293
Atherosclerosis, 24 (1976) 293-299 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
THE FORMATION OF DEOXYCHOLIC ACID IN PATIENTS WITH TYPE II AND IV HYPERLIPOPROTEINAEMIA
MASSIMO CARELLA Department
*, KURT EINARSSON and KJELL
of Medicine,
Serafimerlasarettet,
Stockholm
HELLSTRGM (Sweden)
(Received 21st October, 1975) (Revised, received 9th December, 1975) (Accepted 10th December, 1975)
Summary The formation of deoxycholic acid (D) was studied in 8 patients with Type II hyperlipoproteinaemia and in 6 with Type IV hyperlipoproteinaemia, using orally administered [ 2.4”H] cholic acid and [ 24-14C] deoxycholic acid. The diet was standardized and of natural type. The mean values for fractional turnover, pool size and D synthesis in the patients with Type II pattern were 0.23 days-‘, 331 mg and 75 mg/day, respectively; in Type IV they were 0.39 days-‘, 587 mg and 191 mg/day. Compared with a group of healthy subjects, the pool size and formation of D were normal in Type IV, but significantly reduced in Type II. The mean conversion of cholic acid into the circulating pool of D was calculated to be 37% in Type II, and 38% in Type IV patients. Both these values are within normal limits.
Key words:
Bile acid turnover - Cholesterol - Hyperlipopro teinaemia
metabolism
- Cholic acid - Deoxycholic
acid
Introduction Deoxycholic acid (D) is the major metabolite formed from cholic acid (C) in the intestine. It is effectively absorbed and constitutes one of the major bile acids in human bile, which in healthy subjects contains C, CD (chenodeoxy* Present address: Instituta Clinica Medica Generale II. Faculty of Medicine, University of Naples, Via Pancini, Naples. Italy.
294
cholic acid) and D in the average proportions 1.0 :l.O : 0.6 [l] . Subnormal D concentrations have been observed in duodenal bile of patients with disorders such as liver cirrhosis [ 1,2] and hypercholesterolaemia [ 1,3,4] . The latter observation may have some bearing on the more recent finding of a subnormal formation of C in Type II hyperlipoproteinaemia [ 51. A different pattern of bile acid synthesis, characterized by a high production of C, was encountered in patients with Type IV hyperlipoproteinaemia [ 51. As part of an investigation into the mechanisms responsible for the abnormal bile acid synthesis in Type II and IV hyperlipoproteinaemia, the kinetics of D were studied in patients with these disorders. Materials and methods The patients Fourteen patients, aged between 32 and 59 years, were admitted because of hyperlipoproteinaemia. They were separated into two groups on the basis of their lipoprotein pattern. The first group comprised 8 subjects with a Type IIa (3 females and 2 males) or Type IIb (2 females and 1 male) lipoprotein pattern [6] . All 6 subjects in the other group were males with Type IV hyperlipoproteinaemia. Three of the latter were more than 10% overweight and one had been cholecystectomized. There was no evidence of thyroid, liver, intestinal or kidney diseases. Clinical and laboratory data are presented in Tables 1 and 2. Experimental procedure The patients were hospitalized during the course of the study. For some days before and during the experimental period, they were given a standardized diet of natural type. The total intake of food varied from individual to individual, and was adjusted to keep the body weight constant. About 40% of the calories
TABLE 1 BASAL DATA OF THE PATIENTS Subject No.
1. 2. 3. 4. 5. 6. 7. 8.
