Chylomicron synthesis in experimental nephrotic syndrome

Biochimica et Biophysica Acta. 1005 (1989)20-26

20

Elsevier BBALIP 53191

Chylomicron synthesis in experimental nephrotic syndrome Emile Levy 1, E h u d Ziv 2, H a n o e h B a r - O n 2 a n d Eleazar Shafrir 1 Departments of n Biochemistry and ~ Medicine B, Hadassah University Hospital and Hebrew Unwersity.Hadassah Medical School, Jerusalem (Israel)

(Received 21 February 1989)

Key words: Ngphrotic syndrome: Hyperlipoproteinemia; Lymph flow rate: Chylomicron synthesis: Triacylglycerol: Intestinal apolipoprotein; (Rat)

M ~ t ~ t c lymph was collected for 48 h from rats with aminonucleoside-induced nephrotie syndrome, receiving an intreduodemd infmlon of a trlacylglyeerol emulsion. In nephrosis, the rates of lymph flow and triacylglyeerol transport qJpcox. 2-fold hlllhm', but the transport of total protein and of apoproteins A-I and E was 2- to 3-fold lower than that In control rats, resulting in chylomicrons with a 3-fold approx, elevated trlaeylglycerol/protein ratio. Supplementa. tion ot the trla~lglyeerol infusate with glucose and amino acids did not increase the protein or apoA-! and apoE transpocL PrmIMctton or transpert of B and C apoproteins in nephrotte rats was also reduced, as indicated by tetg~lurea solubility, ineorimraflon of intradnodonully Infused [3Hlleueine and staining of the ehylomieron protelm on SDS-PAGE gels. Apelm~in A.IV was the only ehylomleron component into which the leueine incorporation was elevated, but its relative content was not increased on SDS-PAGE gels. Lymph ehylomlerons of nephrotie rats were ~ in size (14964-37 vs. 1235:1:23 ,A), consistent with the higher trlacylglycerol/protein ratio. The concentration of all lipoiwotein classes was markedly elevated in the plasma of nephrotie rats, as was that of the total A-! and E apopcoteins, lntravenmm injection of taSl.lahelled HDL, followed by tracing of the label in lymph ¢hylmnicrons, Indicated a lower rate of transfer of HDL apoproteins from plasma to lymph in nephrotie rats. We conclude that the intestinal cl~lomicron formation in u e ~ i s is ehacacterised by an enhanced triaeylglycerol transport without the ~ t e ~eln complement. This is probably due to the limited capacity of enterocytes, in marked contrast to hepato~tes, to respond to the hypowoteiuemia of nephrosis with increased production a n d / o r transport of the apowoteim. I n ~

Nephrotic syndrome is well known to be associated with hyperlipoproteinemia [1]. There is ample evidence of i n ~ hq~ttic production of albumin and of the apoprotein and lipid moieties of the lipoproteins [2-7]. The factors trisgeri~ the acceleration of the hepatic lipoproteAn synthesis have not yet been determined with certainty, but replacement of plasma albumin led to diminution of h~rlipoproteinemia [8,9]. Infusion of syl:thetic macromolecuh~ also resulted in amelioration of the hyperlipoproteinemia [9,10] and an increase in medium viscosity attenuated the synthesis of lipopro-

Abbt~.~ti¢~: VLDL, LDL, HDL, very-low-, low- and high-density Upoprmc~ns; SDS.PAGE, sodiom dodecylsulphate polyacrylamide gel elec~; TMU, tetramethylurea. Corn~peadeace: F_ Shafrir, Department of Biochemistry, Hebrew Univets/ty-Hadass~ Med~d School, P.O. Box 12000. Jerusalem 91120, Israel.

