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Original Studies |
Centre de Recherche en Nutrition Humaine (C.M., K.O., R.F., P.M., T.M., M.K.) and Clinique d’Endocrinologie, Maladies Métaboliques et Nutrition (B.C., M.K.), Hôtel Dieu, 44093 Nantes Cedex 01, France
Address all correspondence and requests for reprints to: Dr. Michel Krempf, Clinique d’Endocrinologie, Hôtel Dieu, 44093 Nantes Cedex 01, France. E-mail: mkrempf{at}sante.univ-nantes.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cholesterol-lowering drugs (i.e. HMGCoA reductase inhibitors, bile acid sequestrant) have been effective in most heterozygous FH patients. An additional treatment is the use of LDL-apheresis, consisting of the extracorporeal removal of LDL. This intervention results in a marked reduction of plasma cholesterol, which returns to baseline after several days (8, 9, 10). This treatment is useful for patients who do not respond to drug therapy and may also be a good tool to determine the effect of the large pool size of LDL on the metabolic disturbances found in FH patients.
Kinetic studies performed 5–7 days after LDL-apheresis did not alter the kinetics of apolipoprotein B (apoB)-containing lipoproteins using exogenous (11) or endogenous (12, 13) labeling. These data suggested that the reduction of the plasma LDL pool size did not change apoB metabolism in either heterozygous FH patients (13) or normolipidemic subjects (12) in the long term. However, these kinetic studies were undertaken 1 week after apheresis, when the circulating LDL level had almost returned to baseline. Studies undertaken 1 day after apheresis showed that cholesterol synthesis is stimulated by apheresis (14, 15), suggesting that apheresis could affect apoB metabolism for a very short time period. Only two kinetic studies were performed immediately after apheresis when the apoB and cholesterol levels were significantly decreased compared with baseline levels. In normolipidemic subjects an increased FCR of LDL was found 2 days after apheresis (12). No change in the production rate of VLDL and LDL was observed; however, the nonsteady state of the LDL-apoB pool size was not taken into account in the modeling procedure. In another kinetic study undertaken in heterozygous FH, immediately after apheresis (16), the LDL FCR was significantly increased, with no change in apoB production; however, no information was given about the model used for the data analysis. In addition, a recent study showed that the LDL-apoB level rebound after apheresis cannot be fitted using unchanged LDL-apoB kinetic parameters (17). The researchers suggested that LDL-apheresis may affect LDL-apoB kinetics. Thus, an important question remains unsolved. Is LDL-apheresis able to induce a change in the apoB kinetics of FH patients over a short time period?
In the present study we examined the kinetics of apoB-containing lipoproteins in heterozygous FH patients before and the day after apheresis, when the LDL plasma level was still markedly decreased. These conditions allowed dissection of the role of the receptor defect from the role of the increased LDL pool on apoB metabolism in heterozygous FH. After apheresis we found a tendency for increases in VLDL production and LDL catabolism compared with basal values, whereas direct LDL production decreased.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Five heterozygous FH patients (three men and two women, 53
± 9 yr old) and five normolipidemic subjects (all men; 24 ± 2 yr
old) participated in the study. The characteristics of patients and
controls are shown in Table 1
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A kinetic study was performed on control subjects to determine control values of apoB kinetics. These subjects were not subjected to apheresis. Informed consent was obtained before the study, according to protocols approved by the ethical committee of the University Hospital of Nantes Medical School.
Kinetic study procedure
Subjects were instructed to consume a diet containing 50% of the daily caloric intake as carbohydrate, 35% as fat, and 15% as protein for at least 1 week before the study. [2H3]Leucine (99.8% enrichment; Mass Trace, Woburn, MA) was dissolved in a saline solution and tested for pyrogenicity and sterility before the study. After a 12-h fast a priming dose of 10 µmol/kg [2H3]leucine was administered, immediately followed by a constant infusion of 10 µmol/kg·h over a period of 14 h. The subjects fasted during the entire procedure. Venous blood samples were withdrawn in ethylenediamine tetraacetate tubes (Venoject, France) at baseline, every 15 min during the first hour, then every 30 min during the following 2 h, and finally hourly through the end of the isotope infusion. Plasma was separated by centrifugation for 30 min at 4 C. Sodium azide and gentamicin were added to each plasma sample to final concentrations of 0.05% and 0.005%, respectively.