GB SH KJ AL GN RH RJ KK
9. EA 10. 11. 12. 13. 14.
FB RF GG TW KE
Type of hyper Iipoproteinaemia
Sex/Age
Height
Body weight
Previous history and present
(wars)
(cm)
(kg)
symptoms
IIa
F 56 F 42 M 56 F 59 M 55 M 54 F 58 F 51
160 167 175 150 167 176 169 165
52 64 73 48 66 75 62 55
-
IIa IIa IIa IIa IIb IIb IIb IV
M 54
168
81
IV IV IV IV IV
M M M M M
176 171 180 168 167
105 74 83 72 79
Myocardial infarction, cerebral thrombosis Cholecystectomy
41 32 50 58 43
Xanthelasma Xanthelasma
Myocardial infarction -
Hypertension Hypertension
295
TABLE
2
SERUM
LIPIDS
Subject No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
AND LIPOPROTEIN
PATTERN
(MEAN
+ SEM)
Type of hyperlipoproteinaemia
Cholesterol
Triglycerides
(mg/lOOmll)
(mg/100 ml)
IIa IIa IIa IIa IIa IIb IIb IIb
345 395 383 340 350 340 340 387
f 4 ?r9 f 14 + 31 f 17 k 11 + 6 + 12
IV IV IV IV IV IV
279 370 288 260 255 343
+ * f C + +
13 30 23 16 11 29
Mean Type II Mean Type IV Sign. of diff.
360 + 8 299 ? 19 P < 0.01
Upper normal range
270
56 f 95+130 + 148 * 178 + 221 k 218 k 200 * 266 1,093 338 373 296 629
9 3 20 17 17 38 21 14
?: 21 + 230 ?: 49 f 41 + 27 k 111
156 f 21 499 f 130 P < 0.02 180
Lipoproteins 01W)
pre-P (%)
PC%)
23 24 19 21 19 21 31 19
7+2 13+ 1 9?2 16 f 1 15f 4 22 13 + 3 20 + 5
70 63 72 63 66 57 56 61
14 4+1 13 f 1 15 f 1 19 f 3 5
25 71 38 53 34 77
61 25 50 32 47 18
22+ 1 12 f 2 P < 0.005
14 f 2 50 f 9 P < 0.001
64 f 2 39 f 7 P < 0.005
50
25
60
i 1 + 2 zk3 r 1 t 3 * 4 + 1
f f f f
11 6 0.3 4
f + f f *
2 1 2 1 3
f 7 k 3
+ * f f
9 5 0.3 6
were supplied in the form of fat, most of which contained saturated fatty acids. The daily intake of cholesterol was less than 200 mg. The sodium salts of [3H] cholic acid (15 PCi) and [‘“Cl deoxycholic acid (4 FCi) were dissolved in water; these were given orally to the subjects in the morning before breakfast. Four samples of about 10 ml of duodenal bile were collected at intervals of l-3 days. Cholecystokinin was administered in order to obtain concentrated bile. Serum samples were drawn twice a week, and analyzed for total cholesterol, P-lipoprotein cholesterol (most patients), triglycerides and lipoprotein pattern (lipid electrophoresis). Materials [2.4-‘H]
Cholic acid (9.3 mCi/mg) was obtained from New England Nuclear Corp., Boston, Mass., U.S.A. [24-14C]Deoxycholic acid 18.8 &X/mg) was purchased from Mallingckrodt Nuclear, St. Louis, MO., U.S.A. The radiopurity of the labelled bile acids (>98%) was ascertained by thin layer chromatography (TLC) in several solvent systems. Cholecystokinin was obtained from the Gastrointestinal Hormone Group, Chemical Department, Karolinska Institutet, Stockholm, Sweden.
Methods
The bile samples were hydrolyzed with 1 M KOH in closed steel tubes for 12 h at 110” C. The mixture was acidified with HCI, and extracted with ethyl ether. The ether extract was washed with water until neutral and the solvent was then evaporated. The residue was methylated with diazomethane, and further anal-
296
yzed by TLC using ethyl acetate-trimethylpentane-acetic acid (10 : 10 : 2, v/ v/v) as solvent [7]. The bile acid zones were located by iodine vapor. The bands corresponding to cholic and deoxycholic acid were scraped off separately and eluted with methanol. One aliquot of the methanol extract was treated with trifluoroacetic anhydride, and analyzed by gas-liquid chromatography [S] . Another aliquot was used for determination of radioactivity. The type of hyperlipoproteinaemia was defined on the basis of the serum total cholesterol, /3-lipoprotein cholesterol, triglycerides and the fractionation pattern recorded with lipid electrophoresis on agarose gel. The upper normal levels of serum lipids were 270 mg/lOO ml (total cholesterol), 210 mg/lOO ml (P-cholesterol, determined in all but 3 of the patients) and 180 mg/ml (triglycerides). Details on the techniques used and the method for calculating the bile acid kinetic data have been described in a previous paper [5] . The correlation coefficients for the specific radioactivity decay curves for [3H]cholic acid averaged 0.986 f 0.12 and 0.992 * 0.01 in the patients with Type II and IV hyperlipoproteinaemia, respectively. The corresponding values encountered for [“Cl deoxycholic acid were 0.990 f 0.12 and 0.986 + 0.18. Significance of differences was evaluated with Student’s t-test. Values are given as mean ?SEM.