reins and albumin in isolated hepatocytes [11]. These results suggest that the reduction of plasma viscosity and/or oncotic pressure, resulting from the excessive proteinuria, might provide the stimulus for the hepatic compensatory synthesis of albumin and lipoproteins. The intestine, the site of chylomicron production, is capable of synthesis of apoproteins A-I, A-IV and B, which are integrated together with plasma-derived apoproteins, into the structure of chyiomicrons released to the lymph [12-14]. We have investigated whether the intestinal transport and/or synthesis of chylomicron components is also increased in the nephrotie condition analogously to the enhanced lipoprotein synthesis in the liver. Materials and Methods Male albino rats of Hebrew University strata, weighing 200-250 g and fed a standard pelleted laboratory chow were used. Nephrosis was induced by a single i.v. injection of 3 mg/100 g of aminonucleoside of

0005-2760/89/'$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

21 puromycin (Sigma, St Louis, MO, U.S.A.) as a 30 mg/ml solution in 0.15 mol/l NaCI. The rats were used in the experiments 10-14 days later. Control rats received an appropriate volume of the NaC! solution.

lipoproteins were separated by ultracentrifugation according to Havel et al. [17]. Each fraction was washed once before analysis by recentrifugation at the respective density.

Collection of lymph and isolation of chylomicrons

Incorporation of labelled ieucine

Mesenteric lymph duct cannulation was performed by a modified technique of Bollman et al. [15], using a duodenal fistula for infusion of fluids through silastic tubing. After the closure of the abdominal incision and the exteriorisation of the cannulae and tubings, the rats were maintained in restraining cages. A triacylglyceroi emulsion was infused for 48 h at the rate of 2.8 ml/h. The emulsion consisted of 270 purified glycerol trioleate (Sigma), 270 Intralipid (Vitrum, Stockholm, Sweden), 0.0,~70 purified cholesterol and 0.470 plant phosphatidylcholine and was dispersed in a Polytron sonicator in 0.15 moi,/l NaCI. In several experiments, as specified, the emulsion was fortified by the addition of 570 glucose or 270 amino acid mixture (Freamine, American Hospital Supply Corp., Evanston, IL U.S.A.). After an initial period of flow stabilization, the lymph was collected into a container maintained at 4°C. At specified time intervals, the lymph was removed, freed of any clot by filtration through glass wool and spun in the 50 Ti rotor of Beckman-Spinco ultracentrifuge at 3.10 -3 g - m i n at 4°C. The floating chylomicron layer was gently dispersed in the NaCI solution and recentrifuged, and the chylomicrons were harvested. For the determination of composition, the chylomicrons were purified by passing through a 270 agarose gel column (Bio-Gel A-15, 50-100 mesh, BioRad, CA, U.S.A.), which removed most of the adsorbed albumin, fhe chyiomicrons were eluted with 2 mmol/i solution , f disodium EDTA containing 0.0270 NaN 3 (pH 7.0) at a rate of 7 ml/h. The purified chylomicrons were taken for lipid and apoprotein analysis and for SDS-PAGE [14,16].

3H-labeled leucine (New England Nuclear, Boston, MA, U.S.A.) was added to the intraduodenally infused triacylglycerol emulsion (2 mCi/50 ml). The lymph was collected and processed as described above, and the chylomicrons were isolated and the radioactivity incorporated into the apoproteins was determined by counting segments of the SDS-PAGE gels.

Lipoprotein isolation Disodium EDTA and NaN 3 were added to rat plasma in a final concentration of 1 mg/ml each and the

Administration of labelled HDL HDL, isolated from a pool of control rat serum by ultracentrifugation, was labelled with 1251 as described by Bar-On and Eisenberg [18]. The radio-iodinated HDL contained less than 5% radioactivity in the lipid fraction and the molar ratio I2/HDL-protein was approx. 1.8. The labelled HDL was injected i.v. into control and nephrotic rats 2 h after the start of the intraduodenal triacylglycerol infusion. The chylomicrons were isolated at different time intervals and their total apoprotein radioactivity was determined after lipid extraction, solubilization and counting of the protein moiety.

