The behavior of lipoproteins, cholesterol, triglycerides, free fatty acids and free glycerol in serum of rats with experimental hyperthyroxinemia and hypothyreosis]

In hyperthyroxinemic and hypothyreotic rats the lipoprotein level in serum was investigated using agarosegel-electrophoresis and changes in serum level of cholesterol, triglycerides, free fatty acids and glycerol were determined. In hyperthyroxinemic animals the beta-lipoproteins were found in the same level as the control animals, while the prae-beta-fraction was significantly elevated and the alpha-lipoproteins lowered. The cholesterol was significantly reduced, the triglycerides and the glycerol significantly increased. The free fatty acids were slightly elevated. In hypothyreotic animals the beta-and prae-beta-fraction of lipoproteins was significantly elevated. The alpha-lipoproteins were found diminished. Cholesterol and triglyceride values were also significantly increased. The levels of free fatty acids and glycerol did not differ in both groups of animals.     

Effect of an anti-lipoprotein lipase serum on plasma triglyceride removal.

Anti-lipoprotein lipase sera injected intravenously in roosters blocked quantitatively the catabolism of very low density lipoprotein (VLDL) triglyceride. Antibodies were produced in rabbits immunized with highly purified lipoprotein lipase (LPL, glycerol ester hydrolase, E C 3.1.1.3) prepared from chicken adipose tissue. Following anti-LPL serum injection there was a linear increase in plasma triglyceride concentration. The rate of entry of triglyceride in plasma was estimated from the rate of triglyceride accumulation in the plasma of animals injected with anti-LPL serum, or from the disappearance curve of biologically labelled VLDL. In instances where both measurements were conducted in the same animals there was very close agreement between the two procedures. Inhibition of VLDL triglyceride catabolism of anti-LPL serum provided a way to characterize newly secreted VLDL that exhibited a broad spectrum of particle sizes with a median of 625 A degrees. They contained 76.2 +/- 1.2% triglyceride and had a high ratio of free to ester cholesterol (2.46 +/- 0.45). In control VLDL samples there was 46.1% triglyceride, and the ratio of free to ester cholesterol was 1.19. The complete inhibition of triglyceride removal by an antiserum prepared against adipose tissue LPL demonstrates that the NaCl-inhibited, serum-activated lipase prepared by affinity chromatography on heparin-Sepharose and concanavalin A-Sepharose columns is the enzyme responsible in vivo for the catabolism of VLDL triglyceride. Further, the kinetics of triglyceride accumulation in the plasma provide evidence that the site of degradation of VLDL triglyceride is within the plasma compartment.     

In vitro interactions of phenformin with serum lipoproteins and consequences thereof.

Turbidity developed when phenformin was added to human serum; this turbidity increased in a sigmoidal fashion with rising concentrations of phenformin (5–50 nmole/1). Centrifugation produced clearing of the solution, with collection of particulate matter on the surface of the sera.Extraction of control, and phenformin-treated sera with petroleum ether for 15 min. revealed that cholesterol and triglyceride were responsible for the turbidity. Different sera produced different turbidities with a given concentration of phenformin. No significant simple correlation existed between turbidity and serum cholesterol and/or triglyceride levels. The turbidities, produced by the addition of a constant concentration of phenformin to a series of diluted serum samples, were linearly related to the amount of serum present.The turbidities acquired by purified very-low density (VLDL), low-density (LDL), and high-density lipoprotein (HDL) fractions with phenformin were additive, and the turbidity of phenformin-treated serum was accounted for by these lipoprotein fractions. Serum free of lipoproteins did not become turbid when exposed to phenformin. Phenformin added to serum which had previously been delipidated, failed to produce turbidity. The turbidity produced by phenformin was reversible, because it could easily be cleared by dialysis.No significant differences in quantitative immunochemical reactivities were observed when control serum was compared with the subnatant of phenformin-treated serum, as determined by single radial immunodiffusion with LDL antibodies.These in vitro observations may be related to the in vivo hypolipidemic action of phenformin on hyperlipidemic subjects.     

