Plasma Branched-Chain Amino Acid Concentrations and Glucose Homeostasis in Kidney Transplant Recipients and Candidates.

Background: Post-transplant diabetes mellitus (PTDM) encompasses new-onset and previously unrecognized type 2 diabetes. Kidney failure masks type 2 diabetes. Branched-chain amino acids (BCAA) are closely associated with glucose metabolism. Therefore, understanding BCAA metabolism both in kidney failure and after kidney transplantation may inform PTDM mechanisms. Objective: To understand the impact of present or absent kidney function on plasma BCAA concentrations. Design: Cross-sectional study of kidney transplant recipients and kidney transplant candidates. Setting: Large kidney transplant center in Toronto, Canada. Measurements: We measured plasma BCAA and aromatic amino acid (AAA) concentrations in 45 pre-kidney transplant candidates (15 with type 2 diabetes, 30 without type 2 diabetes) and 45 post-kidney transplant recipients (15 PTDM, 30 non-PTDM), along with insulin resistance and sensitivity by 75 g oral glucose loading for those in each group without type 2 diabetes. Methods: Plasma AA concentrations were analyzed using MassChrom AA Analysis and compared between groups. The insulin sensitivity for oral glucose tolerance tests or Matsuda index (a measure of whole-body insulin resistance), Homeostatic Model Assessment for Insulin Resistance (a measure of hepatic insulin resistance), and Insulin Secretion-Sensitivity Index-2 (ISSI-2, a measure of pancreatic ß-cell response) was calculated from fasting insulin and glucose concentrations, and compared with BCAA concentrations. Results: Each BCAA concentration was higher in post-transplant subjects than pre-transplant subjects (P < .001 for leucine, isoleucine, valine). In post-transplant subjects, each BCAA concentration was higher in PTDM versus non-PTDM (odds ratio for PTDM 3-4 per 1 SD increase in BCAA concentration, P < .001 for each). Tyrosine concentrations were also higher in post-transplant subjects than pre-transplant subjects, but tyrosine did not differ by PTDM status. By contrast, neither BCAA nor AAA concentrations were different in pre-transplant subjects with or without type 2 diabetes. Whole-body insulin resistance, hepatic insulin resistance, and pancreatic ß-cell response did not differ between nondiabetic post-transplant and pre-transplant subjects. Branched-chain amino acid concentrations correlated with the Matsuda index and Homeostatic Model Assessment for Insulin Resistance (P < .05 for each) only in nondiabetic post-transplant subjects-not in nondiabetic pre-transplant subjects. Branched-chain amino acid concentrations did not correlate with ISSI-2 in either pre-transplant or post-transplant subjects. Limitations: The sample size was small, and subjects were not studied prospectively for the development of type 2 diabetes. Conclusions: Plasma BCAA concentrations are higher post-transplant in type 2 diabetic states, but do not differ by diabetes status in the presence of kidney failure. The association of BCAA with measures of hepatic insulin resistance among nondiabetic post-transplant patients is consistent with impaired BCAA metabolism as a characteristic of kidney transplantation.

