Increased catabolism of VLDL-apolipoprotein B and synthesis of bile acids in a case of hypobetalipoproteinemia

Increased

Catabolism of VLDL-Apolipoprotein B and Synthesis in a Case of Hypobetalipoproteinemia

Gloria Lena Vega, Klaus von Bergmann,

Scott M. Grundy, William

of Bile Acids

Beltz, Claus Jahn, and Cara East

A 29-year-old man is described who has reduced concentrations of low density lipoprotein (LDLI-cholesterol seemingly due to an unusual variant of hypobetalipoproteinemia. The patient developed retinitis pigmentosa at age 14. When studied at age 28, his total cholesterol was 104 mg/dL. triglycerides 59 mg/dL. LDL-cholesterol44 mg/dL, and HDL-cholesterol 51 mg/dL. Lipid and lipoprotein levels of his parents and sister were normal. His excretion of bile acids (13.9 mg/kg/d) was markedly elevated at about three times normal, although absorption rates of cholesterol and bile acids appeared to be in the normal range. His high excretion of bile acids equates to a threefold increase in bile acid synthesis. Isotope kinetic studies of his lipoproteins produced unexpected findings. Total production of VLDL-apolipoprotein B (ape B) was estimated to be 20.8 mglkgld. which was in the normal range. Synthesis of VLDL-triglycerides was also normal at 12.0 mg/kg/h. However, 75% of VLDL-apo B was removed directly from the circulation, which was much higher than values for direct removal of VLDL-apo B in control subjects. His production rate of LDL-protein (5.2 mg/kg/d) consequently was below normal, although his fractional catabolic rate for LDL (0.40 pools/d) was not distinctly elevated. These data suggest that the patient’s hypobetalipoproteinemia was due to increased direct removal of VLDL remnants and not to reduced synthesis of VLDL-apo B; this abnormality may have been the result of enhanced activity of LDL receptors, which in turn was secondary to increased synthesis of bile acids. @ 1987 by Grune & Stratton, Inc.

H

YPOBETALIPOPROTEINEMIA is characterized by reduced levels of very low density lipoproteins (VLDL) and low density lipoproteins (LDL).‘,’ Although several forms of hypobetalipoproteinemia have been recognized, the best characterized is familial hypobetalipoproteinemia, in which the disorder is inherited as an autosomal dominant trait. Homozygotes for this disorder have extremely low levels of lipids and suffer from malabsorption of fat, acanthocytosis, atypical retinitis pigmentosa, and neurologic degeneration. Heterozygotes, in contrast, usually are without symptoms and are identifiable only by their moderately reduced levels of plasma lipids. The nature of the mutant allele in familial hypobetalipoproteinemia is not known, but available evidence suggests that synthesis of apolipoprotein B (apo B) is impaired,‘v4 which secondarily results in decreased production of apo B-containing lipoproteins (VLDL, IDL, and LDL). However, all patients with hypobetalipoproteinemia do not conform to the typical familial disorder, and a low level of VLDL and LDL may have other etiologies.sv6 For example, some patients have abnormalities in the structure of the apo B molecule that imparts a

defect in the metabolism of lipoproteins containing apo B.’ In the present report, we present data from a patient with moderate hypobetalipoproteinemia who had characteristics

From the Center for Human Nutrition and the Departments of Biochemistry and Internal Medicine. University of Texas Health Science Center, Dallas, and the Department of Medicine. University of Bonn, West Germany. Supported by the Veterans Administration, Grant HL 29252 (NIH/HDS/DHHS)). NIH Grant MOl-RR00633 (General Clinical Research Center). USPHS training Grant AM07307, the Southwestern Medical Foundation, and the Moss Heart Foundation, Dallas; also, the Sandoz Stiftungfuer therapeutische Forschung. Address reprint requests to Scott M. Grundy, MD, PhD. Center for Human Nutrition, University of Texas Health Science Center, Room Y3.206. S323 Harry Hines Blvd. Dallas, TX 75235. o 1987 by Grune & Stratton. Inc. 0026~495~87/3603-0010$03.00/0

262

previously described. He had retinitis pigmentosa but no fat malabsorption, acanthocytosis, or neurologic defects. He also had an increased excretion of bile acids and a decreased production of LDL. The latter was due to a high rate of direct removal of VLDL, the precursor of LDL, and not to decreased secretion of apo B-containing lipoproteins, as described for classical familial hypobetalipoproteinemia. not

MATERIALS

AND METHODS

Case History

Patient JS, a 29-year-old man, was confirmed to have retinitis pigmentosa at the Ophthalmology Institute, University of Bonn, West Germany. His visual symptoms began as night blindness at age 10, and therefore his vision deteriorated. At age 14 he was diagnosed as having retinitis pigmentosa, and a low level of plasma cholesterol was discovered at age 18. Because his very low concentration of plasma cholesterol was consistent with heterozygous hypobetalipoproteinemia, he was started on high doses of fat-soluble vitamins (A and E) in 1983. To further study his hypocholesterolemia, JS was admitted to the metabolic ward at the University of Bonn in 1984. His physical examination was unremarkable, and his body mass index (BMI) was 19.28 kg/m*. There was no evidence of malnutrition or hepatic or

