Sebelipase alfa in children and adults with lysosomal acid lipase deficiency: Final results of the ARISE study.

BACKGROUND & AIMS: Children and adults with lysosomal acid lipase deficiency (LAL-D) experience cirrhosis and dyslipidemia from lysosomal accumulation of cholesteryl esters and triglycerides. Sebelipase alfa enzyme replacement therapy is indicated for individuals with LAL-D. We report final results from the phase III randomized ARISE study of sebelipase alfa in children aged ≥4 years and adults with LAL-D. METHODS: The study included a 20-week, double-blind, placebo-controlled period; a 130-week, open-label, extension period; and a 104-week, open-label, expanded treatment period. In the open-label periods, all patients received intravenous sebelipase alfa every other week. The primary outcome was alanine aminotransferase (ALT) level normalization; aspartate aminotransferase (AST) levels, lipid parameters, liver histology, liver and spleen volume and fat content, and safety were also assessed. RESULTS: Of 66 patients enrolled, 59 completed the study. Median (range) age at randomization was 13 (4.7-59) years. At the last open-label visit, ALT and AST levels had normalized in 47% and 66% of patients, respectively. Patients who switched from placebo to sebelipase alfa experienced sustained improvements in ALT and AST during the open-label periods that mirrored those observed in the sebelipase alfa group during the double-blind period. Median (IQR) percent changes in lipid levels included a 25% (39%, 6.5%) reduction in low-density lipoprotein cholesterol and a 27% (19%, 44%) increase in high-density lipoprotein cholesterol. Most adverse events during the open-label periods were mild to moderate in severity; 13 patients had infusion-associated reactions (serious in 1 patient). Six patients (9%) developed anti-drug antibodies. CONCLUSIONS: Early and rapid improvements in markers of liver injury and lipid abnormalities with sebelipase alfa were sustained, with no progression of liver disease, for up to 5 years. CLINICAL TRIAL NUMBER: NCT01757184; EudraCT Number: 2011-002750-31 LAY SUMMARY: Sebelipase alfa is used to treat lysosomal acid lipase deficiency (LAL-D), a rare, inherited disease of lipid metabolism. We report the final results of the phase III ARISE clinical study, which show that replacement of the defective LAL enzyme with sebelipase alfa for up to 5 years allows adults and children 4 years of age and older to maintain their initial improvements in liver and cholesterol parameters over the long term, without worsening of liver disease.

Reduced Lysosomal Acid Lipase Activity in Blood and Platelets Is Associated With Nonalcoholic Fatty Liver Disease.

OBJECTIVES: To investigate whether blood total lysosomal acid lipase activity (BT-LAL) levels are uniquely associated with the noncirrhotic and cirrhotic stages of nonalcoholic fatty liver disease (NAFLD) and with protection from NAFLD in metabolically/genetically predisposed subjects and a normal liver. To clarify which enzyme-carrying circulating cells are involved in reduced BT-LAL of NAFLD. METHODS: In a cross-sectional study, BT-LAL was measured by a fluorigenic method in patients with NAFLD (n = 118), alcoholic (n = 116), and hepatitis C virus-related disease (n = 49), in 103 controls with normal liver and in 58 liver transplant recipients. Intracellular platelet and leukocyte LAL was measured in 14 controls and 28 patients with NAFLD. RESULTS: Compared with controls, (i) BT-LAL and LAL in platelets, but not in leukocytes, were progressively reduced in noncirrhotic NAFLD and in nonalcoholic steatohepatitis-related cirrhosis; (ii) platelet and leukocyte counts did not differ in patients with noncirrhotic NAFLD; and (iii) BT-LAL did not differ in alcoholic and hepatitis C virus noncirrhotic patients. BT-LAL progressively increased in controls with metabolic syndrome features according to their PNPLA3 rs738409 steatosis-associated variant status (II vs IM vs MM), and their BT-LAL was higher than that of noncirrhotic NAFLD, only when carriers of the PNPLA3 unfavorable alleles were considered. Liver transplant recipients with de novo NAFLD compared with those without de novo NAFLD had lower BT-LAL. DISCUSSION: LAL in blood and platelets is progressively and uniquely reduced in NAFLD according to disease severity. High BT-LAL is associated with protection from NAFLD occurrence in subjects with metabolic and genetic predisposition. Low LAL in platelets and blood could play a pathogenetic role in NAFLD.

