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Bile acid kinetics, steroid balance and biliary lipids in hyperlipoproteinaemia type III
191
39 (1981) 191+?01 @ Elsevier/North-Holland Scientific Publishers, Ltd.
AtheFOSCleFO6iS,
BILE ACID IUNETICS, STEROID BALANCE HYPERLIPOPROTEINAEMIA TYPE III
EVA ANDERSkN,
KNELL HELLSTRbM
and BENGT
AND BILIARY
LIPIDS
IN
VESSBY
Department of Medicine, St. Erik’s Hospital, S-l 1282 Stockholm Giatrics, University of Uppsala, S- 75012 Uppsala (Sweden)
and Department
of
(Received 24 June, 1980) (Revised, received 20 October, 1980) (Accepted 6 November, 1980)
Summary Bile acid kinetics, steroid balance and biliary lipid composition were studied in 8 patients with hyperlipoproteinaemia (HLP) type III (broad-beta-disease). As a general finding the formation of bile acids exceeded the range encountered in normolipidaemic subjects, but the mean net steroid balance remained normal. On the basis of previous findings of a close correlation between the synthesis of VLDL-triglycerides and bile acids in other types of HLP it is suggested that HLP type III may be associated with an enhanced VLDL production. As a further indication of a disturbed cholesterol metabolism the bile was supersaturated with cholesterol in all subjects. Key words:
Bile acid kinetics - Biliary lipids - Hyperlipoproteinaemia balance
type III -Steroid
Introduction The metabolism of plasma lipoproteins is interrelated and in some way linked to that of cholesterol and bile acids. Having delivered triglycerides (Tg) to extra-hepatic tissues, very low density lipoproteins (VLDL) may be converted to low density lipoproteins (LDL) [1,2]. LDL, possibly responsible for part of the retransport of cholesterol to the liver [3], may ultimately be taken up and degraded by mesenchymal tissues such as skin fibroblasts, arterial The study was supported by grants from the Swedish Medical Research Council and Svenska livfdrsiikringsbolagets niimnd fdr medicinsk forskning.
192
smooth muscle cells and lymphocytes [4]. The turnover of plasma cholesteryl esters and the formation of bile acids both correlate to VLDL-Tg production [ 5,6], indicating that an enhanced synthesis of VLDL requires an increased formation of cholesterol, subsequently transformed into bile acids. Accumulation of VLDL in plasma may be explained by VLDL overproduction, a defective clearance of VLDL-Tg or both [ 71. As a consequence, the rate of bile acid synthesis may vary within a wide range in hyperlipoproteinaemia (HLP) type IV [ 8,9]. However, in consecutive series of patients with this disorder, not only the biosynthesis of bile acids but also the net cholesterol balance exceed the upper normal range in more than 50% of the patients [9,10]. Quantitatively normal values for both parameters are a consistent finding in HLP types IIa and IIb [ 9,101. HLP type III is a puzzling metabolic defect, characterised by an accumulation of lipoprotein particles possibly representing intermediates in the catabolism of VLDL to LDL [ 8,111. Considering the possibility that HLP type III may be associated with VLDL overproduction, it was considered of interest to study bile acid kinetics in this disorder in view of the close relationship between the formation of VLDL-Tg and bile acids in other types of HLP. Material and Methods Patients
Altogether 3 female and 5 male patients, 42-71 years old, admitted to the Department of Geriatrics in Uppsala because of HLP, participated in the investigation. Clinical diagnoses and general data are listed in Table 1. There was no evidence of disease in intestines, kidney, thyroid or liver. None of the patients was addicted to alcohol or narcotics. Three were overweight, defined as a relative body weight > 120%. They were all informed of the nature and purpose of and risks involved in the study before giving their voluntary consent to be investigated. TABLE 1 GENERAL
DATA
ON THE SUBJECTS
Subject NO.
Age (~0 and sex
BW
RBW
(kg)
PJv
1. 2. 3. 4. 6. 6. I. 8.
57 64 52 42 46 71 54 46
54 60 97 86 99 81 71 103
83 97 139 116 121 116 86 120
IE ALH GL so VH AE AS LE
F F F M M M M M
Comments
GBD HT GBD TIA CHD CHD
Abbreviations: F = female, M = male. CHD = coronary heart disease, GBD = %aUbladder disease (cholecystectomy. cholelithiasis. oholecystitis). HT = hypertension, TIA = transient ischemic attacks, BW = body weight, RBW = relative body weight. a RBW = weight (kg) height (cm) -
100
x 100.
193
Experimental protocol diet The patients were hospitalised during the study and fed a standardised in which 45, 43 and 11% .of the energy (2533 kcal) was supplied as fat, carbohydrate and protein, respectively. The average intake of cholesterol was 1.21 mmol/day [12]. Prior to the experiments the subjects were interviewed about their dietary habits by a dietitian and their energy requirements were estimated. Faeces collected for about 10 days were pooled and analysed for neutral steroids. Intravenous glucose [ 131 and fat [ 141 tolerance tests were performed in some of the patients. Lipoprotein analysis Venous blood samples were allowed to clot at room temperature. EDTA was added as a 5% solution and the serum lipoproteins were subsequently separated in an L2 65 B Beckman ultracentrifuge [15]. Triglycerides and cholesterol in serum and in lipoprotein fractions were determined in a Technicon Auto Analyzer type II. Immediately after centrifugation, the top and bottom fractions at d = 1.006, as well as whole serum, were subjected to agarose electrophoresis according to the method of Noble [ 161. A combination of three criteria was required for the diagnosis of HLP type III [ 151: (1) Presence of a lipoprotein fraction with density < 1.006 but with electrophoretic mobility similar to or identical with beta-lipoproteins on agarose gel in the system used; (2) An increased ratio of cholesterol/triglyceride in VLDL (2 0.75 mmol/l/ mmol/l); (3) An increased III-index, i.e. the ratio cholesterol/triglycerides in VLDL X 10 divided by cholesterol/triglyceride in LDL (> 1.30). Bile acid kinetics and faecal steroids [” C] Cholic acid (4 PCi) and [” C] chenodeoxycholic acid (4 PCi) as sodium salts dissolved in water were given orally as a mixture in the morning before breakfast. Four samples of duodenal bile were obtained from each subject at 2-4day intervals. Cholecystokinin was administered