© 1997 American Heart Association, Inc.
Articles |
Subjects With ApoA-I(Lys107
0) Exhibit Enhanced Fractional Catabolic Rate of ApoA-I in Lp(AI) and ApoA-II in Lp(AI With AII)
From the Lipid Metabolism Laboratory and Mass Spectrometry Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Mass, and Helsinki University Central Hospital, Finland (M.T.-K., R.M., M.-R.T.).
Correspondence to Dr Marju Tilly-Kiesi, MD, PhD, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-02900 Helsinki, Finland.
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Abstract Our purpose was to examine HDL metabolism in a Finnish kindred with a 3-bp deletion in the apolipoprotein (apo) A-I gene, resulting in a deletion of Lys107 in the mature apoA-I. Patients with this mutation [apoA-I(Lys107
0)] have reduced plasma HDL cholesterol and lipoprotein (AI with AII) [Lp(AI w AII)] concentrations, but not Lp(AI) levels, compared with unaffected family members. Using primed constant infusions of [5,5,5-2H3]leucine, we determined the residence time (RT) and absolute production rate (APR) of apoA-I and apoA-II entering plasma in two subpopulations of HDL particles: [Lp(AI) and Lp(AI w AII)] in three patients heterozygous for apoA-I(Lys1070) and in seven healthy control subjects. In patients, the mean RT of apoA-I in Lp(AI) (3.75±1.68 days) was less than half that observed in control subjects (8.01±2.51 days, P<.05). The mean RT of apoA-I in Lp(AI w AII) was also lower in patients than in control subjects, but differences were not statistically significant (4.72±2.42 versus 6.50±2.19 days). The mean RT of apoA-II in Lp(AI w AII) was significantly lower in patients (5.24±1.65 days) than in control subjects (9.64±3.57 days, P<.05). The APR of apoA-I into Lp(AI) was twofold higher in patients (5.9±2.1 mg·kg-1·d-1) than in control subjects (2.5±0.9, P<.05). The APRs of apoA-I and apoA-II into Lp(AI w AII) were similar in patients and control subjects. Our results are consistent with the concept that patients heterozygous for the apoA-I(Lys1070) mutation have enhanced fractional catabolism of apoA-I and apoA-II in both HDL subspecies, especially in Lp(AI), and an increase in apoA-I production only into Lp(AI), which may be compensatory. Therefore, only their Lp(AI w AII) levels are decreased.
Key Words: apoA-I(Lys1070) variant • Lp(AI with AII) • kinetics • metabolism • coronary heart disease • HDL cholesterol • Lp(AI)
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Introduction |
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Decreased plasma HDL cholesterol concentrations have been associated with an increased risk of premature CHD.1 The main apolipoproteins of HDL are apoA-I and apoA-II, which constitute 80% to 90% of the total HDL apolipoprotein mass.2 Two distinct subpopulations of HDL particles have been isolated; particles that have apoA-I without apoA-II, or Lp(AI), and particles with apoA-I and apoA-II, or Lp(AI w AII).3 4 5 Puchois et al6 observed that compared with healthy control subjects, patients with coronary artery disease and normal triglycerides had decreased concentrations of Lp(AI) but not Lp(AI w AII) particles. In other studies, CHD patients had decreases in both types of particles compared with control subjects.7 8 9
Several apoA-I gene mutations have been shown to be associated with reductions in HDL cholesterol and apolipoprotein levels.10 11 12 13 14 15 16 17 18 Some are due to large deletions and major DNA rearrangements and some to point mutations.10 11 12 13 14 15 16 17 18 Recently, we have described a Finnish kindred with a 3-bp deletion in the apoA-I gene resulting in removal of the codon for Lys 107 in the mature apoA-I [apoA-I(Lys1070)]. Distinguishing characteristics of the patients with apoA-I(Lys1070) are reduced plasma HDL cholesterol and Lp(AI w AII) concentrations but normal Lp(AI) particle concentrations.19
Previous kinetic studies have revealed that the plasma RT of apoA-I is shorter than that of apoA-II.20 21 22 23 24 25 26 Rader et al26 have reported that the turnover rate of apoA-I in Lp(AI) particles is faster than that of apoA-I in Lp(AI w AII) particles in normal subjects. It has been reported that the FCR of apoA-I is the primary factor controlling plasma apoA-I levels.22 25
The purpose of our study was to investigate the in vivo metabolism of apoA-I and apoA-II in both HDL particles, Lp(AI) and Lp(AI w AII), in subjects heterozygous for the apoA-I(Lys1070) mutation to elucidate the kinetic mechanism accounting for the reduced Lp(AI w AII) levels in these patients.