TABLE 3 THE KINETICS Patient No.
OF CHOLIC ACID AND DEOXYCHOLIC
Cholic acid Type of hyperlipoproteinsemia Pool size (m@
synthesis (mUday)
ACID
Fractional turnover
Deoxycholic
acid
Pool size
Synthesis
(me)
(me/day)
(day-‘)
Fiactionsl turnover (day-‘)
524 1.099 1,438 367
121 241 250 319
0.23 0.22 0.17 0.87
41 267 196 285
7 45 41 152
0.15 0.17 0.21 0.53
423
147 161 216
0.35 0.41 0.33
673 366 414 401
137 73 64 80
0.20 0.20 0.15 0.20
208 f 27
0.37 f 0.09
331 + 65
2.180 869 377 1,508 1.556 789
807 754 216 585 491 433
0.37 0.87 0.57 0.39 0.32 0.56
1197 244 374 464 378 367
278 188 130 139 265 146
Mean f SEM
1,213 * 267
549 * 89
0.51 * 0.08
587 + 151
191+
Significance of differences
NS=
P-C 0.005
NS
NS
P < 0.005
IIa IIa IIa IIa IIa IIb IIb IIb Mean + SEM 9 10 11 12 13 14
IV IV IV IV IV IV
a No significance.
396 646 699 f 156
75 * 17
0.23 + 0.05 0.23 0.77 0.35 0.30 0.30 0.40
27
0.39 f 0.08
NS
297
Results Bile acid kinetics Type II hyperlipoproteinaemia. The pool sizes of C were measured in 7 subjects and ranged from 367 to 1,438 (699 + 156) mg. In all but 2 of these patients, the values for C exceeded those of D by factors of 1.3 to 11 (Table 3). The mean size of the D pool in all 8 subjects was 331 + 65 mg. C synthesis (7 subjects) averaged 208 (range 121-319) mg/day. The corresponding value for D in 8 patients was 75 (range 7-152) mg/day. From these figures, it appeared that 37 f 11 (range 6-93) % of the C was converted into D. In 6 out of the 7 patients in whom comparable results were available, the fractional turnover of C was higher than that of D. Type IV hyperlipoproteinaemia. The C pool size averaged 1.213 + 267 (377-2,180) mg, and was in all instances higher (1.01-3.30 times) than that of D. On average, 39 f 6 (24-60) % of the C synthesized (216-807 mg/day) was transformed into the D (130-278 mg/day) recovered in the enterohepatic circulation. In all subjects the fractional turnover of C exceeded that of D. On a statistical basis, the pool sizes of C in the patients with the Type IV pattern did not differ from those observed for Type II. However, only one subject with Type II hyperlipoproteinaemia had a C pool size which exceeded the mean for type IV, whereas 5 of the 6 patients with Type IV hyperlipoproteinaemia had a larger pool size than the mean for Type II. The synthesis of both C and D was almost 3 times higher in Type IV than in Type II hyperlipoproteinaemia. The two types of disorders also tended to differ with regard to the fractional turnover of both C and D (Table 3). Discussion In the present investigation the pool sizes of C were similar to those previously encountered in larger studies of subjects with Type II and IV hyperlipoproteinaemia [ 51. The formation of C was unexpectedly high in patient No. 4 but, in keeping with earlier findings, subnormal to normal in the other subjects with the Type II pattern. In the patients with Type IV hyperlipoproteinaemia, cholic acid production was normal in subject No. 11, but elevated in the others. The means for both pool size and C synthesis were about twice as high in Type IV as in Type II hyperlipoproteinaemia. The kinetic data recorded for D reflected those for C insofar as the mean synthesis of D in Type II hyperlipoproteinaemia was only 40% of that observed in Type IV. Although not significant, a similar