intravenously and 5-10 ml samples of concentrated bile were collected into ice-chilled tubes. One aliquot of each sample was hydrolysed with 1 M KOH in closed steel tubes for 12 h at 110°C. The deconjugated bile acids were extracted with ethyl ether after acidification and their methyl esters were separated by thin layer chromatography. The amount of bile acids in this extract was quantitated with gas-liquid chromatography (GLC) on a 3% SE-30 column after preparation of the trimethyl silyl ether derivatives. Another aliquot of this extract was analysed for radioactivity by liquid scintihation. On the basis of the specific radioactivity curve, the pool size, half-life and synthesis were determined for cholic acid (C) and chenodeoxycholic acid (CD) [ 171.. The correlation coefficients averaged 0.990 f 0.005 for C and 0.993 rf: 0.003 for CD. Further details of this method have been given previously [9] . The neutral steroids in faeces were analysed with GLC [lo]. Net steroid balance was defined as bile acid formation plus faecal excretion of neutral steroids of cholesterol origin minus cholesterol intake. Biliary lipids Part of the bile samples was extracted
without
delay with 20 vol of chloro-
194
form-methanol (2:1, v/v). The choloroform phase was analysed for cholesterol [ 181 and phospholipids [ 191. The total bile acid concentration in another aliquot of the bile samples was determined enzymatic&y using the 3a-hydroxysteroid dehydrogenase method [ 201. The relative concentrations of cholesterol, phospholipids and bile acids were expressed as molar percentages of total biliary lipids. The cholesterol saturation of bile was expressed in terms of lithogenic indices [ 211 by using the solubility limits of cholesterol given by Hegardt and Dam [22] and Holzbach et al. [ 23[. In the non-cholecystectomised patients, the cholesterol saturation was also calculated according to Carey and Small [ 24 ] using the solubility line for a biliary lipid concentration of 10 g/d1 [25]. Results Blood analyses
The concentrations of cholesterol and Tg in serum and various lipoprotein fractions are listed in Table 2. Specimens from 7 of the 8 patients showed a typical type III lipoprotein pattern, with a broad-p-band upon electrophoresis, increased levels of cholesterol and Tg in the VLDL fraction and an augmented type III index. One patient (No. 8) showed a typical electrophoretic pattern, but the cholesterol/triglyceride ratio in VLDL was below the value considered diagnostic for type III. However, repeated analyses on earlier occasions had shown an increased cholesterol/triglyceride ratio in VLDL and in combination with a clinical history of tuberous xanthomatas and a positive family history for HLP type III, this diagnosis was considered valid. Blood glucose was within normal limits in all instances, as was the glucose tolerance test in the 4 patients studied. The KZ value obtained during the fat tolerance test (n = 5) was below normal or in the low borderline range. Bile acid kinetics
The pool size, synthesis and fractional turnover rate (FTR) of C averaged 1.89 + 0.26 mmol, 1.01 f 0.10 mmol*day-’ and 0.57 + 0.06 day-‘, respectively (Table 3). The mean value recorded for the C pool size in the males exceeded that of the females by a factor of 1.4. Similarly the formation of C in the men was about 1.3 times higher than in the women. The sizes of the C and CD pools were almost. identical but the rate of CD production was in all instances lower than that of C. The synthesis of CD and C + CD in the males was also augmented compared to the findings in the females. The rates of synthesis of the bile acids in the present subjects and in patients with HLP type IIa and IV examined with the same technique [6] are shown in Fig. 1. In all types of HLP, the bile acid formation ranged within fairly wide limits. However, in both females and males the combined production of C and CD in HLP type III was about twice as high as in type IIa. No significant differences were observed between HLP types III and IV. The patients with HLP type IIa were in general less heavy than those with the type III and IV patterns. However, the difference in bile acid biosynthesis was not related to differences in body weight. Thus, in the females, the combined formation of C and CD expressed as Mrnolakg-’ -day-’ averaged 9.8 * 1.2
5.21 1.76
3.20 0.85
6.42 2.73
Female Mean iSEM
Male Mean *SEM
10.57 2.97
8.45 1.00
9.78 1.85
6.76 8.38 10.21 7.00 22.36 7.88 6.78 8.83
5.19 2.39
2.25 0.66
4.09 1.54
1.04 2.37 3.33 2.97 14.38 2.10 1.31 5.18
5.48 3.15
2.28 0.42
4.28 1.98
1.46 2.56 2.81 2.65 18.05 2.41 1.66 2.61
chol t&l (mmoI/lI
chol t5 (mmoI/l)
1.70 3.23 4.66 3.68 17.00 2.84 2.27 6.32
VLDL
SeNm
Total Mean +SEM
1. 2. 3. 4. 5. 6. I. 8.
Subject NO.
0.61 0.06
0.68 0.22
0.64 0.08
0.44 0.48 1.13 0.52 0.73 0.42 0.72 0.65
tg wmoI/D
LDL
LIPOPROTEINLIPIDS,K-VALUEOFIVFTTANDIVGTTANDBLOODGLUCOSE
TABLE2
3.10 0.35
4.69 0.84
3.69 0.45
3.71 4.00 6.35 2.76 2.27 3.97 3.92 2.56
chol
0.31 0.04
0.31 0.02
0.31 0.02
0.21 0.35 0.31 0.26 0.34 0.30 0.20 0.44
(mmolfl)
tg
HDL
1.01 0.13
1.39 0.18
1.15 0.12
1.60 1.54 1.04 1.22 0.69 1.28 0.74 1.12
chol
2.84 0.49
1.70 1.71 4.01
3.75 3.01
%lmin
K*
IVFTT
1.44 0.18
1.02 1.49
1.37 1.86
IVGTT K
4.4 0.12
4.8 4.4
4.6 3.9
4.4 4.3
(mmolll)
Totalblood glucose
f SEM
Mean
2.14 0.36
1.48 0.28
Female Mean + SEM
Male
1.89 0.26
Total Mean +SEM
1.10 0.14
0.86 0.10
1.01 0.10
0.56 0.10
0.60 0.06
0.57 0.06
2.19 0.42
1.05 0.20
1.77 0.33
0.78 1.43 2.14 3.18 1.78 0.90 2.97
0.64 0.11
0.45 0.09
0.57 0.08
0.36 0.35 0.64 0.98 0.72 0.47 0.36 0.66
0.71 0.56 0.53 0.91 0.41 0.56 0.55 0.36
0.65 0.99 0.94 1.57 1.12 1.01 0.68 1.14
0.96
0;91
1.76 1.76 1.73 2.75 1.81 1.23 3.18
0.31 0.05
0.43 0.02
0.36 0.04
0.38 0.45 0.45 0.46 0.23 0.26 0.40 0.22
FTR (day-')
Synthesis (mmol/day)
Pool size WmoI)
F+R (day-')
Pool size (mmoI)
Synthesis (mmol/day)
ChenodeoxychoIicacid(CD)
Cholicacid (C)
1. 2. 3. 4. 5. 6. I. 8.
Subject No.
BILEACIDKINETICS.INDIVIDUALDATA
TABLE3
4.33 0.76
2.53 0.39
3.86 0.57
1.86 2.54 3.19 3.87 5.93 3.59 2.13 6.15
Pool size (mmol)
C+CD
1.74 0.25
1.31 0.17
1.58 0.18
1.01 1.34 1.58 2.55 1.84 1.48 1.04 1.80
Synthesis (mmol/day)
1.03 0.10
1.48 0.40
1.20 0.17
0.96 2.26 1.23 0.81 0.86 1.02 1.37 1.07
Pool size
C/CD
1.79 0.11
2.04 0.41
1.88 0.16
1.81 2.83 1.47 1.60 1.56 2.15 1.89 1.73
Synthesis
P
197 mmol/day 20.