Reduced lysosomal acid lipase activity: A new marker of liver disease severity across the clinical continuum of non-alcoholic fatty liver disease?

Lysosomal acid lipase (LAL) plays a key role in intracellular lipid metabolism. Reduced LAL activity promotes increased multi-organ lysosomal cholesterol ester storage, as observed in two recessive autosomal genetic diseases, Wolman disease and Cholesterol ester storage disease. Severe liver steatosis and accelerated liver fibrosis are common features in patients with genetic LAL deficiency. By contrast, few reliable data are available on the modulation of LAL activity in vivo and on the epigenetic and metabolic factors capable of regulating its activity in subjects without homozygous mutations of the Lipase A gene. In the last few years, a less severe and non-genetic reduction of LAL activity was reported in children and adults with non-alcoholic fatty liver disease (NAFLD), suggesting a possible role of LAL reduction in the pathogenesis and progression of the disease. Patients with NAFLD show a significant, progressive reduction of LAL activity from simple steatosis to non-alcoholic steatohepatitis and cryptogenic cirrhosis. Among cirrhosis of different etiologies, those with cryptogenic cirrhosis show the most significant reductions of LAL activity. These findings suggest that the modulation of LAL activity may become a possible new therapeutic target for patients with more advanced forms of NAFLD. Moreover, the measurement of LAL activity may represent a possible new marker of disease severity in this clinical setting.

Lysosomal Acid Lipase: Can it be a New Non-Invasive Serum Biomarker of Cryptogenic Liver Fibrosis and Cirrhosis?

INTRODUCTION AND AIM: The association between lysosomal acid lipase (LAL) activity and liver steatosis or fibrosis is poorly studied. The aim of our study was to determine the predictive power of LAL for cryptogenic liver steatosis and cryptogenic significant fibrosis/cirrhosis. MATERIAL AND METHODS: Cross-sectional observational study of 101 adult patients with unexplained elevated liver enzymes/hepatomegaly with or without dyslipidemia submitted to the determination of LAL activity and LIPA gene (E8SJM-C.894G^A) mutation. Seventy-one patients underwent liver biopsy or FibroScan®. Patients with an identifiable liver dysfunction cause and well-stablished NAFLD/NASH risk factors were excluded. Predictors for liver steatosis, significant fibrosis (> F2) or cirrhosis (F4) were evaluated. RESULTS: Liver steatosis and fibrosis were mainly assessed by liver biopsy (74.6%; n = 53). Steatosis was present in 62.0% (n = 44), significant fibrosis in 47.9% (n = 34) and cirrhosis in 39.4% (n = 28). The median LAL was 0.36 (0.21-0.46)nmol/spot/h (vs. 0.29 (0.20-0.47); p = 0.558) for liver steatosis, 0.22 (0.11-0.29) nmol/spot/h (vs. 0.40 (0.34-0.51); p <0.001) for significant fibrosis and 0.21 (0.11-0.27) nmol/spot/h (vs. 0.40 (0.32-0.52); p < 0.001) for cirrhosis. No LIPA gene mutations were found. LAL activity was the strongest predictor of significant fibrosis (AUROC: 0.833; p < 0.001) with a cut-off of 0.265 (sensitivity: 85.9%; specificity: 75.0%) and cirrhosis (AUROC: 0.859; p < 0.001) with a cut-off of 0.235 (sensitivity: 86.2%; specificity: 75.0%), being higher than FIB4, GUCI or APRI. However, LAL activity was not associated with liver steatosis (AUROC: 0.536; p =0.558). CONCLUSION: LAL activity can be considered a non-invasive new marker of cryptogenic liver fibrosis with higher accuracy than other known biomarkers. LAL activity < 0.265 nmol/spot/h was strongly associated with cryptogenic significant fibrosis and <0.235 nmol/spot/h with cryptogenic cirrhosis. LAL activity was not associated with cryptogenic liver steatosis.

Specific Substrate for the Assay of Lysosomal Acid Lipase.

