INVESTIGATIONS OF POSTPRANDIAL LIPEMIA

AND GENES MUTATIONS

IN FAMILIAL DYSLIPIDEMIA

Ph.D. dissertations theses

Dr. Reiber István

Pathobiochemical program

Doctor School

Semmelweis University, Budapest

2002

TABLE OF CONTENTS

  1. Introduction
  2. Investigation of postprandial lipemia in several groups of patients
  3. The familial combined hyperlipidemia
  4. The role of the polymorphism of lipoprotein lipase in dyslipidemia
  5. The effect of the polymorphism of the apolipoprotein E on fasting and postprandial lipids and lipoproteins
  6. The mutation of a novel apolipoprotein, the apolipoprotein AV in familial combined hyperlipidemia
  7. The effect of the T-1131C polymorphism of the apolipoprotein AV on the postprandial lipemia
  8. New observations
  9. Own publications in the theme of the dissertation
  10. Own publications in other themes
  11. Own abstracts

1. Introduction

The cardiovascular diseases and their complications are still the leading causes of deaths in Hungary. More than 50 % of the population dies on account of a premature atherosclerotic disease. Plasma lipid abnormalities in dyslipidemia (increased LDL-cholesterol, increased triglyceride, decreased HDL-cholesterol) are important risk factors in development of the progressive atherosclerosis. The results of epidemiological and interventional studies in the last decade show that the level of LDL-cholesterol above 3.0 mmol/l, triglyceride above 1.7 mmol/l and HDL-cholesterol below 1.0 mmol/l is associated with increased vascular risks. Several study confirmed, that improved lipid status alone could decrease the atherosclerotic damage of the vascular system, and therefore, it would decrease significantly the mortality due to ischaemic heart disease or other vascular diseases both in the primary and the secondary prevention.

The importance of fasting triglyceride level as an independent atherogenic risk factor is still questionable. Although epidemiological studies such as PROCAM or Framingham provided obvious data about the association between increased TG value (above 2.0 mmol/l) and vascular disease, many believe that the decreased HDL-cholesterol concentration is the causative agent. Works of JR Patsch proved, that the decreased HDL-cholesterol level (under 1.0 mmol/l) is a marker and a consequence of abnormal triglyceride metabolism. The important discovery of the recent years is the association between the atherogenic small dense LDL and the triglyceride level. We know that small dense LDL is increased, when TG > 1.0 mmol/l. The lipid specialists all agree, that the fasting triglyceride level does not reflect exactly the presence of atherogenic lipids. We spend most time of the day in postprandial state, which is the important finding of Zilversmit about the so-called „postprandial phenomena”.

The familial combined hyperlipidemia (FCH) is a polygenic dyslipidemia associated with the appearance of cardiovascular disease before age of 60 years. The genetic defects underlying FCH are yet unknown. Because FCH is associated with elevated triglyceride level in the early step, genetic factors influencing this may be the contributors for the development of FCH. A number of „modifier” genes including LPL gene and apo AI-CIII-AIV gene cluster could influence the levels of plasma lipids in FCH.

Aim:

- Investigation of:

- postprandial lipemia by standard fat loading test

- familial combined hyperlipidemia in Hungary

- HindIII and PvuII polymorphisms of lipoprotein lipase in familial combined hyperlipidemia and in diabetic dyslipidemia

- effect of apolipoprotein E polymorphism on fasting and postprandial lipid and lipoprotein levels

- a novel apolipoprotein, the apolipoprotein AV T-1131C mutation in familial combined hyperlipidemia

- the effect of the apolipoprotein AV T/C polymorphism on postprandial lipemia

2. Investigation of postprandial lipemia in several groups of patients

Healthy people spend most part of their life in postprandial state, which is the sum of the 6-8 hours after each main meal spanning over 20 or 24 hour per day. After the ingestion of the fat rich meal the intestinal chylomicrons disturbs the balance of lipid metabolism. The disorder of the lipid transport does not always manifest itself in the fasting state, when the lipid transport system is yet at poise. So, the measuring of fasting triglyceride does not reflect exactly the metabolic capacity of the subject. In the past years, considerable amount of evidence has been accumulated showing that larger and delayed postprandial lipemia promote atherosclerosis. The knowledge of characteristics of postprandial lipemia are more useful than just the fasting triglyceride alone, and it is as effective as HDL-cholesterol value in prognosis of development of vascular disease with athero-thrombotic origin.