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Methods |
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Study Subjects
Three heterozygote subjects for the apoA-I(Lys107
0) mutation and seven healthy control subjects (four males and three postmenopausal females) were included in the study. The three subjects with the apoA-I variant were a 53-year-old female (the proband), her 42-year-old brother, and their 52-year-old cousin. All have previously been demonstrated to be heterozygous carriers of a 3-bp deletion in nucleotides 1396-1398 in exon 4 of the apoA-I gene.19 The HDL cholesterol concentrations of these three apoA-I(Lys1070) subjects were abnormally low, 0.31, 0.64, and 0.68 mmol/L, respectively. The 52-year-old female had received medication for essential hypertension and for several years had taken a combination of captopril and indapamid. She reached menopause at the age of 47 and had not received any hormone replacement therapy. The other two subjects were apparently healthy. All subjects underwent a complete physical examination, and blood samples were collected for laboratory analysis. None of the subjects had signs or symptoms of CHD or thyroid or other endocrine diseases. Renal and hepatic disorders were excluded on the basis of laboratory tests and medical history. All female control subjects were postmenopausal, and none of the control subjects were receiving medication or hormone replacement therapy known to influence plasma lipid levels. None of the patients or control subjects consumed more than two to four doses of alcohol weekly before the controlled dietary period. No alcohol was included in the experimental diets. Patient 2 performed physical exercise two to three times a week, while the other two patients and the control subjects reported no physical activity beyond that of normal daily life.
At least 3 weeks before the metabolic studies, the subjects were provided with an isocaloric natural-food diet consisting of 36% fat (15% saturated, 15% monounsaturated, and 6% polyunsaturated), 15% protein, and 49% carbohydrate, with a cholesterol content of about 150 mg/1000 kcal. The study protocol was approved by the Ethical Committee of Helsinki University Central Hospital and by the Human Investigation Review Committee of the New England Medical Center and Tufts University. The clinical characteristics and plasma lipid values of all the participants are given in Table 1
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Study Protocol
The kinetic studies were carried out in each subject in the fed state as previously described.27 The subjects received 20 identical small meals comparable to the prestudy diet, given every hour starting 5 hours before (-5 hours) the stable isotope infusion. Each small meal contained 1/20 of the daily caloric intake. Each study began in the morning (6 AM) after a 12-hour fast. An intravenous line was inserted into one forearm for the infusion solution, and another line was placed into the opposite arm for blood sampling. At zero hour (11 AM), a priming dose of 10 µmol/kg of body weight of [5,5,5-2H3]leucine was given intravenously followed by a constant infusion of [5,5,5-2H3]leucine at a rate of 10 µmol·kg-1·h-1 for the next 15 hours. Blood samples were collected into tubes containing EDTA at 0, 5, 10, 15, 25, 35, and 45 minutes and at 1, 1.5, and 2 hours and every hour thereafter through the 15-hour study period for plasma enrichment measurements. Deuterium enrichment of apoA-I and apoA-II within HDL subspecies was performed from samples taken at 0, 6, 10, 12, and 15 hours in the apoA-I(Lys1070) patients and at 0, 10, 12, and 15 hours in the control subjects. The three patients were studied in the clinical research unit at the Helsinki Central Hospital, Finland, and the control subjects in the metabolic research unit of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Mass. The [5,5,5-2H3]leucine infusion solutions were prepared in Boston and shipped to Helsinki, together with special food items, to assure the identical content of the small meals during the kinetic studies. The isolation of lipoprotein classes from fresh plasma of the three patients was done in Helsinki and shipped with plasma samples to Boston on dry ice. Isolation of lipoprotein classes from fresh plasma of control subjects was done in Boston, where all the laboratory work and data analysis were performed.
Isolation of Plasma Lipoproteins
Plasma lipoprotein fractions were isolated by sequential ultracentrifugation in a Beckman L8-70 ultracentrifuge (Beckman Instruments) using a Beckman 50TI rotor as previously described.28 VLDL, IDL, LDL, and HDL were isolated at densities of 1.006, 1.019, 1.063, and 1.21 g/mL, respectively.