gastrointestinal disease. Tests of hepatic function were normal, and fecal excretion of fat was less than 5 g/d. His hemogram similarly was normal without evidence of acanthocytosis. A duodenal biopsy was performed, and microscopy of the intestinal mucosa revealed no vacuoles indicative of excessive accumulation of fat in mucosal cells. During the 3-week period, six measurements of his plasma lipids and lipoproteins revealed: total cholesterol 104 * 11 (*SD) mg/dL, triglycerides 58 t 10 mg/dL, LDL-cholesterol 44 mg/dL, and HDL-cholesterol 51 mg/dL. JS did not have a family history of retinal disease. The data for his family lipids and lipoproteins are given in Table 1 and are compared to those for JS. Also shown are percentiles for each level for each member’s age and sex, according to the Lipid Research Clinics Population study.’ Both the father and sister had relatively low levels of total cholesterol and LDL-cholesterol, but none of the values were abnormally low, ie, below the 5th percentile. The mother had an abnormally high total cholesterol, but this was due mainly to a marked increase in HDL-cholesterol levels.

Metabolism, Vol36,

No 3 (March), 1987: pp 262-269

263

UNUSUAL CASE OF HYPOBETALlPOPROTElNEMIA

Table 1. Plasma Lipids, Lipoproteins,

and Percentiles

for Family Members Total Cholesterol

Family Member

Total Triglyceride

HDL Cholesterol

LDL Cholesterol

Probrand (JS)

104 (i5th)

58 (1 lth)

44 (<5th)

51 (88th)

Father (55 yrl

182 (19th)

132 (58th)

105 (15th)

52 (76th)

Mother (58 yr) 3 15 (97th)

64 (21st)

179 (89th)

123 (>95th)

156 (20th)

72 (30th)

89 (20th)

Sister (27 yr)

53 (47th)

Values are gwen as mg/dL with percentile in parentheses. *Percentile is by age and sex, according to the Lipid Research Clinics Population Survey.’

To uncover possible abnormalities in metabolism of sterols in the patient, an assessment was made of his balance of cholesterol, excretion of bile acids, and metabolism of biliary lipids; these studies were performed on the metabolic ward, University of Bonn, West Germany. The patient was then relocated to the General Clinical Research Center, Parkland Memorial Hospital, Dallas, where metabolic characteristics of apolipoprotein B and triglycerides were invcstigted. All protocols were approved by appropriate institutional revlew boards, and the patient gave informed consent for study. Utolesteroi

Balance

For determination of cholesterol balance, the patient was studied for 16 days under steady-state conditions on a solid-food diet of low cholesterol content. The diet contained 60% of calories as carbohydrate. 20% as protein, and 20% as fat. The fatty acids were distributed equally among saturated, monounsaturated, and polyunsaturated fatty acids. Daily intakes of cholesterol and sitosterol averaged 137 and 120 mg/d, respectively. During the study the patlent was given chromic oxide (60 mg three times daily) and sitosterol (100 mg three times daily). Feces were collected and combined into 8 pools, one for each two days. Neutral and acidic steroids were determined on each pool by gas-liquid chromatography.8,9 The excretion of sitosterol was used to correct for losses of neutral steroids,” and excretion of acidic steroids was corrected for variation in fecal flow by chromic oxide.”

Biliary Lipids Duodenal contents rich in gallbladder bile were collected three times after duodenal intubation with a single-lumen tube. Cholecystokinin (0.5 Ivy units/kg body weight: Kabi Diagnostika, Studswik, Sweden) was injected intravenously to stimulate contraction of the gallbladder; thereafter, dark gallbladder bile was obtained from the duodenum; it was shaken vigorously, and 3 mL were immediately mixed with chloroform/methanol (2/l, vol/vol); the remainder was returned to the duodenum. Bile was analyzed for content of cholesterol, phospholipids, and total bile acids and composition of individual bile acids according to the methodology of the National Cooperative Gallstone study.” On the last day of the study, measurements were made of hepatic secretion rates of cholesterol, phospholipids, and bile acid as described by Grundy and Metzger”; these measurements allowed the calculation of composition of stimulated hepatic bile. The results obtained in JS are compared to those obtained in 14 middle-aged. normal men.” Since these men were somewhat older and heavier than the current patient, they do not represent matched controls. The bile of these older men might be expected to be somewhat more enriched with cholesterol than thinner, younger men.

Absorption

of Cholesterol

and bile acids. Cholesterol absorption ence between input of cholesterol into bile) and fecal excretion of cholesterol calculation was used for estimating Details of this procedure for calculating age absorption) for both cholesterol described previously.‘4

Kinetic Studies of Lipoproteins Apolipoprotein B

was calculated as the differthe intestine (from diet and and its products. A similar the absorption of bile acids. absorption rates (or percentand bile acids have been

