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Background: The incidence of diaphragmatic hernias caused or exacerbated by diagnostic colonoscopy is not well elucidated at this time, and is believed to be very rare., Case Presentation: We present the case of a 57 year old man with remote history of traumatic injury who first presented with vague left shoulder pain for two weeks, mild anemia, and tested positive for fecal occult blood. Four days post colonoscopy the patient was found to have a strangulated loop of bowel herniated through the diaphragm into the left hemithorax., Conclusions: In patients with previous history of serious traumatic injury and particularly those with previous splenectomy, a thorough history and physical examination before routine colonoscopy is important. A high level of suspicion for post-operative complications should also be maintained when assessing such patients.

Background: A relationship between corneal arcus and atherosclerosis has long been suspected but is controversial. The homozygous familial hypercholesterolemia patients in this study present a unique opportunity to assess this issue. They have both advanced atherosclerosis and corneal arcus., Methods: This is a cross-sectional study of 17 patients homozygous for familial hypercholesterolemia presenting to the Clinical Center of the National Institutes of Health. Plasma lipoproteins, circumferential extent of arcus, thoracic aorta and coronary calcific atherosclerosis score, and Achilles tendon width were measured at the National Institutes of Health., Results: Patients with corneal arcus had higher scores for calcific atherosclerosis (mean 2865 compared to 412), cholesterol-year score (mean 11830 mg-yr/dl compared to 5707 mg-yr/dl), and Achilles tendon width (mean 2.54 cm compared to 1.41 cm) than those without. Corneal arcus and Achilles tendon width were strongly correlated and predictive of each other. Although corneal arcus was correlated with calcific atherosclerosis (r = 0.67; p = 0.004), it was not as highly correlated as was the Achilles tendon width (r = 0.855; p < 0.001)., Conclusion: Corneal arcus reflects widespread tissue lipid deposition and is correlated with both calcific atherosclerosis and xanthomatosis in these patients. Patients with more severe arcus tend to have more severe calcific atherosclerosis. Corneal arcus is not as good an indicator of calcific atherosclerosis as Achilles tendon thickness, but its presence suggests increased atherosclerosis in these hypercholesterolemic patients.

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).

We have used a mathematical/compartmental modeling approach along with a number of rationally designed complementary in vivo and in vitro systems to investigate the effects of administration of various retinoids and/or drug combinations on normal physiological metabolism of native retinoids. The present paper focuses on our studies of the synthetic retinoid 4-HPR and our use of fairly simple mathematical/compartmental modeling techniques to investigate how this retinoid affects the metabolism of native retinoid overall, as well as in two specific tissues, the prostate and the eyes. We have presented our work with this particular retinoid and these tissues as an example of the type of studies we have been doing and to present some of the information that one can obtain using this approach. In addition, an important objective of this paper is to highlight the fact that a great deal of critical information can be derived from fairly simple mathematical/compartmental models. When used appropriately, such models provide a powerful tool to direct the design, conduct, and interpretation of experiments. The models we developed for the prostate and the eyes were used as hypotheses to direct our research efforts in both in vivo and in vitro systems. In the case of the eyes, we were able to elucidate the possible mechanisms involved in one of the most commonly reported complications (i.e., visual function abnormalities) associated with administration of an important chemopreventive and/or chemotherapeutic agent. We are in the process of further expanding our studies with the prostate as well as several other tissues in a similar manner. The immediate clinical relevance and application of our work with the eyes demonstrate the high translational potential of our approach. Without the use of the type of mathematical/compartmental modeling approach we used, which provided the basis for much of this work, we are not aware of any other way that we could have obtained the critical information that we did. We hope that the work presented here demonstrates the usefulness, power, and potential clinical applicability of a modeling approach to investigate different retinoid-based treatments as well as a variety of other chemopreventive and/or chemotherapeutic agents.

Apolipoprotein E (apoE) exists in three common forms in humans: the wild-type apoE3 and two common genetic variants, apoE2 and apoE4. Although previous studies have examined the metabolism of the different apoE isoforms in human subjects, they have not involved direct comparison of two different isoforms in subjects heterozygous for the same two isoforms. We conducted this study to directly compare the catabolism of apoE2 and apoE4 in heterozygous E2/4 subjects in vivo. Iodine 131-labeled apoE2 and iodine 125-labeled apoE4 were simultaneously injected into three E4/2 heterozygous subjects. The mean residence time of apoE4 (0.40 +/- 0.01 day) was found to be one-third that of apoE2 (1.20 +/- 0.18 day). ApoE2 was present primarily in high-density lipoprotein, whereas apoE4 was present equally in very low density and high-density lipoprotein. In all lipoprotein subfractions, apoE4 was catabolized at a much faster rate than apoE2. In conclusion, E4 is catabolized three times faster than apoE2 in heterozygous E2/4 subjects, indicating that these two apoE isoproteins have distinct metabolic pathways.

A family was identified with vertical transmission through three generations with simultaneous increases of apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol, which we have designated familial hyperalphalipoproteinemia and hyperbetalipoproteinemia (HA/HBL). Affected patients develop xanthomas and coronary artery disease (CAD). HA/HBL apoA-I and LDL-apoB were isolated and characterized. The in vivo kinetics of radiolabeled apoA-I and LDL-apoB were evaluated in two HA/HBL probands and three controls. Structural and metabolic characterization showed normal apoA-I and LDL-apoB. The kinetics of metabolism of HA/HBL apoA-I in the HA/HBL subjects showed that elevated apoA-I levels were solely due to an increased synthesis rate (15.2 to 17.6 mg/kg/d v 11.1 to 11.4 mg/kg/d) with a normal apoA-I residence time in plasma (4.2 to 5.4 days v 5.1 to 5.3 days). The elevation of LDL-apoB levels resulted from both an increased synthetic rate (16.6 to 22.9 mg/kg/d v 12.3 to 13.8 mg/kg/d) and a prolonged residence time (3.3 to 3.8 days v 1.4 to 1.9 days). In addition, we evaluated another HA/HBL proband of an unrelated family with HA/HBL to confirm the kinetic data. LDL-receptor binding studies of HA/HBL fibroblasts showed normal binding, uptake, and degradation of LDL isolated from a normolipemic control. The serum concentration of the cholesterol ester transfer protein (CETP) was normal in the studied probands. An apoB 3500 and apoB 3531 mutant, respectively, was ruled out by polymerase chain reaction (PCR). In conclusion, the site of the molecular defect in HA/HBL subjects may be involved in the coordinate regulation of metabolism for both LDL and HDL.

