The dip in BP seen during sleeping hours may be a result of a number of factors such as autonomic nervous system control, function of the local endothelium, or a combination of these two. For people who do not exhibit a night-time dip in BP, irregularities in circadian rhythm or endothelial function could be fundamental to vascular health. Thus, finding non-pharmacological ways to influence non-dippers and mitigate their risk for target end-organ damage is critical.
The present study is the first to assess multiple biomarkers of NO production and oxidative stress in relation to dipping status. It is also the first study to assess these biomarkers in response to AEXT in participants classified as dippers or non-dippers. In the present investigation, we studied a population of 50–75-year-old pre-hypertensive and stage-1 hypertensive individuals and classified them into dipper or non-dipper groups based on their 24-h ABPM profile. Biomarkers were measured under standardized conditions before and after 6 months of AEXT.
Of the many oxidative stress biomarkers, thiobarbituric acid-reactive substances (TBARS) and uNOx have been previously examined in non-dipper populations. In contrast to Pierdomenico et al. (26
), who observed a significantly higher level of oxidative stress in non-dippers as measured by the lipid perioxidation measure TBARS, we saw no significant differences in 8-iso-PGF2α
levels (a measure of lipid peroxidation) between the groups at baseline.
We also report a lack of association between dipping status and uNOx levels at baseline. The results of our study are in opposition to Higashi et al. (7
) who found uNOx levels to be lower in non-dipping hypertensives. Perhaps the discrepancy between the present study and the Higashi et al. study could be related to the population studied and protocol used. Higashi et al. included smokers, and only withheld antihypertensive agents for at least 2 weeks prior to data collection. The current study excluded smokers and also tapered medication such that the subjects were off of their medication over a month before any baseline testing was conducted. Therefore, smoking and medication use could have influenced uNOx levels in the study by Higashi et al. To our knowledge, no other studies have assessed oxidative stress or endothelial function biomarkers following AEXT in dippers and non-dippers.
The primary purpose of our study was to determine how AEXT influenced dipping status, biomarkers of oxidative stress and endothelial function in subjects who were classified as dippers or non-dippers based on their 24-h ABP. The rationale for this was to identify potential differences in AEXT-induced changes in dippers and non-dippers. However, no correlations between any of the oxidative stress biomarkers and dipping status prior to or following AEXT were found. Therefore, subjects were re-classified after AEXT based on our criteria using the 24-h ABP profile that was obtained after AEXT and a separate analysis was performed. Fourteen subjects retained their original dipping status. However, five subjects became non-dippers who were previously dippers, and four subjects became dippers who were previously non-dippers.
In this sub-analysis, we found that total cholesterol showed the greatest reduction in subjects that had a beneficial change from non-dipper to dipper, while subjects that became non-dippers following AEXT increased their total cholesterol. The subjects who remained either non-dipper or dipper (i.e. did not change dipping status) after AEXT, only minimally reduced their total cholesterol levels. These patterns were also true for LDL-cholesterol and ox-LDL. Following AEXT the group that experienced the beneficial status change of non-dipper to dipper showed a large decrease in LDL and ox-LDL levels, while the group that changed from dipper to non-dipper increased their LDL-cholesterol levels and only minimally changed their ox-LDL levels. This difference among groups was significant for LDL-cholesterol changes and approached significance for ox-LDL changes with AEXT. The differences in how these three groups changed their cholesterol levels with AEXT were significant, thus suggesting AEXT-induced changes in total cholesterol levels may be linked to changing ones dipping status with AEXT.
Changes in biomarkers of endothelial function demonstrated inconsistency for people who changed from non-dippers to dippers following AEXT. Changes in uNOx, pNOx, and SOD did not correlate with change in dipping status, and the change in 8-iso-PGF2α levels did not correlate to groups as expected. 8-iso-PGF2α levels were higher in the beneficial non-dipper to dipper group, and the dipper to non-dipper group reduced their levels below baseline values. Given that the non-dipper to dipper change group was associated with a greater levels of 8-iso-PGF2α following AEXT, it demonstrates the need for more investigation into dipping status and endothelial dysfunction. Perhaps other biomarkers of oxidative stress are more appropriate to characterize these changes in a non-dipper population.
LDL-cholesterol and ox-LDL have been shown to have adverse effects on endothelial cell function such as uncoupling of eNOS (27
), increased ROS (28
), and transcriptional regulation of eNOS (29
). While decreases in cholesterol levels highlight the known inverse relationship between endothelial NO production and cholesterol (30
), we did not observe concomitant beneficial changes between biomarkers of endothelial function and cholesterol levels in relation to dipping status. Though exercise-induced reduction of cholesterol levels may have decreased reactive oxygen species (ROS) production (33
), and increased NO bioavailability, we did not observe such changes in these variables. However, restoration of endothelial function may have contributed to the beneficial switch the group that changed from non-dipper to dipper experienced as demonstrated by their change in BP. The group that changed from non-dipper to dipper increased their % Dip MAP while also decreasing their total cholesterol levels and LDL-cholesterol levels. Hence, even though our biomarkers of endothelial function did not demonstrate beneficial changes in relation to the observed changes in total cholesterol, LDL-cholesterol and ox-LDL levels, we observed changes in dipping status that were related to the subject's change in cholesterol levels. In accordance, the group that changed from dipper to non-dipper increased their total cholesterol and LDL-cholesterol levels and decreased % Dip MAP.
There are some limitations of our study worth noting. As with virtually all studies reporting on blood biomarkers in dippers and non-dippers, we were not able to obtain blood samples throughout the 24-h ABPM period. Secondly, it is somewhat difficult to compare the present results with the results of all studies on dipping status because the method for categorizing subjects as dippers and non-dippers is not standardized (34
). In the present study, we used well known published methods to categorize subjects (4
). The methods used account for sleep-awake pattern and have been clinically reproducible (18
). Finally, in the sub-analysis following AEXT, the sample sizes for the groups were small. However, the change in dipping status following an intervention is significant and carries clinical relevance.
In conclusion, AEXT was beneficial in many regards in the present study. However, AEXT did not beneficially affect all participants in regard to dipping status. We initially observed changes in BP that occurred with AEXT in participants that were categorized as dipper or non-dipper were not significantly correlated to the baseline levels of, or changes in, biomarkers of oxidative stress and endothelial function and therefore did not explain dipping status in pre- and stage-1 hypertensives. However, 39% of our study population changed dipping status. We observed a relationship between change in total cholesterol level, change in LDL-cholesterol level, and change in ox-LDL-cholesterol level to the change in dipping status with AEXT. Therefore, to simply assess dipping status only at baseline prior to an intervention may not provide a complete picture as interventions will likely not influence dipping status similarly for all participants.