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Lipoprotein composition in diabetes mellitus
153
Atherosckroais, 30 (1978) 153-162 0 Elsevier/North-Holland Scientific Publishers,
Ltd.
LIPOPROTEIN COMPOSITION IN DIABETES MELLITUS
BARBARA V. HOWARD, PETER J. SAVAGE, PETER H. BENNETT
LYNN J. BENNION
and
Phoenix Clinical Research Section, National Institute of Arthritis, Metabolism, and Digestive Dieeaees, 4212 N. 16th Street, Phoenix, Arizona 85016 (U.S.A.) (Received 27 December, 1977) (Revised received 8 February, 1978) (Accepted 15 February, 1978)
Lipoprotein cholesterol and triglyceride levels have been determined in normal and diabetic Pima Indian women aged 20-35. HDL cholesterol levels were lower, LDL cholesterol levels were higher, and the ratio of HDL cholesterol/LDL cholesterol, a reflection of lipoprotein cholesterol distribution, was lower in the diabetics compared to the normals. VLDL triglyceride levels were also elevated in the diabetics. An analysis of lipoprotein compositiQn suggested that these changes primarily reflect changes in numbers of particles, since lipid composition and lipid/protein ratios were similar in lipoproteins isolated from normals and diabetics. The ratio of ester/free cholesterol in LDL and HDL was lower in normal Pima Indians than in a comparable’ group of Caucasians, although plasma LCAT activity was not significantly different. The data indicate that diabetes may be associated with shifts -in distribution of LDL and HDL, as well as with increases in VLDL. Key words:
Atherosclerosis
- Cholesterol -Diabetes
-Lipoproteins
- Triglycerides
Introduction Examination of blood lipids and lipoproteins in diabetes mellitus is of interest because of the increased prevalence of arteriosclerotic cardiovascular disease associated with this disorder [ 1,2]. There have been many repor@ of abnormalities in blood lipids associated with diabetes mellitus. Hypertriglyceridemia has been frequently observed [3-51 and hypercholesterolemia has also been noted by a number of investigators [ 6-81. More recently there have been This
work was supported in part by the National Institutes of Health Contract No. NOl-AM-B-0219
154
reports of alterations in individual lipoprotein patterns in diabetes [g-11]. Interpretation has been difficult, however, because of the many factors in diabetes that influence blood lipid levels, including insulin levels, obesity, age, sex, the type of diabetes and the genetic heritage of the diabetics under study. The Pima Indians have the highest recorded prevalence of maturity onset diabetes [ 121. They are more genetically homogeneous than the general population [ 131, and there is a relative absence of primary hyperlipemias [ 141. A previous study of plasma triglyceride and cholesterol levels in this population indicated that both cholesterol and triglyceride levels were significantly higher in the diabetics compared to the normals, and that both cholesterol and triglyceride were related in a linear fashion to increasing fasting plasma glucose levels [ 141. We have further investigated these differences by examining cholesterol and triglyceride levels and lipid composition of isolated lipoprotein fractions. In order to minimize the many variables that influence blood lipids, we have selected a group of Pima women, aged 20-35, for a comparison of plasma lipids and lipoproteins between normal and diabetic individuals. Materials and Methods The patients for this study consisted of 14 non-diabetic and 17 diabetic female Pima Indians between 20 and 35 years old. The majority were inpatients in the Phoenix Clinical Research Unit, National Institute of Arthritis, Metabolism and Digestive Diseases. A small number of patient+ in each group were seen as outpatients at the Sacaton Clinic on the Gila River Indian Reservation. The criterion for diagnosis of diabetes was a 2-h plasma glucose level of greater than 200 mg/dl [15] following a 100 g carbohydrate load, and the duration of diabetes ranged from 1 to 14 years. The diabetics were all of maturity-onset type with no overt ketoacidosis. Those classified as non-diabetic subjects were free of symptoms and had 2-h plasma glucose levels less than 150 mg/dl. None of the subjects was taking insulin or any other medication known to affect blood lipids or lipoprotein Ieve@. Clinical data are presented in Table 1. Age
TABLE 1 STUDY OF A-POPULATION
Ale (yr) Height (cm) Weight (kg) Plasma glucose (mn/dl) fastfm3 2-h Insulin (@/ml) fasting 2-h a Mean * SEM. bn=8. Cn=12.
OF PIMA INDIANS NOllIlalS
Diabetics
(14)
(17)
27 158 f 1.0 a 86 f 6.0
28 161 i: 1.0 100 t 4.9
89 f 2.3 124 f 6.1
204 t 21 357 * 28 ,
26 f 4.9 b 228 f 46 b
38 t 5.8 c 100 * 30 c
155
and height in the two populations were comparable, although the diabetics as a group were about 14 kg heavier than the normals. Two-hour plasma glucose levels averaged 357 mg/dl in the diabetics, and 124 mg/dl in the normals, and fasting plasma glucose levels were 204and 89 mg/dl, respectively. Venous blood samples were collected in EDTA after an overnight fast. Plasma was separated after centrifugation at approximately 700 X g for 15 min at 10°C. None of the plasma contained detectable evidence of chylomicrons after overnight incubation in the cold. Lipoproteins were isolated from plasma according to procedures described by Have1 et al. [ 161 and modified by Marsh [ 171. Six ml plasma were overlaid with 2 ml of 0.16 M NaCl, 1 mM EDTA, d 1.006, and very low density lipoproteins (VLDL) were isolated by ultracentrifugation for 16 h at 40,000 rpm in a Beckman model L250 ultracentrifuge using a type 40 rotor. VLDL preparations isolated by migration through overlaid saline contain less than 10% albumin and other proteins [9]. After VLDL were removed the plasma was adjusted to d 1.063 using KBr (d 1.35) and low density lipoproteins (LDL) were isolated by centrifugation for 20 h at 40,000 rpm. The high density lipoprotein (HDL) fraction was the infranatant after removal of the LDL. Efficiency of the ultracentrifugal lipoprotein isolation was monitored by electrophoresis on agarose gels according to the method of Noble [ 181. Triglycerides and cholesterol in plasma and isolated lipoproteins were quantified on an Autoanalyzer II (Technicon), using the cholesterol extract method of Rush et al. [19] and the triglyceride enzymatic method of Bucolo and Davis [20]. The triglyceride assay was standardized using control plasma supplied by the Lipid Standardization Lab, CDC, Atlanta, Georgia. Control plasma for the cholesterol assay was standardized using the CDC Proficiency Testing Service. The coefficient of variation for the cholesterol