Serum lipoprotein abnormalities in renal allograft recipients

97

Atherosclerosis, 30 (1978) 97-107 @ Elsevier/Nortb-Holland Scientific Publishers, Ltd.

SERUM LIPOPROTEIN ABNORMALITIES IN RENAL ALLOGRAFT RECIPIENTS

LYNN K. ABBOTT, CHRISTINE ELLIOT, JANET M. AITKEN and JOHN B. SOMER Department of Medicine, University of New South Wales, Prince Henry Hospital, Little Bay, N.S. W. (Australia) (Received 12 September, 1977) (Revised, received 21 February, 1978) (Accepted 23 February, 1978)

Summary Detailed lipid composition of serum and isolated lipoprotein fractions in male and female transplant recipients has been determined. Results have been compared with appropriate controls and a number of abnormalities have become apparent: (i) Serum total cholesterol, triglyceride and phospholipid levels were significantly elevated in transplant recipients. (ii) All lipid classes in VLDL were significantly increased in both male and female patients. (iii) LDL-total cholesterol, triglyceride and phospholipid were significantly raised in female transplant patients, but this was true only for LDL-triglyceride in male patients. (iv) In female patients, HDL-total cholesterol and -triglyceride were significantly elevated, while in male patients, HDL-total cholesterol, but not HDLtriglyceride, was significantly greater than in controls. (v) The ratio of esterified to free cholesterol was significantly reduced in HDL of female transplant recipients. Results from correlation analysis suggest a relationship between some of the lipid abnormalities and renal function in female patients, while in male patients only immunosuppressive therapy is implicated. Key words: Cholesteryl ester -High density lipoprotein -Lipids ities - Renal transplantation

-Lipoprotein

abnormal-

This work was supported by grants irom The Natioql Health and Medid Research Council, Wellcome Australasia Ltd., Mamickville Holdings. Pty. Ltd., Edible Oils Pty. Ltd. and Merck. Sharpe and Dohme (Australia) Pty. Ltd.

98

Introduction Accelerated cardiovascular disease appears to be a significant clinical complication in renal allograft recipients [l-3]. A greater prevalence of hyperlipidemia, a known coronary risk factor [4,5], has also been demonstrated in these patients [ 6-91. Partial characterization of lipoproteins in renal transplant recipients has been carried out in several laboratories, but results have not always been in agreement. Increased levels of cholesterol and triglyceride have been demonstrated in both very low density lipoprotein (VLDL) and low density lipoprotein (LDL) [7-9]. However, cholesterol in high density lipoprotein (HDL) has been reported as unchanged [ 71 or decreased [8,9 1, and HDL-triglyceride as increased [ 71 or decreased [8]. In these reports no distinction was made between results from male or female transplant recipients [ 7-91. As part of a project designed to examine the nature and mechanisms of hyperlipidemia in renal allograft recipients, the detailed serum - and lipoprotein-lipid composition in a population of renal transplant recipients has been determined. Since male-female differences in lipoprotein-lipid composition have been demonstrated in normal populations [ lO,ll],results from male and female transplant recipients were not combined - as has been done previously [6-9] - but were analyzed separately. A number of changes in lipoproteinlipid composition were observed, the most notable being a significant increase in HDL-cholesterol in both male and female transplant patients. Materials and Methods Renal transplant recipients were drawn from patients attending the Division of Nephrology, Department of Medicine, Prince Henry Hospital for routine follow-up after transplantation of the renal allograft. The dietary pattern of transplant patients was similar to that of the average Australian diet (14% of calories from protein, 41% from fat, 41% from carbohydrate and 4% from alcohol, cf. [ 12]), although total caloric intake was somewhat lower at 2000 calories (cf. [12]) Controls, none of whom were on medication likely to affect blood lipids, were drawn from healthy volunteers among the administrative, medical, maintenance and laboratory staff of the Prince of Wales and Prince Henry Hospitals. Clinical data for patients and controls are presented in Table 1. After an overnight fast, venous blood was drawn by syringe. As we wished to measure free and esterified cholesterol levels, lecithin-cholesterol acyl transferase (LCAT) activity had to be inhibited in the collected sample. To this end, blood was added immediately to a collection bottle containing the sulfhydryl inhibitor N-ethyl maleimide (Sigma Chemical Co., St. Louis, MO., U.S.A.; final concentration 1.5 mM) and allowed to clot. Serum was separated by centrifugation and an aliquot was taken for determination of cholesterol and triglyceride by Auto Analyzer [ 131. Individual lipoproteins were isolated by preparative ultracentrifugation following sequential density adjustments [ 141. Serum and lipoproteins were extracted with chloroform : methanol (2 : 1) and the extract was washed with 0.73% NaCl solution [15]. Free and esterified

1

DATA

aMeanf SD.