We identified a 38-yr-old male patient with the clinical expression of homozygous familial hypercholesterolemia presenting as severe coronary artery disease, tendon and skin xanthomas, arcus lipoides, and joint pain. The genetic trait seems to be autosomal recessive. Interestingly, serum concentrations of cholesterol responded well to diet and statins. We had no evidence of an abnormal low density lipoprotein (LDL)-apolipoprotein B (apoB) particle, which was isolated from the patient using the U937 proliferation assay as a functional test of the LDL-binding capacity. The apoB 3500 and apoB 3531 defects were ruled out by PCR. In addition, we found no evidence for a defect within the LDL-receptor by skin fibroblast analysis, linkage analysis, single-strand conformational polymorphism and Southern blot screening across the entire LDL-receptor gene. The in vivo kinetics of radioiodinated LDL-apoB were evaluated in the proband and three normal controls, subsequently. The LDL-apoB isolated from the patient showed a normal catabolism, confirming an intact LDL particle. In contrast the fractional catabolic rate (d-1) of autologous LDL in the subject and the normal controls revealed a remarkable delayed catabolism of the patient's LDL (0.15 vs. 0.33-0.43 d-1). In addition, the elevation of LDL-cholesterol in the patient resulted from an increased production rate with 22.8 mg/kg per day vs. 12.7-15.7 mg/kg per day. These data indicate that there is another catabolic defect beyond the apoB and LDL-receptor gene causing familial hypercholesterolemia.

The effect on apolipoprotein B kinetics of a diet enriched in either fish oil or safflower oil was investigated in five hypertriglyceridemic (HTG), non-insulin-dependent diabetic subjects. The fish oil diet decreased plasma triglycerides and VLDL-apoB but increased LDL-apoB and LDL-cholesterol. Total plasma apoB concentration did not change, nor did the increased VLDL-apoB secretion present in these HTG subjects, which, accompanied by impaired lipolysis, accounted for their elevated VLDL. The fish oil-induced fall in VLDL resulted from a decrease in secretion without a change in residence time. The IDL fraction, which also contained small VLDL, was the primary site for the secretion of apoB particles in the HTG subjects. On the fish oil diet there was a further, compensatory increase in the secretion of these lipoproteins such that the transport of apoB in IDL remained the same, as did its mass. In the HTG subjects the major portion of IDL lipoproteins was catabolized, with LDL-apoB production comprising the lesser quantity. On the fish oil diet, a shift in the channeling of the lipoprotein output from IDL resulted in a decrease in the catabolic pathway and an increase in conversion to LDL. As the residence time of LDL did not change, this increased input gave rise to the larger mass of LDL-apoB seen in these hypertriglyceridemic subjects when receiving a fish oil diet.

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.

Radioactive tracers are routinely used in investigation of the metabolism of apolipoprotein kinetics. Here, metabolic studies of apolipoprotein tracers labeled with radioiodine were analyzed to determine the absorbed radiation dose received by the subject. This analysis used compartmental modeling techniques to evaluate the radiation dose to various organs and the total body resulting from radioiodinated tracer injection. In this approach, we combined the published kinetic models of iodine and those of specific apolipoproteins. From the solution of the integrated compartmental models, residence times of the radiation in various source organs, in particular the thyroid, whole body, bladder, and red bone marrow, have been determined for the apolipoproteins apoA-I, apoA-II, very-low-density lipoprotein (VLDL)-apoB, and low-density lipoprotein (LDL)-apoB, each labeled with iodine 123, 133, 124, 131, 126, and 125. These tabulated values were used to calculate radiation doses to the different target organs. The thyroid is the organ that receives the largest dose of delivered radiation, and the importance of the duration of administration of iodine salts in blocking radiation to the thyroid is demonstrated. Optimal block times of 28 days for 131I and 42 days for 125I-labeled apolipoprotein tracers are proposed. When such a protocol is followed, the radiation dose to the thyroid and other organs is small by comparison to radiation doses allowed for workers whose occupation exposes them to radiation. The importance of frequent voiding to reduce the radiation dose to the bladder has also been demonstrated.

We have identified a 44-year-old patient with symmetrically excessive xanthomatosis, called Erdheim-Chester disease (ECD), and simultaneously decreased levels of low-density lipoprotein (LDL) cholesterol. Clinically, this patient presents lipoidgranulomatosis of numerous long and flat bones with involvement of the liver, spleen, pericardium, pleura, thyroid, skin, conjunctiva, and gingiva. However, the patient does not have any signs of atherosclerosis. So far, the underlying defect has not been elucidated. We performed a LDL-apolipoprotein B (apoB) kinetic study in the ECD patient and a normal control to determine the etiology of the low LDL level in ECD. LDL was isolated from both subjects, radioiodinated with either 131I or 125I, and injected simultaneously into the ECD patient and the normal control. Normal and ECD LDL was catabolized at the same rate after injection into the control subject (fractional catabolic rate [FCR], 0.43/d and 0.46/d, respectively). Therefore, LDL isolated from an ECD subject is metabolically normal. In contrast, autologous LDL injected into the ECD subject showed a markedly increased catabolism (FCR, 0.69/d) compared with that in the control subject (FCR, 0.43/d). This is the first report about increased catabolism of LDL cholesterol in a patient.

