Mutations in lipoprotein lipase that block binding to the endothelial cell transporter GPIHBP1.

GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, shuttles lipoprotein lipase (LPL) from subendothelial spaces to the capillary lumen. An absence of GPIHBP1 prevents the entry of LPL into capillaries, blocking LPL-mediated triglyceride hydrolysis and leading to markedly elevated triglyceride levels in the plasma (i.e., chylomicronemia). Earlier studies have established that chylomicronemia can be caused by LPL mutations that interfere with catalytic activity. We hypothesized that some cases of chylomicronemia might be caused by LPL mutations that interfere with LPL's ability to bind to GPIHBP1. Any such mutation would provide insights into LPL sequences required for GPIHBP1 binding. Here, we report that two LPL missense mutations initially identified in patients with chylomicronemia, C418Y and E421K, abolish LPL's ability to bind to GPIHBP1 without interfering with LPL catalytic activity or binding to heparin. Both mutations abolish LPL transport across endothelial cells by GPIHBP1. These findings suggest that sequences downstream from LPL's principal heparin-binding domain (amino acids 403-407) are important for GPIHBP1 binding. In support of this idea, a chicken LPL (cLPL)-specific monoclonal antibody, xCAL 1-11 (epitope, cLPL amino acids 416-435), blocks cLPL binding to GPIHBP1 but not to heparin. Also, changing cLPL residues 421 to 425, 426 to 430, and 431 to 435 to alanine blocks cLPL binding to GPIHBP1 without inhibiting catalytic activity. Together, these data define a mechanism by which LPL mutations could elicit disease and provide insights into LPL sequences required for binding to GPIHBP1.

The recovery of dysfunctional lipoprotein lipase (Asp204-Glu) activity by modification of substrate.

Functional deficiency of lipoprotein lipase (LPL) was found in a patient with severe hypertriglyceridemia. The patient was 39-year-old man with a plasma triglyceride level of 2032 mg/dl, and suffered from recurrent pancreatitis. His post heparin plasma LPL mass was almost normal, but the LPL activity was remarkably decreased. Gene analysis showed that homozygote missense mutation (204 Asp (GAC)-Glu (GAG)) exists in exon 5 of LPL gene. The patient LPL purified from post heparin plasma scarcely hydrolyzed VLDL-triglyceride and also triolein emulsified with Triton X-100 or phosphatidylcholine. When phosphatidylethenolamine, phosphatidylserine and cardiolipin were used as an emulsifier for triolein, triolein-hydrolyzing activity of the patient's LPL was observed and was much higher than that of wild-type LPL. Mutant LPL gene (Asp204-Glu) was made by site-direct mutagenesis and was transfected to COS-1 cell. The expressed LPL (Asp204-Glu) also showed the same properties. These results suggested that the LPL (Asp204-Glu) is a functional deficiency, and the activity could be recovered by using acidic phospholipids as an emulsifier.

Elevated lipoprotein lipase activity in skeletal muscle tissue during treatment of hypertriglyceridaemic patients with bezafibrate.

Eleven hypertriglyceridaemic patients were treated with 600 mg bezafibrate daily for 2 months. The mean serum triglyceride concentration decreased by 35% (P less than 0.01) corresponding to a reduction in the very low density lipoprotein triglycerides by 41% (P less than 0.01). The high density lipoprotein cholesterol concentration and the serum concentration of apolipoproteins AI and AII increased by 19% (P less than 0.02), 11% (N.S., P less than 0.08) and 24% (P less than 0.01), respectively. The fractional removal rate (K2) in the intravenous fat tolerance test (IVFTT) increased by 37% (P less than 0.001). This was associated with significant increase of the skeletal muscle lipoprotein lipase activity ( + 39%, P less than 0.05). The mean value for the adipose tissue lipoprotein lipase activity remained unchanged. The K2 at IVFTT was correlated highly significantly to the activity of skeletal muscle lipoprotein lipase (r = 0.85, P less than 0.01). The present study indicates that the main reason for the reduction of the serum triglycerides during treatment with bezafibrate may be an increased activity of the skeletal muscle tissue lipoprotein lipase activity.

