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Articles |
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Bone Biology Laboratory, Faculté de Médecine, 42023 Saint-Etienne cedex 2, France.
2
Nuclear Medicine Department, CHU, 42055 Saint-Etienne,
France.
3
Hopital Necker, 75015 Paris, France.
4
DLR-Institute of Aerospace Medecine, 51147 Köln,
Germany.
5
Institute of Biochemical Problems, Moscow, Russia.
6
Institute of Nutrition Science, University of Bonn,
Germany.
a Address correspondence to this author at: Laboratoire de Biologie du Tissu Osseux, Faculté de Médecine, 15 Rue A Pare, 42023 Saint Etienne cedex 2, France. Fax 33-4-77-57-55-72; e-mail lbto{at}univ-st-etienne.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We measured biochemical markers related to bone remodeling in two cosmonauts before, during, and after 21- and 180-day space flights, respectively.
Results: During both flights, type I procollagen propeptide and bone alkaline phosphatase decreased as early as 8 days after launch. Undercarboxylated osteocalcin percentage increased early and remained high during both flights. Vitamin K supplementation restored carboxylation of osteocalcin during the long-term flight. Urinary and serum C-telopeptide of type I collagen (CTX) increased as early as day 8 of the flights; the increase was greater in serum than in urine. Pyridinoline, free deoxypyridinoline, and N-telopeptide increased less than CTX during the short-term space flight. The circadian rhythm of bone resorption assessed by urine CTX and free deoxypyridinoline was not altered by microgravity.
Conclusion: Vitamin K metabolism or action and bone remodeling may be altered in cosmonauts.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm these results and to further explore changes in bone remodeling that occur under exposure to microgravity, we measured bone formation and resorption markers during the 21-day MIR 97 space mission. BGP is characterized by the presence of three G-carboxyglutamic acid residues derived from the vitamin K-dependent posttranslational modification of glutamic acid residues (10). The carboxylation state of BGP has been shown to be responsive to changes in vitamin K status (11). Serum concentrations of undercarboxylated osteocalcin (Uoc) increase in elderly women (12) and may perhaps relate more to changes in bone quality than to mineral content (13), possibly because carboxylated BGP binds to bone hydroxyapatite, whereas Uoc does not. Thus, we measured the concentrations of Uoc in one cosmonaut from the 1997 MIR mission and from another cosmonaut from the previous 1995 MIR space mission (180 days).
To study resorption, we measured urinary concentrations of pyridinoline (Pyr), Dpd, and CTX. All of these markers were normalized with urine creatinine concentrations, assuming that creatinine excretion was not altered by microgravity (14). To check this possible bias, we measured serum CTX concentrations, for which an assay method was developed recently, in the two cosmonauts from the 1995 and 1997 MIR missions.
Bone remodeling follows a circadian rhythm whose origin is still a matter of controversy (15). BGP circadian variations have been reported to be related to cortisol rhythm. In contrast, the origin of the circadian bone resorption markers remains unknown. Among various possible hypotheses, the role of mechanical strain in nyctohemeral variations (decubitus/procubitus) has been raised (16). Thus, we studied urinary excretion of two resorption markers (CTX and Dpd) throughout 24 h before, during, and after the flight.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples were collected 210, 208, 196, 192, and 71 days before launch for ERE and 71 for LAI, and 12, 145, 147, 149, 151, and 153 days after landing for both cosmonauts. Serum markers were analyzed six times in ERE during the space flight at days 8, 10, 13, 16, 18, and 19. Urine samples were collected 210, 209, 208, 207, 206, 205, 204, 196, 192, 71, 14, and 13 days before launch and 8, 9, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, and 154 days after landing for both cosmonauts. During space flight, urine markers were analyzed 16 times in ERE, on days 5–19.
During the flight, daily calcium intake was 800–1200 mg, and no vitamin K was added to the diet.
We had the opportunity to measure two markers (Uoc and serum CTX) in sera saved from a previous study (9) performed during the Euromir 95 space mission (September 1995 to March 1996, i.e., 180 days) on two cosmonauts: RTO, age 38 years, and ASW, age 40 years. Samples were taken 15 and 60 days before launch and 60 and 90 days after landing. During space flight, markers were analyzed 10 times in RTO, on days 20, 31, 42, 55, 86, 104, 125, 130, 167, and 178. During this flight, daily calcium intake was 800–1200 mg, and vitamin K was given from day 86 to day 130.
Sampling
Before launch.