We performed the fat loading test at 10 normotriglyceridemic obese (BMI > 30 kg/m2) subjects, 10 normotriglyceridemic CHD (after a myocardial infarction) patients, 10 normoglycemic and normotriglyceridemic persons with hyperinsulinemia and 10 normolipidemic (with BMI < 25 kg/m2) subjects. The standard fat rich meal contains 1362 kcal/2 m2 (83 % fat, 14 % CH and 3 % protein). Blood samples of lipid parameters were obtained from subjects while fasting and 2, 4, 6, 8 and 10 hours postprandially after taking of the test meal to determine total cholesterol, triglyceride, HDL-cholesterol, apoA, apoB and glucose levels.

Results: The fat loading test is normal, when the postprandial triglyceride-peak is not later than 3 or 4 hours, and the absolute value of 6 hours TG < 3,0 mmol/l and the 10 hours TG is back to the fasting level. The maximum triglyceride values in 8/10 obese and 8/10 CHD patients appeared at 6 hours. In 7/10 hyperinsulinemic subjects had triglyceride peak between 4 and 6 hours. The Area Under the Curve (AUC) was above 20 in 8 obese, 4 CHD and 6 hyperinsulinemic subjects, there were significantly higher (p < 0.001) than in control.

Conclusion: The three investigated groups (with normal fasting triglyceride levels) exhibited significantly (p < 0.001) higher and extended postprandial lipemia as an important „silent” risk factor in the pathogenesis of atherosclerosis.

3. The familial combined hyperlipidemia

The familial combined hyperlipidemia (FCH) is the most common form of heritable lipid disorder, first described by Goldstein et al. in 1973. The prevalence of FCH is 0.5 to 2.0 % in the general population, and 15 to 20 % in the survivors of myocardial infarction before the age of 60 years.

The genetic defects underlying FCH are yet unknown. Several „modifier” genes have been investigated such as lipoprotein lipase (LPL), apolipoprotein AI-CIII-AIV, apolipoprotein AII, apolipoprotein B, apolipoprotein E, cholesterol ester transport protein (CETP), hepatic lipase (HL), lecithin cholesterol acyl-transferase (LCAT), LDL receptor, microsomal triglyceride transfer protein (MTP), peroxisoma proliferator-activated receptor (PPAR), and magnesium superoxide dismutase (MnSOD).

FCH is characterized by increased apolipoprotein B, triglyceride- and LDL-cholesterol-levels, increased small dense LDL-particles, decreased post-heparin activity of lipoprotein lipase and catabolism of chylomicron remnants, and insulin resistance. Some researcher, found association between FCH, metabolic syndrome and hyperapobetalipoprotein syndrome.

FCH is the most common and remarkable familial dyslipidemia in cardiovascular diseases.

We investigated more than hundreds of FCH subjects. FCH is characterized by variability of phenotype, by age dependence of the dyslipidemia and by the lack of unequivocal diagnostic criteria. We investigated members of a smaller but exactly determined families with FCH.

4. The role of the polymorphism of lipoprotein lipase in dyslipidemia

Lipoprotein lipase (LPL) as a component of the vascular endothelium hydrolyzes triglycerides in large triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins (VLDL)). LPL is a key determinant of the plasma lipid profile. At the same time LPL is a necessary co-determinant for receptor binding of remnants and the LDL-particles.

The gene of the LPL is located on the short arm of chromosome 8, which spans about 30 kb and contains 10 exons. On the basis of the relationship between the enzyme function and the gene polymorphism of LPL it can be suggested that some genotypes could serve as useful markers of atherogenic dyslipidemia. Some population studies have concluded the PvuII and HindIII RFLPs in association with triglyceride levels and coronary heart disease and diabetes mellitus. The role of HindIII and PvuII polymorphisms in FCH is controversial.