3H-leucine administered as a bolus has been widely used as a tracer in kinetic investigations of protein synthesis and secretion. After intravenous injection, plasma specific radioactivity decays over several orders of magnitude during the first half-day, followed by a slow decay lasting a number of weeks that results from recycling of the leucine tracer as proteins are degraded and 3H-leucine reenters the plasma pool. In studies in which kinetic data are analyzed by mathematical compartmental modeling, plasma leucine activity is generally used as a forcing function to drive the input of 3H-leucine into the protein synthesis pathway. 3H-leucine is an excellent tracer during the initial hours of rapidly decreasing plasma activity; thereafter, reincorporation of recycled tracer into new protein synthesis obscures the tracer data from proteins with slower turnover rates. Thus, for proteins such as plasma albumin and apolipoprotein (apo) A-I, this tracer is unsatisfactory for measuring fractional catabolic (FCR) and turnover rates. By contrast, the kinetics of plasma very-low-density lipoprotein (VLDL)-apoB, a protein with a residence time of approximately 5 hours, are readily measured, since kinetic parameters of this protein can be determined by the time plasma leucine recycling becomes established. However, measurement of VLDL-apoB specific radioactivity extending up to 2 weeks provides further data on the kinetic tail of VLDL-apoB. Were plasma leucine a direct precursor for the leucine in VLDL-apoB, the kinetics of the plasma tracer should determine the kinetics of the protein. However, this is not the case, and the deviations from linearity are interpreted in terms of (1) the dilution of plasma leucine in the liver by unlabeled dietary leucine; (2) the recycling of hepatocellular leucine from proteins within the liver, where recycled cellular leucine does not equilibrate with plasma leucine; and (3) a "hump" in the kinetic data of VLDL-apoB, which we interpret to reflect recycling or retention of a portion of the apoB protein within the hepatocyte, with its subsequent secretion. Because hepatocellular tRNA is the immediate precursor for synthesis of these secretory proteins, its kinetics should be used as the forcing function to drive the modeling of this system. The VLDL-apoB tail contains the information needed to modify the plasma leucine data, to provide an appropriate forcing function when using 3H-leucine as a tracer of apolipoprotein metabolism. This correction is essential when using 3H-leucine as a tracer for measuring low-density lipoprotein (LDL)-apoB kinetics. The 3H-leucine tracer also highlights the importance of recognizing the difference between plasma and system residence times, the latter including the time the tracer resides within exchanging extravascular pools. The inability to determine these fractional exchange coefficients for apoA-I and albumin explains the failure of this tracer in kinetic studies of these proteins. For apoB-containing lipoproteins, plasma residence times are generally determined, and these measurements can be made satisfactorily with 3H-leucine.

Familial defective apolipoprotein B-100 (FDB) is a frequently inherited disorder of lipoprotein metabolism. The glutamine-for-arginine substitution at position 3500 of apolipoprotein (apo) B-100 leads to defective binding of apo B-100 to the low density lipoprotein (LDL) receptor and accumulation of LDL in the plasma. We recently identified a patient homozygous for this mutation. His LDL cholesterol and apo B concentrations were approximately twice normal, whereas his apo E plasma level was low. Using a stable-isotope labeling technique ([2H3]leucine-primed constant infusion), we studied lipoprotein turnover in vivo in the fasting state in this patient and three clinically healthy, normolipidemic individuals not carrying the FDB mutation. The residence time of LDL apo B-100 was prolonged 3.6-fold in the FDB homozygote (8.3 vs 2.3 days). The production rate of LDL apo B-100 was decreased (7.4 vs 15 mg per kg per day). In FDB the residence time of very low density lipoprotein (VLDL) apo B-100 was longer (2.6 vs 1.3 hours), whereas the residence time of VLDL apo E was shorter (2.6 vs 4.5 hours) than normal. These data show that the in vivo metabolism of apo B-100-containing lipoproteins in FDB is different from that in familial hypercholesterolemia, in which LDL receptors are defective. In both conditions the residence times of LDL apo B-100 appear to be increased to approximately the same degree. This contrasts with the LDL apo B-100 synthetic rate, which is increased in familial hypercholesterolemia and decreased in FDB. The decreased production of LDL apo B-100 in FDB may originate from enhanced removal of apo E-containing LDL precursors by LDL receptors, which may be upregulated in response to the decreased flux of LDL-derived cholesterol into hepatocytes.

Metabolism of 1-stearoyl-2-arachidonyl-phosphatidyl-choline (SAPC), a major phosphatidylcholine (PC) species in rat plasma, was compared with 1-palmitoyl-2-linoleoyl-PC (PLPC) metabolism. High-density lipoproteins containing SAPC and PLPC tracers labeled in the sn-2 fatty acid with 3H and 14C isotopes, respectively, were administered. The rats were depleted of endogenous bile acids and infused via the ileum with individual bile acids that ranged widely in hydrophobicity. The half-lives for SAPC and PLPC in plasma were 48 and 57 min, respectively. Most of the 3H activity that disappeared from plasma at 1 h was found in the liver in 1-palmitoyl-2-arachidonyl-PC, SAPC, and 1-oleoyl-2-arachidonyl-PC, indicating phospholipase A1 hydrolysis of plasma SAPC forming 2-arachidonyl-lysophosphatidylcholine, which was reacylated in the liver. Plasma PLPC also underwent phospholipase A1 hydrolysis, as reported previously. The fraction of 3H dose that accumulated in plasma cholesteryl arachidonate was two- to threefold higher than the fraction of 14C dose in cholesteryl linoleate. Multicompartmental models for SAPC and PLPC were developed that included lysophosphatidylcholines and cholesteryl esters. Bile acids did not influence plasma PC metabolism. Lecithin-cholesterol acyltransferase and phospholipase A1 (hepatic lipase) hydrolysis accounted for > or = 90% of the SAPC and PLPC that disappeared from plasma; SAPC and PLPC are comparable as substrates for hepatic lipase, but SAPC is preferred by lecithin-cholesterol acyltransferase.