Containing

The kinetics ofapo B were studied in two lipoprotein fractions: (I) VLDL plus intermediate density lipoproteins (VLDL + IDL) and (2) LDL. The experimental methods used for this procedure have been described previously.‘5.‘6 In brief, the patient was maintained throughout the study on an isocaloric diet of solid food consisting of 40% of calories as fat (mostly in the form of lard), 45% as carbohydrate, and 15%as protein. This diet was consumed during turnover studies of both (VLDL + IDL)-apo B and LDL-apo B. After l-week equilibration period on this diet, 250 mL of plasma was obtained by plasmapheresis for isolation of lipoproteins. At this time, the patient was started on supersaturated potassium iodide (0.5 g/d) to inhibit thyroidal uptake of radioactive iodine. The plasma was subjected to sequential ultrancentrifugation at d 1.019g/mL to isolate VLDL + IDL and at d I .067 g/mL to isolate LDL. Each lipoprotein was recentrifuged at its native density for purification and concentration. Thereafter, the lipoproteins were dialyzed extensively against 0.15 mol/L NaCl and 0.01% disodium EDTA, pH 7.4. Approximately 5 to IO mg of each lipoprotein were radioiodinated using the method of McFarlane” as modified by Bilheimer et alI8 and Vega et al.l9 VLDL + IDL was labeled with “‘1, and LDL labeled with “‘I; 30 to 50 &i of each labeled lipoprotein were injected simultaneously into the patient. The rates of disppearance of “‘I-(VLDL + lDL)-apo B from VLDL + IDL and its appearance into LDL-apo B were determined as follows. Blood samples were collected at 10, 20, and 30 minutes and 1,2, 3.4, 6, 9,12, 18, and 24 hours after injection. Thereafter, samples were collected every four hours through 48 hours, every 12 hours for the next two days, and daily thereafter through the 14th day. Radioactivity was counted for each sample of total plasma, and counting was repeated on plasma infranates after sequential isolation of VLDL + IDL and LDL. In addition, the isolated fractions for VLDL + IDL and LDL were counted. Radioactivities in apo B of VLDL + IDL and LDL were determined as follows. The whole

\

out

and Bile Acids

The data from biliary and fecal outputs of neutral and acidic steroids permitted an estimation of absorption of both cholesterol

Fig 1.

described

Kinetic

model for apo B metabolism. and Methods.“

in “Materials

The details

are

264

VEGA ET AL

lipoprotein fraction was treated with 50% isopropanol (IPA) to precipitate apo B.W’ The soluble apoproteins remained in the supernatant. Precipitates were washed with 50% IPA to remove remaining soluble apoproteins. Lipid was then extracted from each precipitate with 100% IPA. All supernatant extracts were pooled and counted for radioactivity. Extracted radioactivity was compared to radioactivity in the whole lipoprotein fraction, and precipitated apo B radioactivity was estimated by difference. Absolute disintegrations per minute in the precipitates containing apo B from each of the lipoprotein fractions were estimated after extraction and washing as follows: (VLDL + IDL)-apo B dpm = (plasma dpm - 1.019 g/mL infranatant dpm) x (fraction of radioactivity in precipitated apo B of total radioactivity in isolated VLDL + IDL) LDL-apo B dpm = (1.019 g/mL infranatant dpm - 1.067 g/mL infranatant dpm) x (fraction of radioactivity in precipitated apo B of total radioactivity in isolated LDL) From the radioactivity in each successive sample, kinetic curves were constructed for fraction of injected dose v time. The I311radioactivity in LDL was counted simultaneously, and curves for ‘“I-LDL were constructed in parallel to those for ‘“‘I-LDL. The concentration of (VLDL + IDL)-apo B was determined as follows. Plasma total cholesterol” and triglyceridez3 was measured enzymatically on each sample of total plasma five times throughout the first 48 hours; another aliquot of the same sample was subjected to ultracentrifugation at d < 1.019 g/mL. Cholesterol was measured on the infranatant, and (VLDL + IDL)-cholesterol concentration was calculated as the difference between total cholesterol and infranatant cholesterol. On the supernatant, measurements were made as previously described for cholesterol,2* total protein, and apo B, with apo B being estimated as the difference between total protein and isopropanol soluble proteins.“(VLDL + IDL)-apo B concentration was then calculated by multiplying total (VLDL + IDL)cholesterol concentration times the (VLDL + IDL)-apo B/cholesterol ratio in the supernatant. Concentration of LDL-apo B was determined as follows. On the same five samples indicated above. LDL-cholesterol concentration was estimated as the difference between cholesterol in the d < 1.019 g/mL infranatant and cholesterol in the HDL fraction obtained by precipitating whole plasma with heparin-manganeseT4 LDL (d I .019 to 1.067 g/mL) also was isolated by ultracentrifugation, and LDL-cholesterol** and LDL-apo B2’were measured to determine the LDL-apo B/cholesterol ratio. The LDL-apo B concentration was Table 2.

Concentrations

Patient(s)

