Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the defective assembly and secretion of apolipoprotein B-containing lipoproteins caused by mutations in the microsomal triglyceride transfer protein large subunit (MTP) gene (MTTP). We report here a female patient with an unusual clinical and biochemical ABL phenotype. She presented with severe liver injury, low levels of LDL-cholesterol, and subnormal levels of vitamin E, but only mild fat malabsorption and no retinitis pigmentosa or acanthocytosis. Our objective was to search for MTTP mutations and to determine the relationship between the genotype and this particular phenotype. The subject exhibited compound heterozygosity for two novel MTTP mutations: one missense mutation (p.Leu435His) and an intronic deletion (c.619-5_619-2del). COS-1 cells expressing the missense mutant protein exhibited negligible levels of MTP activity. In contrast, the minigene splicing reporter assay showed an incomplete splicing defect of the intronic deletion, with 26% of the normal splicing being maintained in the transfected HeLa cells. The small amount of MTP activity resulting from the residual normal splicing in the patient explains the atypical phenotype observed. Our investigation provides an example of a functional analysis of unclassified variations, which is an absolute necessity for the molecular diagnosis of atypical ABL cases.

PAF-acetylhydrolase, predominantly present in LDL in healthy subjects, is associated with HDL in a patient with LDL deficiency.

The degradation of platelet-activating factor (PAF) in plasma is catalyzed by PAF-acetylhydrolase resulting in lyso-PAF which is biologically inactive. Normally, most of the PAF-degrading activity is associated with low-density lipoproteins (LDL). The enzyme activity was measured in the plasma of a patient with abetalipoproteinemia, a disorder characterized by the absence of apolipoprotein-B-containing lipoproteins (chylomicrons, VLDL and LDL). Here we report that the plasma of the patient has a normal activity of PAF-acetylhydrolase. The enzyme activity is bound to high-density lipoproteins (HDL) and shows the kinetic properties of the LDL-associated enzyme of healthy subjects. Following administration of artificial triglyceride-rich particles (ATRP), part of the enzyme activity is found associated with ATRP, indicating that PAF-acetylhydrolase can transfer from HDL to triglyceride-containing lipid complexes in vivo.

Lipoproteins alter the catalytic behavior of the platelet-activating factor acetylhydrolase in human plasma.

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation, allergy, shock, and thrombosis. A specific degradative enzyme, PAF acetylhydrolase (EC 3.1.1.47), is found in plasma and could regulate the concentration of PAF in blood. In plasma, 70% of the PAF acetylhydrolase is found with low density lipoprotein (LDL), and the remainder is in high density lipoprotein (HDL). In previous studies we found that with subsaturating concentrations of PAF the activity in LDL seemed to be the relevant one; e.g., depletion of LDL slowed degradation of PAF, while removal of HDL accelerated the degradation slightly. We have pursued this observation by using plasma from humans with lipoprotein mutations. In abetalipoproteinemia, all of the PAF acetylhydrolase activity was in HDL, whereas in Tangier disease all of the activity was in LDL. In both conditions the total activity measured in an optimized assay was normal or increased. However, when we measured the t1/2 of PAF in plasma, we found that it was prolonged in subjects with abetalipoproteinemia compared to normal controls. Conversely, the t1/2 in Tangier plasma was shortened. We next demonstrated that the PAF acetylhydrolase in HDL was recognized by an antibody to the enzyme purified from LDL, establishing that the enzyme in the two particles is the same protein. Finally, we inactivated the PAF acetylhydrolase in isolated lipoprotein particles and then reconstituted them with enzyme from the opposite particle. The reconstituted particles were used to measure the t1/2 of PAF, and we again found that the LDL particle was more efficient. We conclude that the lipoprotein environment of the PAF acetylhydrolase markedly influences its catalytic behavior. This may be important in pathophysiology and will complicate attempts to assess the role of this enzyme in such circumstances.

Substrate specificity of plasma lecithin: cholesterol acyltransferase in abetalipoproteinemia.

