Collaborative meta-analysis of individual participant data from observational studies of Lp-PLA2 and cardiovascular diseases.

BACKGROUND: A large number of observational epidemiological studies have reported generally positive associations between circulating mass and activity levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) and the risk of cardiovascular diseases. Few studies have been large enough to provide reliable estimates in different circumstances, such as in different subgroups (e.g., by age group, sex, or smoking status) or at different Lp-PLA2 levels. Moreover, most published studies have related disease risk only to baseline values of Lp-PLA2 markers (which can lead to substantial underestimation of any risk relationships because of within-person variability over time) and have used different approaches to adjustment for possible confounding factors. OBJECTIVES: By combination of data from individual participants from all relevant observational studies in a systematic 'meta-analysis', with correction for regression dilution (using available data on serial measurements of Lp-PLA2), the Lp-PLA2 Studies Collaboration will aim to characterize more precisely than has previously been possible the strength and shape of the age and sex-specific associations of plasma Lp-PLA2 with coronary heart disease (and, where data are sufficient, with other vascular diseases, such as ischaemic stroke). It will also help to determine to what extent such associations are independent of possible confounding factors and to explore potential sources of heterogeneity among studies, such as those related to assay methods and study design. It is anticipated that the present collaboration will serve as a framework to investigate related questions on Lp-PLA2 and cardiovascular outcomes. METHODS: A central database is being established containing data on circulating Lp-PLA2 values, sex and other potential confounding factors, age at baseline Lp-PLA2 measurement, age at event or at last follow-up, major vascular morbidity and cause-specific mortality. Information about any repeat measurements of Lp-PLA2 and potential confounding factors has been sought to allow adjustment for possible confounding and correction for regression dilution. The analyses will involve age-specific regression models. Synthesis of the available observational studies of Lp-PLA2 will yield information on a total of about 15 000 cardiovascular disease endpoints.

Insulin-treated Zucker diabetic fatty rats retain the hypertriglyceridemia associated with obesity.

Lipoprotein and apolipoprotein changes were evaluated in 10-week-old Zucker diabetic fatty (ZDF) male rats following 12 weeks of insulin treatment, which normalized blood glucose and maintained weight gaining characteristic of nondiabetic Zucker fatty rats. Compared with untreated ZDF rats (saline-injected), insulin treatment resulted in increased very-low-density lipoprotein (VLDL; d < 1.006 g/mL) and decreased alpha lipoprotein on agarose gel electrophoresis. These findings were consistent with an observed increase in VLDL triglyceride and cholesterol, and decreased high-density lipoprotein (HDL) cholesterol with insulin treatment in isolated lipoproteins. B100 levels were unchanged by insulin treatment, but B48 levels were significantly increased in the VLDL fraction. Insulin treatment depressed apolipoprotein (apo) A-I levels in HDL, but had little effect on total apo E, apo A-IV, or apo C, although apo C was redistributed to the VLDL fraction. These results suggest that insulin treatment of ZDF rats normalizes hyperglycemia and prevents age-related changes in lipoprotein parameters associated with development of insulinopenic diabetes. Insulin therapy in ZDF rats thereby sustains the hyperlipidemic lipoprotein pattern associated with hyperinsulinemia and obesity.

Glucose-stimulated insulin secretion suppresses hepatic triglyceride-rich lipoprotein and apoB production.

The current study assessed in vivo the effect of insulin on triglyceride-rich lipoprotein (TRL) production by rat liver. Hepatic triglyceride and apolipoprotein B (apoB) production were measured in anesthetized, fasted rats injected intravenously with Triton WR-1339 (400 mg/kg). After intravascular catabolism was blocked by detergent treatment, glucose (500 mg/kg) was injected to elicit insulin secretion, and serum triglyceride and apoB accumulation were monitored over the next 3 h. In glucose-injected rats, triglyceride secretion averaged 22.5 +/- 2.1 microg.ml(-1).min(-1), which was significantly less by 30% than that observed in saline-injected rats, which averaged 32.1 +/- 1.4 microg.ml(-1).min(-1). ApoB secretion was also significantly reduced by 66% in glucose-injected rats. ApoB immunoblotting indicated that both B100 and B48 production were significantly reduced after glucose injection. Results support the conclusion that insulin acts in vivo to suppress hepatic very low density lipoprotein (VLDL) triglyceride and apoB secretion and strengthen the concept of a regulatory role for insulin in VLDL metabolism postprandially.