Background: The pharmacokinetics of dacarbazine (DTIC), which has been shown to be an effective therapeutic agent against metastatic melanoma, has not been extensively studied. However, to improve the clinical use of the drug, more information on the kinetics is required., Methods: A pharmacokinetic study was undertaken in six patients with melanoma of an extremity who were undergoing hyperthermic isolation perfusion with DTIC in order to understand better its clinical pharmacokinetics. Plasma was sampled from the arterial and venous lines of an extracorporeal pump during the perfusion with the systemic vein and urine sampled postperfusion. Samples were analyzed for DTIC. 2-azahypoxanthine (2-AZA), and aminoimidazole carboxamide (AIC). 99(m)Tc (Technetium) human serum albumin (HSA) was used in the perfusion circuit to monitor the crossover of the perfusate into the systemic circulation during the procedure. The data were analyzed using a compartmental model of sampled body compartments incorporating the isolated extremity., Results: High tissue DTIC levels were maintained throughout the perfusion, whereas in the systemic circulation, plasma DTIC concentrations, when observed, were 40-100-fold less than those in the perfusate. Almost 70% of the DTIC administered was not recovered in the perfusate after the washout of the extremity., Conclusions: High levels of DTIC can be maintained in an extremity (i.e., arm or leg) during perfusion.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) effectively inhibits cancer in a variety of tissues. In contrast to many other retinoids, the toxicity problems associated with administration of 4-HPR have been found to be minimal or absent. However, the effects of 4-HPR upon normal metabolism of native physiological forms of vitamin A in vivo have not been adequately investigated. To understand better the interaction between 4-HPR and the native physiological forms of vitamin A, the present study examines the effects of long-term administration of 4-HPR upon normal vitamin A metabolism in the eyes. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A (CON group; 0.8 retinol equivalents [RE]/g diet; n = 28) or a CON diet supplemented with 4-HPR (CON + 4-HPR group; 1173 micrograms 4-HPR/g diet; n = 28). Following an i.v. dose of physiologically radiolabelled retinol, associated with its normal plasma transport complex, the vitamin A content and radioactivity of the plasma and eyes were examined at different times over a 41 day period. Mean plasma retinol levels measured during the study period were significantly reduced in the CON + 4-HPR group as compared with the CON group (23.5 +/- 7.0 and 50.3 +/- 5.3 [mean +/- S.D.] micrograms/dl, respectively). From approximately 7 days post-dosing, vitamin A levels in the eyes of the 4-HPR-treated group steadily decreased such that by the end of the study, they were only approximately one-fifth those of the CON group (0.098 +/- 0.075 and 0.50 +/- 0.053 RE, respectively). Kinetic analysis of vitamin A turnover in the eyes indicated that there was no apparent down-regulation of the fraction of vitamin A leaving this tissue on a daily basis; these values were found to be similar in both groups, averaging 0.104 +/- 0.0393 and 0.113 +/- 0.0373 per day (mean +/- fractional standard deviation [F.S.D.]) for the CON and CON + 4-HPR groups, respectively. At the same time, the flow of vitamin A through the eyes was significantly decreased in the CON + 4-HPR group eyes (0.0162 +/- 0.101 microgram/day) as compared with the CON group (0.0604 +/- 0.0672 micrograms/day). Our results suggest that compensatory mechanisms that would normally function to conserve depleting ocular vitamin A stores may be blocked in the 4-HPR-treated animals and further, that the 4-HPR itself appears to be interfering with the normal uptake and/or metabolism of vitamin A in the eye. These findings may help to provide at least a partial explanation for the visual impairment problems that have been reported in human trials that include long-term administration of 4-HPR.

Mutations on the apolipoprotein (apo) B gene that interfere with the full-length translation of the apoB molecule are associated with familial hypobetalipoproteinemia (FHBL), a disease characterized by the reduction of plasma apoB and LDL cholesterol. In this report, we describe an FHBL kindred carrying a unique truncated apoB form, apoB-87Padova. Sequence analysis of amplified genomic DNA identified a single G deletion at nucleotide 12032, which shifts the translation reading frame and causes a termination at amino acid 3978. Two homozygous subjects and seven heterozygous relatives were studied. Although homozygous individuals had only trace amounts of LDL, they were virtually free from the symptoms typical of homozygous FHBL subjects. We investigated the in vivo turnover of radiolabeled normal apoB-100 LDL and apoB-87 LDL in one homozygous patient and two normal control subjects. ApoB-87 LDL showed a similar metabolism in all three subjects, with a fractional catabolic rate more than double that of normal LDL. The rate of entry of apoB-87 in the LDL compartment was also markedly decreased compared with normal apoB-100. The increased in vivo catabolism of apoB-87 LDL was paralleled in vitro by a 2.5-fold increased ability of these particles to inhibit the uptake and degradation of normal apoB-100 LDL by normal human cultured fibroblasts. These results indicate that apoB-87 LDL has an enhanced ability to interact with the LDL receptor, the increased apoB catabolism contributes to the hypobetalipoproteinemia and may explain the mild expression of the disease in the two homozygous individuals.

Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT-induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration.

ApoA-II is a major apolipoprotein constituent of high density lipoproteins (HDL) and may play an important role in lipoprotein metabolism and predisposition to atherosclerosis. Previous radiotracer kinetic studies have suggested that the metabolism of apoA-II in humans may be different than the metabolism of apoA-I, the major HDL apolipoprotein. In the present study, we have used an endogenous labeling technique using stable isotopically labeled amino acids to study apoA-II metabolism and compared the results to those obtained by a simultaneous exogenous radiotracer labeling method. Seven subjects with HDL cholesterol levels ranging from 9 to 93 mg/dl and apoA-II levels from 13 to 60 mg/dl were investigated in this study. [13C6]phenylalanine and 131I-labeled apoA-II were simultaneously administered as a primed-constant infusion and a bolus injection, respectively. In the endogenous labeling study, plateau tracer/tracee ratios of VLDL apoB-100 were used as estimates for the precursor pool tracer/tracee ratios for apoA-II synthesis. Residence times of apoA-II using these two independent methods were found to be highly correlated (r = 0.973, P < 0.0002). These results indicate that the endogenous labeling of apoA-II using stable isotopically labeled amino acids is a reasonable alternative to the conventional exogenous radiotracer labeling method for the investigation of apoA-II turnover. However, under the conditions of our experimental design and modeling strategy, the apoA-II residence times as determined by endogenous labeling were significantly longer (mean 5.33 days) than by exogenous radiotracer (mean 4.65 days). This suggests that apoA-II turnover may be even slower than believed based on radiotracer studies, and further supports the concept that HDL containing apoA-II are metabolized differently than HDL without apoA-II.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.

Metabolism of a 73 mumol oral dose of beta-carotene-d8 in olive oil was determined from plasma beta-carotene-d8 and retinol-d4 concentration-time curves in an adult male. beta-Carotene-d8 and retinol-d4 concentrations in serial plasma were measured using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Plasma beta-carotene-d8 and retinol-d4 concentration-time curves were described by a 5-term and a 3-term polyexponential equation, respectively, using an empirical description of beta-carotene metabolism. A physiologic compartmental model of beta-carotene metabolism was also constructed and tested. This model suggests that 22% of the beta-carotene dose is absorbed: 17.8% as intact beta-carotene and 4.2% as retinoid. Also, it suggests that both liver and enterocyte are important in converting beta-carotene to retinoid; 43% is converted in liver and 57% in enterocyte. Finally, it suggests that the mean residence time for beta-carotene is 51 days and that the 73 mumole dose does not alter the fractional transfer coefficients of the system after absorption takes place. The issue of central versus eccentric cleavage of beta-carotene in humans can be studied with further modeling combined with use of appropriately labeled beta-carotene.