Postprandial adipose tissue lipoprotein lipase activity in primary hypertriglyceridemia.

The fasting activity of adipose tissue lipoprotein lipase has been previously reported to be either normal or reduced in subjects with a primary form of hypertriglyceridemia. The postprandial activity of adipose tissue lipoprotein lipase has not been previously reported in these subjects. In subjects with primary hypertriglyceridemia the fasting lipoprotein lipase activity eluted from pieces of adipose tissue by heparin and the enzyme activity present in extracts of acetone--ether tissue powders were similar to the level of enzyme activity found in normal subjects. There also was no difference in the postprandial adipose tissue heparin-elutable lipoprotein lipase activity between these two groups when measured after high carbohydrate feeding. When the subjects with primary hypertriglyceridemia were further subdivided by genetic diagnosis, there was no difference in the level of adipose tissue lipoprotein lipase of subjects with familial hypertriglyceridemia, familial combined hyperlipidemia, or in those in whom no specific genetic diagnosis could be made. The change in lipoprotein lipase activity after feeding was inversely related to the fasting enzyme level in both the normal subjects (r = -0.58, p less than 0.05, n = 12) and the hypertriglyceridemic subjects (r = -0.92, p less than 0.01, n = 11). In the normal subjects, the plasma triglyceride response to feeding correlated inversely with the postprandial change in lipoprotein lipase activity (r = -0.76, p less than 0.02, n = 12). Adipose tissue lipoprotein lipase activity in patients with primary lipoprotein lipase deficiency was markedly reduced in the fasting state and remained essentially zero after feeding. This suggests that a functional role exists for the enzyme activity as measured.

Effects of slow-release bezafibrate on serum lipids, lipoproteins, apolipoproteins, and postheparin lipolytic activities in patients with type IV and type V hypertriglyceridemia.

The effects of bezafibrate on serum lipids, lipoproteins, apolipoproteins, and post-heparin lipolytic activities were studied in 17 patients with hypertriglyceridemia. All patients received 400 mg of slow-release (SR) bezafibrate daily for four months. In the nine patients with type IV hypertriglyceridemia, mean serum triglyceride (TG) levels decreased significantly, by 53% (P less than 0.01) at two months and 50% (P less than 0.001) after four months of bezafibrate, while in the eight patients with type V, the levels decreased by 61% (P less than 0.001) and 51% (P less than 0.001), respectively. Total cholesterol levels decreased significantly (P less than 0.05) in type V patients at two and four months, by 19% and 18%, respectively, while low-density lipoprotein cholesterol levels increased significantly (P less than 0.05) in type IV patients at two and four months, by 63% and 62%, respectively. High-density lipoprotein (HDL) levels increased significantly (P less than 0.05) at two months in both patient groups. HDL subfraction analysis showed a significant (P less than 0.05) rise in HDL3-cholesterol levels in type V but not in type IV patients. Apolipoprotein (apo) A-I, A-II, and B levels increased, while apo C-II, C-III, and E levels decreased in both groups. The apo A-I/apo A-II ratio decreased significantly (P less than 0.05) at two and four months in type V patients, which also supports increased HDL3 fractions in that group. Lipoprotein lipase and hepatic TG lipase levels tended to rise, and the particle size of TG-rich lipoprotein (TGRL) and the TGRL-apo C-III/TGRL-apo C-II ratio decreased in both patient groups. These data indicate that bezafibrate-induced changes in lipoprotein profiles differed slightly in type IV and type V patients. The results confirm the usefulness of bezafibrate as a lipid-lowering agent.

Serum immunoreactive trypsin in type IV hyperlipoproteinemia.