assay averaged 3.2% and that of the triglyceride assay was 5.8%. Recovery of cholesterol in the isolated lipoprotein fractions averaged 97%, and recovery of the triglycerides in the lipoprotein fractions averaged 92%. VLDL and LDL proteins were quantified by the method of Lowry et al. [21]. One percent sodium dodecyl sulfate was included in the assay mixture in order to eliminate interfering lipid turbidity. Plasma glucose concentrations were measured on the Autoanalyzer using the ferricyanide method [22]. Immunoreactive serum insulin concentrations were measured by a double antibody radioimmunoprecipitation technique [ 231. For the studies of lipoprotein composition, lipid was extracted from the isolated lipoprotein fractions of approximately half of the samples from each group by the method of Bligh and Dyer [24]. The extract was evaporated to dryness in a stream of nitrogen and redissolved in petroleum ether. Lipid subfractions were isolated by thin-layer chromatography on plates of silica gel G, 25 nm, in a solvent system of petroleum ether : ethyl ether : acetic acid (75 : 25 : 1). Lipid subclasses were quantitated by the sulfuric acid charring procedure of Marsh and Weinstein [25] as modified for thin-layer chromatography by Kritchevsky et al. [26]. Details of this method have been described in previous publications [ 27,281. For the determination of ratios of free to esterified cholesterol, lipid was extracted from the isolated lipoproteins, and cholesterol was measured directly
166
in the lipid extract. Esterified cholesterol was measured after saponification of a portion of the extract in 10% ethanolic KOH for 6 h at 60°C. Cholesterol was quantified with a Hewlett-Packard Model 7600A gas-liquid chromatograph equipped with a hydrogen flame ionization detector. A 6 foot column was used packed with 100-120 mesh Gas Chrom and coated with 3% OV 17. Carrier gas flow rate was 35 ml/min, and temperature was 250°C. Sterol was quantified by comparison of peak ratios to an internal standard of P-sitosterol. Lecithin/cholesterol acyl transferase (LCAT) activity in plasma samples was determined by the method of Rose and Juliano [ 291. Results
Table 2 shows cholesterol and triglyceride levels in whole plasma and lipoprotein fractions from normal and diabetic Pima Indian young women. Total plasma cholesterol was increased (P< 0.05) in the diabetic group. There were also significant changes in the distribution of cholesterol among the lipoprotein fractions, with an increase in the amount of LDL cholesterol (P < 0.005) and a decrease in the amount of HDL cholesterol in the diabetic group (P < 0.02). When the distribution of cholesterol in the lipoproteins was expressed as the ratio of HDL cholesterol to LDL cholesterol this ratio was very significantly decreased in the diabetic population (P< 0.001). Plasma triglycerides were significantly higher in the diabetics than in the controls (P< 0.05). This elevation was primarily the result of an increased amount of VLDL triglyceride (P < 0.05). LDL triglyceride levels were also elevated, (P < 0.02). HDL triglyceride levels were not significantly different in the two groups. Correlation coefficients between lipoprotein cholesterol and triglyceride levels and glucose tolerance are shown in Table 3. There were significant correlations between 2-h plasma glucose levels and VLDL triglyceride, and between 2-h plasma glucose and LDL cholesterol. There were significant negative correlations between 2-h plasma glucose levels and HDL cholesterol, and between 2-h glucose and HDL/LDL cholesterol ratio. Since obesity in other populations has been linked with alterations in blood TABLE 2 LIPOPROTEIN INDIANS
CHOLESTEROL
AND TRIGLYCERIDE
LEVELS IN NORMAL
Total VLDL LDL HDL HDL/LDL
cholesterol
a Mean f SEM. b n = 14. cn=17.
176 16 106 51 0.49
f f f r f
6.4 e 2.6 4.1 3.4 0.04
PIMA
Triglyceride (mg/dl)
Cholesterol (mg/dI) Normal b
AND DIABETIC
Diabetic c
Normal b
Diabetic c
202 16 136 41 0.31
136 75 24 13 -
176 114 39 15 -
* 11 f 2.6 f 8.6 r 2.2 * 0.02
f 18 * r 12 _t 5.7 f 4.6
zt 15 f 16 f 3.0 f 2.7
157 TABLE 3 CORRELATION
COEFFICIENTS
Two-hour aucose LDL cholesterol HDL cholesterol HDL/LDL cholesterol VLDL triglyceride
Two-h
LDL
HDL
HDL/LDL
VLDL
Body
gIucose
cholesterol
cholesterol
cholesterol
triglyceride
~WS a
1.00
0.62 b 1.00
-0.39 c 4.07 1 .oo
-0.66
b
1.00
0.52 b 0.72 b -0.10 -0.62 b 1.00
Body mass a
0.19 0.11 -0.23 -0.27 0.09 1.00
a kg/&. b P < 0.01. c P
<
0.05.
lipids [30], the data were analyzed for the effect of weight by determining correlation coefficients between body mass (wt/ht’) and the lipoprotein parameters (Table 3). There were no significant correlations in this group between body mass and any of the lipoprotein parameters. In addition, after covariance analysis was used to make concomitant adjustments for obesity and age, there were still significant shifts in lipoprotein cholesterol distribution and increased VLDL triglycerides in the diabetics. After adjusting for body mass means for VLDL triglycerides in normals and diabetics were 75 and 115 mg/dl (P< 0.05), LDL cholesterol were 105 and 135 mg/dl (P< O.Ol), HDL cholesterol were 50 and 41 mg/dl (P< 0.03) and the HDL/LDL cholesterol ratios were 0.49 and 0.32 (P < 0.001) respectively. To determine whether the shifts in cholesterol and triglyceride levels in the isolated lipoprotein fractions represented increased numbers of lipoprotein particles or alterations in the composition of the lipoproteins the lipid to protein ratios were measured in all samples, and total lipid composition was determined in the lipoproteins of 9 diabetic and 7 normal subjects. Table 4 TABLE 4 LIPOPROTEIN
COMPOSITION
IN NORMAL
AND DIABETIC PIMA INDIANS
VLDL Normal TrigIyceride/protein Cholesterol/protein
4.4 * 0.17 a (13) b -
LDL Diabetic 6.4 f 1.2 (1S) -
HDL
Normal
1.26 f 0.07 a
20.3 f 3.1
28.2
free cholesterol
(7) 10.5 f 3.4
(9) 9.6 i 1.2
(7) 13.2
triglyceride
(7) 57.3 r 2.2
(9) 55.7 f 6.2
(7) 12.2
(7) 9.9 r 1.4
(9) 12.4 f 1.9
(7) 44.6
(7)
(9)
(7)
a Mean
= SEM.
Number of determinations.
Diabetic
-
Lipid fraction (% total) phospholipid 19.7 i 2.2
b
Normal
-
(13)
esterified cholesterol
Diabetic
1.28 f 0.03
-
-
(18) f 4.6
27.3
i: 4.9
46
f 0.8
(9) 14.6
f 1.8
(7) 8.3 f 2.2
(9) 8.5 * 2.0
i 3.4
(9) 13.3
f 2.4
(7) 9.5 f 4.6
(9) 8.1 zt 3.9
2 6.3
(9) 42.0
t 7.2
(7) 37
(9) 36
(9)
(7)
f 3.8 a
* 4.4
48
(9)
;t 2.3
* 3.6
158 TABLE 5 ESTER/FREE
CHOLESTEROL
AND LCAT ACTIVITY
Pima (n = 8) Esterified/free cholesterol VLDL LDL HDL LCAT (/moles/l/h)
1.1 f 0.13 a 2.8 k 0.11 4.4 f 0.20 78.6 f 6.7 b
IN PIMA INDIANS
Caucasian (n = 8)
1.1 1: 0.13 3.2 -+ 0.13 5.0 2 0.18 76.0 -+ 3.9 b
a Mean f SEM. “,=6.
