Isolation and identification of cholesterol esterase and pancreatic lipase inhibitory peptides from brewer's spent grain by consecutive chromatography and mass spectrometry.

The isolation and identification of cholesterol esterase (CE) and pancreatic lipase (PL) inhibitory peptides obtained from the protein hydrolysate of brewer's spent grain (BSG) was performed. BSG peptides were fractionated and purified sequentially by anion exchange, gel filtration (FPLC), and reversed phase high-performance liquid chromatography (RP-HPLC). The fractions obtained from each chromatographic step were collected and the in vitro enzyme inhibitory activity was evaluated. The chromatographic purification process increased the in vitro activities. The most active fractions were evaluated using MALDI-TOF tandem mass spectrometry, which identified three peptides: a peptide with the highest CE inhibition capacity (WNIHMEHQDLTTME) and two peptides with PL inhibition capacity (DFGIASF and LAAVEALSTNG). These three peptides showed hydrophobic and acidic amino acid residues (Asp and Glu) and/or their amines (Asn and Gln), which could be a common feature among lipid-lowering peptides related to CE and PL enzyme inhibition. The in silico studies showed that the three peptides had high hydrophobicity and were susceptible to enzymatic hydrolysis performed by trypsin, pepsin, and pancreatin. The BSG byproduct was a good source of CE and PL inhibitory peptides, thus adding value to this byproduct of the beer industry. This is the first report to demonstrate that BSG peptides can inhibit CE and PL enzymes.

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

Coprinus comatus filtrate extract, a novel neuroprotective agent of natural origin.

In vitro acetylholinesterase (AChE) inhibitory activity of an autochthonous sample of the mushroom Coprinus comatus (encompassing fruiting body FB, mycelia M and filtrate F from the submerged cultivation) was the subject of this study. C. comatus F extract exhibited rather potent anti-AChE activity (73.0 ± 1.5%) at in liquid conditions, comparable to those of the conventional drug donepezil (80.6 ± 1.4%). Also, the same extract exhibited high anti-AChE activity (1 µg) in solid. While its FTIR spectrum indicated the presence of phenolic compounds, quercetin (28.1 µg g-1 d.w.) was found to affect the observed bioactivity (59.8 ± 0.9%). This is the first report of profound anti-AChE activity of any C. comatus extract, a medicinal mushroom that has been successfully cultivated in P.R. China, due to the demanding needs of food industry.

Inhibitory Effect of Condensed Tannins from Banana Pulp on Cholesterol Esterase and Mechanisms of Interaction.

In the present study, the inhibitory effect of condensed tannins (CTs) on cholesterol esterase (CEase) was studied. The underlying mechanisms were evaluated by reaction kinetics, turbidity and particle size analyses, multispectroscopy methods, thermodynamics, and computer molecular simulations. CTs showed potent CEase inhibitory activity with an IC50 value of 64.19 µg/mL, and the CEase activity decreased with increasing CT content in a mixed-competitive manner, which was verified by molecular docking simulations. Fluorescence and UV-vis measurements revealed that complexes were formed from CEase and CTs by noncovalent interaction. Isothermal titration calorimetry indicated that the interaction between CEase and CTs occurred through hydrogen bonding and hydrophobic interactions. Circular dichroism analysis suggested that CTs inhibited the activity of CEase by altering the secondary structure of CEase. The inhibition of CTs on CEase in the gastrointestinal tract might be one mechanism for its cholesterol-lowering effect.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.

We previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5'-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12-/- and CalDAG GEFI-/- mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins.

Novel bioactive peptides from camel milk protein hydrolysates (CMPH) were identified and tested for inhibition of cholesterol esterase (CEase), and their possible binding mechanisms were elucidated by molecular docking. Papain-generated CMPH showed the highest degree of hydrolysis. All CMPH produced upon enzymatic degradation demonstrated a dramatic enhancement of CEase inhibition compared with intact camel milk proteins, with papain-generated hydrolysate P9 displaying the highest inhibition. Peptide identification and their modeling through PepSite 2 revealed that among 20 potential bioactive peptides in alcalase-generated hydrolysate A9, only 3 peptides, with sequences KFQWGY, SQDWSFY, and YWYPPQ, showed the highest binding toward CEase catalytic sites. Among 43 peptides in 9-h papain-generated hydrolysate P9, 4 peptides were found to be potent CEase inhibitors. Molecular docking revealed that WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR from P9 hydrolysates were able to bind to the active site of CEase with good docking scores and molecular mechanics-generalized born surface area binding energies. Overall, this is the first study reporting CEase inhibitory potential of peptides generated from milk proteins.

