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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 9 3056-3061
Copyright © 1998 by The Endocrine Society

Original Studies

Decreased Nitric Oxide Levels and Bone Turnover in Amenorrheic Athletes with Spinal Osteopenia1

E. Stacey, P. Korkia, M. V. J. Hukkanen, J. M. Polak and O. M. Rutherford

Department of Physiology (E.S., P.K., O.M.R.), Imperial College School of Medicine, St. Mary’s Campus, London W2 1PG, United Kingdom; and Department of Histochemistry (M.V.J.H., J.M.P.), Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Dr. Olga M. Rutherford, Department of Physiology, Imperial College School of Medicine, Norfolk Place, London W2 1PG, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Amenorrheic athletes have been likened to postmenopausal women, with low estrogen levels and osteopenia. It has been suggested that estrogen exerts its antiresorptive actions on bone via a nitric oxide (NO)-dependent mechanism. This study investigated whether the mechanism of bone loss in amenorrheic athletes is similar to that of postmenopausal women with reduced NO levels and high bone turnover.

Eleven amenorrheic athletes, 15 eumenorrheic athletes, and 10 sedentary controls were studied. Spine and hip bone mineral density was measured using dual-energy x-ray absorptiometry. Bone turnover was assessed by biochemical markers of formation (osteocalcin and bone-specific alkaline phosphatase) and resorption (deoxypyridinoline). NO metabolites were measured from 24-h urine samples using a chemiluminescence assay.

Spine, but not hip, bone mineral density was reduced in the amenorrheic group, compared with the eumenorrheic (P = 0.0001) and control (P = 0.04) groups. Osteocalcin, bone-specific alkaline phosphatase, and deoxypyridinoline were similar in all groups. NO metabolites were lower in the amenorrheic group, compared with controls (P = 0.035), despite a higher dietary intake of nitrates.

Unlike postmenopausal women, amenorrheic athletes do not have raised bone turnover but do have reduced NO metabolites and spinal osteopenia. The results show, however, that reduced NO production is a common denominator in both conditions and further support the importance of NO in estrogen-mediated protection of skeletal mass and strength.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WEIGHT-BEARING exercise is generally beneficial for bone strength. Intense exercise, however, can lead to menstrual disturbances in female athletes, and this can be accompanied by bone loss, particularly from the lumbar spine (1, 2), although some studies have also demonstrated bone loss at appendicular sites (3). It is estimated that up to 50% of female athletes experience either amenorrhea or oligomenorrhea, compared with about 5% in the nonathletic population (4). The etiology of these menstrual disturbances is not understood and is probably multifactorial, including low body weight and body fat, endocrine changes associated with chronic exercise such as raised cortisol and endorphins, eating disorders, and high training volume and intensity. The menstrual disturbances cause changes in the normal patterns of sex steroid production, with a lowering of estradiol and/or a shortening of the luteal phase (5, 6, 7). It has been assumed that one of the reasons that athletic amenorrhea can lead to bone loss is that the estrogen-deplete younger athlete has a bone turnover pattern similar to a postmenopausal woman. In the first 5–10 yr of the menopause, there is an increase in bone turnover, with resorption exceeding formation, resulting in a net loss of bone.

Biochemical markers of bone turnover are being used increasingly as indicators of bone remodeling status. These markers have been extensively studied in bone disease states, in osteoporosis, and in some longitudinal studies of exercise intervention. In the first decade after the onset of the menopause, there is an increase in levels of osteocalcin, bone-specific alkaline phosphatase (bALP), and pyridinium cross-links, reflecting the increase in bone turnover (8). These levels can be reduced after treatment with hormone replacement therapy (HRT) (9).