Effect of dietary fat on the development of non-insulin dependent diabetes mellitus in obese Zucker diabetic fatty male and female rats.

The obese Zucker diabetic fatty male rat (ZDF/Gmi¿trade mark omitted¿-fa) has become a widely used animal model of NIDDM, in contrast to the obese ZDF females that rarely develop NIDDM. However, preliminary observations suggest that obese ZDF females may become diabetic on high-fat diets. Therefore, we studied the effect of dietary fat on development of NIDDM, dyslipidemia, and alterations in organ-specific serum panels in obese ZDF males and females. Results indicated different effects of dietary fat-content on development of diabetes in males and females. Males, even on low fat-content diets, developed diabetes but the process was accelerated as a function of dietary fat-content, whereas only the highest fat-content diet induced development of NIDDM in obese ZDF females. Additionally, triglyceride/apolipoprotein B ratios demonstrated gender-specific differences in the nature of circulating lipoprotein particles independent of diabetic state with values for females approximately twice those of males indicating more highly triglyceride-enriched lipoprotein particles in females. We conclude that the obese ZDF female rat has the potential to become an important animal model of NIDDM especially in women where few models currently exist.

Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia.

Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.

Liver regrowth and apolipoprotein B secretion by rat hepatocytes following partial hepatectomy.

Apolipoprotein (apo) B is an essential component for the assembly and secretion of lipoproteins. The current report examines apo B production using primary cultures of hepatocytes derived from rats 3 to 21 days after partial hepatectomy (PH) to determine the effects of liver regrowth on apo B. Studies indicate that hepatocytes stimulated by PH have a two-thirds reduction in net apo B production 3 to 7 days after surgery, which coincides with the period of maximum rate of liver regrowth. Both higher (apo BH)- and lower-molecular-weight (apo BL) apo B are synthesized and secreted after PH, indicating the presence of edited apo B mRNA in hepatocytes. Hepatocytes derived from PH rats are more sensitive to insulin inhibition of apo B secretion compared with controls, suggesting an enhanced effect of insulin on newly replicated hepatocytes. Epidermal growth factor (EGF), a key regulator of liver regrowth following PH, potentiates the inhibitory action of insulin on apo B secretion in control hepatocytes and those derived from rats 2 to 3 weeks after PH. However, the potentiating effect of EGF on insulin inhibition of apo B is not discernible in hepatocytes 3 to 7 days after PH. The short-term in vitro hormonal effects occurring even with decreased apo B production suggest that this pathway remains available following PH to balance lipoprotein secretion with lipid and energy requirements necessary for liver regeneration.

Combined serum amylase and lipase determinations for diagnosis of suspected acute pancreatitis.

Serum amylase and lipase measurements are often used to diagnose acute pancreatitis. This study addresses the question of whether it is advantageous to order serum amylase and lipase tests simultaneously. We evaluated performance of the two tests separately and in combination through a retrospective study of patients for whom both amylase and lipase determinations were ordered. Initial analysis of test performance was conducted with a uniformly applied criterion based on determination of optimal sensitivity-specificity pairs. Individual tests and combinations of tests, including the "AND" and "OR" rules and discriminant functions, were examined. Only the discriminant approach demonstrated better performance than the lipase test alone. This finding was subsequently confirmed by logistic regression analysis. We conclude that ordering both tests simultaneously can be advantageous in diagnosing acute pancreatitis when a bivariate approach is used; however, this must be weighed against the difficulties associated with clinical implementation of such approaches.

Cellular heterogeneity in binding and uptake of low-density lipoprotein in primary rat hepatocytes.

Hepatocellular heterogeneity of biochemical function is well established for many aspects of liver metabolism. This study addresses the question of cellular heterogeneity in the catabolism of low-density lipoprotein by rat hepatocytes. Low-density lipoprotein binding (4 degrees C) and uptake (37 degrees C) by rat hepatocytes were studied by use of human low-density lipoprotein labeled with a highly fluorescent lipophilic probe, N,N-dipentadecylaminostyrylpyridinium iodide, recently developed by us. Single-cell suspensions derived from rat hepatocytes in primary culture and from liver perfusion were studied with flow cytometry with and an approximation algorithm for data analysis. These studies show subpopulations of cells negative and positive for the specific binding and uptake of low-density lipoprotein. Dissociation constants for low-density lipoprotein binding and uptake were determined for the total population (18 micrograms/ml, binding; 12 micrograms/ml, uptake) and found to be in good agreement with previously reported values. Additionally, the dissociation constant for binding for the positive subpopulation was determined and found to be 3 micrograms/ml. This lower value is more typical of the values seen in other cell types. These findings are strongly suggestive of functional heterogeneity in the hepatic catabolism of low-density lipoprotein.