We investigated the HindIII and PvuII polymorphisms at 82 FCH subjects (39 females and 43 males, age: 14-70 years), 40 NIDDM patients with apolipoprotein E3/3 genotype (21 females and 19 males) and 100 normolipidemic persons. The DNA PCR products of the LPL genome were digested with two restrictions enzymes and visualized using agarose gels.

Results: The frequencies of HindIII genotypes in FCH patients were as follows: H+/+: 0.66, H+/-: 0.32 and H-/-: 0.02. The presence of H- allele was 0.18 (control H-: 0.33). The distribution of PvuII genotypes were: P+/+: 0.39, P+/-: 0.43 and P-/-: 0.18. The frequency of the rare allele P- was 0.40(vs. control P-: 0.45, NS).

In NIDDM we found 9 homozygotes P+/P+, 22 heterozygotes P+/P- and 9 homozygotes P-/P- genotypes. The PvuII allele frequencies were 0.50 (P+ and P-).

Conclusions: Our data revealed significant difference in the frequency of HindIII genotypes in FCH patients; however, we found no association between the frequency distribution and the fasting lipid values. In the type 2 diabetes mellitus with mild dyslipidemia we did not find characteristic pattern of the LPL PvuII polymorphism independently of apolipoprotein E genotype.

5. The effect of the polymorphism of the apolipoprotein E on fasting and postprandial lipids and lipoproteins

The apolipoprotein E is a glycoprotein containing 299 amino acids, and has a crucial role in the lipid metabolism and atherogenesis. Apolipoprotein E is an ingredient of chylomicrons, chylomicron-remnants, VLDL, IDL and HDL particles. Lipoproteins containing apoE are the ligands for LDL-receptors; LDL-receptor related proteins, VLDL-receptors and scavenger receptors. The natural isoforms of apoE differ with respect to their affinity to cellular lipoprotein receptors. The three alleles of the apoE gene (e2, e3, e4) express six genotypes: E2/2, E2/3, E3/3, E3/4, E2/4 and E4/4. The apo E2 and the apo E4 have only one amino acid difference with the apo E3 form. In healthy subjects is associated with proven that the e4 allele it is higher and e2 allele with lower plasma cholesterol level.

The distribution of apoE genotypes in 402 patients with several dyslipidemia of different origins (86 with metabolic syndrome) and 32 FCH family members has been investigated by PCR-RFLP technique.

Results: The prevalence’s of the most frequent apolipoprotein E genotypes in the whole study group were as follows: E3/3: 45.5%, E4/3: 31.3% and E3/2: 10.2%. The highest prevalence of E4/4 and E4/3 was observed in patients with metabolic syndrome (5.8% and 41.9%). The allele frequencies in this group were: e2: 12.8%, e3: 58.7% and e4: 28.5%.

In FCH 13 persons from the 22 normotriglyceridemic (TG< 2,3 mmol/l) subjects had E3/3 genotype, and 8 from the 10 hypertriglyceridemic subjects had E4/3 genotypes (χ2-test: 4,219 = p < 0.05). In the investigation of the postprandial lipemia we found that 21 subjects had TG above 3.0 mmol/l at 6 hours. In this group 14 subjects were with E4/3 genotype (χ2-test: 4.499 = p < 0.05).

Conclusion: A possible association is suggested between increased cardiovascular disease in metabolic syndrome and the higher prevalence of apo e4 allele in these patients. The presence of apo E4/3 genotypes shows a direct effect on extended postprandial lipemia in FCH.

6. The mutation of a novel apolipoprotein, the apolipoprotein AV in familial combined hyperlipidemia

Pennacchio et al. identified in 2001 a 30 kbp conservation gene sequence proximal to the apoAI-CIII-AIV gene cluster on chromosome 11, which called apoAV. 4 single nucleotide polymorphisms were found of apoAV. Pennacchio et al. found significant associations between both plasma triglyceride levels and VLDL mass and the three neighboring SNPs (SNP1-3). They investigated 500 normolipidemic Caucasian subjects, and independent analysis of the SNPs revealed that plasma triglyceride levels were 20 to 30 % higher in individuals having one minor allele compared with individuals homozygous for the major allele. In an other study a significant overrepresentation of the heterozygous genotype (in T-1131C polymorphism) was found in individuals with high compared with low plasma triglyceride levels (21.7% versus 6.7% respectively). Ribalta et al. reported a potential link between T/C polymorphism in the apoAV and FCH. Among FCH family members carriers of the apoAV minor allele had a higher risk of developing the disease and had an average of 30% higher fasting triglyceride level.

