Cholesterol Ester Hydrolase Research Articles

Regulation by Adrenocorticotropic Hormone and Arachidonate of the Expression of Acyl-CoA Synthetase 4, an Arachidonate-Preferring Enzyme Expressed in Steroidogenic Tissues

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues. We demonstrate that ACS4 expression in steroidogenic tissues in vivo is induced by adrenocorticotropic hormone (ACTH) and suppressed by glucocorticoid. ACTH also induced ACS4 protein but not its mRNA in Y1 adrenocortical tumor cells, whereas both ACS4 mRNA and protein were increased by dibutyryl cAMP (db-cAMP) and forskolin. Furthermore, the levels of ACS4 mRNA and protein in Y1 cells were induced by arachidonate. These data suggest that ACS4 expression in steroidogenic cells is regulated in coordination with induced steroidogenesis and arachidonate released by cholesterol ester hydrolase.

Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL-/-) were superficially normal except that the males were sterile because of oligospermia. HSL-/- mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL-/- mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL-/- mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL-/- mice were neither obese nor cold sensitive. Furthermore, WAT from HSL-/- mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

Endpoint Colorimetric Method for Assaying Total Cholesterol in Serum with Cholesterol Dehydrogenase

Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method. Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of beta-NADH from beta-NAD(+). At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of beta-NADH from the reaction of beta-NAD(+) with cholesterol. We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1-100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0. 29-0.43% and 0.22-0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x - 0.0058 mmol/L; r = 0.997; S(y|x) = 0.117 mmol/L; n = 50. This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.

Regulation of pathways determining cholesterol availability in the baboon placenta with advancing gestation.

Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

Anti-atherogenic effect of 15-lipoxygenase overexpression in rabbit atherosclerosis models

s / Prostaglandins & other Lipid Mediators 59 (1999) 1-235 145 ANTI-ATHEROGENIC EFFECT OF 15LIPOXYGENASE OVEREXPRESSION IN RABBIT ATHEROSCLEROSIS MODELS Frank Trebus and Hartmut Kuhn Institute of Biochemistry, University Clinics Charite, Humboldt University, Hessische Str. 3-4, 10115 Berlin, F.R.G. Oxidative modification of low density lipoprotein has been implicated as patho-physiological process in early atherogenesis and there is a substantial body of experimental data supporting this hypothesis. 15-lipoxygenases (1%LOX) are lipid peroxidizing enzymes, which are capable of oxidizing LDL to an atherogenic form, and thus, these enzymes were suggested to act proatherogenic in vivo. However, we have two lines of experimental evidente suggesting an-antiatherogenic activity of 15LOXs in rabbit atherosclerosis models. i) Transgenic rabbits, which overexpress the 15-LOX specifically in monocyte/macrophages, appear to be protected from development of atherosclerotic lesions when fed a cholesterol rich diet. A similar protection was observed when these transgenic animals were crossed with LDL receptor deficient Watanabe rabbits. ii) Systemic overexpression of the 15-LOX induced by a transient experimental anemia did also protect cholesterol fed rabbits from lipid deposition in the arterial wall. For these experiments a transient experimental anemia was induced in rabbits by phenylhydracine injection. A single treatrnent was followed by a long lasting (up to 10 weeks) systemic (al1 cells and tissues) overexpression of the 15-LOX. After the animals have recovered from the anemie stress (6 days after the injection) they were put on a lipid rich Western-type diet for 10 weeks and the extent of lipid deposition was quantified in the aorta. When 15-LOX overexpressing rabbits (n=lO) were compare with age and sex-matched controls (n=7) we observed a more than 50% reduction of aortic lipid deposition in the 15LOX overexpressing animal. In contrast, the plasma cholesterol levels, the lipoprotein profile, the gain of weight during cholesterol feeding and most of the serum diagnostic parameters were not different between the two groups. As mechanistic reason for the observed anti-atherogenic effect an involvement of the 15-LOX in intracellular cholesterol ester degradation was assumed. In fact, we found that LOX-oxidized cholesterol ester are much better substrates for the neutral cholesterolester hydrolase than their non-oxidized counterparts. These data indicated that specifically oxidized cholesterol esters are more rapidly hydrolyzed providing free cholesterol, which may enter reverse cholesterol transport. Thus, intracellular oxidation of depot cholesterol esters may counteract accumulation of cytosolic cholesterol esters preventing foam cel1 formation.

Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat

Origins of hyperlipidemia and cholestasis that occur during pregnancy were investigated by examining expression of key elements related to plasma and hepatic cholesterol metabolism during pregnancy, lactation, and post-lactation in the rat model. Among major findings were: during pregnancy, the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl coenzyme A:cholesterol acyltransferase, acyl coenzyme A:diacylglycerol acyltransferase, cholesterol 7α-hydroxylase, cholesterol ester hydrolases, low density lipoprotein receptors, LRP, and mdr2 were significantly lower or similar to non-pregnant controls while SR-B1 was elevated. Once lactation began, reductase, cholesterol acyltransferase, 7α-hydroxylase activities, low density lipoprotein receptors, and mdr2 increased while SR-B1 decreased. In later stages of lactation most hepatic elements returned to near control levels. Plasma cholesterol levels were higher than control at birth and during lactation with increase in LDL-size particles. By 24 h post-lactation, plasma triglycerides were 3.7-fold higher while cholesterol remained unchanged. Very large lipoproteins were present while LDL-size particles were now absent. Hepatic cholesterol acyltransferase had decreased to 27“% of control while diacylglycerol acyltransferase increased 3-fold and low density lipoprotein receptors doubled. Most elements were normalized 3 weeks after weaning except for LRP and low density lipoprotein receptors which were elevated. These studies provide an integrated picture of expression of key elements of hepatic and plasma cholesterol metabolism during pregnancy and lactation and advance understanding of hyperlipidemia and cholestasis during these states. —Smith, J. L., S. R. Lear, T. M. Forte, W. Ko, M. Massimi, and S. K. Erickson. Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat. J. Lipid Res. 1998. 39: 2237–2249.

Characterisation of triacylglycerol hydrolase activities in human placenta

Triacylglycerol hydrolase activities were characterised in homogenates, cytosol, and microvillous membranes (MVM) of human placenta. Homogenates of placenta exhibited three distinct triacylyglycerol hydrolase activities with pH optima 4.5, 6.0 and 8.0. On further fractionation, placental cytosol exhibited both acid cholesterol ester hydrolase (pH 4.5) and hormone sensitive lipase (pH 6.0) activities, whereas purified placental MVM exhibited two distinct triacylyglycerol hydrolase activities; a minor activity at pH 8.0 and a second major activity at pH 6.0. Triacylglycerol hydrolase activity at pH 8.0 of MVM appeared to be lipoprotein lipase (consistent with criteria such as serum stimulation and salt inhibition), whereas at pH 6.0 the activity was unique in that it was almost abolished by serum, but was not affected by high NaCl concentrations. Our data, for the first time, demonstrate that human placental MVM, in addition to lipoprotein lipase, contain a newly identified triacylglycerol hydrolase activity at pH 6.0.

Increased acylcoa-cholesterol ester acyltransferase activity in gallbladder mucosa in patients with gallbladder cholesterolosis

Objective: Although cholesterolosis of the human gallbladder is a relatively common disease, its etiology has not been fully understood. The aim of this study was to determine this etiology. Methods: The lipid composition of the gallbladder mucosa and gallbladder bile and the enzyme activities (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) of the gallbladder mucosa were measured in control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease. Results: Levels of cholesterol ester in gallbladder mucosa in patients with cholesterolosis (n = 12) were higher than those in control subjects (n = 8). With regard to the lipid content in gallbladder bile, no differences were found in concentrations of cholesterol, phospholipids, and bile acids among control subjects (n = 11), patients with cholesterolosis (n = 13), and those with cholesterol gallstone disease (n = 15). In gallbladder mucosa, ACAT activity was significantly higher in patients with cholesterolosis (n = 10) than in control subjects (n = 8), whereas CEH activity did not differ between the two groups. As a result, the ACAT/CEH activity ratio was higher in patients with cholesterolosis than in control subjects. Conclusions: It would be suggested that cholesterol ester synthesis of gallbladder mucosa might play an etiological role in the development of cholesterolosis.

