The joint effects of apolipoprotein B, apolipoprotein A1, LDL cholesterol, and HDL cholesterol on risk: 3510 cases of acute myocardial infarction and 9805 controls.

AIMS: Plasma levels of apolipoprotein B (apoB), the main surface protein on LDL particles, and LDL-C, the amount of cholesterol in those particles, are closely correlated and, considered separately, are positive risk factors. Plasma levels of apolipoprotein A(1), the main surface protein on HDL particles, and HDL-C, the amount of cholesterol in those particles, are also closely correlated with each other and, considered separately, are negative risk factors. The interdependence of these four risk factors is unclear. METHODS AND RESULTS: Case-control study among 3510 acute myocardial infarction patients (without prior vascular disease, diabetes, or statin use) in UK hospitals and 9805 controls. Relative risks (age, sex, smoking, and obesity-adjusted) were more strongly related to apoB than to LDL-C and, given apoB, more strongly negatively related to apoA(1) than to HDL-C. The ratio apoB/apoA(1) was uncorrelated with time since symptom onset in cases, was reproducible in samples collected a few years apart in controls (correlation 0.81), and encapsulated almost all the predictive power of these four measurements. Its effect was continuous, substantial throughout the UK normal range [relative risk, top vs. bottom decile of this ratio, 7.3 (95% CI 5.8-9.2)] and varied little with age. The ratio apoB/apoA(1) was substantially more informative about risk (chi(1)(2) = 550) than were commonly used measures such as LDL-C/HDL-C, total/HDL cholesterol, non-HDL cholesterol, and total cholesterol (chi(1)(2) = 407, 334, 204, and 105, respectively). Given apoB and apoA(1), the relationship with risk of LDL-C was reversed, and this reversal was strengthened by appropriate allowance for random measurement errors in two correlated variables. Given usual apoB, lower LDL-C (consistent with smaller LDL particles) was associated with higher risk (P < 0.0001). During the first 8 h after symptom onset HDL-C increased by about 10%, precluding reliable assessment of the joint relationship of apoA(1) and pre-onset HDL-C with risk in such retrospective case-control studies. CONCLUSION: Apolipoprotein ratios are more informative about risk than lipid fractions are. This suggests that, among lipoprotein particles of a particular type (LDL or HDL), some smaller and larger subtypes differ in their effects on risk. Direct measurements of even more specific subtypes of lipoprotein particles may be even more informative about risk.

Is smoking an independent risk factor for invasive cervical cancer? A nested case-control study within Nordic biobanks.

The strong correlation between smoking and exposure to oncogenic human papillomaviruses (HPVs) has made it difficult to verify the independent role of smoking in cervical carcinogenesis. Thus, the authors evaluated this role. Five large Nordic serum banks containing samples from more than 1,000,000 subjects were linked with nationwide cancer registries (1973-2003). Serum samples were retrieved from 588 women who developed invasive cervical cancer and 2,861 matched controls. The samples were analyzed for cotinine (a biomarker of tobacco exposure) and antibodies to HPV types 16 and 18, herpes simplex virus type 2, and Chlamydia trachomatis. Smoking was associated with the risk of squamous cell carcinoma (SCC) among HPV16- and/or HPV18-seropositive heavy smokers (odds ratio=2.7, 95% confidence interval: 1.7, 4.3). A similar risk of SCC (odds ratio=3.2, 95% confidence interval: 2.6, 4.0) was found in heavy smokers after adjustment for HPV16/18 antibodies. The point estimates increased with increasing age at diagnosis and increasing cotinine level. This study confirms that smoking is an independent risk factor for cervical cancer/SCC in women infected with oncogenic HPVs. These findings emphasize the importance of cervical cancer prevention among women exposed to tobacco smoke.

Fibrinogen and coronary heart disease: test of causality by 'Mendelian randomization'.