Separation of Lp(AI) and Lp(AI w AII)
Lp(AI) and Lp(AI w AII) particles were separated from plasma by using immunoaffinity chromatography columns as previously described.5 Briefly, monoclonal antibodies against human apoA-I and apoA-II were conjugated separately to CNBr–Sepharose 4B and cross-linked using glutaraldehyde according to the method described by Kowal and Parsons29 and McConathy.30 One milliliter of plasma from the control subjects and 2 milliliters of plasma from the apoA-I(Lys1070) patients were applied first to the anti–A-I column. The bound lipoproteins were eluted with 3 mol/L NaSCN, pH 8.0, and dialyzed for 20 hours against 0.01 mol/L Tris and 0.01 mol/L NaCl, pH 8.0, at 4°C. Next, the apoA-I–containing lipoproteins isolated with the first column were applied onto an anti–apoA-II column. The unretained fraction containing Lp(AI) and other lipoproteins with apoA-I was collected and dialyzed against 0.01 mol/L Tris and 0.01 mol/L NaCl, pH 8.0, for 20 hours at 4°C. The retained fraction containing Lp(AI w AII) was eluted with 3 mol/L NaSCN and dialyzed against 0.01 mol/L ammonium bicarbonate solution for 20 hours at 4°C. The fraction with Lp(AI) and apoB-containing lipoproteins was applied to an anti-apoB column. The unretained fraction containing Lp(AI) was dialyzed against 0.01 mol/L ammonium bicarbonate solution for 20 hours at 4°C. The Lp(AI) and Lp(AI w AII) fractions were lyophilized, and apoA-I and apoA-II were isolated from Lp(AI) and Lp(AI w AII) by using SDS–polyacrylamide gradient gel (7% to 20%) electrophoresis and a Tris-glycine buffer system.31 The SDS–polyacrylamide gradient gels were loaded by protein content. Apolipoproteins were identified by comparing migration distances with those of known molecular-weight standards.
Determination of Deuterium Enrichment
ApoA-I and apoA-II protein bands were excised from the polyacrylamide gels and hydrolyzed in 12N HCl at 110° for 24 hours and dried under nitrogen. The hydrolysates were converted to N-propyl ester and N-heptafluorobutyramide derivatives and dried under nitrogen as previously described.31 After resolubilization in ethyl acetate, the clear supernatant was placed in autosampler vials (Kimble). For the measurement of deuterium enrichment in plasma, after hydrolysis of the proteins, the free amino acids were isolated using Dowex AG-50W-X8 100- to 200-mesh cation exchange resin (Bio-Rad) and converted to the N-propyl ester and N-heptafluorobutyamide derivatives as described above. All samples were analyzed with a 5985B gas chromatograph–mass spectrometer (Hewlett Packard) using negative chemical ionization and methane as the reagent gas.
Analyses of Kinetic Data
FPRs of apoA-I and apoA-II were calculated by dividing the rate of appearance of deuterated leucine in apoA-I and apoA-II within HDL subspecies by the VLDL–apoB-100 plateau enrichment. This approach was taken on the basis of previously established findings that the VLDL–apoB-100 plateau enrichment was assumed to represent the precursor pool enrichment.32 33 34 Moreover, using appropriate isolation techniques, we have recently documented that the apoB-48 and apoB-100 plateaus within triglyceride-rich lipoproteins are very similar, thus justifying this approach (A.H. Lichtenstein, unpublished observations). The VLDL–apoB-100 plateau enrichment of each individual was calculated from a minimum of three time points, representing the highest isotopic enrichment of VLDL–apoB-100 and a deviation from the linear increase in enrichment observed for the earlier time points. The rate of appearance of deuterated leucine in apoA-I and apoA-II in Lp(AI) and Lp(AI w AII) particles was calculated by linear regression and can be used for estimating kinetic parameters for slowly turning over proteins in primed constant infusion studies.35 A 0.5-hour lag period representing the mean of the study subjects' data on the appearance of total apoA-I was factored into the calculations. Simultaneous measurement of the apoA-I production rates in Lp(AI) and Lp(AI w AII) represents the two compartments in models normally used for calculations of apoA-I kinetic parameters.22 23 APRs of the apolipoproteins were calculated by multiplying the FPRs by the pool size of the apolipoprotein in question and normalizing to body weight. Apolipoprotein pool sizes were calculated by multiplying the plasma apolipoprotein concentrations by the estimated plasma volumes (body weightx0.045). The FPR was assumed to be equal to the FCR. The data are expressed as RT, which is the reciprocal of the FPR.