18. 16. 1.4 12. 10. 0.8
F
M
F
Ila
M
F
III
M IV
Fig. 1. The formation of cholic acid (shaded areas) and chenodeoxycholic acid (white areas) in HLP types Ha [S] , III (current study) and IV 161. F = female, M = male. *Significantly different from HLP type IIa, P < 0.05 or P < 0.02. **Significantly different from HLP type IIa. P < 0.01. *** Significantly different from HLP type Ha. P < 0.001.
TABLE 4 NET STEROID BALANCE IN HLP TYPE III Subject No.
Neutral steroid excretion (mmol/day)
Bile acid synthesis (mmol/day)
Cholesterol intake (mmoI/day)
Net steroid balance (mmol/day)
1. 2. 3. 4. 5. 6. 7. 8.
0.87 0.33 2.45 0.31 1.17 1.75 2.52 2.41
1.01 1.34 1.58 2.55 1.84 1.48 1.04 i.80
0.18 0.81 1.36 1.46 1.55 1.36 0.18 1.55
-
Mean + SEM
1.57 0.33
1.58 0.18
1.21 0.12
- 1.93 0.21
1.10 0.00 2.67 1.46 2.06 1.87 2.78 2.12
in type IIa, 19.1 f 1.7 in-Type III and 21.9 + 8.0 in type IV. The corresponding values (Irmol-kg-’ -day-’ ) in the males were 11.8 f 1.3 (type IIa), 19.8 + 2.6 (type III) and 18.8 + 1.8 (type IV) [6]. Net steroid balance The faecal excretion of neutral steroids of cholesterol origin ranged from 0.3 to 2.5 mmol-day-’ (Table 4). The corresponding values for net steroid balance
198
averaged 1.9 (range 0.8-2.8) mmol. day-’ , i.e. within the range encountered for normolipidaemic subjects (1.8 f 0.22 mmol/day) [lo] . Biliary lipid composition The contributions of cholesterol, bile acids and phospholipids to duodenal bile averaged 12.8, 64.3 and 22.9 molar per cent, respectively. Compared to findings in normolipidaemic controls [ 261, the content of cholesterol was augmented, with a correspondingly reduced content of bile acids. As evidenced by the percentage saturation data [24,25] and lithogenic indices [21-231, all subjects had bile supersaturated with cholesterol in the post-absorptive state (Table 5). Discussion The mechanisms responsible for the development of HLP type III remain controversial. It has been suggested that the metabolic disorder may be linked to the absence of an isoelectric form of apolipoprotein E (apolipoprotein E III) [27] but the importance of such a defect is still unclear. A structural apolipoprotein abnormality does not seem to be a prerequisite for development of the type III pattern, as cholesterol-rich VLDL (beta-VLDL) may also be present in other types of hypertriglyceridaemia [28,29]. HLP type III is reported to be due to overproduction of apolipoprotein-B and its attendant lipids [30] and /or to a defective catabolism of VLDL, most likely at a point between IDL (intermediary density lipoproteins) and LDL [31-331. In the former case the normal capacity for processing VLDL may be saturated and part of the VLDL particles directed to an abnormal pathway with a long halflife, a phenomenon that possibly explains the accumulation of beta-VLDL. The synthesis of bile acids in the present patients with HLP type III was in most cases above the range previously encountered in patients with HLP type II [6,10]. An earlier investigation with a slightly different technique [9] revealed that the combined formation of C and CD in HLP type II was essentially the same as in healthy normolipidaemic controls. Thus, although the diet in this and previous studies [6,9,10] differed slightly, it appears that supranormal amounts of cholesterol were eliminated as bile acids in the present patients with HLP type III. In some patients, this abnormality was associated with a decreased elimination of cholesterol as neutral products, as the mean net steroid balance was within the normal range [lo] . The molar cholesterol level in the bile of the current patients was above the limit commonly found in healthy controls. In all patients the bile samples were oversaturated with cholesterol. This finding opens the possibility that HLP type III, like HLP type IV [34], is associated in the long run with an enhanced risk of developing cholesterol gallstone disease. Further evidence supporting this hypothesis was recently presented in a study where supranormal cholesterol saturation .was demonstrated in gallstone-free patients with the type IV lipoprotein pattern
1261. In summary, HLP types IIa and IV are different entities of disease affecting bile acid and cholesterol metabolism. The augmented formation of VLDL-Tg, cholesterol and bile acids often observed in HLP type IV suggests that a
b
a
6.8 f 0.4
12.8 t 1.44
59.4 18.0 65.4 IQ.3 60.6 61.6 59.8 59.5
(molar %)
Bile acids
SATURATION
71.0 f 1.0
64.3 1- 2.36
CHOLESTEROL
22.3 f 0.9
22.9 _+1.92
27.1 12.8 22.4 20.6 26.6 27.3 18.2 28.1
Phospholipids
OF BILE IN HLP TYPE III
1.85 f 0.22
1.81 1.78 1.73 1.43 1.63 1.41 3.37 1.64
Lithogenic indexa (H-D-H)
According to the limits of cholesterol solubility as defined by Hegardt and Dam [22] and Holzbach et al. [23]. Calculated according to Carey and Small [24,25] using a biliary lipid concentration of 10 g/dI.
Healthy controls (26) Mean f SEM
Mean ? SEM
13.5 9.2 12.2 9.1 12.8 11.1 22.1 12.4
AND
1. 2. 3. 4. 5. 6. 7. 8.
LIPID COMPOSITION
Cholesterol
5
Subject No.
BILIARY
TABLE
ChoIesteroI saturationb
185 _+27
160 137 317 157
163 -
175 -.