Analytical procedures Triacylglycerol and cholesterol were determined by specific enzymatic methods using Boehringer-Mannheim kits (F.R.G.), protein by the method of Lowry et al. [19] and phospholipids by the method of Bartlett [20]. A-I and E apoproteins were radioirnmunoassayed [21,22]. Samples of chylomicrons were taken also for electron-microscopic examination of size distribution [23]. Results

Table I illustrates the pattern of hyperlipoproteinemia in the nephrotic rats which were ,lsed as donors of mesenteric lymph upon cannulation. I'his pattern

TABLE ! Plasma iipoprotein composition and apolipoprotein content in nephrotic rats Values are in mg/dl, obtained from pooled plasma of eight control and eight nephrotic rats (means of triplicates). Values for total plasma lipoproteins represent a recovery of 82-937o of the triacylglycerol and cholesterol concentration in the original plasma pool. Total apolipoprotein levels were determined by immunoassay in the same pool. TG = triacylglycerol; chol. = total cholesterol. Fraction

Nephrotic plasma

Control plasma

TG

chol.

protein

VLDL ( d < 1.006 g / m l ) LDL ( d = 1.006-1.063 g / m l ) HDL ( d = 1.063-1.21 g / m l )

481 50 19

95 208 71

60 126 115

Total lipoproteins

550

374

301

apoA-I

4.11

apoE

10.8

TG

chol.

protein

47.2 8.6 5.1

7.4 15.4 27.6

8.3 10.2 66.8

60.9

50.4

85.3

apoA-I

apoE

0.85

0.73

22

/

30

T

.

0

"

.

.

:

I

!

!

!

.

--,-~

o-- 4

e

~

24

38

'

48

HOURS Fi$, I. R=tes of mescnteric lymph flow, triacylglycerol and total protein transport in nephroti¢ (A) and control (e) rats, infused intraduodenally with a triacylslycerol emulsion. Values are means+ S.E. for the time-course of lymph collection from eight rats.

conforms to that observed previously in our and other laboratories in aminonucleoside nephrosis and documents the marked elevation in all plasma lipoprotein classes. Table I also demonstrates the pronounced increase in plasma levels of total A-! and E apoproteins. Fig. 1 sho,s the rate of mesenteric lymph flow in nephrotic rats. The 2-fold increase in the rate of flow in nephrotic rats was particularly apparent 12 h after the start of lymph collection and persisted until 48 h. An even greater increase was seen in the rate of triacylglycerol transport. This was significant by 8 h after me start of lymph collection (P < 0.02) and persisted for the whole duration of the experiment. However, in contrast to both higher lymph flow and triacyiglycerol transport, there was a consistently low lymph protein transport in nephrotic rats throughout the infusion of triacylglyceroi emulsion. The overall transport rate at 48 h of collection amounted to 38.8 4- 3.0 vs. 15.9 :!: 1.4 m s / h for triacylslycerol in and 9.5 + 1.3 vs. 18.8 :E 1.6 for total protein in the lymph of nephrotic and control rats, respectively, representing an almost 5-fold difference (P < 0.01). The time-course of transport of A-[ and E apoproteins into the lymph is shown in Fig. 2. ]t was persistently lower in nephrotic rats; it remained stable in control rats, while decreasing after 24 h in nephrotic rats. The mean flow rate during the 48 h of collection was 156 4- 17 vs. 290 4- 22 Fg/h for apoA-] and 8 4- 1 vs. 23 4- 2 Fg/h

