Defective Lipolysis Persists in Hearts of Rats With Heymann Nephritis in the Absence of Nephrotic Plasma

Defective Lipolysis Persists in Hearts of Rats With Heymann Nephritis in the Absence of Nephrotic Plasma George A. Kaysen, MD, PhD, X-M. Pan, MD, William G. Couser, MD, and Ilona Staprans, PhD • Catabolism of triglyceride-rich lipoproteins, including chylomicrons (CM), is reduced in the nephrotic syndrome. It has been suggested that hyperlipidemia per se might lead to reduced CM catabolism by saturating catabolic sites. Evidence also implicates disordered high-density lipoprotein function as reducing the activity of lipoprotein lipase (LPL), the final effector of CM lipolysis. To establish whether CM lipolysis would be abnormal in the absence of either abnormal rat lipoproteins or hyperlipidemia, we measured CM lipolysis by isolated perfused hearts of rats with passive Heymann nephritis. We found that lipolysis was significantly reduced by 30% at 30 minutes (246 ± 40 ILmol v 164 ± 10 ILmol fatty acid released/hr, P < 0.05). Uptake of fatty acids was also significantly less in nephrotic hearts than in control hearts (7.25% ± 0.93% of dose v 3.32% ± 0.011% of dose, P < 0.01). Total heart LPL activity was reduced by 40% in hearts of nephrotic animals (368.5 ± 39.4 ILmol v 210.6 ± 25.9 ILmol free fatty acid released/hr/g heart, P < 0.01). The heparin-releasable LPL pool is that pool bound to the vascular endothelium and represents the biologically active fraction. We perfused hearts with heparin and found that heparin-releasable LPL was reduced by an order of magnitude in hearts from nephrotic rats (173 ± 33ILmoi v 19.4 ± 11.7 ILmol free fatty acid released/hr/heart, P < 0.001). The decrease in this pool represented nearly entirely the difference in total heart LPL in the two groups. Thus, Heymann nephritis causes reduced lipolysis of CM by isolated hearts in the absence of other rat lipoproteins and in the absence of increased lipid levels. Decreased lipolysis is associated with reduced activity of LPL on the cardiac endothelium, providing a potential mechanism for the reduced CM clearance in the nephrotic syndrome. © 1993 by the National Kidney Foundation, Inc. INDEX WORDS: Lipoproteins; lipoprotein lipase; chylomicrons; nephrotic syndrome; vascular endothelium.

H

YPERLIPIDEMIA in the nephrotic syndrome is a consequence both of increased synthesis and decreased clearance of lipoproteins. I -3 While there is general agreement that lipogenesis is increased, 1 the relationship between increased production of lipoproteins, their decreased catabolism, and hyperlipidemia in this syndrome has been an area of controversy.4 It has been argued that one mechanism causing defective lipid catabolism is one of saturation of catabolic sites by the persistent hyperlipidemia caused by primary overproduction oflipids. 4 InFrom the Renal Biochemistry Laboratory, Department 0/ Medicine, Division 0/ Nephrology, University 0/ California Davis School 0/ Medicine, Davis, CA; the Department o/Veterans Affairs, Northern California System o/Clinics, Benecia, CA; the Department o/Surgery, University o/California, San Francisco, CA; the Department of Veteran's Affairs Medical Center, San Francisco, CA; and the Department 0/ Medicine, University 0/ Washington, Seattle, WA. Received November 2, 1992; accepted in revised form December 22, 1992. Supported in part by the research service o/the United States Department 0/ Veterans Affairs and in part by National Institutes o/Health Grants No. I-RD1 DK 42297 and DK 39198. Dr Pan was supported by The Pacific Vascular Research Foundation. Address reprint requests to George A. Kaysen, MD, PhD, Division o/Nephrology, University o/California Davis School o/Medicine, 4301 X St, Sacramento, CA 95817. © 1993 by the National Kidney Foundation, Inc. 0272-6386/93/2201-0020$3.00/0 128