(%)
stimulation of VLDL-Tg synthesis is associated with a rise in hepatic cholesterol production, which in turn results in an augmented elimination of cholesterol transformed into bile acids. The elevated formation of such compounds in the present patients suggests that overproduction of VLDL may also be a common phenomenon in HLP type III. Acknowledgements The skilful assistance of Marie Holmberg and Eve-Marie Bergkvist is gratefully acknowledged. The ethical aspects of the investigation were approved by the Ethics Committees of the Karolinska Institutet, Stockholm. References 1 Felts, J.M. and Rudel. L.L.. Mechanisms of hyperlipidemia. In: D. Kritchevsky (Ed.), Hypolipidemic Agents, Springer-Verlag. Berlin. Heidelberg, New York, 1975, pp. 151-189. 2 Eisenberg. S. and Levy, RI., Lipoproteins and lipoprotein metabolism. In: D. Kritchevksy (Ed.), Hypolipidemic Agents, Springer-Verlag, Berlin, Heidelberg, New York, 1975. pp. 191-213. 3 Sniderman. A., Thomas, D., Marpole, D. and Teng. B., Low density lipoprotein - A metabolic pathway for return of cholesterol to the splanchnic bed, J. Chn. Invest., 61 (1978) 867-873. 4 Brown, MS. and .GoIdstein. J.L., Familial hypercholesterolemia - A genetic defect in the low density lipoprotein receptor, N. Engl J. Med., 294 (1976) 1386-1390. 5 Kudchodkar. B.J. and Sodhi. H.S.. Turnover of pksma cholesteryl esters and its relationship to other parameters of lipid metabolism in man. Europ. J. Cbn. Invest., 6 (1976) 285-298. 6 Angelin, B., Einarsson, K., HeRstrom, K. and Leijd, B.. Bile acid kinetics in relation to endogenous triglyceride formation in various types of hyperhpoproteinemia. J. Lipid Res.. 19 (1978) 1004-1016. 7 Sigurdsson. G.. NicoB. A. and Lewis, B.. Metabolism of very low density lipoproteins in hyperlipidaemia - Studies of apolipoprotein B kinetics in man, Europ. J. CIin. Invest., 6 (1976) 167-177. 8 Beaumont, J.-L.. Carlson, L.A.. Cooper, G.R.. Fejfar, 2.. Fredrickson, D.S. and Strasser. T.. Classification of hyperhpidaemias and hyperlipoproteinaemias. BuB. WHO, 43 (1970) 891-915. 9 Einarsson, K.. HeIIstrdm, K. and Kalhaer. M.. Bile acid kinetics in relation to sex. serum lipids, body weights and gailbladder disease in patients with various types of hyperhpoproteinaemia, J. Clm. Invest., 54 (1974) 1301-1311. 10 Angelin. B.. Einarsson, K.. Hehstrom, K. and KaBner, M., Elimination of cholesterol in hyperlipoproteinaemia, Clin. Sci. Mol. Med., 61 (1976) 393-397. 11 Fredrickson, D.S.. Levy, R.I. and Lees, R.S., Fat transport in lipoproteins - An integrated approach to mechanisms and disorders, N. EngI. J. Med., 276 (1967) 3444, 94-103. 148-166, 215-225. 273-281. 12 Gustafsson. L-B.. Determination of the sterol composition of diet used in dietary management of hyperlipoproteinaemiaa, UppsaIa J. Med. Sci., 85 (1980) 35. 13 Ikkos. D. and Luft, R.. On the intravenous glucose tolerance test, Acta &rdocrinol. (Copenhagen), 26 (1967) 312-334: 14 Carlson. L.A. and Rossner, S.A., A methodological study of an intravenous fat tolerance test with IntraIipid@ emulsion, Stand. J. Lab. Invest., 29 (1972) 271-280. 15 Vessby, B., Studies on the serum lipoprotein composition in S&year-old men - A su@estion of chemical criteria for diagnosis of hyperhpoproteinemia type III (broad-@-disease), Clin. Chim. Acta. 69 (1976) 2942. 16 Noble, R.P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 963-700. 17 Lmdstedt, S., Turnover of chohc acid in man, Acta Physiol. Stand., 40 (1957) l-9. 18 Hanel. H.K. and Dam. H.. Determination of smaB amounts of total cholesterol by the Tschugaeff reaction with a note on the determination of lathosterol. Acta Chem. Stand.. 9 (1956) 677-682. 19 Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem.. 234 (1959) 466468. 20 Fausa, 0. and S&egg. B.A., Quantitative determination of bile acids and their conjugates using thinlayer chromatography and a purified 3o-hydroxysteroid dehydrogenase, Stand. J. Gastroenterol., 9 (1974) 249-264. 21 Thomas, P.J. and Hofmann, A.F., A simple cabxdation of lithogenic Index of bile: expressing bihary lipid composition on rectangular coordinates, Gastroenterology, 66 (1973) 698-700.
201 22
Hegardt,
23
Z. Emalnungswiss., 10 (1971) 223-233. Holzbach. R.T.. Marsh, M.. Olzewski, M. and Holan,
F.G. and Dam. H., The solubillty
supersaturated 24 25 26 27
28
29 30 31
32 33 34
bile is frequent
in healthy
of cholesterol
in aqueous
K., Cholesterol
man, J. Clln. Invest.,
solutions solubility
52 (1973)
of bile salts and lecithin, in bile -
Evidence
that
1467-1479.
Carey, M.C. and Small, D.M., The physical chemistry of cholesterol solubility ln bile - Relationship to gallstone formation and dissolution in man. J. Clin. Invest., 61 (1978) 998-1026. Carey, M.C., Critical tables for calculating the cholesterol saturation of native bile. J. Lipid Res.. 5 (1978) 945-955. Ahlberg. J.. Angelin, B., Einarsson, K.. Hellstrom. K. and Leijd, B., Bilisry lipid composition in nonno- and hyperllpoproteinaemia, Gastroenterology. 79 (1980) 90-94. Utermann, G., Jaeschke. M. and Menzel, J.. Familial hyperlipoproteinaemia type III - Deficiency of a specific apolipoprotein (ape E-III) in the very low density lipoproteins, FEBS Lett., 56 (1975) 352355. Hazzard, W.R. and Bierman. E.L.. The spectrum of electrophoretic mobility of very low density lipoproteins - Role of slower-migrating species in endogenous llpemia and broad-o-disease, J. Lab. Clin. Med., 86 (1975) 239-252. Fredrickson. D.S.. Morganroth, J. and Levy. R.J., Type III hyperlipoproteinemia - An analysis of two contemporary definitions, Ann. Intern. Med., 82 (1975) 150-157. Hall, M.H. III. Bllhelmer. D.W., Phalr, R.D.. Levy, R.J. and Berman, M.. A mathematical model for apoprotein kinetics in normal and hyperlipemlc patients, Circulation. 49-50 (1974) (Suppl. III): 114. Hazsard. W.R. and Bierman, E.L.. Delayed clearance of chylomicron remnants following vitamin-Acontaining oral fat loads in broad-o-disease (type III hyperlipoproteinemia), Metabolism, 25 (1976) 277-280. Bilheimer, D.W., Eisenberg. S. and Levy, R.J., Abnormal metabolism of very low density lipoproteins in type III hyperlipoproteinemla, Circulation, 44 (1971) (Suppl. II): 56. Quarfordt, S.H., Levy. RI. and Fredrickson, D.S., The kinetic properties of very low density lipoprotein triglyceride in type III hyperllpoproteinemia. Biochem. Biophys. Acta. 296 (1973) 572-576. Einarsson. K., Hellstrom, K. and Kallner, M.. Gallbladder disease in hyperlipoproteinaemia, Lancet, i (1975) 484-487.