shows data on the composition of lipoproteins isolated from normal and diabetic Pima females. The ratio of triglyceride/protein was not significantly elevated in the diabetic VLDL and the proportion of individual lipid subfractions within VLDL were very similar. The data on VLDL suggest that the elevation of VLDL triglyceride in the diabetic8 primarily represents increased amounts of VLDL with normal composition, rather than the presence of VLDL with increased amounts of triglyceride. LDL cholesterol/protein ratio and the proportions of individual lipids, expressed as percent of total LDL lipids, were similar in the normals and diabetics. The data on LDL suggest that the increased LDL cholesterol observed in the diabetic Pimas represents increased number of LDL particles but that these particles are not enriched in either cholesterol or triglycerides. Since HDL was isolated as the infranatant after LDL flotation no assay of HDL protein could be conducted. However, when the distribution of lipid subclasses was compared it can be seen that they were identical in the two groups. Therefore it is probable that the decreased HDL cholesterol levels observed in the diabetics also reflect a change in total number of HDL particles rather than a change in HDL composition. An examination of the data on cholesterol composition of Pima lipoproteins shown in Table 4 suggested that there might be less cholesterol ester and more free cholesterol in the Pima lipoprotein8 as compared to literature reports for Caucasians [31]. To examine this more closely the ratio of esterified/free cholesterol was measured directly and compared to lipoproteins isolated in this laboratorykfrom Caucasian plasma. Table 5 shows the ratio of esterified to free cholesterol in each lipoprotein class. Whereas the ratio was identical in VLDL fractions from Pimas and Caucasians, in both LDL (P < 0.02) and HDL (P < 0.001) fractions there was a decrease in the ester/free cholesterol ratio in the Pimas as compared to Caucasian controls. Lecithin-cholesterol acyl transferase (LCAT) activity, however, was not significantly different in the two groups. It appears, therefore, that LCAT activity is normal in this population and that the slight decrease in esterlfication of cholesterol ester must be attributed to phenomena other than alterations in LCAT activity. Discussion The results of this study suggest that diabetes mellitus may be associated with change8 in both lipoprotein triglyceride and cholesterol levels. Both total
159
plasma triglyceride and VLDL triglyceride were elevated in the diabetics. Although total plasma cholesterol was only slightly elevated in the diabetics, there were significant shifts in the distribution of lipoprotein cholesterol, with an increase in LDL cholesterol and a decrease in HDL cholesterol. The ratio of HDL cholesterol/LDL cholesterol appears to be a convenient indicator of this shift in proportions of lipoprotein cholesterol, since it reflects cholesterol distribution in the two major cholesterol-containing lipoproteins and is independent of total levels. We found no effect of diabetes on the composition of lipoprotein particles. Although larger particles of similar composition might be present, the shifts in HDL and LDL cholesterol and VLDL triglyceride probably represent changes in total numbers of particles. This could reflect either altered synthesis or clearance of these lipoproteins. Increases in VLDL triglyceride, such as those found in the Pima diabetics, have been a commonly observed concomitant of diabetes [32,33]. Studies of lipoprotein cholesterol in diabetics have yielded less consistent results. Increased LDL cholesterol in uncontrolled diabetics has been reported by Billimoria et al. [lo]. Schonfeld et al. [9] and Lopes-Virella et al. [ 111 have reported decreased HDL levels in Caucasian diabetic populations, whereas Ballantyne et al. [34] found no changes in diabetic HDL or LDL. A recent report from the Framingham study [44] showed diabetes and HDL cholesterol were negatively associated in women, but not in men, between 49 and 82 years. The investigation of aspects of diabetes mellitus using the Pima population has the advantage of a group with a high prevalence of maturity onset diabetes [ 121 and a relatively homogeneous genetic pool [ 131. Furthermore, the present study was limited to females in a narrow age range in order to eliminate the effects of sex [35,36] and age [36] on lipoprotein levels. Thus it is more likely that the specificity of the differences observed can be ascribed to the diabetes per se. Diabetes in the Pima Indians is associated with high incidence of microvascular complications such as nephropathy [37] and retinopathy [38]. In addition, although the prevalence of coronary heart disease is very low among the Pimas [39,40], nevertheless it occurs twice as often in the Pima diabetics as compared to Pima non-diabetics [ 12,391. Recent studies of HDL levels in other populations have suggested that HDL levels may correlate inversely with arteriosclerotic heart disease [ 41-441. It is interesting that even in this population, in which coronary heart disease is relatively infrequent, increased LDL and decreased HDL cholesterol are associated with significantly increased prevalence of coronary heart disease. The diabetic group was significantly heavier than the normals. As with Caucasians, obesity and diabetes appear to be intimately associated in the Pima population. The incidence of diabetes is higher in the more obese sections of the population [ 121 and, conversely, the offspring of two diabetic parents are significantly more obese than those with non-diabetic parents [45]. However, when the data were analyzed for the effect of obesity, correlation coefficients showed no significant correlation in this group between body mass and any of the lipoprotein parameters. However, it must be emphasized that this sample is of a limited age range, and both normals and diabetics were far above ideal body weight. Since there is a high prevalence of obesity in the Pima population, this would be an interesting population for further study,
160
using samples of both males and females of wider age ranges, of the effects of obesity on lipid parameters. Finally, it is of interest to compare the lipoprotein data obtained from the normal Pimas to those reported in the literature for other populations. Values for total plasma triglycerides and their fractions are very similar [35,36]. Total cholesterol levels, however, have been shown to be low in this population [46] and the present data indicate that both LDL and HDL cholesterol levels are decreased as compared to published values for Caucasians. The data of LDL and HDL composition in the present study suggest that although LCAT activity is normal, the amount of esterified cholesterol is slightly low in HDL and LDL fractions of Pimas as compared to Caucasian lipoproteins analyzed in similar fashion. A previous study of LDL turnover in Southwestern American Indians disclosed slower synthetic rates for LDL cholesterol [47] as compared to Caucasians. Further studies of lipoprotein metabolism will be required to understand the interactions of lipoprotein triglyceride and cholesterol and how they are affected by diabetes. Acknowledgements We wish to thank Mrs. Rose Fields and Mr. Michael Davis for excellent technical assistance. References 1 Bradley, R.F., Cardiovascular disease: In: A. Marble, P. White, R.F. Bradley and L.D. Eroll (Ed%), Diabetes Mellitus, Philadelphia, Pa., 1971. pp. 417477. 2 National Heart and Lung Institute Task Force on Arteriosclerosis. Arteriosclerosis, Vol. 2. U.S. Government Prlntlng Office, Washington, D.C., 1971, P. 100. 3 Albrink, M.J., Lavietes, P.H. and Man. E.B.. Vascular disease and serum lipids in diabetes mellltus Observations over 30 years, Ann. Int. Med., 68 (1963) 305-323. 4 Bierman. E.L., Hypertriglyceridemia in early diabetes, Advanc. Metab. Disord.. II Suppl. 2 (1973) 67-72. 6 Nikkill, E.A., Triglyceride metabolism In diabetes meBitus. In: I. MacdonaId (Ed.), Progress in Biochemical Pharmacology, Vol. 8. Karger, New York, N.Y., 1973. PP. 271-299. 6 Lowry, A.D. and Barach, J.H., Predictive value of lipoprotein and cholesterol determination in diabetic patients who developed cardiovascular complications, Circulation. 17 (1958) 14-21. 7 Wilson, D.E., Schreibman, P.H.. Day, V.C. and Arky, R.A., HyperIIpidemia in an adult diabetic POPUIation. J. Chron. Dis., 23 (1970) 501-606. a Garcia, M.J.. McNamara. P.M., Gordon, T. and Kannel. W.B., Morbidity and mortality in diabetes in the FramIngham population. Diabetes. 23 (1974) 106-111. 9 Schonfeld, G.. Birg, G., Miller, J.P., Kessler, G. and Santiago. J.. ApoIIpoprotein B levels and altered lipoprotein composition in diabetes, Diabetes, 23 (1974) 827-834. 10 BiBimoria. J.D., Isaacs. A.J. and Melki. K., A lipid and lipoprotein profile of treated and untreated diabetics, AM. CIin. Biochem.. 13 (1976) 315-321. 11 Lopes-VireIIa, M.D., Stone, P.G. and ColweII. J.A.. High density lipoprotein cholesterol and apoIipoprotein A levels in diabetics with and without macrovascular disease, Diabetologia. 13 (1977) 286-291. 12 Bennett. P.H.. Rushforth, N.B.. Miller, M. and LeCompte. P., EpidlmiologicaI studies of diabetes ln the Phna Indians, Recent Progr. Horm. Res., 32 (1976) 333-376. 13 Matson, G.A.. Burch. T.A. and Polesky. H.F.. Distribution of hereditary factors in the blood of Indians of the Glla River, Arizona. Amer. J. Phys. Anthropol.. 29 (1968) 311-338. 14 Savage, P.S., Bennett, P.H.. Turner, J.N. and Miller. M., Cholesterol and trlglycerlde Levels in Pima Indians over a wide spectrum of glucose tolerance, Submitted for publication. 16 Rushforth, N.B., Bennett, PH., SteInberg. A.G. and Miller, hf.. Comparison of the value of the twoand one-hour gIucose levels of the oral GTT in the diagnosis of diabetes in Pima Indians, Diabetes, 24 (1975) 538-556.