Analytical Methods
Cholesterol and triglyceride levels in plasma and lipoprotein fractions were analyzed with standardized enzymatic methods.31 HDL cholesterol concentration was measured in plasma by using the dextran sulfate–Mg2+ precipitation method.36 Plasma apoA-I and apoB concentrations were assayed with a noncompetitive, enzyme-linked immunosorbent assay using immunopurified polyclonal antibodies.37 38 The concentration of Lp(AI) in the HDL subfraction was measured by using immunoelectrophoresis39 with commercially available kits consisting of hydrated agarose gels and monospecific antisera to apoA-I and apoA-II (Sebia). The concentrations of apoA-I in Lp(AI) and apoA-II in Lp(AI w AII) were determined using standards provided by the manufacturer. The concentration of apoA-I in Lp(AI w AII) particles was calculated by subtracting the Lp(AI) concentration from the total apoA-I plasma concentration as analyzed by enzyme-linked immunosorbent assay. Between- and within-run coefficients of variation for the lipid assays was <5% and for the other assays <10%.
Statistical Analyses
Statistical analyses were done with the BMDP statistical software (University of California Press, 1993). Values are expressed as mean±SD. Comparison of data between the groups was performed using the Mann-Whitney nonparametric test.
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Plasma Lipids and Lipoproteins
The characteristics of the three patients and seven control subjects are shown in Table 1
. The HDL cholesterol concentrations of the three apoA-I(Lys1070) subjects were 0.31, 0.64, and 0.68 mmol/L in patients 1, 2, and 3, respectively, all below the cut point associated with elevated risk of CHD (0.90 mmol/L). The mean HDL cholesterol concentration of the patients (0.54±0.20 mmol/L) was 45% of the mean HDL cholesterol concentration of the control subjects (1.20±0.37 mmol/L, P<.05). Plasma total cholesterol, LDL cholesterol, and triglyceride concentrations were similar in patients and control subjects (Table 1).
Patient 1 had very low plasma apoA-I and apoA-II levels (53 mg/dL and 8 mg/dL, respectively, Table 2). The mean plasma concentrations of apoA-I (93±34 mg/dL) and apoA-II (19±10 mg/dL) in the patients tended to be lower, but not significantly different, from the mean values of apoA-I and apoA-II in control subjects (118±18 and 27±1 mg/dL, respectively, Table 2). The mean apoA-I concentrations in Lp(AI) were similar in patients and control subjects (47±14 and 42±12 mg/dL, respectively). However, the mean apoA-I concentration in Lp(AI w AII) particles was significantly lower in patients than in control subjects (46±20 versus 74±8 mg/dL, P<.05). Similar to our previous findings,19 the mean HDL cholesterol/(apoA-I+apoA-II) ratio in patients (0.19) was significantly lower than in control subjects (0.32, P<.05).
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Kinetics of ApoA-I and ApoA-II
The isotopic enrichment of the apoA-I in Lp(AI) and Lp(AI w AII) of the three apoA-I(Lys1070) subjects and the mean isotopic enrichment of the normal subjects are presented in the Figure (upper and middle panels, respectively). The isotopic enrichment of apoA-II in Lp(AI w AII) of each patient compared with the mean enrichment of the control subjects is shown in the Figure (lower panel). The appearance of deuterium within total apoA-I had a mean lag time of 30 minutes, and this value was factored into the calculations. The isotopic ERs of apoA-I and apoA-II in Lp(AI) and Lp(AI w AII) of each individual subject are given in Table 3. The mean ERs of deuterated leucine of apoA-I in Lp(AI) and apoA-II in Lp(AI w AII) were higher in patients than in control subjects (Table 3).
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), 2 (
), and 3 (
) with apoA-I(Lys107
).