BACKGROUND: Blood concentrations of fibrinogen have been associated with coronary heart disease risk in epidemiological studies, but it is uncertain whether this association is causal or reflects residual confounding by other risk factors. We investigated the relationship between the single nucleotide polymorphism at position -148 in the beta-fibrinogen gene promoter (beta - 148C/T), blood fibrinogen levels, and risk of myocardial infarction (MI) in sufficiently large numbers of coronary disease cases to reliably address this question. METHODS: Genotyping and measurement of blood fibrinogen concentration were carried out in 4,685 cases of confirmed MI and 3,460 controls with no history of coronary disease. A meta-analysis of ISIS and 19 other studies of beta-fibrinogen genotypes involving a total of 12,220 coronary disease cases and 18,716 controls was conducted. RESULTS: Among the ISIS controls, mean plasma fibrinogen concentrations with the C/C, C/T and T/T genotypes were 3.34 (SE 0.015), 3.48 (0.022), and 3.60 (0.064) g/l, respectively, corresponding to an increase of 0.14 (0.024) g/l per T allele (trend P < 0.0001). In the case-control comparison, 0.14 g/l higher usual plasma fibrinogen concentration was associated with an age-adjusted and sex-adjusted risk ratio for MI of 1.17 [95% confidence interval (95% CI) 1.14-1.19; P < 0.0001]. But, after further adjustment for smoking, body mass index, and plasma apolipoprotein B/A(1) ratio, this risk ratio fell to 1.03 (95% CI 1.00-1.05; P = 0.05). Moreover, fibrinogen genotype was not significantly associated with MI incidence: risk ratio of 1.06 (95% CI 0.96-1.16) per higher-fibrinogen allele in ISIS alone and of 1.00 (95% CI 0.95-1.04) per allele in the meta-analysis. CONCLUSIONS: Genotypes that produce lifelong differences in fibrinogen concentrations do not materially influence coronary disease incidence. As these genotype-dependent differences in fibrinogen were allocated randomly at conception (Mendelian randomization), this association is not likely to be confounded by other factors. Consequently, these genetic results provide strong evidence that long-term differences in fibrinogen concentrations are not a major determinant of coronary disease risk.

Maternal circulating nutrient concentrations in pregnancy: implications for birth and placental weights of term infants.

BACKGROUND: Compromised fetal growth may program chronic diseases of adulthood, and it has been suggested that maternal nutrition is a major determinant of fetal growth. We previously found no clinically significant associations between maternal diet and the size of the infant and placenta at birth in a large cohort of white women living in the United Kingdom. OBJECTIVE: The objective was to examine the relations between indexes of maternal nutritional status in pregnancy and the birth and placental weights of infants born at term. DESIGN: We conducted a prospective cohort study of 798 white nulliparous women with singleton pregnancies. Blood samples were obtained at approximately 16 and 28 wk of gestation. RESULTS: The concentration of most nutrients was not associated with pregnancy outcome. High retinol and hemoglobin concentrations in late, but not in early, pregnancy were strongly and independently associated with lower birth weight and smaller placental size at birth. Each 0.1- micro mol increase in retinol predicted a 20.8-g (95% CI: 9.2, 32.5 g) decrease in birth weight (P < 0.001), and each 0.1-g/L increase in hemoglobin predicted a 61.5-g (95% CI: 28.5, 94.4 g) decrease in birth weight (P < 0.001). CONCLUSIONS: We found negative associations between birth and placental weights and maternal retinol and hemoglobin concentrations. These relations may be causal or may reflect an underlying metabolic dysfunction, such as failure of plasma volume expansion. Our results provide no evidence that having high circulating nutrient concentrations, for example, through the use of supplements, would improve infant and placental growth.

Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies.

BACKGROUND: Large blood-based epidemiological studies require simple, cost-effective sample collection methods. Immediate sample separation or rapid transport of chilled blood samples to a central laboratory may be impractical or prohibitively expensive. To assess the feasibility and reliability of transporting blood samples over several days at ambient temperature (e.g. by mail), we evaluated the stability of various plasma analytes in samples stored at room temperature or chilled. METHODS: Multiple vacutainers of blood, containing EDTA and aprotinin as preservative, were drawn from 12 volunteers and stored at 21 degrees C or 4 degrees C. Immediately after collection and 1, 2, 3, 4, and 7 days later, vacutainers stored at each temperature were centrifuged, and the plasma was aliquoted and stored at -80 degrees C. Subsequently, all aliquots from each individual were analysed in one analytical run for a range of chemistries. RESULTS: In whole blood stored at room temperature for up to 7 days, concentrations of albumin, apolipoproteins A1 and B (apoA1 and apoB), cholesterol, high density lipoprotein (HDL), total protein, and triglycerides changed by less than 4%, and low density lipoprotein (LDL) by less than 7%. Whilst alanine transaminase (ALT), creatine kinase (CK), creatinine, and gamma-glutamyl transferase (GGT) concentrations changed substantially at room temperature, there was less than 4% change during chilled storage up to 7 days. By contrast, aspartate transaminase (AST) concentrations increased markedly under both conditions. CONCLUSIONS: A wide range of important analytes, including lipids, change by only a few per cent in whole blood during storage at room temperature for several days. Mailed transport of whole blood samples may, therefore, be a simple and cost-effective option for large-scale epidemiological studies.