for apoE in nephrotic and control rats, respectively, showing a 2- to 3-fold difference in favour of control rats (P < 0.01). Low content of A-I, E and of C apoproteins was also evident from the SDS-PAGE analysis of the lymph chylomicrons of nephrotic rats (Fig. 3). Table II demonstrates that the isolated and purified nephrotic chyiomicrons were richer in triacylglycerol and poorer in protein than those from control rats. This is emphasized by an approx. 3-fold increase in the triacylglycerol/protein ratio in the nephrotic lymph chylomicrons ( P < 0.01). The latter were also slightly richer in cholesterol and significantly poorer in phospholipids (Table ll), Concordant with these changes in composition, the electronmicroscopic appearance (Fig. 4) and size distribution (Fig. 5) revealed that the nephrotic lymph particles were significantly larger. The apoprotein B content of the chylomicrons was estimated by testing its solubility in tetramethylurea (TMU) solution according to the procedure of Kane [24]. in four experiments, the total protein solubility of pools of isolated mesenteric lymph chylomicrons from control rats ranged from 76 to 84% (mean 79%), whereas the nephrotic chylomicron protein was 86 to 93% soluble (mean 90%), indicating a lower content of the TMU-insoluble apoB. The protein content of the

40!

v

:343

&4k,i

J) 0

:

:

;

:

I

I

I

1

I

I

1

4~

T F-

I--

l

HOURS Fig. 2. Rates of transport of A-! and E apolipoproteins in mesenteric lymph of nephrotic ( - ) and control (O) rats, infused intraduodenaily with a triacylslycerol emulsion. Values are means :l: S.E. for the timecourse of lymph collection in four animals.

23 20 -

"---'-

APO B

lo

Albumin

Z

o 1-

A P O A - IV APO E

20

m n," I-I ¢3

lo

I

~

APOA-I 400

800

1200

DIAMETER

l 1600

>1800

(,~)

Fig. 5. Diameter distribution of chylomicrons isolated from mesenteric lymph. Means+ S.E. for 250 measurements are: control 1235 +23 A,; nephrotic 1498 + 37 ,~,, P < 0.005. APO C

C N Fig. 3. SDS-PAGE of purified lymph chylomicrons from control (left) and nephrotic (right) rats illustrating the strong relative decrease in the A-l, E and C groups of apolipoproteins. The apoproteins were identified on the basis of their mobilities by comparison with purified apoproteins run in parallel.

TMU-insoluble fraction was then determined and found to represent 1 7 0 + 2 2 and 1 1 0 + 15 # g / m g of total protein in control and nephrotic chylomicrons, respectively (P < 0.05). The TMU-insoluble protein fraction was also solubilised in 3% sodium dodec)!sulphate and subjected to SDS-PAGE electrophoresis, confirming that

it was almost wholly located in the characteristic origin spot of the B apoprotein. Since the plasma of nephrotic rats is protein-deficient and since they mobilize their amino acid and energy reserves primarily to support the increased hepatic protein synthesis, it seemed necessary to determine whether the decrease in intestinal protein transport may be caused by limited substrate availability to the enterocytes. The mean results of six control and nephrotic rats (not shown) indicated that supplementation of the intraduodenal infusate with amino acids and glucose did not significantly change the lymph chylomicron content, nor did it affect the already aug-

Fig. 4. Representative electron micrographs of lymph chylomicrons from nephrotic (A) and control (B) rats. Original magnification x60000; photomagnification x 102 500.

24 TABLE !!

ConcemrmWn and composition of purified lymph chylomicrons Chylomicrons were isolated from lymph pools of eight control and eight nephrotic rats, collected during 48 h and passed through agarose gel column. Values are means±S.E. Asterisk denotes a significant difference at P < 0.05 at least. Rats

Triacylglycerol

ApoA-!

