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Am J Physiol Heart Circ Physiol 288: H2568-H2573, 2005. First published February 4, 2005; doi:10.1152/ajpheart.00812.2004
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Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m)

Gustavo F. Gonzales,1 Francisco A. Chung,1 Sara Miranda,1 Laura B. Valdez,2 Tamara Zaobornyj,2 Juanita Bustamante,2 and Alberto Boveris2

1Facultad de Ciencias y Filosofía, Departamento de Ciencias Biológicas y Fisiológicas, Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia, Lima, Peru; and 2Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Submitted 9 August 2004 ; accepted in final form 1 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO·min–1·g heart–1 (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.

acclimatization; hypoxia; testosterone; estradiol

EXPOSURE TO HIGH ALTITUDE constitutes a pathophysiological situation that results in both advantageous and disadvantageous effects for the health of humans and animals. Among the deleterious effects of the acute exposure high altitude pulmonary edema (HAPE) (14, 39) is the predominant cause of death due to high altitude illness (47). Among the advantages of life at high altitude is a recognized cardioprotective effect observed after chronic exposure (32) and in natives at high altitude (28). Although many factors have been postulated to play roles in the cardioprotective effect of exposure to high altitude, there is no evidence indicating which is the most important. Between these postulated factors, nitric oxide (NO) has been indicated as one of the main candidates to afford the cardioprotective effect of acclimatization to high altitude (32). In fact, highlanders have increased levels of NO-stable catabolites in blood (46), and L-arginine infusion at high altitude increases arterial O2 saturation and in cases of acute mountain sickness (AMS) decreases AMS score (48). Therefore, the adaptive mechanisms of the cardiovascular system seem to trigger the activation of NO synthesis in specific cells and tissues involved in the adaptation to high altitude. Moreover, subjects that are susceptible to HAPE show decreased pulmonary NO levels during acute hypoxia compared with HAPE-resistant subjects (14), and NO administration exerts beneficial effects at various stages of pulmonary hypertension (42). These facts suggest that NO is involved in the process of acclimatization and adaptation to high altitude.

NO is the first molecule that fulfills the requirement for a physiological modulator of respiration on the basis of its O2 competitive inhibition of cytochrome oxidase activity (3, 12). It is produced in the tissues at a fair rate high enough to exhibit an inhibitory effect on cytochrome oxidase that extends the O2 gradient into the tissues (45, 51). NO may exert its regulatory activities through the inhibition of respiration, respiration-associated functions, and intracellular signaling. This range of actions may afford the molecular mechanisms of acclimatization and adaptation to high altitude (31, 32, 53, 57).

NO is synthesized by the members of the NO synthase (NOS) family by the oxidation of L-arginine and NADPH by O2 to yield L-citrulline and NO (29). Since 1997 to 1998, when the existence of a Ca2+ dependent mitochondrial NOS (mtNOS) was recognized (23, 25), the relevance of this enzyme for mitochondrial bioenergetics and physiology has been under consideration. Liver mtNOS was sequenced and identified as the {alpha}-isoform of neuronal NOS (nNOS), myristylated and phosphorylated at the COOH-terminal end (21), and is now considered a constitutive enzyme of the inner mitochondrial membrane. The biological function of mtNOS is currently discussed in terms of inhibition of tissue respiration (1, 3, 13), modulation of tissue O2 gradients (45, 51), induction of apoptosis (13), and nitrative and nitrosative stress in inflammation (2). Physiological roles for mtNOS have been postulated in cell signaling, mitochondrial pathology, aging, dystrophin deficiency, inflammation, and cancer (17). Mitochondrial production of NO has been determined in heart, liver, brain, kidney, diaphragm, and thymus (2, 10, 15, 19, 30, 35), and mRNA mtNOS transcripts were identified in heart, liver, brain, kidney, muscle, lung, testis, and spleen (21).

Chronic hypobaric hypoxia was observed to significantly increase mtNOS activity in the rat heart (53, 57), but there was no information about the exposure of mammals to high altitudes.