Halofenate and clofibrate: mechanism of hypotriglyceridemic action in the rat.

Rats fed a fat-free diet containing no drug, 0.02% or 0.10% halofenate, or 0.25% clofibrate for 14 days were injected intravenously with equivalent amounts of either [2-3H]glycerol or [1(3)-3H]glycerol. Blood samples were collected at times up to 150 min after injection and serum triglycerides were isolated and assayed for radioactivity. Kinetic analysis of the serum appearance and clearance curves of 3H-labeled triglyceride permits estimation of serum total 3H-labeled triglyceride formation and triglyceride fractional turnover rates. The total amounts of 3H-labeled triglyceride formed from [2-3H] or from [1(3)-3H] glycerol in control-fed animals were very similar. Over 95% of the serum 3H-labeled triglyceride formed from either substrate circulated in a rapidly turning-over triglyceride pool (t1/2 = 8 min). Treatment with 0.10% halofenate or 0.25% clofibrate decreased labeling of serum triglycerides by 75-80% without increasing serum 3H-labeled triglyceride fractional turnover rates. Furthermore, both drugs decreased incorporation in vivo of 14C from [U-14C]glycerol into hepatic but not intestinal triglycerides without significantly decreasing incorporation of 14C into total phospholipids of either tissue. From these observations we suggest that, in the intact normal rat, sustained reduction of serum triglyceride levels produced by treatment with halofenate or clofibrate is due to inhibition of hepatic triglyceride formation.     

Uptake and metabolism of circulating chylomicron triglyceride by rabbit aorta

To determine if chylomicron triglycerides are taken up and metabolized by the arterial wall, rabbit abdominal aortas were perfused in situ for various times up to 2 hr with blood-buffer containing isotopically labeled substrates. Labeled chylomicrons were obtained by feeding [(3)H]palmitic acid or [(3)H]glyceryl trioleate to rats and rabbits with cannulated thoracic ducts. After aortic perfusion with these chylomicrons, more than 85% of aortic lipid ester radioactivity was in triglyceride; when labeled glycerol or palmitic acid was perfused, most aortic ester lipid radioactivity was in diglycerides and phospholipids. This indicated that, during perfusion with chylomicrons, intact triglyceride molecules were taken up by aorta. The rate of triglyceride fatty acid uptake by the inner avascular segment approached maximal values at low concentrations of perfusate triglyceride fatty acids (2 mm), whereas uptake in the outer capillary perfused segment increased with increasing triglyceride fatty acid concentration (0.4-25 mm). By double-radioisotope techniques it was shown that aortic free fatty acid was derived from both perfusate free fatty acids and from hydrolysis of lipoprotein glycerides within the aortic wall. Uptake of chylomicron triglyceride by perfused aorta was independent of triglyceride hydrolysis, which was quantitatively small.     

Effect of p-chlorophenoxyisobutyrate on the antilipolytic action of insulin and insulin binding in isolated adipocytes.

The present study was undertaken to investigate the potentiation by p-chlorophenoxyisobutyrate (CPIB) of the antilipolytic effect of insulin in isolated adipocytes from rats fed a (1) sucrose diet, (2) glycerol-lard diet, or (3) chow diet. CPIB supplementation in the diet consistently resulted in decreased serum triglyceride levels in rats from the three dietary groups. The catecholamine-stimulated glycerol release was significantly depressed to a greater extent by insulin when the fat cells were obtained from rats given CPIB compared to those without drug treatment. The enhanced insulin sensitivity was, however, not accompanied by any changes in insulin binding to adipocytes. These two observations were found in cell preparations from rats fed any one of the diets, although differences among dietary groups could be detected. In an in vitro experiment, epinephrine-stimulated glycerol release was progressively inhibited by increasing concentrations of CPIB in the incubation medium. However, the antilipolytic response to an optimal concentration of insulin (100 muU/ml) was augmented in the presence of CPIB. Thus, it seems that CPIB can potentiate the action of insulin in inhibiting mobilization of free fatty acid from the adipose tissue, and the coordinated effect of both antilipolytic agents is important in lowering serum triglyceride concentration. The mechanism by which CPIB facilitates the effect of insulin is discussed.     