Contexte: Le diabète post-transplantation (DPT) englobe les nouvelles manifestations du diabète de type 2 nouveau et le diabète précédemment non reconnu. L'insuffisance rénale masque le diabète de type 2. Les acides aminés à chaîne ramifiée (AACR) sont étroitement liés au métabolisme du glucose. Par conséquent, la compréhension du métabolisme des acides aminés à chaîne ramifiée (AACR) à la fois dans l'insuffisance rénale et après la transplantation rénale peut informer les mécanismes de DPT. Objectifs: Comprendre l'impact de la présence ou de l'absence de fonction rénale sur les concentrations plasmatiques d'AACR. Type d'étude: Étude transversale portant sur des receveurs d'une greffe rénale et des candidats à une transplantation de rein. Cadre: Un grand centre de transplantation rénale de Toronto (Canada). Mesures: Nous avons mesuré les concentrations plasmatiques d'AACR et d'AA aromatiques (AAA) chez 45 candidats pré-transplantation rénale (15 atteints de diabète de type 2; 30 non-diabétiques) et 45 patients ayant reçu une greffe rénale (15 DPT, 30 non-DPT). Les patients des groupes non-diabétiques ont en outre subi un test de résistance et de sensibilité à l'insuline à la suite de l'administration orale de 75 g de glucose. Méthodologie: Les concentrations plasmatiques d'AA ont été analysées à l'aide de l'appareil Mass Chrom AA Analysis et comparées entre les groupes. La sensibilité à l'insuline pour les tests oraux de tolérance au glucose ou l'indice Matsuda (mesure de la résistance à l'insuline dans tout l'organisme), l'évaluation du modèle homéostatique de la résistance à l'insuline (mesure de la résistance hépatique à l'insuline) et l'indice de sensibilité à la sécrétion d'insuline-2 (mesure de la réponse des cellules ß pancréatiques) ont été calculés à partir des concentrations d'insuline et de glucose à jeun, et comparés aux concentrations d'AACR. Résultats: Chacune des concentrations en AACR était plus élevée chez les sujets post-transplantation que chez les sujets pré-transplantation (p < 0,001 pour la leucine, l'isoleucine, la valine). Chez les sujets post-transplantation, chaque concentration d'AACR était plus élevée chez les sujets DPT que chez le cas des sujets non-DPT (RC pour DPT: entre 3 et 4 pour chaque augmentation de l'écart-type; p < 0,001 pour chacun). Les concentrations de tyrosine étaient également plus élevées chez les sujets post-transplantation que chez les sujets pré-transplantation, mais ne différaient pas selon le statut du DPT. En revanche, ni les concentrations d'AACR ni les concentrations d'AAA n'étaient différentes chez les sujets pré-transplantation qu'ils soient ou non atteints de diabète de type 2. La résistance de tout l'organisme à l'insuline, la résistance hépatique à l'insuline et la réponse des cellules ß pancréatiques ne différaient pas entre les sujets non-diabétiques avant ou après la transplantation. Les concentrations d'AACR étaient corrélées avec l'indice Matsuda et l'évaluation du modèle homéostatique de la résistance à l'insuline (p<0,05 pour chacun) uniquement chez les sujets non-diabétiques après la transplantation, et non chez les sujets non-diabétiques avant la transplantation. Les concentrations d'AACR n'étaient pas en corrélation avec l'ISSI-2, que ce soit chez les sujets avant ou après la transplantation. Limites: L'échantillon était de petite taille et les sujets n'ont pas été étudiés prospectivement pour le développement du diabète de type 2. Conclusion: Les concentrations plasmatiques d'AACR sont plus élevées après la transplantation chez les sujets diabétiques de type 2, mais ne diffèrent pas selon le statut du diabète en présence d'une insuffisance rénale. Les associations entre les AACR et les mesures de la résistance hépatique à l'insuline chez les patients non-diabétiques post-transplantation sont cohérentes avec une altération du métabolisme des AACR comme caractéristique de la transplantation rénale.

Effect of short-term intensive insulin therapy on the incretin response in early type 2 diabetes.

AIMS: Short-term intensive insulin therapy (IIT) and gastric bypass surgery are both interventions that can improve beta-cell function, reduce insulin resistance and induce remission of type 2 diabetes. Whereas gastric bypass yields an enhanced glucagon-like peptide-1 (GLP-1) response that may contribute to its metabolic benefits, the effect of short-term IIT on the incretin response is unclear. Thus, we sought to evaluate the impact of IIT on GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretion in early type 2 diabetes. METHODS: In this study, 63 patients (age 59±8.3 years, baseline A1c 6.8±0.7%, diabetes duration 3.0±2.1 years) underwent 4 weeks of IIT (basal insulin detemir and pre-meal insulin aspart). GLP-1, GIP and glucagon responses were assessed by the area-under-the-curve (AUC) of these hormones on oral glucose tolerance tests at baseline and 1-day after the completion of therapy. Beta-cell function was assessed by Insulin Secretion-Sensitivity Index-2 (ISSI-2), with insulin resistance measured by Homeostasis Model Assessment (HOMA-IR). RESULTS: As expected, comparing the post-therapy oral glucose tolerance test to that at baseline, IIT increased ISSI-2 (P=0.02), decreased HOMA-IR (P<0.001), and reduced AUCglucagon (P<0.001). Of note, however, IIT had no significant impact on AUCGLP-1 (P=0.24) and reduced AUCGIP (P=0.02). CONCLUSION: Despite improving beta-cell function, insulin resistance and glucagonemia, short-term IIT does not change GLP-1 secretion and decreases the GIP response to an oral glucose challenge in early type 2 diabetes. Thus, the beneficial impact of this therapy on glucose homeostasis is not attributable to its effects on incretin secretion.

Docosahexaenoic acid-enriched canola oil increases adiponectin concentrations: a randomized crossover controlled intervention trial.

