Arterial blood analysis of healthy residents in Huamachuco, Peru (3,164 m): a cross-sectional study

Background: Given that arterial blood gas is affected by altitude and ethnicity, establishing reliable reference standards for these values requires analysis of arterial blood at different elevations and locations. Our objective was to measure the arterial blood gases of healthy young volunteers in Huamachuco, Peru, at 3,164 m above sea level. This is likely the first study of arterial blood gas analysis of healthy Northern Peruvians living at high altitude. Methods: Healthy residents of Huamachuco were recruited for this cross-sectional convenience sample study and arterial blood was drawn by standard procedures. People with obesity, diabetes, high levels of physical activity and a history of using selected substances were excluded. The samples were analyzed on-site in less than 15 minutes using a Stat Profile Prime CCS analyzer (Nova Biomedical). Results: Data from 46 participants (17 male, 29 female) were included in the study. The median values for arterial blood pH, oxygen, carbon dioxide, ionized calcium, glucose, lactate, hematocrit, oxygen saturation, and bicarbonate were 7.42, 9.3 kPa (70 mmHg), 4.5 kPa (33.5 mmHg), 1.04 mM, 5.19 mM, 1.8 mM, 50 %, 94 %, and 21.6 mM, respectively. We also found a lower prevalence of diabetes among highlanders compared to the Peruvian population. Conclusions: The results determined here were comparable to other results determined at different altitudes in the Americas, although arterial blood oxygen was slightly higher than predicted. These results indicate that Northern Peruvians have an Andean-style adaptation to high altitude.


Introduction
Approximately 25.2 million people live at an altitude greater than 3,000 meters above sea level, in an environment marked by decreased atmospheric pressure 1 which causes a series of downstream physiological effects that must be compensated to maintain homeostasis.Arterial blood gas analysis (ABGA) offers a window into these mechanisms for residents and sojourners at different altitudes.
ABGA is also an important tool for monitoring and diagnosing cardiopulmonary disfunction, which requires comparison of reference values to a result. 2However, establishing reference intervals is a significant challenge, especially for ABGA at high altitude, for three reasons: (1) few studies have examined reference intervals among healthy people because the test is frequently ordered for unwell patients, 2,3 (2) obtaining arterial blood is a more difficult procedure that causes more pain to the patient than routine venous phlebotomy, 4 and (3) different ethnic groups have different compensating mechanisms for life at high altitude: for instance, South American natives adapt by increasing hemoglobin, while Tibetan natives improve blood circulation. 5,6 provide additional ABGA values at high altitude and to better understand altitude adaptation mechanisms between ethnic groups, we performed arterial blood analysis on young healthy volunteers resident in Huamachuco, Peru (elevation 3,164 m) and compared them with published results at a variety of elevations and locations.We hypothesized that residents in Huamachuco will exhibit similar adaptations seen in other Andean high-altitude natives, regardless of biological sex. 5,6

Ethics statement
Although the research took place at Instituto de Educación Superior Tecnológico Público de Huamachuco (IESTPH) due to its high-altitude location, none of the authors are affiliated with IESTPH, and IESTPH does not have, to the best of our knowledge, an ethics committee capable of approving the study.Because of this, The Training, Teaching and Research Committee (ethical approval committee) of Florencia de Mora -ESSALUD Hospital I approved this study, on 01 April 2019.We sought approval from the Florencia de Mora Hospital as the first author is a doctor based at this hospital.The administration of IESTPH was informed of the project with a formal letter.The National University of Trujillo Postgraduate School also ethically approved this study; VHBZ was affiliated with that institution at the time of the study.
Volunteers were informed of the risks of arterial blood sampling and provided their written informed consent for arterial blood sampling and for the publication of the results.Participation in the study was strictly voluntary with no benefits provided, except for personal analysis results.There was one participant that was under the age of 18 (17 years old).In this case, the participant and the participant's father signed the consent form for use with minors.This cross-sectional convenience-sample study was not preregistered.