The present study has been designed to determine the mtNOS activity in heart and liver after exposure of male rats to high altitude and to relate the changes with hematocrit and serum testosterone and estradiol levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Holtzman rats (body wt, 240–310 g) from the animal facility of the Universidad Peruana Cayetano Heredia were housed under standardized conditions. Rats were divided at random into two groups, one was maintained at about sea level (Lima, Peru, 150 m of altitude) and the second was transported by bus from Lima to Cerro de Pasco (4,340 m of altitude) in a trip that lasted 8 h. Rats, at sea level and at high altitude, were housed six per cage, maintained at environmental temperature (24°C at sea level and 5–10°C at high altitude) under a 12:12-h light-dark cycle, and provided with Purina laboratory chow and tap water ad libitum. At days 0, 7, 14, and 21, the animals at sea level and at high altitude were killed by decapitation and blood was collected from the cervical trunk. Heart and liver were removed immediately after death, deprived from fat and connective tissue, weighed, frozen at 77 K (liquid nitrogen) and kept at 77 K and 195 K (dry ice) until mitochondrial isolation. The experimental protocol was approved by the Ethics Committee for Human and Animal Experimentation of the Universidad Peruana Cayetano Heredia (Lima, Peru) and is in accordance to the American Physiological Society’s "Guiding principles for research involving animals" (2a).

Hematocrit. Hematocrit was determined by the micromethod using nonheparinized capillaries immediately after rat death, and the blood sample was centrifuged at 1,000 g for 5 min.

Serum testosterone and estradiol levels. Serum testosterone and estradiol levels were determined by radioimmunoassay using 125I-labeled testosterone and 125I-labeled estradiol as radioactive markers. The assays were performed by using commercial kits (Diagnostic Products, Los Angeles, CA), and the samples were tested together to minimize assay variation. The within-assay variation was 6.4% for estradiol, and 5.5% for testosterone. The assay sensitivity was 40 ng/dl for testosterone and 8 pg/ml for estradiol.

Mitochondrial isolation and preparation of submitochondrial membranes. Heart and liver were thawed and homogenized in an ice-cold homogenization medium consisting of 0.23 M manitol, 0.07 M sucrose, 1 mM EDTA, and 10 mM Tris·HCl (pH 7.4) (MSTE) with a blade homogenizer (Kendro-Sorvall-DuPont, Asheville, NC). The homogenate was centrifuged at 700 g for 10 min to discard nuclei and cell debris, and the supernatant was centrifuged at 8,000 g for 10 min. The pellet, part of the mitochondrial content of the tissue, was washed and resuspended in MSTE (8). The 8,000-g supernatant was collected as the postmitochondrial supernatant (caveolae and fragments of sarcoplasmic reticulum, mitochondria, and plasma membrane). Submitochondrial membranes were obtained by twice freezing and thawing the mitochondrial preparation and by homogenization by passage through a 25-gauge hypodermic needle. Protein concentration was assayed by the Folin reagent with bovine serum albumin as standard.

NO production. NO production was measured by following spectrophotometrically at 577–591 nm (Beckman DU 7400 diode array spectrophotometer) and the oxidation of oxyhemoglobin to methemoglobin, at 37°C (9). The reaction medium was 50 mM phosphate buffer (pH 7.4), 1 mM L-arginine, 1 mM CaCl2, 0.1 mM NADPH, 10 µM dithiothreitol, 2 µM SOD, 0.1 µM catalase, and 30 µM oxyhemoglobin heme. NO production was determined in heart and liver submitochondrial membranes and in heart postmitochondrial supernatant at 0.72–1.25 mg protein/ml.

Western blot analysis. The proteins of mitochondrial membranes and the postmitochondrial supernatant, 0.1 mg protein each, were separated by SDS-PAGE (7.5%), blotted into nitrocellulose films, and probed with 1/500 diluted rabbit polyclonal anti-iNOS, anti-eNOS, and anti-nNOS antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and with a secondary anti-rabbit antibody conjugated with horseradish peroxidase and revealed by chemiluminescence (15, 35).

Data analysis and statistics. Data are reported as means ± SE. ANOVA was used where applicable to compare between groups. Single linear and nonlinear regression analysis and multiple regression analysis were performed to determine relationships between variables. A P value of ≤0.05 was considered statistically and biologically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body and organ weights. The normal process of weight gain observed in rats at sea level was totally abrogated in the first week, in the phase of acute stress, and markedly reduced by 75 and 60% in the second phase of the two following weeks in agreement with the known anorexic effects of hypoxia (Table 1). Heart and liver weight decreased during the first week of exposure to high altitude and increased thereafter in a second phase up to 21 days (Table 2) in which the organs increased in weight (heart by 27% and liver by 46%) with linear correlations between days of exposure to high altitude and organ weight (heart weight = heart weight at day 7 x [1 + 0.019 (days of exposure – 7)], r = 0.86, P = 0.001; and liver weight = liver weight at day 7 x [1 + 0.033 (days of exposure – 7)], r = 0.82, P = 0.001).


