Fractional and absolute synthetic rate of very low density lipoprotein apo B-100 in lipoprotein lipase deficient patients.
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Adult↗
Biomedical subjects
K A Mitropoulos
Publications and source records attributed to K A Mitropoulos.
The autoactivation of factor XII in the presence of long-chain saturated fatty acids--a comparison with the potency of sulphatides and dextran sulphate.
The incubation of purified human factor XII (Hageman factor [HF]) in the presence of long-chain saturated fatty acids (FA) like stearate (C-18) or behenate (C-22) resulted in a time-dependent increase of amidolytic activity. The HF autoactivation progress curves were sigmoidal. The first order rate for the initial period was constant; this was followed by a period of decreasing rate and a plateau of zero rate. These progress curves were similar to those obtained on the incubation of HF in the presence of sulphatide vesicles or dextran sulphate. The initial rate of autoactivation of HF was dependent on the FA concentration of contact surface and increased with increasing concentration of HF. At constant concentration of contact surface and varying concentration of HF, autoactivation rates in the presence of behenate, sulphatide vesicles or dextran sulphate followed Michaelis-Menten kinetics. The Km values for all three contact surfaces were above the physiological plasma concentration of HF whereas the catalytic efficiency in the presence of behenate (0.034 microM-1s-1) was about 2/3 of that in the presence of sulphatide vesicles (0.053 microM-1s-1) and considerably higher than that in the presence of dextran sulphate (0.004 microM-1s-1). Long-chain saturated FA bound to human serum albumin at the high- or low-affinity sites are ineffective, whereas the crystalline non-bound stearate or behenate provided a potent contact surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Dextran Sulfate↗
Plasma factor VII is activated by postprandial triglyceridaemia, irrespective of dietary fat composition.
Nine adults took two 7-day diets of standardised energy and total fat content, but with a dietary polyunsaturated/saturated fat ratio of less than 0.3 and greater than 3.0 respectively, while adhering to their daily routine. Blood was drawn on 6 occasions between 09.00 and 22.45 h on the final day of each dietary period for factor VII activity (VIIc), factor VII antigen (VIIag) and lipoprotein lipid concentrations. Diurnal variation was described for each variable in terms of its deviation from the individual's daily mean value at each time point across the day. Plasma triglyceride remained low until after the midday meal, whereafter a marked rise was sustained into the later evening. Plasma VIIc declined until early afternoon, but showed a marked rise in the late afternoon. Plasma VIIag showed no significant diurnal variation. Changes in plasma triglyceride concentration during the day were related positively to changes in VIIc about 160 min later, but not to VIIc at other time points. This effect of postprandial triglyceridaemia on VIIc persisted after allowance for the effect of VIIag on VIIc. Dietary fat composition did not influence VIIc or VIIag. The results suggested an acute but evanescent effect of triglyceride-rich lipoproteins on the reactivity of factor VII, irrespective of their lipid core composition.
Antigens↗
The increased rate of activation of factor XII in late pregnancy can contribute to the increased reactivity of factor VII.
The amidolytic activity of enzymes derived from factor XII (XIIa) was 3-fold higher in plasmas collected during pregnancy than from control subjects. Factor VII coagulant activity (VIIc) and XIIa increased in both kinds of plasmas on incubation on ice for 24 h (cold activation). These increases could be attributed to the decreased potency of C1 inhibitor (C1INH). However, variations in the concentration of C1INH and of factor XII could not explain the differences in VIIc and in XIIa between late pregnancy and control plasmas following cold activation under the same conditions. It is concluded that in vitro the increased amount of contact surface in the late pregnancy plasma promotes a higher rate of generation of XIIa and consequently a higher rate of activation of factor VII. The increased amount of contact surface could also be responsible for the increased concentration of XIIa in non-treated plasma from late pregnancy and could contribute in vivo to the higher reactivity of factor VII in this condition.
Amides↗
The prothrombin activation peptide regulates synthesis of the vitamin K-dependent proteins in the rabbit.
The turnover of 125I-bovine prothrombin fragment 1 was studied in the rabbit. The t1/2 of the peptide in the intravascular compartment was 11.5 hours and this compartment accounted for between 7.9 and 14.4% of the injected radioactivity. The rest of the radioactivity was distributed between two compartments in the extravascular space. The injection of the peptide (10 mg/rabbit) was associated with a transient increase in the plasma concentration of prothrombin and of factor X, with maximum concentration of prothrombin between 40 and 66 hours from the injection and between 26 and 40 hours for factor X. It is concluded that the injection of fragment 1 in the rabbit induced a transient increase in the synthesis of the vitamin K-dependent proteins that is compensated for by an increased absolute catabolic rate. It is suggested that the prothrombin activation peptide serves as regulatory message which induces the subsequent restoration of the appropriate concentration of the vitamin K-dependent proteins.
Animals↗
Factor VII and dietary fat intake.
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Adult↗
The activation of the contact phase of coagulation by physiologic surfaces in plasma: the effect of large negatively charged liposomal vesicles.