Study location
IESTPH was chosen for the study site because it was high-altitude location that could accommodate the equipment necessary for arterial blood analysis.The GPS coordinates and altitude of the study location were measured using a Samsung Galaxy A3 with the "Precise Altimeter" application.

Recruitment of participants
A letter from the director or IESTPH was sent to students of the same institution inviting them to participate in the study.Recruitment took place between May to August 2019.On the day of the study, August 26, 2019, the fasting (>8 h)

REVISED Amendments from Version 1
This revision is to incorporate changes suggested by 3 peer reviewers.Aside from minimal edits to improve readability, this version includes: 1. Confidence intervals for arterial blood gas parameters.2. Correlation analysis between bicarbonate blood concentration and other measured blood gas parameters.
3. An improved discussion of the interplay between pH, bicarbonate, and dissolved carbon dioxide.4. Expression of uncertainty regarding adaptation patterns among study participants.5. Since one of the reviewers suggested we colorize the figure, we are uploading an updated figure that presents the same data in colour.
volunteers were informed of the risks of arterial blood sampling.Written informed consent for arterial blood sampling and for the publication of the results was obtained from all participants.Although the study took place in 2019, we are publishing this work now due to delays caused by the COVID-19 pandemic.

Inclusion and exclusion criteria
Inclusion criteria for the entire study were age between 17 and 30 years and residence in Huamachuco district for at least 5 consecutive years prior to the study and no recent travel to low-altitude locations.Exclusion criteria included self-report of strenuous exercise more than 60 minutes per day, use of tobacco, antiplatelet agents, anticoagulants, diuretics, corticosteroids, beta-blockers, or some beta-stimulants, or recent travel to low elevations.Exclusion criteria also included self-report or clinical signs upon evaluation of cardiovascular, pulmonary, or hematologic disease.
Participants with self-reported diabetes and/or diabetes determined by arterial blood test (defined here as arterial blood glucose greater than 7.2 mM to account for the arteriovenous glucose difference 7,8 ), had BMI >30, or had abnormal axillary temperature (not between 35.5 and 37.0°C 9 ) were allowed to continue their participation, but their ABGA results were not used in the aggregated data.However, data from participants with diabetes was combined with other participant data to estimate diabetes prevalence.

Procedure
Volunteers were questioned by a nurse from IESTPH to determine whether the participant met inclusion/exclusion criteria.The nurse measured the height, body mass, pulse rate, blood pressure, and axillary temperature of each participant.Volunteers were also asked to self-report their biological sex.Body mass index (BMI) was calculated by dividing body mass by the square of the height.If the volunteers wished to continue, physicians (authors VHBZ and/or LJFR) obtained one sample of arterial blood (1 mL) from the right brachial artery of the volunteers using standard sterile technique and a heparinized needle (Westmed Pulset 3cc syringe).Blood samples were stored on ice in a cooler prior to analysis.
Personnel from a commercial laboratory (BermanLab, Trujillo, Perú) analyzed blood gas parameters of the samples at IESTPH less than 15 minutes after sampling to avoid contamination during storage and transport.The blood gas analyzer (Stat Profile Prime CCS, Nova Biomedical) passed operational qualification in Huamachuco and was used to measure pH, partial pressure of oxygen (pO 2 ) and carbon dioxide (pCO 2 ), plasma ionized calcium (iCa 2+ ), glucose (Glc), lactate (Lac), and hematocrit (Htc, %).Bicarbonate (HCO 3 À ) and oxygen saturation (sO 2 ) were calculated from the measured parameters.

Bias
The most likely experimental source of bias in this experiment is air contamination of the sample before or during analysis.Air contamination of the sample would artificially elevate pO 2 without causing large changes in other parameters.This bias was addressed by careful sampling technique, rapid analysis of the sample, and removal of suspiciously high pO 2 measurements by statistical methods.A second source of possible bias is due to the inclusion of unhealthy individuals in the ABGA, such as those with diabetes, obesity, or abnormal axillary temperature.If ABGA results were from a patient with diabetes, obesity, or abnormal axillary temperature or if the ABGA results were suspected of air contamination, the results were eliminated from the aggregated arterial blood analyses.However, these results were included in our analysis of the prevalence of diabetes and impaired fasting glucose.

