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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2157-2164.)
© 1995 American Heart Association, Inc.

Articles

Apolipoprotein A-I Deficiency

Biochemical and Metabolic Characteristics

Dominic S. Ng; Camilla Vezina; Thomas S. Wolever; Arnis Kuksis; Robert A. Hegele; Philip W. Connelly

From the Department of Medicine, St Michael's Hospital, University of Toronto, and the C.H. Best Institute (A.K.), Toronto, Ontario, Canada.

Correspondence to Dr P.W. Connelly, J. Alick Little Lipid Research Laboratory, Room 104WA, 38 Shuter St, Toronto, Ontario M5B 1A6, Canada.

	Abstract

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Abstract Familial HDL deficiencies are associated with variable susceptibility to premature coronary heart disease, but the mechanism underlying this association remains poorly understood. Three homozygotes with isolated complete apo A-I deficiency caused by an autosomal codominant apo A-I Q[-2]X mutation and one heterozygote developed coronary heart disease before age 40 years. We characterized the effects of this mutation on lipoprotein metabolism. LDL FC, phospholipid, and apo B were all significantly higher in homozygotes than in heterozygotes. The HDLs of the heterozygotes were apo A-I poor relative to apo A-II. Lecithin-cholesterol acyltransferase activity was 59% lower in homozygotes than in normal subjects or heterozygotes. Cholesteryl ester transfer activity was increased in a homozygote compared with a normolipidemic control subject. Postprandial lipid metabolism was studied in one homozygote and one heterozygote. Postprandial TG response in the homozygote was significantly exaggerated, while residual plasma HDL level remained unaffected. The homozygote also had delayed clearance of retinyl ester, a marker of chylomicron remnant metabolism. Thus, homozygosity and heterozygosity for apo A-I Q[-2]X are associated with qualitative, as well as quantitative, disturbances in plasma HDLs, LDLs, lipid-modifying enzyme activities, and postprandial retinyl ester metabolism. The observed elevation of atherogenic lipoproteins and reduction in antiatherogenic lipoproteins in the affected members of the apo A-I Q[-2]X kindred are consistent with the primary deficiency in apo A-I having pleiotropic effects that markedly enhance susceptibility for coronary heart disease.

Key Words: coronary heart disease • apolipoprotein A-I • reverse cholesterol transport • genetics • postprandial lipemia

	Introduction

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The inverse relationship between plasma levels of HDL cholesterol and the risk of CHD has a strong epidemiological basis,^{1 2 3 4 5} but the mechanism underlying this association remains unclear. In rabbits, infusion of homologous HDL is associated not only with reduction in the rate of formation of fatty streaks⁶ but also with regression of preexisting atherosclerotic lesions.⁷ Transgenic mice that overexpress human apo A-I have elevated levels of HDL and delayed formation of fatty streaks compared with wild-type mice.⁸ Other studies in transgenic mice suggest that the relative abundance of apo A-II in the HDL particles may modulate the cardioprotective effect of HDL.^{9 10} Mice in which the apo A-I gene was disrupted were found not to be prone to atherosclerosis.¹¹ Human kindreds with severe HDL deficiencies have a wide range of expression of premature CHD.^{12 13 14 15 16 17 18 19 20 21 22 23} It appears that the exact genetic defect that causes the depressed HDL may be a major determinant of the extent and nature of predisposition to CHD. Associated secondary factors, such as increased LDL cholesterol, and nonlipid risk factors may aggravate a genetic susceptibility to CHD.