Blood samples were collected in dry glass tubes
at 0900 (Moscow time) in the fasting state, and were
centrifuged for 30 min, aliquoted within 30 min, and kept frozen at
-80 °C until assay.
For spot urine measurements, we collected samples at the same time of the day (around 0800).
During the flight.
Blood samples were collected at 0900
(Moscow time), centrifuged for 30 min, aliquoted within 30 min, and
kept frozen at -20 °C during the flight and then at -80 °C
until assay. Urine sampling modalities were similar to those before
launch.
After the flight.
Bood samples were drawn between 0830 and
1000 and centrifuged, aliquoted, and frozen within 30 min. Urine
sampling modalities were similar to those before launch.
For cycle studies.
An artificial light/dark cycle was
established. Cosmonauts slept for 7 h in the dark during the
subjective night. They did not experience jet-lag because time in the
MIR station was the same as at the launch site. Urines were collected
for 24 h, with each voided sample put in a different container. A
portion was saved in a sample vial on which the time of collection was
recorded. Measurements were performed on every sample, and
corresponding results were pooled in six time periods (4–8, 8–12,
12–16, 16–20, 20–0, and 0–4 h). One cycle was performed during the
preflight period at day -207, during the flight at day 17, and after
the flight at day 166.
These procedures were carried out according to the current revision of the Helsinki Declaration.
Assay methods
Markers of bone formation.
BAP was measured with the IRMA
Tandem-R Ostase (Immunotech International), a two-site
IRMA. The intra- and interassay CVs were <7% and 9%,
respectively (17). Expected values are 8–16.6 µg/L.
The Elsa-OsteoTM IRMA (CIS-bio International) is a two-site IRMA for the determination of intact and fragmented BGP that uses a solid phase (18). The intra- and interassay CVs were <4% and 6%, respectively. Expected values for males (31–50 years) are 13.2–35.5 µg/L.
Uoc measurements were performed using the HAP binding assay (11). Serum (250 µL) was extracted with 7 mg of HAP (tribasic calcium phosphate; Sigma) in an Eppendorf tube, mixed end over end for 1 h, and then centrifuged (1250g for 15 min at 4 °C). BGP measured in the supernatant with the Elsa-Osteo IRMA represents a valid estimate of Uoc. The Uoc was expressed as a percentage of total BGP. The intra- and interassay CVs defined in our laboratory were 5% and 9%, respectively.
The PICP concentration was measured by a radioimmunoassay (Orion Diagnostica). The intra- and interassay CVs were <3% and 5%, respectively (19). Expected values are 38–202 µg/L.
Markers of bone resorption.
All urine and blood samples were
run in duplicate and were corrected for creatinine excretion.
The CrossLapsTM urinary CTX assay is a competitive immunoassay on a microtiter plate (CrossLaps ELISA; Osteometer A/S Rodovre). The CrossLaps antigen coated on the microwell is a synthetic peptide with an amino acid sequence specific for a portion of the C-telopeptide of the a1 chain of type 1 collagen (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg; CrossLaps antigen) (20). The intra- and interassay CVs were <6% and 8%, respectively, and the detection limit was 0.2 g/L.
The serum CrossLaps CTX assay is an immunoassay on a microtiter plate (CrossLaps-S ELISA; Osteometer A/S Rodovre). The CrossLaps antigen coated on the microwell is a synthetic peptide with an amino acid sequence specific for a portion of the C-telopeptide of the a1 chain of type 1 collagen (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg; CrossLaps antigen) (21). The intra- and interassay CVs were <5% and 8.1%, respectively, and the detection limit was 92 pmol/L. Expected values are 302–7208 pmol/L.
N-Telopeptide (NTX; Osteomark; Ortho-Clinical Diagnostics Amersham) was measured with a competitive inhibition ELISA. Microwells serve as the solid phase onto which NTX has been adsorbed. Assay values are expressed in nanomoles of bone collagen equivalents per millimole of creatinine. The intra- and interassay CVs were <10% and 5%, respectively.
Urinary free Dpd was measured with a competitive radioimmunoassay using a monoclonal anti-Dpd antibody coated on to the inner surface of a polystyrene tube (Pyrilinks-D RIA; Metra Biosystems). The intra- and interassay CVs were <5% and 7%, respectively, and the detection limit was 2 nmol/L (22). Expected values for males (25–55 years) are 2.3–5.4 nmol/mmol creatinine.
The PyrilinksTM assay (Metra Biosystems) measures free pyridinium cross-links, Pyr, and Dpd in urine. It is a competitive enzyme immunoassay in a microtiter plate. The interassay CVs were <10%. The detection limit was 7.5 nmol/L. Expected values are 13–25.6 nmol/mmol creatinine.