Identification of Lead Molecules in Garcinia mangostana L. Against Pancreatic Cholesterol Esterase Activity: An In Silico Approach.

Hypercholesterolemia is one of the major risk factors for the development and progression of atherosclerosis. Hence, inhibitors of cholesterol absorption have been investigated for decades as a strategy to prevent and treat cardiovascular diseases associated with hypercholesterolemia. Cholesterol esterase (CEase) in pancreatic juice plays a vital role in the hydrolysis of dietary cholesterol esters to cholesterol and fatty acids. Since inhibition of CEase might lead to a reduction of cholesterol absorption, an attempt is made in this study to identify lead molecules of Garcinia mangostana by the in silico approach. The study employed software applications viz., AutoDock 4.2 and GOLD Suite of Programs 5.2. The study revealed the efficacy of three compounds viz., epicatechin, euxanthone, and 1,3,5,6-tetrahydroxy-xanthone, which exhibited least binding energy in AutoDock and moderate scoring in GOLD. The molecular properties as well as biological activity of these three compounds were predicted by molinspiration prediction tool. The results show the crucial role of polyphenolic compounds to limit the activity of CEase. The drug-likeness prediction revealed the prospects of the identified lead molecules as potential drug candidates.

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Lysosomal acid lipase promotes cholesterol ester metabolism and drives clear cell renal cell carcinoma progression.

OBJECTIVES: Clear cell renal cell carcinoma (ccRCC) is characterized histologically by accumulation of cholesterol esters, cholesterol and other neutral lipids. Lysosomal acid lipase (LAL) is a critical enzyme involved in the cholesterol ester metabolism. Here, we sought to determine whether LAL could orchestrate metabolism of cholesterol esters in order to promote ccRCC progression. MATERIALS AND METHODS: Quantitative reverse-transcription PCR and western blots were conducted to assess the expression of LAL in human ccRCC tissues. We analysed the relationship between LAL levels and patient survival using tissue microarrays. We used cell proliferation assays, colony formation assays, cell death assays, metabolic assays and xenograft tumour models to evaluate the biological function and underlying mechanisms. RESULTS: LAL was up-regulated in ccRCC tissue. Tissue microarray analysis revealed higher levels of LAL in advanced grades of ccRCC, and high LAL expression indicated lower patient survival. Suppressing LAL expression not only blocked the utilization of cholesterol esters but also impaired proliferation and cellular survival. Furthermore, immunohistochemistry staining showed that LAL expression was correlated with Akt phosphorylation. Suppressing LAL expression decreased the phosphorylation level of Akt and Src and reduced the level of 14,15-epoxyeicosatrienoic acids in ccRCC cells. Supplement of 14,15-epoxyeicosatrienoic acids rescued proliferation in vitro and in vivo. CONCLUSIONS: LAL promoted cell proliferation and survival via metabolism of epoxyeicosatrienoic acids and activation of the Src/Akt pathway.

Lipid-lowering effect of bergamot polyphenolic fraction: role of pancreatic cholesterol ester hydrolase.

Bergamot polyphenolic fraction (BPF) has been shown to positively modulate several mechanisms involved in metabolic syndrome, suggesting its use in therapy. In particular, it is able to induce a significant amelioration of serum lipid profile in hyperlipemic patients at different levels. The purpose of our study was to investigate the effect of BPF on cholesterol absorption physiologically mediated by pancreatic cholesterol ester hydrolase (pCEH). An in vitro activity assay was performed to study the effect of BPF on pCEH, whereas the rate of cholesterol absorption was evaluated through in vivo studies. In particular, male, Sprague-Dawley rats (200–225 g) were fed either normal chow or chow supplemented with 0.5% cholic acid, 5.5% peanut oil, and varying amounts of cholesterol (0 to 1.5%). BPF (10 mg/Kg) was daily administrated by means of a gastric gavage to animals fed with lipid supplemented diet for 4 weeks and, at the end of the study, plasma lipids and liver cholesteryl esters were measured in all experimental groups. Our results show that BPF was able to inhibit pCEH activity and this effect was confirmed, in vivo, via detection of lymphatic cholesteryl ester in rats fed with a cholesterol-rich diet. This evidence clarifies a further mechanism responsible for the hypolipemic properties of BPF previously observed in humans, confirming its beneficial effect in the therapy of hypercholesterolemia and in the treatment of metabolic syndrome.