161 16 Havel. R.J.. Eder. H.A. and Bragden, J-H.. Distribution and chemical composition of uItraoentrifugaIIy separated lipoproteins in human serum, J. CIin. Invest., 34 (1955) 1345-1353. 17 Marsh, J.B.. Lipoproteins in a nonrecircuIating perfusate of rat liver, J. Lipid Res., 15 (1974) 544-550. 18 Noble, R.P.. Electrophoratic seepsration of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 693-700. 19 Rush, R.L., Leon, L. and TurreII. J.. Automated simultaneous cholesterol and triglyceride determination on the Auto Analyzer instrument. In: Advances in Automated Analyses, Thurman, Miami, FM., 1970. PP. 503-511. 20 Bucolo, G. and Davis, H., Quantitative determination of serum triglycerides by use of enzymes, Ciin. Chem.. 19 (1973) 475482. 21 Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and Randall. R.J.. Protein measurement with the FoIin phenol reagent, J. Biol. Chem.. 193 (1951) 265-275. 22 Technicon Methodology File N-26, Technicon Instruments Corp., Tarrytown, N.Y., 1965. 23 Hales. CM. and RandIe. P.J.. Immunoassay of msuhn with insuhn-antibody precipitate, Biochem. J.. 88 (1963) 137-146. 24 Bhgh. E.B. and Dyer, W.J.. A rapid method of total lipid extraction and purification. Canad. J. Biochem.. 37 (1959) 911-917. 25 Marsh, J.B. and Weinstein. D.B., Simple charring method for the determination of lipids, J. Lipid Res.. 7 (1966) 574-576. 26 Kritchevsky. D., Davidson, L.M.. Kim, K. and MaIhotra. S., Quantitation of serum lipidsby a simple TLC-charring method, Chn. Chim. Acta. 46 (1973) 63-68. 27 Howard, B.V.. Howard, W.J.. de la Llera. hf. and Kefalides. N.A.. Triglyceride accumulation in cultured fibroblasts - The effects of hypertrighxeridemic serum, Atherosclerosis, 23 (1976) 521-534. 28 Howard, B.V.. Uptake of very low density Iipoprotein triglyceride by bovine aortic endothelial ceBs in culture. J. Lipid Res.. 18 (1977) 561-571. 29 Rosa, H.G. and Juiiano, J.. ReguIation of plasma lecithin : cholesterol acyl transferam in man. Part 1 (Incraaaad activity in primary hypertrigIyceridemia). J. Lab. CIin. Med., 88 (1976) 29-43 30 Bagdade, J.D.. Bierman, E.L. and Porte, D.. Influence of obesity on the relationship between insulin and trigIyceride levels in endogenous hypertriglyceridemia. Diabetes, 20 (1971) 664-672. 31 Hatch, F.T. and Lees, R.S.. Practical method for pIaama lipoprotein analysis. Advanc. Lipid. Res., 6 (1968) l-68. 32 Olefsky, J.M.. Farquhar, J.W. and Reaven. G.M.. Reappraisal of the r6le of insulin in hypertriglyceridemia. Amer. J. Med., 57 (1974) 551-560. 33 Bagdade. J.D.. Porte, D. and Bierman, E.L.. Diabetic iipemia - A form of acquired fat-induced iipemia. New EngI. J. Med., 276 (1967) 427433. 34 BaUantyne. D.. White, C.. Strevens, E.A.. Lawrie, T.D.V.. Lorimer, A.R., Mandemon. W.G. and Morgan, H.G.. Lipoprotein concentration in untreated adult onset diabetes meIIitus and the relationship of tbe fasting pIasma triglyceride concentration and insuhn secretion, CIin. Chim. Acta. 78 (1977) 323-329. 35 Wood, P.D.S.. Stem, M.P.. Silvers. A., Reaven, G.M. and vonder Groeben. Prevalence of plasma iipoprotein abnormalities in a free-living population of the Central Valley. California, J. Circ.. 45 (1972) 114-126. 36 Cadson, L.A. and Ericsson, hf.. Quantitative and qualitative serum lipoprotein anaIysas, Part 1 (Studies in healthy men and women), Atherosclerosis, 21 (1975) 417433. 37 Kamenetsky. S.A.. Bennett, P.H.. Dippe. S.E., MilIer. M. and LeCompte, P., A cIinicaI and histologic study of diabetic nephropathy in the Pima Indians, Diabetes. 23 (1974) 61-68. 38 Dorf. A., BaBantine, E.J., Bennett, P.H. and Miller. M.. Retinopathy in Pima Indians, Diabetes, 25 (1976) 554-560. 39 Ingelfinger, J.A.. Bennett. P.H.. Liebow. I.M. and Miller, M., Coronary heart disease in the Pima Indians, Diabetes, 25 (1976) 561-565. 40 Sievers. ML.. Myocardial infarction among Southwestern American Indians, Ann. Intern. Med.. 67 (1967) 800-807. 41 Berg, K., Borresen. A.L. and DahIen, G.. Serum high density lipoprotein and atherosclerotic heart disease, Lance& 1 (1976) 499601. 42 Miller. N.E., Forde. O.H.. Thelle. D.A. and Mjos, O.D.. The Troms5 heart study. High density Iipoprotein and coronary heart dimasa -A prospective case control study, Lancet. 1 (1977) 965-967. 43 CasteI% W.P.. Doyle, J.T.. Gordon, T.. Haines. C.G.. HiortIand, M.C.. HuIIey, S.B.. Kagen. A. and Zukel. W.J.. HDL cholesterol and other lipids in coronary heart disease, CircuIation. 55 (1977) 767-772. 44 Gordon, T., Caste& W.P., HiortIand. M.C.. Kannel. W.B. and Dawbar. T.R.. Diabetes. blood lipids and the role of obedty in coronary heart dieease risk women, Ann. Int. Med., 87 (1977) 393-397. 45 Savage, P.J.. Bennett, P.H. and MiIIer. M.. FamiIiaI relationships of obesity and hypergIycemta in the Pima Indians, Amer. J. Epidemiol.. 106 (1977) 274.
162 46 Savage, P.J.. Hamman, R.F.,
Bartha, G.. Dippe. S.E., Miller, M. and Bennett, P.H., Serum cholesterol levels in American (Pima) Indian children and adolescents, Pediatrics, 68 (19’76) 274-282. 47 Gamick. M.B., Bennett, P.H. and Lange& T., Low density lipoprotein metabolism and lipoprotein cholesterol composition in American Indians, J. Lipid Res., In press.