Some studies have found higher levels of bone turnover in subjects who are more recreationally active (10, 11, 12). Few studies have specifically looked at bone turnover in amenorrheic athletes. Hetland et al. (13) assessed bone formation by osteocalcin and alkaline phosphatase, and bone resorption by urinary calcium and hydroxyproline, in female runners. They found no difference in bone turnover between amenorrheic and eumenorrheic runners, despite a lower spine bone mineral density (BMD) in the amenorrheic group. In their study, however, no comparisons with sedentary controls were made.

Recently, it has been suggested that the physiological effects of estrogen and mechanical stress on both cardiovascular function and bone turnover are exerted, at least in part, via elevation of nitric oxide (NO) synthesis. In young fertile women with normal menstrual cycles, the plasma nitrite and nitrate, the stable oxidation end products of NO, are at their highest at the midcycle, following closely those of estrogen levels (14). Postmenopausal women have reduced serum NO metabolite levels, and both short- and long-term HRTs elevate these levels in postmenopausal women (15, 16). There is clear evidence that estrogen can induce endothelial NO-synthase (eNOS) messenger RNA, protein, and enzymatic activity in a variety of cells, including those of bone (17, 18).

Recent evidence has established that NO generated from the eNOS isoform is one of the key mediators of mechanical stress-induced increase in bone formation (19, 20), providing for potential involvement in bone resorption associated with osteoporotic disease (21, 22). It is thought that low-level NO production by the eNOS isoform is an absolute requirement for bone cell function (21, 22, 23, 24). Support for the importance of NO on skeletal homeostasis derives from the study by Wimalawansa et al. (25) in rats, who showed that administration of nitroglycerin, an NO donor, in estrogen depletion-induced osteoporosis will suppress osteoporotic bone loss and, conversely, that inhibition of endogenous NO production will suppress the bone-conserving action of estradiol (25).

These observations would offer an attractive explanation of why estrogen restores bone mass, given that NO is involved in the cascade of hormone receptor signaling mechanisms in bone cells. In the present study, we investigated bone turnover and BMD in amenorrheic and eumenorrheic athletes and sedentary controls and hypothesized that amenorrheic athletes with reduced BMD also have decreased NO metabolite levels, compared with the eumenorrheic and sedentary control groups.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

A total of 36 subjects were recruited, of which 11 were amenorrheic athletes, 15 were eumenorrheic athletes, and 10 were sedentary controls. Details of the 3 groups are given in Table 1Go. The athletes were either club runners or triathletes, and amenorrhea was defined as no menstruation in the previous 6 months. The eumenorrheic athletes and sedentary controls all had regular menstrual cycles (10–13 cycles per year) between 25 and 35 days in length, and none of the subjects had any condition known to affect bone metabolism. All subjects were healthy at the time of study. Subjects gave written, informed consent, and the study was approved by the local ethical committee.


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  		Table 1. Details of the groups (mean ± SE, unless otherwise stated)

 
Questionnaires

All subjects completed a detailed questionnaire on past medical and menstrual history, smoking and alcohol consumption, calcium intake, and physical activity levels. The calcium intake section has previously been validated against weighed intake (26). Dietary intake of nitrate was estimated from a food-frequency questionnaire (27). The questionnaire included dietary components rich in nitrates, as well as those eaten in quantity. Full details of training and competition schedules were obtained.

Anthropometry

Height and weight were measured with subjects in minimal clothing and bare feet. Skin-fold thicknesses were measured at 4 sites (suprailiac, subscapular, biceps, and triceps), and the percentage fat was calculated from the equation of Durnin and Womersley (28).

Bone density

BMD of the spine and hip was measured using dual-energy x-ray absorptiometry (Lunar DPX-L). The spine scan was analyzed to give BMD for lumbar 1–4 levels; and the hip scan was analyzed for greater trochanter, neck of femur, and Ward’s triangle. The coefficient of variation of repeat spine scans is less than 1%; and for all sites on the hip, it is less than 1.5% (29).