The endogenous, negatively charged surface that induces activation of the contact coagulation factors was investigated in plasmas taken from women in late pregnancy and control subjects of child-bearing age. The plasmas from the two groups of subjects were incubated at 4 degrees C for 24 hours either in plastic or in glass tubes and the factor VII coagulant activity (VIIc) was assayed in the treated plasmas. The activation of factor VII under these conditions involves the generation of enzymes derived from factor XII (XIIa). The contact surface is rate-limiting for the activation of factor VII in the plasmas in both groups of subjects and can be supplemented by large multilamellar liposomal vesicles carrying the appropriate density of negative charge. The size of these vesicles is within the range of sizes of the large lipoprotein particles (chylomicrons, very low and intermediate-density lipoproteins). The relationship between the density of negative charge on the liposomal vesicles and VIIc was similar in the late pregnancy and the control plasmas incubated in plastic tubes. At a saturating density of negative charge the observed relative VIIc was similar in both sets of plasmas. The incubation of late pregnancy or control plasma in plastic tubes in the presence of sodium stearate caused VIIc to increase with increasing concentration of the added fatty acid. These results suggest that large lipoprotein particles carrying the appropriate free fatty acid at a sufficient density of negative charge could provide the contact surface that induces the generation of factor XIIa and the subsequent activation of factor VII. Moreover, plasmas from women in late pregnancy have a higher concentration of potential surface and a higher density of negative charge than the plasmas from nonpregnant women.
Anions↗
Factor VII coagulant activity is strongly associated with the plasma concentration of large lipoprotein particles in middle-aged men.
A community survey of factor VII coagulant activity (VIIc) and the lipoprotein profile in non-fasting plasma of middle-aged men in NW London was undertaken to search for the determinants of VIIc in the general community. The data demonstrates that associations between VIIc and the plasma concentrations of cholesterol and of triglycerides previously shown in the general population can be explained by the strong and positive associations between VIIc and the large lipoprotein particles, chylomicrons, VLDL and IDL. Consistent with the possibility that the concentration of large lipoproteins determines the in vivo reactivity of factor VII, the association between VIIc and the ratio of lipid in the d greater than 1.019 fraction to the total plasma lipid was also highly significant but negative. The observed correlations between VIIc and lipoproteins smaller than VLDL may be the product of the interrelations that exist between the lipoprotein fractions in plasma. However, the associations between VIIc and the chylomicron lipid concentrations are especially strong when allowance is made for the considerable bias towards zero in the observed correlation, due to large within-person variance in chylomicron concentration.
Age Factors↗
Fat consumption and factor VII coagulant activity in middle-aged men. An association between a dietary and thrombogenic coronary risk factor.
Diet was measured by 5-day weighed inventory to search for an association between fat intake in the general population and factor VII coagulant activity (VIIc), a strong predictor of coronary heart disease. Of 275 men aged 40-59 years registered with a medical practice, 203 (74%) participated and 170 (62%) provided a satisfactory record. After allowance for the increase in fat intake with body size, a statistically significant and positive association was found between dietary fat and VIIc (r = 0.18; P less than 0.05). The correlation coefficient was increased to 0.24 when adjusted for the effect of day-to-day variability in individual fat intake, thereby providing an improved estimate of the true strength of association. The mean difference in VIIc of 12% of standard between men in the highest and lowest quarters of the distribution of fat intake was similar to that reported between men experiencing coronary heart disease and those remaining free. The results support previous experimental fat-feeding studies and suggest that a high fat diet has adverse consequences for blood coagulability and coronary thrombosis.
Adult↗
Hypercoagulability and factor VII in hypertriglyceridemia.
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Animals↗
Turnover of factor X and of prothrombin in rabbits fed on a standard or cholesterol-supplemented diet.
The turnover of prothrombin and of factor X was investigated in rabbits fed on a 1%-cholesterol-supplemented or a standard diet by studying the evolution of radioactivity in blood and in plasma from these animals after the intravenous injection of either 125I-rabbit factor X or 125I-bovine prothrombin. For factor X, half-lives and fractional pool sizes were similar for the two groups of rabbits in the extravascular, intravascular and plasma compartments. However, the equivalent plasma fractional pool size for the two groups of rabbits was only 73% of that in the intravascular compartment. The fractional catabolic rate for the hypercholesterolaemic rabbits [0.064 +/- 0.007 (of the intravascular pool)/h] was not significantly different from that in the rabbits fed on the standard diet (0.074 +/- 0.008/h). However, the absolute catabolic rate, and therefore the rate of synthesis, was significantly higher (1.261 +/- 0.141 mg/day per kg body wt. of rabbit) in the rabbits fed on the cholesterol-supplemented than that in the rabbits fed on the standard diet (0.705 +/- 0.019 mg/day per kg). The prothrombin half-lives and fractional pool sizes were similar for the two groups of rabbits in the extravascular and the intravascular compartments. The fractional catabolic rate for the hypercholesterolaemic rabbits [0.041 +/- 0.003 (of the plasma pool)/h] was not significantly different from that in the rabbits fed on the standard diet (0.035 +/- 0.003/h). However, the absolute catabolic rate and therefore the rate of prothrombin synthesis was significantly higher (3.96 +/- 0.48 mg/day per kg body wt.) in the rabbits fed on the cholesterol-supplemented than that in the rabbits fed on the standard diet (2.24 +/- 0.12 mg/day per kg).