Analytical procedures
Very low density lipoprotein (VLDL; density, <1.006 kg/L), intermediate density lipoprotein (IDL; density, 1.006–1.019 kg/L), and LDL (density, 1.019–1.063 kg/L) were isolated from 3–4 mL plasma by sequential ultracentrifugation using standard methods (18) with a fixed angle rotor at 40,000 rpm for 22 h at 10 C (Himac CP70, Hitachi, Hialeah, FL). Apolipoproteins of the lipoprotein fraction were concentrated (19), and apoB100 was isolated from other apolipoproteins by SDS-PAGE using a discontinuous gel gradient (4%, 5%, and 10%). Apolipoproteins were identified by comparing migration distances to known molecular weight standards. Apolipoprotein bands were excised from polyacrylamide gels dried in vacuo for 2–3 h (RC 10–10, Jouan, Saint Herblain, France). The desiccated gel slices were hydrolyzed with 1 mL 4 mol/L HCl (Sigma, St. Quentin Fallavier, France) at 110 C for 24 h.
Hydrolysates were evaporated to dryness, and the amino acids were purified by cation exchange chromatography using a Temex 50W-X8 resin (Bio-Rad Laboratories, Inc., Hercules, CA). Amino acids were derivatized using heptafluorobutyric anhydride (Fluka, Switzerland) before analysis by electron impact gas chromatography-mass spectrometry (5891A gas chromatograph connected with a 5971A quadrupole mass spectrometer, Hewlett-Packard Co., Palo Alto, CA). The procedures for chromatographic separations and mass spectrometric analyses of leucine were performed as previously described (20). Isotopic abundance was reported as the tracer to tracee mass ratio (21). ApoB concentrations were obtained in lipoprotein fractions using the isopropanol precipitation method (22). The leucine content in precipitated apoB was determined using norleucine as the internal standard. A constant amount of norleucine (6 µmol) was added to precipitated apoB, and the same procedure for hydrolysis, cation exchange chromatography, and derivatization (described above) was undertaken. In mass spectrometry, the ratio of leucine ion abundance to norleucine ion abundance was used to determine the quantity of leucine in the sample by comparison to a standard curve containing different amounts of leucine (0–200 µmol) and a constant amount of norleucine (6 µmol). The corresponding amount of apoB was then calculated according to the number of leucine molecules per apoB molecule (23, 24). The comparison of this internal standard method to the Lowry method (25) for apoB measurement has shown a linear regression slope of 1.1 (r2 = 0.98) on 10 samples of LDL-apoB. The VLDL-, IDL-, and LDL-apoB pool sizes were determined at three to five different times during the kinetic study before apheresis and at all time points during the postapheresis kinetic study.
For the control subjects, apoB concentrations were measured in lipoprotein fractions by immunonephelometry (Behring, Paris, France). Cholesterol and triglycerides were measured using commercially available enzymatic kits (Roche Molecular Biochemicals, Mannheim, Germany). The pool size of apoB (milligrams per kg) was calculated by multiplying the apoB plasma concentration (milligrams per L) in each fraction by 0.045 (plasma volume equal to 4.5% body weight).