deed, unlike the situation for many other plasma proteins,5,6 the fractional clearance oflipoproteins decreases as the plasma concentration increases. It has been hypothesized that this effect is in part a consequence of steric interaction between lipoprotein molecules at the receptor site. 7 We previously reported that the absolute rate of chylomicron (eM) catabolism, whether measured kinetically or by direct tissue uptake of triglycerides (TG), was reduced to approximately 25% of normal in nephrotic rats when measured in the fasting state. 8 This observation was compatible with the existence of a defect in eM catabolism that was not a consequence of hyperlipidemia, but instead a cause of increased lipid levels in the nephrotic syndrome. Thus, defective lipoprotein catabolism would appear to be at least in part independent of an increased rate of lipogenesis. While the enzyme lipoprotein lipase (LPL) is the final effector of eM lipolysis, the biologic activity of this enzyme can only be indirectly determined by measuring its total activity in tissue. Interaction between eM and this enzyme is dependent on steric factors. Hydrolysis of eM is so rapid in vivo that to account for their short halflife Olivecrona and Bengstsson-Olivecrona9 suggested that many LPL molecules must react simultaneously with these large particles during their period of attachment to the vascular en-

American Journal of Kidney Diseases, Vol 22, No 1 (July), 1993: pp 128-134

129

ENDOTHELIAL LPL AND LIPOLYSIS IN NEPHROSIS

dothelium. These investigators suggested that CM must be bound to the vascular endothelium for considerable periods of time to allow their hydrolysis to proceed as rapidly as observed,2,4,1O.11 consistent with the low turnover number ofLPL (104 sec-I) in comparison to the large number of acyl bonds in a single CM. Lipoprotein lipase activity is dependent on the presence of apolipoproteins that may either inhibit the activity of this enzyme (apolipoprotein C-III [apo C-III]) or be required for its activity (apo-C 11).12 These small apolipoproteins in turn are shuttled between high-density lipoprotein (HDL) and both CM and very low-density lipoprotein (VLDL). Thus, interaction between apolipoproteins plays an important role in regulating their rate of catabolism. High-density lipoprotein composition is altered in the nephrotic syndromeY Apolipoprotein A-I is increased/ 4,15 while the concentration of other apolipoproteins is decreased, induding apo C-II and apo A-IV, a lipoprotein recently discovered to be necessary for transfer of apo C-II from HDL to other lipoproteins. 16 Indeed, Furukawa l7 found that lipolysis of rat VLDL by LPL in vitro was significantly greater when incubated in the presence of HDL from normal rats when compared with HDL isolated from nephrotic rats. To determine the effect of the nephrotic syndrome on CM lipolysis in the absence of hyperlipidemia and independently of other lipoproteins, we measured CM lipolysis by isolated perfused hearts of nephrotic rats compared with hearts from normal animals. We chose to study lipoprotein catabolism in the heart in part because this tissue actively metabolized fatty acids and also because LPL has been reported to be normal in this tissue. 4 We had previously reported that fatty acid uptake was reduced in the heart to the same extent as it was in adipose tissue, 10 a tissue in which LPL activity was reduced significantly in the nephrotic syndrome. Our observations, taken in conjunction with those of Levy et al,4 that cardiac LPL was not reduced suggested that lipoprotein-lipoprotein interaction might contribute to defective lipolysis in the nephrotic syndrome. While LPL is synthesized by mesenchymal cells, it is only that pool bound to the vascular endothelium that is able to make effective contact with CM. It might be possible that the LPL pool

associated with the endothelium might be decreased, specifically in the nephrotic syndrome, without a change in total cardiac LPL. For this reason we also measured total cardiac LPL activity and that pool ofLPL released from isolated perfused hearts by heparin. METHODS

Protocol No.1: Measurement o/Chylomicron Catabolism by Isolated Perfused Hearts Male Sprague Dawley rats were maintained on purina rat chow with free access to water. Four were injected intraperitoneally with the sheep anti-FX I A serum 18,19 to induce passive Heymann nephritis (HN); four served as controls. Fifteen days following injection with antiserum, each animal was placed in a metabolic cage for 24-hour urine collection to measure urinary albumin excretion. The animals were fasted overnight, then anesthetized with ether. Their hearts were excised and perfused as described below. Terminal blood was collected for measurement of cholesterol, TGs, albumin, and total protein.

