafiltration achieved. Of course, some targets may be more easily modified  declare that they have no competing interests.LP performed the statistical analyses and drafted the manuscript; NEF participated in the design of the study and helped to draft the manuscript; BGJ assisted in data interpretation and drafting of the manuscript; JHS, NWL, JC, & MJK participated in the design of the study and provided feedback on the manuscript; NRP conceived of the study, participated in its design, assisted in data interpretation, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A goal of systems biology is the quantitative modelling of biochemical networks. Yet for many biochemical systems, parameter values and even the existence of interactions between some chemical species are unknown. It is therefore important to be able to easily investigate the effects of adding or removing reactions and to easily perform a bifurcation analysis, which shows the qualitative dynamics of a model for a range of parameter values.E + S <-> C) into scripts for analytical analysis (Mathematica and Maple), for simulation (XPP and Matlab), and for bifurcation analysis (AUTO). Facile automatically identifies mass conservations and generates the reduced form of a model with the minimum number of independent variables. This form is essential for bifurcation analysis, and Facile produces a C version of the reduced model for AUTO.We present Facile, a Perl command-line tool for analysing the dynamics of a systems biology model. Facile implements the law of mass action to automatically compile a biochemical network (written as, for example, Facile is a simple, yet powerful, tool that greatly accelerates analysis of the dynamics of a biochemical network. By acting at the command-line and because of its intuitive, text-based input, Facile is quick to learn and can be incorporated into larger programs or into automated tasks. Biology is becoming a quantitative science. Driven by the introduction of the Systems Biology Markup Language , an XML-The quantification of the life sciences is also attracting to systems biology members of the mathematical biology and biophysics communities. These scientists are usually mathematically and computationally sophisticated and already have standard computational tools for investigating many questions posed by systems biology. They typically work at the command-line in a UNIX programming environment and prefer text-based rather than GUI-based methods of model entry.Here we introduce Facile, a network compiler for investigating the dynamics of biochemical networks. Facile is aimed specifically at mathematicians, engineers, and physicists working in systems biology. From a textual description, Facile compiles the biochemical network into formats for the standard computational tools used in studies of non-linear dynamics. Facile has several advantages: (1) with one command invocation, it links to all the standard analysis tools used by the mathematical biology and non-linear dynamics communities; (2) it has an intuitive, text-based input, with a format for representing chemical reactions similar to that adopted by most textbooks, making it easy to learn; (3) it is command-line oriented and therefore, like any UNIX command, can be easily incorporated into automated tasks or other software; and (4) it outputs SBML and therefore allows, for example, a mathematical biologist familiar with UNIX to use the SBML tool set with minimal effort.Biochemical networks are non-linear systems. Within non-linear dynamics, three methods have been developed to analyse non-linear behaviour: analytical approaches, simulation, and bifurcation analysis.Facile greatly aids bifurcation analysis. It compiles a version of the model from text into the C programming language to interface directly with AUTO, a standard bifurcation analysis tool . It autoIn addition, Facile converts a network model into a format for analytical analysis by computer algebra packages – Mathematica  or Maple  – and into a format for simulation – XPP  or MatlaFacile is not intended to compete with SBML, but is complementary through its command-line character and its ability to convert an intuitive, text-based form of a model into SBML. Unlike other SBML converters, such as CellDesigner , JDesignFacile is modular and coded in Perl using an object-oriented methodology with a distinct module to generate each output. A new output option or a modification of an existing option can be easily implemented.Facile uses the method proposed by Sauro and Ingalls  to automFacile's input is simple: a system of chemical reactions. Facile automatically calculates reaction rates using the law of mass action. Therefore, for example, the Michaelis-Menten reaction is specified asE + S <-> C ; f = 1.6e7; b = 6C -> P + E ; k = 0.157 M-1 s-1, and the complex, C, dissociates into the product, P, and enzyme with rate 0.15 s-1. Backward reactions can also be written explicitly, e.g.The association rate between the enzyme, E, and the substrate, S, is 1.6 × 10E + S <- C ; b = 6Sources, for example gene expression, and sinks, for example protein degradation, are denoted by null:null -> A ; s = 10A -> null ; d = 0.1-1 and degrades at a rate of 0.1 s-1.implying that protein A is produced at a rate of 10 sFacile also allows explicit specification of reaction velocities, time-varying system parameters, and effective rate expressions, such as Hill functions. A thick arrow, =>, indicates a reaction velocity and overrides the law of mass action. The