Atherosckroais, 30 (1978) 153-162 0 Elsevier/North-Holland Scientific Publishers,
Ltd.
LIPOPROTEIN COMPOSITION IN DIABETES MELLITUS
BARBARA V. HOWARD, PETER J. SAVAGE, PETER H. BENNETT
LYNN J. BENNION
and
Phoenix Clinical Research Section, National Institute of Arthritis, Metabolism, and Digestive Dieeaees, 4212 N. 16th Street, Phoenix, Arizona 85016 (U.S.A.) (Received 27 December, 1977) (Revised received 8 February, 1978) (Accepted 15 February, 1978)
Lipoprotein cholesterol and triglyceride levels have been determined in normal and diabetic Pima Indian women aged 20-35. HDL cholesterol levels were lower, LDL cholesterol levels were higher, and the ratio of HDL cholesterol/LDL cholesterol, a reflection of lipoprotein cholesterol distribution, was lower in the diabetics compared to the normals. VLDL triglyceride levels were also elevated in the diabetics. An analysis of lipoprotein compositiQn suggested that these changes primarily reflect changes in numbers of particles, since lipid composition and lipid/protein ratios were similar in lipoproteins isolated from normals and diabetics. The ratio of ester/free cholesterol in LDL and HDL was lower in normal Pima Indians than in a comparable’ group of Caucasians, although plasma LCAT activity was not significantly different. The data indicate that diabetes may be associated with shifts -in distribution of LDL and HDL, as well as with increases in VLDL. Key words:
Atherosclerosis
- Cholesterol -Diabetes
-Lipoproteins
- Triglycerides
Introduction Examination of blood lipids and lipoproteins in diabetes mellitus is of interest because of the increased prevalence of arteriosclerotic cardiovascular disease associated with this disorder [ 1,2]. There have been many repor@ of abnormalities in blood lipids associated with diabetes mellitus. Hypertriglyceridemia has been frequently observed [3-51 and hypercholesterolemia has also been noted by a number of investigators [ 6-81. More recently there have been This
work was supported in part by the National Institutes of Health Contract No. NOl-AM-B-0219
154
reports of alterations in individual lipoprotein patterns in diabetes [g-11]. Interpretation has been difficult, however, because of the many factors in diabetes that influence blood lipid levels, including insulin levels, obesity, age, sex, the type of diabetes and the genetic heritage of the diabetics under study. The Pima Indians have the highest recorded prevalence of maturity onset diabetes [ 121. They are more genetically homogeneous than the general population [ 131, and there is a relative absence of primary hyperlipemias [ 141. A previous study of plasma triglyceride and cholesterol levels in this population indicated that both cholesterol and triglyceride levels were significantly higher in the diabetics compared to the normals, and that both cholesterol and triglyceride were related in a linear fashion to increasing fasting plasma glucose levels [ 141. We have further investigated these differences by examining cholesterol and triglyceride levels and lipid composition of isolated lipoprotein fractions. In order to minimize the many variables that influence blood lipids, we have selected a group of Pima women, aged 20-35, for a comparison of plasma lipids and lipoproteins between normal and diabetic individuals. Materials and Methods The patients for this study consisted of 14 non-diabetic and 17 diabetic female Pima Indians between 20 and 35 years old. The majority were inpatients in the Phoenix Clinical Research Unit, National Institute of Arthritis, Metabolism and Digestive Diseases. A small number of patient+ in each group were seen as outpatients at the Sacaton Clinic on the Gila River Indian Reservation. The criterion for diagnosis of diabetes was a 2-h plasma glucose level of greater than 200 mg/dl [15] following a 100 g carbohydrate load, and the duration of diabetes ranged from 1 to 14 years. The diabetics were all of maturity-onset type with no overt ketoacidosis. Those classified as non-diabetic subjects were free of symptoms and had 2-h plasma glucose levels less than 150 mg/dl. None of the subjects was taking insulin or any other medication known to affect blood lipids or lipoprotein Ieve@. Clinical data are presented in Table 1. Age
TABLE 1 STUDY OF A-POPULATION
Ale (yr) Height (cm) Weight (kg) Plasma glucose (mn/dl) fastfm3 2-h Insulin (@/ml) fasting 2-h a Mean * SEM. bn=8. Cn=12.
OF PIMA INDIANS NOllIlalS
Diabetics
(14)
(17)
27 158 f 1.0 a 86 f 6.0
28 161 i: 1.0 100 t 4.9
89 f 2.3 124 f 6.1
204 t 21 357 * 28 ,
26 f 4.9 b 228 f 46 b
38 t 5.8 c 100 * 30 c
155
and height in the two populations were comparable, although the diabetics as a group were about 14 kg heavier than the normals. Two-hour plasma glucose levels averaged 357 mg/dl in the diabetics, and 124 mg/dl in the normals, and fasting plasma glucose levels were 204and 89 mg/dl, respectively. Venous blood samples were collected in EDTA after an overnight fast. Plasma was separated after centrifugation at approximately 700 X g for 15 min at 10°C. None of the plasma contained detectable evidence of chylomicrons after overnight incubation in the cold. Lipoproteins were isolated from plasma according to procedures described by Have1 et al. [ 161 and modified by Marsh [ 171. Six ml plasma were overlaid with 2 ml of 0.16 M NaCl, 1 mM EDTA, d 1.006, and very low density lipoproteins (VLDL) were isolated by ultracentrifugation for 16 h at 40,000 rpm in a Beckman model L250 ultracentrifuge using a type 40 rotor. VLDL preparations isolated by migration through overlaid saline contain less than 10% albumin and other proteins [9]. After VLDL were removed the plasma was adjusted to d 1.063 using KBr (d 1.35) and low density lipoproteins (LDL) were isolated by centrifugation for 20 h at 40,000 rpm. The high density lipoprotein (HDL) fraction was the infranatant after removal of the LDL. Efficiency of the ultracentrifugal lipoprotein isolation was monitored by electrophoresis on agarose gels according to the method of Noble [ 181. Triglycerides and cholesterol in plasma and isolated lipoproteins were quantified on an Autoanalyzer II (Technicon), using the cholesterol extract method of Rush et al. [19] and the triglyceride enzymatic method of Bucolo and Davis [20]. The triglyceride assay was standardized using control plasma supplied by the Lipid Standardization Lab, CDC, Atlanta, Georgia. Control plasma for the cholesterol assay was standardized using the CDC Proficiency Testing Service. The coefficient of variation for the cholesterol assay averaged 3.2% and that of the triglyceride assay