BACKGROUND AND AIMS: Little is known about the effect of various dietary fatty acids on pro- and anti-inflammatory processes. We investigated the effect of 5 oils containing various amounts of alpha-linolenic acid (ALA), linoleic acid (LA), oleic acid (OA) and docosahexaenoic acid (DHA) on plasma inflammatory biomarkers and expression levels of key inflammatory genes and transcription factors in whole blood cells. METHODS AND RESULTS: In a randomized, crossover controlled nutrition intervention, 114 adult men and women with abdominal obesity and at least one other criterion for the metabolic syndrome consumed 5 experimental isoenergetic diets for 4 weeks each, separated by 4-week washout periods. Each diet provided 60 g/3000 kcal of different oils: 1) control corn/safflower oil blend (CornSaff; LA-rich), 2) flax/safflower oil blend (FlaxSaff; ALA-rich), 3) conventional canola oil (Canola; OA-rich), 4) high oleic canola oil (CanolaOleic; highest OA content), 5) DHA-enriched high oleic canola oil (CanolaDHA; OA- and DHA-rich). Gene expression in whole blood cells was assessed in a subset of 62 subjects. CanolaDHA increased plasma adiponectin concentrations compared with the control CornSaff oil treatment (+4.5%, P = 0.04) and FlaxSaff (+6.9%, P = 0.0008). CanolaDHA also reduced relative expression levels of interleukin (IL)1B compared with CornSaff and Canola (-11% and -13%, respectively, both P = 0.03). High-sensitivity C-reactive protein concentrations were lower after Canola than after FlaxSaff (-17.8%, P = 0.047). CONCLUSION: DHA-enriched canola oil exerts anti-inflammatory effects compared with polyunsaturated fatty acids from plant sources.

Paraoxonase 1 arylesterase activity and mass are reduced and inversely related to C-reactive protein in patients on either standard or home nocturnal hemodialysis.

UNLABELLED: Human serum paraoxonase (PON1) activity is reduced in standard hemodialysis (SHD) (4 hours, 3 days/week) patients. Home nocturnal hemodialysis (HNHD) (8 hours, 6 days/week), provides a greater dialysis dose resulting in a greater clearance of metabolites. Whether improvements in the metabolic milieu of HNHD patients results in different PON1 activity levels compared to SHD patients is unclear. We determined serum PON1 mass and arylesterase activities in a group of HNHD patients and compared them to SHD patients and a group of healthy controls (HC). PATIENTS AND METHODS: We measured PON1 arylesterase activity and mass, C-reactive protein (CRP), cystatin C, total and high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoproteins A-I and B in 15 HNHD, 15 SHD and 15 HC participants. RESULTS: PON1 arylesterase activity (p < 0.001) and mass (p < 0.05) were significantly higher in HC participants compared to SHD and HNHD participants, although no significant differences were noted between HD groups. CRP (p < 0.05) was significantly higher in SHD compared to HC participants and there were no significant differences noted between HD groups. Cystatin C (p < 0.001) was significantly different among the 3 groups. There were no significant differences noted in any lipoprotein parameters among the groups. PON1 activity (r = -0.636, p < 0.001) and mass (r = -0.425, p = 0.019) were inversely correlated with CRP in HD patients. CONCLUSION: PON1 is reduced in HNHD patients compared to HC subjects, independent of the concentration of HDL cholesterol. Within subjects on HD, the combination of increased CRP and reduced PON1 may identify subjects at a high risk for cardiovascular complications.

Relation of waist circumference and glycemic status to C-reactive protein in the Sandy Lake Oji-Cree.

OBJECTIVE: To determine the prevalence of elevated C-reactive protein (CRP) in the Sandy Lake Oji-Cree, an aboriginal community residing in the Sioux Lookout zone of Northwestern Ontario, Canada, and to determine the associations of obesity and diabetes with CRP in a community with a very high prevalence of type II diabetes. DESIGN: We surveyed 512 community members aged 18 y and older to determine the prevalence and the determinants of elevated CRP in Sandy Lake. MEASUREMENTS: Clinical variables, indices of obesity and serum concentrations of CRP, insulin, serum amyloid A (SAA) and interleukin-6 (IL-6). RESULTS: The prevalence of CRP >or=3.8 mg/l was significantly higher in women than in men (51% vs. 32%, P<0.0001). Regression analysis determined that body mass index and IL-6 were independent determinants of CRP concentration in women and waist circumference and IL-6 were independent determinants of CRP concentration in men. Diabetes was associated with elevated CRP in both sexes, but was only a moderate strong determinant in CRP concentration in multivariate regression analysis. CONCLUSIONS: The prevalence of elevated CRP in this aboriginal community is remarkably high. These data further demonstrate that the association between CRP and specific indices of obesity and metabolism vary according to gender and glycemic status.

Effect of a very-high-fiber vegetable, fruit, and nut diet on serum lipids and colonic function.

