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Clinical Chemistry 47: 1688-1695, 2001;
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(Clinical Chemistry. 2001;47:1688-1695.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Nitrogen Metabolism and Bone Metabolism Markers in Healthy Adults during 16 Weeks of Bed Rest

Kerstin Scheld1, Armin Zittermann1a, Martina Heer2, Birgit Herzog1, Claudia Mika2, Christian Drummer2 and Peter Stehle1

1 Department of Nutrition Science, University of Bonn, 53115 Bonn, Germany

2 DLR-Institute for Aerospace Medicine, 51170 Cologne, Germany

aAddress correspondence to this author at: Department of Nutrition Science, University of Bonn, Endenicher Allee 11-13, 53115 Bonn, Germany. Fax 49-228-733217; e-mail a.zittermann{at}uni-bonn.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The associations between nitrogen metabolism and bone turnover during bed rest are still not completely understood.

Methods: We measured nitrogen balance (nitrogen intake minus urinary nitrogen excretion) and biochemical metabolic markers of calcium and bone turnover in six males before head-down tilt bed rest (baseline), during 2, 10, and 14 weeks of immobilization, and after reambulation.

Results: The changes in nitrogen balance were highest between baseline and week 2 (net change, -5.05 ± 1.30 g/day; 3.6 ± 0.6 g/day at baseline vs -1.45 ± 1.3 g/day at week 2; P<0.05). In parallel, serum intact osteocalcin (a marker of bone formation) was already reduced and renal calcium and phosphorus excretions were increased at week 2 (P <0.05). Fasting serum calcium and phosphorus values and renal excretion of N-telopeptide (a bone resorption marker) were enhanced at weeks 10 and 14 (P <0.05–0.001), whereas serum concentrations of parathyroid hormone, calcitriol, and type I collagen propeptide (a marker of bone collagen formation) were decreased at week 14 (P <0.05–0.01). Significant associations were present between changes of serum intact osteocalcin and 24-h calcium excretion (P <0.001), nitrogen balance and 24-h phosphorus excretion (P <0.001), nitrogen balance and renal N-telopeptide excretion (P <0.05), and between serum osteocalcin and nitrogen balance (P <0.025).

Conclusions: Bone formation decreases rapidly during immobilization in parallel with a higher renal excretion of intestinally absorbed calcium. These changes appear in association with the onset of a negative nitrogen balance, but decreased bone collagen synthesis and enhanced collagen breakdown occur after a time lag of several weeks.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several human studies have provided evidence for a close association between muscle mass and bone mass (1)(2)(3). According to the Utah paradigm of skeletal biology, muscle mass is the primarily mechanical force responsible for adequate formation of bone mass (4). Consequently, the observed loss of bone mass during immobilization (5) might be secondary to a loss of muscle mass and muscle strength (6). In a recent investigation, however, a significant loss of leg lean-body mass during 6 weeks of immobilization was not related to a change in leg-bone mineral density (7). Thus, the association between muscle and bone metabolism during bed rest is not completely understood.

Reliable studies focusing on the relationship between nitrogen and bone metabolism during immobilization require long-term observations using highly sensitive markers. Different bone metabolism markers are now available to sensitively assess osteoblastic and osteoclastic activity: serum concentrations of osteocalcin, bone-specific alkaline phosphatase (BAP), 1 and carboxy-terminal procollagen type I (PICP) are indicators of bone-formation processes (8), whereas N-telopeptide (NTx) is a sensitive marker of bone resorption (9). Measurement of these bone metabolism markers in biologic fluids can provide valuable information about actual changes in bone turnover. Muscle protein and nitrogen metabolism can be assessed by measuring daily nitrogen balance and by analysis of renal 3-methylhistidine excretion (5)(10).

This study was thus aimed at elucidating the interactions between changes in biochemical indices of nitrogen metabolism and in biomarkers of osteoblastic and osteoclastic activity during long-term bed rest.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
Six Caucasian men, 25–42 years of age (mean, 30.3 ± 3.2 years), 1.75–1.90 m in height (mean, 1.81 ± 0.1 m) and 62–114 kg in weight (mean, 79.3 ± 7.6 kg), gave their voluntary consent to participate in the study. Before enrollment, a thorough medical history was taken (questionnaire), and routine medical and laboratory analyses (e.g., electrocardiogram; x-ray of the thorax; blood pressure; gastroscopy; psychological examinations; hematologic indices; serum concentrations of creatinine, alanine aminotransferase, aspartate aminotransferase, and {gamma}-glutamyltransferase; urinary analyses of protein, glucose, bilirubin, leukocytes, and erythrocytes) were used to exclude chronic diseases. None of the participants was on medications known to influence calcium metabolism. Five of the six participants were smokers (>5 cigarettes/day). Physical activity (retrospective questionnaire) was high in two volunteers (>8 h/week) and moderate in three study participants (2–4 h/week). One participant had a sedentary life-style (0 h of physical activity/week). The study participants were fully informed of the purpose of the study and potential risks. All study procedures were in accordance with the Helsinki Declaration.