Statistical analysis
ABGA results with pO 2 outlier values were eliminated from the dataset (Tukey's fence, k = 1.3).Shapiro-Wilk normality tests, Mann Whitney U tests, and linear regression analyses were used to analyze the dataset, compare different groups, and compare these results with previously reported data at a significance level of p < .05using a Bonferroni correction where appropriate.Quantitative variables were reported as means and standard deviations or by quartile depending on the distribution.If the Mann Whitney U test revealed a significant difference between the sexes, data was reported for males and females separately.Confidence intervals were calculated using the t-distribution for ABGA parameters, and the Wilson score interval was calculated for diabetes prevalence values.We used Microsoft Excel for Mac version 16.73 and R version 4.3.0 to analyze the data.Since all participants completed a single blood test on the same day, there was no missing data.

Study location
Huamachuco district has an area of 424 km 2 , and a 2017 population of 66902 including 16456 17 to 30 year old inhabitants. 10A topographic analysis of the district indicates that it varies from 2,200 to 4,600 m, but most of the population lives below 3,400 m.The largest city, also named Huamachuco, is located at about 3,200 m.IESTPH, where the samples were taken, is located at 7.815833 S, 78.03917 W and had a GPS altitude of 3,164 m.

Demographic data of the participants
The study size was based on available IESTPH students that volunteered and met acceptance criteria.A total of 56 participants (21 male and 35 female) volunteered for the study.Ten participants (4 male and 6 female) were excluded from ABGA after blood sampling because they had diabetes, unusual body temperature, obesity, or outlier arterial pO 2 .Some participants excluded at the interview, axillar temperature, or by excessive BMI freely decided to have their blood sampled and analyzed to know their results.
The 46 participants included in our ABGA analysis were between 17 and 28 years old, with a median age of 20 years.Ages were skewed toward lower values: the Shapiro-Wilk tests showed a significant departure from normality for age, W(46) = .885,p < .001.Body height was greater in males than females (Mann Whitney U test, p < 0.001): females had a median height of 1.51 m, and males had a median height of 1.60 m.Statistically significant differences between the sexes in other anthropomorphic parameters were not found.Body mass index had a normal distribution (Shapiro-Wilk, W(46) = .97,p = .31)and had an average of 23.4 and a standard deviation of 2.8.No participant had a BMI > 30, high blood pressure or diabetes.The median blood pressure was 100/60 mmHg, with a variation of less than 20 mmHg.The median pulse rate was 67 with a range of 55 to 89 min -1 .Most of the participants had an axillar temperature of 36.6°C, but the range was 36.0 to 37.0°C.

Blood gas parameters of the participants
The summary values of the resulting analyses are recorded in Table 1.

Differences between sexes for blood parameters
There were significant differences for Htc % (Mann Whitney U test, p < 0.001) and HCO 3 À (Mann Whitney U test, p = 0.0019).Other ABGA parameters failed to reach statistical significance between sexes with Bonferroni correction.