Apo A-I is the major structural apolipoprotein of HDL. It is synthesized by both the small intestine and the liver in the form of a preproprotein.^{24 25} Apo A-I of intestinal origin is secreted with chylomicrons, which are also enriched in apo A-IV. Upon entering the systemic circulation, apo A-I, along with apo A-IV, is transferred to HDL. During this phase of interaction between lipoprotein classes, apo E and apo C's (I, II, and III) are transferred from HDL to the TG-rich chylomicrons. Apo A-I of hepatic origin is secreted in the form of a nascent cholesterol-poor HDL particle. In addition to direct synthesis, HDL precursors can be formed from the excess surface remnant released during hydrolysis of the TG-rich lipoproteins. Apo A-I has been suggested to be the most potent cofactor for activation of LCAT.²⁶ However, in vitro evidence suggests that LCAT can act directly on non-HDL particles.²⁷ In normolipidemic subjects, the majority of plasma LCAT is in the apo A-I–containing lipoprotein particles, with a small proportion found in non–apo A-I–containing particles, including LDL and VLDL.²⁶ In patients with Tangier disease, the total plasma LCAT activity and mass are both only moderately decreased.^{28 29} Substantial redistribution of LCAT activity to non-HDL particles was observed in these subjects, but the LCAT specific activity was normal.²⁶ Whole plasma LCAT activities in patients homozygous for apo A-I/C-III deficiency were 38% of control values, with an LCAT mass that was 30% of control.³⁰ Subbaiah et al³¹ measured LCAT activity from two apo A-I/C-III–deficient subjects by three different methods and reported LCAT activities that ranged from 36% to 63% of the respective control means. On the basis of these studies, the moderate decreases in LCAT deficiency in the apo A-I/C-III–deficient subjects have been attributed to the reduction in the whole-plasma LCAT mass. Lackner et al²² found that LCAT activity distribution in a 7-year-old Turkish girl with isolated apo A-I deficiency due to homozygosity for an exon 3 cytosine insertion was not substantially different from normal. More LCAT activity was seen in the lipoprotein-free fraction, and LCAT activity was not detected in the non-HDL lipoprotein particles.²²

CETP mediates the transfer of CE to the apo B–containing particles, VLDL, and chylomicrons in exchange for TGs. Subbaiah et al³¹ studied the CET activity in subjects with apo A-I/C-III deficiency using two methods: (1) mass transfer assay and (2) isotope CE transfer assay using normal exogenous HDL with labeled CE. With the mass transfer assay, there was an increase in the percentage of HDL-CE transferred to VLDL+LDL, although the absolute CE transferred was much lower than in the control subjects. Using the latter method, the authors also reported the percentage of HDL-CE transferred in the affected subjects to have been increased up to fourfold compared with the control subjects.

Delayed clearance of postprandial lipoproteins has been suggested to contribute to atherogenesis.³² Although there is a positive correlation between postprandial hyperlipemia and fasting TGs,³³ an association with fasting HDL is uncertain. Patsch et al³⁴ suggested that low fasting HDL may be caused by postprandial hyperlipemia. However, studies by O'Meara et al³⁵ and Ooi et al³⁶ found little correlation between fasting HDL and postprandial hyperlipemia.

We reported a large kindred with complete isolated apo A-I deficiency caused by a nonsense mutation at codon [-2] of the apo A-I gene, leading to severe HDL deficiency.²³ To date, we have identified a total of six homozygotes and seven heterozygotes with the same mutation. We report here that there is a high prevalence of premature CHD among the affected subjects. Among the subjects with clinical CHD, a concomitant moderate elevation of LDL cholesterol was identified, which might also have contributed to the risk of atherosclerosis. However, other potentially atherogenic metabolic disturbances as a result of the apo A-I deficiency required additional study. We now report in vitro characterization of the disturbances of apo A-I deficiency in reverse cholesterol transport and postprandial lipoprotein metabolism.

	Methods

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Plasma Lipids and Lipoproteins
The plasma lipid and lipoprotein profiles were determined in the J.A. Little Lipid Research Laboratory according to the Lipid Research Clinics protocol.³⁷ Blood samples were obtained from subjects after a 12- to 14-hour fast and collected into tubes containing Na₂EDTA. VLDL was isolated by ultracentrifugation of plasma at 45 000 rpm for 16.5 hours at 10°C with a Beckman 50.3-Ti rotor in an L8-80 centrifuge (Beckman Instruments). LDL (d=1.006 to 1.063 g/mL) was prepared by ultracentrifugation at 45 000 rpm for 20 hours. HDL (d=1.063 to 1.21 g/mL), HDL₂ (d=1.063 to 1.125 g/mL), and HDL₃ (d=1.125 to 1.21 g/mL) were prepared by sequential ultracentrifugation at 45 000 rpm for 40 hours at 10°C.

