Measurement Of Lipoprotein Lipase Activity Research Articles

Using lipase activity to probe milkfat globule membranes (MFGM) or interfaces

Measurement of lipoprotein lipase activity (as free fatty acid change or accumulation) is accepted as indicative of milkfat globule membrane (MFGM) disruption. However, measurement is confounded by variables unrelated to the MFGM, e.g., microbial quality. To resolve this, a modified approach to using lipase activity to probe MFGM was developed. Methanol (1%, w/w) and lipoprotein lipase (2%, w/w, raw milk) were added to pasteurised milk samples resulting in formation of methyl esters that therefore served as a new and unique signal for lipase gaining access to interfacial fat. Analysis conditions minimised product formation to minimise sample perturbation. The method could detect 1% (w/w) of homogenised milkfat globules blended with native globules, with linear response to 10% (w/w) and saturation above 20% (w/w). The approach was applied to assess MFGM in samples subjected to disruptive treatments (shear, aeration) under different conditions; findings were consistent with previous research, but with greater sensitivity.

A novel nonsense mutation in the LPL gene in a Chinese neonate with hypertriglyceridemia

Background Lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder characterized by hypertriglyceridemia. The genetic defect lies in a mutation of the LPL gene. Methods A Chinese neonate with non-consanguineous parents was incidentally found to have hypertriglyceridemia. Mutation in her LPL gene was screened by using polymerase chain reaction and direct DNA sequencing. Results Homozygous missense mutations (L252V) were detected in the LPL gene of the patient. A novel nonsense mutation (C27X) was also identified. Conclusion Our finding supports L252V mutation in the LPL gene is a common mutation in Chinese with familial hyperchylomicronemia syndrome. DNA-based diagnosis in this syndrome is definitive. It saves the need for heparin-infusion test, which carries the risk of hemorrhage, and the measurement of LPL activity, which is tedious and is not widely available.

Calibration, specificity and trueness of a postheparin plasma lipoprotein lipase assay.

Measurement of lipoprotein lipase activity in postheparin plasma is generally accompanied by moderate within-run variation CV(W-R) (<10%) and higher between-run variation CV(B-R) (5-25%). A calibration system was introduced in order to improve the reproducibility of measurements and to compare lipoprotein lipase activities from different days. Every day a calibration curve for lipoprotein lipase activity was constructed. Fifteen calibration curves designed over 2 years, show linearity over the whole biological spectrum and a considerable reduction of between-run variation in lipoprotein lipase activity, from 42% to 5.3% as estimated from two control postheparin plasma samples. The lipoprotein lipase calibration system is an easy and very cheap arrangement, which makes it possible to compare lipoprotein lipase activities achieved over years. When the lipoprotein lipase control values are compared with reference lipoprotein lipase samples determined in other lipase laboratories, the calibration-control system becomes an important tool for reducing analytical bias. The article reviews the original analytical criteria of catalytic measurement of lipoprotein lipase activity and describes the implementation of the calibration-control system. We describe a model for reduction of the analytical variability in the measurement of lipoprotein lipase activity. Other standardization efforts need to be made in the future, especially to define the reference material for calibration.

Androgen Effect on Body Composition and Fat Metabolism

Recognizing the adverse metabolic effects of predominantly visceral body fat distribution associated with low testosterone levels, researchers have investigated the effects of androgen treatment on body fat distribution in men. This presentation reviews the results of research on acute and longer-term effects of treatment with testosterone and dihydrotestosterone. Methods for investigating these results, including measurement of lipoprotein lipase activity and of radiolabeled fat uptake and turnover, are described and discussed.

Studies on equine lipid metabolism. 2. Lipolytic activities of plasma and tissue lipases in large horses and ponies.