Type III hyperlipoproteinemia is characterized by delayed chylomicron and VLDL remnant catabolism and is associated with homozygosity for the apoE-2 allele. We have identified a kindred in which heterozygosity for an apoE mutant, apoE-1 (Lys146-->Glu), is dominantly associated with the expression of type III hyperlipoproteinemia. DNA sequence analysis of the mutant apoE gene revealed a single-point mutation that resulted in the substitution of glutamic acid (GAG) for lysine (AAG) at residue 146 in the proposed receptor-binding domain of apoE. The pathophysiological effect of this mutation was investigated in vivo by kinetic studies in the patient and six normal subjects, and in vitro by binding studies of apoE-1 (Lys146-->Glu) to LDL receptors on human fibroblasts and to heparin. The kinetic studies revealed that apoE-1 (Lys146-->Glu) was catabolized significantly slower than apoE-3 in normals (P < 0.005). In the proband, the plasma residence times of both apoEs were substantially longer and the production rate of total apoE was about two times higher than in the control subjects. ApoE-1 (Lys146-->Glu) was defective in interacting with LDL receptors, and its ability to displace LDL in an in vitro assay was reduced to 7.7% compared with apoE-3. The affinity of apoE-1 (Lys146-->Glu) to heparin was also markedly reduced compared with both apoE-2 (Arg158-->Cys) and apoE-3. These abnormal in vitro binding characteristics and the altered in vivo metabolism of apoE-1 (Lys146-->Glu) are proposed to result in the functional dominance of this mutation in the affected kindred.

The kinetics of apolipoproteins A-I and A-II were examined in human subjects using leucine tracers administered intravenously. High density lipoproteins were separated and apoA-I and A-II were isolated. The specific activity or enrichment data for these apolipoprotein were analyzed by mathematical compartmental modeling. In 11 of 14 subjects studied with a bolus-injected [3H]leucine tracer, in 3 subjects studied similarly with [3H]leucine, and in one subject studied by primed dose, constant infusion of [3H]leucine, a rapidly turning-over apoA-I fraction was resolved. A similar component was observed in 7 of 10 studies of apoA-II. The apoA-I data were analyzed using a compartmental model (Zech, L.A. et al. 1983. J. Lipid Res. 24: 60-71) modified to incorporate plasma leucine as a precursor for apoprotein synthesis. The data permitted resolution of two apoA-I pools, one, C(2), turned-over with a residence time of less than 1 day, the other, C(1), a slowly turning-over pool, appeared in plasma after a delay of less than half a day. C(1) comprised the predominant mass of apoA-I and was also the primary determinant of the residence time of apoA-I. Although the mass of the fast pool, C(2), was considerably less than that of C(1), because of its rapid turnover, the quantities of apoA-I transported through this fast pathway were 2- to 4-fold greater. These kinetic studies indicate that apoA-I is secreted into both fast and slowly turning-over plasma pools. The latter is predominantly measured with radioiodinated apoA-I tracers. The data can be analyzed by postulating either separate input pathways to each of the pools or by assuming the fast pool is the precursor to the slow pool. Thus, apoA-I could be initially secreted as a family of particles that are rapidly cleared from plasma, and a portion of this apoprotein then reappears in a slowly turning-over pool that constitutes the major mass of apoA-I. The physiologic identity of these kinetically distinct apoA-I species is unknown; however, the fast pool of apoA-I demonstrated in these studies is strikingly similar to that seen in subjects with Tangier disease who lack the slow pool.

Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.

HDLs are heterogeneous in their apolipoprotein composition. Apolipoprotein (apo) A-I and apoA-II are the major proteins found in HDL and form the two major HDL subclasses: those that contain only apoA-I (LpA-I) and those that contain both apoA-I and apoA-II (LpA-I:A-II). Substantial evidence indicates that these two subclasses differ in their in vivo metabolism and effect on atherosclerosis, with LpA-I the more specifically protective subfraction against atherosclerosis. The purpose of this study was to investigate the effect of apoA-I and apoA-II production and catabolism on plasma LpA-I and LpA-I:A-II levels. Fifty normolipidemic subjects (those with HDL cholesterol levels in the top and bottom tenth percentiles were excluded) underwent kinetic studies with radiolabeled apoA-I and apoA-II, and the kinetic parameters of apoA-I and apoA-II were correlated with LpA-I and LpA-I:A-II levels. ApoA-I levels were strongly correlated with apoA-I residence times and less strongly correlated with apoA-I production rates. In contrast, apoA-II levels were correlated only with apoA-II production rates and not with apoA-II residence times. Levels of apoA-I in LpA-I were correlated with apoA-I residence times, whereas levels of apoA-I in LpA-I:A-II were correlated primarily with apoA-II production rates. The fraction of apoA-I in LpA-I was highly inversely correlated with apoA-II production rate (r = -.67, P < .001). In multiple regression analysis, apoA-II production rate was the most significant independent variable determining percent apoA-I in LpA-I among all the kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL). The role of the LDL receptor in the catabolism of Lp(a) has been controversial. We therefore investigated the in vivo catabolism of Lp(a) and LDL in five unrelated patients with homozygous familial hypercholesterolemia (FH) who have little or no LDL receptor activity. Purified 125I-Lp(a) and 131I-LDL were simultaneously injected into the homozygous FH patients, their heterozygous FH parents when available, and control subjects. The disappearance of plasma radioactivity was followed over time. As expected, the fractional catabolic rates (FCR) of 131I-LDL were markedly decreased in the homozygous FH patients (mean LDL FCR 0.190 d-1) and somewhat decreased in the heterozygous FH parents (mean LDL FCR 0.294 d-1) compared with controls (mean LDL FCR 0.401 d-1). In contrast, the catabolism of 125I-Lp(a) was not significantly different in the homozygous FH patients (mean FCR 0.251 d-1), heterozygous FH parents (mean FCR 0.254 d-1), and control subjects (mean FCR 0.287 d-1). In summary, absence of a functional LDL receptor does not result in delayed catabolism of Lp(a), indicating that the LDL receptor is not a physiologically important route of Lp(a) catabolism in humans.