39 (1981) 191+?01 @ Elsevier/North-Holland Scientific Publishers, Ltd.
AtheFOSCleFO6iS,
BILE ACID IUNETICS, STEROID BALANCE HYPERLIPOPROTEINAEMIA TYPE III
EVA ANDERSkN,
KNELL HELLSTRbM
and BENGT
AND BILIARY
LIPIDS
IN
VESSBY
Department of Medicine, St. Erik’s Hospital, S-l 1282 Stockholm Giatrics, University of Uppsala, S- 75012 Uppsala (Sweden)
and Department
of
(Received 24 June, 1980) (Revised, received 20 October, 1980) (Accepted 6 November, 1980)
Summary Bile acid kinetics, steroid balance and biliary lipid composition were studied in 8 patients with hyperlipoproteinaemia (HLP) type III (broad-beta-disease). As a general finding the formation of bile acids exceeded the range encountered in normolipidaemic subjects, but the mean net steroid balance remained normal. On the basis of previous findings of a close correlation between the synthesis of VLDL-triglycerides and bile acids in other types of HLP it is suggested that HLP type III may be associated with an enhanced VLDL production. As a further indication of a disturbed cholesterol metabolism the bile was supersaturated with cholesterol in all subjects. Key words:
Bile acid kinetics - Biliary lipids - Hyperlipoproteinaemia balance
type III -Steroid
Introduction The metabolism of plasma lipoproteins is interrelated and in some way linked to that of cholesterol and bile acids. Having delivered triglycerides (Tg) to extra-hepatic tissues, very low density lipoproteins (VLDL) may be converted to low density lipoproteins (LDL) [1,2]. LDL, possibly responsible for part of the retransport of cholesterol to the liver [3], may ultimately be taken up and degraded by mesenchymal tissues such as skin fibroblasts, arterial The study was supported by grants from the Swedish Medical Research Council and Svenska livfdrsiikringsbolagets niimnd fdr medicinsk forskning.
192
smooth muscle cells and lymphocytes [4]. The turnover of plasma cholesteryl esters and the formation of bile acids both correlate to VLDL-Tg production [ 5,6], indicating that an enhanced synthesis of VLDL requires an increased formation of cholesterol, subsequently transformed into bile acids. Accumulation of VLDL in plasma may be explained by VLDL overproduction, a defective clearance of VLDL-Tg or both [ 71. As a consequence, the rate of bile acid synthesis may vary within a wide range in hyperlipoproteinaemia (HLP) type IV [ 8,9]. However, in consecutive series of patients with this disorder, not only the biosynthesis of bile acids but also the net cholesterol balance exceed the upper normal range in more than 50% of the patients [9,10]. Quantitatively normal values for both parameters are a consistent finding in HLP types IIa and IIb [ 9,101. HLP type III is a puzzling metabolic defect, characterised by an accumulation of lipoprotein particles possibly representing intermediates in the catabolism of VLDL to LDL [ 8,111. Considering the possibility that HLP type III may be associated with VLDL overproduction, it was considered of interest to study bile acid kinetics in this disorder in view of the close relationship between the formation of VLDL-Tg and bile acids in other types of HLP. Material and Methods Patients
Altogether 3 female and 5 male patients, 42-71 years old, admitted to the Department of Geriatrics in Uppsala because of HLP, participated in the investigation. Clinical diagnoses and general data are listed in Table 1. There was no evidence of disease in intestines, kidney, thyroid or liver. None of the patients was addicted to alcohol or narcotics. Three were overweight, defined as a relative body weight > 120%. They were all informed of the nature and purpose of and risks involved in the study before giving their voluntary consent to be investigated. TABLE 1 GENERAL
DATA
ON THE SUBJECTS
Subject NO.
Age (~0 and sex
BW
RBW
(kg)
PJv
1. 2. 3. 4. 6. 6. I. 8.
57 64 52 42 46 71 54 46
54 60 97 86 99 81 71 103
83 97 139 116 121 116 86 120
IE ALH GL so VH AE AS LE
F F F M M M M M
Comments
GBD HT GBD TIA CHD CHD
Abbreviations: F = female, M = male. CHD = coronary heart disease, GBD = %aUbladder disease (cholecystectomy. cholelithiasis. oholecystitis). HT = hypertension, TIA = transient ischemic attacks, BW = body weight, RBW = relative body weight. a RBW = weight (kg) height (cm) -
100
x 100.
193
Experimental protocol diet The patients were hospitalised during the study and fed a standardised in which 45, 43 and 11% .of the energy (2533 kcal) was supplied as fat, carbohydrate and protein, respectively. The average intake of cholesterol was 1.21 mmol/day [12]. Prior to the experiments the subjects were interviewed about their dietary habits by a dietitian and their energy requirements were estimated. Faeces collected for about 10 days were pooled and analysed for neutral steroids. Intravenous glucose [ 131 and fat [ 141 tolerance tests were performed in some of the patients. Lipoprotein analysis Venous blood samples were allowed to clot at room temperature. EDTA was added as a 5% solution and the serum lipoproteins were subsequently separated in an L2 65 B Beckman ultracentrifuge [15]. Triglycerides and cholesterol in serum and in lipoprotein fractions were determined in a Technicon Auto Analyzer type II. Immediately after centrifugation, the top and bottom fractions at d = 1.006, as well as whole serum, were subjected to agarose electrophoresis according to the method of Noble [ 161. A combination of three criteria was required for the diagnosis of HLP type III [ 151: (1) Presence of a lipoprotein fraction with density < 1.006 but with electrophoretic mobility similar to or identical with beta-lipoproteins on agarose gel in the system used; (2) An increased ratio of cholesterol/triglyceride in VLDL (2 0.75 mmol/l/ mmol/l); (3) An increased III-index, i.e. the ratio cholesterol/triglycerides in VLDL X 10 divided by cholesterol/triglyceride in LDL (> 1.30). Bile acid kinetics and faecal steroids [” C] Cholic acid (4 PCi) and [” C] chenodeoxycholic acid (4 PCi) as sodium salts dissolved in water were given orally as a mixture in the morning before breakfast. Four samples of duodenal bile were obtained from each subject at 2-4day intervals. Cholecystokinin was administered intravenously and 5-10 ml samples of concentrated bile were collected into