Abstract
There is a significant genetic influence on arterial oxygen saturation (SaO2) in high-altitude (HA) residents. It is not known whether this is true of lowlanders ascending to HA. The I allele of the angiotensin-converting enzyme (ACE) gene is associated with low ACE activity and elite endurance performance. An excess of the I allele has also been reported in South American natives living over 3,000 m and among elite HA mountaineers who demonstrate extreme endurance in a hypoxic environment, where maintenance of SaO2 is crucial to performance. We postulated that the I allele may confer an advantage at HA through genotype-dependent alterations in SaO2. Rapid ascent (n = 32) and slow ascent groups (n = 40), ascending to approximately 5,000 m over 12.0 and 18.5 days, respectively, had their SaO2 assessed throughout and compared with their ACE genotype. Resting SaO2 was independent of the ACE genotype and remained so for the slow ascent group, in whom the fall in SaO2 with ascent was genotype independent. However, SaO2 with ascent was significantly associated with the ACE genotype in the rapid ascent group (p = 0.01) with a relatively sustained SaO2 in the II subjects. These data are the first to report an association of the I allele with the maintenance of SaO2 at HA.
As part of the circulating renin-angiotensin system, angiotensin-converting enzyme (ACE) generates vasoconstrictor angiotensin II (hence stimulating aldosterone release) and degrades vasodilator kinins. It is also a key component of the local renin-angiotensin systems, which have been identified in diverse tissues including adipose tissue (1), skeletal muscle (2), heart (3), and lung (4). A polymorphism of the human ACE gene has been identified in which the presence (insertion, I allele) rather than the absence (deletion, D allele) of a 287-bp fragment is associated with lower serum (5) and tissue (6, 7) ACE activity.
An excess frequency of the I allele has recently been noted in elite distance runners (8) and rowers (9) when compared with control subjects. Genotype-dependent improvements in the mechanical efficiency of trained skeletal muscle (10) may underlie these observations.
There is also an excess of the I allele compared with whites in the Quechua-speaking natives of South America who live over 3,000 m (11). As the authors highlight, although the higher frequency of the I allele might have facilitated the migration of the ancestral Quechua to the highlands, the I allele has not been subsequently selected for in this high-altitude (HA) population. There is no excess when compared with lowland Native American populations. However, there is an excess of the I allele and II genotype among residents of the Ladakh region of India living above 3,600 m with an even greater excess in lowlanders who migrated to Ladakh (12).
An I allele excess has also been reported among elite HA mountaineers (13). These athletes perform extreme endurance exercise in a hypoxic environment where maintenance of arterial oxygen saturation (SaO2) becomes crucial to performance. There is marked interindividual variation in SaO2 at HA with a significant genetic influence noted in Tibetan HA residents (14). In a separate cohort of 354 Tibetan residents at 3,800–4,065 m, the average SaO2 was found to be 89.4 ± 0.2%, with a range of 76–97% (15). A major gene influencing SaO2 may explain 21% of this phenotypic variation. The authors postulate that homozygotes for the low-SaO2 allele have a mean SaO2 of 83.6%, whereas heterozygotes and homozygotes for the high-SaO2 allele have means of 87.6% and 88.3%, respectively. The latter, therefore, have a selective advantage in their HA hypoxic environment. It is not known whether this is true of lowlanders ascending to HA.
The renin-angiotensin system might play a role in the maintenance of SaO2 in a number of ways. Angiotensin II modulates the pulmonary vasoconstrictive response to hypoxia (16). This is thus attenuated by ACE inhibition (17) and angiotensin II type 1 (AT1) receptor antagonism (16). Second, angiotensin II stimulates the hypertrophic and hyperplastic growth of vascular smooth muscle cells involved in pulmonary vascular remodeling. A reduction in ACE also decreases the development of chronic hypoxic pulmonary hypertension and the degree of vascular remodeling in established pulmonary hypertension (18). Third, angiotensin II increases vascular permeability (19, 20) and might thus increase V/Q mismatch and offer a diffusion barrier to oxygen transport. Fourth, angiotensin II may have a role in the regulation of hypoxic respiratory drive through modulation of carotid body chemoreceptor activity (21, 22) and its central transduction (23–26).