Data and statistical analyses
In Figs. 1
, 3
, 4
, and 5
, data are expressed in Z-scores
calculated with the mean value and SD of the preflight data for ERE
(21-day flight) and RTO (180-day flight). The percentages of variation
given in the results during flight were calculated with the mean values
during flight in relation to the mean preflight values, and the
percentages of variation after flight were calculated with the mean
postflight values in relation to the mean values during flight. In Fig. 2
, Uoc variations are expressed as percentage of total BGP.
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Circadian variations of Dpd and CTX.
Data were plotted as Dpd
or CTX concentrations/urinary creatinine concentrations or as the
percentage of the 24-h mean value. The percentage of variation between
two individual values at one time point (preflight value/flight
value/postflight value) was compared to 2
x the
interindividual variation coefficient of CTX or Dpd measurements (8%
and 7%, respectively) (23).
The 24-h cyclic changes in markers of bone turnover were calculated for
the three-period analysis with sine and cosine as independent variables
[y = ß1 sin (t) + ß2 cos
(t), where t = 2p x time
(hours)/24]. The coefficients ß1 and ß2 to sine and cosine were
then used as summary measures of the circadian variation and compared
to zero by multiple analysis of variance. Amplitudes were calculated as
(24):
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone formation markers
The Z-scores for BGP, BAP, and PICP are shown in Fig. 1
, and the percentages of Uoc are shown in Fig. 2
. During the preflight period, bone formation markers remained
within the reference intervals. No overlapping between
preflight and in-flight concentrations was observed except for
BGP. PICP and BAP decreased by -40% and -18%, respectively, during
the flight compared with preflight concentrations and increased by 36%
and 22%, respectively, after the flight compared with concentrations
during flight. The decreases in BAP and PICP occurred as early as the
first sampling (Z-score = -2 and -2.5 at day 8,
respectively), whereas trends to a decrease in BGP occurred only at day
19. In contrast, the percentage of Uoc was already increased at day 8
of the flight and remained substantially above preflight values
throughout the flight. Uoc decreased 12 days after landing and reached
preflight concentrations 100 days later.
We also measured the percentage of Uoc in the cosmonaut who flew for 180 days (RTO). The percentage of Uoc was higher (22.5%) during the first 72 days of flight compared with the preflight values (14.5%). The percentage of Uoc decreased immediately after vitamin K supplementation down to 9% at day 125; it increased again at day 130 (20%) and remained at this concentration until the end of the flight (18% and 20% at days 146 and 178, respectively). After RTO returned to Earth, the percentage of Uoc was 8% at day 30 and remained at this concentration 90 days after landing.
Bone resorption markers
Changes in Z-scores for the bone resorption markers are
shown in Figs. 3
and
4. During the preflight period, bone resorption markers remained
within the reference intervals. Pyr, Dpd, and NTX increased by
44%, 50%, and 17%, respectively, during the flight compared with
preflight concentrations and decreased by -44%, -50%, and -12%,
respectively, when measured after landing compared with concentrations
during the flight. Pyr and Dpd showed a trend to increase, beginning at
day 7, and remained just above preflight values during the flight
except for 2 days (days 14 and 15) and had returned to preflight
concentrations 12 days postflight. In contrast, NTX exhibited a wide
range of variations (Z-score, -1 to 2.1) and remained
within preflight values except for 5 days.
Urinary and serum CTX concentrations increased by 122% and 40%, respectively, during the flight and decreased by -39% and -17%, respectively, postflight. Urine and serum CTX were increased as early as the eighth day of flight (Z-score = 2 and 5, respectively) and increased regularly (Z-score up to 4.5 and 9.5, respectively) throughout the flight. Therefore, serum CTX followed variations similar to those of urine CTX, but its amplitude of response was greater. Serum CTX had not returned to preflight concentrations 12 days after landing and was normalized 5 months later. To check whether CTX could further increase with the duration of space flight, we measured this marker in serum from cosmonaut RTO, who flew 180 days. CTX increased progressively from the preflight values and peaked at day 72 with a 60% increase compared with preflight values. Serum CTX concentrations then decreased regularly but remained above the preflight concentrations and finally normalized during postflight recovery (4200 pmol/L).