tendency was observed for the D pool size. The subjects with the 2 disorders also appeared to differ with regard to the D fractional turnover, this being lowest in the patients with the Type II lipoprotein pattern. As compared to the findings of normolipidaemic subjects studied under the same dietary conditions as those used in this investigation [9], the pool size and D synthesis were normal in the present subjects with Type IV hyperlipoproteinaemia, but decreased by about 50% in those with the Type II pattern (Figs. 1 and 2). The mean conversion of C into the circulating pool of D was about the same in all groups of subjects studied, being 41% [25561] in controls, 37% (6-93), and 38% (2440) respectively in patients
298 POOL SIZE(mg)
SYNTHESIS (mg/day)
300
r
COITROL
n-6
1 -
200
100
-
TVPEII
TVPEIV
n=6
n-6
COWVROL n=6
TYPE II
TVPEIV
n-6
n-6
Fig. 1. The pool size of deoxycholic trols [91.
acid in hyperlipoproteinaemic
patients and in normolipidaemic con-
Fig. 2. The synthesis of deoxycholic trols [91.
acid in hyperlipoproteinaemic
patients and in normolipidaemic con-
with Type II and IV hyperlipoproteinaemia. Bile acid formation is regulated by a negative feed-back mechanism triggered by the amount of bile acids reaching the liver, i.e., the bile acid pool size times the number of its enterohepatic circulations. The composition of the bile acid pool may influence this feed-back control, since studies in rata [lo] indicate that C, CD and D are not equally effective in inhibiting various steps involved in cholesterol and bile acid biosynthesis. Furthermore, CD and D as compared to C are more effectively absorbed in the upper snall intestine of both normoand hyperlipidaemic subjects [ll], and as a result the three bile acids rotate at different rates in the enterohepatic circulation. These circumstances focus interest on the possibility that abnormalities in the formation and excretion of D may be responsible for the abnormal bile acid kinetics in hyperlipoproteinaemia. However, evidence to support such a concept was not obtained in the present study. The subnormal poolsize and formation of D in the patients with the Type II pattern appear to reflect the more primary defect in the biosynthesis of C. Studies aimed at investigating this defect are now in progress. References 1 Sjijvall, J., Bile acids in man under normal and pathological conditions. Clin. Chim. Acta, 5 (1960) 33. 2 Vlahcevic, Z.R., Juttijudata, P.. Bell, Jr., CC. and Swell, L., Bile acid metabolism in patients with cirrhosis, Part 2 (Cbolic and chenodeoxycholic acid metabolism). Gastroenterol., 62 (1972) 1174. 3 Blomstrand, R., Gas-liquid chromatography of human bile acids, Proc. Sot. Exp. Biol. Med., 107 (1961) 126. 4 Hellstrkim. K. and Lindstedt, S.. Studies on the formation of cholic acid in subjects given standardized diet with butter or corn oil as dietary fat, Amer. J. Clin. Nutr.. 18 (1966) 46.
299
5 Einarsson. weights
K.. Hellstrom.
K. and Kallner.
and gallbladder
disease
vest., 54 (1974) 1301. 6 Beaumont, J.L., Carlson. tion of hyperlipidemias
L.A.,
M., Bile acid kinetics
in patients Cooper,
with various
G.R.,
Felfar.
and hyperlipoproteinaemias,
types
in relation
to sex, serum lipids, body
of hyperlipoproteinaemia.
2.. Frederikson,
S. and Strasser,
WHO Bull., 43 (1970)
J. Clin. InT.. Classifica-
891.