Finally, there is a reduced resting plasma aldosterone concentration (PAC) at HA (27–34). A relatively greater drop in PAC to plasma renin activity (PRA) increases the PRA:PAC ratio and reflects a reduced PAC response to PRA. This reduction in PAC to PRA is a beneficial adaptation that permits a natriuresis and diuresis and reduces the formation of edema. This uncoupling may reduce the exercise-related sodium and fluid retention that occurs at HA and may otherwise contribute to acute mountain sickness and pulmonary interstitial edema (34, 35). In support of this hypothesis, a greater resting and exercise-stimulated PAC at HA, although still suppressed compared with SL, is associated with acute mountain sickness and with lower SaO2 both before and during exercise (34). The I allele, with low ACE activity, may potentially increase this uncoupling and benefit elite mountaineers through genotype-dependent alterations in SaO2 at HA.
We have thus tested the hypothesis that the I allele excess among elite mountaineers might be partly due to genotype-dependent alterations in SaO2 at HA. This would also clarify whether a significant genetic influence on SaO2 also occurs in lowlanders ascending to HA.
The study had appropriate ethics committee approval. Written informed consent was obtained from all participants.
Two groups of Caucasian subjects were studied. The first group (rapid ascent [RA]) comprised members of the 1994 British Mount Everest Medical Expedition. These subjects flew from 1,250 m (Kathmandu) to 2,800 m (Lukla) and then ascended to 5,180 m over a 12-day period. The second group (slow ascent [SA]) comprised members of the 1998 Medex Kanchenjunga Expedition who similarly ascended from 1,250 to 5,100 m but took over 50% longer to do so, ascending over 18.5 days.
In all subjects, SaO2 was assessed (Nellcor N-20P pulse oximeter; Nellcor Puritan Bennett Ltd., Coventry, UK) on warmed hands, under gloves if necessary, and after at least 1 hour of rest. Recordings were made on arrival at any new altitude and again the following morning before departure.
All subjects were nonsmokers with no history of cardiorespiratory disease and were not taking regular medications. In particular, subjects were excluded if they used prophylactic medication against acute mountain sickness.
DNA was extracted from mouthwash samples as previously described (36) with ACE genotype determined using a three-primer method (37), yielding amplification products of 65 bp (I allele) and 84 bp (D allele). These were separated by electrophoresis on a 7.5% polyacrylamide gel and visualized using ethidium bromide. Genotyping was performed by experienced staff blind to subject data.
Baseline (sea level) respiratory function values were assessed in the RA cohort only. These were performed according to guidelines by the British Thoracic Society (38). The best of three attempts was recorded for each subject using an accurate (39) handheld Micro Medical Microplus turbine spirometer (Micro Medical Ltd., Rochester, Kent, UK).
Allele frequencies were determined by gene counting. A chi-squared test was used to compare the observed numbers for each genotype with those expected for a population in Hardy-Weinberg equilibrium. Gene frequencies were compared with two large control groups, the first consisting of 1,248 military recruits, age 19.7 ± 2.5, the second a control group of 615 from the ECTIM study (40), age 53.5 ± 0.3, where the genotype distributions were (II, ID, DD) 0.24, 0.49, 0.27 and 0.21, 0.50, 0.29, respectively. Statistical analysis of the SaO2 data was conducted using the Stata package, version 6.0. Repeated-measures analysis of variance was conducted, with saturation as the response variable. Using this method, the possible effects of the ACE genotype and altitude and also the possibility of an interaction between ACE genotype and altitude on these responses were considered.
The RA group comprised 32 individuals (23 males: mean ± SD, age 39.6 ± 11.2 years, height 174.9 ± 9 cm, and mass of 73.4 ± 12.1 kg: 10 DD, 15 ID, 7 II). The SA group comprised 40 individuals (31 males) who were slightly but not significantly older and of heavier build (mean ± SD, age 41.7 ± 12.2 years, height 180.4 ± 8.8 cm, and mass 75.3 ± 12.5 kg: 14 DD 22 ID, 4 II). The null hypothesis that the ACE genotype distributions in both the RA and SA groups were in Hardy-Weinberg equilibrium could not be rejected (p = 0.76 and p = 0.27, respectively). There was no significant difference in the genotype distributions either between the two groups (p = 0.38) or compared with the two control groups (p = 0.93 and p = 0.84 for the RA group and p = 0.26 and p = 0.1 for the SA group compared with the ECTIM and recruit control groups, respectively). In both groups, sex distribution, subject age, height, and weight were independent of genotype.