ApoE

Composition (weight%)

(mg/di)

(pg/dl)

(pg/dl)

triacylglycerol

total chol.

phosphoiipids

total protein

protein

Control

7.6 ±0.6

38.9 ±5.2

2.9 ±0.3

80.5 ±2.1

4.1 ±0.5

9.8 ±0.9

5.6 ±1.1

14.4 ±1.4

N~hmti¢

11.9" ±0.5

22.5* ±!.3

0.9* ±0.3

87.6* ±1.~

5.0 ±0.4

5.4* ±0.6

2.0* ±0.4

43.8* ±0.8

mented triacyl$1ycerol transport, nor did it improve the low lymph protein secretion in the nephrotic rats. Addition of 81ucose and amino acids tended to support the lymph flow rate in nephrotic rats during the later hours of collection, but essentially the extent of increase in the overall flow rate of lymph components was not different from the results of Fig. 1. To examine whether the reduced content of apoproteins A-! and E in the nephrotic lymph chylomicrons was due to decreased transport or production, we have coinfused labelled leucine with triacylglycerol emulsion. The results of Table III show that the incorporation of label into apoproteins A-I, B, C and E was significantly lower compared with control rats. On the other hand, the incorporation of leucine into A-IV apoprotein was unexpectedly higher, even if the relative amount of A-IV apoprotein in the chylomicrons did not appear to be increased, ~s seen in the SDS-PAGE gels

(Fi8. 3). To further investigate whether a limitation in the supply of preformed apoi.:oteins was the important reason for apoproteita deficiency in the nephrotic lymph chylomicrons, we have i,v, injected apoprotein-labelled

Triacylglycerol/

HDL into the triacylglycerol-infused lymph donor rats. The rate of label appearance in the total chylomicron apoproteins isolated from the lymph was significantly lower in nephrotic rats (Table IV). It should be stressed that this result was obtained even if twice as much HDL was injected into nephrotic than control rats, to offset the dilution of specific activity in view of the approx. 2-fold higher HDL-protein concentration in their circulation (Table I). Determination of the label distribution among the individual apoproteins was not feasible due to the low amount of total radioactivity in the chylomicrons. Discussion

The mesenteric lymph flow and transport of ¢hylomicrons in the control rats showed a pattern similar to previous observations [14,22]. However, in the nephrotic rats, the lymph flow and triacylglycerol transport were increased, whereas the apoprotein incorporation into the chylomicrons by the intestinal cells did not match the triacylglycerol transport. Consequently, larger chyiomicrons with an approx. 3-fold higher triacyl-

TABLE II!

l,eucme iscorpor~ioee into lymph ckylomicron apoproteins ~H-lahelled leucine was added to the triacylalycerol infusate. Chylomicron yields were collected at the indicated times from the start of the infusion: the chyiomicmn$ were isolated, delipidated and subjected to SDS-PAGE. Values are dpm x 10- j per 50 p8 protein applied to the gel. * Silwificant dilTegence nephrotic vs. control at P < 0.02 at least. Time (h): 2 ApoB

Control Nephrotic AlmA-iV Control Nephrotic ApoE Control Nephrotic ApoAol Control Nephrotic ApoC Control Nephrotic

4,81 3,50 3.55 4.40 0.65 0.34 17.73 8.17 Z56 1.68

3

4

5

6

3-6 a

12,54 9,26 12.90 15.60 2.81 1.42 62.27 33.33 7.57 4.86

13,89 9,82 12.16 15.69 4.31 1.25 61.09 32.76 9.63 6.35

16,05 10.20 i 1.33 19.19 3.55 1.22 69.99 36.23 11.22 4.96

15.10 11.95 13.68 18.95 5.31 1.65 73.50 42.15 12.31 5.45

14.40 + 0.76 10.31 ± 0.58 12.52 + 0.44 17.36 + 0.86 4.00 + 0.46 1.39 + 0.10 66.71 + 3.00 36.12 :t: 2.15 10.18 + 1.03 5.40 + 0.34

• Mean :l:S.E. of label incorporation in four hourly collections.