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  		Table 1. Body weight of adult male rats exposed to high altitude (4,340 m)

 

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  		Table 2. Rat heart and liver weight during exposure to high altitude (4,340 m)

 
Hematocrit. Hematocrit was significantly higher at high altitude than at sea level (day 0) at all time points with a time-dependent rate of increase highest in the first week of exposure to high altitude (Table 3).


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  		Table 3. Hematocrit values and serum testosterone and estradiol levels and testosterone-to-estradiol ratio in adult male rats after exposure to high altitude (4,340 m)

 
Serum sex hormone levels. Serum testosterone levels were significantly increased at day 14 of exposure to high altitude, whereas values at days 7 and 21 were similar to those at sea level (day 0) (Table 3). The biphasic behavior may reflect the involved complex regulatory mechanisms. Serum estradiol levels were not significantly modified by exposure to high altitude (Table 3), with a slight (10%) decrease after 1 wk at high altitude. The testosterone-to-estradiol ratio was increased during the first 2 wk of exposure to high altitude driven by the increased testosterone values and reflecting a different regulation for the two sexual hormones during adaptation to high altitude.

Heart and liver mtNOS activity of rats exposed to high altitude. Heart mtNOS activity was markedly and significantly increased after 7, 14, and 21 days of exposure to high altitude (Table 4). At day 21, mtNOS activity was 58% higher than at sea level (day 0). Control animals (sea level) showed unchanged values of heart mtNOS activity. The NO production of heart postmitochondrial supernatant, calculated as heart cytosolic eNOS activity, was not affected by exposure to high altitude (Table 4), which confers specificity to the hypoxia-induced upregulation of heart mtNOS. Liver mtNOS activity was also not affected in the rats exposed to high altitude (Table 4), which gives organ specificity to the observed increase in heart mtNOS activity.


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  		Table 4. Effect of exposure to high altitude (4340 m) on the enzymatic activities of heart mtNOS, heart cytosolic eNOS and liver mtNOS in adult male rats

 
Western blots of mitochondria and the postmitochondrial supernatant. In the conditions of the assay, a protein of ~130 kDa of heart mitochondrial membranes, identified as mtNOS, clearly reacted with the anti-iNOS antibody (Fig. 1). It is worth noting the very weak reaction with the anti-eNOS antibody and the absence of reaction with the anti-nNOS antibody. The heart postmitochondrial supernatant showed the reactions of a 130-kDa protein with the anti-iNOS antibody and of a 135-kDa protein with the anti-eNOS antibody. No reaction with the anti-nNOS antibody was observed. The 130-kDa protein is identified with the mtNOS of the mitochondrial fragments of the postmitochondrial supernatant (18), whereas the 135-kDa protein is identified as the eNOS of the caveolae and plasma membrane (4, 6, 37). The densitometric quantitation of the Western blot spots of Fig. 1 is consistent with the biochemical data of Table 4, and both indicate a high mitochondrial participation in the total heart NOS activity.



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  		Fig. 1. Western blot analysis of the reactivity of heart mitochondria and the postmitochondrial supernatant to anti-nitric oxide synthase (NOS) antibodies. Total content of densitometric units (U) is 407.65 kU/g heart wt, corresponding 186.3 kU/heart to mitochondrial NOS (mtNOS; 3.45 kU/mg protein x 54 mg mitochondrial protein/g heart wt; 46% of total) and 221.5 kU/g heart to endothelial NOS (eNOS; 2.33 kU/mg protein x 95 mg cytosolic protein/g heart wt; 54% of total). nNOS, neuronal NOS.

 
Regression analysis. The data points corresponding to individual experimental animals were used for regression analysis. Estradiol and testosterone serum levels showed a biphasic quadratic regression with heart mtNOS activity. Serum estradiol levels from 12 to 17 pg/ml were associated with increased heart mtNOS activity, whereas estradiol levels over 18 pg/ml were associated with a reduced heart mtNOS activity (Fig. 2A). In the case of serum testosterone, levels in the range of 40 to 100 ng/dl were associated with a decreased heart mtNOS activity, and testosterone concentrations over 120 ng/dl were associated with an increased heart mtNOS activity (Fig. 2B).



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  		Fig. 2. A: quadratic relationship between serum estradiol levels and heart mtNOS activity in adult male rats (R2 = 0.26; Y = –0.021 x2 + 0.706 x – 4.997, P = 0.018). B: quadratic relationship between serum testosterone levels and heart mtNOS activity in adult male rats (R2 = 0.25, Y = 7E-05 x2 – 0.016 x + 1.580, P = 0.02).