Bone turnover

Deoxypyridinoline (Dpd) was measured from 24-h urine samples from which a 50-ml aliquot was taken and frozen at -20 C until analyzed. Osteocalcin and bALP were measured from a blood sample taken the day of completion of the urine collection. Blood samples were collected in the afternoon, and assays were carried out in triplicate and in two batches. Samples were collected in either a heparanized tube with 100 µl Tresylol (for osteocalcin) or a plain tube (bALP). The tubes were immediately centrifuged at 1,500 rpm for 10 min, and aliquots of serum or plasma were frozen at -20 C until analyzed. All menstruating subjects had the samples collected within the first 4 days of the cycle, i.e. early follicular phase. Subjects were asked to refrain from training during the collection phase.

All bone marker assays were carried out using immunoassay kits from Metra Biosystems Inc. (CA). These were Pyrilinks-D, NovoCalcin, and Alalphase-B for Dpd, osteocalcin, and alkaline phosphatase respectively. The assays used a microtitre stripwell format using monoclonal antibodies and a b-nitrophenyl phosphate substrate. Dpd values were standardized for urinary creatinine.

Nitric oxide (NO)

NO was measured in 24-h urine samples by the chemiluminescence assay. Triplicate 50-µL samples were injected into a purge vessel, and urinary nitrate and nitrite were converted to NO in a reducing mixture of 0.1 mol/L vanadium(III) chloride in 1.0 mol/L HCl at 96.5 C. NO was detected by its chemiluminescence reaction with ozone, generated from pure oxygen using a Sievers NOA 270B NO analyzer (Sievers Instruments, Boulder, CO). Comparisons were made against standard curves generated by known concentrations of sodium nitrate.

Statistics

Data is expressed as mean ± SE, unless otherwise stated. Between-group comparisons were carried out using ANOVA with post hoc analysis by Student’s t test. Relationships between parameters were investigated using Spearman’s rank correlation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subject details

The amenorrheic athletes were significantly lighter than the other two groups (P = 0.0025) and had the lowest body fat percentage (P = 0.001; Table 1Go). There was no difference in the age of menarche or calcium intake among the groups. The average length of amenorrhea was 42.2 months (range, 6–120).

Bone density

Table 2Go shows the group mean data for BMD at each site. BMD of the spine, neck of femur, and greater trochanter has also been expressed as a percentage of age- and weight-matched controls taken from the large Lunar reference database. Spine BMD was significantly lower in the amenorrheic athletes, compared with both eumenorrheic athletes (P = 0.0001) and sedentary controls (P = 0.04) and were also significantly lower than predicted from the database (P = 0.01; Fig. 1Go). There was no relationship between the length of amenorrhea and BMD of the spine.


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  		Table 2. BMD, expressed as an absolute value and percentage of age-matched controls

 


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  		Figure 1. Percentage difference between group mean BMD of the lumbar spine and femoral neck and predicted age-matched controls from the Lunar database (**, P < 0.01).

 
There were no significant differences between the groups at any site on the hip, but when compared with the database, the eumenorrheic athletes had a significantly higher BMD at the greater trochanter and neck of femur (P = 0.005, Fig. 1Go).

Bone turnover

There was no significant difference between levels of osteocalcin, bALP, or Dpd among the three groups. Individual values are shown in Fig. 2Go. Most of the values in each group fell within the normal ranges for premenopausal women, as supplied by the manufacturers of the assay kits. One amenorrheic subject had an extremely high bALP, but this was not associated with a high osteocalcin or Dpd, although this subject did have one of the lowest body fat levels (8.5%) and spine BMD (75% of age-matched).



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  		Figure 2. Individual values for biochemical markers of bone turnover. SED, sedentary controls.

 
There was no significant correlation between the markers and BMD at any site. The only significant correlation between markers was between Dpd and bALP (r2 = 0.27, P = 0.0012).