7 Eneroth. P., Thin-layer chromatography of bile acids, J. Lipid Res., 4 (1963) 11. 8 SjGvall, J.. Qualitative analysis of bile acids by gas chromatography, Acta Chem. Stand., 16 (1962) 1761. 9 Einarsson. K. and Hellstrom, K.. The formation of deoxycholic acid and chenodeoxycholic acid in man. Clfn. Sci. Mol. Med., 46 (19 74) 183. 10 Shefer, S., Hauser, S.. Lapar, V. and Mosbach, E.H., Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methyl_taryl CoA reductase and ‘lo-hydroxylase in the rat, J. Lipid Res., 14 (1973) 573. 11 Angelin. B.. Einarsson, K. and Hellstrom, K., Evidence for the absorption of bile acids in the proximal small intestine of normo- and hyperlipidaemic subjects, Gut, In press.
Atherosclerosis, 24 (1976) 293-299 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
THE FORMATION OF DEOXYCHOLIC ACID IN PATIENTS WITH TYPE II AND IV HYPERLIPOPROTEINAEMIA
MASSIMO CARELLA Department
*, KURT EINARSSON and KJELL
of Medicine,
Serafimerlasarettet,
Stockholm
HELLSTRGM (Sweden)
(Received 21st October, 1975) (Revised, received 9th December, 1975) (Accepted 10th December, 1975)
Summary The formation of deoxycholic acid (D) was studied in 8 patients with Type II hyperlipoproteinaemia and in 6 with Type IV hyperlipoproteinaemia, using orally administered [ 2.4”H] cholic acid and [ 24-14C] deoxycholic acid. The diet was standardized and of natural type. The mean values for fractional turnover, pool size and D synthesis in the patients with Type II pattern were 0.23 days-‘, 331 mg and 75 mg/day, respectively; in Type IV they were 0.39 days-‘, 587 mg and 191 mg/day. Compared with a group of healthy subjects, the pool size and formation of D were normal in Type IV, but significantly reduced in Type II. The mean conversion of cholic acid into the circulating pool of D was calculated to be 37% in Type II, and 38% in Type IV patients. Both these values are within normal limits.
Key words:
Bile acid turnover - Cholesterol - Hyperlipopro teinaemia
metabolism
- Cholic acid - Deoxycholic
acid
Introduction Deoxycholic acid (D) is the major metabolite formed from cholic acid (C) in the intestine. It is effectively absorbed and constitutes one of the major bile acids in human bile, which in healthy subjects contains C, CD (chenodeoxy* Present address: Instituta Clinica Medica Generale II. Faculty of Medicine, University of Naples, Via Pancini, Naples. Italy.
294
cholic acid) and D in the average proportions 1.0 :l.O : 0.6 [l] . Subnormal D concentrations have been observed in duodenal bile of patients with disorders such as liver cirrhosis [ 1,2] and hypercholesterolaemia [ 1,3,4] . The latter observation may have some bearing on the more recent finding of a subnormal formation of C in Type II hyperlipoproteinaemia [ 51. A different pattern of bile acid synthesis, characterized by a high production of C, was encountered in patients with Type IV hyperlipoproteinaemia [ 51. As part of an investigation into the mechanisms responsible for the abnormal bile acid synthesis in Type II and IV hyperlipoproteinaemia, the kinetics of D were studied in patients with these disorders. Materials and methods The patients Fourteen patients, aged between 32 and 59 years, were admitted because of hyperlipoproteinaemia. They were separated into two groups on the basis of their lipoprotein pattern. The first group comprised 8 subjects with a Type IIa (3 females and 2 males) or Type IIb (2 females and 1 male) lipoprotein pattern [6] . All 6 subjects in the other group were males with Type IV hyperlipoproteinaemia. Three of the latter were more than 10% overweight and one had been cholecystectomized. There was no evidence of thyroid, liver, intestinal or kidney diseases. Clinical and laboratory data are presented in Tables 1 and 2. Experimental procedure The patients were hospitalized during the course of the study. For some days before and during the experimental period, they were given a standardized diet of natural type. The total intake of food varied from individual to individual, and was adjusted to keep the body weight constant. About 40% of the calories
TABLE 1 BASAL DATA OF THE PATIENTS Subject No.