The metabolism of high density lipoproteins (HDL) is tightly linked to the metabolism of apoB-containing lipoproteins through the exchange and transfer of lipids and apolipoproteins within the plasma compartment. Abetalipoproteinemia (ABL), a genetic disease in which apoB is absent from the plasma and HDL are the sole plasma lipoproteins, is a model for the investigation of HDL metabolism without modification by apoB-containing lipoproteins. Apolipoproteins A-I and E are two of the major apolipoproteins in HDL. Plasma apoA-I levels, but not apoE levels, have been reported to be decreased in patients with ABL. Furthermore, HDL from ABL patients is enriched in apoE compared with normal subjects. The purpose of the present study was: 1) to elucidate the metabolic basis of the low apoA-I levels in ABL; 2) to determine whether in vivo apoE production rates are normal in the absence of apoB-lipoprotein secretion; and 3) to test the hypothesis that apoE influences apoA-I and HDL catabolism in ABL. 131I-labeled apoA-I and 125I-labeled apoE were reassociated with autologous lipoproteins and injected into two unrelated ABL patients and control subjects. The mean residence time of apoA-I in ABL (2.4 days) was significantly decreased by nearly 50% compared with control subjects (4.7 +/- 0.6 days). ApoA-I production rates were also significantly decreased by 40% in ABL (7.1 mg/kg-d) compared with control subjects (11.8 +/- 1.7 mg/kg-d). The mean residence time of apoE in ABL (0.50 days) was somewhat shorter than that of control subjects (0.66 +/- 0.15 days), whereas the mean apoE production rate in ABL (2.14 mg/kg-d) was not substantially different from that of control subjects (1.55 +/- 0.62 mg/kg-d). HDL subfractions LpA-I and LpA-I:A-II were isolated using immunoaffinity chromatography. In contrast to the normal metabolism, apoA-I in LpA-I:A-II particles was catabolized at a faster rate than apoA-I in LpA-I, accounting for the greater decrease of plasma LpA-I:A-II relative to LpA-I in the ABL patients. HDL subfractions with and without apoE were also isolated using anti-apoE immunoaffinity chromatography. Labeled apoA-I in apoE-containing HDL was catabolized faster than that in HDL without apoE. Among the three different forms of apoE, the apoE monomer was catabolized at the fastest rate, the apoE homodimer at an intermediate rate, and the apoE-A-II heterodimer had the slowest rate of catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

The efficacy of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been demonstrated in the inhibition of cancers in a variety of tissues. Moreover, toxicity effects following administration of 4-HPR have been found to be reduced or absent when compared to other retinoids. Pharmacokinetic studies in both animals and humans have focused on the metabolism of 4-HPR and its metabolites, and relatively little information has been published detailing the effects of long-term administration of 4-HPR upon normal endogenous vitamin A metabolism. Thus, the present study was carried out to examine the effects of long-term administration of 4-HPR upon plasma and tissue vitamin A kinetics. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A [CON group; 1.0 retinol (ROH) equivalents/g diet] or a CON diet supplemented with 4-HPR (CON+4HPR group; 1173 micrograms 4-HPR/g diet). Following i.v. injection of a physiologically radiolabeled dose of ROH, ROH tracer and tracee kinetics were monitored in plasma and tissues over a 41-day period. Kinetic parameters were determined using the SAAM/CONSAM computer modeling programs to carry out graphical analysis of the tracer concentration curves. Mean plasma ROH levels measured for the CON+4HPR group were reduced to one-third of those of the CON group. Most of the kinetic parameters calculated were found to be significantly altered by the inclusion of 4-HPR in the diet. The fraction of the plasma ROH being catabolized per day (fractional catabolic rate) was nearly twice as high in the CON+4HPR treated group (3.61 +/- 0.49 day-1; mean +/- SD) as compared to the CON group (2.00 +/- 0.68 day-1). The amount of time that vitamin A molecules spent in the body before being lost irreversibly from the system (system residence time) was decreased by half in the CON+4HPR group (19.20 +/- 7.13 days) versus the CON group (38.63 +/- 9.62 days). Despite the increased catabolic rates and decreased system residence times measured for the CON+4HPR group, the estimated vitamin A use in these animals (11.01 +/- 3.10 micrograms/day) was 33% less than that used by the CON group (16.31 +/- 2.47 micrograms/day). Studies investigating the mechanisms by which 4-HPR alters vitamin A kinetics are presently under way in our laboratory. Nevertheless, these results suggest that long-term administration of 4-HPR markedly perturbs normal vitamin A metabolism in rats. Whether 4-HPR similarly alters human vitamin A metabolism with untoward clinical consequences deserves careful evaluation.

Lipoprotein(a) (Lp[a]) is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL) but contains an additional protein called apolipoprotein(a) (apo[a]). Apo(a) is highly polymorphic in size, and there is a strong inverse association between the size of the apo(a) isoform and the plasma concentration of Lp(a). We directly compared the in vivo catabolism of Lp(a) particles containing different size apo(a) isoforms to establish whether there is an effect of apo(a) isoform size on the catabolic rate of Lp(a). In the first series of studies, four normal subjects were injected with radio-labeled S1-Lp(a) and S2-Lp(a) and another four subjects were injected with radiolabeled S2-Lp(a) and S4-Lp(a). No significant differences in fractional catabolic rate were found between Lp(a) particles containing different apo(a) isoforms. To confirm that apo(a) isoform size does not influence the rate of Lp(a) catabolism, three subjects heterozygous for apo(a) were selected for preparative isolation of both Lp(a) particles. The first was a B/S3-apo(a) subject, the second a S4/S6-apo(a) subject, and the third an F/S3-apo(a) subject. From each subject, both Lp(a) particles were preparatively isolated, radiolabeled, and injected into donor subjects and normal volunteers. In all cases, the catabolic rates of the two forms of Lp(a) were not significantly different. In contrast, the allele-specific apo(a) production rates were more than twice as great for the smaller apo(a) isoforms than for the larger apo(a) isoforms. In a total of 17 studies directly comparing Lp(a) particles of different apo(a) isoform size, the mean fractional catabolic rate of the Lp(a) with smaller size apo(a) was 0.329 +/- 0.090 day-1 and of the Lp(a) with the larger size apo(a) 0.306 +/- 0.079 day-1, not significantly different. In summary, the inverse association of plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the catabolic rates of Lp(a) but rather to differences in Lp(a) production rates.

