A possible mechanism for accelerated atherogenesis in male versus female rats.

Dietary fat and cholesterol enter the circulation as chylomicrons. They are removed from the circulation by attachment to lipoprotein lipase located on the endothelial surfaces. As the result of lipoprotein lipase action, chylomicrons are partially hydrolyzed and then reenter the circulation as remnants, which are rapidly cleared by the liver. We investigated the fate of 3H-retinol- and 14C-cholesterol-labeled chylomicrons injected into male and female rats. The disappearance curves of chylomicrons from the circulation were not significantly different in males and females, which suggests that translocation from plasma to endothelium is similar for both sexes. However, in male rats, the "dwell time" of chylomicrons on the endothelium was significantly prolonged. At 10 and 20 minutes after chylomicron injection, more label was found in the livers of female than male rats. The opposite was true for hearts. Male hearts contained significantly more endothelium-bound chylomicrons when compared with female hearts. This increase in dwell time may allow greater cholesterol deposition in the endothelium of male rats. The more rapid processing of chylomicrons was associated with a 300% greater postheparin lipoprotein lipase in female rats, which suggests a greater enzyme density at chylomicron attachment points on endothelium.

Kinetic characterization, stereoselectivity, and species selectivity of the inhibition of plant acetyl-CoA carboxylase by the aryloxyphenoxypropionic acid grass herbicides.

The selective grass herbicides diclofop, haloxyfop, and trifop were found to be potent reversible inhibitors of acetyl-CoA carboxylase from the susceptible species barley, corn, and wheat. Kis values with variable concentrations of acetyl-CoA ranged from 0.01 to 0.06 microM at pH 8.5 depending on the species of grass. Inhibition of the wheat enzyme by diclofop was noncompetitive versus acetyl-CoA with Kis less than Kii and noncompetitive versus MgATP and bicarbonate, but with Kis approximately equal to Kii. Since the apparent inhibition constant was most sensitive to the level of acetyl-CoA, these compounds probably interact with the transcarboxylase site rather than the biotin carboxylation site. With the wheat enzyme the Kis value for the R-(+)-enantiomer of trifop was 1.98 +/- 0.22 times lower than that of the racemic mixture. This confirms the stereoselectivity observed in the whole plant. The enzyme from tolerant broadleaf species (spinach and mung bean) was much less sensitive to these herbicides (Kis values varied from 16 to 515 microM). These data confirm that acetyl-CoA carboxylase is the site of action for the aryloxyphenoxypropionic acid herbicides and may explain their selectivity for monocotyledenous species.

Cyclohexanedione Herbicides Are Selective and Potent Inhibitors of Acetyl-CoA Carboxylase from Grasses.

Biochemical studies of plant species susceptible to the cyclohexanedione herbicides, alloxydim, sethoxydim, and clethodim, have demonstrated that these selective grass herbicides inhibit acetyl-coenzyme A carboxylase, the second enzyme common to both fatty acid and flavonoid biosynthetic pathways. The K(i)s for the cyclohexanediones tested ranged from 0.02 to 1.95 micromolar, depending on the species. The enzyme isolated from broadleaf plants was much less sensitive to inhibition by these herbicides (K(i)s from 53 micromolar to 2.2 millimolar). These results may explain the mechanism of action of these herbicides and their selectivity for monocotyledonous species.

The disappearance from the circulation of chylomicrons obtained from control and diabetic rats.

Previous studies have demonstrated that cholesterol synthesis is increased in the small intestine of diabetic animals and that there is an increased transport of this newly synthesized cholesterol from the small intestine into the circulation. Chylomicrons play an important role in the transport of cholesterol from the small intestine into the circulation, and the present study compared the rates of disappearance from the circulation and the fate of chylomicron cholesterol obtained from control and diabetic animals when administered to either intact control or diabetic rats. Thoracic duct lymph was collected from normal and diabetic rats after the administration of [14C]cholesterol and [3H]vitamin A1. The labeled chylomicrons were isolated by centrifugation and then administered to either control or diabetic rats. The major observation of this study is that chylomicron-associated sterols obtained from diabetic animals were cleared from the circulation in a normal manner. If one compares the rate of disappearance of either [3H]vitamin A1 or [14C] cholesterol-labeled normal chylomicrons administered to control animals with that of labeled diabetic chylomicrons administered to diabetic animals, the half-times in the circulation are almost identical (control: [3H]vitamin A1 t 1/2, 3.6 min; [14C]cholesterol t 1/2, 5.7 min; diabetic: [3H]vitamin A1 t 1/2, 3.5 min; [14C]cholesterol t 1/2, 4.4 min). In both experimental situations the rapid disappearance of [14C]cholesterol was associated with the appearance of [14C]cholesterol in the liver. Very little [14C]cholesterol was present in tissues other than liver, indicating that the rapid removal of labeled chylomicron remnants from the circulation was accounted for by hepatic uptake. These results demonstrate that in diabetic animals chylomicron-associated sterols are cleared from the circulation normally and that the bulk of intestinally derived cholesterol carried in the chylomicron lipoprotein fraction is rapidly delivered to the liver, where it could potentially influence lipoprotein metabolism.