1. 2. 3. 4. 5. 6. 7. 8.
GB SH KJ AL GN RH RJ KK
9. EA 10. 11. 12. 13. 14.
FB RF GG TW KE
Type of hyper Iipoproteinaemia
Sex/Age
Height
Body weight
Previous history and present
(wars)
(cm)
(kg)
symptoms
IIa
F 56 F 42 M 56 F 59 M 55 M 54 F 58 F 51
160 167 175 150 167 176 169 165
52 64 73 48 66 75 62 55
-
IIa IIa IIa IIa IIb IIb IIb IV
M 54
168
81
IV IV IV IV IV
M M M M M
176 171 180 168 167
105 74 83 72 79
Myocardial infarction, cerebral thrombosis Cholecystectomy
41 32 50 58 43
Xanthelasma Xanthelasma
Myocardial infarction -
Hypertension Hypertension
295
TABLE
2
SERUM
LIPIDS
Subject No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
AND LIPOPROTEIN
PATTERN
(MEAN
+ SEM)
Type of hyperlipoproteinaemia
Cholesterol
Triglycerides
(mg/lOOmll)
(mg/100 ml)
IIa IIa IIa IIa IIa IIb IIb IIb
345 395 383 340 350 340 340 387
f 4 ?r9 f 14 + 31 f 17 k 11 + 6 + 12
IV IV IV IV IV IV
279 370 288 260 255 343
+ * f C + +
13 30 23 16 11 29
Mean Type II Mean Type IV Sign. of diff.
360 + 8 299 ? 19 P < 0.01
Upper normal range
270
56 f 95+130 + 148 * 178 + 221 k 218 k 200 * 266 1,093 338 373 296 629
9 3 20 17 17 38 21 14
?: 21 + 230 ?: 49 f 41 + 27 k 111
156 f 21 499 f 130 P < 0.02 180
Lipoproteins 01W)
pre-P (%)
PC%)
23 24 19 21 19 21 31 19
7+2 13+ 1 9?2 16 f 1 15f 4 22 13 + 3 20 + 5
70 63 72 63 66 57 56 61
14 4+1 13 f 1 15 f 1 19 f 3 5
25 71 38 53 34 77
61 25 50 32 47 18
22+ 1 12 f 2 P < 0.005
14 f 2 50 f 9 P < 0.001
64 f 2 39 f 7 P < 0.005
50
25
60
i 1 + 2 zk3 r 1 t 3 * 4 + 1
f f f f
11 6 0.3 4
f + f f *
2 1 2 1 3
f 7 k 3
+ * f f
9 5 0.3 6
were supplied in the form of fat, most of which contained saturated fatty acids. The daily intake of cholesterol was less than 200 mg. The sodium salts of [3H] cholic acid (15 PCi) and [‘“Cl deoxycholic acid (4 FCi) were dissolved in water; these were given orally to the subjects in the morning before breakfast. Four samples of about 10 ml of duodenal bile were collected at intervals of l-3 days. Cholecystokinin was administered in order to obtain concentrated bile. Serum samples were drawn twice a week, and analyzed for total cholesterol, P-lipoprotein cholesterol (most patients), triglycerides and lipoprotein pattern (lipid electrophoresis). Materials [2.4-‘H]
Cholic acid (9.3 mCi/mg) was obtained from New England Nuclear Corp., Boston, Mass., U.S.A. [24-14C]Deoxycholic acid 18.8 &X/mg) was purchased from Mallingckrodt Nuclear, St. Louis, MO., U.S.A. The radiopurity of the labelled bile acids (>98%) was ascertained by thin layer chromatography (TLC) in several solvent systems. Cholecystokinin was obtained from the Gastrointestinal Hormone Group, Chemical Department, Karolinska Institutet, Stockholm, Sweden.