Serum immunoreactive trypsin (IRT) under basal conditions and after pancreatic stimulation with secretin was studied in 10 patients with type IV hyperlipoproteinemia (HLP) and in 10 control subjects. No significant difference was observed between basal values of the two groups (p = NS). The increase of serum IRT was already significant 5 minutes after secretin administration (p < 0.01) and persisted with significance for one hour in both groups. The integrated trypsin output (ITO) was significantly greater in type IV HLP than in controls (510.3 +/- 17.8 and 72.2 +/ 17.6 respectively, mean +/- SEM, p < 0.0125). Only 2 (20%) of 10 patients with HLP had an ITO in the range of the controls. No significant correlation was found between ITO and triglyceride levels (p = NS). The response of serum IRT to secretin in HLP patients appears comparable to that observed in alcoholics and in patients with chronic pancreatitis with mild to moderate exocrine dysfunction, in whom there may be an abnormal regurgitation of trypsin-like material into the blood stream after secretin stimulation, probably due to an obstruction to pancreatic secretory flow. A similar obstructive mechanism may be hypothesized also in patients with HLP, but no data concerning the exocrine pancreatic secretion and the histological features of the pancreas are available to confirm this hypothesis.

Effect of cimetidine on trypsin-like immunoreactivity in serum.

Serum trypsin-like immunoreactivity (TLI) was studied after administration of secretin (GIH, 75 CU i.v.) in 10 alcoholics and in 3 patients with type IV hyperlipoproteinemia. A significant increase of TLI was observed after secretin administration and persisted throughout the test. In order to assess the possibility of an acid-sensitive effect, the test was repeated on a different day, one hour after the administration of cimetidine (400 mg. p.o.). Cimetidine did not affect serum TLI for one hour, but did reduce significantly the response of serum TLI to injection of secretin (p < 0.01). Some studies were performed, which failed to show any effect of cimetidine on pancreatic exocrine function. However, they were based on the measurement of pancreatic secretion into the duodenum, which required aspiration of gastric secretion from the stomach before entering the duodenum. Therefore, the reduction of TLI response to injection of secretin may be tentatively attributed to the inhibition of gastric acid secretion and delivery to the duodenum caused by cimetidine. If an endogenous acid-sensitive mechanism is involved, it is unlikely to be mediated by secretin.

[Lipoprotein lipase activity of ischemic heart disease patients with different types of hyperlipoproteinemia on the basis of fat loading]. / Aktivnost' lipoproteidlipazy u bol'nykh ishemicheskoi bolezn'iu serdtsa s razlichnymi tipami giperlipoproteidemii na fone zhirovoi nagruzki.

Lipolytic activity was distinctly increased in blood plasma of patients with ischemic heart impairment and various types of hyperlipoproteinemia after a test breakfast. The increase in lipolysis was due to elevation in the lipoprotein lipase activity in response to loading with lipids. Attempts to distinguish among the ischemic patients some subgroups on the basis only of the initial level of triglycerides in their blood plasma did not exhibit any differences in the enzymatic activity after the loading with lipids. This relationship was clearly noted in classification of the patients on the basis of the types of hyperlipoproteinemia. The earliest and the most pronounced increase in lipolytic activity in response to the loading with lipids was observed in the 11a type of hyperlipoproteinemia.

A novel substitution at the translation initiator codon (ATG-->ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis.

A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.

Activation of plasma prorenin by plasminogen activators in vitro and increase in plasma renin after stimulation of fibrinolytic activity in vivo.

Plasminogen can be activated by intrinsic activators that circulate in plasma in a precursor form, by extrinsic activator originating from tissues or the vessel wall and by the exogenous activators, urokinase and streptokinase. Tissue activator and vascular activator are probably identical. Dialysis of plasma against pH 4.0 buffer causes denaturation of the plasmin inhibitors, alpha 2-antiplasmin and C1-inhibitor, while alpha 2-macroglobulin is left intact. Incubation of pH 4.0-pretreated plasma with urokinase or streptokinase at pH 7.5 led to activation of plasminogen and prorenin. Incubation of a plasma fraction, which contained plasminogen and prorenin but no alpha 2-antiplasmin and renin, with highly purified tissue plasminogen activator also led to activation of prorenin. The vasopressin analogue, 1-desamino-8-D-arginine vasopressin (DDAVP), is a potent stimulant for the release of extrinsic activator into the bloodstream. After infusion of DDAVP, 0.4 micrograms/kg, into normal subjects, parallel increments in plasma fibrinolytic activity and renin were observed. Infusion of DDAVP into patients with type IV hyperlipoproteinaemia had little effect on plasma fibrinolytic activity and the response of plasma renin was also subnormal. These observations warrant further studies on a possible role for plasminogen activators in prorenin activation in vivo.