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The values for the RTs of apoA-I and apoA-II in Lp(AI) and Lp(AI w AII) of the patients and control subjects are shown in Table 4. In the patients, the mean RT of apoA-I in Lp(AI) (3.75±1.68 days) was less than half the mean RT of control subjects (8.01±2.51 days, P<.05). The mean RT of apoA-I in Lp(AI w AII) in patients (4.72±2.42 days) tended to be lower, but not significantly, than the mean RT of apoA-I in Lp(AI w AII) in control subjects (6.50±2.19 days). The mean RT of apoA-II in Lp(AI w AII) of the patients, 5.24±1.65 days, was clearly shorter than the mean RT of apoA-II in Lp(AI w AII) in the control subjects, 9.64±3.57 days (P<.05, Table 4).
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The individual APRs of apoA-I and apoA-II into Lp(AI) and Lp(AI w AII) particles are given in Table 5. The mean APR of apoA-I into Lp(AI) particles was markedly higher in patients (5.89±2.09 mg·kg-1·d-1) than in control subjects (2.49±0.85 mg·kg-1·d-1, P<.05), but the mean APR of apoA-I into Lp(AI w AII) particles was similar in patients (4.53±0.83 mg·kg-1·d-1) and control subjects (5.61±1.74 mg·kg-1·d-1, Table 5). The mean APRs of apoA-II in patients (1.58±0.59 mg·kg-1·d-1) and control subjects (1.46±0.65 mg·kg-1·d-1) were similar. The ratio of the APR of apoAI into Lp(AI) and Lp(AI w AII) particles (APR ratio) was found to be the most discriminating parameter between patients and control subjects. The mean APR ratio of the patients (1.29±0.11) was more than twofold higher than the APR ratio of the control subjects (0.46±0.12) (P<.01). These data indicate that in patients with apoA-I(Lys1070), 60% of the newly synthesized apoAI enters plasma within Lp(AI) particles, while in control subjects this value is only 30%, the remainder entering in Lp(AI w AII).
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Our results indicate that the FCR of apoA-I within Lp(AI) and apoA-II within Lp(AI w AII) is enhanced in patients who are heterozygous for the apoA-I(Lys107
0) mutation compared with control subjects. It should be noted that for Patient 1, the proband, all these differences were much enhanced. Also, her HDL cholesterol level was much lower than that of other patients, but according to her medical records this reduction was only partly due to her increased obesity in the past years.40 In patients, the APR of total apoA-I was higher than in control subjects and that of apoA-II was similar to control subjects. Therefore, the reduced plasma apoA-I and apoA-II concentrations in apoA-I(Lys1070) heterozygotes are not consequences of defects in the production of apoA-I or apoA-II but are due to enhanced fractional catabolism. HDL is a polydisperse collection of lipoprotein particles of various sizes. In addition to apoA-I and apoA-II, HDL particles have been reported to contain apoA-IV, apoE, and C apolipoproteins, all of which may play an important role in lipoprotein metabolism. In HDL deficiency secondary to Tangier disease, there is hypercatabolism of HDL protein constituents, with apoA-I being catabolized much more rapidly than apoA-II.41 It is now well established that apoA-I has a separate metabolic fate from apoA-II and that apoA-I is generally catabolized more rapidly than apoA-II.20 21 22 23 24 25 26 Kinetic modeling of apoA-I revealed two plasma compartments of apoA-I and only one for apoA-II.22 23 It has been documented that HDL particles consist of two major subclasses of apoA-I–containing particles; those without apoA-II [Lp(AI)] and those with apoA-II [Lp(AI w AII)]. Rader et al26 have reported a shorter plasma RT for apoA-I in Lp(AI) than in Lp(AI w AII) in normal subjects. Some evidence suggests that Lp(AI) particles are better acceptors of cellular cholesterol than Lp(AI w AII) particles,42 43 44 although differences in efficiency of reverse cholesterol transport have not been consistently observed.45 46 47 Recent findings by Ohta et al48 have suggested that small Lp(AI) particles are more effective acceptors of cellular cholesterol than large Lp(AI) particles. Most of the plasma cholesteryl ester transfer protein activity is associated with Lp(AI).49 Lp(AI w AII) particles have been shown to be better substrates for hepatic lipase, and plasma levels of Lp(AI w AII) have been reported to correlate positively with lecithin: cholesterol acyltransferase activity.50
It has also been documented that the FCR of apoA-I is a major determinant of apoA-I levels in normal subjects.22 25 51 Recently, Ikewaki et al52 have shown that the rate of catabolism of apoA-I is an important factor determining Lp(AI) levels and that the rate of apoA-II production is a major determinant of the distribution of apoA-I between Lp(AI) and Lp(AI w AII). Many patients with HDL deficiency have abnormalities in triglyceride metabolism. In studies of CHD kindreds with HDL deficiency, most also had hypertriglyceridemia.53 54 Patients with hypertriglyceridemia have been reported to have enhanced catabolism of HDL apoA-I and apoA-II.24 Interestingly, the production of apoA-I into Lp(AI) was significantly increased in our patients versus the control subjects. This may be a compensatory mechanism in response to the markedly enhanced rate of catabolism of Lp(AI). However, despite increases in the catabolism of both apoA-I and apoA-II in Lp(AI w AII), no such compensatory increase was noted in apoA-I or apoA-II production into Lp(AI w AII). The net result of these different metabolic patterns between the patients and control subjects is that the patients have a deficiency of Lp(AI w AII) but not Lp(AI). The data support the concept that in the setting of enhanced catabolism of apoA-I due to the mutation apoA-I(Lys1070), there is an attempt to preserve Lp(AI) but not Lp(AI w AII) levels by increasing production.