Methods
The bile samples were hydrolyzed with 1 M KOH in closed steel tubes for 12 h at 110” C. The mixture was acidified with HCI, and extracted with ethyl ether. The ether extract was washed with water until neutral and the solvent was then evaporated. The residue was methylated with diazomethane, and further anal-
296
yzed by TLC using ethyl acetate-trimethylpentane-acetic acid (10 : 10 : 2, v/ v/v) as solvent [7]. The bile acid zones were located by iodine vapor. The bands corresponding to cholic and deoxycholic acid were scraped off separately and eluted with methanol. One aliquot of the methanol extract was treated with trifluoroacetic anhydride, and analyzed by gas-liquid chromatography [S] . Another aliquot was used for determination of radioactivity. The type of hyperlipoproteinaemia was defined on the basis of the serum total cholesterol, /3-lipoprotein cholesterol, triglycerides and the fractionation pattern recorded with lipid electrophoresis on agarose gel. The upper normal levels of serum lipids were 270 mg/lOO ml (total cholesterol), 210 mg/lOO ml (P-cholesterol, determined in all but 3 of the patients) and 180 mg/ml (triglycerides). Details on the techniques used and the method for calculating the bile acid kinetic data have been described in a previous paper [5] . The correlation coefficients for the specific radioactivity decay curves for [3H]cholic acid averaged 0.986 f 0.12 and 0.992 * 0.01 in the patients with Type II and IV hyperlipoproteinaemia, respectively. The corresponding values encountered for [“Cl deoxycholic acid were 0.990 f 0.12 and 0.986 + 0.18. Significance of differences was evaluated with Student’s t-test. Values are given as mean ?SEM.
TABLE 3 THE KINETICS Patient No.
OF CHOLIC ACID AND DEOXYCHOLIC
Cholic acid Type of hyperlipoproteinsemia Pool size (m@
synthesis (mUday)
ACID
Fractional turnover
Deoxycholic
acid
Pool size
Synthesis
(me)
(me/day)
(day-‘)
Fiactionsl turnover (day-‘)
524 1.099 1,438 367
121 241 250 319
0.23 0.22 0.17 0.87
41 267 196 285
7 45 41 152
0.15 0.17 0.21 0.53
423
147 161 216
0.35 0.41 0.33
673 366 414 401
137 73 64 80
0.20 0.20 0.15 0.20
208 f 27
0.37 f 0.09
331 + 65
2.180 869 377 1,508 1.556 789
807 754 216 585 491 433
0.37 0.87 0.57 0.39 0.32 0.56
1197 244 374 464 378 367
278 188 130 139 265 146
Mean f SEM
1,213 * 267
549 * 89
0.51 * 0.08
587 + 151
191+
Significance of differences
NS=
P-C 0.005
NS
NS
P < 0.005
IIa IIa IIa IIa IIa IIb IIb IIb Mean + SEM 9 10 11 12 13 14
IV IV IV IV IV IV
a No significance.
396 646 699 f 156
75 * 17
0.23 + 0.05 0.23 0.77 0.35 0.30 0.30 0.40
27
0.39 f 0.08
NS
297
Results Bile acid kinetics Type II hyperlipoproteinaemia. The pool sizes of C were measured in 7 subjects and ranged from 367 to 1,438 (699 + 156) mg. In all but 2 of these patients, the values for C exceeded those of D by factors of 1.3 to 11 (Table 3). The mean size of the D pool in all 8 subjects was 331 + 65 mg. C synthesis (7 subjects) averaged 208 (range 121-319) mg/day. The corresponding value for D in 8 patients was 75 (range 7-152) mg/day. From these figures, it appeared that 37 f 11 (range 6-93) % of the C was converted into D. In 6 out of the 7 patients in whom comparable results were available, the fractional turnover of C was higher than that of D. Type IV hyperlipoproteinaemia. The C pool size averaged 1.213 + 267 (377-2,180) mg, and was in all instances higher (1.01-3.30 times) than that of D. On average, 39 f 6 (24-60) % of the C synthesized (216-807 mg/day) was transformed into the D (130-278 mg/day) recovered in the enterohepatic circulation. In all subjects the fractional turnover of C exceeded that of D. On a statistical basis, the pool sizes of C in the patients with the Type IV pattern did not differ from those observed for Type II. However, only one subject with Type II hyperlipoproteinaemia had a C pool size which exceeded the mean for type IV, whereas 5 of the 6 patients with Type IV hyperlipoproteinaemia had a larger pool size than the mean for