We investigated the prevalence of minor allele of apoAV T-1131C polymorphism in 30 FCH subjects from four FCH families (8 probands, 7 siblings, 15 children).

Results: We found 13 carriers of the minor allele from the 30 FCH family members, out of which two were homozygous. Overrepresentation of the minor allele (0.25) was found in FCH individuals compared with general population (0.067). With unvaried analysis, the minor allele carrier status was significantly associated with increased concentrations of total-cholesterol (7.12 vs. 5.84 mmol/l, p< 0.05), fasting triglyceride (3.74 vs. 1.44 mmol/l, p< 0.001), apoB (1.47 vs. 1.17 g/l, p< 0.01) and apo CIII (19.0 vs. 12.3 mg/dl, p< 0.01). Conversely, carriers had decreased concentration of HDL-cholesterol (1.13 vs. 1.5 mmol/l, p< 0.05).

Conclusion: In the investigated Hungarian FCH patients we found overrepresentation of minor allele of apolipoprotein AV T/C polymorphism. The carrier status was associated with abnormal fasting lipid levels.

7. The effect of the T-1131C polymorphism of the apolipoprotein AV on the postprandial lipemia

In FCH family members with apo E4/3 genotype we have previously reported an extended and delayed postprandial response. The postprandial effect of the polymorphism of apolipoprotein AV described by Pennacchio et al. has not been yet investigated.

Aim of our further investigation was to determine the postprandial effect of apolipoprotein AV T/C polymorphism in FCH.

We evaluated the polymorphism of apo AV T-1131C by PCR technique and the fat loading test by Patsch at previously defined 30 FCH subjects. The subjects were divided in to three groups according to their fasting triglyceride level: low-triglyceridemic (LT): TG < 1.7 mmol/l, increased-triglyceridemic (IT): TG 1.7-2.3 mmol/l and hypertriglyceridemic (HT): TG > 2.3 mmol/l.

Results: We found higher and extended postprandial lipemia at 13 minor allele carriers compared to non-carriers. Carriers of the minor allele had higher AUC value as compared to non-carriers (17.3±1.8 vs. 7.5±1.5 mmol/l*h, p< 0.001). We found no difference between genders. According to fasting TG the fat loading curve of LT group was normal (= minor allele non-carriers) and in HT group we confirm extended abnormal remnant phase (all of the 9 subjects were minor allele carriers). The most important observation made we in the IT group, was a clear postprandial effect of carriers compared to non-carriers at the same fasting TG levels (with two times larger AUC value). In spite of the similar representation of apoE and apoAV genotypes we found definite effects of carrier/non-carrier status on postprandial TG values in the apoE 3/4 group.

Conclusion: Our results suggest a decreased and extended catabolism of the remnants in FCH caused by apolipoprotein AV T-1131C promoter variation that seems to have a more direct effect on the postprandial status than that of apoE 3/3-3/4 polymorphisms.

8. New observations

- In the daily practice the accepted normal fasting TG level (≤ 2.3 mmol/l) could result in higher and extended postprandial lipemia.

- In selected FCH families the primary prevention is easier and more effective.

- In FCH presence of mutations of LPL gene can cause functional and structural modification of enzyme.

- In metabolic syndrome and in FCH we found more frequent prevalence of apolipoprotein e4 allele compared to normal population.

- In FCH we described an association between apoE 4/3 genotypes and extended, higher postprandial lipemia.

- At the investigated Hungarian FCH subjects we confirm higher frequency of apolipoprotein AV T/C minor allele with higher fasting lipid levels.

- To the best of our knowledge we are the first to investigate the postprandial effect of the apolipoprotein AV T/C polymorphism in FCH and we found decreased and extended catabolism of the triglyceride-rich particles at minor allele carriers.