Effect of temperature and light on the stability of fat-soluble vitamins in whole blood over several days: implications for epidemiological studies.

BACKGROUND: Biochemical measurement of fat-soluble vitamins would allow direct assessment in epidemiological studies of their association with disease. However, the perceived instability of these compounds and typically high cost of collection and analysis may make their measurement impractical, particularly in large-scale studies. Using a high performance liquid chromatography assay developed in-house, we have investigated the separate effects of temperature and light on the stability of vitamins in whole blood over several days. METHODS: Multiple blood samples from 10 volunteers were stored at 20 degrees C or 4 degrees C and in dark or light conditions. Immediately after collection and 1, 2, 3, 4, and 7 days later, samples stored under each condition were centrifuged, and the plasma was aliquoted and stored at -80 degrees C. Subsequently, all aliquots from each individual were analysed in one analytical run. RESULTS: In whole blood stored under any of the four conditions for up to 7 days, concentrations of alpha-carotene, beta-carotene, lutein, lycopene, retinol, and alpha-tocopherol changed by less than 8%, and cryptoxanthin and gamma-tocopherol by less than 11%. Although significant temperature effects were observed for alpha-carotene, and alpha- and gamma-tocopherol, and a significant effect of light was observed for alpha-carotene, cryptoxanthin, and lycopene, these analytes changed by less than 1% per day under all conditions. CONCLUSIONS: Concentrations of these fat-soluble vitamins change by only a few per cent in whole blood during storage at room temperature for several days. Hence, delayed separation of blood samples (which may be required for practical reasons in large-scale epidemiological studies) does not preclude reliable measurement of fat-soluble vitamins.

Lipid-related genes and myocardial infarction in 4685 cases and 3460 controls: discrepancies between genotype, blood lipid concentrations, and coronary disease risk.

BACKGROUND: Blood lipid concentrations are causally related to the risk of coronary heart disease (CHD). Various associations between CHD risk and genes that moderately affect plasma lipid levels have been described, but previous studies have typically involved too few 'cases' to assess these associations reliably. METHODS: The present study involves 4685 cases of myocardial infarction (MI) and 3460 unrelated controls without diagnosed cardiovascular disease. Six polymorphisms of four 'lipid-related' genes were genotyped. RESULTS: For the apolipoprotein E epsilon2/epsilon3/epsilon4 polymorphism, the average increase in the plasma ratio of apolipoprotein B to apolipoprotein A(1) (apoB/apoA(1) ratio) among controls was 0.082 (s.e. 0.007) per stepwise change from epsilon3/epsilon2 to epsilon3/epsilon3 to epsilon3/epsilon4 genotype (trend P < 0.0001). The case-control comparison yielded a risk ratio for MI of 1.16 (95% CI: 1.06, 1.27; P = 0.001) per stepwise change in these genotypes. But, this risk ratio was not as extreme as would have been expected from the corresponding differences in plasma apoB/apoA(1) ratio between genotypes. Hence, following adjustment for the measured level of the plasma apoB/apoA(1) ratio, the direction of the risk ratio per stepwise change reversed to 0.83 (95% CI: 0.74, 0.92; P < 0.001). Similarly, for the apolipoprotein B Asn4311Ser and Thr71Ile polymorphisms, genotypes associated with more adverse plasma apolipoprotein concentrations were associated with significantly lower risk of MI after adjustment for the apoB/apoA(1) ratio. The B2 allele of the cholesteryl ester transfer protein TaqIb polymorphism was associated with a significantly lower plasma apoB/apoA(1) ratio, but with no significant difference in the risk of MI. Finally, the lipoprotein lipase Asn291Ser and T4509C (PvuII) polymorphisms did not produce clear effects on either the plasma apoB/apoA(1) ratio or the risk of MI. CONCLUSIONS: It remains unresolved why some of these genetic factors that produce lifelong effects on plasma lipid concentrations have significantly less than the correspondingly expected effects on CHD rates in adult life.

Self-reported and serum cotinine-validated smoking in pregnant women in Estonia.