Influence of perinatal factors and sampling methods on TSH and thyroid hormone levels in cord blood.

To evaluate the effect of perinatal factors and sampling methods on thyroid stimulating hormone (TSH) and thyroid hormone levels in cord blood, serum TSH, free thyroxine (FT4) and free triiodothyronine (FT3) concentrations were measured in 124 healthy term neonates. Eighty-eight infants were born in normal vaginal deliveries, 25 were delivered by vacuum extractor and 11 by Cesarean section. There was no significant difference among the three infant groups in the mean TSH levels. Birth weight, the infant's sex, duration of labor and uterotonic agents had no effect on cord serum TSH and free thyroid hormone levels in the neonates born by normal vaginal delivery. To assess the adequacy of specimen collection, mixed cord blood samples, obtained by a direct application of cord on a filter paper, and venous blood withdrawn with a plastic syringe were collected in another 200 infants. There was a significant linear correlation in the TSH concentration in mixed cord blood and cord venous serum from the same individuals, while a poor correlation was found in T4 values from two specimens. Our results suggest that the TSH value in cord blood is less influenced by perinatal factors, including the sampling method, and the mixed cord blood collected by this technique might be a feasible alternative specimen for a TSH screening program with cord blood which is useful in countries where neonatal blood is not available.     

Alterations of lipid metabolism in mice injected with endotoxin.

Some alterations in lipid metabolism in mice were observed by the intraperitoneal injection of endotoxin from Salmonella typhimurium. The content of serum triglyceride increased markedly in poisoned mice 16-24 hr postintoxication. The level of free fatty acid (FFA) in the serum of endotoxin-administered mice decreased in inverse proportion to an increase in the injected dose of endotoxin. The electrophoretic analysis of the serum lipoprotein on cellulose acetate membrane showed that pre beta-lipoprotein increased markedly and that FFA fraction in the poisoned mice sera disappeared 18 hr postintoxication. The activity of hormone-sensitive lipase in adipose tissue was elevated appreciably 2 hr after injection, but decreased more significantly after 18 hr than that in fasted control mice. On the other hand, the activity of lipoprotein lipase decreased in the post-heparin serum and adipose tissue 3 hr postintoxication, and decreased significantly after 16 hr. There were no significant differences between changes in the formation of active glycerol (alpha-GP) and in the activity of alpha glycerophosphate dehydrogenase (alpha-GPDH) in the mice liver with or without administration of endotoxin, and after 16 hr levels of both hepatic alpha-GP content and alpha-GPDH activity in poisoned mice showed a tendency to be slightly lower than those in fasted control mice.     

Nerve growth factor levels in mouse serum: Variations due to stress

The presence of nerve growth factor (NGF) in the serum of adult male mice was assayed using the chick embryo dorsal root ganglion (DRG) bioassay technique in a serum free N1 supplemented medium. Wide variations in the serum-induced nerve fiber outgrowth response were observed when serum was obtained from animals maintained four per cage. Of 64 mice tested, sera of 7 animals induced a profound nerve fiber outgrowth response while the sera of 57 mice failed to show a similar response. In animals kept in isolation for 7 days prior to the start of the experiment, aggression provoked a marked increase in serum NGF levels. In contrast to the sera of aggression-unprovoked mice, the sera of all aggression-provoked mice stimulated a dense nerve fiber outgrowth. The sera of both groups of mice stimulated an intense proliferation and migration of nonneuronal cells. The neurite outgrowth responses elicited by sera from aggression-provoked and unprovoked mice were completely inhibited by the rabbit anti-NGF antiserum. In conclusion, both crowded housing and aggression in mice may provoke an elevation in the serum NGF levels that can be confirmed by the ganglion bioassay technique.     