reaction velocity may be a constant or an expression enclosed in double quotes. For example, the Michaelis-Menten equations could be specified asE + S < = > C; f = "1.6e7*E*S"; b = "6*C"C = > P + E; k = "0.15*C"or asS = > P; f = "S*V/(K + S)"in the quasi-steady state approximation. Variables can also be defined, such asvariable V = 0.15*0.2variable K = (0.15+6)/16μM. Once a variable has been specified, its value is available throughout the rest of the input file.for the quasi-steady state approximation with a total enzyme concentration of 0.2 Time can be included in a reaction rate by using t. For example, the expression of a gene may be periodically modulated by the cell cycle:null -> A ; s = "10*(1+cos(2*pi*t/2400))"for a cell cycle of 40 minutes or 2400 seconds.INIT keyword in the input file. Any concentration not specified is set to zero. For the Michaelis-Menten example, we haveTo run simulations, initial concentrations are also needed. Concentrations are specified beneath the INITE = 0.2 uMS = 3P is initially zero, S is initially 3 M, and E is initially 0.2 μM. The substrate could also be initialized as S = 3 M.implying that Mathematica and Maple are two standard computer algebra packages. They can be used to derive analytical expressions for steady-state concentrations, for the eigenvalues determining the stability of the steady-state, or to evaluate perturbation expansions, such as the method of multiple scales.The commandfacile.pl -M modelconverts the textual input for the Michaelis-Menten system shown earlier (and contained in the file model) into Mathematica format. Since Facile automatically applies the law of mass action, it generates the expressionsdEdt = + b C - f E S + k C ;dSdt = + b C - f E S ;dCdt = + f E S - b C - k C ;dPdt = + k C ;-L option generates an equivalent format for Maple.These expressions can be cut-and-pasted or loaded into Mathematica and then algebraically manipulated. For example, they can be solved for the quasi-steady state solution. The -m option, or XPP, via the -x option. These software packages provide tools for numerically integrating ordinary differential equations and visualizing their solutions. Two files are created for Matlab: one describing the model as a system of ordinary differential equations and another (driver) file for the user. An ode file is created for XPP.Analytical solutions are usually only possible for an approximate form of a model, if at all. Facile provides an ordinary differential equation version of the model for input to Matlab and Octave , via theNevertheless, intracellular molecular numbers are often small, and significant stochastic fluctuations can exist in biochemical networks . ThroughA bifurcation analysis is frequently used to show how the qualitative behaviour of a system changes when parameter values are altered. It is particularly important in systems biology where many parameters are unknown and where the dynamical behaviour of the systems is often expected to be robust to parameter changes .AUTO is a standard tool for numerical bifurcation analysis . Like anE or C form, and the total amount of substrate is conserved, in either the S, C, or P form. Running Facile with the -rM option, produces a Mathematica version of the model in the reduced form:Facile automatically finds any mass conservations and incorporates them into a reduced system model which is only a function of the independent variables . For example, consider again the Michaelis-Menten system. It has two mass conservations: the total amount of enzyme is conserved, in either the E = - C + E_tot ;S = - P - C + S_tot ;dCdt = + f E S - b C - k C ;dPdt = + k C ;E_tot is the total amount of enzyme and S_tot is the total amount of substrate. Facile determines the numerical value of these two constants from the initial conditions specified in the input file.where C and P could be related to E_tot and S_tot in the Michaelis-Menten example, and E and S would then be the independent variables. In the input file to Facile, the user can specify the chemical species that should be independent and those that should be dependent. Any chemical species not specified will have its dependency assigned automatically. The MOIETY section in the input file is used for these specifications. For example,Once the mass conservations have been identified, there is not a unique reduced form for the system. For example, MOIETYdependent E, Sensures that the Michaelis-Menten equations are reduced to the form above. So, too, doesMOIETYindependent C, PA combination of independent and dependent variables is also valid.Although XPPAUT does allow bifurcation analysis (and is supported by the -x option in Facile), the complex biochemical networks studied in systems biology usually require the stand-alone version of AUTO because it allows the user to tune more of AUTO's parameters. Via its -a option, Facile produces a C version of the reduced model in the format expected by AUTO. AUTO also needs to know the parameters to be varied for the bifurcation analysis. These are specified in the BIFURC_PARAM section of the Facile input file and are incorporated into the C version of the reduced model. For example, if the user wished to investigate whether the system bifurcates as the association rate, f, and dissociation rate, b, for the Michaelis-Menten reaction are varied, Facile requiresBIFURC_PARAMf, bin its input file.