There was no difference by genotype in sea level FEV1 (4.4 ± 0.4, 4.0 ± 0.7, 4.1 ± 0.9, mean ± SD for DD, ID, and II subjects, respectively, p = 0.63 by analysis of variance) or FVC (5.9 ± 0.7, 5.2 ± 1, 5.1 ± 1.3, mean ± SD for DD, ID, and II subjects, respectively, p = 0.4 by analysis of variance). Peak expiratory flow (PEF) similarly showed no association with genotype (633 ± 144, 621 ± 100, 633 ± 106, mean ± SD for DD, ID, and II subjects, respectively, p = 0.98 by analysis of variance).
Of the 640 possible data points in the SA cohort, 90% of saturations were recorded, in the RA cohort, 86% of the 448 possible data points were recorded. Baseline resting oxygen saturations were independent of both ACE genotype and the presence of the I allele. This remained so for the SA group, in whom the fall in SaO2 in the group overall (93.4 ± 1.9 to 81.8 ± 4.2 mean ± SD from 1,900 to 5,100 m, respectively) was allele and genotype independent (Figure 1)
Figure 1. Oxygen saturation (mean ± SEM) at various altitudes by ACE genotype during the 1998 Kanchenjunga expedition (SA group). The initial saturation on arrival at a higher altitude is demonstrated. There was no difference in oxygen saturation by ACE genotype (p = 0.69).
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Figure 2. Oxygen saturation (mean ± SEM) at various altitudes by ACE genotype during the 1994 British Mount Everest Medical Expedition (RA group). The initial saturation on arrival at a higher altitude is demonstrated. SaO2 with ascent was significantly associated with the ACE genotype (p = 0.01) with a relatively sustained SaO2 in the II-allele subjects.
[More] [Minimize]British Mount Everest Medical Expedition (3,870 m, RA) | Kanchenjunga 1998 (4,050 m, SA) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ACE | DD | ID | II | DD | ID | II | ||||
| Mean | 85.4 | 87.5 | 90.1 | 86.9 | 88.6 | 91.7 | ||||
| n | 9 | 15 | 7 | 13 | 22 | 3 | ||||
| SD | 1.9 | 3.3 | 3.2 | 4.6 | 3.7 | 4.2 | ||||
| Median | 86 | 88 | 90 | 88 | 89 | 93 | ||||
| Minimum | 83 | 83 | 84 | 80 | 76 | 87 | ||||
| Maximum | 89 | 94 | 94 | 94 | 95 | 95 | ||||
These data are the first to report an association of low ACE activity, as marked by the I allele of the ACE gene, with the maintenance of SaO2 during hypobaric hypoxia. These data may help account for the previously reported significant I allele excess among elite mountaineers (13).
Whether the genotype association with SaO2 is related to differences in endocrine ACE activity or to differences in tissue ACE activity is unclear. Such a distinction is difficult to make, given that endocrine renin-angiotensin system activity may mark that of paracrine systems. Both may, in fact, play a role.
Reduced ACE may be a beneficial adaptation to HA, as ACE activity is reduced in dogs (41) and rats (42) in response to acute hypoxia. The same response does not appear to occur in humans, however (27, 43–45). Reduced transpulmonary angiotensin I conversion does occur in hypoxia (46, 47), with a temporary (up to 11 days) reduction in angiotensin II in humans (33). The decreased level of ACE associated with the I allele could have advantageous effects in a number of ways.
PRA has been variably reported to increase (29, 32, 48) or remain unchanged (27, 45, 49) during hypobaric hypoxia. Most workers (30, 31, 34, 50, 51), however, have demonstrated a reduction in PRA at HA (differences in study results may depend on whether sodium balance is controlled during exposure and the degree of rest). More consistent has been the finding of a reduced resting PAC at HA (27–34). A relatively greater drop in PAC to PRA increases the PRA:PAC ratio and reflects a reduced PAC response to PRA. In vitro studies suggest this may be partly due to direct inhibition of aldosterone synthesis in the adrenal cortex by hypoxia (52, 53). This reduction in PAC to PRA is a beneficial adaptation that permits a natriuresis and diuresis and reduces the formation of edema. Exercise at HA still produces a rise in PRA and PAC (33, 48, 49, 54), but of a lesser magnitude than at SL (34, 55).
The I allele, through association with lower baseline ACE activity, may potentially increase such uncoupling between PRA and PAC at HA, during both rest and exercise. The uncoupling of PRA and PAC may reduce the exercise-related sodium and fluid retention that occurs at HA and may otherwise contribute to acute mountain sickness and pulmonary interstitial edema (34, 35). In support of this hypothesis, a greater resting and exercise stimulated PAC at HA, although still suppressed compared with SL, is associated with acute mountain sickness and with lower SaO2 both before and during exercise (34).