The total plasma and lipoprotein cholesterol and TGs were measured by automated enzymatic procedures with the Technicon RA1000. The FC, phospholipid, CE, and TG in lipoprotein fractions were determined by automated injection and temperature-programmed gas-liquid chromatography adapted to use a capillary column.³⁸

Apolipoproteins
Apo A-I, A-II, B, C-II, C-III, and E were measured by ELISA techniques.^{39 40} Apo A-I and B were also measured by nephelometry (Behring). Plasma Lp A-I and apo A-II were determined by a differential electroimmunodiffusion assay (Sebia). The provided standard, calibrated for Lp A-I and apo A-II concentration by the manufacturer, and the plasma samples were diluted and applied to the electrophoretic gel according to the manufacturer's instructions. Rocket heights of both the apo A-II–containing particles and the Lp A-I particles were measured, and concentrations were calculated from the four-point standard curve with curve-fitting software (INPLOT, GraphPad).

LCAT Activity
Plasma LCAT activities of the subjects were determined by two different methods: (1) an endogenous self-substrate method, modified from Stokke and Norum,⁴¹ and (2) an exogenous substrate–based method. Briefly, in the first method, the endogenous cholesterol in fresh plasma was equilibrated at 37°C with [¹⁴C]cholesterol-albumin mix in the presence of 1.4 mmol/L of DTNB. Subsequently, excess mercaptoethanol (12 mmol/L) was added to reverse the inhibition of LCAT. CER, the rate of conversion of labeled FC to CE, was determined after 30 minutes of incubation at 37°C. The reaction was stopped by addition of methanol, and the lipid was extracted with methanol and chloroform. The CE and FC were separated by silica gel thin-layer chromatography (Gelman Science Inc) with acetic acid:diethyl ether:n-hexane (1:10:90) as the eluting solvent. The FC and CE spots were cut out, and the radioactivity was determined in a liquid scintillation counter. To establish the linearity of the CER over time, CER was determined serially for incubation times of 5, 10, 20, 30, 40, and 60 minutes each for one of the homozygotes, one of the heterozygotes, and a control subject. For the second method, the exogenous substrate assay, an apo A-I–containing proteoliposome (apo A-I, egg lecithin, [¹⁴C]cholesterol) was used as an exogenous substrate.⁴² In this procedure, the plasma was incubated with the proteoliposome at 37°C for 30 minutes. The reaction was stopped by addition of methanol. The lipid extraction and determination of rate of conversion from FC to CE were as described above. LCAT activity was expressed as nmol cholesterol esterified·h^-1·mL plasma^-1.

CET Activity
CET activities of one homozygote and a control subject were quantified by use of endogenously labeled HDL (d=1.063 to 1.21 g/mL) with a slight modification of the method by Guerin et al.⁴³ One half to 1 mCi of [¹⁴C]cholesterol in 10 mL of ethanol was injected into the d>1.063-g/mL plasma fraction. The radiolabeled d>1.063-g/mL fraction was then incubated at 37°C for 18 hours to allow esterification of the labeled cholesterol by endogenous LCAT. For both the control and the homozygous subject, 38% of the [¹⁴C]cholesterol was esterified, according to thin-layer chromatographic quantification. The labeled HDL was incubated with the subject's own plasma for a variable length of time (0, 2, and 4 hours) at 37°C and as a control at 4°C for 18 hours. The incubation reaction was stopped by chilling the tubes at 4°C. The lipoproteins from each incubation mix were then separated by step gradient-density ultracentrifugation. Serial 500-µL fractions were collected with a precision pipette from the top. Lipid extraction was performed on each fraction, and the degree of esterification rate in each was determined by analysis of the radioactivities associated with the CE and FC bands after thin-layer chromatographic separation as described earlier.