Discussion
Living in the reduced atmospheric pressure of a high-altitude environment causes a series of physiological adaptations to maintain adequate blood oxygenation.These adaptations have been shown to vary between ethnic groups 5,6 making it necessary to sample different healthy populations to establish reference intervals.To our knowledge, this is the first systematic ABGA of healthy northern Peruvians living at high altitudes.Furthermore, this study includes analysis of arterial iCa 2+ , Glc, and Lac, which are also not frequently measured among healthy high-altitude residents.
pO 2 A growing body of ABGAs of healthy residents of the Americas at different elevations makes a systematic comparison of pO 2 and altitude possible.Figure 1A visualizes the published pO 2 for healthy residents of the Americas living at different altitudes.The results collected over the past half century [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] reveal that the inverse relationship between meters above sea level and pO 2 (kPa) can be modeled with a linear equation up to about 4,500 m (R 2 = 0.87, F(1,40) = 273.13,p < .001,β = -0.00133,p < .001,α = 12.5, p < .001).Inspection of Figure 1A reveals that there is a lack in published ABGA results between 100 to 1000 m.It is likely that data in this range will be useful in confirming or rejecting a linear model for pO 2 and altitude.The median value of pO 2 determined here is 1 kPa (7.50 mmHg) higher than the predicted value of the regression, although the data range overlaps the trendline and the point is within the spread of the data.
One possible explanation is that arterial blood oxygen decreases with age and the ages of the participants in this study were skewed toward young adults. 2,11When possible, only subjects that resembled the ages of the participants were included in the regression, but selection criteria and age range reporting differed between studies; older participants in the published studies included in the regression would systematically lower the regression line (Figure 1A).A second possibility was hyperventilation during sample acquisition, but breathing rate among participants was not monitored.Changes in equipment use over the years may also have added systematic errors to the data.
Andeans have been shown to have elevated oxygen saturation compared to Tibetan highlanders. 5,6In fact, the results of this study confirm higher pO 2 for Andean residents, as two studies that measured similar parameters in Tibet 31,32 fell slightly below the regression line (Figure 1A, diamonds).Furthermore, the results from Tibet were approximately 2 kPa (15 mmHg) below our result.Therefore, these results suggest support for the hypothesis that Andeans have higher oxygen saturation than Tibetans at similar altitudes, although more experimental data from random population samples at different altitudes in Tibet and Peru more would help confirm this observation.
pH and acid-base balance Blood pH is tightly regulated through several buffering systems, the most important of which is the bicarbonate-carbonic acid system, which is derived from dissolved carbon dioxide.Like pO 2 , pCO 2 (kPa) has an inverse relationship with altitude in m (R 2 = .57,F(1,40) = 53.27,p < .001.β = -.0003,p < .001,α = 5.05, p < .001, Figure 1B), although the slope of the correlation is less steep and altitude explains less of the variation in pCO 2 than it does with pO 2 (Compare Figure 1A  and B).This decrease in pCO 2 as altitude increases is usually explained by hyperventilation to compensate for decreased oxygen partial pressure at high altitude.We find that our results largely follow this trend.Unlike some other studies, 2,12,33 a statistically significant difference between sexes for pCO 2 was not observed.
Although altitude and pCO 2 were moderately correlated, no such relationship is present for pH, which remains almost constant regardless of altitude in m (R 2 = .025,F(1,40) = 1.01, p = .320,β = .00000264,p = .320,α = 7.41, p < .001, Figure 1C).The results determined here nearly exactly overlap the regression line.Given that dissolved CO 2 has an inverse relationship with pH, the pH should also increase with altitude, but this is compensated by renal excretion of nonvolatile bases. 34,35These changes also require modification of the Siggard-Anderson acid-base chart to account for altitude.Our data support moving the normal target of a plot of pH vs pCO 2 downward, but the variance of our results suggests a widening of the vertical size of the target. 34is increased bicarbonate excretion is also reflected in an inverse relationship between HCO 3 À (mM) and altitude in m (Figure 1D; R 2 = 0.43, F (1,27) = 20.13,p < .001,β = -.00095,p < .001,α = 23.16,p < .001).Since HCO 3 À is derived from pH and pCO 2 , it follows that if pH is relatively stable, HCO 3 À would also decrease if pCO 2 also decreases.We found no correlation between blood pH and actual HCO 3 À or pH and pCO 2 .This is likely because maintenance of pH within a narrow range result ultimately in a lower concentration of oxygen, carbon dioxide and bicarbonate, creating a new equilibrium at a lower buffer concentration but the same pH.A study at sea level did find weak correlations between HCO 3 À , pH and pCO 2 , with higher pH having lower pCO 2 . 2 It is likely that this study was able to find these correlations because of its larger size, but the difference is over 0.1 pH unit.The HCO 3 À concentration determined here is higher than the regression line, but the spread of the data overlaps the trendline, and males had statistically significantly higher HCO 3 À than females.