-A option will automatically load the values of the steady-state concentrations from a text file, generated by Matlab or XPP, and incorporate them into the C version of the model required by AUTO.As a starting point for generating the bifurcation diagram, bifurcation analysis software uses the concentrations of the chemical species in a system at an attractor of the system. Usually a starting point is found by integrating the system to steady-state. Facile with its facile.pl -rx model at the command-line to generate model.ode. This XPP file is a reduced form of the model in the form of ordinary differential equations: rates are automatically calculated by the law of mass action and any mass conservations are found and used to reduce the model to the minimum number of independent variables. We then integrate the model to a steady-state and use the XPP data browser to save the corresponding time course data to a space-delimited text file, output.dat. Alternatively, we could run XPP without an interface with the command xppaut model.ode -silent. This command runs XPP and saves the time series generated in output.dat. By running facile.pl -aA output.dat model, we create a C version of the model, which includes the steady-state concentrations taken from the last time point of output.dat. We then directly load into AUTO the model.c file created. Matlab could also be used to integrate the model equations (with the -rm option in Facile). In summary, the single-line command facile.pl -rx model; xppaut model.ode -silent; facile.pl -aA output.dat model builds both the C model expected by AUTO and an XPP simulation file.Facile greatly speeds up bifurcation analysis of biochemical models. Here we illustrate the steps that a user could follow to set up a bifurcation analysis. Facile can be used to generate both the simulation file and the C AUTO file. The simulation file is useful for finding the initial point for the bifurcation analysis and for exploring the dynamics of the model in different regions of the bifurcation diagram. As an example, we will use XPP as the integrator. We specify the model as a system of chemical reactions in the text file model. For XPP output, we run SBML export is enabled by the -S option and uses the libSBML package, which must also be installed.As mentioned, variables or parameters can be defined in the Facile input file using the variable command. The input file also has a CONFIG section where if desired the user can specify the time interval to run simulations, the particular differential equation solver to be used by Matlab, and XPP configuration commands. See the online Facile manual.Facile is a tool to quickly create models of biochemical networks and to analyse their dynamics. Although specifically aimed at systems biologists from the non-linear dynamics community, Facile should be useful to any computational biologist modelling biochemical networks. It links directly to the standard analysis tools used for non-linear systems and indirectly to many more via SBML.A major feature of Facile is its ability to generate a reduced form of a model in C for the bifurcation analysis software AUTO. Bifurcation analysis gives the global behaviour of non-linear systems for ranges of parameter values. It is particularly important in systems biology where many parameter values are unknown or are only known approximately. It is essential for bifurcation analysis that any mass conservations in a model are identified and used to write the model in a form with a minimum number of independent variables. In the Michaelis-Menten system, for example, the sum of the free enzyme and complex is equal to the total amount of enzyme. If this mass conservation is not identified, varying the initial conditions for the free enzyme and for the complex, will create a system with a different amount of total enzyme that has its own steady-state. AUTO will therefore find a line of steady-states rather than the expected single steady-state. Facile automatically identifies and uses mass conservations to produce a reduced form of any model.The input to Facile is a text file of chemical reactions written in a notation very similar to that used by most undergraduate text books. Facile automatically calculates reaction rates and generates rate equations. Exploring changes in model structure with Facile is therefore straightforward. Chemical reactions can be added to or removed from the input file, and Facile ensures that the rate equations are generated consistently.By using a text file as input and because it operates at the command-line, Facile can be run automatically and embedded into larger programs. In addition, there are several problems in systems biology where a GUI interface would not be suitable to build a model. In signal transduction, for example, the number of proteins that can both exist in different phosphorylated states and bind together in complexes rapidly leads to a large number of distinct chemical species. An example is the Ste5 scaffold protein in the yeast pheromone response pathway which is involved in 1300 different chemical species . A GUI bWritten in Perl, Facile fits naturally into the UNIX programming environment. It is an easily learned addition to a systems biologist's toolbox. [see also Additional file Facile runs in any programming environment that can support Perl, such as Linux, Mac OS X, and Windows. Facile and its manual are available and can be used online at FSN and JFO wrote Facile; PSS wrote an early version. JFO wrote the online manual. FSN, JFO, and PSS wrote the paper.Facile source code. the Perl source code for the Facile command-line tool.Click here for file