ice-chilled tubes. One aliquot of each sample was hydrolysed with 1 M KOH in closed steel tubes for 12 h at 110°C. The deconjugated bile acids were extracted with ethyl ether after acidification and their methyl esters were separated by thin layer chromatography. The amount of bile acids in this extract was quantitated with gas-liquid chromatography (GLC) on a 3% SE-30 column after preparation of the trimethyl silyl ether derivatives. Another aliquot of this extract was analysed for radioactivity by liquid scintihation. On the basis of the specific radioactivity curve, the pool size, half-life and synthesis were determined for cholic acid (C) and chenodeoxycholic acid (CD) [ 171.. The correlation coefficients averaged 0.990 f 0.005 for C and 0.993 rf: 0.003 for CD. Further details of this method have been given previously [9] . The neutral steroids in faeces were analysed with GLC [lo]. Net steroid balance was defined as bile acid formation plus faecal excretion of neutral steroids of cholesterol origin minus cholesterol intake. Biliary lipids Part of the bile samples was extracted
without
delay with 20 vol of chloro-
194
form-methanol (2:1, v/v). The choloroform phase was analysed for cholesterol [ 181 and phospholipids [ 191. The total bile acid concentration in another aliquot of the bile samples was determined enzymatic&y using the 3a-hydroxysteroid dehydrogenase method [ 201. The relative concentrations of cholesterol, phospholipids and bile acids were expressed as molar percentages of total biliary lipids. The cholesterol saturation of bile was expressed in terms of lithogenic indices [ 211 by using the solubility limits of cholesterol given by Hegardt and Dam [22] and Holzbach et al. [ 23[. In the non-cholecystectomised patients, the cholesterol saturation was also calculated according to Carey and Small [ 24 ] using the solubility line for a biliary lipid concentration of 10 g/d1 [25]. Results Blood analyses
The concentrations of cholesterol and Tg in serum and various lipoprotein fractions are listed in Table 2. Specimens from 7 of the 8 patients showed a typical type III lipoprotein pattern, with a broad-p-band upon electrophoresis, increased levels of cholesterol and Tg in the VLDL fraction and an augmented type III index. One patient (No. 8) showed a typical electrophoretic pattern, but the cholesterol/triglyceride ratio in VLDL was below the value considered diagnostic for type III. However, repeated analyses on earlier occasions had shown an increased cholesterol/triglyceride ratio in VLDL and in combination with a clinical history of tuberous xanthomatas and a positive family history for HLP type III, this diagnosis was considered valid. Blood glucose was within normal limits in all instances, as was the glucose tolerance test in the 4 patients studied. The KZ value obtained during the fat tolerance test (n = 5) was below normal or in the low borderline range. Bile acid kinetics
The pool size, synthesis and fractional turnover rate (FTR) of C averaged 1.89 + 0.26 mmol, 1.01 f 0.10 mmol*day-’ and 0.57 + 0.06 day-‘, respectively (Table 3). The mean value recorded for the C pool size in the males exceeded that of the females by a factor of 1.4. Similarly the formation of C in the men was about 1.3 times higher than in the women. The sizes of the C and CD pools were almost. identical but the rate of CD production was in all instances lower than that of C. The synthesis of CD and C + CD in the males was also augmented compared to the findings in the females. The rates of synthesis of the bile acids in the present subjects and in patients with HLP type IIa and IV examined with the same technique [6] are shown in Fig. 1. In all types of HLP, the bile acid formation ranged within fairly wide limits. However, in both females and males the combined production of C and CD in HLP type III was about twice as high as in type IIa. No significant differences were observed between HLP types III and IV. The patients with HLP type IIa were in general less heavy than those with the type III and IV patterns. However, the difference in bile acid biosynthesis was not related to differences in body weight. Thus, in the females, the combined formation of C and CD expressed as Mrnolakg-’ -day-’ averaged 9.8 * 1.2
5.21 1.76
3.20 0.85
6.42 2.73
Female Mean iSEM
Male Mean *SEM
10.57 2.97
8.45 1.00
9.78 1.85
6.76 8.38 10.21 7.00 22.36 7.88 6.78 8.83
5.19 2.39
2.25 0.66
4.09 1.54
1.04 2.37 3.33 2.97 14.38 2.10 1.31 5.18
5.48 3.15
2.28 0.42
4.28 1.98
1.46 2.56 2.81 2.65 18.05 2.41 1.66 2.61
chol t&l (mmoI/lI
chol t5 (mmoI/l)
1.70 3.23 4.66 3.68 17.00 2.84 2.27 6.32
VLDL
SeNm
Total Mean +SEM
1. 2. 3. 4. 5. 6. I. 8.
Subject NO.
0.61 0.06
0.68 0.22
0.64 0.08
0.44 0.48 1.13 0.52 0.73 0.42 0.72 0.65
tg wmoI/D
LDL
LIPOPROTEINLIPIDS,K-VALUEOFIVFTTANDIVGTTANDBLOODGLUCOSE
TABLE2
3.10 0.35
4.69 0.84
3.69 0.45
3.71 4.00 6.35 2.76 2.27 3.97 3.92 2.56
chol
0.31 0.04
0.31 0.02
0.31 0.02
0.21 0.35 0.31 0.26 0.34 0.30 0.20 0.44
(mmolfl)
tg
HDL
1.01 0.13
1.39 0.18
1.15 0.12
1.60 1.54 1.04 1.22 0.69 1.28 0.74 1.12
chol
2.84 0.49
1.70 1.71 4.01
3.75 3.01
%lmin
K*
IVFTT
1.44 0.18
1.02 1.49
1.37 1.86
IVGTT K
4.4 0.12
4.8 4.4
4.6 3.9
4.4 4.3
(mmolll)
Totalblood glucose
f SEM
Mean
2.14 0.36
1.48 0.28
Female Mean + SEM
Male
1.89 0.26
Total Mean +SEM
1.10 0.14
0.86 0.10
1.01 0.10
0.56 0.10
0.60 0.06
0.57 0.06
2.19 0.42
1.05 0.20
1.77 0.33
0.78 1.43 2.14 3.18 1.78 0.90 2.97
0.64 0.11
0.45 0.09
0.57 0.08
0.36 0.35 0.64 0.98 0.72 0.47 0.36 0.66
0.71 0.56 0.53 0.91 0.41 0.56 0.55 0.36
0.65 0.99 0.94 1.57 1.12 1.01 0.68 1.14
0.96
0;91
1.76 1.76 1.73 2.75 1.81 1.23 3.18
0.31 0.05
0.43 0.02
0.36 0.04
0.38 0.45 0.45 0.46 0.23 0.26 0.40 0.22
FTR (day-')
Synthesis (mmol/day)
Pool size WmoI)
F+R (day-')
Pool size (mmoI)
Synthesis (mmol/day)
ChenodeoxychoIicacid(CD)
Cholicacid (C)
1. 2. 3. 4. 5. 6. I. 8.
Subject No.
BILEACIDKINETICS.INDIVIDUALDATA
TABLE3
4.33 0.76
2.53 0.39
3.86 0.57
1.86 2.54 3.19 3.87 5.93 3.59 2.13 6.15
Pool size (mmol)
C+CD
1.74 0.25
1.31 0.17
1.58 0.18
1.01 1.34 1.58 2.55 1.84 1.48 1.04 1.80
Synthesis (mmol/day)
1.03 0.10
1.48 0.40
1.20 0.17
0.96 2.26 1.23 0.81 0.86 1.02 1.37 1.07
Pool size
C/CD
1.79 0.11
2.04 0.41
1.88 0.16
1.81 2.83 1.47 1.60 1.56 2.15 1.89 1.73
Synthesis
P
197 mmol/day 20.