Angiotensin II, which may be increased in DD subjects (56), is a human pulmonary vasoconstrictor (57) that is capable of modulating the hypoxic pulmonary vasoconstrictive response (17) via the AT1 receptor (16). Lower ACE levels thus blunt the hypoxic pulmonary vascular response (17), whereas elevated levels are associated with an exaggerated pulmonary arterial hypertensive response to exercise in patients with chronic lung disease (58). Of note, a greater hypoxic pulmonary vasoconstrictive response, which also occurs in DD subjects, is present in HA pulmonary edema-sensitive subjects compared with control subjects (59). The D allele, via the effect of angiotensin II on carotid body chemoreceptor activity (20, 21) and its central transduction (23–26), may contribute to a reduced respiratory drive at an altitude with a consequent reduction in SaO2.
The association of genotype with SaO2 in only one of the two study groups does warrant further comment. First, the SA group was slightly older and of marginally heavier build. However, these differences were not statistically significant, and no association between body mass index and SaO2 at altitude has previously been reported. Although Figure 1 appears to demonstrate higher SaO2 in the II subjects, this was not significant. There were also, nonsignificantly, fewer II subjects in the SA group than the RA group. However, the statistical model used accounts for the effect of each saturation for each subject by ACE genotype at each altitude. This thereby incorporates a great deal of data for each genotype throughout the ascent and minimizes the effect of the differences in group size. Most obviously, the SA group ascended over 18.5 days, compared with the 12 days of the RA group. It seems likely that this may play a key role in explaining the differences between this and the RA group.
Any beneficial effect from low ACE associated with the I allele in terms of increased uncoupling of PRA and PAC may be short lived, as the reduced PAC/PRA ratio seen at HA may normalize within 12–20 days (60). The response of PRA and PAC to exercise has also been found to be less reduced with chronic (14–16 days), rather than acute, HA exposure (49). The time scale for the improved exercise and resting recovery of the reduced PAC/PRA ratio corresponds with the ascent duration in the SA group and may reflect a period of reduced influence from ACE activity. Furthermore, downregulation of the AT1 receptor is known to occur with sustained HA exposure (42, 61). The downregulation of AT1 receptors with chronic exposure and the normalization of the resting and reduced exercise response of PRA/PAC may explain the lack of an effect of the ACE genotype on SaO2 with gradual ascent.
Another factor preventing an influence of the ACE genotype with gradual ascent may be the fact that after 2–3 weeks at HA lowlanders develop pulmonary hypertension (62). This is not completely reversed by 100% oxygen breathing and may suggest some degree of vascular smooth muscle cell proliferation and pulmonary vascular remodeling (62). Furthermore, in established pulmonary hypertension, the acute hypoxic vascular response is virtually lost, and lowering serum ACE no longer has an effect (18).
The downregulation of AT1 receptors with chronic exposure may explain the apparent contradictory findings of a sixfold increase in II genotype frequency among subjects with HA pulmonary hypertension in the Kyrghyz republic (63). Downregulation of AT1 receptors may be a chronic adaptation to HA, rendering any difference in serum ACE conferred by the I allele inconsequential in certain HA natives.
The ACE genotype may also be associated with a variety of different physiologic responses (skeletal muscle function [11, 64], cardiac adaptation to exercise [65], changes in body morphology [66], metabolic efficiency [11]), in addition to pulmonary vascular responses (16, 58). It is quite conceivable that one genotype would be associated with advantageous short-term changes in one parameter and perhaps disadvantageous changes in another.
Our data support the contention that low ACE activity, as marked by the I allele of the ACE gene, may be of benefit to HA mountaineers via the maintenance of SaO2 during hypobaric hypoxia, albeit only during RA. Further experiments are required to confirm these observations and to explore the physiologic mechanisms that underlie them. This effect, in addition to previously reported improvements in muscle efficiency, may account for the excess of the I allele in elite HA mountaineers.
The authors gratefully acknowledge the assistance of the organizers and members of the 1994 British Mount Everest Medical Expedition and the 1998 Medical Expeditions (Charitable Company) Kangchenjunga Expedition.
Supported in part by the British Heart Foundation, Dr. David Williams, and the late Professor Donald Heath through the University of Liverpool; D. W. is supported by the Royal Army Medical Corps.
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