Modeling
ApoB leucine tracer to tracee ratios in VLDL, IDL, and LDL were
analyzed by multicompartmental modeling. The model used was a minimal
compartmental model for apoB metabolism (Fig. 1). In our experimental conditions a more
complex compartmental model, previously used with data obtained for a
longer time experiment (26), did not provide a
statistically better fit of tracer to tracee ratio data
(27). Our model included a precursor pool (compartment 1)
from which apoB enters the VLDL compartment (compartment 10), IDL
compartment (compartment 20), or LDL compartment (compartment 30) after
a delay compartment (compartment 2). VLDL-apoB is converted into
IDL-apoB or directly into LDL-apoB. ApoB can be removed from any
compartment. Our model can estimate 1) the FCR of VLDL as the sum of
the rate constants of conversion of VLDL into IDL and LDL and direct
removal of VLDL; 2) the FCR of IDL as the sum of rate constants of IDL
conversion into LDL and direct removal of IDL, and 3) the FCR of LDL as
the rate constant of e removal of LDL from plasma. Compared with our
previously published model developed using the same experimental
conditions (27, 28), direct input of IDL-apoB and LDL-apoB
was added. The direct conversion process of VLDL-apoB into LDL-apoB was
added to fit some patient data. The introduction of this shunt pathway
for control subjects resulted in multiple solutions for the model;
therefore, the shunt pathway process was not included for controls. The
SAAM-II program (29) was used to determine the parameters
of the model. The plasma free leucine tracer to tracee ratios were used
as a forcing function describing the tracer to tracee ratios of the
precursor pool (compartment 1) during the course of the infusion. The
forcing function was created in SAAM-II, by linear interpolation
between sequential data of plasma leucine tracer to tracee ratios.
During the preapheresis kinetic study, we assumed that each subject
remained in the steady state condition regarding transfer coefficients
and apoB pool sizes in each compartment. The apoB production rate in
milligrams per kg/day was calculated as the product of FCR and the pool
size of apoB in the lipoprotein fraction. For the postapheresis kinetic
study, the model had to take into account the nonsteady state of tracee
mass (apoB pool size) of VLDL, IDL, and especially LDL, which was
increasing during the 14-h study. The nonsteady state condition was
modeled using two experiments in the SAAM-II program of the model shown
in Fig. 1. One experiment (tracer experiment) corresponded to the time
course of tracer in each compartment, and the second experiment (tracee
experiment) represented the time course of apoB (tracee) in each
compartment. The tracer to tracee ratio data provided the link between
these two experiments; the tracer experiment provided the numerator of
the ratio, and the tracee experiment provided the denominator of the
ratio. The tracee experiment was also associated with the VLDL-, IDL-,
and LDL-apoB concentrations measured during the study.
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Data are expressed as the mean ± SD unless
otherwise stated. Paired Student’s t test was used for
comparison of data obtained before and after the apheresis kinetic
study. Unpaired Student’s t test was used to compare the
data for control subjects with the data for patients. The correlation
between the change in LDL cholesterol and the LDL FCR was examined by
linear regression analysis. P 
0.05 was considered
significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, apheresis treatment decreased the
concentrations of plasma cholesterol and LDL cholesterol in the five
patients (65 ± 6% and 77 ± 5%, respectively). In the
postapheresis kinetic study, the plasma and LDL cholesterol
concentrations were not significantly different between patients and
controls. The plasma triglyceride concentration of patients was
significantly higher than that of controls and was not different in the
two kinetic studies. ApoB concentrations obtained pre- and
postapheresis kinetic studies for patients and controls are shown in
Table 2. The VLDL-apoB
concentration of patients did not change significantly between the pre-
and postapheresis kinetic studies and was not statistically different
from the control value. The IDL-apoB concentration of patients was
significantly higher than the control value and was significantly
decreased in the postapheresis kinetic study, with no significant
difference between patients and controls. The LDL-apoB concentration of
patients was significantly higher than the control value and was
significantly decreased in the postapheresis kinetic study, but
remained significantly higher than the control value. In the
postapheresis kinetic study, VLDL-apoB and IDL-apoB reached a steady
state, whereas LDL-apoB was increasing during the entire study period
(Fig. 2).