Time since transplantation (months)

48.3

14.1 1.7 119.2

Prednisone dose (mg/day) Betamethasone dose (me/day) Azathioprine dose (mg/day)

-

f 34.8

0.8 n.d. n-d.

+

0.1

30 39.0 ? 10.5 23.78 r 3.68

Control

+ 9.1 (n = 24) f 0_6(n=14) + 36.6

f. 0.4 f 21.4 ,. 0.27

33 43.1 + 11.6 a 23.71 + 4.21

Transplant

Female-

RECIPIENTS

1.1 67.1 0.26

TRANSPLANT

Serum creatinine (mg/lOO ml) Creatinine elearauce (mI/min) Urlnaw protein (g/day)

Number Age (years) Weight/height*

CLINICAL

TABLE

52.5

11.8 1.9 131.5 f 30.3

t 7.5 (n = 20) f 0.4(n=7) f 34.6

1.6 f 0.8 66.6 + 31.7 0.34 f 0.60

27 38.0 + 14.6 24.15 f 3.03

Transplant

Male

-

-

0.8 n-d. n.d.

+

0.1

34 37.8 k 13.1 24.46 f 2.11

Control

8

100

cholesterol were separated by thin-layer chromatography on silica gel H (H. Merck, A.G., Darmstadt, West-Germany) in a hexane : diethyl ether : glacial acetic acid (146 : 50 : 4, v/v/v) solvent system [16]. Free cholesterol and cholesteryl ester were visualized with iodine; the bands were scraped into tubes, extracted with 5.0 ml of isopropanol and their content of cholesterol determined [13]. The triglyceride content of serum- and lipoprotein extracts was measured by AutoAnalyzer [13], and phospholipid was measured by the method of Bartlett [17]. Means were statistically compared using Student’s t-test on the log derivatives of the appropriate lipid or lipid ratio data. Results Serum lipids The serum levels of 3 major lipid classes (total

cholesterol, triglyceride and phospholipid) were significantly elevated in both male and female transplant recipients (Table 2). The greatest increase relative to controls occurred in serum triglyceride (approximately 145% in females and 51% in males), while smaller increases were noted in serum cholesterol (about 31% in females and 17% in males) and serum phospholipid (around 20% in females and 13% in males). In females, the ratios of serum esterified to free cholesterol were 2.73 f 0.51 in transplant recipients and 2.80 r 0.41 in controls, while in males the ratios were 2.72 t 0.30 in transplants and 2.66 + 0.44 in controls. None of these values differed significantly. Lipid composition of isolated lipoprotein fractions. (I) Very low density lipoprotein (VLDL) As shown in Table 3, VLDL-cholesterol,

-triglyceride and -phospholipid levels were all significantly elevated in transplant recipients when compared with controls. In female transplants the increases in VLDL-cholesterol and VLDL-triglyceride were of similar magnitude, being approximately 3-fold, while the increase in VLDL-phospholipid was somewhat less, being approximately 130% greater than in controls (Table 3). The increases in VLDL-lipids in male

TABLE 2 SERUM LIPIDS IN RENAL ALLOGRAFT

RECIPIENTS AND CONTROLS

Cholesterol (mg/lOO ml serum)

Triglyceride (mg/lOO ml serum)

Phospholipid (mg/lOO ml serum)

Female transp1ent control

263 f 67 ** 201 f 36 a

162 * 62t

291 f 54 ** 243 f 33

Male transplant control

241 f 64 * 208 f 40

166 f. 75*** 110 f 116

e Mean t SD. *P < 0.06. **

P<

0.01.