* * * * *

25 TABLE IV

Apoprotein transfer from plasma HDL to lymph chylomicrons 12Si-labelled H D L was injected into the tail vein of rats, 2 h after the start of intraduodenal triacylglycerol infusion. Control rats received 0.5 ml of the solution containing 2-10 6 dpm and nephrotic rats received I mi of the solution, in view of the approximately twice as large plasma H D L pool in the latter. Chyiomicron yields were collected at the indicated time intervals after the start of infusion, isolated by centrifugation as described in Materials and Methods. The radioactivity of the protein moiety was determined and expressed as dpm/100 lag total chylomicron protein. Time (h): 4 Experiment I Control Nephrotic Experiment il Control Nephrotic

5

6

7

8

4-8 a

294 153

229 150

261 170

235 161

210 142

246 + 15 155:1:5 *

400 268

302 231

295 218

315 206

395 223

341 ± 23 229 ± 11 *

a Mean ~ S.E. of label incorporation in five hourly collections.

glycerol/protein ratio were secreted into the lymph. Our findings that the content of apoproteins A-l, E, C and B was decreased in the lymph chylomicrons, in contrast to the markedly raised concentration of these components ~n the circulating lipoproteins of the same animals (Table I), indicates that their availability to the enterocytes a n d / o r their production were suppressed in the nephrotic condition. Thus, the intestinal chylomicron secretion pattern does not conform to that of hepatic lipoprotein overproduction. This effect of nephrosis has been amply demonstrated in intact animals [4-7], in perfused liver [5,6] and by simulating the nephrotic conditions in a medium batlfing isolated hepatocytes [11], and is consistent with a general stimulation by hypoproteinemia of hepatic lipoprotein synthesis. No evidence for such a stimulatory effect on the enterocyte chylomicron synthesis was found in the present study. The decreased transport of apoA-I in the nephrotic lymph should be regarded as decreased intestinal production since it has been demonstrated that more than 90~ of this apoprotein originates from enterocyte synthesis [25]. Amino acid and glucose supplementation did not enhance the production of chylomicron protein production, pointing out that the defect was in the enterocyte function rather than in substrate availability. The finding of reduced incomoration of labelled leucine into apoA-I and other chylomicron apoproteins of nephrotic rats (Table III) also leads to this conclusion. On the other hand, the decreased apoE content of chylomicrons may be linked to a reduced supply from the circulation since, according to previous observations, apoE is not extensively synthesised by enterocytes [25]. It should be remarked, however, that some leucine incorporation into apoE was evident, suggesting that

the supply of this apoprotein is not absolutely dependent on transfer into the lymph by lipoprotein carriers. With respect to the reduced apoproteins B and C of chylomicrons (Table Ill, and TMU solubility), the former is predominantly derived from enterocytes, and the latter also appears to be produced, at least in part, in the intestine. It is then probable that the synthesis of these apoproteins in the intestine was reduced in nephrosis in addition to their transfer from plasma. The observation that total protein levels, including albumin, were low in the nephrotic lymph, as is the case in the nephrotic plasma, seems also of importance. Enterocytes do contribute only small amounts of specific proteins to the lymph, the bulk is derived from the plasma. The equilibration of albumin between lymph and vascular compartments appears to be rapid due to its relatively low molecular weight, whereas the diffusion of the large lipoprotein carriers of apoproteins appears to be slower in nephrosis. This is suggested by uniformly reduced transfer of the HDL apoprotcins into lymph chylomicrons, as shown with labelled HDL introduced into the circulation (Table IV). This experiment should be interpreted with reservation, as no elimination and degradation kinetics could be measured. However, its results, together with the findings of reduced incorporation of leucine, warrant the assumption that both the transfer of preformed apoproteins and the de novo apoprotein synthesis may contribute to the diminished apoprotein complement of chylomicrons in nephrosis. The share of these two sources in the deficiency of individual chylomicron apoproteins remains to be determined. As discussed above, the stimulus of hypoproteinemia in nephrosis differs in the intestine, compared with the liver, as the enterocytes responded solely with increased lymph flow and triacylglycerol transport. To explain the low enterocyte apoprotein transport, it may be speculated that in the hepatocyte, multiple functions are affected by the encompassing membrane contact with the surrounding extracellular hypoproteinemic fluid. In the intestine, the hypoproteinemia is present only on the lacteal side from which the lymph is secreted. The luminal side is exposed to the duodenal fluid, which may be important for regulation of apoprotein synthesis, but is not hypoproteinemic. Alternatively, the enterocytes may be at the peak of chylomicron production on triacylglycerol loading, without being able to respond to additional stimuli of hypoproteinemia, whereas the liver, the main site of apoprotein synthesis, appears to be liable to wide adaptation in its rate of apoprotein elaboration.