[Evaluation of the pseudocholinesterase activity in hyperlipoproteinemia type II and type IV]. / Valutazione dell'attivita' pseudocolinesterasica (PCE) nelle iperlipoproteinemie tipo IIa e tipo IV.

Authors have evaluated pseudocholinesterase activity in patients with type IIa and type IV hyperlipoproteinemia. Significative correlation has been found between PCE and C in type IV hyperlipoproteinemia. Authors suggest PCE activity can be proposed as useful biochemical marker of hyperlipoproteinemia.

Paradoxical esterification of plasma cholesterol in fish eye disease.

The activity of lecithin: cholesterol acyl transferase (LCAT), the enzyme which catalyses the esterification of human plasma cholesterol, has been measured by two independent methods in plasma from the two known living Swedish patients with fish eye disease. The enzyme activity was in both cases about 15% of that of normal plasma. Paradoxically, however, the percentage of plasma cholesterol which was esterified was almost normal in both patients. In addition, a normal spectrum of the fatty acids of the cholesteryl esters was present indicating a normal cholesterol esterification pathway in vivo. Incubation experiments in vitro of plasma from the two patients also yielded normal cholesterol esterification rates when measured by two different methods. These paradoxical results for cholesterol esterification are discussed on the basis of the present biochemical knowledge of fish eye disease and LCAT deficiency.

[Pseudohypertriglyceridemia in glycerokinase deficiency]. / Pseudo-Hypertriglyceridämie bei Glycerokinase-Mangel.

Falsely high serum triglyceride concentrations (410-850 mg/dl) were measured in four members of a family of five from Franconia in Germany. The cause was hyperglyceridaemia on the basis of glycerol kinase deficiency. None had any symptoms and no other metabolic anomaly was demonstrated. The possibility of glycerol kinase deficiency should be considered in any case of elevated serum triglyceride concentration but with clear serum, normal lipid electrophoresis and lack of response to lipid-lowering measures.

Role of triglyceride-rich lipoproteins and hepatic lipase in determining the particle size and composition of high density lipoproteins.

These studies examined the proposition that the small particle size of HDL3 in the plasma of hypertriglyceridemic subjects is the consequence of the sequential actions of lipid transfer protein and hepatic lipase on HDL. Incubation of unmodified total HDL or HDL3 in the presence of hepatic lipase resulted in a depletion of phospholipid, but little change in the size of the particles. On the other hand, HDL3 that had first been depleted of cholesteryl ester and enriched with triglyceride and phospholipid, during prior incubation with Intralipid and a source of lipid transfer protein, were much more susceptible to the action of hepatic lipase. When these modified HDL3 were incubated with hepatic lipase there was a depletion of the triglyceride and phospholipid content and a conversion into much smaller particles the same size as those predominant in hypertriglyceridemic subjects. These very small particles were derived from a population of modified particles that were larger than the original HDL3 and were within the size range of HDL2. It is proposed, therefore, that in the plasma of hypertriglyceridemic subjects there exists a dynamic balance between the formation of enlarged triglyceride-rich HDL and a secondary conversion of these particles by hepatic lipase to form populations of very small HDL.

Prevalence of alleles encoding defective lipoprotein lipase in hypertriglyceridemic patients of French Canadian descent.