18. 16. 1.4 12. 10. 0.8
F
M
F
Ila
M
F
III
M IV
Fig. 1. The formation of cholic acid (shaded areas) and chenodeoxycholic acid (white areas) in HLP types Ha [S] , III (current study) and IV 161. F = female, M = male. *Significantly different from HLP type IIa, P < 0.05 or P < 0.02. **Significantly different from HLP type IIa. P < 0.01. *** Significantly different from HLP type Ha. P < 0.001.
TABLE 4 NET STEROID BALANCE IN HLP TYPE III Subject No.
Neutral steroid excretion (mmol/day)
Bile acid synthesis (mmol/day)
Cholesterol intake (mmoI/day)
Net steroid balance (mmol/day)
1. 2. 3. 4. 5. 6. 7. 8.
0.87 0.33 2.45 0.31 1.17 1.75 2.52 2.41
1.01 1.34 1.58 2.55 1.84 1.48 1.04 i.80
0.18 0.81 1.36 1.46 1.55 1.36 0.18 1.55
-
Mean + SEM
1.57 0.33
1.58 0.18
1.21 0.12
- 1.93 0.21
1.10 0.00 2.67 1.46 2.06 1.87 2.78 2.12
in type IIa, 19.1 f 1.7 in-Type III and 21.9 + 8.0 in type IV. The corresponding values (Irmol-kg-’ -day-’ ) in the males were 11.8 f 1.3 (type IIa), 19.8 + 2.6 (type III) and 18.8 + 1.8 (type IV) [6]. Net steroid balance The faecal excretion of neutral steroids of cholesterol origin ranged from 0.3 to 2.5 mmol-day-’ (Table 4). The corresponding values for net steroid balance
198
averaged 1.9 (range 0.8-2.8) mmol. day-’ , i.e. within the range encountered for normolipidaemic subjects (1.8 f 0.22 mmol/day) [lo] . Biliary lipid composition The contributions of cholesterol, bile acids and phospholipids to duodenal bile averaged 12.8, 64.3 and 22.9 molar per cent, respectively. Compared to findings in normolipidaemic controls [ 261, the content of cholesterol was augmented, with a correspondingly reduced content of bile acids. As evidenced by the percentage saturation data [24,25] and lithogenic indices [21-231, all subjects had bile supersaturated with cholesterol in the post-absorptive state (Table 5). Discussion The mechanisms responsible for the development of HLP type III remain controversial. It has been suggested that the metabolic disorder may be linked to the absence of an isoelectric form of apolipoprotein E (apolipoprotein E III) [27] but the importance of such a defect is still unclear. A structural apolipoprotein abnormality does not seem to be a prerequisite for development of the type III pattern, as cholesterol-rich VLDL (beta-VLDL) may also be present in other types of hypertriglyceridaemia [28,29]. HLP type III is reported to be due to overproduction of apolipoprotein-B and its attendant lipids [30] and /or to a defective catabolism of VLDL, most likely at a point between IDL (intermediary density lipoproteins) and LDL [31-331. In the former case the normal capacity for processing VLDL may be saturated and part of the VLDL particles directed to an abnormal pathway with a long halflife, a phenomenon that possibly explains the accumulation of beta-VLDL. The synthesis of bile acids in the present patients with HLP type III was in most cases above the range previously encountered in patients with HLP type II [6,10]. An earlier investigation with a slightly different technique [9] revealed that the combined formation of C and CD in HLP type II was essentially the same as in healthy normolipidaemic controls. Thus, although the diet in this and previous studies [6,9,10] differed slightly, it appears that supranormal amounts of cholesterol were eliminated as bile acids in the present patients with HLP type III. In some patients, this abnormality was associated with a decreased elimination of cholesterol as neutral products, as the mean net steroid balance was within the normal range [lo] . The molar cholesterol level in the bile of the current patients was above the limit commonly found in healthy controls. In all patients the bile samples were oversaturated with cholesterol. This finding opens the possibility that HLP type III, like HLP type IV [34], is associated in the long run with an enhanced risk of developing cholesterol gallstone disease. Further evidence supporting this hypothesis was recently presented in a study where supranormal cholesterol saturation .was demonstrated in gallstone-free patients with the type IV lipoprotein pattern
1261. In summary, HLP types IIa and IV are different entities of disease affecting bile acid and cholesterol metabolism. The augmented formation of VLDL-Tg, cholesterol and bile acids often observed in HLP type IV suggests that a
b
a
6.8 f 0.4
12.8 t 1.44
59.4 18.0 65.4 IQ.3 60.6 61.6 59.8 59.5
(molar %)
Bile acids
SATURATION
71.0 f 1.0
64.3 1- 2.36
CHOLESTEROL
22.3 f 0.9
22.9 _+1.92
27.1 12.8 22.4 20.6 26.6 27.3 18.2 28.1
Phospholipids
OF BILE IN HLP TYPE III
1.85 f 0.22
1.81 1.78 1.73 1.43 1.63 1.41 3.37 1.64
Lithogenic indexa (H-D-H)
According to the limits of cholesterol solubility as defined by Hegardt and Dam [22] and Holzbach et al. [23]. Calculated according to Carey and Small [24,25] using a biliary lipid concentration of 10 g/dI.
Healthy controls (26) Mean f SEM
Mean ? SEM
13.5 9.2 12.2 9.1 12.8 11.1 22.1 12.4
AND
1. 2. 3. 4. 5. 6. 7. 8.
LIPID COMPOSITION
Cholesterol
5
Subject No.
BILIARY
TABLE
ChoIesteroI saturationb
185 _+27
160 137 317 157
163 -
175 -.
