The main feature of observational studies is the representation of naturalistic treatment conditions. In contrast to clinical trials, they allow the evaluation and quantification of adverse event profiles of drugs under “real life” conditions. The price for this unquestionable chance is the proneness to distorting factors, which may aggravate the interpretation of the study results. Analysis of observational study results therefore has to control for potentially influential factors and reconsider possible alternatives explaining observed associations. The most important distorting factors, which should be taken into account during analysis and interpretation are under-reporting, event selection, bias, confounding and misusage? Authors and readers of such study results should be aware of this possible sources of error, in order to derive optimal benefit from this study approach.

A previous analysis of the Alpha-Tocopherol Beta-Carotene (ATBC) Study on male smokers found that β-carotene supplementation increased the risk of pneumonia 4-fold in those who started smoking at the age of ≥21 years and smoked ≥21 cigarettes/d (a subgroup of 7 % of the study population). The present study hypothesised that β-carotene increases mortality in the same subgroup. The ATBC Study (1985–1993) recruited 29 133 Finnish male smokers (≥5 cigarettes/d) aged 50–69 years. Cox regression models were constructed to estimate the effect of β-carotene supplementation in subgroups. β-Carotene increased mortality (risk ratio 1·56; 95 % CI 1·06, 2·3) in those who started to smoke at ≥21 years and smoked ≥21 cigarettes/d. Within this subgroup, there was strong evidence of further heterogeneity. The effect of β-carotene supplementation was further modified by dietary vitamin C intake, fruit and vegetable intake (P = 0·0004), and by vitamin E supplementation (P = 0·011). Thus, harm from β-carotene was not uniform within the study population. Interactions between β-carotene and vitamins C and E were seen only within a subgroup of 7 % of the ATBC participants, and therefore should not be extrapolated to the general population. Heterogeneity of the β-carotene effect on mortality challenges the validity of previous meta-analyses that have pooled many diverse antioxidants for one single estimate of effect using the assumption that a single estimate equally applies to all antioxidants and all people. Trial registration: ClinicalTrials.gov NCT00342992.

We sought to examine the potential modifiers in the association between long-term low-dose folic acid supplementation and the reduction of serum total homocysteine (tHcy) among hypertensive patients, using data from the China Stroke Primary Prevention Trial (CSPPT). This analysis included 16 867 participants who had complete data on tHcy measurements at both the baseline and exit visit. After a median treatment period of 4·5 years, folic acid treatment significantly reduced the tHcy levels by 1·6 μmol/l (95 % CI 1·4, 1·8). More importantly, after adjustment for baseline tHcy and other important covariates, a greater degree of tHcy reduction was observed in certain subgroups: males, the methylenetetrahydrofolate reductase (MTHFR) 677TT genotype, higher baseline tHcy levels (≥12·5 (median) v. <12·5 μmol/l), lower folate levels (<8·0 (median) v. ≥8·0 ng/ml), estimated glomerular filtration rate (eGFR) <60 ml/min per 1·73 m2 (v. 60–<90 and ≥90 ml/min per 1·73 m2), ever smokers and concomitant use of diuretics (P for all interactions <0·05). The degree of tHcy reduction associated with long-term folic acid supplementation can be significantly affected by sex, MTHFR C677T genotypes, baseline folate, tHcy, eGFR levels and smoking status.