We tested the effects of feeding a diet very high in fiber from fruit and vegetables. The levels fed were those, which had originally inspired the dietary fiber hypothesis related to colon cancer and heart disease prevention and also may have been eaten early in human evolution. Ten healthy volunteers each took 3 metabolic diets of 2 weeks duration. The diets were: high-vegetable, fruit, and nut (very-high-fiber, 55 g/1,000 kcal); starch-based containing cereals and legumes (early agricultural diet); or low-fat (contemporary therapeutic diet). All diets were intended to be weight-maintaining (mean intake, 2,577 kcal/d). Compared with the starch-based and low-fat diets, the high-fiber vegetable diet resulted in the largest reduction in low-density lipoprotein (LDL) cholesterol (33% +/- 4%, P <.001) and the greatest fecal bile acid output (1.13 +/- 0.30 g/d, P =.002), fecal bulk (906 +/- 130 g/d, P <.001), and fecal short-chain fatty acid outputs (78 +/- 13 mmol/d, P <.001). Nevertheless, due to the increase in fecal bulk, the actual concentrations of fecal bile acids were lowest on the vegetable diet (1.2 mg/g wet weight, P =.002). Maximum lipid reductions occurred within 1 week. Urinary mevalonic acid excretion increased (P =.036) on the high-vegetable diet reflecting large fecal steroid losses. We conclude that very high-vegetable fiber intakes reduce risk factors for cardiovascular disease and possibly colon cancer. Vegetable and fruit fibers therefore warrant further detailed investigation.

Common and rare ABCA1 variants affecting plasma HDL cholesterol.

Mutations in ABCA1, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of ABCA1 can affect plasma HDL cholesterol in the general population. To address this issue, we developed a screening strategy to find common ABCA1 variants. This strategy involved long-range amplification of genomic DNA by using coding sequences only, followed by deep sequencing into the introns. This method helped us to characterize a new set of amplification primers, which permitted amplification of virtually all of the coding sequence of ABCA1 and its intron-exon boundaries with a single DNA amplification program. With these new sequencing primers, we found 3 novel ABCA1 mutations: a frameshift mutation (4570insA, A1484S-->X1492), a missense mutation (A986D) in a TD family, and a missense mutation (R170C) in aboriginal subjects with FHA. We also used these sequencing primers to characterize 4 novel common amino acid variants in ABCA1, in addition to 5 novel common silent variants. We tested for association of the ABCA1 I/M823 variant with plasma HDL cholesterol in Canadian Inuit and found that M823/M823 homozygotes had significantly higher plasma HDL cholesterol compared with subjects with the other genotypes. The results provide proof of principle of the effectiveness of this approach to identify both rare and common ABCA1 genomic variants and also suggest that common amino acid variation in ABCA1 is a determinant of plasma HDL cholesterol in the general population.

Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein bind to separate domains of CD36.

The cytoadherence of erythrocytes (red blood cells) infected with Plasmodium falciparum (pRBCs) to endothelial cells and the uptake of oxidized low-density lipoprotein (oxLDL) by macrophages are both mediated, in part, by the glycoprotein receptor CD36. The interaction of lipoproteins and pRBCs competing for the human CD36 receptor was examined by use of Chinese hamster ovary cells expressing human CD36. OxLDL competitively inhibits the adherence of pRBCs to CD36, but native LDL and high-density lipoprotein do not. Modification of Lys residues in CD36 inhibits both oxLDL and pRBC binding; however, only oxLDL binding is inhibited by receptor iodination, and only pRBC binding is influenced by pH variations and receptor reduction. Furthermore, peptide inhibitors of the pRBC/CD36 interaction do not influence oxLDL binding. These results suggest that, although oxLDL competitively inhibits the adherence of pRBCs, these ligands interact with distinct domains on the CD36 receptor.

The prevalence of hyperlipidemia in women and its association with use of oral contraceptives, sex hormone replacement therapy and nonlipid coronary artery disease risk factors. Canadian Heart Health Surveys Research Group.