Protocol No.2: Determination o/Cardiac Lipoprotein Lipase Activity in Nephrotic Rats A total of 10 HN and nine control animals were studied. Nephrotic rats were each placed in a metabolic cage for 24 hours for urine collection. All animals were fasted overnight prior to study. Four controls and five HN animals were anesthetized with ether as in protocol no. I and their hearts were perfused with heparin as described below. Terminal blood was collected for measurement of TGs, cholesterol, and albumin. Lipoprotein lipase activity was determined both in the perfusate and in the heparin-perfused heart as described below. Total LPL activity was the sum ofLPL activity obtained from assay of the perfusate and activity obtained from assay of the heparin-perfused heart. Total LPL activity was also measured in five hearts from HN animals and from five control hearts without prior heparin perfusion to determine whether the perfusion procedure artifactually reduced LPL activity. These hearts were frozen in liquid N2 without prior perfusion for analysis of LPL in tissues that had not been either subjected to postmortem warming or exposed to heparin.

Heart Perfusion Retrograde aortic perfusion of isolated rat hearts was carried out by the method of Langendorff as described by Fielding. 2o Hearts were rapidly removed. They were initially perfused without recirculation with buffered Krebs-bicarbonate (0.118 mol/L NaC!, 0.025 mol/L NaHC0 3 , 0.0035 mol/L CaCI 2, 0.005 mol/L KCI, 0.001 mol/L KH 2P04 , 0.001 mol/L MgS04 , pH 7.4), gassed with 95% O2 and 5% CO 2, at a flow rate of6 mL/min for 15 minutes to establish viability. The hearts were then perfused with medium containing human serum albumin (I %) and glucose (1 mg/mL). The total perfusion volume of 30 mL was used when we measured CM catabolism, but the volume of perfusate was reduced to 15 mL when the hearts were perfused with heparin in order to release LPL.

KAYSEN ET AL

130

When CM catabolism was measured perfusion was carried out with a recirculating system at 6 mL/min and duplicate 0.2-mL samples were withdrawn every 5 minutes for analysis. Lipids were then extracted from each blood sample by the Dole method, as modified by Trout et al. 21 The samples were each extracted with the Dole method (acid-heptane) to determine residual activity in unhydrolyzed TGs. After extraction, the heptane phase was mixed with 0.5 mL of ethanolic thymol blue solution and made alkaline with 0.05 mL of 0.0 I mol/ L NaOH. The heptane phase containing nonhydrolyzed CM was counted in a scintillation counter (Beckman Instruments, Palo Alto, CAl for 10 minutes and the dose remaining in the perfusate was calculated. The aqueous phase was acidified and re-extracted with Dole's mixture and counted to examine the generation of free fatty acids. Individual CM clearance curves were calculated from the slopes of serum counts per minute using the method of least squares. The rate constant for CM clearance was determined for each animal as In2/tl/2. A mean CM disappearance curve was determined for each group of animals by regression analysis using the means of each time point (based on the percentage of remaining counts) from all animals within each group. To verify that labeled CM were not lost due to sticking to the apparatus, leakage, or other nonspecific causes, the perfusion was also carried out as described above, but without the heart.

Tissue Extraction Hearts were extracted with chloroform/methanol in a dilution of 2: I. Tissues were homogenized with a polytron tissue homogenizer (Brinkman Instruments, Westbury, NY) so that the ratio of organic phase (chloroform/methanol) to aqueous phase was 20: 1. The clear organic phase was then filtered through glass wool. Five milliliters of the filtrate was allowed to evaporate to dryness and was then dissolved in heptane and counted in a liquid scintillation counter for 20 minutes.1O In protocol no. 2, heparin (50 V/15 mL perfusate) was added to the perfusate after the IS-minute period of initial stabilization. The entire IS mL of perfusate was allowed to pass through the heart a single time and was collected for analysis. Both the heart and perfusate were rapidly frozen on dry ice and stored overnight at -70°C until analysis the following day.