The lecithin: cholesterol acyltransferase (LCAT) activity of lipoprotein-depleted plasma from a patient with abetalipoproteinemia has been assayed in a modified Glomset-Wright incubation system with three different normal lipoprotein substrates consisting of an authentic mixture of very low (VLDL), low (LDL) and high (HDL) density lipoproteins for the assay of total LCAT activity, HDL to assay alpha-LCAT activity and combined VLDL and LDL to assay beta-LCAT activity, respectively. Although reduced to about half the normal control values, both alpha- and beta-LCAT activities were present in the patient's plasma. It has been shown earlier that secretion of LCAT is linked to that of VLDL, but since patients with abetalipoproteinemia cannot form either chylomicrons or VLDL, our results suggest that a secretion of these triglyceride-rich lipoproteins do not seem to be a prerequisite for a basal secretion of beta-LCAT.

Lipoprotein lipase and hepatic lipase activity after heparin administration in abetalipoproteinemia and hypobetalipoproteinemia.

The purpose of this study was to examine whether an absence of triglyceride-rich lipoproteins (chylomicrons and very-low-density lipoproteins) in plasma is associated with any changes in the enzyme activity of lipoprotein lipase or hepatic lipase after heparin administration. To study this, the activities of hepatic lipase and lipoprotein lipase were determined in control subjects, in two patients with heterozygous hypobetalipoproteinemia, and in three patients with phenotypic abetalipoproteinemia after administration of heparin. Both enzymes showed normal activity in the patients with hypobetalipoproteinemia, but showed consistently reduced activity in the patients with abetalipoproteinemia. Hepatic lipase activity in plasma samples from these three patients obtained 15 minutes after intravenous injection of heparin was 55%, 87%, and 46% of that of the controls, whereas corresponding values in plasma samples obtained 30 minutes after heparin were 47%, 70%, and 57%, respectively. Lipoprotein lipase activity in the three patients with abetalipoproteinemia was 46%, 29%, and 34% of that of the controls in the samples obtained 15 minutes after heparin injection, whereas the values obtained after 30 minutes were 53%, 64%, and 47% of that of the controls. We conclude that an inherent absence of triglyceride-rich lipoproteins, as occurs in abetalipoproteinemia, is associated with reduced enzyme activity of both hepatic lipase and lipoprotein lipase in plasma after heparin administration.

Requirement of low density lipoproteins for the lysolecithin acyl transferase activity in human plasma: assay of enzyme activity in abetalipoproteinemic patients.

Previous studies from this laboratory have shown that the lecithin:cholesterol acyl transferase (LCAT) of normal human plasma can convert lysolecithin to lecithin in the presence of low density lipoproteins (LDL). In order to study the importance of LDL for the lysolecithin acyl transferase (LAT) activity, the LCAT and LAT activities were assayed in two patients with abetalipoproteinemia. The plasma from both patients had only 5%-6% of the LAT activity present in normal plasma. This activity was stimulated up to 22-fold by the addition of normal LDL but not very low density lipoproteins (VLDL) or high density lipoproteins. The LAT activity of normal plasma was only stimulated by two-threefold by LDL. The LCAT activity in both patients was reduced to 42%-46% of the control values. This activity was stimulated up to fourfold by the addition of LDL as well as VLDL which is comparable to the activation obtained with control plasma. These results therefore suggest that LDL is physiological activator of LAT reaction in normal human plasma.

[Intestinal enzymopathies of children (author's transl)]. / Intestinale Enzymopathien beim Kinde

In recent years it has been successfully demonstrated that, in relation to congenital metabolic abnormalities, every enzyme of the gastro-intestinal tract may be absent or inactive.. In serious diseases of the intestinal tract, secondary inactivation of numerous enzymes may result. In this comprehensive presentation the pathology of the disaccharidases, the peptidases and lypolysis is described, the physiology being gone into briefly in each case. Furthermore, all known disturbances of absorption of sugar, aminoacids and fats are briefly dealt with. Finally, congenital chloridorrhea is described.

3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human hair roots.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (1.1.1.34) was measured in isolated human hair roots. In growing scalp hair roots, the amount of enzyme activity per cell was surprisingly high and was of the same order of magnitude as has been reported for human liver cells and for human fibroblasts cultured in the absence of low density lipoproteins. Hair root enzyme activity showed no diurnal variation, was unaffected by cholestyramine feeding, and was similar in 14 normal subjects, one patient with abetalipoproteinemia, and one homozygote and three heterozygotes with familial hypercholesterolemia. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human hair roots appears to be unrelated to the level of low density lipoproteins in the plasma.