Determination of free-insulin in antibody containing sera: comparison of polyethylene glycol and Staphylococcus aureus cells

Polyethylene glycol (PEG) and killed Staphylococcus aureus cells (S. aureus) were used as agents to separate free insulin from antibody-bound insulin in diabetic sera. Insulin was determined by conventional double antibody radioimmunoassay. The free insulin values after PEG treatment were almost half of those after S. aureus treatment. The free insulin levels in high-antibody containing sera preincubated at 37 degrees C, 2 h were double the value of fresh sera. PEG treatment caused about 40% loss of total serum protein. The addition of bovine serum albumin (BSA) to the PEG-treated serum greatly increased the immuno-reactive insulin values. This may suggest that protein concentration plays a role in insulin radioimmunoassay. PEG treatment may also enhance the interaction between free insulin and free antibodies resulting in underestimation of free insulin level.     

Correlation between apolipoprotein A-IV and triglyceride concentrations in human sera

Apolipoprotein A-IV concentration was measured by a newly developed competitive enzyme immunoassay in sera from fasted human subjects (n = 105) whose triglyceride concentrations ranged from 20 to 474 mg/dl (total cholesterol below 260 mg/dl) and in which chylomicrons could not be detected. Mean (+/- SD) apolipoprotein A-IV concentration was 13.0 +/- 2.6 mg/dl in sera with triglyceride levels ranging from 20 to 100 mg/dl, 16.9 +/- 3.7 mg/dl in sera with triglyceride levels ranging from 101 to 250 mg/dl, and 22.7 +/- 6.7 mg/dl in sera with triglyceride levels ranging from 251 to 474 mg/dl. The differences among the three groups were highly significant (P less than 0.001). Moreover, variations of apolipoprotein A-IV concentrations according to the triglyceride levels were noted within the normo-triglyceridemic population. Apolipoprotein A-IV concentration was 12.8 +/- 2.1 mg/dl for triglyceride levels ranging from 20 to 75 mg/dl and 16.4 +/- 3.8 mg/dl for triglyceride levels ranging from 76 to 150 mg/dl (P less than 0.01). In the entire population that was studied there was a significant linear correlation (r = 0.61, P less than 0.001) between the concentrations of serum apolipoprotein A-IV and triglyceride. Although the hypothesis of an unknown factor independently influencing both very low density lipoproteins and apolipoprotein A-IV cannot be ruled out, and although no apolipoprotein A-IV was found in the triglyceride-rich lipoprotein fraction after separation by gel filtration, these data suggest that, in fasting subjects, the secretion of very low density lipoproteins could contribute to the plasma apolipoprotein A-IV level.     

Measurement of serum total glycerides and free glycerol by high-performance liquid chromatography

Serum levels of total glycerides and free glycerol are important indices of lipid metabolism and cardiovascular disease risk. Convenient enzymatic methods of measurement have been available, but they are susceptible to interference. Situations exist in both research and clinical laboratories in which more specific and precise methods are needed. We developed HPLC methods for the measurement of serum total glycerides and free glycerol. For total glycerides, serum was mixed with an internal standard (1,2,4-butanetriol) and treated with alcoholic sodium hydroxide to hydrolyze glycerides to glycerol. After deproteinization with tungstic acid, the glycerol was benzoylated with an optimized Schotten-Baumann reaction and analyzed by HPLC. For free glycerol, serum was equilibrated with the internal standard and deproteinized with tungstic acid to remove the glycerides. The glycerol was benzoylated and analyzed as for total glycerol. Various factors were investigated, and no significant sources of interference were detected. The total coefficients of variation ranged from 0.7% to 2.0% for total glycerides and from 1.7% to 3.2% for free glycerol. The analytical recoveries ranged from 98.5% to 101.6%. In conclusion, simple and reliable HPLC methods for serum total glycerides and free glycerol have been developed. The methods may also be used for the analyses of glycerol or glycerides in other biological samples.     