It has previously been estimated that due to genetic "founder effects," 97% of lipoprotein lipase (LPL) gene alleles conferring type I hyperlipoproteinemia (HLP) in French Canadians encode one of the following mutant LPL forms: Gly188-->Glu, Pro207-->Leu, or Asp250-->Asn. Although the genetic basis of type I HLP is known to be homozygosity for LPL deficiency, that for other forms of HLP, especially types IV, and V HLP, is not clear. It is also unclear whether hypertriglyceridemia due to very low density lipoprotein (VLDL) overproduction can be distinguished phenotypically from that due to defective catabolism of plasma lipoprotein triglycerides. The present study took advantage of the unique circumstances inherent in the relatively genetically isolated French Canadian population to address these questions. This study was carried out in order to determine the prevalence of these three mutant LPL alleles, and of a fourth encoding LPL Asn291-->Ser, in French Canadian patients with hypertriglyceridemia. The prevalence of heterozygosity for one of the four LPL mutant alleles in nondiabetic, nonobese hypertriglyceridemic subjects was 16 of 95 type IV HLP (17%) and 4 of 26 type V HLP cases (15%). These alleles were not found in over 150 normotriglyceridemic subjects, supporting the likelihood that the mutant alleles were at least partially responsible for HLP. In addition, heterozygosity for LPL deficiency due to one of these mutations apparently did not contribute to hypoalphalipoproteinemia, and was observed in 3 of 39 subjects with type III HLP. The results suggest that in French Canadians, 15-20% of type IV and V HLP cases are associated with these genetic defects in plasma triglyceride catabolism.

Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-alpha-triglyceridemia.

Hyper-alpha-triglyceridemia is a rare dyslipoproteinemia characterized by a pronounced increase in the concentration of triglycerides in the plasma high density lipoprotein (HDL) fraction. One case with this condition, an apparently healthy 61-year-old man, has been studied. Additional lipoprotein abnormalities were present, such as abnormally cholesterol-rich very low density lipoproteins (VLDL) with retarded electrophoretic mobility (beta-VLDL) and triglyceride enrichment of low density lipoproteins (LDL). The patient's plasma concentration of apolipoproteins A-I, A-II and B were normal and those of C-I, C-II, C-III and E were elevated. No abnormal forms of the soluble apolipoproteins of VLDL and high density lipoproteins (HDL) were found after analysis by isoelectric focusing. Lecithin:cholesterol acyltransferase activities, plasma cholesterol esterification rates and lipid transfer protein activities were normal. Post-heparin plasma activity of hepatic lipase was virtually absent and that of lipoprotein lipase was reduced by 50%. In plasma of this patient, HDL was almost exclusively present as large triglyceride-rich particles corresponding in size to particles of the HDL2 density fraction. The only brother of the patient also had hyper-alpha-triglyceridemia together with the other lipoprotein abnormalities described for the index case and deficiency of postheparin plasma activity of hepatic lipase. The findings presented below support the hypothesis that one primary function of hepatic lipase is associated with degradation of plasma HDL2. Deficiency of this enzyme activity thus causes accumulation of HDL2 in plasma leading to hyper-alpha-triglyceridemia. The results further suggest that the abnormal chemical and electrophoretic properties of VLDL and LDL in plasma from the patient, reminiscent of type III hyperlipoproteinemia, are secondary to the lack of the action of hepatic lipase on the HDL particles.

A newly identified heterozygous lipoprotein lipase gene mutation (Cys239-->stop/TGC972-->TGA; LPLobama) in a patient with primary type IV hyperlipoproteinemia.

We investigated measures for identification of heterozygous lipoprotein lipase (LPL) deficiency in unrelated subjects with primary type IV hyperlipoproteinemia in order to acquire a helpful clue for understanding the correlation between hypertriglyceridemia and the status of being a heterozygous carrier of an LPL gene variant. Identification of heterozygous LPL deficiency was performed by monitoring the immunoreactive LPL mass in postheparin plasma (PHP) using our developed sandwich-enzyme immunoassay technique for first screening. Then, in subjects found to have half or less than half of the control LPL mass value in PHP, the polymerase chain reaction-single strand conformation polymorphism method was used to detect LPL gene aberrations as a second screening. This approach was evaluated as being useful as it succeeded in identifying a subject (proband KD) with heterozygous LPL deficiency. The mutation in the LPL gene of proband KD was newly characterized as a nucleotide C972 to A transversion in exon 6, resulting in substitution of a premature termination codon (TGA) for Cys239 (TGC). This nonsense mutation, designated as LPLobama, creates an MboI restriction site and eliminates an HgiAI restriction site, and this allows rapid screening of subjects with type IV as well as type I hyperlipoproteinemia for the mutation. The homozygous state for the LPLobama allele resulted in neither detectable LPL activity nor immunoreactive LPL mass in PHP, and this was seen in two of proband KD's siblings.