Design, synthesis, and pharmacological evaluation of a novel series of hormone sensitive lipase inhibitor.

HSL inhibition is a promising approach to the treatment of dyslipidemia. As a result of re-optimization of lead compound 2, we identified novel compound 25a exhibiting potent inhibitory activity against HSL enzyme and cell with high selectivity for cholinesterases (AChE and BuChE). Reflecting its potent in vitro activity, compound 25a exhibited antilipolytic effect in rats at 1mg/kg p.o., which indicated that this novel compound is the most potent orally active HSL inhibitor. Moreover, compound 25a did not show bioactivation liability.

Antioxidant and Cholesterol Esterase Inhibitory Properties of Supplementation with Coconut Water in Submerged Cultivation of the Medicinal Chinese Caterpillar Mushroom, Ophiocordyceps sinensis CS1197 (Ascomycetes).

We investigated the potential use of coconut water to supplement potato dextrose broth (PDB) in the production of Ophiocordyceps sinensis CS1197 by submerged cultivation. The basal PDB medium was modified by supplementation with tender coconut water (TCW) and mature coconut water (MCW) at 10% and 5% (v/v), respectively; these mixtures were cultured at 28°C for 14 days, with a pH of 7 and an inoculum volume of 10%. The addition of optimized levels of TCW and MCW improved the biomass yield by 2.2- and 2.5-fold, respectively, and adenosine, cordycepin, and polysaccharide content by 58% and 69%, 50% and 55%, and 19% and 27%, respectively. Antioxidant and cholesterol esterase (CE) inhibitory activities of the aqueous extract from O. sinensis CS1197 mycelia supplemented with TCW and MCW were high compared with those of the control, indicating that coconut water has a positive correlation with the enhanced antioxidant and CE inhibitory activities. These antioxidant and CE inhibitory responses were dependent on concentration, and the larger amounts of bioactives in O. sinensis CS1197 are beneficial in pharmaceutical formulations.

Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of LAL resulted in reduced release of 18:2- and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages. We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.

Informe de evaluación científica basada en la evidencia disponible: angioedema hereditario / Scientific evaluation report based on available evidence: hereditary angioedema

INTRODUCCIÓN: El Angioedema Hereditario debido al déficit de inhibidor de C1 esterasa es un enfermedad genética rara que se presenta con episodios recurrentes de edema subcutáneo o submucoso localizado, particularmente en sistema digestivo, vías respiratorias y piel. La presentación clásica incluye crisis de dolor abdominal por la oclusión del lumen intestinal. Las manifestaciones más graves incluyen edema de vía aérea, que puede llevar a la asfixia. TECNOLOGÍAS SANITARIAS ANALIZADAS: El proceso de evaluación fue iniciado por resolución N°1062, de septiembre de 2017, de Subsecretario de Salud Pública, para las tecnologías Inhibidor de C1 Esterasa Humana (Berinert®) e Icatibant. Comenzada la evaluación, se constató que al 31 de octubre de 2017, según lo informado por el Instituto de Salud Pública de Chile, para el medicamento Icatibant no se había iniciado proceso de registro en esa Institución, por lo que en virtud de lo dispuesto en el artículo 9°, del decreto supremo N°13, de 2017, del Ministerio de Salud, que aprueba "Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con sistema de protección financiera, según lo establecido en los artículos 7° y 8° de la ley N° 20.850", no se continúa la evaluación para dicho producto farmacéutico EFICACIA DE LOS TRATAMIENTOS: El inhibidor de C1 esterasa humano (Berinert®) probablemente reduce el tiempo de inicio de remisión de síntomas, además, probablemente implica una alta reducción de tiempo hasta la casi completa remisión de síntomas. ANÁLISIS ECONÓMICO: De la evidencia de costo efectividad encontrada se aprecia que el tratamiento puede ser costo efectivo, pero es conveniente disminuir su costo lo mayor posible para mayor certidumbre. Además, la incertidumbre respecto al costo del medicamento refiere a la cantidad de dosis de Inhibidor de la C1 esterasa que se debe usar por episodio, la cual depende del peso del paciente. Una línea de exploración podría ser la bonificación por parte del proveedor cuando sea necesario el uso de más de tres viales en un ataque. CONCLUSIÓN: Para dar cumplimiento al artículo 28° del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 del Ministerio de Salud, se concluye que el presente informe de evaluación se considera favorable, de acuerdo a lo establecido en el Título III. de las Evaluaciones Favorables de la Norma Técnica N° 0192 de este mismo ministerio.