was 5.8%. Recovery of cholesterol in the isolated lipoprotein fractions averaged 97%, and recovery of the triglycerides in the lipoprotein fractions averaged 92%. VLDL and LDL proteins were quantified by the method of Lowry et al. [21]. One percent sodium dodecyl sulfate was included in the assay mixture in order to eliminate interfering lipid turbidity. Plasma glucose concentrations were measured on the Autoanalyzer using the ferricyanide method [22]. Immunoreactive serum insulin concentrations were measured by a double antibody radioimmunoprecipitation technique [ 231. For the studies of lipoprotein composition, lipid was extracted from the isolated lipoprotein fractions of approximately half of the samples from each group by the method of Bligh and Dyer [24]. The extract was evaporated to dryness in a stream of nitrogen and redissolved in petroleum ether. Lipid subfractions were isolated by thin-layer chromatography on plates of silica gel G, 25 nm, in a solvent system of petroleum ether : ethyl ether : acetic acid (75 : 25 : 1). Lipid subclasses were quantitated by the sulfuric acid charring procedure of Marsh and Weinstein [25] as modified for thin-layer chromatography by Kritchevsky et al. [26]. Details of this method have been described in previous publications [ 27,281. For the determination of ratios of free to esterified cholesterol, lipid was extracted from the isolated lipoproteins, and cholesterol was measured directly
166
in the lipid extract. Esterified cholesterol was measured after saponification of a portion of the extract in 10% ethanolic KOH for 6 h at 60°C. Cholesterol was quantified with a Hewlett-Packard Model 7600A gas-liquid chromatograph equipped with a hydrogen flame ionization detector. A 6 foot column was used packed with 100-120 mesh Gas Chrom and coated with 3% OV 17. Carrier gas flow rate was 35 ml/min, and temperature was 250°C. Sterol was quantified by comparison of peak ratios to an internal standard of P-sitosterol. Lecithin/cholesterol acyl transferase (LCAT) activity in plasma samples was determined by the method of Rose and Juliano [ 291. Results
Table 2 shows cholesterol and triglyceride levels in whole plasma and lipoprotein fractions from normal and diabetic Pima Indian young women. Total plasma cholesterol was increased (P< 0.05) in the diabetic group. There were also significant changes in the distribution of cholesterol among the lipoprotein fractions, with an increase in the amount of LDL cholesterol (P < 0.005) and a decrease in the amount of HDL cholesterol in the diabetic group (P < 0.02). When the distribution of cholesterol in the lipoproteins was expressed as the ratio of HDL cholesterol to LDL cholesterol this ratio was very significantly decreased in the diabetic population (P< 0.001). Plasma triglycerides were significantly higher in the diabetics than in the controls (P< 0.05). This elevation was primarily the result of an increased amount of VLDL triglyceride (P < 0.05). LDL triglyceride levels were also elevated, (P < 0.02). HDL triglyceride levels were not significantly different in the two groups. Correlation coefficients between lipoprotein cholesterol and triglyceride levels and glucose tolerance are shown in Table 3. There were significant correlations between 2-h plasma glucose levels and VLDL triglyceride, and between 2-h plasma glucose and LDL cholesterol. There were significant negative correlations between 2-h plasma glucose levels and HDL cholesterol, and between 2-h glucose and HDL/LDL cholesterol ratio. Since obesity in other populations has been linked with alterations in blood TABLE 2 LIPOPROTEIN INDIANS
CHOLESTEROL
AND TRIGLYCERIDE
LEVELS IN NORMAL
Total VLDL LDL HDL HDL/LDL
cholesterol
a Mean f SEM. b n = 14. cn=17.
176 16 106 51 0.49
f f f r f
6.4 e 2.6 4.1 3.4 0.04
PIMA
Triglyceride (mg/dl)
Cholesterol (mg/dI) Normal b
AND DIABETIC
Diabetic c
Normal b
Diabetic c
202 16 136 41 0.31
136 75 24 13 -
176 114 39 15 -
* 11 f 2.6 f 8.6 r 2.2 * 0.02
f 18 * r 12 _t 5.7 f 4.6
zt 15 f 16 f 3.0 f 2.7
157 TABLE 3 CORRELATION
COEFFICIENTS
Two-hour aucose LDL cholesterol HDL cholesterol HDL/LDL cholesterol VLDL triglyceride
Two-h
LDL
HDL
HDL/LDL
VLDL
Body
gIucose
cholesterol
cholesterol
cholesterol
triglyceride
~WS a
1.00
0.62 b 1.00
-0.39 c 4.07 1 .oo
-0.66
b
1.00
0.52 b 0.72 b -0.10 -0.62 b 1.00
Body mass a
0.19 0.11 -0.23 -0.27 0.09 1.00
a kg/&. b P < 0.01. c P
<
0.05.
lipids [30], the data were analyzed for the effect of weight by determining correlation coefficients between body mass (wt/ht’) and the lipoprotein parameters (Table 3). There were no significant correlations in this group between body mass and any of the lipoprotein parameters. In addition, after covariance analysis was used to make concomitant adjustments for obesity and age, there were still significant shifts in lipoprotein cholesterol distribution and increased VLDL triglycerides in the diabetics. After adjusting for body mass means for VLDL triglycerides in normals and diabetics were 75 and 115 mg/dl (P< 0.05), LDL cholesterol were 105 and 135 mg/dl (P< O.Ol), HDL cholesterol were 50 and 41 mg/dl (P< 0.03) and the HDL/LDL cholesterol ratios were 0.49 and 0.32 (P < 0.001) respectively. To determine whether the shifts in cholesterol and triglyceride levels in the isolated lipoprotein fractions represented increased numbers of lipoprotein particles or alterations in the composition of the lipoproteins the lipid to protein ratios were measured in all samples, and total lipid composition was determined in the lipoproteins of 9 diabetic and 7 normal subjects. Table 4 TABLE 4 LIPOPROTEIN
COMPOSITION
IN NORMAL
AND DIABETIC PIMA INDIANS
VLDL Normal TrigIyceride/protein Cholesterol/protein
4.4 * 0.17 a (13) b -
LDL Diabetic 6.4 f 1.2 (1S) -
HDL
Normal
1.26 f 0.07 a
20.3 f 3.1
28.2
free cholesterol
(7) 10.5 f 3.4
(9) 9.6 i 1.2
(7) 13.2
triglyceride
(7) 57.3 r 2.2
(9) 55.7 f 6.2
(7) 12.2
(7) 9.9 r 1.4
(9) 12.4 f 1.9
(7) 44.6
(7)
(9)
(7)
a Mean
= SEM.
Number of determinations.
Diabetic
-
Lipid fraction (% total) phospholipid 19.7 i 2.2
b
Normal
-
(13)
esterified cholesterol
Diabetic
1.28 f 0.03
-
-
(18) f 4.6
27.3
i: 4.9
46
f 0.8
(9) 14.6
f 1.8
(7) 8.3 f 2.2
(9) 8.5 * 2.0
i 3.4
(9) 13.3
f 2.4
(7) 9.5 f 4.6
(9) 8.1 zt 3.9
2 6.3
(9) 42.0
t 7.2
(7) 37
(9) 36
(9)
(7)
f 3.8 a
* 4.4
48
(9)
;t 2.3
* 3.6
158 TABLE 5 ESTER/FREE
CHOLESTEROL
AND LCAT ACTIVITY
Pima (n = 8) Esterified/free cholesterol VLDL LDL HDL LCAT (/moles/l/h)
1.1 f 0.13 a 2.8 k 0.11 4.4 f 0.20 78.6 f 6.7 b
IN PIMA INDIANS
Caucasian (n = 8)
1.1 1: 0.13 3.2 -+ 0.13 5.0 2 0.18 76.0 -+ 3.9 b
a Mean f SEM. “,=6.