*** P < 0.001.

51** 24

274 f 47** 242 f 56

101 TABLE 3 VERY LOW DENSITY AND CONTROLS

LIPOPROTEIN-LIPID

COMPOSITION

24 f 12 *** 8t 4*

Male transplant control

25 + 12 *** 17 f: 22

* Mean c SD. * P < 0.05, ** P < 0.01,

94k 31r

ALLOGRAFT

RECIPIENTS

Phospholipid (mg/lOO ml serum)

Triglyceride (mg/lOO ml serum)

Cholesterol (mg/lOO ml serum) Female transplant control

IN RENAL

46*** 20

30 f 13 *** 13+ 6

112 ?. 62*** 69 f 105

33 it 17 ** 24 f 30

*** P < 0.001.

transplants were less than females, being 62% for VLDL-triglyceride, 47% for VLDLcholesterol and 31% for VLDL-phospholipid (Table 3). Results presented in Table 4 again indicate that the increases in the different VLDL-lipids were not proportional. Thus, in both male and female transplant recipients the ratios of total cholesterol to phospholipid and of triglyceride to phospholipid were significantly increased (Table 4). These data indicate that the increases in VLDL-cholesterol and VLDL-triglyceride were greater than the increase in VLDL-phospholipid. The ratio of total cholesterol to triglyceride in female transplant recipients was not different from that in controls (Table 4), suggesting that VLDLcholesterol and -triglyceride increased in proportion. However, in males this ratio was decreased (0.24 in transplants, 0.28 in controls, P < 0.05, Table 4), suggesting that in males the increase in VLDL-triglyceride was greater than the increase in VLDL-cholesterol. Ratios of esterified to free cholesterol were similar in the VLDL fractions of all groups of subjects (Table 4).

(ii) Low density lipoprotein In female transplant recipients, LDL-cholesterol,

-triglyceride and -phospho-

TABLE 4 VERY LOW DENSITY LIPOPROTEIN-LIPID RECIPIENTS AND CONTROLS

INTERRELATIONSHIPS

IN RENAL

ALLOGRAFT

Esterified cholesterol

Total cholesterol

Total cholesterol

Triglyceride

Free cholesterol

Triglyceride

Phospholipid

Phospholipid

1.08 t 0.28 a 1.11 f 0.36

0.27 f 0.06 0.28 f 0.08

0.84 f 0.14 *** 0.64 f 0.18

3.11 f 0.48 *** 2.44 f 0.90

0.96 f 0.24 1.15 r 0.38

0.24 i 0.05 * 0.28 f 0.07

0.79 f 0.14 * 0.72 f 0.18

3.40 f 0.67 *** 2.58 f 0.53

Female transplant control Male transplant control

a Meanf

SD. * P < 0.05. ***p

< 0.001.

102 TABLE 5 LOW DENSITY CONTROLS

LIPOPROTEIN-LIPID

COMPOSITION

IN RENAL

ALLOGRAFT

RECIPIENTS

Cholesterol (mg/lOO ml serum)

Triglyceride (mg/lOO ml serum)

Phospholipid (mg/lOO ml serum)

146 f 48 ** 111 f 26 a

47 f 14 *** 23r 6

99 i 30 ** 18 r 17

115??43 117 f 35

35 f 11 ** 26 f 10

90 + 38 81 t 23

AND

Female transplant control Male transplant control

aMean

f SD. * P < 0.05, ** P < 0.01.

*** P < 0.001.

lipid were all significantly greater than in controls (P < 0.01, P < 0.001 and P < 0.01, respectively, Table 5). In male transplants, however, only LDL-triglyceride was significantly elevated (P < 0.01, Table 5) and, in contrast to the findings in females, LDL-cholesterol and -phospholipid levels did not significantly differ from controls (Table 5). Data shown in Table 6 indicate that, as for VLDL-lipids, the increases in LDL-lipids in transplant recipients were not proportional. In female transplants, the ratio of total cholesterol to triglyceride was decreased (P< 0.001, Table 6), while that of triglyceride to phospholipid was increased (P< 0.001, Table 6), indicating a greater increase in LDL-triglyceride than in LDL-cholesterol and LDL-phospholipid. The unchanged ratio of cholesterol to phospholipid suggests that the increases in LDL-cholesterol and LDLphospholipid were in proportion (Table 6). The changes in LDL lipid ratios observed in male transplant recipients, as shown in Table 6, reflect the observation that only LDL-triglyceride was increased in these patients (cf. Table 5). In both male and female patients the ratio of LDL-cholesteryl ester to LDLfree cholesterol did not significantly differ from that in controls (Table 6). TABLE 6 LOW DENSITY LIPOPROTEIN-LIPID IENTS AND CONTROLS

INTERRELATIONSHIPS

IN RENAL

ALLOGRAFT

RECIP-

Esterified cholesterol

Total cholesterol

Total cholesterol

Triglyceride

Free cholesterol

Triglyceride

Phospholipid

Phospholipid

Female transplant control

2.76 f 0.32 a 2.65 f 0.39

3.22 f 1.04 *** 4.98 f 1.16

1.44 * 0.15 1.41 f 0.09

0.48 ?: 0.13 *** 0.30 ?: 0.01

Male transp1ent control

2.73 i 0.41 2.86 r 0.46

3.35 f 0.91 *** 5.00 r 1.62

1.38 r 0.22 1.46 f 0.15

0.44 f 0.14 ** 0.32 _+0.13

e Mean r SD. ** P < 0.01, *** P < 0.001.

