Lewis Fall

Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood–brain barrier function

We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood–brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia‐mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O2) and following 6 h passive exposure to hypoxia (12% O2). Blood flow velocity in the middle cerebral artery (MCAv) and mean arterial blood pressure (MAP) were measured for determination of CA following calculation of transfer function analysis and rate of regulation (RoR). Nine subjects developed clinical AMS (AMS+) and were more hypoxaemic relative to subjects without AMS (AMS–). A more marked increase in the venous concentration of the ascorbate radical (A•−), lipid hydroperoxides (LOOH) and increased susceptibility of low‐density lipoprotein (LDL) to oxidation was observed during hypoxia in AMS+ (P < 0.05 versus AMS–). Despite a general decline in total nitric oxide (NO) in hypoxia (P < 0.05 versus normoxia), the normoxic baseline plasma and red blood cell (RBC) NO metabolite pool was lower in AMS+ with normalization observed during hypoxia (P < 0.05 versus AMS–). CA was selectively impaired in AMS+ as indicated both by an increase in the low‐frequency (0.07–0.20Hz) transfer function gain and decrease in RoR (P < 0.05 versus AMS–). However, there was no evidence for cerebral hyper‐perfusion, BBB disruption or neuronal–parenchymal damage as indicated by a lack of change in MCAv, S100β and neuron‐specific enolase. In conclusion, these findings suggest that AMS is associated with altered redox homeostasis and disordered CA independent of barrier disruption.

Systemic oxidative–nitrosative–inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity

What is the central question of this study? Exercise performance is limited during hypoxia by a critical reduction in cerebral and skeletal tissue oxygenation. To what extent an elevation in systemic free radical accumulation contributes to microvascular deoxygenation and the corresponding reduction in maximal aerobic capacity remains unknown. What is the main finding and its importance? We show that altered free radical metabolism is not a limiting factor for exercise performance in hypoxia, providing important insight into the fundamental mechanisms involved in the control of vascular oxygen transport.

Haemostatic response to hypoxaemic/exercise stress: the dilemma of plasma volume correction

Objective Patients with arterial occlusive disease are typically hypoxaemic, and exercise is prescribed for rehabilitation. Both stressors independently contract plasma volume (PV), which may influence clinical interpretation of a patients thrombogenicity. The aim of the study was to emphasise the conceptual significance of PV correction. Methods and Results Venous plasma samples were obtained from 18 healthy men at rest in normoxia for the measurement of fibrinogen, prothrombin (PT), thrombin (TT) and activated partial thromboplastin (aPTT) times. Additional samples were obtained in hypoxia (12% oxygen) after 6 h of rest and immediately after a maximal exercise challenge. Haemostatic parameters were expressed before and after volume-shift correction. Passive hypoxia reduced PV by 3±5% (p<0.05 vs normoxia), with a further decrease observed during exercise (14±5%, p<0.05). The latter increased the absolute concentration of fibrinogen and shortened aPTT (p<0.05), but these changes were no longer apparent after PV correction (p>0.05). Likewise, the lack of change in absolute values for PT and TT (p>0.05) translated into an elongation after correction (p<0.05). Conclusions These findings highlight the important, but previously ignored, interpretive implications of PV correction when haemostasis is assessed.

Arterial hypoxaemia and its impact on coagulation: significance of altered redox homeostasis

Aims Arterial hypoxaemia stimulates free radical formation. Cellular studies suggest this may be implicated in coagulation activation though human evidence is lacking. To examine this, an observational study was designed to explore relationships between systemic oxidative stress and haemostatic responses in healthy participants exposed to inspiratory hypoxia. Results Activated partial thromboplastin time and international normalised ratio were measured as routine clinical biomarkers of coagulation and ascorbate free radical (A•−) as a direct global biomarker of free radical flux. Six hours of hypoxia activated coagulation, and increased formation of A•−, with inverse correlations observed against oxyhaemoglobin saturation. Conclusions This is the first study to address the link between free radical formation and coagulation in vivo. This ‘proof-of-concept’ study demonstrated functional associations between hypoxaemia and coagulation that may be subject to redox activation of the intrinsic pathway. Further studies are required to identify precisely which intrinsic factors are subject to redox activation.