Glycosaminoglycans and chylomicron metabolism in control and nephrotic rats.

Nephrotic patients and rats with experimentally induced nephrotic syndrome have elevated plasma triglycerides and impaired triglyceride removal. This may be due to a defective interaction of chylomicrons and very low density lipoproteins with lipoprotein lipase. Since the glycosaminoglycan, heparan sulfate, was found to stimulate the lipoprotein lipase reaction in vitro, we investigated the plasma heparan sulfate content and measured the urinary excretion of heparan sulfate in control rats and rats with experimentally induced nephrotic syndrome. In addition, we studied the effect of heparan sulfate on the rate of removal of radiolabeled chylomicrons in nephrotic rats. Glycosaminoglycan concentrations in plasma were the same in control and nephrotic rats, although 35S incorporation in high charge glycosaminoglycans was markedly reduced. In addition, in nephrotic rats there is a marked reduction in the urinary excretion of heparan sulfate and chondroitin sulfate suggesting a markedly reduced turnover of these glycosaminoglycans. This was associated with increased plasma triglycerides in nephrotic rats. Nephrotic rats showed a reduced rate of clearance of injected chylomicrons. Intravenous administration of heparan sulfate completely and immediately corrected the chylomicron removal defect. We also noted a log-dose response effect of administered heparan sulfate on chylomicron removal. This effect was not due to a release of soluble lipoprotein lipase by heparan sulfate. These findings suggest that a rapidly turning over fraction of plasma heparan sulfate may play an important role in chylomicron clearance.

The quantitative separation of chylomicrons and chylomicron remnants by column chromatography.

Chylomicrons and chylomicron remnants cannot be separated by classical techniques of ultracentrifugation or gel filtration, since there is a marked overlap of density and size. Chylomicron remnants develop a high negative surface charge during their formation presumably due to surface-oriented free fatty acids. Two chromatographic matrices have been identified which separate these two lipoprotein species based on the charge differences. Both DEAE-Sephacel and protamine-Affi-Gel 10 effect a quantitative separation. These techniques may be useful in studies of chylomicron metabolism both in vivo and in vitro.

Analysis of selected plasma constituents in continuous ambulatory peritoneal dialysis effluent.

Patients maintained on continuous ambulatory peritoneal dialysis (CAPD) lose plasma constituents into the dialysis effluent. We have analyzed 24-hour CAPD effluents for selected components--total protein, a typical glycoprotein (alpha 1-acid glycoprotein), a typical lipoprotein (high density lipoprotein), and glycosaminoglycans. Our findings suggest that the plasma constituents found in CAPD effluent are similar to those found in urine from nephrotic patients. The loss of one or more of these plasma constituents into the dialysis solution may be linked to the hypertriglyceridemia observed in these patients.

Mechanisms and consequences of proteinuria.