Total Cholesterol

JS

115 + 10

Normal men

179 f 2

then obtained by multiplying the LDL-cholesterol concentration by the LDL-apo B/cholesterol ratio just as was done for VLDL + IDL measurements. The radioactivity curves for (VLDL + IDL)-apo B and LDL-apo B derived in the above analyses were subjected to multicompartmental analysis according to Beltz et al.” The model employed in the current analysis is shown in Fig 1. This model makes the following assumptions: (I) all apo B enters plasma as VLDL-apo B in a precursor pool (V,); (2) the ape B in V, can have three fates: (a) direct removal from the circulation, (b) direct conversion to LDL, or (c) degradation by the delipidation chain for VLDL (V,); (3) apo B in the VLDL delipidation can pass through a variable number of delipidation steps to IDL (VlaA--Vln-I); (4) a small fraction of apo B in the delipidation chain may be diverted into a slowly catabolized pool (V,), which can either be removed directly or converted to LDL, (5) the remaining apo B in the dehpidation chain can either be convererd to LDL or removed directly from the circulation (shown here to exit from IDL but may exit anywhere along the chain); (6) IDL represents the last compartment in the delipidation chain; and lastly (7) the kinetics of LDL conform to the two-pool model of Matthews. ” Evidence for the validity of this overall model has been discussed in depth recently.‘5~‘6*26 Results obtained in our previous collaborative studies’s,‘6.26are consistent with the concept that IDL is essentially the terminal pool in the delipidation chain of VLDL-apo B. Although more investigation is needed to fully define the kinetics of IDL, we conclude that the previous findings justify our combining VLDL and IDL into a single fraction. For comparison of the results of JS with normal, we have included the turnover data from the study of Kesaniemi et al’? the results for VLDL-apo B and IDL-apo B were combined in a way to closely approximate the analysis done for (VLDL + IDL)-ape B in JS. Kinetics of VLDL-TG The turnover of VLDL-TG was determined after completion of the turnover study for apo B. The method employed is that described by Zech et al. x During this study, the patient received a fat-free diet which contained 60% of maintenance calories as described previously.” Thirty-six hours after starting the fat-free diet, ‘H-glycerol was injected intravenously. The resulting radioactivity curve for VLDLTG was subjected to multicompartmental anlaysis.” The model for the delipidation chain for VLDL-TG is similar to that for VLDLapo B (Fig 1); the model for VLDL-TG, however, has in addition fast- and slow-synthetic pathways for conversion of ‘H-glycerol to VLDL-TG.” It should be noted that the VLDL fraction used for this

of Plasma Lipids and Lipoproteins Total Triglycerides 80+

13

104 f 4

in Patient JS During LDL Turnover Study VLDL Cholesterol

LDL Cholesterol

13 + 3’

51 56t

17 + l$

117 *2§

HDL Cholesterol 50*

13

45 + 1

Values are given as mg/dL (mean k SD) for seven determinations for patient JS and mg/dL (mean + SE) for 855 normal men, ages 25 to 29 years, of the Lipid Reserach Clinic survey.‘r

lVLDL-cholesterol represents VLDL of d < 1 ,019 g/mL. This VLDL had the following percentage composition from seven determinations: total cholesterol 12 + 1%. triglycerides 32 k 30/o, phospholipids 39 + 3%, and total protein 16 k 2%. Apo 8 was determined to be 43 k 4% of total protein. This composition can be compared to that obtained for VLDL (d < 1 .O 19 g/mL) for 12 normal men by Mattson and Grundy’“: total cholesterol 14 + 2%. triglycerides 35 -t 5%. phospholipids 32 r 5%. and total protein 13 2 1%. TLDL-cholesterol represents LDL of d = 1 ,019 to 1.063

g/mL. This LDL had the following percentage composition: total cholesterol 3 1 + 1%,

triglycerides 6 k 3%. phospholipids 36 k 3%, and total protein 26 2 2%. Apo B was determined to be 98% of total protein. These results can be compared to the composition for LDL obtained by Mattson and Grundy”: total cholesterol 33 + 1%. triglycerides 6 r 1%. phospholipids 23 + 2%, and total protein 22 + 1%. SVLDL represents d -C 1.006 g/mL. SLDL represents d = 1.006 to 1.019 g/mL.

UNUSUAL

265

CASE OF HYPOBETALlPOPROTElNEMIA

Table 3.

Lipid Composition

of Gallbladder

of bile with cholesterol thus was relatively low in JS. Compared to middle-aged men (Table 4), hourly hepatic secretion rates of cholesterol and phospholipids in JS were relatively low, while secretion rates for bile acids were relatively high. The distribution among the individual bile acids was in the normal range (Table 4).

and Hepatic Bile

Lipid Composition Bile Cholesterol

Bile Acids

Phospholipids

Saturation

lmolar %I

(molar %)

(molar %I

(%I

Gallbladder Bile

3.6

84

12

77

Hepatic

3.1

86

11

79

77 i 4

18 t 3

92 * 14

PatientIs)

-

JS Bile

Cholesterol Balance and Sterol Absorption

Controls* Hepatic

Bile

5.0

i

0.9

The results of gallbladder bile are the mean of three determinations. For hepatic bile obtained during measurements of hepatic secretion rates, six samples were analyzed. Cholesterol saturation was calculated according to Carey and Smal13susing the critical tables of Careyse and assuming a total lipid content of gallbladder bile of 10% solids and hepatic bile of 5% solids. *Composition of stimulated hepatic bile is for 14 control men.”

of density less than 1.006 g/mL, which was the same as that of our normal subjects used for comparison.” study was

Results for cholesterol balance and absorption of cholesterol and bile acids for JS are shown in Table 5. Data for cholesterol balance in 14 normal, middle-aged men are shown for comparison. The patient’s excretion of neutral steroids was in the normal range. In contrast, excretion of bile acids (acidic steroids) was increased strikingly, being elevated almost threefold above normal. As a result, fecal balance (total steroid output minus cholesterol intake) was approximately twice normal. Values for percentage absorption of both cholesterol and bile acids were in the normal range, compared to percentages from nine normal subjects. Apo B Kinetics