Postheparin Lipolytic Activity
Subjects were fasted overnight for 14 hours. Postheparin lipolytic activity was determined in plasma samples obtained from the subjects one-half hour after an intravenous injection of heparin (100 U/kg) as previously described.⁴⁴ Postheparin lipoprotein lipase activity was estimated by the difference between postheparin total lipase and hepatic lipase activities. Lipolytic activities were expressed as mmol free fatty acids hydrolyzed·h^-1·mL plasma^-1.

Fat Tolerance Tests
Fat tolerance tests were performed on the proband, one of her heterozygous daughters, and six female normolipidemic control subjects, all with apo E genotype E3/3. None of the subjects were taking any lipid-lowering agent for at least 2 weeks before the study.

Fat tolerance tests were performed after a 12-hour fast and consisted of 5 mL water, 0.5 g corn oil, 1.28 g skim milk powder (0.5 g protein), and 0.5 g glucose, all per kilogram of body weight, to which was added 50 000 IU RP (Hoffmann–La Roche Ltd). After a fasting blood sample was drawn, subjects consumed the test meal within 15 minutes and had further blood samples drawn at hourly intervals for 10 hours. Blood samples were protected from light during removal, transport, and processing of the plasma. After removal of chylomicrons by centrifugation at 20 000 rpm for 30 minutes (Beckman rotor 50.3), the remainder of the plasma was separated by ultracentrifugal flotation into d<1.006-g/mL and d>1.006-g/mL fractions. RP was measured in total plasma and in the chylomicron d<1.006-g/mL and d>1.006-g/mL fractions.

During the fat tolerance tests, hourly plasma and lipoprotein samples were measured for lipids and for apo C-II, C-III, A-I, A-II, and E. Apo A-I and apo A-II were also measured on the same samples after additional ultracentrifugation was used to separate HDL₂ and HDL₃ subfractions.

Statistical Analysis
Student's t test was used for comparison of the means between groups.

	Results

Top Abstract Introduction Methods Results Discussion References

 
An abbreviated pedigree of the apo A-I Q[-2]X kindred is shown in Fig 1. Four of the 10 study subjects from the fourth generation developed initial clinical manifestations of CHD in the third and fourth decades of life. Subject IV-003, a sister of the proband homozygous for the mutation (referred to as II-c in Reference 23), suffered an inferior myocardial wall infarction at age 34 years and underwent coronary bypass surgery at age 37 years. Subject IV-002 (referred to as II-b in Reference 23), a sister of the proband heterozygous for the mutation, developed angina at age 35 years and had reversible myocardial ischemia on a thallium stress test at age 39 years. Subject IV-017, a homozygous female first cousin of the proband, suffered an anterior myocardial wall infarction at age 33 years. Subject IV-011 (referred to as II-j in Reference 23), a homozygous brother of the proband, developed angina at age 26 years but had a negative stress thallium test. Symptoms worsened, however, and he had four-vessel coronary bypass surgery at age 30 years. Two obligate heterozygous men from the third generation (III-001 and III-004) developed CHD in the sixth decade of life. Subject III-001 died of a myocardial infarction at age 64 years. The other subjects studied to date have been asymptomatic for CHD.

View larger version (21K): [in this window] [in a new window]   Figure 1. Pedigree of the apo A-I Q[-2]X kindred. The homozygosity for the rare apo A-I Q[-2]X mutation in the two contemporary branches must have arisen from consanguinity in the small community of origin for this family, with the likely common relatives indicated by the broken line.