Nitric oxide metabolites and nitrate intake

Group mean NO levels and nitrate intakes are shown in Table 3Go. The amenorrheic athletes had the lowest urinary NO, and the sedentary controls had the highest, the difference between these two groups being significant (P = 0.035; Fig. 3Go). Because urinary levels of NO could be affected by dietary nitrate intake, we assessed intake, to investigate any group differences. Nitrate intake was highest in the amenorrheic athletes and lowest in the sedentary controls, the difference again being significant (P = 0.0329; Fig. 3Go). There was a significant negative correlation between nitrate intake and urinary NO levels (r2 = -0.62; P = 0.042).


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  		Table 3. NO metabolite excretion and dietary nitrate intake in the three groups

 


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  		Figure 3. Group mean levels of urinary NO metabolites and nitrate intake (*, Significant difference between amenorrheic athletes and sedentary controls; P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has shown that amenorrheic athletes are at risk of bone loss from the lumbar spine, in agreement with previous studies on these types of athletes (1, 2, 7, 30). Unlike the spine, hip BMD was relatively well preserved in the amenorrheic athletes. Previous studies have also found the spine to be the site most prone to osteopenia in this group. Drinkwater et al. (31) found no differences between BMD of eumenorrheic and amenorrheic athletes at appendicular sites such as the radius, neck of femur, tibia, and fibula, although the amenorrheic group did have a significantly lower density in the upper shaft of femur. Rutherford (2) found no difference in total body BMD between groups or in the individual regions of the scan (arms, legs, and pelvis); however, Myburgh et al. (3) did find bone loss from appendicular weight-bearing sites. Athletes who begin intensive exercise before puberty often experience a delay in menarche. Low bone density, later in life, could be caused by a reduction in bone accretion during these early years, rather than by bone loss later. The average age of menarche was not different among the three groups in this study, and all but one of the amenorrheic athletes had developed amenorrhea at least 4 yr after menarche, with the majority being in their mid-twenties. Bone accretion and consolidation continues until about the age of 30; and so, lack of accretion, rather than bone loss, could explain some of the lower BMD values in the amenorrheic group.

Load-bearing exercise is known to be beneficial for bone density (32), and this is illustrated by the eumenorrheic athletes, who had the highest bone densities at all sites measured. When compared with the Lunar database, this group had significantly higher BMD at the greater trochanter and neck of femur. Despite their equally high level of activity, these sites were not increased in the amenorrheic group but were similar to the controls. That BMD was not low at the hip in the amenorrheic group suggests that the pattern of loading imparted by the athletic activities may be having a protective effect on the hip but not the spine. Some forms of exercise are able to give a protective effect to the spine in amenorrheic athletes; for example, amenorrheic rowers have higher spine bone densities than amenorrheic runners (33). The skeletal effects of athletic amenorrhea therefore depend, to a certain extent, on the types of activity performed.

Despite the spinal osteopenia in the amenorrheic group, there was no difference in markers of bone formation or resorption between the groups. We originally hypothesized that the amenorrheic athletes might have an increased bone turnover, particularly resorption, similar to that seen in postmenopausal women. Inspection of Fig. 2Go clearly shows that resorption, as indicated by Dpd excretion, is not increased in the amenorrheic athletes, compared with the other two groups. There was no relationship between the length of amenorrhea and any of the markers, although the numbers may have been too small to detect such a relationship. Therefore, despite apparently normal bone formation and resorption, bone loss from the spine is occurring in amenorrheic athletes. This agrees with the findings of Hetland et al. (13), who also found normal formation and resorption in amenorrheic athletes. Because the amenorrhea seems to particularly affect the spine, the bone turnover status of the rest of the skeleton may mask any localized imbalance. Systemic markers of bone turnover should reflect total body BMD, which we have previously shown to be normal in the amenorrheic athletes (2).