Effect of corticosterone on epididymal lipids in pubertal rat

Serum corticosterone excess was induced by the administration of corticosterone acetate to adrenal intact rats. Different lipid classes were studied in unwashed and washed (epididymal sperm and fluid free) caput and cauda epididymides. The unwashed caput epididymidis registered a significant decrease in total lipid, cholesterol and phospholipid while total glyceride glycerol and its fractions were not altered after corticosterone treatment. Among phospholipid fractions phosphatidyl inositol, choline and ethanolamine showed a significant decrease. Unlike the unwashed caput epididymidis, the washed caput region recorded a marked increase in total lipid, glyceride glycerol and its fractions. However, total lipid in the washed cauda region significantly increased and the increase was mainly due to triacyl glycerol. Though the phospholipid fractions phosphatidyl choline and ethanolamine showed an increase, the total phospholipid was not altered significantly. Serum testosterone and prolactin registered a significant decrease while gonadotropins were unaltered. On the withdrawal of corticosterone treatment, all the lipid classes turned to normalcy along with serum testosterone and prolactin. It is concluded that corticosterone excess favours lipid accumulation in the sperm free epididymal tissue and its influence on epididymis is region specific and reversible.     

Serum Triglyceride: Method of Estimation and Levels in Normal Humans

Previously described glycerol methods for estimation of serum triglyceride were modified. Conditions during saponification and colour development were altered so as to minimize the possibility of glycerol loss. Using this modification, serum triglyceride was determined in 100 healthy men and women, aged 15 to 79 years. There was a log normal distribution. In both sexes the level increased up to the sixth decade and then decreased. In each decade men had higher levels than women. The geometric mean (and 95% limits) for men was 129 mg. % (68-248); for women, 105 mg. % (54-206); and for the entire group, 117 mg. % (59-233). Comparison of results from several laboratories using different methods showed wide variation in serum triglyceride levels.     

Biological effects of Spirometra erinacei plerocercoids in several species of rodents

Observations were made of the biological effects on infection with plerocercoids of Spirometra erinacei on normal female Snell mice, male chinese hamsters, golden hamsters, normal and hypox rats. Plerocercoid infection caused the strongest growth-promoting effect on normal Snell mice. In mice, this effect appears to be independent of strain. Chinese hamsters infected with these larvae showed similar growth. The infected normal rats and golden hamsters, however, showed a weight increase in the skeletal muscle only, while the hypox rats exhibited no effect at all. The elevation in the concentration of serum triglyceride was observed in all the animals investigated except for rats. Golden hamsters, in particular, exhibited a marked increase in the concentration of serum free fatty acids and total cholesterol. There was close correlation between the concentrations of serum triglyceride and free fatty acids, and the regression coefficient of the resulting linear regression equation for the experimentals was higher than that for the controls. This suggests that serum triglyceride results from an increased concentration of serum free fatty acids derived from stimulated lipolysis. The total cholesterol concentration in the serum decreased in chinese hamsters infected with larvae. The serum glucose concentration increased in normal Snell mice but decreased in chinese and golden hamsters. No difference in glycerol and free fatty acid concentration was observed in infected animals except for golden hamsters.     

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.     

The relationship between serum and cell type on the development of rat and pig cultured preadipocytes

Stromal-vascular cells from rats and pigs were isolated from adipose tissue and used to measure preadipocyte proliferation and differentiation. Cells from rats and pigs were grown in either 2.5% pig serum or 2.5% rat serum. Cells were either supplemented or unsupplemented with insulin after five days of growth in culture. In these cultures, pig fat cells developed as discrete clusters while rat fat cells developed as loose clusters or as individual cells. Rat cells had greater levels of sn-glycerol phosphate dehydrogenase activity compared to pig cells. Rat serum increased soluble protein in plated cells when compared to cells grown in pig serum. Pig serum increased glycerol phosphate dehydrogenase specific activity when compared to rat serum. In this system, there was no response to insulin. The cells grown in rat serum did not resemble adipocytes in regard to the presence of large lipid droplets (oil red 0 staining). These results demonstrate that rat and pig stromal-vascular cells in culture are morphologically different. Cells from both species, however, responded similarly to sera from either species showing that cells from rats and pigs responded to the growth and differentiation factors present in these sera.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Lecithin-Agar Assay for Lecithinase Antibodies in Serum