Acknowledgements Sincere thanks are due to Dr. M. Fainaru from the Department of Medicine of the Kaplan Hospital, Re-

26 hovoth, for the performance of apoprotein A-I and E immunoassays. This work was supported by a grant from the Joint Research Fund of the Hebrew University and Hadassah to E.L. and represents a partial fulfillment of the requirements for his Ph.D. degree. References 1 Baxter, J.H. (1%2) Arch. Intern. Med. 109, 742-757. 2 Marsh, J.B. and Drabkin, D.L. (1960) Metabolism 9, 946-955. 3 ReAding, C.M. and Steinberg, D. (1960) J. Clin. Invest. 39, 1560-1569. 4 Shafrir, E. and Brenner, T. (1979) Lipids 14, 695-702. 5 Brenner, T. and Slutfrir, E. (1980) Lipid3 15, 637-643. 6 Marsh, J.B. and Sparks, C.E. (1979) J. ,21in. Invest 64, 1229-1237. 7 Calandra, S., Oherardi, E., Faina:u, M., Quaitani, A. and Bartosek, !. (1981) Biochem. Biophys. Acta 065, 331-338. 8 Baxter, J.H., Goodman, H.C. and Allen, J.C. (1961) J. Clin. Invest. 40, 490-498. 9 Allen, J.C., Baxter, J.H. and Goodman, H.C. (1961) J. Clin. Invest. 40, 499-508. 10 Yed~r. S.. Eilam, O. and Shafrir. E. (1984) Am. J. Physiol. 248, EIO-E14. I ! YedlPtr, S., Weinstein, D.B., Patsch, W., ~honfeld, G., Casanada. F.E. and Steinberg, D. (1982) J. Biol. Chem. 257, 2188-2192.

12 Windmueller, H.G., Herbert, P.N. and Levy, R.I. (1973) J. Lipid Res. 14, 215-223. 13 Rooke, J.A. and Skinner, K. (1976) Biochem. Soc. Trans. 4, 1144-1145. 14 lmaizumi, K., Fainaru, M. and Havel, R.J. (19/8) J. Lipid Res. 17, 7L~.-722. 15 Boilman, £L., Cain, J.C. and Grindlay, J.H. (1948) J. Lab. Clin. Med. 33, 1349-1352. 16 Felker, T.E., Fainaru, M., Hamilton, R.L. and Havel, R.J. (1977) J. Lipid Res. 18, 465-473. 17 Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. 18 Bar-On, H. and Eisenberg, S. (1978) Diabetologia 14, 65-69. 19 Lowry, O.H., Rosebrough. N.J., Farr, A.L. and Randall, R.J. (1952) J. Biol. Chem. 193, 265-275. 20 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 21 Fainaru, M., Havel, R.J. and Felker, T.E. (1976) Biochim. Biophys. Acta 446, 56-68. 22 Fainaru, M., Havel, R.J. and Imaizumi, K., (1977) Biochim. Biophys. Acta 490, 144-155. 23 Oschry, Y. and Eisenberg, S. (1982) J. Lipid Res. 23, 1099-1106. 24 Kane, J.P. (1973) Anal. Biochem. 53, 350-364. 25 lmaizumi, K., Havel, R.J., Fainaru, M. and Vigne, J.L. (1978) J. Lipid Res. 19, 1038-1046.