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for one patient, and apoB turnover
rates calculated for each model pathway are shown in Fig. 1. Kinetic
parameters calculated for patients in the pre- and postapheresis
kinetic studies and in control subjects are shown in Table 3. In the preapheresis study a
significant decrease in the FCRs of VLDL, IDL, and LDL and a
significant increase in LDL production were observed in patients
compared with controls. The increased LDL production was mainly due to
a high direct LDL production in patients compared with controls
(9.2 ± 7.8 vs. 1.4 ± 1.2 mg/kg·day;
P = 0.057). For heterozygous FH patients, VLDL and LDL
direct production accounted for 51 ± 7% and 33 ± 15% of
the total apoB production, respectively, whereas in controls the
corresponding values were 80 ± 7% and 8 ± 7%
(P < 0.01). No statistical differences were found
between kinetic parameters obtained before and after apheresis, as
indicated by paired t test. However, there was a trend for
an increase in the FCR of LDL induced by apheresis that was correlated
with the decrease in LDL cholesterol (r2 = 0.76;
P < 0.05), indicating that the more LDL cholesterol
decreases the more the FCR of LDL increases. There was also a trend for
an increase in the production of VLDL-apoB (Table 3) in the
postapheresis study compared with the preapheresis study, resulting in
a significant increase in the fraction of VLDL-apoB synthesis of the
total apoB synthesis (76 ± 13 vs. 51 ± 7%;
P < 0.05).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A previous kinetic study performed 7 days after apheresis in heterozygous FH patients did not show any difference in the production rate or FCR of apoB-containing lipoproteins (13). The apoB kinetic experiment was performed when the plasma LDL cholesterol level was close to baseline. These data demonstrated that apheresis treatment did not induce a long-term effect on apoB kinetics in heterozygous FH patients.
To dissect out the metabolic perturbations related to the LDL-R genetic deficiency from the perturbations secondary to the elevated LDL plasma level, we performed our kinetic study on the day immediately after the apheresis procedure to study its short-term effect and assess the lipoprotein metabolism changes in an acute LDL cholesterol reduction. These specific experimental settings allowed characterization of the apoB metabolism of heterozygous FH patients in the absence of a large LDL pool size. A nonsteady state modeling procedure was required to take into account the change in apoB pool size during the postapheresis study. We used a short-term protocol consisting of an 14-h infusion of tracer and assumed that there were linear turnover rates during the study period. This approach is supported by a recent study that demonstrated that the endogenous labeling method could detect changes in LDL FCR when undertaken as soon as possible after the apheresis procedure (9).
Before apheresis, the FCR of LDL was decreased by about 60% in patients compared with controls. The values we found are similar to those in heterozygous FH patients using exogenous (see Ref. 1 for review) or endogenous labeling (6, 13). This is consistent with a LDL-R defect. After the apheresis we observed a 45% increase in the LDL FCR of patients. These results suggested that despite the genetic defect, LDL-R activity could be stimulated in heterozygous FH patients. The apheresis treatment dramatically diminished LDL cholesterol and, therefore, the net cholesterol uptake by the liver. Apheresis thus may stimulate LDL-R synthesis by a decrease in the stored cholesterol within the cell (30). This improvement in FCR may represent a reversible part of the defect in LDL catabolism in heterozygous FH.