OBJECTIVES: Although widely used in epidemiological studies, self-report has been shown to underestimate the prevalence of smoking among pregnant women. Objectives of this study were to examine the discrepancy between self-reported and cotinine-validated smoking status, and the sociodemographic characteristics associated with the misclassification of real smoking status among pregnant women in Tallinn, the capital of Estonia. METHODS: Serum cotinine assays were performed on a subsample (n = 1360) of the pregnant women, who had participated in a recent study of human papillomavirus type 16 (HPV-16) seroprevalence in Estonia. In the present study, serum concentrations > or = 15 ng/ml were used to distinguish current smokers from nonsmokers. The serum-validated smoking level was compared with the self-reported level in the records of the Estonian Medical Birth Registry. For the group of self-reported non-smokers, the differences between the cotinine-validated smokers and the cotinine-validated nonsmokers, with respect to their sociodemographic characteristics (age, ethnicity, educational level, employment status, marital status, parity), were estimated by logistic regression. RESULTS: Of 1239 women who reported being nonsmokers, 259 (20.9%) had serum cotinine levels > or = 15 ng/ml, and can be regarded as current smokers. Among self-reported nonsmokers, nondisclosure of current smoking was significantly more frequent in non-Estonian, less educated, socially inactive, cohabiting and multiparous women. CONCLUSIONS: Self-reported data on smoking in pregnant women underestimates the real smoking prevalence in Estonia. Maternal unwillingness to declare smoking during pregnancy needs to be taken into account in the practice of maternal and child health to better target prenatal smoking cessation interventions.

Large-scale evidence that the cardiotoxicity of smoking is not significantly modified by the apolipoprotein E epsilon2/epsilon3/epsilon4 genotype.

Results from two small studies, involving a total of only 174 cases, have suggested that the increased risk of coronary heart disease conferred by cigarette smoking is substantially affected by genotype at the apolipoprotein E (APOE) epsilon2/epsilon3/epsilon4 polymorphism. We have established APOE genotypes in 4484 patients with acute myocardial infarction diagnosed before the age of 55 years for male and 65 years for female patients, and in 5757 controls with no history of cardiovascular disease. On average, the hazard ratio for myocardial infarction was 1.17 (95% CI 1.09-1.25; p<0.00001) per stepwise change from epsilon3/2 to epsilon3/3 to epsilon3/4 genotype. Among individuals in this study with known cigarette smoking status, the hazard ratio for myocardial infarction in smokers versus non-smokers was 4.6 (4.2-5.1). There was, however, no significant difference between the smoker/non-smoker hazard ratios for those with different APOE genotypes (chi2(2)=0.69; p=0.7). When differences in risk between different genotypes are not extreme (as with this APOE polymorphism), reliable assessment of hypothesised gene-environment interactions will often require the study of many thousands of disease cases.

Herpes simplex virus and risk of cervical cancer: a longitudinal, nested case-control study in the nordic countries.

Human papillomaviruses (HPVs) play the major role in cervical carcinogenesis. The authors reevaluated the role of herpes simplex virus type 2 (HSV-2) in this multistage process by conducting a longitudinal, nested case-control study using 1974-1993 data and comparing the results with those from a meta-analysis of studies. A Nordic cohort of 550,000 women was followed up for an average of 5 years, after which 178 cervical carcinoma cases and 527 controls were identified. HSV-2; HPV-16, HPV-18, and HPV-33; and Chlamydia trachomatis antibodies were determined at baseline by HSV-2 glycoprotein gG-2 and HPV virus-like-particle enzyme immunoassays and by using the microimmunofluorescence method. The relative risk of cervical carcinoma was calculated by conditional logistic regression. Longitudinal studies on HSV-2 and cervical neoplasia were identified through MEDLINE (National Library of Medicine, Bethesda, Maryland), and weighted mean relative risks were calculated. Smoking (relative risk = 1.6, 95% confidence interval (CI): 1.1, 2.3) and HPV-16/HPV-18/HPV-33 (relative risk = 2.9, 95% CI: 1.9, 4.3) were both associated with cervical carcinoma. The smoking- and HPV-16/HPV-18/HPV-33-adjusted relative risks for HSV-2 were 1.0 (95% CI: 0.6, 1.7) and 0.7 (95% CI: 0.3, 1.6), respectively, for HPV seropositives. In the meta-analysis, the relative risk for HSV-2 was 0.9 (95% CI: 0.6, 1.3). In both sets of data, HSV-2 did not play a role in cervical carcinogenesis.