103 TABLE I HIGH DENSITY CONTROLS

LIPOPROTEIN-LIPID

COMPOSITION

IN RENAL

ALLOGRAFT

RECIPIENTS AND

Cholesterol (mg/lOO ml serum)

TrigIyceride (mg/lOO ml serum)

PhosphoIipid (mg/lOO ml serum)

Female transplant control

80 f 18 ** 67 k 14

18 r 5 *** 14 * 3

180 k 33 151 f 26

Male Transplant control

78 c 18 *** 62 + 15

14 f 4 15 r 9

142 r 34 141 f 28

* P < 0.05,

**p

< 0.01,

***p

<

0.001.

(iii) High density lipoproteins In female transplant recipients, levels of HDL-cholesterol and HDL-trigly ceride were significantly elevated (P < 0.01 and P < 0.001, respectively,

Table 7), but HDL-phospholipid levels were similar to controls. In male transplant patients, only HDL-cholesterol was significantly elevated (P < 0.001, Table 7), with HDL-triglyceride and HDL-phospholipid levels being similar to those in controls (Table 7). As shown in Table 8, the ratio of HDL-cholesterol to HDL-triglyceride was not significantly altered in female transplant recipients, indicating that the increase8 in cholesterol and triglyceride in HDL, as demonstrated in Table 7, were in proportion. The increases in the ratios of total cholesterol to phospholipid and of triglyceride to phospholipid reflect the observations (cf. Table 7) that cholesterol and triglyceride, but not phospholipid, level8 were raised in HDL in female transplant patients. The changes in HDL-lipid ratios in male transplant recipients, a8 shown in Table 8, reflect the observation that only HDL-cholesterol was significantly elevated (cf. Table 7). Of particular note is the finding that in female transplant patients the ratio of esterified to free cholesterol in HDL was significantly reduced (3.67 k 0.61 TABLE 8 HIGH DENSITY LIPOPROTEIN-LIPID IENTS AND CONTROLS

INTERRELATIONSHIPS

IN RENAL

ALLOGRAFT

RECIP-

Esterified cholesterol

Total cholesterol

Total cholesterol

Triglyceride

Free cholesterol

TrigIycerIde

PhosphoIIpid

PhosphoIipid

Female transplant control

3.67 f. 0.61 ** 4.10 + 0.65 a

4.82 r 1.44 5.23 f. 1.82

0.51 f. 0.10 ** 0.45 f 0.09

0.11 i 0.04 * 0.09 f 0.02

Male transplant control

3.71 f 0.69 4.02 ? 0.69

6.62 k 3.42 * 4.98 f 1.64

0.66 + 0.17 ** 0.44 f 0.11

0.11 f 0.04 0.10 f 0.06

aMeant

SD. * P < 0.05, **

P<

0.01.