9. Own publications in the theme of the dissertation

  1. Reiber I., Gógl Á. A low-density lipoprotein-cholesterin csökkentése apheresis segítségével. Orvosi Hetilap 1994; 135:563-568.
  2. Reiber I., Schaper J., Eckardt H., Bimmermann A., Steinhagen-Thiessen E. Increased prevalence of e4 allele inpatients with the plurimetabolic syndrome. In: Woodford FP, Davignon J, Sniderman A, eds. Atherosclerosis X. Amsterdam: Elsevier Science BV; 1995; 533-536.
  3. Kalina Ákos, Czeizel Endre, Romics László, Pados Gyula, Reiber István, Dósa András, Hermányi István, Lakatos Zoltán, Tarján Jenő, Kollega-Tarsoly Ella, Kovács Margit, Szalai Csaba, Császár Albert. A familiáris defektív apolipoprotein B-100 előfordulási gyakorisága klinikailag familiáris hypercholesterinaemiásnak tartott betegek között. Orvosi Hetilap 1998; 139:755-759.
  4. Ákos Kalina, Albert Császár, Andrew E. Czeizel, László Romics, Ferenc Szabóki, Csaba Szalai, István Reiber, Attila Németh, Susan Stephenson, Roger R. Williams. Frequency of the R3500Q mutation of the apolipoprotein B-100 gene in a sample screened clinically for familial hypercholesterinaemia in Hungary. Atherosclerosis 2001; 154:247-251.
  5. Kalina A, Szalai C, Prohaszka Z, Reiber I, Csaszar A. Association of plasma lipid levels with apolipoprotein E polymorphism in Type 2 diabetes. Diabetes Res Clin Pract 2002; 56:63-68.
  6. Reiber I, Mezo I, Kalina A, Palos G, Romics L, Csaszar A. Postprandial triglyceride levels in familial combined hyperlipidemia. The role of apolipoprotein E and lipoprotein lipase polymorphisms. J Nutr Biochem (in press).
  7. István Reiber, Izabella Mező, Ákos Kalina, Csaba Szalai, László Romics, Albert Császár. ApoAV T-1131C and not apoE3/3-3/4 genetic variation influences postprandial triglyceride levels in familial combined hyperlipidemia. Atherosclerosis (közlésre beadva).

10. Own publications in other themes

  1. Reiber I., Schaper J., Eckardt H., Bimmermann A., Steinhagen-Thiessen E. Die medikamentöse Langzeitbehandlung der Dyslipoproteinämie. In Endothelfunktion und Arteriosklerose, Kiadó: H. Heinle, H. Schulte, H.K. Breddin, Tübingen 1994; 239-243.
  2. Reiber I., Tóth G.T., Gógl Á. Association between cardiovascular risk factors and level of uric acid. In 22nd Congress of ISIM Proceedings Book (Editors: Varró-de Chatel), Bologna 1995; 827-830.
  3. Reiber I. Fluvasztatin-(LESCOL) kezelés hatása a szérumlipidekre. Praxis 1995; 9:33-37.
  4. Reiber I., Gógl Á. A zsíranyagcsere-zavarok gyógyszeres kezelésének gazdasági vonatkozásai (Költség-hatásosság elemzés). Orvosi Hetilap 1995; 136:2827-2831.
  5. Reiber I., Gógl Á. Prävalenzen von kardiovaskulären Risikofaktoren bei Patienten mit einer Hyperlipidämie. In Arteriosklerose zerebraler Gefässe, Kiadók: H. Heinle, H. Schulte, H. Kaffarnik, Tübingen 1995; 341-345.
  6. Reiber I., Szabó P. A rendszeres testmozgás hatása a lipidekre és lipoproteinekre koszorúérbetegeknél. Rehabilitáció 1996; 4:14-15.
  7. Reiber I., Gógl Á. A máj és a szénhidrátok. A lipidanyagcsere. Varró Vince: Gastroenterológia könyvben, Medicina Könyvkiadó Rt., Budapest 1997. (könyvfejezet).
  8. Reiber I. Hiperlipidémia. Az MTA Magyarország az ezredfordulón sorozatának „Egészségügy és Piacgazdaság” kötetében (Szerkesztő: Glatz Ferenc). Budapest, Magyar Tudományos Akadémia 1998;179-182.
  9. Garmendia F, Brown AS, Reiber I, Adams PC. Attaining United States and European guideline LDL-cholesterol levels with simvastatin in patients with coronary heart disease (the GOALLS study). Curr Med Res Opin 2000; 16:208-219.
  10. Reiber I. Hiperlipoproteinémiák diétás kezelése. Háziorvosi Szemle 2001; 6:12-14.
  11. Reiber I. A familiáris kevert hyperlipidaemiáról. Orvostovábbképző Szemle 2001; 40:19-20.
  12. Tokodi I, Molnar A, Reiber I, Simon G. Nem alkoholos steatohepatitis. Orvosi Hetilap 2002; 143:1899-1903.
  13. Reiber István. Hiperlipidémia: a következmény és a megelőzés költség-hatás elemzése. Egészségmegtartás, betegségmegelőzés MTA kiadványban. Budapest, 2002; 57-66.