Circadian variations of resorption markers
Urinary CTX and Dpd peaked at the end of the night and reached
nadir between 1600 and 2000 during the three periods (Fig. 5
). Interestingly, urinary CTX nadir values were similar between
the three periods, whereas the increase in Dpd concentrations during
the flight was observed throughout the nyctohemeral period. The
amplitudes of the cycles are reported in Table 2
. The increase in amplitude between preflight and flight was
~160% for both markers. In contrast, when preflight, in-flight, and
postflight values, which summarize the cycles, were taken together, we
found that they were significantly different from 0 for urinary CTX
(P <0.01) but not for Dpd (P = 0.052).
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Total creatinine 24-h excretion during the flight increased by 50% (day 17) compared with preflight values (day -207) and decreased by -25% after the flight (day 166) compared with values during the flight.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surprisingly, we observed that the percentage of Uoc increased rapidly, as early as the eighth day of flight, and was maintained at high concentrations through the entire flight (23%). Uoc progressively returned to preflight concentrations after landing. It has been shown that the Uoc percentage was predictive of the femoral neck fracture in elderly women independent of bone mass, suggesting that higher Uoc concentrations somehow reflect a loss in bone quality (11). Thus, the decrease in carboxylation of BGP might be an additional cause of bone fragility in cosmonauts after they return to Earth. The percentage of carboxylated BGP is vitamin K dependent (13), and Sokoll et al. (27) stressed that the percentage of Uoc reflects vitamin K status. Moreover, it recently was shown that usage of vitamin K antagonists was associated with an increase in fracture risk of vertebral and rib in a time-dependent manner (28). We found that carboxylation of BGP was restored by vitamin K supplementation during the long-term flight. The percentage of Uoc compared with preflight values was similar during both flights before vitamin K supplementation, suggesting that the duration of the flight did not influence this phenomenon. Impairment of vitamin K metabolism could be a consequence of either a decrease in vitamin K intake or an increase in daily vitamin K needs or degradation. As far as we know, fresh green vegetables, which are the main source of alimentary vitamin K, are scarce in cosmonauts’ diet, and it is likely that the lack of carboxylated BGP is a consequence of a low daily intake. However, it seems worthy to study vitamin K metabolism during the next flights to rule out other causes.
We observed that cycling of resorption markers was maintained during space flight with a peak early in the morning and a nadir at the end of the afternoon. Several studies have attempted to define the cause of the circadian pattern of bone turnover (29)(30)(31). Posture might be a cause because decubitus is known to increase bone resorption. However, Schlemmer et al. (32) showed that urinary Pyr and Dpd circadian variations remained unchanged after a 5-day bed rest. Furthermore, Monk et al. (33) showed that a 17-day space flight did not alter the circadian rhythm of urinary free cortisol and melatonin in four astronauts. This might be related to the fact that the "circadian timekeeping system was properly aligned in performing its role of supporting sleep during the subjective night and wakefulness during the subjective day" (33). Thus, we showed that the severe skeleton unloading induced by microgravity was not associated with alteration of the circadian cycle of resorption, although it dramatically increased its amplitude. Interestingly, we found that this increase in urinary CTX depended on the peak, whereas the nadir was unchanged. A better understanding of this phenomenon could improve our knowledge on the circadian rhythm of bone resorption.
Finally, it is commonly accepted to express urinary concentrations of bone resorption markers, measured on spot urine, as a ratio of creatinine excretion. Our concern was that microgravity might alter muscle mass, which is reflected by urinary creatinine excretion, and thus introduce a bias in evaluating the variations in bone resorption markers. Interestingly, Smith et al. (26) showed that astronauts lost weight and were in a negative energy balance during space flight, leading to a likely decrease in muscle mass. In our cosmonauts, we found a 50% increase in 24-h creatinine excretion during the flight (day 17) compared with preflight values (day -207) that might lead to underestimation of the variations of resorption markers. Therefore, we measured CTX concentrations in serum, and all measurements were performed on samples taken between 0750 and 0900 to avoid the variations related to the circadian cycling of this marker. We found similar variations of CTX concentrations in urine and serum with higher amplitude of variations in serum, thus validating the measurement of CTX in serum for evaluation of bone resorption.
In conclusion, we found that microgravity induced early uncoupling of bone remodeling in one human. Moreover, we showed an increase in undercarboxylation of BGP during short- and long-term space flights, suggesting an impairment of vitamin K metabolism, the origins of which remain to be further investigated. The increase in serum CTX concentrations reflected the increase observed in urine. Finally, the circadian rhythm of bone resorption assessed by urinary Dpd and CTX excretion was not altered by space flight.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, preflight; 
, during flight; 
, postflight; *, significantly
different from preflight values.
, cosmonaut
RTO. (B), Euromir 1997: •, cosmonaut LAI;