study protocol
Participants were admitted to a Metabolic Ward Unit at the Institute of Biomedical Problems, Moscow, Russia. The experiment was part of a comprehensive, international collaborative study, where specific examination periods were assigned to each study group. The study protocol was divided into three phases. In the ambulatory control period, the participants already lived in the research unit and baseline measurements were performed. The volunteers were then placed on head-down tilt [(HDT); -6°] bed rest for a period of 16 weeks from February to May (Fig. 1 ).



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  		Figure 1. Study design.

BI, 6 weeks before immobilization; RA, 3.5 weeks after reambulation.

During the bed rest, all activities, including showering, were performed either in HDT or horizontal position. The participants were allowed to raise themselves on one elbow for eating. After the bed rest period, the subjects remained in the metabolic unit for additional 4 weeks for post-bed-rest testing.

During the five examination periods on days 1–7 [before immobilization (BI)], days 56–62 [immobilization week 2 (IW2)], days 111–117 (IW10), days 144–153 (IW14), and days 186–191 (after reambulation), participants received a specifically prepared diet providing 10 500–11 400 kJ/day and 1000 mg (25 mmol) calcium/day. Calcium-rich foods were bought in Germany and analyzed by a German food chemistry laboratory (Hermann-Kutscher-Kollach, Cologne, Germany). Foods were then transported to Moscow. All meals were prepared in the kitchen of the Metabolic Ward Unit in Moscow by staff members of the German investigator group. Results of calcium analysis in foods were used to calculate the dietary regimen of the participants. During all examination periods, a comparable daily menu was given. Energy requirements were calculated for each individual. To maintain body weight, the energy content of the diets was reduced during the immobilization periods. All foods were exactly weighted for each participant, and participants were asked to consume the complete meal. Intake of mineral water low in calcium (<10 mg/L) was allowed ad libitum. Meals were served at 0800 (breakfast), at 1330 (lunch), at 1530 (snack), at 1900 (dinner), and at 2100 (snack). Fasting blood samples were collected from the antecubital vein (serum monovettes) on day 5 (BI), day 60 (IW2), day 113 (IW10), day 148 (IW14), and day 190 (reambulation). After blood sampling, body weight was measured. Urine samples were collected quantitatively during the entire five examination periods on a void-by-void basis. Aliquots of all samples were frozen consecutively and stored at -80 °C until analysis. Body composition was assessed by bioimpedance analysis on days 7 and 151 (during BI and IW14). Throughout the remaining time of their stay in the unit, participants were on a typical Russian diet (Fig. 1Up ).

analyses
Biochemical analyses of serum hormone concentrations and of bone metabolism markers are listed in Table 1 . The CVs are in the range of published data (11)(12). Blood and urine calcium and phosphorus concentrations were measured on a Hitachi automated analyzer by routine methods. CVs were below 3.8%. Plasma amino acids were quantified by reversed-phase HPLC after precolumn derivatization with o-phthaldialdehyde-3-mercaptopropionic acid (13)(14) and fluorescence detection. The urinary 3-methylhistidine concentrations were determined by reversed-phase HPLC after precolumn derivatization with dansyl chloride (15) and fluorescence detection. CVs of the amino acid analysis were 2–5%. Total urinary nitrogen was determined in 24-h urine pools by highly sensitive chemiluminescence (16) with an Antek automated nitrogen analyzer (Antek 7000V). The CV of this method was 2.8% within a series of analyses performed on 1 day using freshly prepared calibrators. Serum strontium was measured by means of graphite furnace atomic absorption spectrometry (HGA-600; Perkin Elmer). The within- and intra-day CVs were 4.8% and 3.9%, respectively. Bioimpedance analysis was performed using a single frequency 50-kHz, 800-µA device (BIAMED). A tetrapolar electrode placement was used, with electrodes placed on the dorsal surfaces of the right hand and foot. The other electrodes were placed at the distal metacarpals and metatarsals respectively, between the distal prominences of the radius and the ulna at the wrist, and the medial and lateral malleoli at the ankle. The CV of the method was 1.3%.