Increased acylcoa-cholesterol ester acyltransferase activity in gallbladder mucosa in patients with gallbladder cholesterolosis

Objective: Although cholesterolosis of the human gallbladder is a relatively common disease, its etiology has not been fully understood. The aim of this study was to determine this etiology. Methods: The lipid composition of the gallbladder mucosa and gallbladder bile and the enzyme activities (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) of the gallbladder mucosa were measured in control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease. Results: Levels of cholesterol ester in gallbladder mucosa in patients with cholesterolosis (n = 12) were higher than those in control subjects (n = 8). With regard to the lipid content in gallbladder bile, no differences were found in concentrations of cholesterol, phospholipids, and bile acids among control subjects (n = 11), patients with cholesterolosis (n = 13), and those with cholesterol gallstone disease (n = 15). In gallbladder mucosa, ACAT activity was significantly higher in patients with cholesterolosis (n = 10) than in control subjects (n = 8), whereas CEH activity did not differ between the two groups. As a result, the ACAT/CEH activity ratio was higher in patients with cholesterolosis than in control subjects. Conclusions: It would be suggested that cholesterol ester synthesis of gallbladder mucosa might play an etiological role in the development of cholesterolosis.

Hormone-sensitive lipase overexpression increases cholesteryl ester hydrolysis in macrophage foam cells.

Atherosclerosis is a complex physiopathologic process initiated by the formation of cholesterol-rich lesions in the arterial wall. Macrophages play a crucial role in this process because they accumulate large amounts of cholesterol esters (CEs) to form the foam cells that initiate the formation of the lesion and participate actively in the development of the lesion. Therefore, prevention or reversal of CE accumulation in macrophage foam cells could result in protection from multiple pathological effects. In this report, we show that the CE hydrolysis catalyzed by neutral cholesterol ester hydrolase (nCEH) can be modulated by overexpression of hormone-sensitive lipase (HSL) in macrophage foam cells. For these studies, RAW 264.7 cells, a murine macrophage cell line, were found to be a suitable model of foam cell formation. HSL expression and nCEH activity in these cells and in peritoneal macrophages were comparable. In addition, antibody titration showed that essentially all nCEH activity in murine macrophages was accounted for by HSL. To examine the effect of HSL overexpression on foam cell formation, RAW 264.7 cells were stably transfected with a rat HSL cDNA. The resulting HSL overexpression increased hydrolysis of cellular CEs 2- to 3-fold in lipid-laden cells in the presence of an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Furthermore, addition of cAMP produced a 5-fold higher rate of CE hydrolysis in cholesterol-laden, HSL-overexpressing cells than in control cells and resulted in nearly complete hydrolysis of cellular CEs in only 9 hours, compared with <50% hydrolysis in control cells. Thus, HSL overexpression stimulated the net hydrolysis of CEs, leading to faster hydrolysis of lipid deposits in model foam cells. These data suggest that HSL overexpression in macrophages, alone or in combination with ACAT inhibitors, may constitute a useful therapeutic approach for impeding CE accumulation in macrophages in vivo.

Molinate: Rodent Reproductive Toxicity and Its Relevance to Humans—A Review

Molinate is a thiocarbamate herbicide used on rice. During the evaluation of the compound for regulatory compliance, an adverse effect on male reproduction in rats was observed. This led to extensive investigations in rats, mice, rabbits, dogs, monkeys, and humans, resulting in a description of the cause of the effect and establishing an empirical case for rodent specificity. More recent investigations have also shown an effect on the ovaries in rodents. A series of investigations into the mechanism of action to support the view that the effect was specific to rodents has been concluded. The results from this investigation are drawn together and show that the mode of action is via a metabolite, the sulfoxide, which is primarily found in rodents, and that the lesion in both rodent sexes is elicited via inhibition of the enzyme neutral cholesterol ester hydrolase, resulting in interference with mobilization of cholesterol from high-density lipoprotein, a path specific to rodents. Thus, the mechanism of action is such that the effect cannot be elicited in other species including humans.