iCa 2+
Among healthy individuals, calcium is maintained within a narrow range.Departures from this range lead to several severe symptoms, including tetany, vomiting, coma, and neurological disturbances. 362][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] The red square data point is the median determined here, with whiskers representing the interquartile range.Data from native Tibetans are represented as blue diamonds. 31,32Previously published results from the Americas (green circle data points) were used to calculate the regression lines in the figure and regression statistics in the text.between 1.05 and 1.30 mM, which is slightly higher than the range (0.95 to 1.13 mM) found here. 36Similar studies in the Andes also reported average results below the lower limit of the reference range. 13,14Since iCa 2+ is dependent on several factors, including albumin content, vitamin D, and blood pH, it is not yet possible to determine whether lower iCa 2+ is a result of altitude or another confounding factor.Correcting iCa 2+ for pH did not change values significantly: the largest adjustment was not greater than 0.02 mM.

Lac and Glc
Lac concentration is a measure of anaerobic respiration and tissue hypoxia and has a reference range of between 0.3-0.8mM, but this has been called into question. 36,37The range of values determined here is both wider and has a higher median than the reference range.A study at higher altitude also reported higher Lac. 14These results may reflect a variety of activity levels among study participants, especially considering that the test site was in mountainous terrain.Alternatively, reduced oxygen at high altitude can promote anaerobic metabolism, but high-altitude residents have been shown to have Lac levels similar to those of lowlanders after exercise. 38We found a positive correlation with Lac and Glc concentration, which may be because of their relationship in the Cori cycle, but this hypothesis requires further testing.

Diabetes and impaired fasting glucose prevalence
Due to the selection criteria, only subjects with normoglycemia and reasonable ABGA results were included in the data analysis.However, it is unlikely that fasting arterial blood Glc would be impacted by outlier ABGA results, which provides an opportunity to measure diabetes and impaired fasting blood glucose (IFBG) prevalence in the entire study population of 56 people.Diabetes and IFBG are usually defined in terms of glucose concentration in venous blood plasma; arterial blood was measured here.For fasting individuals, the arteriovenous glucose difference is on the order of 0.2 mM, which revises the cutoff to ≥ 7.2 mM for diabetes and between 5.8 and 7.2 mM for IFBG. 7,8Given these criteria, the prevalence was 2/56 (3.6%, 95% CI [0.98%, 12%]) for diabetes, and 10/56 (17.9%, 95% CI [10%, 30%]) for IFBG.Aggregated diabetes prevalence in Peru is approximately 6% and IFBG prevalence is approximately 15-20%.However, highland prevalence is lower for both diabetes and IFBG, where it is estimated to be approximately 4.5% and 17.4%, respectively. 39These results are almost exactly what we observe as well, further supporting that living at high altitude correlates with lower diabetes prevalence.
Htc. Htc, a measure of the red blood cell content of blood, has been shown to vary between biological males and females, as well as between different high-altitude resident populations.Andeans and acclimatized Europeans tend to respond to high altitude by increasing hemoglobin and Htc% in a dose-response manner. 5,6,40The hematocrit determined here (Table 1) falls on the high end of the sea-level reference interval (42-52% and 37-47% for males and females respectively), suggesting similar adaptations seen in other Andean high-altitude natives. 5,6,36

Conclusions
We observe that in resident Andean highlanders pO 2 and pCO 2 decrease, Htc% increases, and pH and iCa 2+ do not change very much with altitude.Thus, Northern Peruvians seem to have similar adaptations seen in other Andean high-altitude natives.However, these results show higher than expected pO 2 and pCO 2 for the altitude of the analysis.This is likely due to the young age range of the participants and the small study sample.
This study used a convenience sample cross section of young and healthy Andeans, which means it is not likely applicable to older people or those with chronic conditions, those with different altitude adaption profiles, or sojourners to the highaltitude environment.Additional sample taking of a more demographically balanced cross section and controls preventing air exposure of the sample are necessary to confirm these results and make the generalizable to the population of Huamachuco.Furthermore, comparing the results as a function of barometric pressure rather than altitude can eliminate some variability.
We hope that these results can be helpful in further establishing altitude-and ethnicity-dependent data-supported values for ABGA as well as Glc, Lac, and iCa 2+ , which will aid in the diagnosis and treatment of cardiopulmonary disease at high altitude.
The authors have excluded diabetic volunteers from the study.Hence, a comment like "lower prevalence of diabetes among highlanders...." is little misleading.