Animals↗
Increased factor VII coagulant activity in the rabbit following diet-induced hypercholesterolaemia. Evidence for increased conversion of VII to alpha VIIa and higher flux within the coagulation pathway.
Factor VII coagulant activity (VIIc) is considerably higher in rabbits fed a 1% cholesterol-supplemented diet than in rabbits fed a standard diet. This increase was statistically significant 4-6 days from the beginning of treatment and rose to about 300% during the 100 days of treatment. Treatment is also associated with a 20-fold increase in plasma cholesterol concentration with the major fraction of excess cholesterol associated with the very low and intermediate density lipoprotein fractions. In both groups of rabbits, the direction and extent of variation in VIIc generally coincided with variation in cholesterol, so that over time there were significant and positive correlations between plasma cholesterol concentration in both the rabbits fed the standard diet and the rabbits fed the cholesterol-supplemented diet. The increase in VIIc was due to a higher proportion of the more active alpha VIIa in the plasma of hypercholesterolaemic rabbits rather than to an increase in the concentration of the single-chain protein. The plasma concentration of factor X and prothrombin had increased in the hypercholesterolaemic rabbits by 10 days from the beginning of treatment and both proteins were maintained at 150-200% of the concentrations in the plasma of rabbits fed the standard diet. However, these differences were only seen when the factor X and prothrombin were assayed using synthetic substrates. The specific coagulation assays for these two factors revealed no differences between the groups of animals up to 100 days.
Animals↗
Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.
Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.
Animals↗
Lysosomal accumulation of cholesterol and sphingomyelin: evidence for inhibition of acid sphingomyelinase.
The subcellular site of accumulation of non-esterified cholesterol and sphingomyelin in lipid-laden livers and spleens from rats given multiple intravenous injections with liposomes made up of these lipids were investigated by morphological and biochemical techniques. The subcellular fractionation of liver homogenates from cholesterol-sphingomyelin treated rats followed by lipid and enzymatic analyses of the fractions revealed that most of the accumulating lipid was present in very low density lysosomes floating in the post-microsomal supernatant fraction. The low density lysosomes exhibited good latency and had a very much lower relative activity of sphingomyelinase compared with values for N-acetyl-beta-glucosaminidase. Multiple injections of sphingomyelin-cholesterol liposomes resulted in splenomegaly. The spleen homogenates of the treated rats showed a many-fold increase in the concentration of sphingomyelin, of non-esterified cholesterol and in the activity of N-acetyl-beta-glucosaminidase over corresponding values for rats injected with saline. Electron microscopy of liver and spleen sections from treated rats revealed distinctive polymorphic intracellular inclusions bound by a membrane and containing numerous osmiophilic bodies. Structures identical to the storage inclusions seen in fixed liver sections from treated rats were also seen by electron microscopy in the post-microsomal fraction of these livers. The results suggest that sphingomyelin and non-esterified cholesterol accumulate in lysosomes when they occur in cells in excess of that structurally associated with cellular membranes.
Acetylglucosaminidase↗
Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro.
The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.
Acyltransferases↗
The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA:cholesterol acyltransferase substrate pool in liver microsomal fraction.
The incubation at 37 degrees C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA:cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA:cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these various was also the activation energy for the increase in acyl-CoA:cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.
Acyltransferases↗
Stability and clearance of small unilamellar liposomes. Studies with normal and lipoprotein-deficient mice.
The effect of high density lipoproteins (HDL) on the stability and clearance of injected liposomes was investigated under conditions of abnormal lipoprotein metabolism in vivo. Small unilamellar liposomes composed of phosphatidylcholine and containing quenched carboxyfluorescein were injected intravenously or intraperitoneally into normal mice or mice previously made lipoprotein deficient with 4-aminopyrazolo[3,4d]pyrimidine (4-APP). As evidenced from quenched carboxyfluorescein values in the blood, levels of stable liposomes in the circulation were increased and clearance rates reduced considerably in lipoprotein-deficient animals indicating increased bilayer integrity. This was confirmed by the demonstration that transfer of liposomal phosphatidylcholine to HDL, occurring in the presence of normal mouse plasma, was virtually abolished in the presence of plasma from lipoprotein deficient mice. The role of other lipoprotein species in destabilizing liposomes was also investigated. Plasma from lipoprotein-deficient mice was supplemented with increasing amounts of HDL, LDL + IDL or VLDL (to cover the physiological range of lipoprotein concentrations in mouse blood) prior to the addition of phosphatidylcholine liposomes, and incubated at 37 degrees C. It was shown that among the lipoprotein species studied only HDL was detrimental to liposomal stability under the conditions employed. Our results indicate that use of liposomal drugs in the treatment of patients must take into account HDL fluctuations in their blood as these could after liposomal membrane permeability to the drugs and thus upset therapeutic efficiency.
Animals↗
On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone.
The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.
Acyltransferases↗