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  		Table 1. Biochemical analyses of serum hormone concentrations and of bone metabolism markers.

calculations
Total body fat was calculated by bioimpedance analysis according to the formula of Hodgon and Fitzgerald (17). Muscle mass was determined on the basis of the bioimpedance analysis measurements by computer software developed by the manufacturer. The validity of this measurement has recently been verified by the 24-h renal creatinine excretion method (18).

Nitrogen balance was estimated by nitrogen intake minus urinary nitrogen and did not include fecal or integumental nitrogen losses. Urinary nitrogen was directly measured on days 3–5 of the investigation periods; in parallel, nitrogen intake was calculated from food logs (protein intake/6.25). Renal calcium, phosphorus, and NTx excretions were expressed in mmol/day, as the mean of the five examination periods. Fractional calcium absorption was assessed by the use of a stable strontium test as described previously (19). Calculation of absorption rates was based on net serum strontium concentrations (change in strontium concentration, t240 - t0) and on distribution volume. Because of the findings of Finlay et al. (20) and Milsom et al. (21), it was assumed that the extracellular distribution volume of strontium was 15% of body weight.

statistics
Statistical analysis was performed with the Statistical Package for the Social Science (SPSS Inc). The data were tested by use of univariate repeated-measure ANOVA, mean contrasts, and a Greenhouse-Geiser adjustment factor with significance set at P <0.05. The Student t-test for paired samples was used to evaluate the statistical significance of the differences. To assess interrelationships between variables, Pearson’s bivariate correlation coefficient and the partial correlation coefficient were used. All statistical tests were two sided. P values <0.05 were considered significant, and P values between 0.05 and 0.10 were considered borderline significant. Data are presented as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
energy and nutrient intake, body weight, and body composition
Energy, protein, calcium, and phosphorus intake did not differ during the examination periods (Tables 2 and 4 ). Total body mass remained unchanged. Immobilization, however, led to a decrease in muscle mass (-3.5 kg at IW14) compared with the pre-bed-rest period. In parallel to the changes in muscle mass, a concomitant increase in fat mass was observed (3.3 kg at IW14; Fig. 2 ).


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  		Table 2. Energy and protein intake, nitrogen balance, and body weight of six healthy males during a pre-bed-rest period, during immobilization, and after reambulation.1


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  		Table 4. Biochemical markers of calcium, phosphorus, and bone metabolism in six healthy male subjects during 16 weeks of bed rest and during reambulation.1



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  		Figure 2. Muscle mass and fat mass of six healthy males before immobilization (BI) and after 14 weeks of immobilization (IW14).

**, P <0.01; *, P <0.05; mean ± SE.

nitrogen metabolism
Nitrogen balance was lower at all occasions during bed rest compared with pre-bed-rest and reambuluation periods (Table 2Up ). Total plasma amino acids were enhanced at IW10 and IW14 and after reambulation compared with BI. The plasma concentrations of branched-chain amino acids (BCAAs) were increased at IW2 (Table 3 ). Renal 3-methylhistidine excretion and circulating testosterone concentrations did not differ from baseline values during immobilization (Table 3 ). However, a significant increase in serum testosterone concentrations was observed after reambulation compared with IW14 (Table 3 ).


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  		Table 3. Effects of immobilization and reambulation on serum concentrations of testosterone and free amino acids, as well as on urinary 3-methylhistidine excretion in six healthy male adults.1

calcium, phosphorus, and bone metabolism
Hypercalciuria (renal calcium excretion >7.5 mmol/day) was present at all occasions of immobilization, the extent of which being dependent on time of bed rest (Table 4Up ). Significant increases in renal phosphorus excretion were observed at IW2, the values being ~110% above the pre-bed-rest concentrations. Enhanced serum calcium and phosphorus values were observed at IW10 and IW14. Intact parathyroid hormone (PTH) and calcitriol were diminished 42% and 22%, respectively, at the end of immobilization. Fractional calcium/strontium absorption was reduced 18% at IW10 in comparison with BI. Serum 25-hydroxyvitamin D (25-OHD) concentrations were kept constant at the lower limit of the reference interval (reference interval, 25–150 nmol/L). Serum intact PTH, calcitriol, 25-OHD, and fractional calcium/strontium absorption markedly increased after remobilization to concentrations above pre-bed-rest values. Intact osteocalcin was already reduced at IW2 and remained low during the entire period of immobilization. PICP was decreased at IW14, whereas BAP did not change during immobilization, but increased after remobilization. NTx was enhanced at IW10 and IW14 and returned to pre-bed-rest values after reambulation.

relationship between variables
In Table 5 , correlation coefficients among the biochemical markers assessed are summarized; significant associations were observed between renal calcium and phosphorus excretion, between intact osteocalcin and 24-h urinary calcium excretion, between nitrogen balance and serum concentrations of BCCAs, between nitrogen balance and 24-h urinary phosphorus excretion, and between nitrogen balance and renal NTx excretion. A borderline significance (P = 0.060) was present between nitrogen balance and 24-h calcium excretion. Moreover, serum intact osteocalcin was associated with nitrogen balance (r = 0.480; P <0.025) after we adjusted for renal phosphorus and calcium excretion.