Lipoprotein Lipid and Apolipoprotein Composition
The means and SDs for lipoprotein lipids and apolipoproteins for four homozygous (X/X), four heterozygous (N/X), and two normal (N/N) family members are shown in the Table. Analyses for differences between homozygotes and heterozygotes using both a nonparametric test and a t test were nearly identical, and thus only the results of the t test will be presented. VLDL FC, CE, phospholipid, TG, apo C-III, apo C-II, and apo E tended to be lower in X/X versus N/X subjects, but these differences did not reach statistical significance. VLDL apo E and apo B values were available only for three homozygotes. The results showed a trend for lower ratios of VLDL apo C-III, apo C-II, and apo E to VLDL apo B. LDL CE (P=.07) and TG (P=.15) were higher in X/X versus N/X subjects, but the differences did not reach statistical significance. LDL FC (P=.02), LDL phospholipid (P=.02), and LDL apo B (P=.008) were significantly higher in X/X versus N/X subjects. The LDL FC/CE ratio (wt/wt) was 0.31±0.04 for X/X subjects versus 0.29±0.05 for N/X subjects. As previously reported,²³ all classes of HDL lipids, apo A-I, and apo A-II were significantly lower in X/X versus N/X subjects. In contrast, HDL apo C-III and apo C-II tended to be lower in X/X versus N/X subjects, but this difference was not statistically significant. There was no evidence that HDL apo E was different between X/X and N/X subjects. It is common to find apo A-I and apo E in the d>1.21-g/mL fraction after sequential ultracentrifugation for the fractionation of lipoproteins. The concentration of apo E in this fraction was not different between homozygous and heterozygous subjects.

View this table: [in this window] [in a new window]   Table 1. Lipoprotein Lipid and Apolipoprotein Concentrations in the Apo A-I Deficiency Kindred

HDL Lp A-I Versus Lp A-I–A-II
The HDL apolipoprotein compositions in three female heterozygotes and four unaffected female control subjects evaluated by differential electroimmunodiffusion were expressed as the ratio of apo A-II to Lp A-I–apo A-I levels. The heterozygotes had a mean ratio of 0.92±0.12, compared with a mean of 0.61±0.18 for the control subjects (P=.02).

Postheparin Lipase Activities
The proband and her heterozygous daughter had hepatic lipase activities of 8.7 and 11.2 mmol · h^-1·mL^-1, respectively, compared with a control mean±SD of 6.7±4.9 mmol·h^-1·mL^-1 for five female unrelated control subjects. The lipoprotein lipase activities of the same two affected subjects were 12.2 and 8.6 mmol·h^-1·mL^-1, respectively, compared with a control mean±SD of 10.6±3.7 mmol·h^-1·mL^-1.

LCAT Activity
Fig 2a shows mean LCAT activities of the 4 homozygous, 3 heterozygous, and 8 control subjects determined by the "endogenous substrate" method.³⁵ The homozygotes had a mean LCAT activity of 48% of the LCAT activity of the control group (27.3±5.4 versus 58.4±17.9 nmol·h^-1·mL^-1, P<.002). The heterozygotes had a mean LCAT activity of 63.2±16.9 nmol·h^-1·mL^-1, which was not significantly different from the control mean (P=.35). The validity of using a 30-minute incubation time for estimation of CER was established by the observed linear correlations between percent cholesterol esterified and incubation time up to 60 minutes for subjects with each genotype (slope=0.027 min^-1, r=.999 for the homozygote; slope=0.054 min^-1, r=.995 for the heterozygote; and slope=0.051 min^-1, r=.995 for an unaffected subject).

View larger version (31K): [in this window] [in a new window]   Figure 2. Bar graphs showing (a) mean±SD of CER of plasma from the homozygous (X/X), heterozygous (N/X), and control subjects (N/N) by the endogenous self-substrate method. b, Mean±SD LCAT activities of whole plasmas from the same three groups of subjects with an apo A-I–containing proteoliposome as exogenous substrate.

Mean LCAT activities by the "common substrate" method³⁶ for three subjects of each genotype are shown in Fig 2b. The homozygotes had a mean LCAT activity of 55.5% of the mean LCAT activity of the control group (52.5±11.8 versus 94.6±16.0 nmol·h^-1·mL^-1, P=.01). The heterozygotes had a mean LCAT activity (96.7±15.4 nmol·h^-1·mL^-1) that, again, was not significantly different from the control mean (P=.44).

CETP Mass
Plasma CETP concentrations determined by radioimmunoassay on 5 homozygous, 2 heterozygous, and 4 unaffected subjects were all found to be within the normal range (Ruth McPherson, MD, PhD, personal communication, 1994).