The association between low plasma HDL cholesterol levels and the in vivo metabolism of apoA-I and apoA-II has been intensively studied. Ginsberg et al55 found a higher apoA-I FCR but similar apoA-I production rates in subjects with isolated low HDL cholesterol or low HDL cholesterol associated with increased plasma triglyceride concentration compared with normolipidemic subjects. Similarly, Gylling et al56 have reported a normal apoA-I production rate and decreased plasma RT of apoA-I for normolipidemic subjects with decreased HDL cholesterol levels. Brinton et al57 have demonstrated an inverse correlation between plasma HDL cholesterol level and the FCR of both apoA-I and apoA-II. Taskinen et al58 and Brinton et al25 have shown that the plasma concentration of Lp(AI w AII) particles is primarily dependent on the rate of synthesis of these particles. Contrary to previous observations, the synthesis of Lp(AI) is increased and the synthesis of Lp(AI w AII) is normal in patients with apoA-I(Lys1070).
The in vivo metabolism of apoA-I has been studied in two mutant forms of apoA-I by using radioiodinated apoA-I. One of these apoA-I variants, apoA-IMilano, is the first described variant form of apolipoprotein A-I, in which cysteine is substituted for arginine at amino acid 173 of the mature apoA-I protein.10 ApoA-IMilano subjects are characterized by reduced HDL cholesterol and apoA-I levels. The other apoA-I variant, apoA-IIowa, is a variant in which a glycine to arginine substitution at residue 26 of apoA-I is associated with hypoalphalipoproteinemia and systemic amyloidosis.11 In apoA-IMilano subjects, the reduced apoA-I concentration was demonstrated by Roma et al59 to be a consequence of increased catabolism of both abnormal apoA-I and normal apoA-I, while the total apoA-I production rate was normal. In the apoA-IIowa subjects, Rader et al60 reported an increased clearance rate of radiolabeled apoA-IIowa compared with control subjects. The production rate of apolipoprotein A-I in the patients with apoA-IIowa was normal. The metabolism of apoA-II was not investigated in either the apoA-IMilano or apoA-IIowa subjects.
Over 20 different apoA-I variants caused by point mutations, deletions, or rearrangements of the apoA-I gene have been described. Some of these apoA-I variants have clearly been associated with reduced plasma HDL cholesterol and apolipoprotein A-I concentrations. The results of this study demonstrate that an increased rate of clearance and not a decreased rate of production of apoA-I and apoA-II accounts for the markedly lower levels of Lp(AI w AII) particles and HDL cholesterol in patients with the apoA-I(Lys1070) variant. In the patients, a higher proportion of newly synthesized apoA-I enters plasma within Lp(AI) than in Lp(AI w AII), which may explain the normal Lp(AI) levels.
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Acknowledgments |
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Dr Tilly-Kiesi and the research in Finland were supported by the Helsinki University Central Hospital and by a grant from the Meilahti Foundation, Helsinki, Finland. The research in the United States was supported by grant HL-39326 from the National Institutes of Health and contract 53-3K06-5-10 from the US Department of Agriculture Research Service. The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. We are grateful to Leena Lehikoinen, Sirkka-Liisa Runeberg, Jennifer Jenner, Kari Wojtanik, and Zhiyang Sun for the excellent technical assistance.
Received November 19, 1995; accepted July 22, 1996.
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References |
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