 
Linear regression analysis showed that heart mtNOS activity was linearly related to days of exposure (r = 0.59, P = 0.001) and to hematocrit (r = 0.89, P = 0.0001). Multivariate regression analysis showed that heart mtNOS activity, chosen as the determinant variable, was associated with days of exposure to 4340 m with heart weight and with the testosterone-to-estradiol ratio (Table 5). Multivariate analysis also showed that days of exposure at 4,340 m (R2 = 0.001, P = NS), liver weight (R2 = 0.06, P = NS), hematocrit (R2 = 0.09, P = NS), serum testosterone (R2 = 0.002, P = NS), and serum estradiol (R2 = 0.01, P = NS) were not associated with either heart eNOS or liver mtNOS.


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  		Table 5. Multiple regression analysis for heart mtNOS activity and serum testosterone-to-estradiol ratio, heart, weight and days of exposure to high altitude (4,340 m) in adult male rats

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Acute exposure to high altitude causes hyperventilation, higher heart rate, and high hemoglobin and hematocrit values as the primary physiological mechanisms of acclimatization (27). There is also an accompanying endocrine response: testosterone, which is able to increase erythropoiesis (36), increases in the plasma of male rats exposed to 4,340 m altitude (26), whereas there is no information on the effects of high altitude on estradiol, which was reported to reduce erythropoiesis (41). It is unknown how the changes in hematocrit, the cardiovascular system, the NOS activities, and sexual hormone serum levels are related during exposure to high altitude.

The arrest and reduction in body weight and the hematocrit increase, which constitute the basic biological response after exposure to environmental hypoxia (5, 43, 55), were observed after 7 days of exposure to high altitude indicating an effective acclimatization of the experimental animals.

The most salient observation of the present study is the marked (58%) increase in heart mtNOS activity of rats exposed for 21 days to the high altitude of Cerro de Pasco (4,340 m, 460 Torr, 61.3 kPa). This upregulation of heart mtNOS activity has a perfect correlation with hematocrit values, which suggests a common O2-dependent regulation for erythropoietin and heart mtNOS mediated by the hypoxia-inducible transcription factor (HIF-1{alpha}) (49, 53). Although it was reported that exposure to hypobaric hypoxia increased the activity of heart mtNOS (53, 57), the evidence presented here is the first indication that mtNOS plays a role in the natural condition of exposure, acclimatization, and adaptation to high altitude. Rats submitted to chronic hypobaric hypoxia at 53.8 kPa air pressure for 2–18 mo showed an increase of 20–60% in heart mtNOS activity (53, 57). Acute hypoxia (8% O2, 30 min) was also reported as a cause for a marked (82%) increase in liver mtNOS activity (33); however, in the present study, liver mtNOS was not affected by exposure to high altitude for 21 days.

Production of NO by mtNOS appears as a regulatory process that modulates mitochondrial and heart O2 uptake (1, 3). Under physiological and pathophysiological conditions in which heart perfusion and O2 levels become limiting for ATP production and contractility, the NO-inhibited respiration lowers the steepness of the O2 gradient in the normoxic/anoxic transition, allows O2 to diffuse further along its gradient, and extends the space of adequate tissue oxygenation away from the blood vessel (45, 51). The NO-supplemented condition will be associated with more areas with high enough ATP levels to sustain an homogeneous and synchronic contraction of the myofibrils.

It has been recognized that mtNOS activity can be regulated by thyroid hormones and by angiotensin II. Downregulation of liver mtNOS activities constitutes a substantial part of thyroid hormone effects on mitochondrial O2 uptake (16), and downregulation of liver and heart mtNOS by angiotensin II has been inferred from the effects of angiotensin-converting enzyme inhibitors (7). In addition, liver mtNOS is upregulated in cold acclimatization (44), a fact that may have acted synergistically with high altitude in the rats used in this study, taking into account the relatively low temperature at Cerro de Pasco. Increased heart NO production and NO steady-state levels seem essential for the development of the cardioprotection (4, 32); increased mtNOS activity and NO levels are likely involved in the signaling and improved contractility described after exposure to environmental hypoxia (32, 57). Rat left ventricle responded to the hypoxia induced by controlled ventilation with 10% O2 with an increased expression of the mRNA of iNOS after 2 h of hypoxia (22), and increased left ventricle mtNOS activity was associated with a higher papillary muscle contractility in chronic hypoxic rats (57).

