Tracking of plasma lipids and lipoproteins, within-person and year-to-year variability in plasma cholesterol levels among participants from age 8 to 18 in Project HeartBeat!

Coronary heart disease remains the leading cause of death in the United States and increased blood cholesterol level has been found to be a major risk factor with roots in childhood. Tracking of cholesterol, i.e., the tendency to maintain a particular cholesterol level relative to the rest of the population, and variability in blood lipid levels with increase in age have implications for cholesterol screening and assessment of lipid levels in children for possible prevention of further rise to prevent adulthood heart disease. In this study the pattern of change in plasma lipids, over time, and their tracking were investigated. Also, within-person variance and retest reliability defined as the square root of within-person variance for plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides and their relation to age, sex and body mass index among participants from age 8 to 18 years were investigated. ^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. We examined the relationship between repeated observations by Pearson's correlations. Age- and sex-specific quintiles were calculated and the probability of participants to remain in the uppermost quintile of their respective distribution was evaluated with life table methods. Plasma total cholesterol, HDL-C and LDL-C at baseline were strongly and significantly correlated with measurements at subsequent visits across the sex and age groups. Plasma triglyceride at baseline was also significantly correlated with subsequent measurements but less strongly than was the case for other plasma lipids. The probability to remain in the upper quintile was also high (60 to 70%) for plasma total cholesterol, HDL-C and LDL-C. ^ We used a mixed longitudinal, or synthetic cohort design with continuous observations from age 8 to 18 years to estimate within person variance of plasma total cholesterol, HDL-C, LDL-C and triglycerides. A total of 5809 measurements were available for both cholesterol and triglycerides. A multilevel linear model was used. Within-person variance among repeated measures over up to four years of follow-up was estimated for total cholesterol, HDL-C, LDL-C and triglycerides separately. The relationship of within-person and inter-individual variance with age, sex, and body mass index was evaluated. Likelihood ratio tests were conducted by calculating the deviation of −2log (likelihood) within the basic model and alternative models. The square root of within-person variance provided the retest reliability (within person standard deviation) for plasma total cholesterol, HDL-C, LDL-C and triglycerides. We found 13.6 percent retest reliability for plasma cholesterol, 6.1 percent for HDL-cholesterol, 11.9 percent for LDL-cholesterol and 32.4 percent for triglycerides. Retest reliability of plasma lipids was significantly related with age and body mass index. It increased with increase in body mass index and age. These findings have implications for screening guidelines, as participants in the uppermost quintile tended to maintain their status in each of the age groups during a four-year follow-up. The magnitude of within-person variability of plasma lipids influences the ability to classify children into risk categories recommended by the National Cholesterol Education Program. ^

The relationship between alcohol consumption and HDL cholesterol: A study of Hispanic drinking patterns

There has been very little research that has studied the effects of alcohol on biochemical markers in ethnic populations. This particular study is designed to identify the association, if any, between drinking patterns and high density lipoprotein cholesterol (HDL-C) levels in a Hispanic population. Most of what we know about the association between alcohol and HDL-C deals specifically with volume of alcohol consumed on a daily basis. Frequency, or how often alcohol consumption occurs within a given time period, is a variable that has rarely been studied. The results of this study showed how both volume and frequency of alcohol consumption affect HDL-C levels in a predominantly middle-aged Hispanic population. Ultimately, we will be able to apply these findings to future studies concerning risk of CHD in Hispanics. ^

Genome-wide association study of LDL-cholesterol over ApoB ratio

Genome-Wide Association Study analytical (GWAS) methods were applied in a large biracial sample of individuals to investigate variation across the genome for its association with a surrogate low-density lipoprotein (LDL) particle size phenotype, the ratio of LDL-cholesterol level over ApoB level. Genotyping was performed on the Affymetrix 6.0 GeneChip with approximately one million single nucleotide polymorphisms (SNPs). The ratio of LDL cholesterol to ApoB was calculated, and association tests used multivariable linear regression analysis with an additive genetic model after adjustment for the covariates sex, age and BMI. Association tests were performed separately in African Americans and Caucasians. There were 9,562 qualified individuals in the Caucasian group and 3,015 qualified individuals in the African American group. Overall, in Caucasians two statistically significant loci were identified as being associated with the ratio of LDL-cholesterol over ApoB: rs10488699 (p<5 x10-8, 11q23.3 near BUD13) and the SNP rs964184 (p<5 x10-8 11q23.3 near ZNF259). We also found rs12286037 ((p<4x10-7) (11q23.3) near APOA5/A4/C3/A1 with suggestive associate in the Caucasian sample. In exploratory analyses, a difference in the pattern of association between individuals taking and not taking LDL-cholesterol lowering medications was observed. Individuals who were not taking medications had smaller p-value than those taking medication. In the African-American group, there were no significant (p<5x10-8) or suggestive associations (p<4x10-7) with the ratio of LDL-cholesterol over ApoB after adjusting for age, BMI, and sex and comparing individuals with and without LDL-cholesterol lowering medication. Conclusions: There were significant and suggestive associations between SNP genotype and the ratio of LDL-cholesterol to ApoB in Caucasians, but these associations may be modified by medication treatment.^