Substrate specificity of retinyl ester hydrolase activity in retinal pigment epithelium

In the eye, hydrolysis of stored retinyl esters is catalyzed by retinyl ester hydrolase (REH) activities in retinal pigment epithelium (RPE) membranes. In the present study, biochemical analyses were conducted to determine the substrate specificity of these activities. Specific activities determined for hydrolysis of various retinol isomers of retinyl palmitate (9-cis-, 11-cis-, 13-cis-, and all-trans-retinyl palmitates) indicated that 11-cis-retinyl palmitate is preferentially hydrolyzed (1.7 nmol/min/mg) compared to the other isomers (0.1–0.3 nmol/min/mg). Examination of the specificity of REH activity for 11-cis-retinyl esters of varied acyl chain length (-myristate, -palmitate, and -stearate) and degree of saturation (-oleate and -linoleate) further demonstrated that palmitate is the preferred fatty acyl moiety. Notably, retinyl esters possessing chain lengths which more closely approximate that of the palmitate ester exhibited higher rates of hydrolysis. Similar results were obtained in retinyl ester-plasma membrane fusion studies in which hydrolysis took place within the membrane domain rather than at the lipid–water interface. REH substrate specificity was further assessed in competition studies in which 11-cis-retinyl palmitate hydrolysis was monitored in the presence of 13-cis-, 9-cis-, or all-trans-retinyl palmitate. Results show that addition of these retinyl palmitate isomers does not affect the rate of hydrolysis of 11-cis-retinyl palmitate. However, the hydrolytic rates associated with other retinyl palmitate isomers were significantly reduced in the presence of 11-cis-retinyl palmitate. Finally, cholesterol ester hydrolase activity was found to be distinct from the observed 11-cis-REH activity and the presence of cholesterol oleate did not affect the rate of 11-cis-retinyl palmitate hydrolysis. Collectively, these data support the hypothesis that a distinct, membrane-associated, 11-cis-retinyl palmitate-specific retinyl ester hydrolase activity exists in the retinal pigment epithelium.—Mata, J. R., N. L. Mata, and A.T.C. Tsin. Substrate specificity of retinyl ester hydrolase activity in retinal pigment epithelium.

Molecular Cloning of the Promoter for Rat Hepatic Neutral Cholesterol Ester Hydrolase: Evidence for Transcriptional Regulation by Sterols

Neutral cholesterol ester hydrolase is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5′-untranslated region as a primer, we amplified and cloned 1.3 Kb of promoter sequence upstream of the ATG initiation codon. Analysis of the sequence revealed the presence of a consensus GC-box, which can bind the positive transcription factor Sp1, 35 bases upstream from the transcription start site. Transcriptional regulation by agents perturbing cholesterol metabolism was studied in HepG2 cells by transient transfection assays of the promoter activity in deletion constructs linked to the luciferase reporter gene. Three functional sterol response sequences were identified at positions -92, -160, and -280 of the CEH promoter. The sterol response sequence at position -92 was shown to bind SREBP-2. Therefore, the CEH gene is similar to other genes involved in regulation of cholesterol homeostasis, in that it appears to be transcriptionally regulated by sterols.

Effect of trifluoperazine on certain arterial wall lipid-metabolizing enzymes inducing atherosclerosis in rhesus monkeys.

The effect of trifluoperazine (TFP) was investigated on arterial wall lipid-metabolizing enzymes like acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol ester hydrolase (CEH) in rhesus monkeys. The activity was determined in aortic wall homogenates obtained from rhesus monkeys fed an atherogenic diet coupled with intramuscular injections of adrenaline and TFP. Although TFP had no significant effect on serum cholesterol and triglycerides, it decreased significantly the formation of atherosclerotic lesions by decreasing the esterification of cholesterol, by inhibiting ACAT and enhancing its utilization by activating CEH. Hence, the preventive effect of TFP on the development of atherosclerosis in rhesus monkeys is mediated through its ability to influence the activities of arterial wall lipid-metabolizing enzymes like ACAT and CEH.

Solubilization of a cholesterol ester hydrolase activity from rat liver microsomes.

Solubilization of a cholesterol ester hydrolase activity from rat liver microsomes.