RESULTS

Plasma Lipids and Lipoproteins

Plasma concentrations of total cholesterol, triglycerides, and cholesterol in each lipoprotein fraction (VLDL, LDL, and HDL) as determined during the turnover study of apo B for patient JS, are presented in Table 2. Corresponding values are presented for these parameters for men of the same age range obtained by the Lipid Research Clinics Prevalence study.’ Total cholesterol and LDL-cholesterol in JS were reduced markedly, both below the 5th percentile level for age and sex.’ Plasma triglycerides were well below 100 mg/dL, but VLDL-cholesterol and HDL-cholesterol were in the normal range. The patient’s relatively high concentration of HDL presents a striking contrast to his low level of LDL. The chemical compositions of the patient’s VLDL and LDL were normal (Table 2). Biliary Lipid Metabolism

The compositional data for lipids in gallbladder bile and stimulated hepatic bile for JS are given in Table 3, and mean values for stimulated hepatic bile from 14 normal, middleaged men are given for comparison. The molar percentages of cholesterol and phospholipids were lower and percentage of bile acids higher for JS than control values. The saturation

Table 4.

Wary

Lipid Secretion

Kinetic data for apo B in JS are presented in Table 6, and they are compared to results recently reported for five menI In JS, the concentration of LDL-apo B (29 mg/dL) was well below the mean for normal men [87 2 9 (SEM) mg/dL]. His production rate for LDL-apo B was 5.2 mg/kg/d, which likewise was below the normal mean of 13.1 * 0.8 mg/kg/d; however, his FCR for LDL-apo B (0.04 pools/d) was only minimally above the mean for controls, which was 0.36 k 0.01 pools/d. In control subjects, the delipidation chain for VLDL-apo B contributed a mean of 8.0 k 0.8 mg/kg/d to turnover of LDL-apo B, while 5.1 -r- 0.9 mg/kg/d was derived from “direct” input, ie, directly from compartment V,. In JS, only 2.2 mg/kg/d of apo B reached LDL though the delipidation chain, whereas 3.0 mg/kg/d of apo B entered LDL “directly.” The lower production rate for LDL in JS can be explained from his metabolism of VLDL. Input into the delipidation chain for VLDL-apo B (V, - Vi,) was somewhat less in the patient (8.8 mg/kg/d) than in controls (12.5 + 2.2 mg/ kg/d). In contrast, the quantity of (VLDL t. IDL)-apo B removed directly from the circulation in JS (ie, 6.6 mg/ kg/d) greatly exceeded the value for controls (3.6 + 0.2 mg/kg/d). Whereas only 29% of VLDL-apo B in the delipidation chain was removed directly from the circulation in

and Individual Bile Acid Composition

LipidComposition* (w/kg/h)

~__^

BileAcid Compositionf (molar%) BileAcids

LCA

DCA

CDCA

CA

JS

0.41

t 0.03

3.11

* 0.33

15.1 k 2.8

1.4

15

39

45

0.7

Controls~

0.63

+ 0.07

4.81

-t 1.21

13.1 r 2.4

1 .o

23

38

36

2.4

Patient(s)

Cholesterol

Phospholipids

UDCA

Abbreviations: LCA, lithocholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid: UDCA. ursodeoxycholic acid. *Hourly outputs were measured during a period of a six-hour formula infusion (steady state) after an initial infusion period of four hours (equilibration period). TMean composition throughout the infusion study. fResults in controls have been published previously fn = 14).13

266

VEGA ET AL

Table 5. Cholesterol

Balance and Absorption

Rates of Cholesterol

FsCtll Neutral Steroids (mglkgld)

F.Xsl Acidic Steroids (melkeld)

JS

6.1 + 0.6

13.9 r 2.1

17.9 k 2.2

Controls’

6.5 + 1.8

4.9 * 1.1

9.6 f 2.2

Patient

Cholesterol BslsnlX (r&kg/d)

and Bile Acids

Cholesterol Absorption (%) 49 40-84

(range)

Bile Acid

Absorption (%) 96.2 95.8-99.3

(range)

Values are given as mean * SD. *Results in controls (14 normal middle-aged men) for cholesterol balance13 and absorption rates of cholesterol and bile acids in nine normal subjects have been presented previously.‘4

controls, fully 75% was cleared directly in JS. Essentially, no VLDL-apo B entered the slowly removed compartment (V,) in JS. The rate of entry of apo B into compartments V, in JS was estimated to be 20.8 mg/kg/d compared to 19.6 + 1.7 mg/kg/d for controls. If it is assumed that direct removal of VLDL-apo B from V, is equal to that from the chain pathway (V,, - I), then the patient’s direct clearance from V, of 9.0 mg/kg/d greatly exceeded that of controls (2.3 f 0.3 mg/kg/d). However, since compartment V, is a hypothetical, rapidly turning-over pool of VLDL, which was not labeled directly, undue emphasis cannot be put on the fraction of apo B removed directly from this pool. In our view, the conclusion that there is an increase in direct removal of VLDL is amply demonstrated from the proportion of apo B removed directly from the delipidation chain. VLDL-TG

KiPretics

During the study of VLDL-TG kinetics, the patient’s concentration of VLDL-TG averaged 37 mg/dL, which was much lower than the control value of 137 mg/dL. His FCR

Table 6. Kinetics of Apo B in JS Compared

to Five Normal Men

JS

Normal Subjects

VLDL + IDL metabolism Apo B concentration (mg/dL)