OBJECTIVE: To report the prevalence of lipid and nonlipid coronary artery disease risk factors in women classified by use of oral contraceptives or sex hormone replacement therapy. DESIGN, SETTING AND PARTICIPANTS: A population-based cross-sectional survey in nine Canadian provinces (not including Nova Scotia) between 1988 and 1992 invited 13,506 women aged 18 to 74 years to participate. During a clinic visit after a home interview, a blood sample was obtained following a fast of 8 h or more from 8637 women. OUTCOME MEASURES: Fasting plasma total cholesterol, triglycerides, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, blood pressure, smoking status, self-reported diabetes, and self-reported use of oral contraceptive or sex hormone replacement therapy pills. MAIN RESULTS: The prevalence of oral contraceptive use was 41% for women 18 to 24 years old and 20% for women 25 to 34 years old. The prevalence of sex hormone replacement therapy was 4% for women 35 to 44 years old, 20% for women 45 to 64 years old and 11% for women 65 to 74 years old. Users of sex hormone replacement therapy aged 35 to 44 years had slightly higher mean LDL cholesterol than nonusers (3.04 versus 2.89 mmol/L). Users and nonusers aged 45 to 54 years had similar LDL cholesterol levels, and users aged 55 to 64 and 65 to 74 years had lower LDL cholesterol and higher HDL cholesterol levels, respectively, than nonusers. Triglyceride levels were higher in oral contraceptive users and in younger women on sex hormone replacement therapy than in nonusers. In the general population of Canada the use of oral contraceptives in women less than age 35 years had only a marginal effect on the prevalence of lipid and nonlipid risk factors. Women aged 18 to 24 years using oral contraceptives had a higher mean LDL cholesterol level of 2.73 versus 2.35 mmol/L for nonusers. The prevalence of lipid and nonlipid risk factors in women using sex hormone replacement therapy increased slightly for those aged 35 to 54 years and decreased in women aged 55 to 74 years. A lower percentage of women using sex hormone replacement therapy, aged 55 to 74 years, had high risk LDL cholesterol levels (21% versus 36% for nonusers). A larger percentage of women using sex hormone replacement therapy had low risk HDL cholesterol levels (54% versus 29% for nonusers). The nonlipid risk factor profile for women aged 35 to 54 years on sex hormone replacement therapy was less favourable than for nonusers: obesity was more common (36% versus 28%, respectively), hypertension was higher (22% versus 12%, respectively), and the proportion of women with one or more nonlipid risk factors was higher. The nonlipid risk factor profile for women 55 to 74 years of age who were using sex hormone replacement therapy was more favourable than for nonusers: obesity was lower (31% versus 47%, respectively), smoking was lower (7% versus 16%, respectively), sedentary behaviour was lower (28% versus 37%, respectively), and fewer women had two or more of these risk factors (31% versus 52%, respectively). CONCLUSION: The findings suggest that women at higher risk for coronary artery disease tend to have a lower prevalence of use of sex hormone replacement therapy.

Prevalence of high plasma triglyceride combined with low HDL-C levels and its association with smoking, hypertension, obesity, diabetes, sedentariness and LDL-C levels in the Canadian population. Canadian Heart Health Surveys Research Group.

OBJECTIVE: To report the associations of plasma triglyceride, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) with nonlipid coronary artery disease risk factors. In particular, the associations for persons with high triglyceride and low HDL-C levels were examined. DESIGN: A stratified random probability sample of 29,855 men and women aged 18 to 74 years from the Canadian Heart Health Surveys (1986 to 1992) in 10 provinces. Blood samples were obtained from 18,555 participants who had fasted for 8 h or more. Plasma lipids were determined at the J Alick Little Lipid Research Laboratory, Toronto, Ontario, with standardization of the Centers for Disease Control Lipid Standardization Program, Atlanta. OUTCOME MEASURES: Fasting plasma total cholesterol, triglyceride, LDL-C and HDL-C levels. MAIN RESULTS: The prevalence of men with triglyceride levels above 1.7 mmol/L and HDL-C levels below 0.9 mmol/L was 10%, compared with 3% for men with triglyceride levels below 1.7 mmol/L and HDL-C levels below 0.9 mmol/L. The prevalence of women with triglyceride levels above 1.7 mmol/L and HDL-C levels below 0.9 mmol/L was 3% compared with a prevalence of less than 1% for women with triglyceride levels below 1.7 mmol/L and HDL-C levels below 0.9 mmol/L. Even when plasma LDL-C was low at less than 3.4 mmol/L, there was an age trend for increasing prevalences of the combination of triglyceride levels 2.3 mmol/L or greater and HDL-C levels less than 0.9 mmol/L in both sexes. The prevalence of a triglyceride levels 2.3 mmol/L or greater combined with an HDL-C level below 0.9 mmol/L was increased in groups who were cigarette smokers, diabetic, hypertensive, obese or sedentary, or who had higher LDL-C levels in both sexes, and the increase was even greater in the presence of two or more of these other risk factors. CONCLUSIONS: Among men or women with low HDL-C and high triglyceride levels, smoking, diabetes, sedentariness, hypertension and obesity were much more prevalent than among those at low risk with high HDL-C and low triglyceride levels.

Hepatic lipase deficiency.