The common factor between postmenopausal women and amenorrheic athletes is a lowering of estrogen levels. The mechanism of this reduction is, however, different. In amenorrheic athletes, the low estrogen levels are secondary to a reduction in pituitary LH production, because of an inhibition of GnRH release from the hypothalamus (34). In postmenopausal women, the decline in estrogen is caused by a depletion of ovarian follicles, and the lack of negative feedback of estrogen on the hypothalamic-pituitary axis results in raised LH production. The mechanism behind a lowering of GnRH in amenorrheic athletes is still unknown. There is some evidence for a raised cortisol production in this group (34, 35), and this may play a role in inhibiting hypothalamic GnRH release. Raised glucocorticoid levels are also associated with bone loss. The mechanism differs from that seen in postmenopausal women, with a depression of bone formation and an unaltered bone resorption (36, 37). Other endocrine changes have also been implicated in athletic amenorrhea, such as a raised endorphin release and altered thyroid status (38, 39). These additional hormonal changes may also be influencing bone turnover in a manner different from that of low estrogen.

This finding of no increase in bone turnover is important when considering treatment options for amenorrheic athletes with a low bone density. HRT is sometimes used when natural menses cannot be regained (40). HRT acts mainly by inhibiting bone resorption (41). Although successful in retarding bone loss in postmenopausal women, where resorption is increased, its effects in the amenorrheic athlete may be limited. If resorption is normal, and depressed by HRT, this may have the overall effect of depressing bone turnover and reducing the ability of the bone to repair microfractures. The incidence of stress fractures is much greater in amenorrheic (compared with eumenorrheic) athletes (42), and these often take a long time to heal. If bone remodeling is suppressed, then it is possible that this problem could be further exacerbated.

Despite the lack of similarity between bone turnover in amenorrheic athletes and postmenopausal women, concentrations of NO metabolites were affected as predicted, with the lowest being in the amenorrheic group. A confounding factor when measuring NO metabolites is the dietary intake of nitrates. These were assessed by questionnaire and found to be the reverse of the NO metabolite levels, i.e. amenorrheic athletes had the highest dietary intake of nitrates. It is therefore extremely unlikely that the low levels of nitrite/nitrate found in the amenorrheic group were caused by a low dietary intake. Exercise has been shown to increase NO synthesis (43, 44), and this is thought to play a role in arteriolar vasodilatation during exercise and the cardioprotective effect of chronic exercise. Levels of NO metabolites might then be expected to be high in athletes (43); this was not the case for the eumenorrheic group, who had a urinary NO metabolite level similar to that of the sedentary controls in the present study. The amenorrheic athletes, although also extremely active, had the lowest urinary NO levels. The low levels of NO metabolites in the amenorrheic athletes are most likely caused by chronic low estrogen levels. NO is generated in many estrogen-sensitive tissues, and the urinary levels will reflect the contribution from many of these sites. It is unlikely, however, that NO synthesis in bone would be unaffected by low estrogen if production in other tissues has been reduced. At a cellular level in bone, eNOS-derived NO is thought to mediate mechanical stress stimuli by osteocytes (19, 20), activate osteoblast function (45), and suppress osteoclast resorption activity (23, 24).

In conclusion, the results show that amenorrheic athletes do not resemble postmenopausal women in terms of bone turnover but do have reduced urinary concentrations of NO metabolites and spinal osteopenia. The question, therefore, arises as to the mechanism behind bone loss in these young women. Longitudinal studies of athletes who experience amenorrhea for part of the year, because of variations in training intensity, would enable more detailed study within an individual of the pattern of bone turnover and NO synthesis status with changing endocrine status. Subtle changes in bone formation and resorption, which may be obscured by group comparisons, might then become apparent. Further attention needs to be paid to treatment options for these women. Restoration of NO synthesis by dietary arginine supplementation could be one approach (46).


   Footnotes
 
1 This work was supported by grants from the National Osteoporosis Society, Dunhill Medical Trust, Medical Research Council, PPPhealthcare Medical Trust, and the Wellcome Trust. Back

Received November 7, 1997.

Revised March 13, 1998.

Accepted April 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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