shows data on the composition of lipoproteins isolated from normal and diabetic Pima females. The ratio of triglyceride/protein was not significantly elevated in the diabetic VLDL and the proportion of individual lipid subfractions within VLDL were very similar. The data on VLDL suggest that the elevation of VLDL triglyceride in the diabetic8 primarily represents increased amounts of VLDL with normal composition, rather than the presence of VLDL with increased amounts of triglyceride. LDL cholesterol/protein ratio and the proportions of individual lipids, expressed as percent of total LDL lipids, were similar in the normals and diabetics. The data on LDL suggest that the increased LDL cholesterol observed in the diabetic Pimas represents increased number of LDL particles but that these particles are not enriched in either cholesterol or triglycerides. Since HDL was isolated as the infranatant after LDL flotation no assay of HDL protein could be conducted. However, when the distribution of lipid subclasses was compared it can be seen that they were identical in the two groups. Therefore it is probable that the decreased HDL cholesterol levels observed in the diabetics also reflect a change in total number of HDL particles rather than a change in HDL composition. An examination of the data on cholesterol composition of Pima lipoproteins shown in Table 4 suggested that there might be less cholesterol ester and more free cholesterol in the Pima lipoprotein8 as compared to literature reports for Caucasians [31]. To examine this more closely the ratio of esterified/free cholesterol was measured directly and compared to lipoproteins isolated in this laboratorykfrom Caucasian plasma. Table 5 shows the ratio of esterified to free cholesterol in each lipoprotein class. Whereas the ratio was identical in VLDL fractions from Pimas and Caucasians, in both LDL (P < 0.02) and HDL (P < 0.001) fractions there was a decrease in the ester/free cholesterol ratio in the Pimas as compared to Caucasian controls. Lecithin-cholesterol acyl transferase (LCAT) activity, however, was not significantly different in the two groups. It appears, therefore, that LCAT activity is normal in this population and that the slight decrease in esterlfication of cholesterol ester must be attributed to phenomena other than alterations in LCAT activity. Discussion The results of this study suggest that diabetes mellitus may be associated with change8 in both lipoprotein triglyceride and cholesterol levels. Both total
159
plasma triglyceride and VLDL triglyceride were elevated in the diabetics. Although total plasma cholesterol was only slightly elevated in the diabetics, there were significant shifts in the distribution of lipoprotein cholesterol, with an increase in LDL cholesterol and a decrease in HDL cholesterol. The ratio of HDL cholesterol/LDL cholesterol appears to be a convenient indicator of this shift in proportions of lipoprotein cholesterol, since it reflects cholesterol distribution in the two major cholesterol-containing lipoproteins and is independent of total levels. We found no effect of diabetes on the composition of lipoprotein particles. Although larger particles of similar composition might be present, the shifts in HDL and LDL cholesterol and VLDL triglyceride probably represent changes in total numbers of particles. This could reflect either altered synthesis or clearance of these lipoproteins. Increases in VLDL triglyceride, such as those found in the Pima diabetics, have been a commonly observed concomitant of diabetes [32,33]. Studies of lipoprotein cholesterol in diabetics have yielded less consistent results. Increased LDL cholesterol in uncontrolled diabetics has been reported by Billimoria et al. [lo]. Schonfeld et al. [9] and Lopes-Virella et al. [ 111 have reported decreased HDL levels in Caucasian diabetic populations, whereas Ballantyne et al. [34] found no changes in diabetic HDL or LDL. A recent report from the Framingham study [44] showed diabetes and HDL cholesterol were negatively associated in women, but not in men, between 49 and 82 years. The investigation of aspects of diabetes mellitus using the Pima population has the advantage of a group with a high prevalence of maturity onset diabetes [ 121 and a relatively homogeneous genetic pool [ 131. Furthermore, the present study was limited to females in a narrow age range in order to eliminate the effects of sex [35,36] and age [36] on lipoprotein levels. Thus it is more likely that the specificity of the differences observed can be ascribed to the diabetes per se. Diabetes in the Pima Indians is associated with high incidence of microvascular complications such as nephropathy [37] and retinopathy [38]. In addition, although the prevalence of coronary heart disease is very low among the Pimas [39,40], nevertheless it occurs twice as often in the Pima diabetics as compared to Pima non-diabetics [ 12,391. Recent studies of HDL levels in other populations have suggested that HDL levels may correlate inversely with arteriosclerotic heart disease [ 41-441. It is interesting that even in this population, in which coronary heart disease is relatively infrequent, increased LDL and decreased HDL cholesterol are associated with significantly increased prevalence of coronary heart disease. The diabetic group was significantly heavier than the normals. As with Caucasians, obesity and diabetes appear to be intimately associated in the Pima population. The incidence of diabetes is higher in the more obese sections of the population [ 121 and, conversely, the offspring of two diabetic parents are significantly more obese than those with non-diabetic parents [45]. However, when the data were analyzed for the effect of obesity, correlation coefficients showed no significant correlation in this group between body mass and any of the lipoprotein parameters. However, it must be emphasized that this sample is of a limited age range, and both normals and diabetics were far above ideal body weight. Since there is a high prevalence of obesity in the Pima population, this would be an interesting population for further study,
160
using samples of both males and females of wider age ranges, of the effects of obesity on lipid parameters. Finally, it is of interest to compare the lipoprotein data obtained from the normal Pimas to those reported in the literature for other populations. Values for total plasma triglycerides and their fractions are very similar [35,36]. Total cholesterol levels, however, have been shown to be low in this population [46] and the present data indicate that both LDL and HDL cholesterol levels are decreased as compared to published values for Caucasians. The data of LDL and HDL composition in the present study suggest that although LCAT activity is normal, the amount of esterified cholesterol is slightly low in HDL and LDL fractions of Pimas as compared to Caucasian lipoproteins analyzed in similar fashion. A previous study of LDL turnover in Southwestern American Indians disclosed slower synthetic rates for LDL cholesterol [47] as compared to Caucasians. Further studies of lipoprotein metabolism will be required to understand the interactions of lipoprotein triglyceride and cholesterol and how they are affected by diabetes. Acknowledgements We wish to thank Mrs. Rose Fields and Mr. Michael Davis for excellent technical assistance. References 1 Bradley, R.F., Cardiovascular disease: In: A. Marble, P. White, R.F. Bradley and L.D. Eroll (Ed%), Diabetes Mellitus, Philadelphia, Pa., 1971. pp. 417477. 2 National Heart and Lung Institute Task Force on Arteriosclerosis. Arteriosclerosis, Vol. 2. U.S. Government Prlntlng Office, Washington, D.C., 1971, P. 100. 3 Albrink, M.J., Lavietes, P.H. and Man. E.B.. Vascular disease and serum lipids in diabetes mellltus Observations over 30 years, Ann. Int. Med., 68 (1963) 305-323. 4 Bierman. E.L., Hypertriglyceridemia in early diabetes, Advanc. Metab. Disord.. II Suppl. 2 (1973) 67-72. 6 Nikkill, E.A., Triglyceride metabolism In diabetes meBitus. In: I. MacdonaId (Ed.), Progress in Biochemical Pharmacology, Vol. 8. Karger, New York, N.Y., 1973. PP. 271-299. 6 Lowry, A.D. and Barach, J.H., Predictive value of lipoprotein and cholesterol determination in diabetic patients who developed cardiovascular complications, Circulation. 17 (1958) 14-21. 7 Wilson, D.E., Schreibman, P.H.. Day, V.C. and Arky, R.A., HyperIIpidemia in an adult diabetic POPUIation. J. Chron. Dis., 23 (1970) 501-606. a Garcia, M.J.. McNamara. P.M., Gordon, T. and Kannel. W.B., Morbidity and mortality in diabetes in the FramIngham population. Diabetes. 23 (1974) 106-111. 9 Schonfeld, G.. Birg, G., Miller, J.P., Kessler, G. and Santiago. J.. ApoIIpoprotein B levels and altered lipoprotein composition in diabetes, Diabetes, 23 (1974) 827-834. 10 BiBimoria. J.D., Isaacs. A.J. and Melki. K., A lipid and lipoprotein profile of treated and untreated diabetics, AM. CIin. Biochem.. 13 (1976) 315-321. 11 Lopes-VireIIa, M.D., Stone, P.G. and ColweII. J.A.. High density lipoprotein cholesterol and apoIipoprotein A levels in diabetics with and without macrovascular disease, Diabetologia. 13 (1977) 286-291. 12 Bennett. P.H.. Rushforth, N.B.. Miller, M. and LeCompte. P., EpidlmiologicaI studies of diabetes ln the Phna Indians, Recent Progr. Horm. Res., 32 (1976) 333-376. 13 Matson, G.A.. Burch. T.A. and Polesky. H.F.. Distribution of hereditary factors in the blood of Indians of the Glla River, Arizona. Amer. J. Phys. Anthropol.. 29 (1968) 311-338. 14 Savage, P.S., Bennett, P.H.. Turner, J.N. and Miller. M., Cholesterol and trlglycerlde Levels in Pima Indians over a wide spectrum of glucose tolerance, Submitted for publication. 16 Rushforth, N.B., Bennett, PH., SteInberg. A.G. and Miller, hf.. Comparison of the value of the twoand one-hour gIucose levels of the oral GTT in the diagnosis of diabetes in Pima Indians, Diabetes, 24 (1975) 538-556.
