Value of plasma chitotriosidase to assess non-neuronopathic Gaucher disease severity and progression in the era of enzyme replacement therapy.

Gaucher disease (GD) is caused by deficiency of the enzyme glucocerebrosidase catalysing the regular lysosomal degradation of glucosylceramide. In the common non-neuropathic variant of GD, glucosylceramide-laden macrophages (Gaucher cells) accumulate in various tissues. Gaucher cells secrete chitotriosidase, an active chitinase, resulting in increased plasma chitotriosidase levels, which can be sensitively monitored by an enzyme activity assay. Plasma chitotriosidase is a rough estimate of body burden of Gaucher cells. Non-neuronopathic GD is presently treated by enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). We addressed the question whether plasma chitotriosidase acts as (predictive) marker of clinical manifestations in non-neuronopathic GD patients receiving treatment. Reductions in plasma chitotriosidase during therapy correlated with corrections in liver and spleen volumes and showed positive trends with improvements in haemoglobin and platelet count and bone marrow composition. The occurrence of long-term complications and associated conditions such as multiple myeloma, bone complications, Parkinson's disease, hepatocellular carcinoma and pulmonary hypertension positively correlated with the plasma chitotriosidase level pre-therapy, the average plasma chitotriosidase during 3 years of ERT and the residual plasma chitotriosidase after 2 years of ERT. In summary, plasma chitotriosidase is a valuable marker in the assessment and follow-up of GD patients.

Acid sphingomyelinase (Asm) deficiency patients in The Netherlands and Belgium: disease spectrum and natural course in attenuated patients.

Niemann-Pick disease (NPD) is a neurovisceral lysosomal storage disorder caused by acid sphingomyelinase (ASM) deficiency, which can be categorized as either Niemann-Pick disease type A [NPD-A], with progressive neurological disease and death in early childhood, or as Niemann-Pick disease type B [NPD-B], with a more variable spectrum of manifestations. Enzyme replacement therapy (ERT) with recombinant sphingomyelinase is currently studied as potential treatment for NPD-B patients. The objective of this study is to characterize the clinical features of patients with ASM deficiency in the Netherlands and Belgium with focus on the natural disease course of NPD-B patients. Prospective and retrospective data on ASM deficient patients were collected in The Netherlands and part of Belgium. Patients with NPD-B that could be followed prospectively were evaluated every 6-12 months for pulmonary function tests, 6 minute walk test (6 MWT), imaging (bone marrow infiltration measured by QCSI, organ volumes by MRI and CT scan of the lungs) and biochemical markers. Twenty-five patients with ASM deficiency were identified (13 males, 12 females, median age 13years, range 1-59 years). Nine patients had died at the time of the study, including four NPD-A patients at the age of 1,1, 2, 3 and five NPDB patents at the age of 5, 6, 43, 56 and 60 years. There was a high prevalence of homozygosity and compound heterozygosity for the common p.Arg608del mutation in 43% and 19% of NPD-B patients, respectively. In NPD-B patients, thrombocytopenia was present in most, while anemia and leucopenia were less common (33% and 6 % respectively). HDL cholesterol was reduced in most patients. Pulmonary disease was severe in several patients. Follow-up up to 11 years revealed a gradual decrease in platelet count. Detailed investigations in 6 NPD-B patients with follow-up in 4 patients revealed remarkable stable disease parameters up to 6 years, with some decline in pulmonary function and 6 MWT. Bone marrow fat fractions were decreased, indicating the presence of storage macrophages. Lung involvement was not related to the extent of visceromegaly, cytopenia or bone marrow involvement. In conclusion, in NPD-B patients pulmonary disease is the most debilitating feature. Disease manifestations are mostly stable in attenuated patients. Bone marrow infiltration is a less prominent feature of the disease.