CET Activity
For the homozygous subject, at 2 hours of incubation, 75% of the radiolabeled CE had transferred from HDL to VLDL and LDL. In contrast, for a normolipemic control subject, we observed a 10% reduction in CE-associated radioactivity in HDL and a corresponding increase in CE in the VLDL and LDL fractions at 2 hours.

Fat Tolerance Test
Fig 3 shows the deviation of postprandial plasma TG from fasting TG (TG) after the fat challenge. The heterozygote's plasma TG response was only marginally different from those of the control subjects, whereas the homozygote's postprandial TG showed a slight delay in peaking but a marked delay in its return to baseline.

View larger version (17K): [in this window] [in a new window]   Figure 3. Graph of postprandial plasma TG after an oral fat challenge corrected for TG concentration at 0 time ( Triglyceride). Results are plotted for a homozygous subject (), a heterozygous subject (), and a normolipidemic control subject ().

Retinyl Palmitate
RP excursions in total plasma are shown in Fig 4. The total plasma RP excursion in the heterozygote had a temporal profile comparable to that of the control subject. In contrast, the homozygous subject had a delayed peak at 8 hours after the fat load. This deviation from the normal subjects was even more evident after study of both the chylomicron and the d<1.006-g/mL ("chylomicron remnant") fractions (Fig 5). The RP in the chylomicron fraction in the homozygote showed an exaggerated excursion and had not returned to baseline by 10 hours. More dramatically, the RP in the d<1.006-g/mL fraction in the homozygote showed an initial response to the fat load similar to that of the control subjects, but 6 hours after the fat load and for the period following, RP in this fraction continued to accumulate and at 10 hours appeared not yet to have peaked (Fig 5b).

View larger version (19K): [in this window] [in a new window]   Figure 4. Graph of postprandial excursions of total plasma RP levels in a homozygous subject (), a heterozygous subject (), and the mean±SD of six normolipidemic control subjects () after an oral fat challenge.

View larger version (26K): [in this window] [in a new window]   Figure 5. Graphs of postprandial RP levels in (a) chylomicron fractions and (b) d<1.006-g/mL fractions in a homozygous subject () and a heterozygous subject () compared with mean±SD of six normolipidemic control subjects () after an oral fat challenge.

Apo E
Apo E levels in the chylomicron fraction (Fig 6) in the homozygote showed a delayed peak at 8 hours and, unlike the control subjects, did not return to the baseline at 10 hours. In the d<1.006-g/mL fraction, apo E excursion of the homozygote showed a prompt initial rise that peaked early, at about 4 hours, but was followed by a rapid fall toward the baseline. The d<1.006-g/mL apo E levels were within 1 SD of the control means (Fig 6). This returned to baseline levels at 10 hours. The homozygote began with a higher apo E in the d>1.006-g/mL fraction but exhibited a decrease of a similar magnitude with a minimum at 6 hours. The apo E concentration had not returned to baseline levels at 10 hours (Fig 6).

View larger version (19K): [in this window] [in a new window]   Figure 6. Graphs of apo E levels in the Sf>400 (chylomicron), d<1.006-g/mL (chylomicron remnants plus VLDL), and d>1.006-g/mL (IDL plus LDL plus HDL) fractions of a homozygous subject (X/X), a heterozygous subject (N/X), and mean±SD of 8 control subjects (N/N) (3 E3/3 and 5 E3/4).

Apo C-II and Apo C-III
Apo C-II and apo C-III excursions after the fat load in the homozygote were also within 1 SD of control means at all time points (data not shown).

HDL₂ and HDL₃
There was no detectable change in HDL₂ or HDL₃ lipid concentrations in the homozygous subject during the 10 hours after fat challenge (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have identified a number of structural and functional disturbances in lipoprotein metabolism in the family with the apo A-I Q[-2]X mutation. Some of these alterations may contribute to the development of the accelerated atherosclerosis observed in this family.