BACKGROUND: Deficiency of lysosomal acid lipase (LAL) causes Wolman disease and cholesterol ester storage disease. With the recent introduction of enzyme replacement therapy to manage LAL deficiency comes the need for a reliable assay of LAL enzymatic activity that can be applied to dried blood spots (DBS). METHODS: We prepared and tested a library of analogs of palmitoyl 4-methylumbelifferyl esters to find a highly active and specific substrate for LAL in DBS. The LAL assay was optimized leading to both LC-MS/MS and fluorometric assay of LAL. We tested the new assay on DBS from healthy and LAL-deficient patients. RESULTS: The ester formed between palmitic acid and 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) was found to be >98% selective for LAL in DBS based on the sensitivity of its activity to the LAL-specific inactivator Lalistat-2 and the fact that the activity was close to zero using DBS from patients previously shown to be LAL-deficient. Use of P-PMHC and heavy isotope-labeled internal standard with optimized assay conditions led to an approximately 2-fold increase in the specific activity of LAL compared with the previously reported LAL assay. Patients deficient in LAL were readily distinguished from normal persons with the new LAL assay using UPLC-MS/MS or fluorometric assay platforms. CONCLUSIONS: The new assay can measure LAL in DBS with a single measurement compared with the previous method involving 2 assays done in parallel.

Identification of rare diseases by screening a population selected on the basis of routine pathology results-the PATHFINDER project: lysosomal acid lipase/cholesteryl ester storage disease substudy.

AIMS: Lysosomal acid lipase deficiency (LALD) is an autosomal recessive disorder of cholesterol ester storage associated with hepatic disease, cirrhosis and accelerated atherosclerosis. Its prevalence in the general population, patients with dyslipidaemia and raised transaminases is unclear. This study attempted to identify the prevalence of LALD from patients with abnormal results in laboratory databases. METHODS: Electronic laboratory databases were interrogated to identify from clinical biochemistry records patients with a phenotype of low high-density lipoprotein-cholesterol (≤0.85 mmol/L; 33 mg/dL) and with elevated alanine or aspartate transaminases (≥60 IU/L) on one occasion or more over a 3-year time interval. Patients were recalled, and a dried blood spot sample was collected for lysosomal acid lipase determination by a fluorimetric enzyme assay. Histopathology databases of liver biopsies were interrogated for patients with features of 'microvesicular cirrhosis' or 'cryptogenic cirrhosis' in the report. Histological blocks were sampled, and samples were analysed by next-generation sequencing for the presence of mutations in the LAL gene. RESULTS: Samples were obtained from 1825 patients with dyslipidaemia and elevated transaminases. No cases of LALD were identified. Liver biopsies were obtained from six patients. DNA extraction was successful from four patients. Two patients were homozygous for the LAL c.46A>C;p.Thr16Pro unclassified variant in exon 2. CONCLUSIONS: Pathology databases hold routine information that can be used to identify patients with specific patterns of results or those who had biopsies to allow targeted testing for possible causes of disease. Biochemical screening suggests that the gene frequency of LAL deficiency in adults is less than 1 in 100.

High Fat Diet with a High Monounsaturated Fatty Acid and Polyunsaturated/Saturated Fatty Acid Ratio Suppresses Body Fat Accumulation and Weight Gain in Obese Hamsters.

The aim of this study was to investigate the effect of a high fat diet with experimental oil consisting of 60% MUFAs (monounsaturated fatty acids) with a P/S ratio of 5 on fat deposition and lipid metabolism in obese hamsters. Hamsters were randomly assigned to a control group and a diet-induced obesity group for nine weeks. Then an additional eight-week experimental period began, during which obese hamsters were randomly divided into three groups and fed different amounts of the experimental oil mixture in their diets as follows: 5%, 15%, and 20% w/w (OB-M5, OB-M15, and OB-M20 groups, respectively). The results showed that the OB-M15 and OB-M20 groups had significantly lower blood cholesterol and higher insulin levels. Compared to the control group, the three obese groups exhibited higher hepatic fatty acid synthase activity; however, the acyl-CoA oxidase activities were also enhanced. Although dietary fat content differed, there were no differences in energy intake, final body weights, and epididymal fat weights among the four groups. These results suggest that regardless of whether the specimens had a high fat intake or not, dietary fat containing high MUFAs with a high P/S ratio had beneficial effects on maintaining blood lipid profiles and may not result in body fat accumulation in obese hamsters, possibly by promoting lipolytic enzyme activities.