Type II. The synthesis of both C and D was almost 3 times higher in Type IV than in Type II hyperlipoproteinaemia. The two types of disorders also tended to differ with regard to the fractional turnover of both C and D (Table 3). Discussion In the present investigation the pool sizes of C were similar to those previously encountered in larger studies of subjects with Type II and IV hyperlipoproteinaemia [ 51. The formation of C was unexpectedly high in patient No. 4 but, in keeping with earlier findings, subnormal to normal in the other subjects with the Type II pattern. In the patients with Type IV hyperlipoproteinaemia, cholic acid production was normal in subject No. 11, but elevated in the others. The means for both pool size and C synthesis were about twice as high in Type IV as in Type II hyperlipoproteinaemia. The kinetic data recorded for D reflected those for C insofar as the mean synthesis of D in Type II hyperlipoproteinaemia was only 40% of that observed in Type IV. Although not significant, a similar tendency was observed for the D pool size. The subjects with the 2 disorders also appeared to differ with regard to the D fractional turnover, this being lowest in the patients with the Type II lipoprotein pattern. As compared to the findings of normolipidaemic subjects studied under the same dietary conditions as those used in this investigation [9], the pool size and D synthesis were normal in the present subjects with Type IV hyperlipoproteinaemia, but decreased by about 50% in those with the Type II pattern (Figs. 1 and 2). The mean conversion of C into the circulating pool of D was about the same in all groups of subjects studied, being 41% [25561] in controls, 37% (6-93), and 38% (2440) respectively in patients
298 POOL SIZE(mg)
SYNTHESIS (mg/day)
300
r
COITROL
n-6
1 -
200
100
-
TVPEII
TVPEIV
n=6
n-6
COWVROL n=6
TYPE II
TVPEIV
n-6
n-6
Fig. 1. The pool size of deoxycholic trols [91.
acid in hyperlipoproteinaemic
patients and in normolipidaemic con-
Fig. 2. The synthesis of deoxycholic trols [91.
acid in hyperlipoproteinaemic
patients and in normolipidaemic con-
with Type II and IV hyperlipoproteinaemia. Bile acid formation is regulated by a negative feed-back mechanism triggered by the amount of bile acids reaching the liver, i.e., the bile acid pool size times the number of its enterohepatic circulations. The composition of the bile acid pool may influence this feed-back control, since studies in rata [lo] indicate that C, CD and D are not equally effective in inhibiting various steps involved in cholesterol and bile acid biosynthesis. Furthermore, CD and D as compared to C are more effectively absorbed in the upper snall intestine of both normoand hyperlipidaemic subjects [ll], and as a result the three bile acids rotate at different rates in the enterohepatic circulation. These circumstances focus interest on the possibility that abnormalities in the formation and excretion of D may be responsible for the abnormal bile acid kinetics in hyperlipoproteinaemia. However, evidence to support such a concept was not obtained in the present study. The subnormal poolsize and formation of D in the patients with the Type II pattern appear to reflect the more primary defect in the biosynthesis of C. Studies aimed at investigating this defect are now in progress. References 1 Sjijvall, J., Bile acids in man under normal and pathological conditions. Clin. Chim. Acta, 5 (1960) 33. 2 Vlahcevic, Z.R., Juttijudata, P.. Bell, Jr., CC. and Swell, L., Bile acid metabolism in patients with cirrhosis, Part 2 (Cbolic and chenodeoxycholic acid metabolism). Gastroenterol., 62 (1972) 1174. 3 Blomstrand, R., Gas-liquid chromatography of human bile acids, Proc. Sot. Exp. Biol. Med., 107 (1961) 126. 4 Hellstrkim. K. and Lindstedt, S.. Studies on the formation of cholic acid in subjects given standardized diet with butter or corn oil as dietary fat, Amer. J. Clin. Nutr.. 18 (1966) 46.
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