Hepatic lipase (HL) is an enzyme that is made primarily by hepatocytes (and also found in adrenal gland and ovary) and hydrolyzes phospholipids and triglycerides of plasma lipoproteins. It is secreted and bound to the hepatocyte surface and readily released by heparin. It is a member of the lipase superfamily and is homologous to lipoprotein lipase and pancreatic lipase. The enzyme can be divided into an NH2-terminal domain containing the catalytic site joined by a short spanning region to a smaller COOH-terminal domain. The NH2-terminal portion contains an active site serine in a pentapeptide consensus sequence, Gly-Xaa-Ser-Xaa-Gly, as part of a classic Ser-Asp-His catalytic triad, and a putative hinged loop structure covering the active site. The COOH-terminal domain contains a putative lipoprotein-binding site. The heparin-binding sites may be distributed throughout the molecule, with the characteristic elution pattern from heparin-sepharose determined by the COOH-terminal domain. Of the three N-linked glycosylation sites, Asn-56 is required for efficient secretion and enzymatic activity. HL is hypothesized to directly couple HDL lipid metabolism to tissue/cellular lipid metabolism. The potential significance of the HL pathway is that it provides the hepatocyte with a mechanism for the uptake of a subset of phospholipids enriched in unsaturated fatty acids and may allow the uptake of cholesteryl ester, free cholesterol, and phospholipid without catabolism of HDL apolipoproteins. HL can hydrolyze triglyceride and phospholipid in all lipoproteins, but is predominant in the conversion of intermediate density lipoproteins to LDL and the conversion of post-prandial triglyceride-rich HDL into the postabsorptive triglyceride-poor HDL. HL plays a secondary role in the clearance of chylomicron remnants by the liver. Human post-heparin HL activity is inversely correlated with intermediate density lipoprotein cholesterol concentration only in subjects with a hyperlipidemia involving VLDL. This is consistent with intermediate-density lipoproteins being a substrate for HL. HDL cholesterol has been reported to be inversely correlated to HL activity, and on this basis it has been suggested that lowering HL would increase HDL cholesterol. However, the correlation could also be due to a common hormonal factor such as estrogen, which has been shown to up-regulate apoAI and HDL cholesterol and lower HL. A striking feature of severe deficiency of HL is the increase in HDL cholesterol and apolipoprotein AI and an approximately 10-fold increase in HDL triglyceride. Hyper-alpha-triglyceridemia is not a feature of antiatherogenic HDL. HL binds not only to heparan, but also to the LDL receptor-related protein. It has been suggested that enzymatically inactive HL can play a role in hepatic lipoprotein uptake, forming a "bridge" by binding to the lipoprotein and to the cell surface. This raises the interesting possibility that production and secretion of mutant inactive HL could promote clearance of VLDL remnants. We have described a rare family with HL deficiency. Affected patients are compound heterozygotes for a mutation of Ser267 to Phe that results in an inactive enzyme and a mutation of Thr383 to Met that results in impaired secretion and reduced specific activity. Human HL deficiency in the context of a second factor causing hyperlipidemia is strongly associated with premature coronary artery disease. Recently, it has been reported that mutations affecting the structure of HL (e.g., T383M) are relatively frequent in the Finnish population. A C-to-T polymorphism in the promotor region of the HL gene is associated with lowered HL activity and less strongly with increased HDL cholesterol. In summary, there is a good understanding of what HL does in lipoprotein metabolism; however, there is little understanding of its physiological importance, that is, why HL does what it does. (ABSTRACT TRUNCATED)

Common genomic variation in the APOC3 promoter associated with variation in plasma lipoproteins.

We hypothesized that common genomic variation that affected the expression and/or function of the products of the APOC3, APOE, FABP2, and PON1 genes would be associated with variation in biochemical phenotypes in a previously unstudied human sample. We determined genotypes of functional genomic variants of APOC3, APOE, FABP2, and PON1 in 509 adult aboriginal Canadians from an isolated community in Northern Ontario. We tested for genotype associations with plasma lipoprotein traits. We found that (1) common variation at nucleotide -455 of the APOC3 promoter was associated with variation in plasma triglycerides (P = .006) and (2) common variation of APOE determining plasma isoforms of apo E was associated with variation in plasma apo B (P = .009). Analysis of subjects classed by APOC3 markers showed that homozygosity for presence of a C at nucleotide -455 and a T at nucleotide -482 was associated with significantly increased plasma triglycerides in both men and women. Furthermore, this allele was approximately twice as frequent in subjects within the highest quartile of plasma triglycerides as in subjects within the lowest quartile. Since the DNA variation detected by the APOC3 markers affects in vitro expression of the gene product, it is possible that the marker itself caused the associations. However, the associations could also have resulted from linkage disequilibrium with other functional variants in APOC3 or the closely linked APOA1 and/or APOA4 genes.

Intestinal fatty acid-binding protein variation associated with variation in the response of plasma lipoproteins to dietary fibre.