Plasma globotriaosylsphingosine: diagnostic value and relation to clinical manifestations of Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-Galactosidase A, causing accumulation of globotriaosylceramide and elevated plasma globotriaosylsphingosine (lysoGb3). The diagnostic value and clinical relevance of plasma lysoGb3 concentration was investigated. All male and adult female patients with classical Fabry disease could be discerned by an elevated plasma lysoGb3. In young pre-symptomatic Fabry heterozygotes, lysoGb3 levels can be normal. Individuals carrying the R112H and P60L mutations, without classical Fabry symptoms, showed no elevated plasma lysoGb3. Multiple regression analysis showed that there is no correlation of plasma lysoGb3 concentration with total disease severity score in Fabry males. However, plasma lysoGb3 concentration did correlate with white matter lesions (odds ratio: 6.1 per 100 nM lysoGb3 increase (95% CI: 1.4-25.9, p=0.015). In females, plasma lysoGb3 concentration correlated with overall disease severity. Furthermore, plasma lysoGb3 level was related to left ventricular mass (19.5+/-5.5 g increase per 10 nM lysoGb3 increase; p=0.001). In addition, it was assessed whether lifetime exposure to lysoGb3 correlates with disease manifestations. Male Fabry patients with a high lysoGb3 exposure (>10,000 U), were moderately or severely affected, only one mildly. Female patients with a low exposure (<1000 U) were asymptomatic or mildly affected. A large proportion of the females with an exposure >1000 U showed disease complications. Plasma lysoGb3 is useful for the diagnosis of Fabry disease. LysoGb3 is an independent risk factor for development of cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in females. Disease severity correlates with exposure to plasma lysoGb3.

Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: correlations with disease severity and response to therapeutic intervention.

The concentrations of plasma glucosylceramide (GlcCer) and ceramide (Cer) were determined in a cohort of type 1 Gaucher disease patients. In plasma of untreated patients, GlcCer concentrations were on average 3-fold increased (median Gaucher: 17.5 nmol/ml, range: 6.5-45.5 (n=27); median control: 5.9 nmol/ml, range 4.0-8.6 (n=15)). Although plasma Cer concentrations were not significantly different between the two groups (median Gaucher: 7.2 nmol/ml, range: 4.2-10.9 (n=27); median control: 7.8 nmol/ml, range 5.7-11.9 (n=15)) in individual patients plasma GlcCer/Cer ratio yields slightly better discrimination between Gaucher disease patients and normal individuals than the GlcCer levels. Positive correlations were detected between plasma GlcCer concentration and GlcCer/Cer ratio and severity of disease, plasma chitotriosidase and CCL18, surrogate markers of storage cells. Gaucher disease is treated by enzyme replacement and substrate reduction therapy. Both therapies were found to result in decreases in plasma GlcCer already within 6 months, without causing abnormal plasma GlcCer or Cer concentrations. The corrections in plasma GlcCer were most robust in patients with a pronounced clinical response. In conclusion, plasma GlcCer concentration and GlcCer/Cer ratio is of value to monitor Gaucher disease manifestation and response to therapeutic intervention.

Short-term manipulation of plasma free fatty acids does not change skeletal muscle concentrations of ceramide and glucosylceramide in lean and overweight subjects.

CONTEXT: Increased plasma free fatty acid (FFA) concentrations may be in part responsible for the increased levels of ceramide in skeletal muscle of obese subjects. OBJECTIVE: We studied the effect of lowering and increasing plasma FFA levels on muscle ceramide and glucosylceramide concentrations in lean and obese subjects. DESIGN: Plasma FFAs were either increased or decreased for 6 h by infusing a lipid emulsion or using Acipimox, respectively. Muscle biopsies were performed before and after the intervention for measurements of ceramide and glucosylceramide. STUDY SUBJECTS: Eight lean [body mass index 21.9 (range, 19.6-24.6) kg/m2] and six overweight/obese [body mass index 34.4 (27.8-42.5) kg/m2] subjects without type 2 diabetes mellitus participated in the study. MAIN OUTCOME MEASURE: Differences in muscle ceramide and glucosylceramide upon manipulation of plasma FFAs were measured. RESULTS: There were no differences in muscle ceramide and glucosylceramide between lean and obese subjects, respectively. Increasing or decreasing plasma FFAs for 6 h had no effect on ceramide [high FFAs: 24 (19-25) vs. 24 (22-27) pmol/mg muscle, P=0.46; and 22 (20-28) vs. 24 (18-26) pmol/mg muscle, P=0.89 in lean and obese, respectively; low FFAs: 26 (24-35) vs. 23 (18-27) pmol/mg muscle, P=0.17 and 24 (15-44) vs. 24 (19-42) pmol/mg muscle, P=0.6 in lean and obese, respectively] and glucosylceramide [high FFAs: 2.0 (1.7-4.3) vs. 3.4 (2.1-4.6) pmol/mg muscle, P=0.17; and 3.0 (1.3-6.7) vs. 2.6 (1.2-3.9) pmol/mg muscle, P=0.89 in lean and obese, respectively; low FFAs: 2.2 (1.0-4.4) vs. 1.7 (1.4-3.0) pmol/mg muscle, P=0.92; and 6.6 (1.0-25.0) vs. 4.3 (1.3-7.6) pmol/mg muscle, P=0.7 in lean and obese, respectively] concentrations in skeletal muscle. CONCLUSION: Short-term manipulation of plasma FFAs has no effect on ceramide and glucosylceramide concentrations in skeletal muscle from lean and obese subjects.