Preparation of Chylomicrons Labeled CMs were obtained from a thoracic duct cannula by a modification of the technique of Bollman et al 22 as previously described. 2 The rats were gavage fed with a 2-mL mixture of corn oiljmilk (1:2 dilution) emulsion sonicated together with 0.15 mCi of glycerol tri-(9-1O[3H])-0Ieate (Amersham Searle Corp, Arlington Heights, IL). The chyle was collected in iced tubes for 18 hours and CMs were isolated by layering the chyle under 0.15 moljL NaCI and by flotation in a SW40 rotor (Beckman Instruments) at 5 X 106 g/min at 16°C for I hour.

Lipoprotein Lipase To assay LPL in heart perfusate a total reaction mixture of 3.5 mL was prepared using 1.55 mL of the 15 mL of perfusate, to which was added 0.32 mL of 1.3 moljL Tris-HCI

(Sigma Chemical Co, St Louis, MO), pH 8.6; 0.50 mL of 0.025 moljL NH 40H, pH 8.6; 0.1 mL of 10% Intralipid (Vitrum, Stockholm Sweden); 0.03 mL VLDL (0.8 mg protein/ mL); and 1.00 mL 15% (w/v) crystalline bovine serum albumin (Sigma). The reaction mixture was then incubated at 37°C. One milliliter was removed at time 0, and duplicate 1.0-mL samples were removed from the reaction mixture at 45 minutes. Samples were analyzed for free fatty acids (FFAs) as described below. Free fatty acids were extracted and their content determined by titration with tetrabutyl ammonium hydroxide. Lipoprotein lipase activity was defined in units as micromoles of FFA released per hour per gram of tissue. For measurement of total cardiac LPL activity, 0.4 g of heart was first homogenized in 0.025 moljL ammonia, adjusted to pH 8.2 with HCI, containing 5 mmoljL ethylenediaminotetraacetic acid (EDTA) and 0.8 mg Triton X-IOO, 0.4 mg sodium dodecyl sulfate, I /Lg pepstatin, 10 /Lg leupeptin, 25 KIE Trasylol, and 50 V heparin/mL. 23 Total volume of the extract was 4.0 mL. To assay LPL in heart extract, a total reaction mixture of 3.5 mL was prepared using 0.2 mL of the 4 mL of cardiac extract, to which was added 0.375 mL of 1.3 moljL Tris-HCI, pH 8.6; 0.5 mL of 0.025 moljL NH 40H, pH 8.6; 0.1 mL of 10% Intralipid (Uppsala, Sweden); 0.03 mL VLDL (0.8 /Lg protein/mL); 1.125 mL of 15% (w/v) crystalline bovine serum albumin; and 1.17 mL of 0.154 moljL NaCl. The reaction mixture was then incubated as described for the heart perfusate samples.

Analytic Methods Albumin was measured by electroimmunodiffusion as described previously. I I Both TGs and cholesterol in plasma were determined using kits obtained from Sigma (Cholesterin 50, catalog #352-50; triglycerides-GPO-TRINDER 20, catalog #339-20). Total protein was measured by the method of Lowry et al. 24 Statistical analysis was performed using a one-way analysis of variance. 25 P < 0.05 was considered to be statistically significant.