The enzymatic fundamentals of lipid metabolism of equine have not been thoroughly investigated at this point in time. It is still unclear why ponies in contrast to horses may become hyperlipaemic when coming negative energy balance. In this study, the activities of the triglyceride-cleaving key enzymes of ponies are large bred horses were investigated in order to obtain insight into the aetiology of the syndrome. The objective of the study was to measure the activities of hormone-sensitive lipase (HSL), lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in ponies and horses in ex vivo in vitro assays. Norepinephrine (NE) stimulated pony adipocytes to release FFA in a linear fashion (4.57 +/- 2.09 nmol FFA.10(5) cells-1.min-1). This was not observed in horses. Lipolysis was significantly higher in fat cells of ponies than in horses when adenosine deaminase (ADA) and NE were added (12.71 +/- 3.12 vs. 1.96 +/- 1.22 nmol FFA.10(5) cells-1.min-1). Relative inhibition of lipolysis by the action of insulin was comparable in adipocytes of horses and ponies. However, absolute FFA release in pony fat cells was as high as the maximal NE and ADA stimulated lipolysis in horse adipocytes. Postheparin plasma lipase activities in ponies and horses did not differ between the sub-species. This finding was supported by the results obtained from measurement of LPL activity in adipose and muscle tissue showing only a tendency of increased activities in pony explants when compared to horse tissue incubations. This study further supports the hypothesis that differences in regulation of TG release from fat stores rather than clearance of TG from plasma is causative for the development of hyperlipaemia in ponies. Abbreviations used: ADA, adenosine deaminase; BW, body weight; FFA, free fatty acid; HSL, hormone-sensitive lipase; HTGL, hepatic triglyceride lipase; LPL, lipoprotein lipase; NE, norepinephrine; SDS, sodium dodecyl sulfate; TG, triglyceride; VLDL, very low density lipoprotein.

A practical approach to the laboratory diagnosis of dyslipidemia.

Disorders in lipoprotein metabolism (dyslipidemia) can result in premature atherosclerosis or pancreatitis. Dyslipidemias can be classified as hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, and low levels of high density lipoprotein (HDL) cholesterol. All of the dyslipidemias can be primary or secondary. Both elevated levels of low density lipoprotein cholesterol and decreased levels of HDL cholesterol predispose to premature atherosclerosis. Triglyceride levels greater than 1,000 mg/dL increase the risk for pancreatitis. In the appraisal of the dyslipidemias, measurement of serum cholesterol, triglycerides, HDL-cholesterol and obtaining the LDL cholesterol by Friedewald equation is usually sufficient in the majority of patients. However, in some cases, such as the diagnosis of the Type III dyslipidemia and when triglycerides are > or = 400 mg/dL, ultracentrifugation is required to determine the VLDL or LDL cholesterol. Lipoprotein electrophoresis can be useful in the diagnosis of Type III dyslipidemia (broad beta band) and also to detect chylomicrons. In young subjects with coronary artery disease with a normal LDL cholesterol an apolipoprotein B-100 level may be a useful test. In children and young adults with severe hypertriglyceridemia, measurement of lipoprotein lipase activity or assaying apolipoprotein C-II levels can be useful in elucidating the cause. Also, laboratory tests are useful in excluding a secondary cause of dyslipidemia (urinalysis, plasma creatinine, TSH, glucose, protein electrophoresis, alkaline phosphatase and transaminases). Thus, laboratory investigations play an important role in the management of dyslipidemia.

Low dose continuously infused growth hormone results in increased lipoprotein(a) and decreased low density lipoprotein cholesterol concentrations in middle-aged men.

Animal studies have shown that slight increases in basal GH concentrations may result in changes in lipoprotein metabolism. Such changes in GH secretion have been observed in physiological and pathophysiological states such as fasting, uncontrolled diabetes and during oestrogen treatment. The aim of this study was to investigate the possible effects of increases in basal plasma GH concentrations on lipoprotein concentrations. Recombinant human growth hormone (rhGH) was given as a continuous subcutaneous infusion in a low dose (0.02 U/kg/day) in an open study. Eight middle-aged (42-59 years) overweight (body mass index: 26.1-33.8 kg/m2) but otherwise healthy men were studied over a period of 14 days. Blood samples were obtained after an over-night fast before and after 2, 7 and 14 days of treatment. Plasma and serum were separated and used for subsequent measurements of hormone and lipoprotein concentrations. On days 0, 7 and 14 of treatment, post-heparin plasma was also obtained for determinations of plasma lipoprotein lipase and hepatic lipase activities. In addition, a hyperinsulinaemic euglycaemic glucose clamp was performed on days 0 and 13 of the study. Fat biopsies from abdominal and gluteal fat depots were obtained for measurement of lipoprotein lipase activities on days 0 and 14 of the study. Serum GH concentrations increased to a steady level of 2-4 mU/l during treatment. Serum insulin-like growth factor-I (IGF-I) concentrations increased throughout the treatment period to twice the pretreatment levels. Plasma insulin and blood glucose concentrations increased on day 2 of treatment. After 7 and 14 days of treatment blood glucose concentrations were not different from pretreatment levels, but plasma insulin concentrations were still elevated. Serum cholesterol and low density lipoprotein (LDL) cholesterol concentrations had decreased after 7 and 14 days of treatment. High density lipoprotein (HDL) cholesterol concentrations were not affected, but very low density lipoprotein (VLDL) cholesterol and triglyceride concentrations increased transiently at day 2 of treatment. Serum apolipoprotein (apo) A-I, apoB and apoE concentrations were not significantly affected. Serum lipoprotein(a) concentrations had increased by days 7 and 14 to 147 and 142% of pretreatment concentrations, respectively. Lipoprotein lipase and hepatic lipase activities in post-heparin plasma, as well as abdominal and gluteal adipose tissue lipoprotein lipase activities, were not affected. There was no significant change in glucose disposal rate estimated from the glucose clamp studies. A low dose infusion of GH results in marked changes in lipoprotein concentrations with a transient increase in VLDL cholesterol and thereafter in a decrease in LDL cholesterol. In addition, this low dose of GH resulted in marked increases in lipoprotein(a) concentrations. The observed effects of GH may partly involve changes in IGF-I and insulin secretion.