The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels.

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked lysosomal storage disorder with characteristic vascular, renal, cardiac and cerebral complications. Globotriaosylceramide (Gb(3)) accumulates in Fabry patients as a result of alpha-galactosidase A deficiency. The phenotypic variability is high, but the relationship between clinical symptoms in individual Fabry patients has not been uniformly documented. Also, the relation between the most prominent biochemical abnormalities, elevated Gb(3) levels in plasma and urine, and clinical symptoms is not firmly established. METHODS: Clinical and biochemical characteristics of 96 (25 deceased) Dutch Fabry patients were collected retrospectively and before the initiation of enzyme therapy. RESULTS: Clinical assessment revealed that median life expectancy was 57 years for male and 72 years for female patients. Cerebral complications, acroparaesthesias and gastrointestinal complications, but not cardiac and auditory complications, were all seen more frequently in male than female patients. Glomerular filtration rate (GFR) was highly variable in male patients, including 2 patients with GFR < 30 ml/min, but median GFR did not differ between males and females (103 and 101 ml/min, respectively). Hyperfiltration was more frequently observed in the female patient group. Microalbuminuria was present in 60% of males and 45% of females. No specific pattern of combined symptoms existed except for a relationship between left ventricular hypertrophy (LVH) and cerebral complications (males 36%, females 32%), or proteinuria (males 35%, females 31%). Gb(3) was found to be more elevated in plasma samples from male (n = 26; median 6.27 micromol/L (1.39-9.74)) than female Fabry patients (n = 37; median 2.16 (0.77-4.18)). This was also observed for urinary Gb(3): males (n = 22) median 1851 nmol/24 h (40-3724); females (n = 29) median 672 (86-2052). Plasma and urinary Gb(3) levels correlated with each other in both males (r = 0.4, p = 0.05) and females (r = 0.4, p = 0.03), but no correlation between elevated Gb(3) levels and clinical symptoms could be detected. CONCLUSION: Analysis of the characteristics of the Dutch Fabry cohort has revealed that a limited relationship between various disease manifestations exists and that individual symptoms do not correlate with elevated urinary or plasma Gb(3) levels, limiting their value as surrogate disease markers.

Evidence for the existence of only one triacylglycerol lipase of rat liver active at alkaline pH.

There have been numerous reports suggesting the existence of two or more lipases in liver capable of hydrolyzing triacylglycerols at neutral to alkaline pH. We set out to determine if rat liver contains an alkaline triacylglycerol lipase, in addition to heparin-releasable lipase, which has an intracellular localization. We report here the results of studies concerning the pH dependence, subcellular localization and kinetic analysis of the alkaline lipase(s) of rat liver. Homogenates and cytosolic, microsomal and plasma membrane-enriched subfractions all exhibited an optimum of lipase activity at approx. pH 8.0. In no case was there evidence of multiple pH optima in the alkaline ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the com- and subfractions prepared from control livers with those prepared from livers perfused with collagenase. The loss (93%) of lipase activity from both the cytosolic and microsomal subfractions after collagenase perfusion was identical to the loss (93%) of activity from the homogenates, suggesting a common origin with the collagenase-sensitive alkaline lipase on plasma membrane. The characteristics of hydrolysis in vitro of triacylglycerol contained in artificial and natural substrate preparations by the alkaline lipase of rat liver were examined. The artificial substrate preparation was emulsified tri[1-14C]oleoylglycerol prepared by sonication and the natural substrate preparation was a triacylglycerol-rich lipid fraction ('liver fat') prepared from rat liver homogenates. Although the curves were complex, apparent Km values (mean +/- S.W., n = 3-6) over the limited concentration ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the complexity of these kinetics was related to changes in the products of lipolysis, we examined the products after incubations of plasma membrane-enriched fractions with lower and higher concentrations of triacylglycerol. In either case, the products of lipolysis were diacylglycerol, fatty acids and glycerol; no monoacylglycerol accumulated under any circumstances. At the lower concentrations of either tri[1-14C]oleoylglycerol or liver fat, most triacylglycerol hydrolyzed was degraded fully to fatty acids and glycerol. At the higher triacylglycerol concentrations, while complete degradation continued, virtually all of the increased lipolysis of triacylglycerol (over the lipolysis at the lower substrate concentrations) yielded diacylglycerol. The data indicated that the hydrolysis of diacylglycerol by the alkaline lipase of rat liver occurred at a rate slower than that of triacylglycerol. If the same enzyme catalyzes the lipolysis of both tri- and diacylglycerols, triacylglycerols would appear to be preferred...