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  		Table 5. Correlation coefficients between time-dependent changes ({Delta} values) in variables of nitrogen metabolism, renal mineral excretion, and bone metabolism markers in males who underwent 16 weeks of bed rest.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes the time-dependent changes in nitrogen, calcium, and bone metabolism during long-term immobilization.

Within the first 2 weeks of HDT bed rest, a rapid loss of body nitrogen followed the reduced mechanical stress of the musculoskeletal system (Table 3Up ). Although fluid shifts might have influenced bioimpedance measurements under HDT conditions, data indicate that changes in nitrogen balance are accompanied by a loss of muscle mass (Fig. 2Up ). Our results are in agreement with a previous study that used 17 weeks of horizontal bed rest, which indicated a decrease in lean body (muscle) mass of 3.9 kg (SD, 2.1 kg) (5). Our data suggest a more pronounced loss of nitrogen in the first weeks of bed rest compared with IW10 and IW14 (Table 3Up ), thereby confirming previous results of a rapid change in muscle mass and muscle strength within 4–6 weeks of bed rest (22). The unchanged renal excretion of 3-methylhistidine during bed rest (Table 2Up ) can be interpreted that HDT immobilization does not induce a higher muscle protein breakdown. Consequently, the loss of muscle mass must be dependent on decreased endogenous synthesis. This interpretation is verified by studies with 15N-labeled amino acids in study participants who had 14 days of HDT bed (23). Furthermore, the increase in plasma concentrations of BCAAs at IW2 (Table 3Up ) support our hypothesis of a decreased muscle protein synthesis. The skeletal muscle contains 70% of the BCAAs (24), and although plasma concentrations of amino acids are not an indicator for the intracellular concentration, it can be assumed that diminished utilization of and/or release from skeletal muscle during immobilization might have influenced plasma BCAA concentrations. It can be ruled out that the loss of muscle mass and body nitrogen is the result of inadequate energy or protein intake. Both caloric and protein intake were above actual recommendations (25). Moreover, the maintenance of body mass (Table 2Up ) supports the assumption that caloric intake was adequate during bed rest.

For the first time, an early decrease in serum intact osteocalcin in association with a markedly decreased nitrogen balance could already be observed after 2 weeks of bed rest (Tables 4Up and 5Up ), indicating a reduction of bone formation processes. Others have found no change or even an increase in serum osteocalcin concentrations during immobilization (7)(26)(27). Possible explanations for these inconsistent results are different timing of sample collection (28), the use of different assays (29), and the lack of sample batching. Our data of an early decrease in serum intact osteocalcin may be indicators of suppressed bone mineralization. In vitro studies indicate increased osteocalcin expression of osteoblast synthesis during the phase of bone matrix mineralization (30). Human iliac crest histomorphometric studies have demonstrated a significant reduction in the mineral apposition rate within 1 week of HDT bed rest (31). Moreover, animal studies confirm that matrix mineralization is already disturbed after 1 week of mechanical unloading (32), most likely because of reduced gene expression for osteocalcin synthesis (33). A substantial reduction of serum intact osteocalcin concentrations has also been observed in patients suffering from acute anorexia nervosa (34), a situation that is known to lead to reduced fat-free mass and muscle mass (35). Muscle loss and a disturbed synthesis of intact osteocalcin may both be caused by a decreased activity of specific cytokines such as insulin-like growth factor-I (IGF-I) (36)(37). Immobilization leads to target cell resistance to IGF-I (38), whereas anorexia nervosa is associated with reduced circulating IGF-I concentrations (39).

The increase in renal calcium excretion had already occurred during early immobilization, when both the fractional calcium absorption rate and bone resorption processes were still unaffected by immobilization (Table 4Up ). Consequently, the increased renal calcium excretion must be the result of decreased retention of intestinally absorbed calcium. The early increase in renal calcium excretion obviously follows suppression of bone-formation processes, as indicated by the negative correlation between serum intact osteocalcin and 24-h renal calcium excretion (Table 5Up ). Moreover, the associations among nitrogen balance, intact osteocalcin, and 24-h renal calcium excretion (Results and Table 5Up ) further strengthen the assumption that the changes in calcium and bone metabolism are induced by a loss of muscle protein.