The heterozygous subjects, with one-half normal plasma levels of apo A-I and HDL, had HDL particles that were relatively enriched in apo A-II. In conjunction with previously reported size distribution of the HDL particles,²³ HDL particles in the fasting state in the heterozygotes were characterized by a population of dense particles predominantly in the HDL₃ density range that were relatively enriched in apo A-II.

Our data showed that five of the apo A-I Q[-2]X homozygotes had uniform reduction in whole-plasma LCAT activities. The observed comparable reduction in LCAT activities with both endogenous and exogenous substrates suggests that the decrease in LCAT activity with complete apo A-I deficiency is most likely to be due to a reduction in LCAT mass. Taken together with the relatively normal LCAT activity and CER observed in the heterozygotes, our data support the concept that apo A-I is not absolutely essential for LCAT action, since one-half the normal level of apo A-I was sufficient to maintain normal levels of LCAT. It is of interest to note that the total plasma FC/CE ratio in the homozygotes was significantly different from that of heterozygotes (0.39±0.03 versus 0.29±0.04, P=.01), while the LDL FC/CE ratio was not significantly different.

The present study reports the initial examination of CET activity in an apo A-I–deficient subject by use of endogenous HDL labeled with [¹⁴C]cholesterol, as reported by Guerin et al.⁴³ As shown earlier, the HDL (d=1.063 to 1.21-g/mL) fraction of the homozygous subject showed a significant endogenous LCAT activity despite complete apo A-I deficiency, suggesting that LCAT is associated with the residual abnormal apo A-II–containing HDL particles and possibly with other non–apo A-I HDL particles. Furthermore, CE formed in this fraction was more efficiently transferred to the VLDL and LDL particles compared with the control subject.

The accelerated relative CE transfer activity observed in the homozygote may be a consequence of the relative instability of the CE-enriched HDL. Alternatively, it may reflect the preferential transfer of the CE to VLDL and LDL due to a lack of a normal HDL pool for transfer. Given these observations, it remains to be established whether the small number of residual HDL particles may acquire cell-derived unesterified cholesterol efficiently enough to promote effective reverse cholesterol transport. An understanding of CE transfer in these patients would require a complete time course analysis and control for the relative concentrations of acceptor and donor lipoproteins. Nevertheless, the studies of LCAT activity and CET demonstrate that it is conceivable that the HDL present in apo A-I deficiency is a primary site for esterification and is an excellent substrate for the normal amounts of CETP present in plasma.

We observed a marked postprandial increase of TG in the homozygote, with a delayed return to baseline (Fig 3). This deviation from normal persists even after adjustment for fasting TG level by calculating the ratio of TG to fasting TG (data not shown). Analysis of subfractions of the plasma samples revealed that elevation of TG levels occurred predominantly in the chylomicron fraction (data not shown). The chylomicron fraction was also characterized by a delay of TG in reaching peak level (6 hours after fat challenge) and a delayed return of TG to baseline. Concomitantly, we noted exaggerated postprandial accumulation of plasma RP (Fig 4), which was mainly due to RP accumulation in the S_f>400 fraction. Chylomicron apo E levels rose and peaked within the normal range but appeared to have a delayed return to baseline (Fig 6, top). Chylomicron apo B excursion was characterized by a moderate rise that peaked at 4 hours, with a prompt return to baseline by hour 10. These data suggest that the apo A-I–deficient homozygote has, after a fat load, a selective accumulation of chylomicron-remnant lipoprotein particles in the S_f>400 fraction. Postprandially, both apo B-48– and apo B-100–containing TG-rich lipoprotein particles compete for the hydrolytic action of LPL, a saturable pathway.⁴⁵ It has recently been shown that the rate of hydrolysis of TG-rich lipoprotein particles is strongly affected by the particle composition, most notably the TG content.⁴⁶ This may contribute partially to the observed heterogeneous rate of clearance of the various chylomicron subfractions.