Our data show that heterozygous FH patients have increased LDL production compared with controls. An increased plasma LDL level in heterozygous FH patients is related to both a low LDL FCR and a high LDL production (se Ref. 2 for review). Assessment of the respective contributions of VLDL conversion and direct LDL production to the total LDL overproduction is not simple and requires the use of multicompartmental modeling to include the VLDL, IDL, and LDL in an integrated model. The conversion of VLDL to LDL was determined by the injection of radiolabeled VLDL and a multicompartmental approach in which the mass of LDL that was not accounted for by VLDL conversion was attributed to direct LDL production (3, 4, 5). Endogenous labeling is a more direct method to assess direct LDL production, because the early part of the LDL tracer to tracee ratio curve can be analyzed. Using this method, the direct production of IDL and LDL was directly measured in normolidemic subjects (31, 32) and was markedly increased in heterozygous FH (6). The use of a very rapid turnover pool of VLDL directly converted to LDL was also reported (13). However, this latter option provided very high turnover rates of the VLDL shunt pathway, which do not correlate to physiological processes (32, 33). Despite the fact that our model includes a direct conversion of VLDL to LDL, direct LDL production is required to fit the LDL tracer to tracee curve and the steady state mass of LDL. Our data show that about 50% of FH patient LDL synthesis comes from direct production, whereas it is about 10% for controls. The overproduction of LDL resulting from increased direct LDL production may play an important role in the atherogenesis of heterozygous FH by increasing the amount of circulating LDL with an increased retention time in plasma due to the receptor defect. This direct LDL production was not directly demonstrated in humans, but data support the direct LDL production pathway. Cultured hepatocytes synthesized LDL (23, 34, 35, 36), and these in vitro data suggested that hepatocytes may have the in vivo ability to secrete LDL. In vivo data in guinea pigs suggested that direct production of LDL could be decreased by statins (37). Direct LDL production in heterozygous FH may be the result of a high cholesterol content in the liver, which may secrete LDL density particles (2, 5, 6, 38). Despite the LDL-R genetic deficiency, the absolute flux of cholesterol entering the liver of FH patients is higher than that in normolipidemic subjects because of the large pool size of circulating LDL (2, 7, 39). Thus, the decrease in cholesterol content in the liver after apheresis could explain the reduction of LDL direct production observed in three of the five FH patients.
We observed a 57% increase in VLDL production induced by apheresis in heterozygous FH patients. The elevated endogenous synthesis of cholesterol after apheresis (14, 40, 41) may be involved in the increased VLDL production. A correlation between VLDL production and cholesterol synthesis previously found in normolipidemic (42) and hyperlipidemic (43) subjects suggests that in heterozygous FH patients the liver may produce more VLDL-apoB in response to cholesterol synthesis stimulation. The VLDL production rate may be partly dependent on endogenous cholesterol synthesis that has been stimulated by apheresis. Apheresis changed the balance between these two distinct hepatic cholesterol pools, resulting in a coordinate change in the pattern of apoB secretion.
Statins are poorly effective in some heterozygous FH patients (8). We postulate that this treatment failed in these patients because of the high direct LDL production that increased the hepatic cholesterol content and subsequently down-regulated LDL-R activity (7, 39), preventing the effect of HMGCoA reductase inhibitors. These data support the hypothesis that in heterozygous FH, LDLR activity is substantially regulated by hepatic cholesterol content (2). On the other hand, LDL-apheresis, by the marked plasma LDL removal, is able to stimulate LDL-R activity as well as endogenous cholesterol synthesis. The increased production rate of VLDL observed after apheresis is probably a key factor in the fast relapse of the patients secondary to these combined effects. Furthermore, our data suggested a key role played by the increased LDL production in the pathophysiology of heterozygous FH enhancing the genetic deficiency of LDL-R. It would be of interest to study statin-treated patients to determine whether the drug can offset the increase in VLDL-apoB production following apheresis. These findings pointed out the relevance of the development of apoB production-lowering drugs in heterozygous FH, such as microsomal triglyceride transfer protein inhibitors targeting VLDL as well as direct LDL production.
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Acknowledgments |
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Footnotes |
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2 Present address: Hoffman-LaRoche Inc.,
Pharmaceuticals Division, Basel, Switzerland.
Received March 13, 2000.
Revised December 1, 2000.
Accepted December 6, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) and IDL (
) in the upper
panel and for LDL (•) in the lower
panel.