RESULTS

The rate of hydrolysis of TGs from CM was reduced in hearts from nephrotic animals. Both the t1/2 ofTG removal from CM (Fig 1, Table 1) and the release of FFA into the perfusate (Fig 2) were reduced to approximately 40% of control. Chylomicron hydrolysis by the hearts of nephrotic animals was proportional to the fasting premortem serum TG concentration (r = 0.9696, P < 0.05). The uptake of fatty acids by normal hearts was also significantly greater than the uptake by hearts from nephrotic animals (Table I). The sum of eH]oleate remaining in CM, released as FFA and taken up in the hearts, remained unchanged throughout the period of perfusion in both normal control Sprague Dawley (SO) and HN hearts, demonstrating that [3H]0Ieate hydrolyzed from CM was not bound

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DISCUSSION

nonspecificaUy to the perfusion apparatus and that disappearance of counts from CM was a consequence of lipolysis. Total LPL activity was reduced by less than 50% in the hearts of nephrotic rats when compared with controls (Fig 3). Both total LPL activity and heparin releasable LPL activity were significantly less in hearts of HN rats (Table 2, Fig 3). However, there was no significant difference in the fraction ofLPL activity extracted from the hearts after perfusion with heparin. Total cardiac LPL activity was also significantly less in HN rats than in controls when hearts were not subjected to perfusion with heparin prior to extraction (Table 3). Indeed, total cardiac LPL activity was not affected by prior perfusion with heparin, suggesting that perfusion at 37°C did not inactivate the enzyme.

The hydrolysis and clearance of plasma CM is regulated by the endothelium-bound enzyme LPL. 26 Lipoprotein lipase is synthesized in parenchymal cells, but its biologic activity is expressed only by that fraction that is bound on the vascular endothelium and has access to circulating lipoproteins. Lipoprotein lipase is tethered to the vascular endothelium by the glycosaminoglycan heparan sulfate.27 It is predominantly this fraction that is solubilized when heparin is administered or added to the perfusate. It has been reported that the activity of this enzyme is reduced in nephrosis.3 .4. lo, II ,28 Our findings that the activity of this enzyme is reduced in hearts of HN rats, specifically and only in the heparin labile compartment, are compatible with the hypothesis that depletion of LPL from the vascular endothelium contributes to reduced clearance of

Table 1. Characteristics of Rats in Which Cardiac Chylomicron Lipolysis Was Determined Group Control animals (n = 4) HN animals (n = 4)

Weight (g)

Heart Weight (g)

TG (mg/dL)

Chol

Alb (mg/mL)

TP (mg/mL)

UalbV (mg/24 hr)

220 ± 3.7

1.005 ± 0.0039

63 ± 5

102 ± 12

33 ± 1.7

59 ± 3.7

NM

194 ± 5

0.9325 ± 0.0236

388 ± 85'

452 ± 53'

13± 1.9'

49 ± 7

440 ± 53

CM II (In,ft,,,)

1.2 X ± 0.4 4.36 X ± 1.24

10-' X10- ' 10-3 X 10-3•

FFA Uptake (% ofDose)

7.25 ± 0.93 3.32 ± 0.11'

Note. Comparison of animals used to measure CM catabolism in isolated perfused hearts. Weight refers to total body weight on the day of study. Data are presented as the means ± SEM. Abbreviations: TG, serum triglycerides measured in terminal blood; ChoI, serum cholesterol measured in terminal blood; Alb. serum albumin measured in terminal blood; TP. total protein measured in terminal blood; UalbV. 24-hour urinary albumin excretion determined in the 24-hour period preceding the experiment; CM II. the rate constant of TG removal from CMs in the perfusion system; FFA uptake. the percentage of counts in the perfusate that is present in hearts at the end of the 30minute perfusion period. , P < 0.05 versus control.

KAYSEN ET AL

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Fig 3. Lipoprotein lipase activity solubalized by heparin in the solution used to perfuse isolated hearts of HN and control rats (open area of bars) and LPL activity remaining in the perfused heart (cross-hatched area of bar, hearts from HN rats; diagonal-shaded area of bar, hearts from control animals). The upward-pointing error bars are the SEM of measurement of LPL activity in the perfusate and the differences are significant at P < 0.01. The downward-pointing error bars are the SEM of measurement of LPL activity in the residual myocardium and the differences between these measurements are not significantly different. Total cardiac LPL activity for each of these two groups is the sum of these two values and is represented by the total height of the bars. n = 5 for nephrotic animals and n = 4 for control animals. *P < 0.05 v control.