Effect of fasting and feeding a high-sucrose, fat-free diet on the synthesis of hepatic glycerolipids in vivo and in isolated hepatocytes.

1. The synthesis of glycerolipids from a number of radioactive precursors, such as [2-3H] glycerol, [32P]phosphate, [Me-14C]choline and [1,2-14C2]ethanolamine proceeds in enzymatically isolated hepatocytes with a specificity that agrees very well with that observed in the liver in vivo. 2. The nutritional state of the rat has a profound influence on the glycerolipid metabolism of isolated hepatocytes. Fasting strongly decreased the incorporation of glycerol via sn-glycerol 3-phosphate into triacylglycerols whereas the formation of phosphatidylcholine and, particularly, phosphatidylethanolamine was much less affected by food deprivation. Refeeding a high-sucrose, fat-free diet caused a tremendous increase in the synthesis of diacylglycerols and triacylglycerols to values exceeding those found in hepatocytes from normally fed animals. The formation of phosphatidylcholine increased more slowly and the synthesis of phosphatidylethanolamine was even depressed by refeeding a high-sucrose, fat-free diet. 3. Alteration of the nutritional state resulted in similar changes in the amounts and metabolism of hepatic glycerolipids in vivo. The formation and, consequently, the amount of diacyglycerol and triacylglycerol were decreased by fasting and increased to values above normal in rats refed a high-sucrose, fat-free diet. The formation and the amounts of phospholipids in the liver decreased slightly by fasting which was mainly due to a decrease in phosphatidylcholine. Refeeding caused a significant increase in the formation and amount of phosphatidylcholine. The amount of phosphatidylethanolamine was even further diminished by feeding a high-sucrose, fat-free diet to 48-h-fasted rats. 4. These results show that the alterations, induced in the in vivo metabolism of hepatic glycerolipids, by changes of the dietary state, are also reflected in the isolated hepatocytes. This finding strengthens the potential significance of isolated hepatocytes in studies on the regulation of hepatic lipid metabolism.

Lysosomal phospholipase activity is decreased in mucolipidosis II and III fibroblasts.

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.

Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase.

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat.

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.

Cholesteryl ester transfer protein gene polymorphism is a determinant of HDL cholesterol and of the lipoprotein response to a lipid-lowering diet in type 1 diabetes.

The TaqIB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 +/- 0.38 vs. 1.24 +/- 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated:saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqIB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.

Identification and use of biomarkers in Gaucher disease and other lysosomal storage diseases.

UNLABELLED: The value of biomarkers in the clinical management of lysosomal storage diseases is best illustrated by the present use of plasma chitotriosidase levels in the diagnosis and monitoring of Gaucher disease. The enzyme chitotriosidase is specifically produced and secreted by the pathological storage macrophages (Gaucher cells). Plasma chitotriosidase levels are elevated on average 1000-fold in symptomatic patients with Gaucher disease and reflect the body burden on storage cells. Changes in plasma chitotriosidase reflect changes in clinical symptoms. Monitoring of plasma chitotriosidase levels is nowadays commonly used in decision making regarding initiation and optimization of costly therapeutic interventions (enzyme replacement therapy or substrate reduction therapy). A novel substrate has been developed that further facilitates the measurement of chitotriosidase in plasma samples. Moreover, an alternative Gaucher-cell marker, CCL18, has been very recently identified and can also be employed to monitor the disease, particularly in those patients lacking chitotriosidase due to a genetic mutation. There is a need for comparable surrogate markers for other lysosomal storage diseases and the search for such molecules is an area of intense investigation. CONCLUSION: The use of biomarkers can provide valuable insight into the molecular pathogenesis of LSDs, such as Gaucher disease and Fabry disease.