Spleen dimensions are inversely associated with lysosomal acid lipase activity in patients with non-alcoholic fatty liver disease.

Fatty liver and splenomegaly are typical features of genetic lysosomal acid lipase (LAL) deficiency. No data in adult patients with non-genetic reduction of LAL activity are available. We investigate the association between spleen dimensions and LAL activity in non-alcoholic fatty liver disease (NAFLD) patients, in whom a reduced LAL activity has been reported. We include 425 consecutive patients who underwent abdominal ultrasound to evaluate hepatic steatosis and spleen dimensions. LAL activity was measured with dried blood spot method (Lalistat2). NAFLD was present in 74.1% of screened patients. Higher median spleen longitudinal diameter (10.6 vs. 9.9 cm; p < 0.001) and spleen area (SA) (32.7 vs. 27.7 cm2; p < 0.001), together with a higher and proportion of splenomegaly (17.8 vs. 5.5%, p = 0.001), are present in patients with NAFLD compared to those without. In NAFLD patients, median LAL activity is 0.9 nmol/spot/h. LAL activity is lower in 56 patients with splenomegaly, as compared to those without (p = 0.009). At multivariable logistic regression analysis, age (above median, OR 0.344; p = 0.003), LAL activity (below median, OR 2.206, p = 0.028), and platelets (OR 0.101, p = 0.002) are significantly associated with splenomegaly. NAFLD patients disclose a relatively high prevalence of spleen enlargement and splenomegaly, which are significantly associated with a reduced LAL activity, suggesting that LAL may contribute to spleen enlargement in this setting.

Best practice in the measurement and interpretation of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.

Lysosomal acid lipase deficiency (LAL-D) is an inherited, autosomal recessive lysosomal storage disorder characterized by progressive damage in multiple organ systems. Diagnosis is especially important in infants, in whom the course of disease is rapidly lethal without treatment. The recent regulatory approval of recombinant human lysosomal acid lipase (LAL), sebelipase alfa, merits rapid diagnosis in clinical routine, particularly in infants. A method for measuring LAL activity in dried blood spot (DBS) samples using the highly specific LAL inhibitor Lalistat 2 is available. This method is shown to effectively discriminate between individuals with LAL-D and unaffected controls. With the increase in DBS LAL testing since the original publication of this method, a need to optimise assay performance has been identified. Here, we describe refinements to the DBS assay, including technical modifications, quality control measures and best-practice guidance for interpreting and reporting results. Particular attention is paid to alternatives to the use of mercuric chloride as the stop reagent and the choice of excitation wavelength for 4-methylumbelliferone palmitate under assay conditions at pH4.0. In addition, a simpler method of reporting results is proposed using cutoffs based on percentage mean normal enzyme activity.

Severe reduction of blood lysosomal acid lipase activity in cryptogenic cirrhosis: A nationwide multicentre cohort study.

BACKGROUND AND AIMS: Blood lysosomal acid lipase (LAL) is reduced in non-alcoholic steatohepatitis, which is the major cause of cryptogenic cirrhosis (CC); few data on LAL activity in CC do exist. We investigated LAL activity in a cohort of patients with liver cirrhosis. METHODS: This is a multicentre cohort study including 274 patients with liver cirrhosis of different aetiology from 19 centres of Internal Medicine, Gastroenterology and Hepatology distributed throughout Italy. Blood LAL activity (nmol/spot/h) was measured with dried blood spot extracts using Lalistat 2. RESULTS: Overall, 133 patients had CC, and 141 patients had cirrhosis by other causes (61 viral, 53 alcoholic, 20 alcoholic + viral, 7 autoimmune). Mean age was 64.2 ± 13.4 years, and 28.5% were women. Patients with CC were older compared to other aetiology-cirrhosis, with a lower Child-Turcotte-Pugh (CTP, p=0.003) and MELD (p=0.009) score, and a higher prevalence of cardio-metabolic risk factors and previous ischemic events. In the whole cohort, median LAL activity value was 0.58 nmol/spot/h, 0.49 and 0.65 in the groups of CC and known-aetiology cirrhosis, respectively (p=0.002). The difference remained significant after adjustment for white blood cells count (p=0.001). Multivariable linear regression analysis showed that CC (vs. known aetiology, Beta = -0.144, p=0.018), platelet count (Beta = 0.398, p < 0.001) and CTP score (Beta = -0.133, p=0.022) were associated with log-LAL activity. Similar results were found using MELD as covariate. CONCLUSIONS: We found a marked reduction of LAL activity in patients with cryptogenic cirrhosis compared to the other known aetiologies. A prospective study will clarify the role of LAL in chronic liver diseases.