The biology of the low-density lipoprotein (LDL) receptor has been examined in detail, and a paradigm for LDL metabolism has evolved from comparative studies of cholesterol metabolism in a variety of cells cultured from normal individuals and subjects with familial hypercholesterolemia (FH). Cultured cells from patients with homozygous FH lack a functional LDL receptor and show diminished LDL clearance, induction of the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, increased cholesterol synthesis, decreased cholesterol ester production, and depleted cholesterol ester stores. The observed decrease in the fractional catabolic rate (FCR) of LDL is attributed to the mutated LDL receptor gene. However, in the experimental animal model of this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, cholesterol ester stores are increased, while hepatic cholesterol synthesis is decreased. Furthermore, in humans HMG-CoA reductase is suppressed, and the LDL apolipoprotein (apo) B production rate is increased in patients with FH. These findings raise questions about the adequacy of the paradigm in understanding hepatic cholesterol metabolism in vivo. In humans, apoB metabolism is believed to be principally determined by the liver, where apoB is both synthesized and catabolized. Assuming the neutral lipid content of the liver is the major determinant of apoB metabolism, we postulated that the changes in apoB metabolism in FH are predictable when based on the assumption of an increase in hepatic cholesterol and cholesterol ester content, as observed both in the WHHL rabbit and in humans. We examined this hypothesis in vivo in patients with heterozygous FH by using tracer kinetic methodology and have used similar data from normal and hypertriglyceridemic (HTG) subjects as controls. Whereas normal and HTG subjects secrete apoB primarily as large, triglyceride-enriched very-low-density lipoprotein (VLDL), heterozygous FH patients have an absolute decrease in apoB production and secrete almost 40% of apoB as smaller intermediate-density lipoprotein (IDL)/LDL. In normal humans, about half of secreted apoB is catabolized rather than being converted to LDL. In HTG subjects two thirds of apoB follows this same route, by which VLDL remnants remaining after triglyceride hydrolysis are largely returned to the liver. In contrast, in FH subjects secreted apoB is fully converted to LDL. Thus, although total apoB secretion is reduced in FH subjects, total LDL production is greater than in either normal or HTG subjects. Under basal conditions the elevated LDL in heterozygous FH is due to both decreased LDL receptor-mediated catabolism and increased LDL production. However, the number of LDL receptors actually expressed is suppressed below the number of potentially functional receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein A-I is the major apolipoprotein constituent of high density lipoproteins (HDL). Methods used to investigate in vivo kinetics of apoA-I include exogenous labeling with radioiodine and endogenous labeling with stable isotopically labeled amino acids. We report here a direct comparison of these methods to determine the in vivo kinetics of apoA-I in four normal subjects. Purified apoA-I was labeled with 125I, reassociated with autologous plasma, and injected into study subjects. At the same time, [13C6]phenylalanine was administered as a primed constant infusion for up to 14 hours. The kinetic parameters of apoA-I were determined from the 125I-labeled apoA-I plasma curves. For the analysis of data from stable isotope studies, very low density lipoprotein (VLDL) apoB-100, VLDL apoB-48, and total apoA-I were isolated by ultracentrifugation and subsequent preparative NaDodSO4-PAGE, hydrolyzed, and derivatized. The tracer/tracee ratio was determined by gas chromatography-mass spectrometry. Monoexponential function analysis was used to determine the tracer/tracee curves of VLDL apoB-100 and VLDL apoB-48, and total apoA-I. The mean plateau tracer/tracee ratio of VLDL apoB-100 (primarily liver-derived) was 5.19%, whereas that of VLDL apoB-48 (intestinally derived) was only 3.74%. Using the VLDL apoB-100 plateau tracer/tracee ratio as the estimate of the precursor pool enrichment for apoA-I, the mean apoA-I residence time (RT) was 5.14 +/- 0.41 days, compared with 4.80 +/- 0.30 days for the exogenous labeling method. The apoA-I RTs using these two methods were highly correlated (r = 0.874).(ABSTRACT TRUNCATED AT 250 WORDS)

Familial hyperalphalipoproteinemia (FHA) is a heritable trait associated with elevated plasma concentrations of high-density lipoprotein (HDL) cholesterol and possibly with longevity and protection against coronary heart disease (CHD). The metabolic basis and molecular etiology of FHA have not been established in most kindreds. The proband of a kindred with FHA and possible longevity was found to have elevated plasma levels of HDL cholesterol, apolipoprotein (apo) A-I, and lipoproteins containing apo A-I without apo A-II (Lp A-I), but normal levels of apo A-II and lipoproteins containing apo A-I with apo A-II (Lp A-I:A-II). The in vivo kinetics of apo A-I and apo A-II were studied in the FHA proband and in control subjects using both exogenous radiotracer (125I-apo A-I and 131I-apo A-II) and endogenous stable isotope (primed constant infusion of 13C6-phenylalanine) labeling techniques. The production rate (PR) of apo A-I was markedly increased in the FHA subject (28.9 mg/kg.d) compared with the control subjects (12.0 +/- 2.1 mg/kg.d), whereas the apo A-II PR was not substantially increased. The primary sequence of the proband's apo A-I gene, including 1.2 kb of the 5'-flanking sequence, was normal. We conclude that a selective upregulation of apo A-I production is one metabolic cause of FHA, and results in high plasma concentrations of HDL cholesterol, apo A-I, and Lp A-I and possibly in protection from atherosclerotic CHD.