Increased dietary fibre intake is a component of prudent dietary advice, although the mechanism of its beneficial effect is unclear. Furthermore, plasma lipoprotein response to dietary fibre seems to vary both between individuals and according to the type of fibre consumed. Two common genetic variants, A54 and T54, of the intestinal fatty acid-binding protein gene (FABP2) have different in vitro binding affinities for long-chain fatty acids. We have hypothesized that variation in FABP2 would be associated with interindividual variation in the response of plasma lipoproteins to either dietary soluble or insoluble fibre. We studied 43 subjects who participated in a year-long cross-over study of the effect of insoluble and soluble fibre on plasma lipoproteins. We tested for associations between FABP2 genotypes and the response of plasma lipoproteins to dietary fibre. When compared with subjects homozygous for FABP2 A54, we found that subjects with FABP2 T54 had significantly greater decreases in plasma total and low-density lipoprotein (LDL)-cholesterol and apoB during the period when the diet was high in soluble fibre than during the period when the diet was high in insoluble fibre. Furthermore, compared with subjects with the FABP2 A54 allele, subjects with the FABP2 T54 allele had significantly lower secretion of total fecal bile acids, but this did not increase with dietary soluble fibre. Genetic variation in FABP2 may thus contribute to interindividual variation in the response of plasma lipoproteins to different dietary fibres, but the mechanism does not appear to be related to increases in fecal bile acid secretion.

Apolipoprotein E R112; R251G: a carboxy-terminal variant found in patients with hyperlipidemia and coronary heart disease.

A 49 year-old hypercholesterolemic male with marked electrocardiographic ST segment depression on exercise testing was found to have an apo E E3/3 phenotype by isoelectric focusing, but an APOE E4/3 genotype using HhaI restriction isotyping. DNA sequence analysis of the proband's APOE gene found a G-->C point mutation at codon 251. This predicted a change in the amino acid encoded by codon 251, from arginine to glycine. The mutation occurred on an allele that encoded arginine at position 112 and this variant was named APOE R112; R251G. The R251G change altered a recognition site for the endonuclease StuI and was the basis for a restriction isotyping method to rapidly screen for this mutation. In relatives of the proband, APOE R112; R251G was consistently found in subjects with both hyperlipidemia and atherosclerosis. Apo E R112; R251G-containing very low density lipoproteins bound normally to macrophages in vitro. However, the proband had an abnormal post-prandial lipoprotein response to a dietary fat challenge. The association of APOE R112; R251G with abnormal phenotypes suggests that the amino acid change in the carboxy-terminal, perhaps in combination with the common amino acid polymorphism at codon 112, has a functional impact upon lipoprotein metabolism in members of this family.

Common genomic variants associated with variation in plasma lipoproteins in young aboriginal Canadians.

We hypothesized that common genomic variants would be associated with variation in lipoprotein phenotypes in young subjects. We determined genotypes of FABP2, PON, APOC3, and APOE in 188 aboriginal Canadians, aged 9 to 17 years. We found that 13 of 32 possible genotype-phenotype associations were significant: (1) the FABP2 codon 54 genotype was associated with variation in plasma triglycerides (P = .045); (2) the PON codon 192 genotype was associated with variation in plasma total and LDL cholesterol and apoB (P = .0099, P = .0088, and P = .016, respectively); (3) the APOC3 insulin-response-element genotype was associated with variation in plasma triglycerides, HDL cholesterol, apoA-I, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .0014, P = .0069, P = .045, P = .0021, and P = .0081, respectively); and (4) the APOE restriction isotype was associated with variation in plasma LDL cholesterol, apoB, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .025, P = .034, P = .045, and P = .047, respectively). The average young age and relative absence of age-dependent secondary environmental factors could have eased the identification of small genetic effects on lipoprotein phenotypes in this study sample.

Are Canadian Inuit at increased genetic risk for coronary heart disease?

The Keewatin Inuit of the Northwest Territories of Canada have a very low age-adjusted mortality rate from coronary heart disease. We hypothesized that this apparent protection from disease has a genetic basis. We determined the prevalence of the disease-associated alleles of five candidate genes for atherosclerosis-related phenotypes. Surprisingly, four of the five alleles studied, namely AGT T235, FABP2 T54, PON R192 and APOE E4, were significantly more frequent in a sample of 175 Keewatin Inuit than among a representative control sample of whites living in the region. The high frequencies of these disease-associated alleles suggests either that they have no relationship with disease susceptibility in the Inuit, or that some unmeasured genetic and/or environmental factors mitigate disease susceptibility that is associated with these alleles. This highlights the difficulty in extrapolating findings from one population to another. Also, very modest genotype-phenotype associations were observed between APOE genotype (P = 0.016) and plasma low-density lipoprotein cholesterol concentration and between FABP2 genotype and plasma 2-h postprandial, glucose concentration (P = 0.048). The relationship between APOE alleles and plasma low-density lipoprotein cholesterol was the same as has been previously reported in many study samples. However, the relationship between FABP2 alleles and plasma 2-h postprandial glucose concentrations was the opposite to that reported in other studies. This suggests that differences in environment, such as the type of fatty acid consumed, interacts with functional differences in gene products involved in candidate metabolic pathways to produce phenotypic differences.