Insulin and insulin-like growth factor-1 modulate the lipolytic action of growth hormone by altering signal pathway linkages.

Growth hormone (GH) has many actions in vertebrates, including the regulation of two disparate metabolic processes: growth promotion (anabolic) and the mobilization of stored lipids (catabolic). Our previous studies showed that GH stimulated IGF-1 production in hepatocytes from fed rainbow trout, but in cells from fasted fish GH stimulated lipolysis. In this study, we used rainbow trout (Oncorhynchus mykiss) to elucidate regulation of the mechanisms that enable cells to alter their lipolytic responsiveness to GH. In the first experiment, cells were removed from either fed or fasted fish, conditioned in medium containing serum (10%) from either fed or fasted fish, then challenged with GH. GH stimulated the expression of hormone sensitive lipase (HSL), the primary lipolytic enzyme, in cells from fasted fish conditioned with "fasted serum" but not in cells from fasted fish conditioned in "fed serum." Pretreatment of cells from fed fish with "fasted serum" resulted in GH-stimulated HSL expression, whereas GH-stimulated HSL expression in cells from fasted fish was blocked by conditioning in "fed serum." The nature of the conditioning serum governed the signaling pathways activated by GH irrespective of the nutritional state of the animals from which the cells were removed. When hepatocytes were pretreated with "fed serum," GH activated JAK2, STAT5, Akt, and ERK pathways; when cells were pretreated with "fasted serum," GH activated PKC and ERK. In the second study, we examined the direct effects of insulin (INS) and insulin-like growth factor (IGF-1), two nutritionally-regulated hormones, on GH-stimulated lipolysis and signal transduction in isolated hepatocytes. GH only stimulated HSL mRNA expression in cells from fasted fish. Pretreatment with INS and/or IGF-1 abolished this lipolytic response to GH. INS and/or IGF-1 augmented GH activation of JAK2 and STAT5 in cells from fed and fasted fish. However, INS and/or IGF-1 eliminated the ability of GH to activate PKC and ERK from fasted cells. These results indicate that INS and IGF-1 determine the signaling pathways activated by GH and whether or not a lipolytic response ensues. Such hormone-receptor-signal pathway linkages provide insight into the molecular basis of GH multifunctionality and into how cellular responses to GH can be adjusted to meet physiological (e.g., nutritional), developmental, or other conditions.

Platelet count may impact on lysosomal acid lipase activity determination in dried blood spot.

BACKGROUND: We aimed to evaluate the influence of white blood cell (WBC) and platelet (PLT) counts on dried blood spot (DBS)-determined lysosomal acid lipase (LAL) activity in a large group of healthy subjects. METHODS: One-hundred-and-seventy-two healthy subjects aged ≥18 were enrolled. Complete clinical biochemistry and LAL activity in DBS were determined. In 35 subjects, WBCs and PLTs were isolated, and LAL activity was measured in both blood cell populations. Univariate and multivariate analyses to DBS-LAL activity were performed. RESULTS: Mean age of subjects was 44.8±17.2years, 43.6% were males, and mean DBS-LAL activity was normal (1.0±0.3nmol/spot/h). LAL activity in WBCs was significantly higher than in PLTs (458.9±133.6 vs 235.0±88.3nmol/mg/h, p<0.001). However, LAL activity in DBS correlated more strongly with that in PLTs (r=0.65, p<0.001) than with that in WBCs (r=0.49, p<0.01). Consistently, in the multivariate model, DBS-LAL activity was independently associated only with PLT count (ß=0.39, p<0.001). CONCLUSIONS: PLT number may impact on the result of the DBS-LAL test, and a consideration of PLT count is recommended before interpreting LAL activity in DBS.

Synergistic effects of caffeine and catechins on lipid metabolism in chronically fed mice via the AMP-activated protein kinase signaling pathway.