(%)
stimulation of VLDL-Tg synthesis is associated with a rise in hepatic cholesterol production, which in turn results in an augmented elimination of cholesterol transformed into bile acids. The elevated formation of such compounds in the present patients suggests that overproduction of VLDL may also be a common phenomenon in HLP type III. Acknowledgements The skilful assistance of Marie Holmberg and Eve-Marie Bergkvist is gratefully acknowledged. The ethical aspects of the investigation were approved by the Ethics Committees of the Karolinska Institutet, Stockholm. References 1 Felts, J.M. and Rudel. L.L.. Mechanisms of hyperlipidemia. In: D. Kritchevsky (Ed.), Hypolipidemic Agents, Springer-Verlag. Berlin. Heidelberg, New York, 1975, pp. 151-189. 2 Eisenberg. S. and Levy, RI., Lipoproteins and lipoprotein metabolism. In: D. Kritchevksy (Ed.), Hypolipidemic Agents, Springer-Verlag, Berlin, Heidelberg, New York, 1975. pp. 191-213. 3 Sniderman. A., Thomas, D., Marpole, D. and Teng. B., Low density lipoprotein - A metabolic pathway for return of cholesterol to the splanchnic bed, J. Chn. Invest., 61 (1978) 867-873. 4 Brown, MS. and .GoIdstein. J.L., Familial hypercholesterolemia - A genetic defect in the low density lipoprotein receptor, N. Engl J. Med., 294 (1976) 1386-1390. 5 Kudchodkar. B.J. and Sodhi. H.S.. Turnover of pksma cholesteryl esters and its relationship to other parameters of lipid metabolism in man. Europ. J. Cbn. Invest., 6 (1976) 285-298. 6 Angelin, B., Einarsson, K., HeRstrom, K. and Leijd, B.. Bile acid kinetics in relation to endogenous triglyceride formation in various types of hyperhpoproteinemia. J. Lipid Res.. 19 (1978) 1004-1016. 7 Sigurdsson. G.. NicoB. A. and Lewis, B.. Metabolism of very low density lipoproteins in hyperlipidaemia - Studies of apolipoprotein B kinetics in man, Europ. J. CIin. Invest., 6 (1976) 167-177. 8 Beaumont, J.-L.. Carlson, L.A.. Cooper, G.R.. Fejfar, 2.. Fredrickson, D.S. and Strasser. T.. Classification of hyperhpidaemias and hyperlipoproteinaemias. BuB. WHO, 43 (1970) 891-915. 9 Einarsson, K.. HeIIstrdm, K. and Kalhaer. M.. Bile acid kinetics in relation to sex. serum lipids, body weights and gailbladder disease in patients with various types of hyperhpoproteinaemia, J. Clm. Invest., 54 (1974) 1301-1311. 10 Angelin. B.. Einarsson, K.. Hehstrom, K. and KaBner, M., Elimination of cholesterol in hyperlipoproteinaemia, Clin. Sci. Mol. Med., 61 (1976) 393-397. 11 Fredrickson, D.S.. Levy, R.I. and Lees, R.S., Fat transport in lipoproteins - An integrated approach to mechanisms and disorders, N. EngI. J. Med., 276 (1967) 3444, 94-103. 148-166, 215-225. 273-281. 12 Gustafsson. L-B.. Determination of the sterol composition of diet used in dietary management of hyperlipoproteinaemiaa, UppsaIa J. Med. Sci., 85 (1980) 35. 13 Ikkos. D. and Luft, R.. On the intravenous glucose tolerance test, Acta &rdocrinol. (Copenhagen), 26 (1967) 312-334: 14 Carlson. L.A. and Rossner, S.A., A methodological study of an intravenous fat tolerance test with IntraIipid@ emulsion, Stand. J. Lab. Invest., 29 (1972) 271-280. 15 Vessby, B., Studies on the serum lipoprotein composition in S&year-old men - A su@estion of chemical criteria for diagnosis of hyperhpoproteinemia type III (broad-@-disease), Clin. Chim. Acta. 69 (1976) 2942. 16 Noble, R.P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 963-700. 17 Lmdstedt, S., Turnover of chohc acid in man, Acta Physiol. Stand., 40 (1957) l-9. 18 Hanel. H.K. and Dam. H.. Determination of smaB amounts of total cholesterol by the Tschugaeff reaction with a note on the determination of lathosterol. Acta Chem. Stand.. 9 (1956) 677-682. 19 Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem.. 234 (1959) 466468. 20 Fausa, 0. and S&egg. B.A., Quantitative determination of bile acids and their conjugates using thinlayer chromatography and a purified 3o-hydroxysteroid dehydrogenase, Stand. J. Gastroenterol., 9 (1974) 249-264. 21 Thomas, P.J. and Hofmann, A.F., A simple cabxdation of lithogenic Index of bile: expressing bihary lipid composition on rectangular coordinates, Gastroenterology, 66 (1973) 698-700.
201 22
Hegardt,
23
Z. Emalnungswiss., 10 (1971) 223-233. Holzbach. R.T.. Marsh, M.. Olzewski, M. and Holan,
F.G. and Dam. H., The solubillty
supersaturated 24 25 26 27
28
29 30 31
32 33 34
bile is frequent
in healthy
of cholesterol
in aqueous
K., Cholesterol
man, J. Clln. Invest.,
solutions solubility
52 (1973)
of bile salts and lecithin, in bile -
Evidence
that
1467-1479.
Carey, M.C. and Small, D.M., The physical chemistry of cholesterol solubility ln bile - Relationship to gallstone formation and dissolution in man. J. Clin. Invest., 61 (1978) 998-1026. Carey, M.C., Critical tables for calculating the cholesterol saturation of native bile. J. Lipid Res.. 5 (1978) 945-955. Ahlberg. J.. Angelin, B., Einarsson, K.. Hellstrom. K. and Leijd, B., Bilisry lipid composition in nonno- and hyperllpoproteinaemia, Gastroenterology. 79 (1980) 90-94. Utermann, G., Jaeschke. M. and Menzel, J.. Familial hyperlipoproteinaemia type III - Deficiency of a specific apolipoprotein (ape E-III) in the very low density lipoproteins, FEBS Lett., 56 (1975) 352355. Hazzard, W.R. and Bierman. E.L.. The spectrum of electrophoretic mobility of very low density lipoproteins - Role of slower-migrating species in endogenous llpemia and broad-o-disease, J. Lab. Clin. Med., 86 (1975) 239-252. Fredrickson. D.S.. Morganroth, J. and Levy. R.J., Type III hyperlipoproteinemia - An analysis of two contemporary definitions, Ann. Intern. Med., 82 (1975) 150-157. Hall, M.H. III. Bllhelmer. D.W., Phalr, R.D.. Levy, R.J. and Berman, M.. A mathematical model for apoprotein kinetics in normal and hyperlipemlc patients, Circulation. 49-50 (1974) (Suppl. III): 114. Hazsard. W.R. and Bierman, E.L.. Delayed clearance of chylomicron remnants following vitamin-Acontaining oral fat loads in broad-o-disease (type III hyperlipoproteinemia), Metabolism, 25 (1976) 277-280. Bilheimer, D.W., Eisenberg. S. and Levy, R.J., Abnormal metabolism of very low density lipoproteins in type III hyperlipoproteinemla, Circulation, 44 (1971) (Suppl. II): 56. Quarfordt, S.H., Levy. RI. and Fredrickson, D.S., The kinetic properties of very low density lipoprotein triglyceride in type III hyperllpoproteinemia. Biochem. Biophys. Acta. 296 (1973) 572-576. Einarsson. K., Hellstrom, K. and Kallner, M.. Gallbladder disease in hyperlipoproteinaemia, Lancet, i (1975) 484-487.