Elevated cholesteryl ester transfer protein activity in IDDM men who smoke. Possible factor for unfavorable lipoprotein profile.

OBJECTIVES: To determine the effect of cigarette smoking on the activity of cholesteryl ester transfer protein (CETP) and high-density (HDL), low-density (LDL), and very-low-density (VLDL) lipoproteins in insulin-dependent diabetic (IDDM) men with microvascular complications. RESEARCH DESIGN AND METHODS: We performed a case-control study in a referral-based diabetes clinic on a sequential sample of 9 cigarette-smoking and 12 nonsmoking IDDM men with microvascular complications and 12 nonsmoking control men. CETP activity was determined in each serum with an isotope assay with exogenous cholesteryl ester-labeled LDL and HDL. The method is independent of the endogenous lipoprotein present in serum. RESULTS: The HDL-cholesterol (VLDL and LDL) ratio was lower in the smoking diabetic men than in the other groups (P less than 0.05 vs. the nonsmoking diabetic men and P less than 0.01 vs. the control subjects). CETP activity was 70% higher in the smoking diabetic men than in the control subjects (P less than 0.01) and 30% higher than in the nonsmoking diabetic men (P less than 0.05). The HDL-cholesterol (VLDL and LDL) ratio and the apolipoprotein A-I-B ratio were inversely correlated to CETP activity in the diabetic patients (r = -0.52, P less than 0.02 and r = -0.45, P less than 0.05, respectively). CONCLUSIONS: CETP activity is increased in cigarette-smoking IDDM men with microvascular complications. High CETP activity may contribute to the unfavorable lipoprotein profile in these patients.

Hypercholesterolaemia and hepatosplenomegaly: two manifestations of cholesteryl ester storage disease.

Cholesteryl ester storage disease (CESD) is a rare autosomal recessive disease caused by mutations in LIPA. Here we describe two different clinical presentations of this disease: one case with a clear phenotype of familial hypercholesterolaemia and one case with hepatosplenomegaly from childhood onwards. These two cases exemplify the diversity of clinical phenotypes of patients with CESD. Knowledge on the phenotypic variability of the disease is of clinical relevance in light of enzyme replacement therapy (sebelipase alpha) for patients with mutations in LIPA, which is currently under development.

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency.

Different locations of cholesteryl ester transfer protein and phospholipid transfer protein activities in plasma.

Activities of cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) were measured in plasma of four vertebrate species: man, rabbit, pig, and rat. The activities were measured in the absence and presence of antibodies raised against purified human CETP. PLTP activities were present in all four species with highest values in pig (11.7 +/- 1.2 U/ml) and human plasma (9.2 +/- 1.6 U/ml). Considerable lower activities were found in rabbit (3.5 +/- 0.6 U/ml) and rat plasma (1.6 +/- 0.7 U/ml). These activities were not affected significantly by antibody against human CETP. CETP activities could be measured in human (0.23 +/- 0.05 U/ml) and in rabbit plasma (0.19 +/- 0.03 U/ml). CETP activity in human plasma was inhibited over 97% by antibody against human CETP. Plasma was chromatographed on a Superose 6 gel filtration column. Average HDL particle sizes in the four species differed notably and decreased in the order: rat HDL greater than rabbit HDL greater than human HDL greater than pig HDL. A separation of the two lipid transfer activities was evident after gel filtration chromatography. The peak of the PLTP activity coeluted with a fraction of HDL particles with the size of human HDL2 (particle weights 300-375 kDa). CETP activity in human and rabbit plasma coeluted largely with relatively small HDL particles (particle weights 140-180 kDa). These results show that CETP and PLTP activities are located in different macromolecular complexes.

Cholesteryl ester transfer activity. Localization and role in distribution of cholesteryl ester among lipoproteins in man.