PURPOSE: To investigate the mechanistic effects of combined exposure to caffeine and catechins on lipid metabolism in mice. METHODS: Seventy mice were randomly assigned to seven groups and fed diets containing varying doses of caffeine and catechins for 24 weeks. Body weight gain, intraperitoneal adipose tissue (IPAT) weight, serum biochemical parameters, and enzymatic activities, mRNA and protein expression levels of lipid metabolism-related enzymes in the liver and IPAT were analyzed. RESULTS: Following administration of caffeine and catechins, body weight gain, IPAT weight, serum and liver concentrations of total cholesterol and triglyceride were markedly reduced. Lipase activities, including that of AMP-activated protein kinase (AMPK), acyl-CoA oxidase, carnitine acyltransferase, adipose triglyceride lipase, and hormone-sensitive lipase, were significantly upregulated; however, fatty acid synthase (FAS) activity in the liver was suppressed. Combined exposure to caffeine and catechins significantly upregulated mRNA and protein expression levels of lipases while downregulating FAS mRNA expression and protein expression of peroxisome proliferator-activated receptor γ2. CONCLUSIONS: The combination of caffeine and catechins regulated the enzymatic activities, mRNA, and protein expression levels of lipid metabolism-related enzymes, resulting in suppression of body weight gain and IPAT weight in mice, potentially through activation of the AMPK signaling pathway. This study indicates that chronic intake of both caffeine and catechins can synergistically contribute to prevention of obesity and lifestyle-related diseases.

Association between adjunctive metformin therapy in young type 1 diabetes patients with excess body fat and reduction of carotid intima-media thickness.

INTRODUCTION    Lipoprotein-associated phospholipase A2 (Lp-PLA2) and cholesteryl ester lipase (CEL) may oxidize low-density lipoproteins (oxLDL). OBJECTIVES    The aim of the study was to determine the influence of metformin on the metabolism of atherogenic lipid fractions in relation to Lp-PLA2 and CEL levels, as well as assess consequent improvement in the intima-media thickness (IMT) of the common carotid artery in young type 1 diabetes patients with excess body fat. PATIENTS AND METHODS    It was an open-label randomized clinical trial that lasted 6 months. It included a total of 84 people with metabolic decompensation (glycated hemoglobin >7.5%, >58.5 mmol/mol) of diabetes. Adjunctive metformin therapy (in addition to insulin) was administered in 42 patients, and the remaining 42 patients received insulin alone. Glycated low-density lipoproteins (LDLs), oxLDL, Lp-PLA2, and CEL were assessed by commercially available enzyme-linked immunosorbent assay kits. Cartoid IMT was measured using the Carotid Analyser for Research tool. Biochemical analyses were performed using routine laboratory techniques. RESULTS    The reduction of mean carotid IMT was observed in young type 1 diabetic adults treated additionally with metformin (0.6 ±0.1 cm vs 0.53 ±0.1 cm; P = 0.002). This effect was probably due to weight reduction (90 ±16 kg vs 87 ±15 kg, P = 0.054) and the decrease in atherogenic glycated LDL levels (1.5 ±0.5 mg/dl vs 1.6 ±1.046 mg/dl, P = 0.006). No such correlations were observed in patients treated with insulin alone. Additionally, in patients receiving metformin, glycated LDL levels were inversely correlated with Lp-PLA2 levels (r = -0.31, P <0.05). CONCLUSIONS    Additional use of metformin in young type 1 diabetic patients with excess body fat leads to a significant reduction of mean IMT in the common carotid artery. Concentrations of CEL and Lp-PLA2 were significantly increased in both study arms despite improved glucose metabolism.

Reduced lysosomal acid lipase activity - A potential role in the pathogenesis of non alcoholic fatty liver disease in pediatric patients.

BACKGROUND: Within the spectrum of nonalcoholic fatty liver disease (NAFLD), recent evidence suggests that adult patients with nonalcoholic steatohepatitis (NASH) have significantly lower blood lysosomal acid lipase (LAL) activity than those with steatosis. This has not been studied in pediatric patients with NAFLD. AIM: Investigate blood LAL activity in pediatric patients with NAFLD and assess its correlation with histological severity. METHODS: We collected data on consecutive children with biopsy-proven NAFLD including demographics, anthropometrics, and routine laboratory tests. The histological features were graded according to the NAFLD activity scoring proposed by Kleiner et al. Blood LAL activity was measured prospectively using Lalistat 2. RESULTS: A total of 168 children were included for analysis. Mean age was 12.6±8.5 years, 60.1% were males and 52.4% had NASH. Children with significant fibrosis (stage 2-3, n=64) had a significantly lower LAL activity compared to those with mild fibrosis (stage 0-1, n=104). There was no significant difference in LAL activity between children with NASH compared to those without NASH. CONCLUSION: Reduced blood LAL activity correlates with severity of liver fibrosis in children with NAFLD indicating a potential role of reduced LAL activity in the pathogenesis of NAFLD-induced fibrosis.