Effect of Prolactin (PRL) on Lipoprotein Lipase (LPL) Activity in the Rat Fetal Liver

In order to clarify the possible involvement of PRL in the regulation of lipid metabolism, the influence of PRL on the levels of LPL activity in rat fetal liver was investigated. Rat fetuses at 18th day of gestation in one uterine horn were injected with o-PRL while those in the opposite horn with saline. Forty-eight hr later, the livers were removed from the fetuses, homogenized, defatted, dried overnight, and further homogenized to the enzyme suspension. The measurement of LPL activity in these suspensions revealed that LPL activity in the fetal liver treated with o-PRL was significantly higher than that of control. LPL activities in the rat fetal liver gradually increased until the time of birth as the gestation proceeded. These results suggest that PRL may be one of the factors regulating fetal lipid metabolism.

Detection and characterization of the heterozygote state for lipoprotein lipase deficiency.

Because there are no characteristic clinical or biochemical manifestations, the heterozygote state for lipoprotein lipase (LPL) deficiency has been difficult to detect. Measurements of postheparin plasma LPL activity and of LPL mass were performed in six families of probands with LPL deficiency to characterize the heterozygote state. LPL mass was measured in a sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (5D2) that had been produced against bovine milk LPL. Thirteen obligate heterozygotes from these families had reduced LPL activity and mass below the 95th percent confidence limits of 34 normal controls, while one obligate heterozygote had LPL activity and mass between the 90th and 95th percent confidence limits. Potential heterozygotes in these families were identified as normal (n = 8) or heterozygotes (n = 6) by comparison to the 95th percent confidence limits of the controls. Some relatives in four of the six families exhibited mild hyperlipidemia, similar to the pattern seen in familial combined hyperlipidemia (FCHL). The hyperlipidemia segregated with the heterozygote state for LPL deficiency in these families (p less than 0.03). High density lipoprotein (HDL) cholesterol was significantly reduced in the heterozygotes for LPL deficiency (p less than 0.01). The measurement of LPL activity and mass allows identification of the heterozygote state for LPL deficiency, which is characterized by variable expressions of hyperlipidemia and reduced HDL cholesterol. These results suggest that the heterozygote state for LPL deficiency may form one subset of FCHL.

Biochemical indicators of fatness in meat-type chickens: lack of correlation between lipoprotein lipase activity in post-heparin plasma and body fat.

1. Possible relationships between fatness and lipoprotein lipase activity in adipose tissue and plasma from heparinised birds were examined in 7-week-old male and female broilers. 2. Total lipoprotein lipase activity in abdominal fat was significantly correlated (r = 0.5) with fat pad weight, but there was no correlation between specific activity of the enzyme and fat pad weight. 3. Lipoprotein lipase activity in post-heparin plasma showed no correlation with either abdominal fat or total body fat content. 4. The results indicate that measurements of lipoprotein lipase activity in biopsy samples or in post-heparin plasma are of no value in predicting the fat content of live birds.

The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme.

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.