104

in transplants, 4.10 f 0.65 in controls, P < 0.01, Table 8), whereas the apparent reduction in this ratio in males did not quite reach statistical significance (3.71 ?:0.69 in transplants, 4.02 + 0.69 in controls, P < 0.1,Table 8). Discussion The present study has demonstrated a number of abnormalities in serum and lipoprotein lipids in renal allograft recipients, confirming some - but not all and considerably extending results from studies reported earlier [6-9,18]. Serum cholesterol and triglyceride levels were raised, in accord with previously reported observations [6-9,18]. In addition, serum phospholipid levels were shown to be elevated in the present report. In VLDL, all lipid classes measured were significantly elevated in transplant patients, while analysis of lipid ratios indicated that the increases observed were disproportionate. Nonuniform increases in all LDL lipids were also observed in female transplant patients, while in males only LDL-triglyceride was significantly increased. Finally, HDL-cholesterol was significantly elevated in both male and female patients, while HDL-triglyceride was increased only in female patients. The ratio of HDL esterified to free cholesterol was reduced in transplant patients, significantly so in females; a change not observed in serum, VLDL or LDL. A few studies have appeared recently, reporting partial lipoprotein-lipid characterization in combined groups of male and female transplant patients [7,8]. In accord with results presented above, increased levels of VLDL-chol[ 7,8], ester01 and triglyceride and of LDL-triglyceride were demonstrated However, increased levels of HDL-cholesterol (as shown above) were not observed in these studies [ 7,8]. The reason for these apparent discrepancies is not clear. LCAT activity was inhibited in the present study by adding the blood to the sulfhydryl inhibitor N-ethyl maleimide immediately after collection. This was not done in the earlier studies [ 7,8 J. Whether this difference in procedure can explain the difference in results with regard to HDL-cholesterol levels remains to be determined. HDL-unesterified cholesterol appears to be the preferred substrate for LCAT [19,20], and at least some of the resulting cholesteryl ester is transferred to the other lipoprotein fractions [20,21]. Reports of lipoprotein-lipid levels in normal subjects have shown considerable variation, For example, published values for VLDL-cholesterol range from 11 to 32 mg/lOO ml [7,8,11,22-241, for LDL-cholesterol from 94 to 173 mg/lOO ml [7,8,11,22-251, and for HDL-cholesterol from 35 to 83 mg/lOO ml [ 7,8,11,22-251. Similar variation is also apparent in lipoprotein-triglyceride levels [ 7,8 ,!1,26]. Whether this variability reflects population differences or is the result of methodological differences is not known. However, Miller et al. have recently observed HDL-cholesterol levels of 63 mg/lOO ml in urban Jamaican males (predominantly of European origin), values similar to those obtained in the present study, and 83 mg/lOO ml in rural Jamaican males, primarily of African origin, but levels of only 48 mg/lOO ml in Edinburgh males [25]. It remains to be determined whether the abnormal lipoprotein fractions isolated from renal transplant patients represent new pathological species of lipoprotein or result from a redistribution of lipoprotein sub-fractions within each major lipoprotein class. A number of differences have been described in lipid

105

composition between sub-fractions of VLDL, LDL and HDL. It has been shown that as VLDL sub-fractions decrease in density they become richer in triglyceride. The ratios of triglyceride to phospholipid and of esterified to free cholesterol increase, while the total cholesterol to phospholipid ratio decreases [27]. In VLDL isolated from renal transplant recipients, as shown in Table 4, the triglyceride to phospholipid ratio was indeed increased, but the ratio of total cholesterol to phospholipid was also increased, while that of esterified to free cholesterol was not significantly altered. LDL sub-fractions, comparatively enriched in triglyceride, have been shown to be poorer in cholesteryl ester relative to free cholesterol [ 281. However, LDL isolated from renal transplant patients was comparatively richer in triglyceride, but normal in cholesteryl ester content relative to free cholesterol (cf. Table 6). The lessdense subfraction of HDL in normal populations shows a decrease in the esterified to free cholesterol ratio, with increases in the total cholesterol to phospholipid and total cholesterol to triglyceride ratios [29]. As shown in Table 8, in HDL isolated from renal transplant patients, the esterified to free cholesterol ratio is decreased, the total cholesterol to phospholipid ratio is increased, as is the ratio of total cholesterol to triglyceride in male patients. It could be suggested then that the changes observed in transplant HDL might be explained by postulating increased amounts of HDL plus a shift in the distribution of HDL sub-fractions. The mechanisms responsible for the various serum- and lipoprotein-lipid TABLE 9 SIGNIFICANT CORRELATIONS BETWEEN AND LIPOPROTEIN - LIPID VALUES Variables

VARIOUS

CLINICAL

AND SERUM -

Correlation coefficient Female

Male Red&one

PARAMETERS

dose versus

VLDL-TC VLDL-TG

0.54 * 0.46 *

-0.06 0.02

Betamethasone dose versus

LDLPL HDL-PL

0.49 * 0.65 **

-0.24 0.22

Azathioprine dose versus

Serum-TC S--TG VLDL-TC VLDLPL VLDGTG LDL-TG

0.36 0.45 0.48 0.41 0.40 0.28

Serum creatiie

Serum-TG VLDGTC VLDGPL HDL-TC HDL-PL

0.05 0.29 0.30 -0.18 a.14

0.59 0.49 -0.32 -0.52

*** ** * **

Serum-TG VLDL-PL VLDL-TG HDtTC

-0.12 4.17 -0.14 0.13

-0.34 -0.64 a.44 0.31

* ** ** *

versus

Creatinine clearance verses

* ** ** * *

0.21 0.21 0.04 0.08 0.18 0.36 * 0.44 **

Abbreviations wad: VLDL = very low density ltpoprotein. LDL = low density lipoProtein. HDL - high density lipoprotein. TC = total cholesterol, PL = phospholtpid, TG = triglyceride. ??P < 0.05, ** P < 0.01, *** P < 0.001.