The glomerulus is a complex structure containing a remarkable capillary bed which is freely permeable to water and solutes up to the size of inulin. Many small proteins are filtered, reabsorbed, and catabolized by the kidney; but most large proteins, such as albumin or immunoglobulins, are almost entirely excluded from the glomerular ultrafiltrate due to the charge-size permselectivity of the glomerular capillary basement membrane. These large proteins appear in the urine when diseases reduce the charge selectivity or result in the development of large pores in this membrane. The reabsorptive capacity of the renal tubules for these proteins is overwhelmed. Hypoalbuminemia results when increased synthetic and decreased catabolic rates of albumin fail to compensate for the urinary loss of the protein. The resulting decrease in serum oncotic pressure increases the flux of fluid out of systemic capillaries into the interstitial space, a process that increases lymphatic flow and returns the relatively protein-poor ultrafiltrate to the plasma compartment. Interstitial proteins are swept into the plasma by the increased lymphatic flow, leading to a depletion of the extravascular pool of albumin even more severe than the depletion of albumin in the plasma compartment. The rate of albumin synthesis is increased but not sufficiently to replace losses and restore the serum concentration to normal. The rate of albumin catabolism is decreased. This decrease from the normal catabolic rate is as important as the increased rate of albumin synthesis in maintenance of albumin homeostasis in nephrosis. Whereas the reduced serum oncotic pressure certainly contributes to edema formation, sodium retention may result from processes intrinsic to the kidney itself; and plasma volume may actually be expanded despite hypoalbuminemia. The hyperlipemia that occurs in nephrosis is due to a combined defect in lipoprotein metabolism: increased hepatic synthesis of VLDL and decreased removal of TG and highly atherogenic remnants of incompletely metabolized CMs. The defects in lipoprotein metabolism may in part be the end result of the urinary loss of highly negative-charged macromolecules of the mucopolysaccharide called orosomucoid, which carries with it heparan sulfate, and important cofactor for LPL.

Isolation and characterization of glycosaminoglycans in human plasma.

We have described methodology for the isolation and quantitation of glycosaminoglycans present in human plasma. Plasma glycosaminoglycans can be quantitatively adsorbed on a DEAE-Sephacel ion exchanger and eluted with a salt gradient as two groups: a low-charge fraction and a high-charge fraction. The low-charge fraction consists of chondroitin sulfate with a low sulfate content and the high-charge fraction consists of heparan sulfate, chondroitin sulfate, and keratan sulfate (type I). We have determined the plasma concentration of each of these glycosaminoglycans in six normal human subjects. We have established that none of the glycosaminoglycans in plasma are covalently linked to plasma proteins. All are isolated as complexes with plasma proteins in noncovalent linkages. The glycosaminoglycans in the low-charge fraction are bound with high affinity to a single plasma glycoprotein by a lectin-type bond that can be disrupted by a simple glycoside. The high-charge fraction contains three major proteins and several minor proteins associated with the glycosaminoglycans by both lectin-type and ionic bonding. The plasma proteins associated with glycosaminoglycans represent less than 0.5% of the total plasma proteins. Little is known about the physiologic role of the plasma glycosaminoglycans as components of metabolic processes. Because glycosaminoglycans have been implicated in lipid metabolism and atherosclerosis, we tested all of these compounds, isolated in free form, on the in vitro hydrolysis of triglycerides by lipoprotein lipase. Plasma heparan sulfate stimulated the rate of this reaction severalfold. All other plasma glycosaminoglycans were inactive. Thus, plasma heparan sulfate may play an important role in plasma lipoprotein metabolism.

Quantitative determination of individual glycosaminoglycans in plasma by concanavalin A rocket electrophoresis.

A new one-dimensional agarose gel electrophoresis method for the quantitation of glycosaminoglycans in biological samples has been described. In this procedure, concanavalin A, suspended in agarose gel, interacts with glycosaminoglycans such that rocket-like precipitin lines are formed. The area of the rocket is directly proportional to the glycosaminoglycan content of the sample. This procedure permits measurement of glycosaminoglycans in amounts as low as 0.5 nmol uronic acid equivalents with a coefficient of variation of only 8%. The described method has been applied to the determination of free heparan sulfate in plasma. This method can also be used to measure all high-charge glycosaminoglycans of biological interest.

A glycosaminoglycan activator of lipoprotein lipase in human plasma.

By use of ion exchange chromatography we have isolated two discrete classes of "free" glycosaminoglycans (GAG) from human plasma. The GAG fractions were tested for their effects on two lipoprotein lipase (LPL) enzyme systems containing an apolipoprotein C-II activated emulsion as the triglyceride substrate and bovine serum albumin as the free fatty acid acceptor. The lowcharge GAG (Fraction I) had essentially no effect on the LPL reaction. The high-charge GAG (Fraction II) stimulated the LPL reaction 100 to 300%. The GAG composition of each fraction was investigated with chemical and enzymatic techniques. Fraction I consisted of low-charge chondroitin sulfate noncovalently bound to protein. Fraction II consisted of a mixture of high-charge GAG non-covalently bound to protein. Degradation with nitrous acid eliminated the ability of high-charge GAG to stimulate LPL. This and other evidence suggests that the high-charge GAG in human plasma responsible for LPL activation is heparan sulfate (HS). We suggest that plasma HS may modulate triglyceride clearance mechanisms in vivo by its interaction with LPL.