Chemical genetics screening reveals KIAA1363 as a cytokine-lowering target.

Inflammation is a hallmark of many human diseases, including pain, arthritis, atherosclerosis, obesity and diabetes, cancer, and neurodegenerative diseases. Although there are several successfully marketed small molecules anti-inflammatory drugs such as cyclooxygenase inhibitors and glucocorticoids, many of these compounds are also associated with various adverse cardiovascular or immunosuppressive side effects. Thus, identifying novel anti-inflammatory small molecules and their targets is critical for developing safer and more effective next-generation treatment strategies for inflammatory diseases. Here, we have conducted a chemical genetics screen to identify small molecules that suppress the release of the inflammatory cytokine TNFα from stimulated macrophages. We have used an enzyme class-directed chemical library for our screening efforts to facilitate subsequent target identification using activity-based protein profiling (ABPP). Using this strategy, we have found that KIAA1363 is a novel target for lowering key pro-inflammatory cytokines through affecting key ether lipid metabolism pathways. Our study highlights the application of combining chemical genetics with chemoproteomic and metabolomic approaches toward identifying and characterizing anti-inflammatory smal molecules and their targets.

Cardiotrophin-1 stimulates lipolysis through the regulation of main adipose tissue lipases.

Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.

Synthesis and evaluation of 2-(1H-indol-3-yl)-4-phenylquinolines as inhibitors of cholesterol esterase.

A series of 2-(substituted) phenyl and 2-indolyl quinoline derivatives (10a-l) was synthesized by an efficient microwave-assisted, trifluoroacetic acid-catalyzed, solvent-free method. Evaluation of the inhibitory activity led to the identification of two quinoline inhibitors of cholesterol esterase. 2-(1H-Indol-3-yl)-6-nitro-4-phenylquinoline (10l; IC50=1.98µM) was characterized as a mixed-type inhibitor with a pronounced competitive binding mode.

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p<0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann-Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.

A practical fluorometric assay method to measure lysosomal acid lipase activity in dried blood spots for the screening of cholesteryl ester storage disease and Wolman disease.

Fluorometric measurements of 4-methylumbelliferone (4-MU) are generally used to screen lysosomal storage diseases (LSDs) using dried blood spots (DBSs). However, in DBS, it is difficult to measure lysosomal acid lipase (LAL) activity due to the influence of other lipases in whole blood. Recently, Hamilton used a fluorometric enzyme assay with 4-MU derivatives to measure the LAL activity in DBS. This method requires mercury chloride as stopping reagent, and the fluorescence intensity of 4-MU was measured at an acidic pH. We report a revised method to measure the LAL activity without using toxic mercury chloride and to measure the fluorescence intensity of 4-MU at a basic pH. For this measurement, we established a more practical method that does not require mercury chloride. The LAL activity in DBS was measured in 51 normal controls, seven obligate carriers and seven patients with CESD. The average LAL activities ± SD in the DBS from the normal, obligate carriers and CESD patients were 0.68 ± 0.2 (range: 0.3-1.08), 0.21 ± 0.1 (range: 0.11-0.41) and 0.02 ± 0.02 (range: 0-0.06) nmol/punch/h, respectively. There was a significant difference between the normal and the CESD. Our method does not require toxic mercury chloride and is an appropriate revised enzyme assay using DBS for screening patients with CESD.

Cholesteryl ester storage disease: a rare and possibly treatable cause of premature vascular disease and cirrhosis.

Cholesteryl ester storage disease (CESD) is an autosomal recessive lysosomal storage disorder caused by a variety of mutations of the LIPA gene. These cause reduced activity of lysosomal acid lipase, which results in accumulation of cholesteryl esters in lysosomes. If enzyme activity is very low/absent, presentation is in infancy with failure to thrive, malabsorption, hepatosplenomegaly and rapid early death (Wolman disease). With higher but still low enzyme activity, presentation is later in life with hepatic fibrosis, dyslipidaemia and early atherosclerosis.Identification of this rare disorder is difficult as it is essential to assay leucocyte acid phosphatase activity. An assay using specific inhibitors has now been developed that facilitates measurement in dried blood spots. Treatment of CESD has until now been limited to management of the dyslipidaemia, but this does not influence the liver effects. A new enzyme replacement therapy (Sebelipase) has now been developed that could change treatment options for the future.