Deficiency of the cholesteryl ester transfer protein (CETP) in humans is characterized by markedly elevated plasma concentrations of HDL cholesterol and apoA-I. To assess the metabolism of HDL apolipoproteins in CETP deficiency, in vivo apolipoprotein kinetic studies were performed using endogenous and exogenous labeling techniques in two unrelated homozygotes with CETP deficiency, one heterozygote, and four control subjects. All study subjects were administered 13C6-labeled phenylalanine by primed constant infusion for up to 16 h. The fractional synthetic rates (FSRs) of apoA-I in two homozygotes with CETP deficiency (0.135, 0.134/d) were found to be significantly lower than those in controls (0.196 +/- 0.041/d, P < 0.01). Delayed apoA-I catabolism was confirmed by an exogenous radiotracer study in one CETP-deficient homozygote, in whom the fractional catabolic rate of 125I-apoA-I was 0.139/d (normal 0.216 +/- 0.018/d). The FSRs of apoA-II were also significantly lower in the homozygous CETP-deficient subjects (0.104, 0.112/d) than in the controls (0.170 +/- 0.023/d, P < 0.01). The production rates of apoA-I and apoA-II were normal in both homozygous CETP-deficient subjects. The turnover of apoA-I and apoA-II was substantially slower in both HDL2 and HDL3 in the CETP-deficient homozygotes than in controls. The kinetics of apoA-I and apoA-II in the CETP-deficient heterozygote were not different from those in controls. These data establish that homozygous CETP deficiency causes markedly delayed catabolism of apoA-I and apoA-II without affecting the production rates of these apolipoproteins.

Apolipoprotein (apo) A-IV is a polymorphic, intestinally derived apolipoprotein that is genetically linked to and similar in structure to apoA-I, the major apolipoprotein in high density lipoproteins (HDL). ApoA-IV plays a potentially important role in lipoprotein metabolism and reverse cholesterol transport, but its in vivo metabolism is poorly understood. In order to gain insight into factors modulating apoA-IV metabolism in humans, the in vivo kinetics of the two major human apoA-IV isoproteins apoA-IV-1 and apoA-IV-2 were investigated in normolipidemic human subjects. 131I-apoA-IV-1 and 125I-apoA-IV-2 were reassociated with autologous plasma and injected into study subjects. Analysis of the kinetic data revealed a rapid mean fractional catabolic rate (FCR) for apoA-IV-1 of 2.42 +/- 0.11 d-1. The mean production, or transport, rate of apoA-IV-1 was 16.3 +/- 1.4 mg/kg per d. Plasma apoA-IV concentrations were highly correlated with apoA-IV production rate (r = 0.84, P < 0.001) and not correlated with apoA-IV fractional catabolic rate (r = 0.25, P = NS). The mean FCR of apoA-IV-2 was 2.21 +/- 0.10 d-1. In the ten subjects in whom 131I-apoA-IV-1 and 125I-apoA-IV-2 were simultaneously injected, the FCR of apoA-IV-2 was significantly slower by paired t test (P = 0.003). The FCR of apoA-IV-2 in an apoA-IV-2/2 homozygote was only 1.49 d-1, substantially slower than in all other subjects. We conclude that: (a) apoA-IV is a rapidly catabolized apolipoprotein in humans, with a fractional catabolic rate more than 10 times greater than that of apoA-I; (b) apoA-IV has a high absolute transport rate similar to that of apoA-I; (c) plasma levels of apoA-IV are primarily determined by apoA-IV production rate in normolipidemic subjects; and (d) the fractional catabolic rate of the common variant apoA-IV-2 is slower than that of the wild-type apoA-IV-1.

Apo A-IMilano is a mutant form of apo A-I in which cysteine is substituted for arginine at amino acid 173. Subjects with apo A-IMilano are characterized by having low levels of plasma HDL cholesterol and apo A-I. To determine the kinetic etiology of the decreased plasma levels of the apo A-I in these individuals, normal and mutant apo A-I were isolated, radiolabeled with either 125I or 131I, and both types of apo A-I were simultaneously injected into two normal control subjects and two subjects heterozygous for apo A-IMilano. In the normal subjects, apo A-IMilano was catabolized more rapidly than the normal apo A-I (mean residence times of 5.11 d for normal apo A-I vs. 3.91 d for apo A-IMilano), clearly establishing that apo A-IMilano is kinetically abnormal and that it has a shortened residence time in plasma. In the two apo A-IMilano subjects, both types of apo A-I were catabolized more rapidly than normal (residence times ranging from 2.63 to 3.70 d) with normal total apo A-I production rates (mean of 10.3 vs. 10.4 mg/kg per d in the normal subjects). Therefore, in the subjects with apo A-IMilano, the decreased apo A-I levels are caused by rapid catabolism of apo A-I and not to a decreased production rate, and the abnormal apo A-IMilano leads to the rapid catabolism of both the normal and mutant forms of apo A-I in the affected subjects.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to, but metabolically distinct from, LDL. Factors regulating plasma concentrations of Lp(a) are poorly understood. Apo(a), the protein that distinguishes Lp(a) from LDL, is highly polymorphic, and apo(a) size is inversely correlated with plasma Lp(a) level. Even within the same apo(a) isoform class, however, plasma Lp(a) concentrations vary widely. A series of in vivo kinetic studies were performed using purified radiolabeled Lp(a) in individuals with the same apo(a) isoform but different Lp(a) levels. In a group of seven subjects with a single S4-apo(a) isoform and Lp(a) levels ranging from 1 to 13.2 mg/dl, the fractional catabolic rate (FCR) of 131I-labeled S2-Lp(a) (mean 0.328 day-1) was not correlated with the plasma Lp(a) level (r = -0.346, P = 0.45). In two S4-apo(a) subjects with a 10-fold difference in Lp(a) level, the FCR's of 125I-labeled S4-Lp(a) were very similar in both subjects and not substantially different from the FCRs of 131I-S2-Lp(a) in the same subjects. In four subjects with a single S2-apo(a) isoform and Lp(a) levels ranging from 9.4 to 91 mg/dl, Lp(a) concentration was highly correlated with Lp(a) production rate (r = 0.993, P = 0.007), but poorly correlated with Lp(a) FCR (mean 0.304 day-1). Analysis of Lp(a) kinetic parameters in all 11 subjects revealed no significant correlation of Lp(a) level with Lp(a) FCR (r = -0.53, P = 0.09) and a strong correlation with Lp(a) production rate (r = 0.99, P < 0.0001). We conclude that the substantial variation in Lp(a) levels among individuals with the same apo(a) phenotype is caused primarily by differences in Lp(a) production rate.