106

abnormalities observed in renal transplant recipients are not known. However, the results presented in Table 9, showing the significant correlations found between serum- and lipoprotein-lipids and various clinical parameters, should provide some insight. Thus, steroid therapy affected relatively few lipid values in male patients and none in female patients. Azathioprine therapy appeared to influence serum cholesterol and triglyceride and all VLDL-lipids in males, but only LDL-triglyceride in females. Renal function, as monitored by serum creatinine and creatinine clearance, appeared to be related to serum triglyceride, VLDL lipid levels and to the major HDL lipids in females only. Overall, it would appear that in female patients lipid values were related primarily to kidney function, with drug therapy being a negligible contributory factor. In male patients, on the other hand, only drug therapy appeared to be a contributing factor to the raised lipid levels observed. Thus, it could be suggested that at least some of the mechanisms leading to hyperlipidemia may be different in male and female transplant recipients. Acknowledgements We thank Drs. Graham McDonald, John Charlesworth and Malcolm Robertson of the Division of Nephrology, Prince Henry Hospital for their ready cooperation and allowing access to the transplant patients. We also thank Sister Gwen Banks for cooperation in organizing the blood collections and Mrs. Jan Bakalich for repeatedly typing the manuscript. The continued support and helpful discussion of Professor R.B. Blacket are also greatly appreciated. References 1 Lowrie. E.G., Lazarus, J.M.. Mocelin. A.J.. Bailey. G.L.. Hampers. C.L., Wilson, RX and Merrill, J.P., Survival of patients undergoing chronic hemodialysis and renal transplantation, New Engl. J. Med., 288 (1973) 883-88’7. 2 Ibels. L.S.. Stewart, J.H.. Mahony. J.F. and She& A.G.R.. Deaths from occlusive arterial disease in renal allograft recipients, Brit. Med. J., 3 (1974) 552-564. 3 Ibels, L.S.. Stewart, J.H., Mahony, J.F.. Neale. F.C. and She& A.G.R.. Occlusive arterial disease in uremic and hemodialyais patients and renal transplant recipients, Quart, J. Med.. 46 (1977) 197-214. 4 Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, IipoProteias and the risk of coronary heart disease, Ann. Int. Med., 74 (1971) l-12. 5 Carlson, L.A. and Bbttiger. L.E.. Ischemic heart disease in relation to fasting values of Plasma MYcerides and cholesterol, Lancet. 1 (1972) 865-868. 6 Casaretto. A., Marchioro, T.L., Goldsmith, R. and Bagdade, J.D., Hyperlipidemia after successful renal transplantation, Lancet, l(1974) 481484. 7 Ibels. L.S.. Simons, L.A., King. J.O., Williams, P.F.. Neale, F.C. and Stewart, J.H.. Studies on the nature and causes of hyperlipidemia in uremia, maintenance dialysisand renal transplantation, Quart. J. Med., 44 (1976) 601-614. 8 Bagdade. J., Casaretto, A. and Albers, J.. Effects of chronic uremia, hemodialysis and renal transplantation on plasma lipids and lipoproteins in man, J. Lab. Clin. Med., 87 (1976) 3748. 9 Bagdade. J. and Albers. J.J., PIasma high-density lipoprotein concentrations in chronic-hemodialyds and renal-transplant patients. New EngI. J. Med., 296 (1977) 1436-1439. 10 Nichols, A.V.. Functions and interrelationships of different classes of plasma lipoprotein. Proc. Nat. Acad. Sci. (Wash.), 64 (1969) 1128-1137. 11 Carlson. L.A. and Ericsson. M.. Quantitative and qualitative sermn lipoprotein anal~ds. Part 1 (Studies in healthy men and women), Atherosclerosis, 21 (1975) 417433. 12 Leelarthaepin, B.. Woodhill, J.M., Palmer, A.J. and Blacket. R.B., Obesity, diet and type II hyperUpidemia. Lance& 2 (1974) 1217-1219. 13 Methods N-24a and N-78. Technlcon Instrument Corporation Auto Analyzer Manual, Tarrytown, N.Y.. 1968.

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