Immunological identification of cholesterol ester hydrolase in the steroidogenic tissues, adrenal glands and testis

Monoclonal antibodies were generated against the purified pancreatic cholesterol ester hydrolase (CEH, EC 3.1.1.13) to examine the expression of CEH in various bovine tissues. The presence of CEH isozyme antigenically indistinguishable from pancreatic enzyme in the steroidogenic tissues, adrenal glands and testis has been first demonstrated here using the immunoprecipitation method. These results suggest that CEH isozyme, similar to pancreatic CEH, might be involved in the cholesterol metabolism in the steroidogenic tissue.

Age‐related changes in catalytic activity, enzyme mass, mRNA, and subcellular distribution of hepatic neutral cholesterol ester hydrolase in female rats

Activity and protein mass of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in liver cytosol and washed microsomes of female Sprague-Dawley rats aged 3, 4, 7, 9, 13, and 16 wk. CEH mRNA was also measured. The microsomal component varied with age and contributed a greater fraction of total activity in females than previously reported in males. Nevertheless, the cytosolic component accounted for 62-80% of activity and 77-94% of immunoreactive protein in postmitochondrial fractions. Cytosolic and microsomal CEH specific activities, relative to total protein, decreased 94 and 83%, respectively, from 3 to 4 wk, prior to onset of puberty at 5 wk, and increased 360 and 137%, respectively, from 12 to 16 wk. These results contrast with an earlier study, in which cytosolic CEH activity of males increased with puberty and declined after 12 wk. Although cytosolic CEH was activated by protein kinase A and inhibited by alkaline phosphatase treatment at all ages, protein kinase activation peaked at 4 wk, coinciding with the initial decrease in specific activity. Specific activity in cytosol and microsomes correlated with CEH mass at all ages, suggesting that this CEH accounts for most variation in cellular activity. In contrast, CEH mRNA varied little from 3-16 wk, indicating that transcriptional regulation does not make a major contribution to the variation in CEH activity and mass in females, although it may make an important contribution to male-female differences in CEH expression. Specific activities of cytosolic and microsomal CEH, relative to immunoreactive CEH protein mass, exhibited changes consistent with posttranslational regulation. These results indicate gender-specific multivalent regulation of hepatic CEH by posttranslational mechanisms during development of female rats.

DIAGNOSIS OF EXOCRINE PANCREATIC INSUFFICIENCY IN CHILDREN BY USE OF PANCREOLAURYL TEST

In adult studies, the urine pancreolauryl test (PLT) is used as an indirect measure of pancreatic exocrine function: the fluorescein dilaurate is cleaved by the pancreas specific cholesterol ester hydrolase and the liberated fluorescein is absorbed and excreted in the urine. Duodenal intubation and stimulation of pancreatic secretion is complicated and unpleasant in children. We present here a modified version of PLT based on plasmatic fluorescein concentration, previously validated in adult patients, in the aim to measure exocrine pancreatic insufficiency (EPI) in children. Methods: 2 capsules containing 174,5 mg of fluorescein dilaurate were ingested with a standard meal. Plasmatic fluorescein concentration was determined spectrophotometrically (492 nm) on blood samples collected through a small catheter at time 0 and every 30 mn during 6 hours. In our adult series PLT has been validated by comparison with the results of duodenal intubation after pancreatic stimulation; 58 patients have been studied: 10 healthy subjects, 7 with chronic digestive disease without pancreatic involvement, 18 patients with moderate EPI and 23 with severe EPI. Calculation of the experimental area under curve (AUC) in mg/ml-1/h showed statistically significant differences (< 0.0001) between controls (57,39 ± 15,67) and EPI (moderate = 19,24 ± 23,93 and severe = 8,05 ± 9,50) and also between digestive diseases (35,75 ± 36,62) and severe EPI. Choosing 30 mg/ml-1/h as the cut off limit: sensitivity was 72% for moderate EPI and specificity was 100% for severe EPI. The PLT was carried out on 18 children (5-16 years): 8 with cystic fibrosis (CF), 2 with Swachman's syndrome (SS). 2 with chronic pancreatitis (CP), 3with chronic diarrhoea (CD),1 with constipation, 1 with failure to thrive, and 1 with recurrent pneumonia. Results: 13 children presented severe EPI (AUC ranging from 0,02 to 0,11): 7 with CF, 2 with SS, 2 with CP and 2 with CD. Fat absorption coefficient was below 90% in 4 of the 9 children tested. The calculated AUC for the child with failure to thrive was 16,95 expressing a moderate EPI and 4 children had normal AUC ranging from 38,08 to 52,63. Conclusion: the modified PLT eliminates the need for timed urine collections. It provides a simple, reliable, and tubeless test of exocrine pancreatic function, more sensitive than the fat absorption coefficient in children. PTL can be used as a diagnostic test to distinguish infants and children with EPI from normal children and those with other causes of diarrhoea and failure to thrive.