Acute effects of dichloroacetate in the depancreatized dog: glucose synthesis and turnover.

Blood glucose turnover (entry and removal rates) and the rate of recycling of radiolabelled glucose carbon into newly synthesized blood glucose have been evaluated before and acutely after the administration of dichloroacetate to depancreatized dogs. Blood glucose concentration began to decline immediately after dichloroacetate administration and fell to new steady state levels within 1.5-3 h. Analysis of blood glucose kinetics during the decline demonstrated a 52% (average) reduction in the rate of hepatic glucose supply. Glucose supply remained reduced over the duration of these studies (3-4.5 h). Glucose turnover in the steady state following dichloroacetate administration averaged 62% of pretreatment values. Cori cycle activity was depressed by 63% after dichloroacetate administration. The results of these studies are consistent with the hypothesis that a major mechanism underlying the hypoglycaemic action of this drug is the inhibition of glucose synthesis.

Characterization of glycosaminoglycans in urine from patients with nephrotic syndrome and control subjects, and their effects on lipoprotein lipase.

Previously we found that alpha 2-acid glycoprotein fraction from urine of patients with the nephrotic syndrome stimulated the lipoprotein lipase reaction in vivo and in vitro. The activator was separated from the alpha 1-acid glycoprotein and identified as a glycosaminoglycan. The studies reported here were undertaken to characterize and quantify the glycosaminoglycans contained in urine of patients with the nephrotic syndrome and to compare these to the glycosaminoglycans in urine of the control subjects. We found that free low molecular weight glycosaminoglycans, heparan sulfate and chondroitin 4-sulfate, are excreted in both patients with the nephrotic syndrome and controls however, patients with the nephrotic syndrome excreted much less of both glycosaminoglycans. The free form of heparan sulfate was found to be the activator which stimulated the lipoprotein lipase reaction in vitro in the presence of apolipoprotein CII. In addition, the urine from patients with the nephrotic syndrome contained a protein-glycosaminoglycan complex which was absent in control urine. Glycosaminoglycans in the complex could be released by papain digestion or by trichloroacetic acid. Our evidence indicates that this glycosaminoglycans fraction is a law charge form of chondroitin sulfate.

Lipoprotein spectrum analysis of uremic patients maintained on chronic hemodialysis.

It is known that patients maintained on chronic hemodialysis have elevated plasma lipids. In order to establish whether the type of kidney pathology is related to a specific lipoprotein abnormality, we measured plasma lipoprotein in eight patients with glomerulonephritis, two patients with polycystic kidney disease and nine patients that had been surgically nephrectomized. The concentration and composition of plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) in patients were compared to plasma lipoproteins in a control group. In all patient groups, the lipoprotein alterations appeared identical. VLDL were elevated 3 to 4 fold, LDL were decreased 15--35% and HDL were decreased 30--45% when compared to values of our control group. Since no differences in the lipoprotein spectrum were found among the patient groups, it appears that the hypertriglyceridemia of chronic uremia is due to the uremic state per se and is not related to a specific pathology of the kidney.

Effect of haematocrit value and pO2 on the redox state and metabolism of the perfused liver.

1. The haematocrit value and pO2 of blood perfusing the isolated liver were varied. Provided O2 content of the blood was not rate-limiting, O2 consumption was related to haemotocrit value rather than O2 saturation or pO2. 2. Hypoxia caused the blood-glucose concentration and ketogenesis to increase and the output of very-low-density (d less than 1.006) lipoproteins to decrease. 3. A decrease in pO2 caused an increase in both the (lactate)/(pyruvate) and (3-hydroxybutyrate)/(acetoacetate) and a decrease in (ATP)/(ADP) ratios, independently of O2 consumption. 4. The more reduced redox state was associated with a shift in the balance between the oxidation and esterification of free fatty acids in favour of oxidation. 5. Acetoacetate may be an important hydrogen acceptor during hypoxia of the liver.