CM in the nephrotic syndrome. Reduced LPL activity alone, however, may not be sufficient to cause reduced CM lipolysis. Chylomicron and VLDL clearance is normal in rats with hereditary analbuminemia, II even though LPL activity is also greatly reduced in postheparin plasma in these animals. Lipoprotein clearance only becomes abnormal on the development of proteinuria in these animals, suggesting that urinary protein loss plays a necessary role in the pathogenesis of the abnormality in

lipoprotein clearance, independent of any effect on serum albumin concentration or in postheparin LPL activity. Although LPL activity is reduced in the nephrotic syndrome, this may therefore not be the sole cause of defective lipoprotein catabolism in vivo. The ratio of protein to lipid is reduced in nephrotic lipoproteins,4,29 and the relative concentration of specific apolipoproteins within each lipoprotein class is also abnormal. 30 While plasma apo A-I levels are increased in proportion to HDL levels in nephrosis, the concentration of virtually all other apolipoproteins (A-II, A-IV, E, and CII) is reduced in proportion to HDL. 13 ,14 While total plasma apo C-II is normal in both nephrotic rats l3 ,14 and patients,31,32 the concentration of this important LPL activator is reduced per CM or VLDL particle 32 and the concentration of the competitive inhibitor of LPL, apo C-III, is increasedY Apolipoprotein C-II is a necessary LPL co-factor and apo A-IV is required for efficient release of apo C-II from either HDL or VLDL,16 a process necessary for LPL-mediated hydrolysis of TG in nascent CMs. Thus, abnormalities in the apolipoprotein composition of nephrotic lipoproteins could contribute in their decreased rate of catabolism. Furukawa et all? reported that VLDL was hydrolyzed by LPL at a lower rate in vitro in the presence of HDL from nephrotic rats compared with HDL from normal rats, suggesting that LPL might perform suboptimally in vivo because of an abnormality in HDL and not because of a reduction in LPL.

Table 2. Characteristics of Rats in Which Heparin Releasable Lipoprotein Lipase Activity Was Determined Heparin-releasable LPL ("mol FFA Released/hr)

Group Control (n ~ 4) HN (n

~

5)

Heart Weight (g)

TG (mg/dL)

Chol

Alb (mg/mL)

0.925 ± 0.028

64 ±7

69 ±3

36.6 ± 0.4

0.82 ± 0.026

434 ± 40"

434 ± 39"

12.6 ± 0.15"

UalbV (mg/24 hr)

NM 377 ± 20

Tissue-bound LPL ("mol/hr) Per Gram Heart

Per Heart

Total LPL Activity &tmol FFA Released/hr)

Per Gram of Heart

Per Heart

Per Gram Heart

Per Heart

173 ± 33

159 ± 29

226 ± 28

207.7 ± 23

385 ± 51

353.5 ±43.4

19.4 ± 11.7"

15.6 ± 9.8"

156.8 ± 38.6

129.4 ± 32.5

172.4 ± 36"

142.0 ± 30.4"

Note. Comparison of animals used to measure heparin-labile LPL activity in isolated perfused hearts. Data are presented as means ± SEM. Abbreviations: TG, serum triglycerides measured in terminal blood; Chol, serum cholesterol measured in terminal blood; Alb, serum albumin measured in terminal blood; UalbV, 24-hour urinary albumin excretion determined in the 24-hour period preceding the experiment; NM, not measured. Total LPL activity is the sum of LPL activity released by heparin during infusion and LPL activity extracted from the perfused heart. " P < 0.05 versus control.

