Statistical methods Directory UMM :Data Elmu:jurnal:A:Atherosclerosis:Vol152.Issue1.Sep2000:

3. Laboratory methods

Plasma cholesterol and triglycerides were determined by an enzymatic procedure Vitalab, Vital Scientific, Diesen, Netherlands. HDL-cholesterol HDL-C con- centrations were determined after precipitation of apo- B-containing lipoproteins with phosphotungstic acid. Low-density lipoprotein cholesterol LDL-C was esti- mated by the Friedewald equation [38], which has been validated in our population [39]. The apo B insertiondeletion site in exon 1, and the XbaI, MspI, and EcoRI RFLPs at the 3 end of the apo B gene were studied. The polymorphism at the 5 end of the apo B gene involves the insertiondeletion of 9 bp in exon 1 which results in the addition or deletion of the three amino acids in the signal peptide [40]. The XbaI RFLP involves the third base of threonine codon 2488 ACCACT without affecting the amino acid sequence, the MspI RFLP changes codon 3611 from arginine CGG to glutamine CAG and the EcoRI RFLP changes codon 4154 from glutamic acid GAA to lysine AAA [41]. The three different alleles o2, o3 and o4 of the apo E gene were studied after PCR amplification of the targeted region within the apo E gene, digestion with the HhaI restriction enzyme, electrophoretic separation on 8 polyacrylamide gels, and staining with ethidium bromide [42]. The E2 and E4 isoforms differ from the E3 common allele by a single amino substitution at position 112 and 158. Identification of carriers of the T-93G mutation in the promoter of the LPL gene was performed by PCR using primers: 5-GGCAGGGTTGTTCCTCATTACT- GTT-3 sense and 5-GACACTGTTT TCACGC- CAAGGCTGC-3 anti-sense [43]. The reaction mixture included 15 pmol of each primer, 0.5 mg genomic DNA, 0.2 mM of each dNTP, 10 mM Tris – HCl; pH 9.0, 1.5 mM MgCl2, 50 mM KCl, 0.01 wv gelatin, 0.1 Triton X-100 and 1 unit Taq polymerase in a total volume of 50 ml. Amplification was performed for 32 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C, with an initial denaturation period of 3 min. Some 20 ml of PCR products was digested with the restriction enzyme A6aII according to recommenda- tions of the supplier Pharmacia. Thereafter, fragments were separated on a 4 MP agarose gel Boehringer Mannheim, FRG and stained with ethidium bromide. In the case of the mutant allele an A6aII restriction site is abolished and therefore digestion of the PCR product will reveal one fragment of 189 bp for the mutant allele, and two fragments of 137 and 52 bp for the normal allele. To identify carriers with the S447stop mutation at the LPL gene, PCR analysis was performed using pri- mers: 5-CATCCATTTTCTTCCACAGGG-3 sense; 5-GCCCAGAATGCTCACC AGACT-3 anti-sense. After amplification, the PCR product was digested with HinfI, and subsequently fragments were separated on a 4 agarose gel. The 3-primer was elongated with a 20 base-pair AT-tail for optimal visualization of the di- gested fragment. In the case of the S447stop mutation, two fragments could be detected. The LPL mutation N291S was detected by the PCR method as previously described [44]. Exon 6 of the LPL gene was amplified with a 5-PCR primer located in intron 5 near the 5 boundary of exon 6 5-GCCGA- GATACAATCTTGGTG-3. The 3 mismatch PCR primer was located in exon 6 near the Asn291Ser mutation 5-CTGCTTCTTTTGGCTCTGACTGTA- 3. PCR amplification reactions were performed; 20 ml of the PCR product was digested with 10 U of Rsa1, and the digested fragments were separated on 2 agarose gel. Identification of the CETP I405V RFLP was per- formed as described previously [45]. This RFLP in- volves an A “ G nucleotide substitution in exon 14 which does not result in the creation or disappearance of an enzyme restriction site. Therefore, a mismatch promoter that introduced an RsaI restriction site in the presence of the mutation, was used. Target sequences were amplified with a 5-PCR primer 5-CTGTTTC- CAACTTGACTGAG-3. The 3 mismatch PCR primer was 5-CGCCCGCCGCGCCCCGCGCCCGT- CCCGCCGCCCCCGCCC CCATTGACTGCAGGA- AGCTCTGTA-3. The amplification reaction mixture included 10 mmoll Tris – HCl; pH 9.0, 50 mmoll KCl, 0.1 wv gelatin, 1.5 mmoll MgCl 2 , 1 Triton X-100 and 20 mgdl BSA containing 0.1 – 0.5 mg genomic DNA and final concentrations of 100 – 200 mmoll dNTPs and 0.5 mmoll primers in a total volume of 50 m l. After initial denaturation 10 min, 95°C, 0.3 – 0.5 U thermostable DNA polymerase was added, followed by 30 amplification cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, with a final extension step of 10 min at 72°C. Some 20 ml of the PCR reaction product was digested with the restriction enzyme ac- cording to the instructions of the manufacturer in a total volume of 20 ml for 2 h at 72°C. Thereafter, fragments were separated on 1 – 2 agarose gel Boehringer Mannheim, FRG and stained with ethid- ium bromide. DNA restriction fragments were visual- ized and analyzed on transilluminator.