7.4

14.5

8.8

12.5 + 2.2

2.2

8.0 + 0.8

6.6

3.6 + 0.2

0

0.9 * 0.2

2.6

2.3 t 0.3

Input into chain pathway (V, - V,,l(mg/kg/d) Conversion to LDL N,, -

I-

LDL)(mg/kg/d)

Direct removal V,, -

I-

out)(mg/kg/d)

Conversion to slow path (V,. - V,, - V,Hmg/kg/d) FCR for (VLDL + IDL)-apo B (pools/d) LDL metabolism Apo B concentration (mg/dL) Total input (mg/kg/d)

29 5.2

87 f 9 13.1 + 0.8

“Direct” input (V, - LDLHmg/kg/d) FCR for LDL-apo 8 (pools/d) Direct removal from V, (mg/kg/d) Total Apo 8 production

3.0 0.40

5.1 + 0.9 0.36

& 0.01

9.0

2.3 + 0.3

20.8

19.6 f 1.7

Values for five normal subjects are presented as mean + SEM.

for VLDL-TG was 0.67 pools/h, which was markedly higher than the normal value of 0.21 * 0.02 (SEM) pools/h obtained from 27 normal subjects.28 The transport rate (ie, production rate) of VLDL-TG in JS was 12.0 mg/kg/h, compared to a normal value of 11.4 + 0.7 mg/kg/h.28 DISCUSSION

Patient JS has an unusual disorder characterized by a high rate of excretion of bile acids and a low concentration of plasma LDL-cholesterol. Although the clinical features of JS in many ways resemble those reported previously for heterozygous familial hypobetalipoproteinemia, our metabolic data suggest that our patient probably did not have the classical disorder.‘*2 Neither of his parents had abnormally low levels of plasma cholesterol; furthermore, the patient had retinitis pigmentosa. These findings are not characteristic of the heterozygous condition, although we cannot be certain that the retinitis was related to his low levels of LDL. These discrepancies, together with the presence of an abnormality in metabolism of bile acids, suggest that JS did not have typical heterozygous familial hypobetalipoproteinemia, but rather a variant form of hypobetalipoproteinemia. The increased synthesis of bile acids in JS could have resulted from a defect in reabsorption of bile acids or from a primary increase in bile acid synthesis. The percentage absorption of bile acids in JS was not abnormally low, but it is difficult to rule out a minor defect in reabsorption of bile acids that would enhance fecal outputs. Still, hourly outputs of biliary bile acids were relatively high, suggesting that the enterohepatic circulation was not depleted of bile acids. If increased losses of bile acids into feces were not the result of malabsorption, then there must have been a primary increase in the activity of cholesterol-7a-hydroxylase, the ratelimiting enzyme in the synthesis of bile acids. The reason for a persistent oversynthesis of bile acids in the face of an apparently normal pool of bile acids in the enterohepatic circulation is puzzling. Perhaps the patient had a defect in control systems for induction and repression of bile acid synthesis. Conceivably, he could have deletion of an operator gene that normally would switch off the operon controlling the formation of cholesterol-7cu-hydroxylase in the presence of an adequate bile acid pool. Such a defect, of course, would be difficult to prove but is consistent with the observed data of this patient. A high rate of conversion of cholesterol into bile acids should have consequences for the metabolism of LDL; this is

UNUSUAL

CASE OF HYPOBETALIPOPROTEINEMIA

revealed by the LDL-lowering actions of bile acid sequestrams and ileal dysfunction. With either means of interrupting the enterohepatic circulation, the transformation of cholesterol into bile acids is enhanced; this reduces hepatic pools of cholesterol and thereby stimulates the synthesis of LDL receptors.29 As a result, plasma levels of LDL decline. The reduction in concentration of LDL seemingly occurs by two mechanisms: (1) the fractional clearance of LDL is increased, and (2) uptake of VLDL remnants, the precursors of LDL is enhanced. 29 The latter response, which should cause a fall in production of LDL, occurs because VLDL remnants also are removed by LDL receptors.29.30 Indeed, VLDL remnants may bind to LDL receptors better than LDL itself because remnants contain apo E, recent studies indicate that apo E has a greater affinity for LDL receptors than does apo B3’ and the presence of apo E on apo B-containing lipoproteins thus promotes their clearance by receptors. Did our patient actually have an increase in activity of LDL receptors, as might be anticipated from his high synthetic rate of bile acids? Certainly, his very low concentration of LDL is compatible with a high activity of receptors, and so was his reduced input of LDL. Another possible cause for a decrease in production rate of LDL could have been a reduction in the synthesis of VLDL. According to our calculations, however, the patient’s synthesis of VLDL-apo B was not reduced. Production rates for VLDL-apo B were estimated to be in the normal range. On the other hand, the percentage of direct removal of VLDL-apo B was enhanced at least twofold above normal. The two levels of LDL in JS thus appear to be mainly due to a high rate of clearance of VLDL remnants and not to reduced synthesis of VLDL. As indicated above, an accelerated removal of VLDL remnants is consistent with an enhanced activity of LDL receptors. We cannot fully explain why JS did not have a higher FCR for LDL, if he had an increased activity of LDL receptors. One possible explanation for the high rate of removal of VLDL without a comparable increase in FCR for LDL could be that the increase in LDL receptors in this patient was confined to the liver. Since VLDL remnants are cleared exclusively by receptors on hepatocytes, while LDL can be removed by either receptor or nonreceptor pathways in liver or extrahepatic tissues, an increase in hepatic LDL receptors should have a greater impact on the rate of removal of VLDL remnants than LDL on the fractional clearance of LDL. In JS, the actual mass of apo B entering the LDL fraction via the delipidation chain for VLDL was extremely low. A larger fraction of LDL-apo B seemingly was derived from “direct” input of LDL. We have postulated recently that most of the direct input of LDL comes from very rapid catabolism of newly secreted VLDL,15 but it also could come from direct secretion of IDL or of triglyceride-rich LDL, both of which should be catabolized rapidly to native LDL. As shown in Fig 1, the assumption was made that the precursor for the “direct” input of LDL (ie, compartments V,) also has a direct removal pathway, and the magnitude of direct removal pathway was assumed to approximate that of