ENDOTHELIAL lPl AND LIPOLYSIS IN NEPHROSIS

133

Table 3. Lipoprotein Lipase Activity in Hearts Obtained From Rats With Heymann Nephritis LPL Activity (j.mol FFA Released/hr) Group Control (n = 5) HN (n = 5)

Weight (g)

Heart weight (g)

215 ± 7

0.882 ± 0.049

187 ±

r

0.75

± 0.052

TG (mg/dL)

34.6 ±

8.7

274.0 ± 54.5"

Chol (mg/dL)

68.2 ± 11.5 369 .2 ± 77.6"

Alb (mg/mL)

Per Gram Heart

Per Heart

33.1 ± 2.4

368.5 ± 39.4

323.9 ± 35.3

11.0 ± 1.04"

210 .6 ± 25.9"

154.7 ± 14.9"

Note. Comparison of animals used to measure LPL activity in unperfused hearts. Data are presented as the means ± SEM . Abbreviations: Weight. total body weight on the day the animals were killed; TG. serum triglycerides measured in terminal blood; Chol. serum cholesterol measured in terminal blood; Alb. serum albumin measured in terminal blood; UalbV. 24-hour urinary albumin excretion determined in the 24-hour period preceding the experiment; LPL is lipoprotein lipase activity; NM. signifies not measured . " P < 0.05 versus control.

We initiated these studies because we had previously found that the rate of fatty acid uptake was similarly decreased in heart and adipose tissue. IO The decrease in TG uptake was not only a consequence of dilution of labeled TGs in a large unlabeled pool since absolute uptake offatty acids was decreased. We found this especially interesting in light of the report of Levy et at,4 which indicated that LPL activity in the heart was normal in nephrotic rats. This finding, were it verified, would exclude a role for decreased LPL per se as being responsible for decreased eM lipolysis in the nephrotic syndrome and would support Furakawa et ai's l7 observations that HDL may be dysfunctional in nephrotic animals. The finding that decreased lipolysis occurred in the isolated perfused heart despite the absence of plasma from nephrotic animals is direct evidence that local endothelial factors necessary for eM lipolysis are abnormal in the nephrotic rat, but do not exclude abnormalities in lipoprotein-lipoprotein interactions as well. Indeed, the difference in eM lipolysis by isolated hearts for nephrotic animals compared with controls was much less than the differences in cardiac TG uptake observed in vivo. IO The difference between our observations and those of Levy et al may be explained by the differences in methods used to determine LPL activity. Levy et al 4 extracted LPL from an acetone powder, while we used a more gentle detergent extraction from frozen hearts. It is possible that Levy et al extracted an especially tightly bound LPL pool that we did not, or that their method inactivated LPL while ours did not. Nevertheless, the relatively small, but biologically important LPL pool releasable by heparin is reduced by

nearly an order of magnitude in hearts from nephrotic animals and the methods used by Levy et al could not have revealed the differences that we observed, even if total LPL activity is indeed nearly identical in nephrotic and normal myocardium. It is not known why the heparin labile pool of LPL is so reduced in the nephrotic syndrome. Karpe et al 34 recently established that plasma LPL activity increased in normal human subjects after an oral fat load. They suggested that this response might be a consequence of release of LPL mediated by its interaction with either TGs or FFAs. They did not measure postheparin LPL activity in these patients to establish whether any depletion of the heparin labile pool also occurred. Saxena et al 3s found that LPL bound to endothelial cells in tissue culture could be released by exposure to the TG lipoproteins VLDL and eM. Thus, hyperlipidemia, as occurs in both the nephrotic syndrome and in the condition of hereditary analbuminemia, might be in part responsible for reduced heparin labile LPL activity. ACKNOWLEDGMENT The authors acknowledge the expert assistance of Agnes Frank.

REFERENCES I. Marsh JB: Lipoprotein metabolism in experimental nephrosis. J Lipid Res 25:1619-1623, 1984 2. Staprans I, Felts JM, Couser WG: Glycosaminogiycans and chylomicron metabolism in control and nephrotic rats. Metabolism 36:496-501, 1987 3. Garber OW, Gottlieb BA, Marsh lB, Sparks CE: Catabolism of very low density Ii po-proteins in experimental nephrosis. J Clin Invest 74: 1375-1383, 1984 4. Levy E, Ziv E, Bar-On H, Shafrir E: Experimental nephrotic syndrome: Removal and tissue distribution of chy-

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