After a time lag of several weeks, higher fasting serum calcium and phosphorus concentrations were observed, suggesting an enhanced release of calcium and phosphorus from endogenous body stores (Table 4Up ). Other investigators have observed an increase in ionized but not in total serum calcium already after 7 days of HDT bed rest (31). Obviously, longer immobilization is necessary to induce a more pronounced change in fasting serum calcium and, thus, in bone-derived calcium. In agreement with this assumption is a significant increase in NTx, a sensitive marker of osteoclastic activity (9) and a predictor of osteoporotic fracture risk (40), occurred at first during IW10 and IW14. The NTx concentrations during these time periods were ~69% higher compared with BI, whereas a minimal increase of only 26% was present during IW2 (Table 3Up ). Our data support previous results of a 20% increase in renal NTx after 1–2 weeks bed rest (41).

PICP is released into the circulation from the precursor procollagen in stoichiometric amounts (42), and serum PICP concentrations are associated with histomorphometric indices of bone formation (43). Thus, both the enhanced NTx concentrations and the reduced serum PICP concentrations at IW14 (Table 4Up ) indicate an uncoupling of bone-collagen synthesis and breakdown after long-term bed rest. These alterations thus occur in addition to the reduced serum concentrations of intact osteocalcin (Table 4Up ). The combined results strongly indicate that the changes in bone turnover are modest during the first weeks of immobilization and more pronounced during long-term immobilization. Our data are in agreement with the observation that after 6 weeks of bed rest, only trends toward a decrease in total-body bone mineral density, as well as decreases in the lumbar spine, trunk, and legs have been found (7), whereas substantial decreases in total-body, lumbar spine, femoral neck, trochanter, tibia, and calcaneus bone mineral densities were present after 17 weeks of immobilization (6). The time lag of the initiation of bone resorption processes during strict bed rest (Table 4Up ) is in contrast to the rapid occurrence of a negative nitrogen balance and of enhanced plasma BCAA (Table 3Up ). However, our data confirm clinical studies using densitometric measurements. These former investigations have demonstrated that bone loss is relatively small during the first 2 months of muscle loss and that bone loss continues even when a new steady state of fat-free mass has already been achieved (44).

During remobilization, the increase in circulating testosterone may have contributed at least in part to an increase in muscle protein synthesis (45)(46) and, thus, to the positive nitrogen balance (Table 3Up ). The increase in calciotropic hormones and in the calcium/strontium absorption rate during reambulation were more rapid and more pronounced than the decrease of these markers during long-term bed rest (Table 4Up ). The increase in serum 25-OHD, together with the enhanced PTH, may induce an increase in renal calcitriol production and in intestinal calcium absorption (47). Moreover, testosterone can lead to an increase in serum PTH and a decrease of serum calcium (46). Fasting serum concentrations of bone formation markers and 24-h renal NTx excretion also showed a rapid normalization (Table 4Up ). However, with the exception of an increase in serum BAP, indices did not differ from pre-bed-rest values. Thus, it is feasible that no distinct regain in bone mass does occur during reambulation, a suggestion that is in agreement with substantial decrements in bone mineral density of the lumbar spine, femoral neck, and calcaneus observed in able-bodied men after bed rest (22). In that study, bone loss was not fully reversed after 6 month of normal weight-bearing activity (22).

In summary, this study provides new information about the time-dependent alterations in nitrogen and bone turnover during long-term bed rest. Data indicate an early onset of an excessively negative nitrogen balance in parallel with a decrease in specific bone-formation processes. However, there is a lag time of several weeks until the onset of pronounced bone-resorption processes. Consequently, rapid mobilization after bed rest should is mandatory to minimize the onset of excessive bone loss.


   Acknowledgments
 
This work was supported by the European Space Agency and by the German Aerospace Center, Grant 50 WB 96260. The study was part of the doctoral thesis of K. S.


   Footnotes
 
1 Nonstandard abbreviations: BAP, bone-specific alkaline phosphatase; PICP, carboxy-terminal procollagen type I; NTx, N-telopeptide; HDT, head-down tilt; BI, before immobilization; IW, immobilization week; BCAA, branched-chain amino acid; PTH, parathyroid hormone; 25-OHD, 25-hydroxyvitamin D; and IGF-I, insulin-like growth factor-I.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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