4. Statistical methods

Blood lipids and lipoproteins were each adjusted for sex and age effects by multiple linear regression. In addition, the association between change in lipid and lipoprotein variables with BMI, were examined. The hypothesis of effect of the diet, the existence of a period effect, and the possible interaction between dietary Y . Friedlander et al . Atherosclerosis 152 2000 239 – 248 Table 2 Baseline values and lipid changes adjusted for sex and age by apo B polymorphic sites Signal peptide SP G4154L EcoRI A3611G MspI T2488 XbaI GG AA AG GLLL SP2724 −− SP2424 +− SP2727 ++ n = 21 n = 184 n = 80 n = 131 n = 4 n = 88 n = 75 n = 107 n = 15 n = 104 Total cholesterol 216.8 9 37.8 211.9 9 30.5 214.1 9 34.3 215.1 9 36.4 217.3 9 36.9 214.8 9 33.9 221.3 9 42.2 214.9 9 34.4 215.1 9 35.1 217.8 9 37.3 Baseline 15.1 9 17.9 1.1 9 21.8 8.0 9 22.4 a 16.2 9 22.7 10.6 9 22.3 12.1 9 24.1 − 5.7 9 34.3 11.5 9 23.3 11.2 9 22.4 10.4 9 23.4 Change LDL cholesterol 172.9 9 37.9 170.3 9 33.2 173.4 9 39.9 168.1 9 32.5 170.5 9 35.0 174.5 9 39.2 180.6 9 42.7 170.6 9 38.7 172.9 9 35.2 171.7 9 37.0 Baseline 6.2 9 22.4 a 15.8 9 21.1 9.8 9 21.7 11.4 9 22.6 − 7.2 9 39.8 16.1 9 16.4 9.7 9 23.0 Change 0.3 9 23.4 11.2 9 21.5 9.4 9 22.8 HDL cholesterol 37.1 9 7.7 38.4 9 7.6 37.3 9 7.5 Baseline 37.5 9 7.6 38.7 9 8.6 35.3 9 6.4 37.6 9 7.9 37.2 9 7.4 37.6 9 7.7 37.5 9 7.6 1.5 9 5.2 1.0 9 7.0 0.7 9 5.3 2.1 9 6.7 0.7 9 6.2 1.8 9 5.1 Change 1.3 9 6.0 1.5 9 6.5 1.0 9 5.5 1.3 9 4.7 Triglyceride 152.7 9 95.3 144.1 9 77.8 154.0 9 76.5 149.5 9 100.0 154.4 9 66.3 98.2 9 47.4 146.6 9 100.0 148.6 9 87.6 153.4 9 101.5 Baseline 147.0 9 116.0 4.2 9 62.1 16.7 9 67.6 2.0 9 55.9 2.3 9 45.3 3.4 9 53.5 0.7 9 54.7 17.3 9 38.4 − 1.4 9 50.4 4.8 9 51.2 1.3 9 51.2 Change a P50.01. Table 3 Baseline values and lipid changes adjusted for sex and age by apo E genotypes o 3 o3 n = 132 o 3 o4 n = 29 o 2 o3 n = 25 Total cholesterol Baseline 234.87 9 39.15 212.78 9 32.52 a 204.48 9 39.68 11.42 9 21.60 Change 7.28 9 26.60 10.06 9 26.15 LDL cholesterol Baseline 194.21 9 42.42 169.02 9 32.72 a 159.14 9 42.58 10.97 9 23.48 Change 8.52 9 26.80 9.93 9 21.31 HDL cholesterol Baseline 35.72 9 6.10 37.35 9 7.63 39.61 9 8.96 − 0.16 9 6.24 1.09 9 7.71 Change 1.68 9 5.63 Triglyceride Baseline 171.83 9 89.37 149.22 9 90.01 142.38 9 72.78 Change − 4.16 9 46.82 5.95 9 55.68 − 11.30 9 45.89 a P50.01. Table 5 Baseline values and lipid changes adjusted for sex and age by CETP I405 polymorphic site V¯V n = 34 V¯I n = 88 I¯I n = 72 Total cholesterol 219.23 9 39.61 Baseline 216.15 9 30.41 212.86 9 38.35 10.48 9 26.01 Change 11.02 9 20.99 10.88 9 23.02 LDL cholesterol 176.45 9 37.64 Baseline 172.06 9 33.26 170.00 9 40.06 Change 9.28 9 25.95 10.30 9 21.11 10.08 9 21.69 HDL cholesterol 38.08 9 8.30 36.76 9 7.26 36.99 9 6.64 Baseline Change 0.34 9 4.73 1.33 9 6.43 2.02 9 5.93 Triglyceride 158.14 9 88.34 152.68 9 89.13 146.83 9 85.07 Baseline Change 4.48 9 51.41 − 1.27 9 53.37 4.00 9 54.02 were performed on sex and age adjusted values. Our results from the crossover study indicate an overall significant effect of the diets on plasma cholesterol, LDL-C and triglyceride concentrations data not shown. For all variables, no carry over effect was demonstrated, and the order of the given diets had no effect on the response of the lipid phenotypes. Due to these results, the mean values of the two determinations made at the end of the LSC diet compared with that determined at the end of the HSC diet, irrespective of the order of the diets, were used in order to test the null hypothesis that phenotypic change was not associated with the genetic variation at the gene locus under study Tables 2 – 5. The frequency of apoB polymorphisms is similar to those previously described in the Israeli population [14,35]. The frequency distribution of apoE genotype treatment and period in this basic two-period crossover design were assessed by means of t-tests according to Fleiss [46]. Analysis of variance was performed in order to examine the homogeneity of baseline levels and changes across the genotype groups.

5. Results