The unique aspect of the fat tolerance test in the homozygote was the accumulation of RP in the d<1.006-g/mL fraction of the homozygous subject (Fig 5b). However, there was not a proportional accumulation of apo E (Fig 6), apo B, TG, or cholesterol in the same fractions (data not shown). Such accumulation may have resulted from abnormal transfer of RP between chylomicrons and the apo B– and/or apo E–containing remnant particles. Alternative explanations, such as formation of novel remnant lipoprotein particles or recycling of RP after hepatic uptake, may need to be considered. Fazio and Yao,⁴⁷ on the basis of in vitro cell culture studies, suggested that it is possible for VLDL to acquire apo E before secretion. In contrast, chylomicrons should have to acquire apo C-III, apo C-II, and apo E via transfer from the existing plasma pool. Goldberg et al⁴⁸ suggested that apo A-IV can displace apo C-II from HDL and increase the net transfer to model chylomicrons. Human chylomicrons contain apo A-I and apo A-IV that are stably associated with chylomicrons but readily dissociate during lipolysis and associate with or form HDL. It is possible that, in normal subjects, chylomicron-associated apo A-I, lost during lipolysis, normally displaces HDL apo E, enhancing transfer of apo E to chylomicrons. The temporal changes in chylomicron apo C-III paralleled the TG changes, with apo C-III returning to baseline by 10 hours. These changes were not different from other hyperlipidemic subjects, consistent with their normal postheparin lipolytic activity. The temporal changes in chylomicron apo E were similar to those of the control group at times up to 6 hours. However, although the concentration of apo E did not increase after 6 hours, it had not returned to baseline by 10 hours.

Alternatively, the dramatic accumulation of retinyl ester in d<1.006-g/mL lipoproteins could be independent of the deficiency of apo A-I. It could also be independent of the particle content of apo E and apo C-II ascertained from venous plasma. An indeterminate proportion of chylomicron clearance may result from a "secretion-capture" mechanism in which hepatocytes secrete apo E into the space of Disse, followed by acquisition by chylomicrons and sequestering of these particles by glycosaminoglycans, LDL receptor–related protein, and the LDL receptor.⁴⁹ We do not have the tools at this time to measure the relative contribution to chylomicron clearance in humans of "secretion-capture" versus acquisition of apo E from the preexisting plasma pools.

In summary, affected members in the kindred with the apo A-I Q[-2]X mutation exhibited a number of significant disturbances in lipoprotein metabolism. Plasma HDL and apo A-I levels were reduced, and the proportion of Lp A-I HDL particles among the heterozygotes was significantly altered. Plasma LCAT activity in the homozygotes was reduced to approximately one-half normal levels, with no detectable effect on the LDL FC/CE ratio. LDL FC, phospholipid, and apo B concentrations were elevated in homozygotes versus heterozygotes. Preliminary results are consistent with an increase in plasma CET activity in a homozygous subject. Complete apo A-I deficiency appeared to be associated with abnormalities in chylomicron metabolism, which we speculate is due to impaired apo E–dependent clearance. Homozygotes have higher LDL than heterozygotes, suggesting that additional lipoprotein abnormalities contribute to the excess CHD observed in this family. The results are consistent with the theory that primary apo A-I deficiency is a metabolic state that is abnormally susceptible to early CHD in both women and men.

	Selected Abbreviations and Acronyms

 

CE = cholesteryl ester CER = cholesterol esterification rate CET = cholesteryl ester transfer CETP = cholesteryl ester transfer protein CHD = coronary heart disease FC = free cholesterol LCAT = lecithin-cholesterol acyltransferase RP = retinyl palmitate TG = triglyceride

	Acknowledgments

 
This study was supported by operating grants from the Medical Research Council of Canada and the Heart and Stroke Foundations of Ontario and Canada. Dr Hegele is a McDonald Scholar of the Heart and Stroke Foundation of Canada. Dr Ng is a postdoctoral fellow supported by the Heart and Stroke Foundation of Canada. We gratefully acknowledge the technical contributions of Teresa Lippingwell, Graham Maguire, K. Yun Lam, and Lieba Snitman.

Received May 15, 1995; accepted August 18, 1995.

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