Total cholesterol and anthropometric measurements distributions in a cross-sectional study of school children

Cross-sectional age and sex specific distributions of serum total cholesterol were described for 1091 children age 6-18 years, in The Woodlands, Texas. Associations of serum total cholesterol with five anthropometric measurements (weight, height, body mass index, arm circumference, and triceps skinfold thickness) were examined by correlation and regression analyses. Examination of serum total cholesterol distributions showed lower levels in boys than in girls for most of the age groups studied. Mean levels of total cholesterol peaked at age 9 for boys and 8 for girls. Serum total cholesterol leveled off until age 14 for boys and 11 for girls, and then dropped through age 18 for both boys and girls. These results support the hypothesis that serum total cholesterol concentration drops at pre-adolescence.^ Age adjusted correlations were observed between serum total cholesterol and triceps skinfold thickness for both boys and girls. This association was stronger in boys. Triceps skinfold thickness and arm circumference were consistently the strongest correlates for serum total cholesterol in boys. Weight and arm circumference were consistently the strongest correlates for serum total cholesterol in girls. ^

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

THE RELATIONSHIP BETWEEN SERUM CHOLESTEROL AND CANCER INCIDENCE IN THE POPULATION OF THE HYPERTENSION DETECTION AND FOLLOW-UP PROGRAM

The relationship between serum cholesterol and cancer incidence was investigated in the population of the Hypertension Detection and Follow-up Program (HDFP). The HDFP was a multi-center trial designed to test the effectiveness of a stepped program of medication in reducing mortality associated with hypertension. Over 10,000 participants, ages 30-69, were followed with clinic and home visits for a minimum of five years. Cancer incidence was ascertained from existing study documents, which included hospitalization records, autopsy reports and death certificates. During the five years of follow-up, 286 new cancer cases were documented. The distribution of sites and total number of cases were similar to those predicted using rates from the Third National Cancer Survey. A non-fasting baseline serum cholesterol level was available for most participants. Age, sex, and race specific five-year cancer incidence rates were computed for each cholesterol quartile. Rates were also computed by smoking status, education status, and percent ideal weight quartiles. In addition, these and other factors were investigated with the use of the multiple logistic model.^ For all cancers combined, a significant inverse relationship existed between baseline serum cholesterol levels and cancer incidence. Previously documented associations between smoking, education and cancer were also demonstrated but did not account for the relationship between serum cholesterol and cancer. The relationship was more evident in males than females but this was felt to represent the different distribution of occurrence of specific cancer sites in the two sexes. The inverse relationship existed for all specific sites investigated (except breast) although a level of statistical significance was reached only for prostate carcinoma. Analyses after exclusion of cases diagnosed during the first two years of follow-up still yielded an inverse relationship. Life table analysis indicated that competing risks during the period of follow-up did not account for the existence of an inverse relationship. It is concluded that a weak inverse relationship does exist between serum cholesterol for many but not all cancer sites. This relationship is not due to confounding by other known cancer risk factors, competing risks or persons entering the study with undiagnosed cancer. Not enough information is available at the present time to determine whether this relationship is causal and further research is suggested. ^

MARITAL STATUS AND CARDIOVASCULAR RISK (SERUM, CHOLESTEROL, CIGARETTE, SMOKING, BLOOD, PRESSURE, SYSTOLIC)

There were three purposes of this study. The first was to describe the association between stable marital status and serum cholesterol, systolic blood pressure and cigarette smoking. The second purpose was to determine whether individuals who were married at one point and became widowed or divorced/separated had higher serum cholesterol, higher systolic blood pressure or were more likely to smoke prior to the change in marital status compared with individuals who did not change marital status. The third purpose was to determine whether the changes in marital status described above were related to increases in serum cholesterol or in cigarette smoking behavior. The rationale for the study was to determine whether previously reported associations between marital status categories and cardiovascular mortality may be mediated through higher values of risk correlates for cardiovascular disease among unmarried individuals.^ The study group selected for this dissertation was a sample from the Hypertension Detection and Follow-up Program (HDFP) population. The HDFP population was aged 30-69 years at the initial visit and included blacks and whites, males and females. The population was followed five years after the initial visit and periodic measurements of serum cholesterol, blood pressure and cigarette smoking behavior were obtained.^ Serum cholesterol was not associated with stable marital status category or with marital status prior to change. Changes in serum cholesterol were associated with marital status categories after change but the serum cholesterol values deceased rather than increased. Married individuals were shown to have higher serum cholesterol values compared with unmarried. Selection of the HDFP population may have influenced an ability to detect a significant association between marital status and serum cholesterol but it is doubtful that use of a general population would alter the direction of the association.^ Systolic blood pressure was significantly higher at the initial visit among unmarried white males and females compared with their married counterparts. No association between systolic blood pressure was found among black males or females. Those individuals who were married at the initial visit who experienced a change in marital status were found to have higher systolic blood pressure prior to the change in marital status. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^