11. Own abstracts

  1. Reiber I., Steinhagen-Thiessen E., Gógl Á. Az LDL-apheresis lehetőségei a hyperlipoproteinémia kezelésében. Magyar Belorvosi Archivum 1992; 2. Suppl. 69.
  2. Reiber I., Steinhagen-Thiessen E., Gógl Á. A metabolikus syndroma és az apolipoprotein E polymorphismusa. Diabetologia Hungarica 1994; Suppl. 1:39.
  3. Reiber I., Schaper J., Steinhagen-Thiessen E., Gógl Á. Statinok tartós adása monotherápiában vagy ioncserélő gyantával kombinálva dyslipoproteiné-miában. Magyar Belorvosi Archivum 1994; 47: Suppl. 1:33.
  4. Reiber I., Schaper J, Eckardt H, Bimmermann A, Steinhagen-Thiessen E. Increased prevalence of e4 allele in patients with metabolic syndrome. Atherosclerosis 1994; 109: 180.
  5. Reiber I., Gógl Á. Effects of long-term lipid lowering therapy on lipids and apolipoprotein B and A-I. Atherosclerosis 1995; 115:( Suppl.): 94.
  6. Reiber I., Szilágyi O., Gógl Á. Májbetegségek hatása a lipidanyagcserére és a lipidcsökkentők hatása a májra. Magyar Belorvosi Archivum 1996; Suppl.1: 43.
  7. Reiber I., Szalai Cs., Gógl Á., Romics L., Császár A. Lipoprotein lipase gén polimorfizmusa familiáris kombinált hyperlipidémiásoknál. Magyar Belorvosi Archivum 1996; Suppl. 2: 196.
  8. Reiber I., Gógl Á. The Combined Therapy with Fenofibrate and Statin. Atherosclerosis 1997; 129: 142.
  9. Reiber I., Szalai Cs., Gógl Á., Romics L., Császár A. DNA polymorphisms of lipoprotein lipase gene in familial combined hyperlipidemia. Atherosclerosis 1997; 130 (Suppl): 32.
  10. Reiber I. Fibrátok és statinok. Magyar Belorvosi Archivum 1997; 6: 673.
  11. Reiber I., Gógl Á., Szalai Cs., Császár A., Romics L. Trigliceriddús lipoproteinek és az atherosclerosis. Magyar Belorvosi Archivum 1997; Suppl. 2: 97.
  12. Reiber I., Szalai Cs., Gógl Á., Császár A., Romics L. Genpolymorhis-men der Lipoprotein-Lipase bei Patienten mit familiärer kombinierten Hyperlipidämie. Perfusion 1997; 2: 72.
  13. Reiber I., Szalai Cs., Gógl Á., Romics L., Császár A. DNA polymorphisms of lipoprotein lipase gene and apolipoprotein E in Hungarian patients with familial combined hyperlipidemia. Atherosclerosis 1999; 138 (Suppl. 1): 14.
  14. Reiber I., Mező I., Kalina Á., Császár A. Abnormal postprandial plasma triglyceride levels in normotriglyceridemic children of familial combined hyperlipidemic parents. Atherosclerosis 2000; 151: 177.
  15. Mező I., Reiber I. Simvastatin treatment lowers postprandial lipemia in CHD patients. Atherosclerosis 2000; 151: 46.

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