Redox‐regulation of haemostasis in hypoxic exercising humans: a randomised double‐blind placebo‐controlled antioxidant study

In vitro evidence has identified that coagulation is activated by increased oxidative stress, though the link and underlying mechanism in humans have yet to be established. We conducted the first randomised controlled trial in healthy participants to examine if oral antioxidant prophylaxis alters the haemostatic responses to hypoxia and exercise given their synergistic capacity to promote free radical formation. Systemic free radical formation was shown to increase during hypoxia and was further compounded by exercise, responses that were attenuated by antioxidant prophylaxis. In contrast, antioxidant prophylaxis increased thrombin generation at rest in normoxia, and this was normalised only in the face of prevailing oxidation. Collectively, these findings suggest that human free radical formation is an adaptive phenomenon that serves to maintain vascular haemostasis.

Post-prandial hyperlipidaemia results in systemic nitrosative stress and impaired cerebrovascular function in the aged

Post-prandial hyperlipidaemia (PPH) acutely impairs systemic vascular endothelial function, potentially attributable to a free radical-mediated reduction in vascular nitric oxide (NO) bioavailability (oxidative-nitrosative stress). However, it remains to be determined whether this extends to the cerebrovasculature. To examine this, 38 (19 young (≤35 years) and 19 aged (≥60 years)) healthy males were recruited. Cerebrovascular function (middle cerebral artery velocity, MCAv) and cerebrovascular reactivity to hypercapnea (CVRCO2Hyper) and hypocapnea (CVRCO2Hypo) were determined via trans-cranial Doppler ultrasound and capnography. Venous blood samples were obtained for the assessment of triglycerides (photometry), glucose (photometry), insulin (radioimmunoassay), ascorbate free radical (A•-, electron paramagnetic resonance spectroscopy) and nitrite (NO2-, ozone-based chemiluminescence) in the fasted state prior to and 4 h following consumption of a standardized high-fat meal (1362 kcal; 130 g of fat). Circulating triglycerides, glucose and insulin increased in both groups following the high-fat meal (P<0.05), with triglycerides increasing by 1.37 ± 1.09 mmol/l in the young and 1.54 ± 1.00 mmol/l in the aged (P<0.05). This resulted in an increased systemic formation of free radicals in the young (P<0.05) but not the aged (P>0.05) and corresponding reduction in NO2- in both groups (P<0.05). While the meal had no effect on MCAv in either age group, CVRCO2Hyper was selectively impaired in the aged (P<0.05). These findings indicate that PPH causes acute cerebrovascular dysfunction in the aged subsequent to systemic nitrosative stress.

Interpretive limitations associated with plasma volume shifts in the clinical assessment of hemostasis.