The total cardiomyocyte capacity for NO production, calculated from the NO production rates measured with the subcellular fractions fully supplemented with the NOS substrates (Table 4) increased from 57 nmol NO·min–1·g heart wt–1 (sea level) to 79 nmol NO·min–1·g heart wt–1 (4,340 m) after high altitude exposure. These total capacities may constitute an overestimation of the in vivo rates, but they point out to the adaptive mechanism. The contribution of mtNOS to the total cardiomyocyte capacity for NO production also increased after high altitude exposure, from 62% (sea level) to 71% (4,340 m).

The activity of cytosolic NOS, assayed in the postmitochondrial supernatant, consisting of the eNOS activity of the caveolae of sarcoplasmic reticulum and plasma membranes, did not change after exposure to high altitude. The specificity of the mtNOS response to environmental hypoxia becomes interesting, considering the skepticism of the consideration of mtNOS activity as a contamination of mitochondria with other cellular NOS (11, 38). The determination of mtNOS activity in a series of organs with similar specific activities, the sequencing of liver mtNOS (21), the different mtNOS activity in mitochondrial states 4 and 3 (56), and the sensitivity of the enzyme activity to environmental hypoxia certainly point out to mtNOS as a biologically important component of the inner mitochondrial membrane.

Different effects of high altitude on NOS activities, as reported here for NOS heart and liver mtNOS, have been described. Chronic exposure of pregnant sheep to an altitude of 3,820 m increased eNOS protein and mRNA levels in uterus endothelium but not in femoral and renal arteries (54). Chronic hypoxia increased pulmonary eNOS protein (33) but decreased aortic eNOS (52). The mechanisms of organ specificity of the NOS responses to high altitude are far from being understood. Vascular remodeling and hemodynamic changes in localized areas at high altitude were associated with upregulated NOS activities (54). Increased NO tissue levels in highlanders would allow them to keep an adequate tissue perfusion and an effective O2 supply despite the decreased O2 partial pressure in the inhaled air (46). High NO levels were observed in expired air during acute exposure to high altitude (14), and, interestingly, high-altitude pulmonary (14) and cerebral edema (20) were related to a reduced capacity for NO synthesis.

Cell and tissue NO homeostasis, in other words, NO steady-state concentrations in cells and tissues, are attained through a complex balance between its production and consumption rates. Under hypoxic conditions, NO production by mtNOS will be biochemically limited by cell O2 due to its high KMO2 values, in the range of 37–73 µM O2 (1). The increased hematocrit of animals exposed to high altitude may also increase NO catabolism by providing more hemoglobin perfusion to the tissues and a more efficient utilization of NO.

The results presented here show that heart mtNOS activity is linearly related to hematocrit and that has a biphasic and quadratic relationship with serum estradiol and testosterone. Estradiol and testosterone plasma levels had opposed associations with heart mtNOS activity, similarly to the opposite effects of the sexual hormones in erythropoiesis and ventilation. Testosterone increases erythropoiesis and decreases ventilation (27, 36), whereas estradiol decreases erythropoiesis and increases ventilation (40, 50).

Estradiol augments NO synthesis and attenuates the induction of erythropoietin in hypoxia (40, 41), but it does not induce eNOS expression in the kidney despite the enhanced hypoxia-induced increases in plasma nitrates (40), indicating estradiol organ-specific effects on NO metabolism. The present study shows that serum estradiol levels are not associated with heart mtNOS activity or hematocrit after exposure to high altitude. Concerning testosterone and the testosterone-to-estradiol ratio, statistic multivariable analysis showed them associated with exposure to high altitude, hematocrit, and heart mtNOS activity. The observed time-dependence profiles of the serum levels of the sex hormones and of the testosterone-to-estradiol ratio after high altitude exposure are different from the profiles of hematocrit and heart mtNOS activity in the same process and indicate that they are subjected to different regulatory mechanisms.

In summary, the present study shows that heart mtNOS is upregulated after exposure to high altitude for periods of 7 to 21 days. The effect is specific because heart eNOS and liver mtNOS were not affected in the same conditions. Simultaneously, lower body weight increase, higher hematocrit, and higher serum testosterone-to-estradiol ratios were observed as part of the complex physiological acclimatization to high altitude.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Vicerectorate of Investigation of the Universidad Peruana Cayetano Heredia (Perú), and by Grants PICT 01-08710 from Agencia Nacional de Promoción Científica y Tecnológica, PIP-02272 from Consejo Nacional de Investigaciones Científicas y Técnicas, and 14156-18 from Fundación Antorchas (Argentina).


   ACKNOWLEDGMENTS
 
The authors thank Sharon Castillo for technical assistance and Dr. Carmen Goñez for help with the radioimmunoassay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Boveris, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina (E-mail: aboveris{at}ffyb.uba.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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