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the delipidation chain.” If so, a significant quantity of newly secreted VLDL-apo B would have been cleared directly from the precursor compartment in JS; however, we had no direct measurement of the magnitude of this pathway. Previous investigators4 have reported that low concentrations of LDL in heterozygous familial hypobetalipoproteinemia result from decreased synthesis of VLDL-apo B. Unexpectedly, in JS the reduced level of LDL apparently occurred because of enhanced clearance of precursors to LDL, and not because of a low synthetic rate for VLDL. Unfortunately, this conclusion is “model dependent,” ie, it depends on the validity of the model used for analyzing the metabolism of lipoproteins containing apolipoprotein B. Nonetheless, the data for JS when compared to those for normal subjects strongly suggest that JS had a high direct removal of VLDL-apo B. Our data do not support the concept that the low plasma LDL in JS was secondary to reduced synthesis of VLDL-apo B. Further evidence that the production of VLDL was normal in JS comes from the kinetics of VLDL-TG. His production rate for VLDL-TG was in the normal range (12 mg/kg/h). In contrast, a previously reported patient with a variant of familial hypobetalipoproteinemia demonstrated a reduced synthesis of VLDL-TG (2.8 mg/kg/h).5 Studies in both patients were carried out in the same laboratory by the same technique. The method employed an endogenous tracer to estimate turnover of VLDL-TG, which eliminated potential artifacts from exogenous labeling. Thus, the kinetics of VLDL-TG in JS, which differed from those of the previous patient,’ bolster our contention that JS had a normal production rate for VLDL. Our findings in JS differ from those reported for heterozygous familial hypobetalipoproteinemia; in the latter, synthesis of VLDL appeared to be reduced.4 Our patient’s concomitant abnormality in bile acid synthesis may be another manifestation of his unique condition. If so, we should consider whether low concentrations of LDL in general can occur by either of two processes: (1) decreased synthesis of VLDL and (2) increased activity of LDL receptors with enhanced clearance of VLDL remnants. We propose that JS had the latter abnormality. One argument against this hypothesis might be that the FCR for LDL-apo B was not high enough to signify a marked increase in activity of LDL receptors. However, we recently noted that the quantity of VLDL converted to LDL, or the rate of production of LDL, may be a more sensitive indicator of activity LDL receptors activity than the FCR for LDL.3’ This may be so because VLDL remnants have a greater affinity for hepatic LDL receptors than LDL itself. Thus, JS appears to represent one form of hypobetalipoproteinemia having an increased clearance of VLDL remnants, which would be secondary to enhanced activity of LDL receptors. An increase in LDLreceptor activity in this patient may have been due to a defect in the synthesis of bile acids, which led to enhanced conversion of cholesterol to bile acids. A search for the biochemical basis of the defect in this patient could lead to a better understanding of the regulation of bile acid synthesis and the relation of bile acids to LDL metabolism.

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ACKNOWLEDGMENT

The authors express their appreciation to Carolyn Croy, Ruth Jiles-Jackson, Deidra Lewis, Cinthia Stenoien, Marjorie Whelan, and the nursing and dietetics staffs of the General Clinic Research Center, Parkland Memorial Hospital, Dallas; also, to Petra Pitters and Staniak Weiner of the Department of Medicine, University of Bonn. REFERENCES 1. Fredrickson DS, Gotto AM, Levy RI: Familial Lipoprotein