We read with interest the recent publication by Austin et al. (1), where the authors analyzed blood from healthy males before and after the Tier Social Stress Test for markers of coagulation activation. They then reconstituted the samples for changes in plasma volume (PV) using participants’ prestress plasma and saline and mathematically using the methods of Dill and Costill (2). They concluded that observed changes in coagulation were, in part, a consequence of stress and hemoconcentration and that the method of correction by Dill and Costill is not optimal for PV correcting coagulation times. They concluded by suggesting that saline reconstitution was a more ‘‘biologically relevant’’ method of correction. Our laboratory has performed an experiment examining the same phenomenon (3), using significantly more potent PV stressors to ‘‘prove the same concept.’’ We also used the correction technique of Dill and Costill used by the present authors, although it yielded very different results. To that end, we have identified several discrepancies that warrant consideration for interpretation of these findings. We noted an intriguing finding where mathematical correction resulted in further shortening of coagulation times after the experimental stress-induced hemoconcentration. This is, as already stated, contrary to our findings and is also contrary to research they cited in their study (4). Darlington and colleagues (4) found that progressive dilutions from 0 to 90% of citrated plasma elongated coagulation times, which is in agreement with the results of our experiment and makes intuitive sense. We feel that the present authors’ divergent findings could be explained by mathematical error. The equation used by the authors, as stated on page 290 of the article, is correct and indeed proper for correcting the concentrations of plasma coagulation proteases and molecules such as fibrinogen and D-dimer; however, it is not the correct equation to use for the correction of coagulation times. The problem lies in data interpretation; an ‘‘increase’’ in coagulation results in a decrease in coagulation time across the contact factor pathway or tissue factor pathway and common pathway. Therefore, time needs to be ‘‘given back’’ to correct for loss of PV. The correct equation should incorporate a plus sign (+) and not, as is the case with the current publication, a minus (j) sign. This would then correctly account for the shift in PVand remove the artifactual decrease in activated partial thromboplastin time (aPTT) and percentage of prothrombin time (PT%) noted by the authors in the present publication. Indeed, if the reported

Redox-regulation of haemostasis in hypoxic exercising humans: a randomised double-blind placebo-controlled antioxidant study: Redox-regulation of haemostasis

PV from baseline to poststress is used (3.5% T ,2.3%) and apply the correction mentioned earlier, the aPTT only reduces by 0.1 second (34.3 T 0.8 second from 34.4 T 0.8 second), thereby abrogating the reduction. Furthermore, ‘‘significant’’ shortening from baseline to uncorrected values was reported as 33.1 T 0.8 second, which is comfortably within the boundaries of a normal aPTT (5). Even with the incorrect mathematical correction artifactually shortening aPTT further still to 31.1 T 0.8 second, it remains clinically insignificant because it has long been established that normal values are 27 to 37 second (5) on nonheparinized participants. We feel that the authors fail to express this important fact adequately in their study, which is especially relevant given that the clinical focus underlying their publication is a possible link between acute stress and cardiovascular disease. The PT data were expressed as PT%, which is confusing given that the standard clinical expression for PT in the patient population is the international normalized ratio (INR). The study would be more accessible if the authors performed the INR calculation, or even expressed their PT data as raw times, given that their participants were free from coagulopathy. This would have made their data more comparable for the clinical reader, given that most point of care devices express PT in INR. However, their PT% data unfortunately suffer from the same artifactual error as aPTT and, again, did not fall outside the boundaries of normality (6). Beyond themathematical oversight, we have some additional, mostly biochemical, concerns that may further influence the interpretation of the findings. The authors used the participants’ own un-stressed plasma as a method of reconstitution. It has been suggested that acute psychological stress increases inflammation (7), and it is known that there is a bidirectional relationship between coagulation and inflammation (8). Furthermore, acute psychological stress has been shown to increase oxidative stress (9), and a relationship between oxidative stress and coagulation activation has long been suggested (10). Thus, reconstituting with the participants’ plasma-harvested prestress is actually inappropriate. Furthermore, the authors state that this study forms part of a larger flavanol intervention trial and take great care to point out that the intervention would not influence the present data. We are not sure that this will be the case, for the reasons outlined in the previous paragraph. Flavanols are potent phenolic antioxidants and, as such, have an outstanding capacity to scavenge free radicals, alter nitric oxide bioavailability (11), and thus affect shear stress and intravascular pressures. Because the brain noadrenergic system is activated by acute stress (12), an additional aspect to consider for reconstitution may have been to measure norepinephrine levels and to treat the prestress plasma with a norepinephrine ‘‘spike’’ to try and achieve a reconstitution fluid more similar in composition to the poststress plasma. Also, given the potentially exciting mechanistic value of these data, it is possible that the patient population may benefit from drugs that have the potential LETTERS TO THE EDITOR