Initial diagnosis of lipoprotein lipase deficiency in a 75-year-old man

Lipoprotein lipase deficiency, characterized by recurrent pancreatitis, profound hypertriglyceridemia, and delayed clearance of chylomicrons, is generally first diagnosed in childhood. Although patients with this condition have died during episodes of acute pancreatitis in the fourth and fifth decades, no patient older than 50 years has been previously reported. The de novo diagnosis of lipoprotein lipase deficiency in a 75-year-old man illustrates important points about this disease. This inborn error in metabolism may have a relatively benign clinical course resulting in normal life span, particularly if there is strict adherence to a low-fat diet and abstinence from alcohol. Moreover, measurement of lipoprotein lipase activity in persons with severe hypertriglyceridemia and recurrent abdominal pain, even in elderly patients, should lead to the correct diagnosis and treatment of this condition.

Lipoprotein lipase activity of rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro.

1. Isolated cardiac-muscle cells from the hearts of adult rats were shown to retain a high amount of viability during 4 h of incubation when viability was assessed by Trypan Bue stain exclusion and intracellular enzyme leakage. 2. The cells also retained their ability to take up O2 and utilize added substrates over the period of incubation at both 25 and 30 degrees C. 3. When cells from the hearts of fed rats were incubated in a buffered-salts solution at pH 7.4 in the presence of amino acids and heparin, lipoprotein lipase activity in the medium increased progressively. 4. During these incubations the intracellular activity of the enzyme remained constant and the total activity of lipoprotein lipase in the system (cells plus medium) increased by 80% over the 4 h of incubation at 25 degrees C. 5. In the absence of heparin only low amounts of enzyme activity were detectable in the medium and the total lipoprotein lipase activity in the system remained constant. 6. The measurement of lipoprotein lipase activity in either fresh homogenates of the cells or in homogenates of acetone/diethyl ether-dried powders of the cells had no effect on the overall pattern of activity change during the incubations, although as reported previously the total activity detected with acetone/diethyl either-dried preparations was approx. 3-fold higher than with fresh cell homogenates. 7. The observations were compared with published data on lipoprotein lipase activity changes in neonatal heart cell cultures maintained in vitro.

Clofibrate and nafenopin (SU-13437): effects on plasma clearance and tissue distribution of chylomicron triglyceride in the dog.

AbstractAdult male dogs, which received orally for 21 days either no drug, 75 mg/kg/day of clofibrate or 25 mg/kg/day of nafenopin, were injected iv with14C‐labeled chylomicron‐lipid, essentially chylomicron‐triglyceride fatty acid, at 0.2 g of fat per kilogram body weight. At times from 2–160 min after injection, samples of blood, liver, skeletal muscle and adipose tissue were removed, weighed and assayed for14C‐lipid content. Also, in other dogs treated for 14 days, samples of skeletal muscle and adipose tissue were removed before and after treatment by biopsy for measurement of lipoprotein lipase activity. Chylomicron‐14C was cleared more rapidly from the plasma of nafenopin‐treated and to a lesser extent of clofibrate‐treated dogs than controls. Thet0.5 for clearance was ca. 31 min for control, 12 min for nafenopin‐treated and 20 min for clofibrate‐treated dogs. The total skeletal muscle mass of nafenopintreated animals took up 40–50% of the injected chylomicron‐triglyceride during the first 20 min after injection, as compared with ca. 25–30% for clofibratetreated and 20–25% for control dogs. Also in treated dogs lesser percentages of the cleared chylomicron‐14C appeared in the liver soon after injection and more in adipose tissue at later times. These observations could be related partially to a measured ability of clofibrate to increase adipose tissue lipoprotein lipase activity (ca. 50%) and of nafenopin to increase a lipase activity of skeletal muscle (ca. 20%). These data suggest that clofibrate and particularly nafenopin lower plasma triglycerides by increasing their clerance by the peripheral tissues.

Measurement of lipoprotein lipase activity in post heparin plasma: description of technique

A method has been described for the quantitative assay of lipoprotein lipase activity in post heparin plasma. The conditions of the assay were subjected to critical study. The technique described permits the reaction to proceed in the test tube as a zero order reaction. It is believed that this technique will permit accurate quantitative study of lipoprotein lipase activity in different groups of patients. The rate of lipolysis was compared with the rate of clearing of lipemic plasma. The ratio of the rate of lipolysis to the rate of clearing was quite variable in normal subjects. This suggests that the use of the clearing technique to study lipoprotein lipase activity in different groups of patients, or under different experimental conditions, may give misleading results.