Low Serum Lysosomal Acid Lipase Activity Correlates with Advanced Liver Disease.

Fatty liver has become the most common liver disorder and is recognized as a major health burden in the Western world. The causes for disease progression are not fully elucidated but lysosomal impairment is suggested. Here we evaluate a possible role for lysosomal acid lipase (LAL) activity in liver disease. To study LAL levels in patients with microvesicular, idiopathic cirrhosis and nonalcoholic fatty liver disease (NAFLD). Medical records of patients with microvesicular steatosis, cryptogenic cirrhosis and NAFLD, diagnosed on the basis of liver biopsies, were included in the study. Measured serum LAL activity was correlated to clinical, laboratory, imaging and pathological data. No patient exhibited LAL activity compatible with genetic LAL deficiency. However, serum LAL activity inversely predicted liver disease severity. A LAL level of 0.5 was the most sensitive for detecting both histologic and noninvasive markers for disease severity, including lower white blood cell count and calcium, and elevated γ-glutamyltransferase, creatinine, glucose, glycated hemoglobin, uric acid and coagulation function. Serum LAL activity <0.5 indicates severe liver injury in patients with fatty liver and cirrhosis. Further studies should define the direct role of LAL in liver disease severity and consider the possibility of replacement therapy.

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.

Changes in glucose and carnitine levels and their transporters in utero-tubal junction in relation to sperm storage in the vespertilionid bat, Scotophilus heathi.

Prolonged sperm storage over winter is a common feature of reproduction in some bats. In order to understand how sperm storage in the female genital tract of the vespertilionid bat, Scotophilus heathi (Greater yellow bat), is controlled, we compared concentrations of glucose and the fatty-acid carrier carnitine in the blood, and carnitine concentrations and levels of expression of the glucose transporters (GLUTs) and the carnitine transporter OCTN2 in the utero-tubal junction of females during non-storage (early winter) and sperm-storage periods (late winter-early spring). During the sperm-storage period (December-January) blood glucose concentrations declined, as did the expression of GLUT3 and GLUT5 in the utero-tubal junction. At the same time there were increases in the concentration of carnitine, and expression of OCTN2 and hormone-sensitive lipase (HSL) in the utero-tubal junction. These results suggest that prolonged sperm storage is enhanced by decreased glucose availability but increased free fatty acid availability at the site of sperm storage. Increases in expression of GLUT4 and GLUT8 in late winter suggest a role for these GLUTs in increasing sperm motility prior to fertilization.

A new method for the measurement of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.

BACKGROUND: Cholesterol ester storage disease (CESD) and Wolman Disease (WD) are due to deficiency of lysosomal acid lipase (LAL). A new method is described for the measurement of LAL in dried blood spots (DBS) using Lalistat 2 an inhibitor of LAL. METHODS: LAL activity in DBS extracts was measured using the substrate 4-methylumbelliferyl palmitate. LAL activity was determined by measuring total lipase activity and lipase activity in the presence of Lalistat 2. The specificity of Lalistat 2 was investigated using human recombinant LAL (hrLAL) and human pancreatic lipase (hPL). RESULTS: Lalistat 2 inhibited hrLAL with 1% residual activity at 1 µM inhibitor but had no effect on hPL up to 10 µM. LAL activity in DBS samples obtained from normal controls (n=140) was 0.50-2.30 nmol/punch/h and in patients with CESD was <0.03 nmol/punch/h (n=11). Activity in carriers showed intermediate activity: 0.15-0.40 nmol/punch/h (n=15). CONCLUSIONS: Measurement of LAL using DBS is made difficult by the presence of other lipases in whole blood. Lalistat 2 is a specific inhibitor of LAL which allows the determination of LAL in DBS. Results show the method differentiates clearly between normal controls, carriers and affected cases.