2.
The conclusion statement "Northern Peruvians have and Andean-style adaptation to high altitude" based on arterial blood gas analysis a big claim.

3.
In such field studies involving blood collection, I personally expect some other methods like ELISA/western blotting -based evidences for the original hypothesis.

4.
It is now a well known fact that hypoxia response of males and females are distinct.Thus, combing male and female volunteer data sets for deciphering common hypoxia response is unwarranted.

5.
Is there any effect of elevation (Study location section of result section) on blood gas parameters?What are the authors opinion about the same parameters at 2,200 m or 4,600 m (the lowest and highest elevations of the studied district)?I have some thoughts on the use of the term "Andean-style adaptation".One could predict that some of the adaptations or maladaptations (such as excessive erythrocytosis and blunted ventilation/respiratory drive) in Andean high-altitude natives may be the normal response expected from other ancestral groups when exposed to a lifetime of high-altitude hypoxia, and not a specific phenotype expected only to appear in Andean individuals.However, since we don't have much data from individuals of sea-level ancestry living for very long periods at high altitude, we can not test this idea yet.In my opinion, I would pull back from using this term and phrase this as something along the lines of "similar to adaptations seen in Andean high-altitude natives", but it's up to the authors.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: High-altitude medicine, pulmonary physiology, control of breathing I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
think that the measured blood pressure is reasonable.
We updated the figure to take advantage of color to make the different groups clearer.
Perhaps we were too bold in concluding Andean style adaptation, so we changed the wording where appropriate to suggest a greater level of uncertainty.

Latika Mohan
All India Institute of Medical Sciences, Rishikesh, utt, India The article presents valuable data, is topical and useful.The study examines arterial blood gas analysis in adapted young adults residing at 3000m above sea level and discusses trends in ABGA with change in altitude.It is acknowledged that it is difficult to do such studies on normal subjects at altitude locations, but a larger sample size would have yielded more robust analysis.Was a sample size calculation done?
The methodology has been described in detail and all procedures documented well.Although the authors admit that most of the ABGA parameters failed to reach statistical significance, the trends can be observed in table 1, and follow the expected trends for pO 2 , pCO 2 .In contrast the pH and Bicarbonate values do not seem to follow the inverse relationship.This needs to be discussed in greater detail, as these are long term residents and there may be physiological compensatory mechanisms coming into play.
The regression model presented for predicting pO2 is very interesting, and larger population studies are required.As the altitude adaptation strategies are different in Tibetians and the Andeans, there may be merit in analysis of trends by ethnicity.
Overall, well written and useful study.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Figure 1 :
Figure 1: It is a bit difficult to see differences in the three point types for American, This Study, and Tibet.Can this figure be made in color or in some other way show the differences more clearly?

Competing Interests:
None Reviewer Report 04 September 2023 https://doi.org/10.5256/f1000research.147631.r198150© 2023 Mohan L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 1
. ABGA results for 46 healthy young adults in Huamachuco (3164 m).Values are expressed as minimum, first quartile, median, third quartile, maximum and 95% confidence interval (95% CI) as calculated using the t-distribution, as some results showed a statistically significant departure from normality.Males and females are separated where a statistically significant difference was found.

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Huamachuco-calculations-110623.xlsx (Dataset for the article "Arterial blood gas analysis of healthy residents in Huamachuco, Peru (3,164 m)".All raw data collected, most calculations, and results reported in the figure and table are included in this spreadsheet.)

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly
6. References 1. Suresh K, Chandrashekara S: Sample size estimation and power analysis for clinical research studies.J Hum Reprod Sci.2012; 5 (1): 7-13 PubMed Abstract | Publisher Full Text Is Reviewer Expertise: Hypoxia, High altitude biology, Human response to hypoxia I confirm that I