A technique for assay of lecithinase antibodies in serum was developed in this laboratory by using a lecithin-agar plate diffusion procedure based on a combination of described plate assays. Egg yolk lipoprotein composed primarily of lecithin was used as a substrate for reaction with free or non-neutralized lecithinase C after incubation of known amounts of lecithinase C with various dilutions of control and test sera. It was found that the size of the reaction zone was a function of enzyme concentration and inversely proportional to the antibody concentration. Accuracy and precision of the assay were determined. In addition, lecithinase antibody levels in sera from experimentally inoculated rats and rabbits and sera from randomly selected human patients were studied.     

17 beta-Hydroxysteroid oxidoreductase activity in human maternal and umbilical cord sera

The specific activity of 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) in human umbilical cord arterial serum has been reported to be similar to that of maternal serum and 5- to 15-times higher than that of cord venous serum. Based on these findings, it was proposed that 17 beta-HSOR in cord arterial serum arises from fetal tissue sources other than placenta. In the course of studies of the role of 17 beta-HSOR in the modulation of bioactive estrogen levels in the human fetus, we determined that: (i) the specific activity of 17 beta-HSOR in maternal serum is 2.1- to 55-times higher than that in either umbilical cord venous serum or cord arterial serum; (ii) the specific activity of 17 beta-HSOR in umbilical cord venous and cord arterial sera are similar; (iii) anti-human placental cytosolic 17 beta-HSOR antibody inactivates the 17 beta-HSOR in maternal, umbilical cord arterial, and cord venous sera but not in maternal or fetal erythrocytes; (iv) the specific activity of 17 beta-HSOR in maternal serum (expressed per mg protein) is higher than that in umbilical cord serum and maternal and fetal erythrocytes, and is approximately 700-times lower than that of the placental microsomal enzyme; (v) the preferred cofactor for maternal serum 17 beta-HSOR is NADP+; (vi) 17 beta-HSOR is associated with the high speed supernatant fraction of maternal serum rather than with the particulate fraction; and, (vii) the patterns of binding of [3H]estradiol-17 beta to proteins in maternal and umbilical cord arterial sera and those of 17 beta-HSOR activity, determined in corresponding fractions obtained after sucrose density gradient centrifugation, are approximately coincidental at S20, omega 4.6-5. The findings of higher 17 beta-HSOR levels in maternal serum compared with umbilical cord arterial serum and the inactivation of the cord arterial serum enzyme by an antibody that recognizes human placental cytosolic 17 beta-HSOR is suggestive that 17 beta-HSOR in cord arterial serum is of placental origin.     

A novel method for measurement of triglyceride lipase activity: suitable for microgram and nanogram quantities of tissue

The measurement of triglyceride lipase activity in microgram and nanogram quantities of tissue is reported. The method involves quantitation of glycerol released from a triglyceride substrate, which is shown to provide a value of approximately one-third of that obtained by quantitation of free fatty acid release. Influences on glycerol release, including pH optimum, NaCl inhibition, and activation by serum and heparin are characterized. Two separate assays are described for the measurement of glycerol that yield identical results with nanogram quantities of tissue. The advantage of one assay is its simplicity, while the advantage of the other is that it can be adjusted to measure very small tissue samples (nanogram) with the use of microanalytical procedures (i.e., enzymatic amplification of the NAD+ product of glycerol analysis). Sensitivity of the method is demonstrated by the analysis of triglyceride lipase activity in nanogram samples of single soleus muscle fibers. Measurement of picomole quantities of glycerol produced by lipase activity in single muscle fibers represents at least a 1,000-fold increase in sensitivity compared to currently available methods.