Suppression of Neutral Cholesterol Ester Hydrolase Activity by Antisense DNA of Hormone-Sensitive Lipase

In order to investigate the role of the hormone-sensitive lipase (HSL) gene in the activity of neutral cholesterol ester hydrolase (NCEH) from a molecular perspective, Chinese hamster ovary (CHO) cells were transfected with rat antisense hormone-sensitive lipase cDNA, and three different cell lines, designated as anti-HSL, were established. NCEH activity in anti-HSL cells was reduced to approximately 50% of that in control CHO cells. The concentration of cellular esterified cholesterol increased and the concentration of free cholesterol decreased in the anti-HSL cell lines. These results suggest that the HSL gene has a function of NCEH as well.

Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse

Acute inflammation results in a profound change in the apolipoprotein composition of high density lipoprotein (HDL). Several isoforms of the serum amyloid A (SAA) family, SAA 1 and SAA 2, become major components of HDL. This structural relationship has suggested that acute phase SAA plays some as yet unidentified role in HDL function, possibly related to cholesterol transport, during the course of acute inflammation. Using subcutaneous AgNO 3 to induce a sterile abscess changes in plasma cholesterol and SAA were monitored over the subsequent 144 h. Total plasma cholesterol began to increase within 12 h of the induction of inflammation and reached a peak in 24 h. Thereafter its plasma levels fell returning to normal values by 96–120 h. The bulk of the increase in plasma cholesterol was found in the free cholesterol fraction of HDL. This pattern of cholesterol increase corresponds to the established temporal changes for acute phase SAA (AP-SAA). AP-SAA levels increased within 8 h of the induction of inflammation and reached a peak at 24 h. They began to decrease by 48 h with small quantites still present 120 h later. In concert, but inversely, with the changes in AP-SAA the apoA-I, apoA-II, and apo-E, content of HDL decreased during the AP-SAA increases and increased as AP-SAA levels fell. The plasma appearance of cholesterol from the periphery, and central parts of the inflammatory site was assessed by the use of radiolabelled cholesterol. The peripherally placed cholesterol rapidly reached a peak plasma concentration within 24 h of injection. Cholesterol placed in the central part of the sterile abscess, a site relatively inaccessible to the vasculature required 48 h to reach its peak and was 5-times lower than that placed peripherally. The influence of AP-SAA on neutral cholesterol ester hydrolase (nCEH) activity in mouse liver homogenates, mouse peritoneal macrophage homogenates, and a purified porcine pancreatic enzyme with nCEH activity was also assessed. Following optimization with regard to pH, bile salt concentration, protein concentration and incubation time, mouse peritoneal macrophages had a significantly higher nCEH specific activity than that found in liver (7–8 fold). Purified AP-SAA, assessed over a concentration range of 0–10 μg/ml, enhanced nCEH activity at concentrations above 2 μg/ml. The nCEH activity, regardless of its source, increased by 3–7 fold in the presence of AP-SAA. Equivalent concentrations of apolipoprotein A-I (apo A-I) and bovine serum albumin (BSA) failed to alter the activity of nCEH. The effect of AP-SAA on a purified form of nCEH suggests that AP-SAA may have a direct effect on the activity of this enzyme. The temporal correlation of circulating AP-SAA and plasma cholesterol and the significant stimulation of nCEH by AP-SAA (but not apoA-I or BSA) provides further evidence that AP-SAA plays a role in cholesterol metabolism during the course of acute inflammation.