Deficiency (Abetalipoproteinemia, Hypobetalipoproteinemia, and Tangier Disease), in Stanbury JB, Wyngaarden JB, Fredrickson DS (eds): The Metabolic Basis of Inherited Disease (ed 3): New York, McGraw-Hill, 1972, pp 493-530 2. Herbert PN, Assman G, Gotto AM, et al: Familial lipoprotein deficiency: Abetalipoproteinemia, hypobetalipoproteinemia, and Tangier Disease, in Stanbury JB, Wyngaarden JB, Fredrickson DS, et al (eds): The Metabolic Basis of Inherited Diseases (eds). New York, McGraw-Hill, 1983, pp 589-621 3. Levy RI, Langer T, Gotto AM, et al: Familial hypobetalipoproteinemia, a defect in lipoprotein synthesis. Clin Res 18:539, 1970 4. Sigurdsson G, Nicoll A, Lewis B: Turnover of apolipoprotein-B in two subjects with hypobetalipoproteinemia. Metabolism 26:2531,1977 5. Steinberg D, Grundy SM, Mok HYI, et al: Metabolic studies in an unusual case of asymptomatic familial hypobetalipoproteinemia with hypoalphalipoproteinemia and fasting chylomicronemia. J Clin Invest 64:292-301, 1979 6. Malloy MJ, Kane JP, Hardman DA, et al: Normotriglyceridemic abetalipoproteinemia. Absence of the B-100 lipoprotein. J Clin Invest 67:1441-1450, 1981 7. The Lipid Research Clinics Population Studies Data Book, volume 1. The Prevalence Study. US Department of Health, Education, and Welfare. NIH Publication No. 79-1527, July 1979 8. Miettinen TA, Ahrens EH Jr, Grundy SM: Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 6:4l l-424, 1965 9. Grundy SM, Ahrens EH Jr, Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 6:397-4 10, 1965 10. Grundy SM, Ahrens EH Jr, Salen G: Dietary beta-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J Lipid Res 10:91-107, 1969 11. Hofmann AF, Grundy SM, Lachin JM, et al: Pretreatment biliary liquid composition in white patients with radiolucent gallstones in the National Cooperative Gallstone Study. Gastroenterology 83:738-752, 1982 12. Grundy SM, Metzger AL: A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62:1200-I 217,1972 13. von Bergmann K, Mok HY, Hardison WGM, et al: Cholesterol and bile acid metabolism in moderately advanced, stable cirrhosis of the liver. Gastroenterology 77:1183-l 192, 1979 14. Mok HYI, von Bergmann K, Grundy SM: Effect of continuous and intermittent feeding on biliary liquid outputs in man: Application for measurements of intestinal absorption of cholesterol and bile acids. J Lipid Res 20:389-398, 1979 15. Beltz WF, Kesaniemi YA, Howard BV, et al: Development of

an integrated model for analysis of the kinetics of apolipoprotein B in plasma VLDL, IDL, and LDL. J Clin Invest 76:575-585,1985 16. Kesaniemi YA, Beltz WF, Grundy SM: Comparison of metabolism of apolipoprotein B in normal subjects, obese patients, and patients with coronary heart disease. J Clin Invest 76:586-595, 1985 17. McFarlane AS: Efficient trace-labeling of proteins with iodine. Nature 182:53, 1958 18. Bilheimer DW, Eisenberg S, Levy RI: The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. Biochim Biophys Acta 260:212-221, 1972 19. Vega GL, Beltz WF, Grundy SM: Low density lipoprotein metabolism in hypertriglyceridemic and normolipidemic patients with coronary heart disease. J Lipid Res 26: 115-I 26, 1985 20. Egusa G, Brody DW, Grundy SM, et al: Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies at very low density lipoprotein and low density lipoprotein apolipoprotein B. J Lipid Res 24:1261-1267, 1983 21. Vega GL, Grundy SM: Comparison of apolipoprotein B to cholesterol in low density lipoproteins of patients with coronary heart disease. J Lipid Res 25:58&592, 1984 22. Roeschlau P, Bernt E, Gruber W: Enzymatic determination of total cholesterol in serum. Z Klin Chem Klin Biochem 12:226, 1974 23. Wahlefeld AW: Triglyceride determination after enzymatic hydrolysis, in Methods of Enzymatic Analysis. Bergmeyer HU (ed), Orlando, FL, Academic, 1974, p 1931 24. Lipid and lipoprotein analysis, in Manual of Laboratory Operations. Lipid Research Clinic Program. DHEW publication No. NIH/75-628. Washington DC, Government Printing Office. 25. Matthews CME: The theory of tracer experiments with “‘I-labelled plasma proteins. Phys Med Biol 2:36-53, 1957 26. Egusa G, Beltz WF, Grundy SM, et al: The influence of obesity on the metabolism of apolipoprotein B in man. J Clin Invest 76:596-603,1985 27. Zech LA, Grundy SM, Steinberg D, et al: A kinetic model for production and metabolism of very low density lipoprotein triglycerides: Evidence for a slow production pathway and results for normolipidemic subjects. J Clin Invest 62: 125 1-l 273, 1979 28. Beil U, Grundy SM, Crouse JR, et al: Triglyceride and cholesterol metabolism in primary hypertiglyceridemia. Arteriosclerosis 2:44-57, 1982 29. Brown MS, Goldstein JL: Lipoprotein receptors in the liver: Control signals for plasma cholesterol traffic. J Clin Invest 72:743747.1983 30. Grundy SM, Vega GL, Bilheimer DW: Influence of combined therapy with mevinolin and interruption of bile-acid reabsorption on low density lipoproteins in heterozygous familial hypercholesterolemia. Ann Intern Med 103:339-343, 1985 3 1. Maley RW, Innerarity TL: Lipoprotein receptors and cholesterol homeostasis. Biochim Biophys Acta 737:197-222, 1983 32. Grundy SM, Vega GL: Influence of mevinolin on metabolism of low density lipoproteins in primary moderate hypercholesterolemia. J Lipid Res 26:1464-1475, 1985 33. Salen G, Shefer S, Berginer VM: Familial disease with storage of sterols other than cholesterol: Cerebrotendinous xanthomatosis and sitosterolemia with xanthomatosis, in Stanbury JB, Wyngaarden JB, Fredrickson DS, et al teds): Metabolic Basis of Inherited Disease (ed 5). New York, McGraw-Hill, 1983, pp 713-730 34. Ballantyne CM, Vega GL, East C, et al: Low density

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