Immunolocalization, quantitation and cellular heterogeneity of apolipoprotein B in rat hepatocytes.

Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.

Effect of weight loss on serum lipoprotein(a) concentrations in an obese population.

To assess the distribution of serum concentrations of the atherogenic lipoprotein(a) [Lp(a)] in an obese population and the possible effects of weight reduction, we determined Lp(a) in 52 white, moderately obese subjects (16 men and 36 women). The subjects were participating in a weight-reduction program of diet, exercise, and behavior modification plus combination anorectic drug therapy (fenfluramine and phentermine). This placebo-controlled, double-blind study lasted 104 weeks. We also determined concentrations of fasting insulin, triglycerides, total cholesterol, high-density lipoprotein cholesterol, apolipoprotein (apo) A-I, low-density lipoprotein cholesterol, and apo B. Results showed the following: a highly skewed, nongaussian distribution of serum Lp(a) values in the obese population, virtually identical to that reported for healthy adults; no clinically significant change in Lp(a) concentrations with weight loss; and further evidence of normalization of insulin, lipid, and lipoprotein concentrations with weight loss.

The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye.

N,N-dipentadecylaminostyrylpyridinium iodide is a dye that is approximately 100-fold more intensely fluorescent in a lipid than aqueous environment. This observation suggests its potential as a fluorescence stain for lipoproteins. This work reports the staining of LDL with this dye for use in studies of cellular binding. The staining procedure is simple, resulting in stable attachment of the dye as determined by transfer experiments, physical properties essentially identical to native LDL as demonstrated by virtually identical electrophoretic mobility, and consistent results in studies of cellular binding using flow cytometry. Increased signal to noise ratio over other dyes used for lipoprotein staining including the widely used Dil (3,3'-dioctadecylindocarbocyanine iodide) allows determinations of greater sensitivity and precision to be made. This is demonstrated by the flow cytometric determination of the 4 degrees C binding curve of LDL with freshly isolated human peripheral blood lymphocytes (i.e., cells not LDL receptor upregulated). Mediation of binding by the LDL receptor is demonstrated by correspondence between the LDL receptor dissociation constant derived from this work and literature values; increased specific binding in lymphocytes cultured in lipoprotein-deficient media to up-regulate the LDL receptor; and decreased specific binding in lymphocytes cultured in the presence of 25-hydroxy cholesterol for 48 h to suppress the LDL receptor.

Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence.

A method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary. Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.

A rapid and accurate single-drop modification of the acid-elution technique for detecting fetomaternal hemorrhage.

A single-drop modification of the acid-elution technique (Kleihauer-Betke) for quantitating fetomaternal hemorrhage is described. It obviates the need for the tedious and time-consuming manual counting of background adult cells. Rather, this is achieved by automated red-blood cell counting of the initial specimen and delivery of a standard volume (1 microliter) of a standard dilution (1:1,000) in the form of a droplet to a microscope slide. The droplet is left to dry undisturbed at room temperature and then stained. The fetal cells are manually counted while the total number of cells is calculated from the initial red-blood cell count, standard volume, and standard dilution. Determinations on 4 different concentrations of fetal/adult red cell mixtures are performed. Results indicate improved accuracy and precision relative to the standard technique in significantly less time for volumes of fetomaternal hemorrhage requiring more than the standard dose of Rho(D)-immune globulin.

Comparison of quantitative acid-elution technique and flow cytometry for detecting fetomaternal hemorrhage.

A study of the Kleihauer-Betke acid-elution technique for quantitating fetomaternal hemorrhage was performed to assess intra- and inter-technologist accuracy and precision as well as to delineate the statistically valid domain of the test as usually performed. The results were then compared to a parallel study quantitating fetomaternal hemorrhage by flow cytometry. Additionally, a statistical model for estimating efficacy of treatment with Rh immune globulin in the prevention of pregnancy-associated Rh(D) isoimmunization was developed. The results indicate that the acid-elution technique can be performed in a reproducible manner with acceptable accuracy and precision with whole blood fetomaternal hemorrhages 25 ml and higher if a background correction for false positive identification of fetal cells is included. Flow cytometric determination reveals significantly increased accuracy in comparison to the corresponding Kleihauer-Betke results.