161 16 Havel. R.J.. Eder. H.A. and Bragden, J-H.. Distribution and chemical composition of uItraoentrifugaIIy separated lipoproteins in human serum, J. CIin. Invest., 34 (1955) 1345-1353. 17 Marsh, J.B.. Lipoproteins in a nonrecircuIating perfusate of rat liver, J. Lipid Res., 15 (1974) 544-550. 18 Noble, R.P.. Electrophoratic seepsration of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 693-700. 19 Rush, R.L., Leon, L. and TurreII. J.. Automated simultaneous cholesterol and triglyceride determination on the Auto Analyzer instrument. In: Advances in Automated Analyses, Thurman, Miami, FM., 1970. PP. 503-511. 20 Bucolo, G. and Davis, H., Quantitative determination of serum triglycerides by use of enzymes, Ciin. Chem.. 19 (1973) 475482. 21 Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and Randall. R.J.. Protein measurement with the FoIin phenol reagent, J. Biol. Chem.. 193 (1951) 265-275. 22 Technicon Methodology File N-26, Technicon Instruments Corp., Tarrytown, N.Y., 1965. 23 Hales. CM. and RandIe. P.J.. Immunoassay of msuhn with insuhn-antibody precipitate, Biochem. J.. 88 (1963) 137-146. 24 Bhgh. E.B. and Dyer, W.J.. A rapid method of total lipid extraction and purification. Canad. J. Biochem.. 37 (1959) 911-917. 25 Marsh, J.B. and Weinstein. D.B., Simple charring method for the determination of lipids, J. Lipid Res.. 7 (1966) 574-576. 26 Kritchevsky. D., Davidson, L.M.. Kim, K. and MaIhotra. S., Quantitation of serum lipidsby a simple TLC-charring method, Chn. Chim. Acta. 46 (1973) 63-68. 27 Howard, B.V.. Howard, W.J.. de la Llera. hf. and Kefalides. N.A.. Triglyceride accumulation in cultured fibroblasts - The effects of hypertrighxeridemic serum, Atherosclerosis, 23 (1976) 521-534. 28 Howard, B.V.. Uptake of very low density Iipoprotein triglyceride by bovine aortic endothelial ceBs in culture. J. Lipid Res.. 18 (1977) 561-571. 29 Rosa, H.G. and Juiiano, J.. ReguIation of plasma lecithin : cholesterol acyl transferam in man. Part 1 (Incraaaad activity in primary hypertrigIyceridemia). J. Lab. CIin. Med., 88 (1976) 29-43 30 Bagdade, J.D.. Bierman, E.L. and Porte, D.. Influence of obesity on the relationship between insulin and trigIyceride levels in endogenous hypertriglyceridemia. Diabetes, 20 (1971) 664-672. 31 Hatch, F.T. and Lees, R.S.. Practical method for pIaama lipoprotein analysis. Advanc. Lipid. Res., 6 (1968) l-68. 32 Olefsky, J.M.. Farquhar, J.W. and Reaven. G.M.. Reappraisal of the r6le of insulin in hypertriglyceridemia. Amer. J. Med., 57 (1974) 551-560. 33 Bagdade. J.D.. Porte, D. and Bierman, E.L.. Diabetic iipemia - A form of acquired fat-induced iipemia. New EngI. J. Med., 276 (1967) 427433. 34 BaUantyne. D.. White, C.. Strevens, E.A.. Lawrie, T.D.V.. Lorimer, A.R., Mandemon. W.G. and Morgan, H.G.. Lipoprotein concentration in untreated adult onset diabetes meIIitus and the relationship of tbe fasting pIasma triglyceride concentration and insuhn secretion, CIin. Chim. Acta. 78 (1977) 323-329. 35 Wood, P.D.S.. Stem, M.P.. Silvers. A., Reaven, G.M. and vonder Groeben. Prevalence of plasma iipoprotein abnormalities in a free-living population of the Central Valley. California, J. Circ.. 45 (1972) 114-126. 36 Cadson, L.A. and Ericsson, hf.. Quantitative and qualitative serum lipoprotein anaIysas, Part 1 (Studies in healthy men and women), Atherosclerosis, 21 (1975) 417433. 37 Kamenetsky. S.A.. Bennett, P.H.. Dippe. S.E., MilIer. M. and LeCompte, P., A cIinicaI and histologic study of diabetic nephropathy in the Pima Indians, Diabetes. 23 (1974) 61-68. 38 Dorf. A., BaBantine, E.J., Bennett, P.H. and Miller. M.. Retinopathy in Pima Indians, Diabetes, 25 (1976) 554-560. 39 Ingelfinger, J.A.. Bennett. P.H.. Liebow. I.M. and Miller, M., Coronary heart disease in the Pima Indians, Diabetes, 25 (1976) 561-565. 40 Sievers. ML.. Myocardial infarction among Southwestern American Indians, Ann. Intern. Med.. 67 (1967) 800-807. 41 Berg, K., Borresen. A.L. and DahIen, G.. Serum high density lipoprotein and atherosclerotic heart disease, Lance& 1 (1976) 499601. 42 Miller. N.E., Forde. O.H.. Thelle. D.A. and Mjos, O.D.. The Troms5 heart study. High density Iipoprotein and coronary heart dimasa -A prospective case control study, Lancet. 1 (1977) 965-967. 43 CasteI% W.P.. Doyle, J.T.. Gordon, T.. Haines. C.G.. HiortIand, M.C.. HuIIey, S.B.. Kagen. A. and Zukel. W.J.. HDL cholesterol and other lipids in coronary heart disease, CircuIation. 55 (1977) 767-772. 44 Gordon, T., Caste& W.P., HiortIand. M.C.. Kannel. W.B. and Dawbar. T.R.. Diabetes. blood lipids and the role of obedty in coronary heart dieease risk women, Ann. Int. Med., 87 (1977) 393-397. 45 Savage, P.J.. Bennett, P.H. and MiIIer. M.. FamiIiaI relationships of obesity and hypergIycemta in the Pima Indians, Amer. J. Epidemiol.. 106 (1977) 274.
162 46 Savage, P.J.. Hamman, R.F.,
Bartha, G.. Dippe. S.E., Miller, M. and Bennett, P.H., Serum cholesterol levels in American (Pima) Indian children and adolescents, Pediatrics, 68 (19’76) 274-282. 47 Gamick. M.B., Bennett, P.H. and Lange& T., Low density lipoprotein metabolism and lipoprotein cholesterol composition in American Indians, J. Lipid Res., In press.