The cholesteryl ester exchange/transfer protein is involved in the transport of cholesteryl ester from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Localization of cholesteryl ester transfer activity (CETA) in plasma was studied by measuring CETA in various delipidated fractions from a single step density ultracentrifugation gradient of plasma. CETA was measured in an in vitro system by calculating the exchange of cholesteryl ester in a standard mixture of [3H]CE-HDL and LDL. The method used for the delipidation of plasmas and fractions to be tested was critical. Optimal results were obtained by delipidation with diisopropylether-butanol (60: 40, v/v) at O degrees C. The bulk of CETA was detected in HDL3 (1.125 less than d less than 1.210 g/ml) when the lipoproteins were separated by single-step density gradient ultracentrifugation and in the 'lipoprotein-free' fraction (d greater than 1.250 g/ml) when the lipoproteins were separated by flotation ultracentrifugation including two washes. To determine whether CETA plays a role in the distribution of cholesteryl ester among the various lipoproteins, it was measured in whole plasma from normal and hyperlipidemic subjects. Plasma was delipidated before the assay in order to prevent bias due to variation of cholesterol content. CETA was higher in delipidated plasma of hyperlipidemic subjects (117.3 +/- 36.5 nmol CE/ml/h) than in delipidated plasma of normolipidemic controls (68.7 +/- 17.6 nmol CE/ml/h) (P less than 0.005). A positive correlation (r = 0.80, P less than 0.005) was found between CETA and (VLDL + LDL) cholesterol levels. A negative correlation (r = 0.57, P less than 0.05) existed between CETA and HDL cholesterol. This correlation was found both in the group as a whole and within the normal and the hyperlipidemic groups separately. The activity of the cholesteryl ester transfer appears to be a regulatory factor in the distribution of cholesteryl ester over the various lipoproteins.

Diet-induced alteration in the activity of plasma lipid transfer protein in normolipidemic human subjects.

Studies were performed to investigate the effect of diets rich in oleic or linoleic acids on the activity of plasma cholesteryl ester transfer protein (CETP) in normolipidemic subjects. Previous to the test diets, all subjects consumed a baseline diet rich in saturated fatty acids ("sat-diet") for 17 days. The test diets, rich in either monounsaturated fatty acids ("mono-diet") or rich in polyunsaturated fatty acids ("poly-diet"), were given for 5 weeks to 52 normolipidemic healthy volunteers. The activity of CETP was measured, using a method independent of endogenous plasma lipoproteins, as the rate of exchange of radioactive cholesteryl oleate between labelled LDL and unlabelled HDL. The "mono-diet" induced a statistically significant decrease in CETP activity (from 115 +/- 20 to 102 +/- 19 units/ml plasma, P less than 0.01), while the small decrease on the "poly-diet" (from 111 +/- 23 to 107 +/- 22 units/ml plasma) did not reach significancy. The percentual decrease in CETP activity induced by the "mono-diet" was higher than that induced by the "poly-diet" as was also found for the decrease in LDL cholesterol. In both diet groups a positive correlation was found between changes in CETP activity and changes in plasma total or (VLDL + LDL) cholesterol. The results suggest that high levels of dietary monounsaturated fatty acids may result in decreased plasma CETP activity, as well as LDL cholesterol levels. The mechanisms of these effects, and their possible interrelations, remain to be established.

Delayed increase in high density lipoprotein-phospholipids after ingestion of a fat load in normolipidemic patients with coronary artery disease.

We studied the effect of a single oral fat load, supplemented with retinyl palmitate (RP), on high density lipoprotein (HDL) lipids in six normolipidemic men with coronary artery disease (CAD) and in six age- and lipid-matched controls. All subjects were selected from a study group which underwent the same protocol 2 years earlier. Post-prandial total plasma lipids, plasma RP levels, and HDL lipids were evaluated at 2-h intervals up till 10 h after the meal. In most subjects the post-prandial response of plasma triglyceride (TG) and plasma RP was identical in the first and second tests. Following the fat load, control subjects showed no change in HDL total cholesterol (TC) or HDL cholesteryl ester (CE) and showed an increase in HDL-TG. CAD subjects however showed a decrease in HDL-TC and HDL-CE and an increase in HDL-TG, similar to the increase in control subjects. In control subjects an increase in HDL phospholipid (PL) was apparent between 0 and 8 h after the fat load. By contrast, in CAD subjects the increase in HDL-PL was only found after as long as 6 h. The magnitude of the post-prandial response of HDL-PL measured during the test was significantly lower in the CAD group. The effects of the fat load on HDL free cholesterol (FC) were similar to the changes in HDL-PL. These data support the hypothesis that PL and FC released during the degradation of chylomicrons as surface remnants are taken up by HDL. This process is clearly delayed in normolipidemic CAD subjects compared with controls. The data suggest that differences in the post-prandial response to an oral fat load in normolipidemic CAD patients and control subjects are not confined to the clearance of TG-rich lipoproteins, but also involve a difference in the uptake of chylomicron surface material by HDL.