Genetic variation of intestinal fatty acid-binding protein associated with variation in body mass in aboriginal Canadians.

We hypothesized that genomic variation affecting the primary amino acid sequence of the intestinal fatty acid-binding protein would be related to variation in body mass index and associated clinical phenotypes in aboriginal Canadians. We studied 507 adult native Canadians from an isolated community in Northern Ontario. We found that the frequency of the T54 variant of the intestinal fatty acid-binding protein gene was 0.14 in this sample. The presence of this variant was associated with significant increases in body mass index, percent body fat, and fasting plasma triglyceride concentration (P = 0.012, 0.019, and 0.012, respectively). However, the variant was not associated with the presence of diabetes mellitus. These findings suggest that the T54 variant of the intestinal fatty acid-binding protein is associated with differences in fat metabolism in this aboriginal population.

Genetic diagnosis of familial hypercholesterolemia in affected relatives using pedigree tracing.

OBJECTIVES: To describe a process of diagnosing familial hypercholesterolemia (FH) at the DNA level in selected family members of affected individuals. DESIGN AND METHODS: A 63-year-old male patient presented with cholesterol elevations consistent with heterozygous familial hypercholesterolemia. Through participation with the international "MEDPED FH" project to detect affected relatives and to identify their LDL-receptor mutation, the patient was discovered to carry the Lebanese mutation, whereby the codon for cysteine at residue 660 instead codes for a premature termination (C660X), thus truncating the protein product. This mutation also created a new restriction recognition site for the endonuclease Hinfl, which permitted rapid detection of the mutation in selected family members using restriction fragment-length polymorphisms. RESULTS: The patient's son, who had cholesterol levels consistent with heterozygous FH, was also found to be a heterozygote for the C660X variant of the LDL-receptor. CONCLUSIONS: Diagnosis of familial hypercholesterolemia at the DNA level is possible as a relatively rapid screening technique in families with a known LDL-receptor mutation, established through participation with the MED-PED FH project.

Restriction isotyping of apolipoprotein E R145C in type III hyperlipoproteinemia.

BACKGROUND: An adolescent female who presented with type III hyperlipoproteinemia was found to have an E3/2 phenotype by isoelectric focusing while restriction isotyping using HhaI revealed a pattern compatible with classical E3/3. Studies were carried out to determine the nature of the patient's apolipoprotein E abnormality. METHODS: A 244 bp fragment of exon 4 of apolipoprotein E was amplified by the polymerase chain reaction (PCR). Restriction isotyping was carried out with BbvI and Fnu4HI and results were confirmed by direct sequencing. RESULTS: The patient was found to be heterozygous for a C-->T transition at the first base of codon 145, resulting in a substitution of cysteine for arginine (R145C) which completely explained the discrepancy between the isoelectric focusing and HhaI restriction isotyping. We noted that the DNA change altered palindromic recognition sites for the endonucleases BbvI and Fnu4HI. CONCLUSIONS: Digestion with BbvI or Fnu4HI allows for rapid restriction isotyping for the rare apolipoprotein E R145C mutation associated with hyperlipidemia.

Detection of a new apolipoprotein-E mutation in type III hyperlipidemia using deoxyribonucleic acid restriction isotyping.

While determining the apolipoprotein-E (apo-E) genotype of 22 patients with type III hyperlipidemia (HLP III) by restriction isotyping, we identified a new mutant form of apo-E by its unusual DNA restriction fragment length polymorphism pattern. DNA sequence analysis of a polymerase chain reaction-amplified portion of the proband's apo-E gene revealed the substitution of cysteine (TGC) for arginine (CGC) at position 136 in the mutant allele (designated R136C). Lipoproteins containing this mutant protein bound defectively to macrophages in vitro, confirming the contribution of R136C to the expression of HLP III in the proband. The proband's two siblings carried the mutant allele and were also heterozygous for E2. Each also had dysbetalipoproteinemia (indicated by the presence of beta-very low density lipoprotein), but neither was hyperlipidemic, attesting to the importance of other factors for the full expression of HLP III. The mutant allele appears to contribute to the inheritance of HLP III in a recessive fashion. Restriction isotyping facilitates the diagnosis of subjects with HLP III, aids in the identification of affected individuals through family screening, and can contribute to the discovery of new mutations that help explain the pathogenesis of HLP III.