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Technical Briefs |
Department of Clinical Biochemistry, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany
a author for
correspondence: fax 49-228/287-5028
Measurements of molecules of bone resorption or formation can be used for estimating the rate of bone turnover. Bone turnover, as assessed by several bone metabolic markers, has been reported to undergo a diurnal rhythm. Thus far, urinary pyridinoline and deoxypyridinoline, serum osteocalcin, bone specific alkaline phosphatase, serum type I collagen cross-linked N-telopeptides (NTx), the C-terminal pyridinoline cross-linked telopeptide of type I collagen, and urinary excretion of NTx were reported to undergo circadian periodicities with high values at night (1)(5).
Degradation products derived from a sequence (EKAHD-ß-GGR)
specific for a part of the C-telopeptide 
1-chain of type I collagen
(CTx) in urine and serum could be quantified by the enzyme-linked
immunosorbent assay, CrossLapsTM. It had been
demonstrated that such fragments are sensitive markers of bone
resorption (6)(8). The purpose of this study was
to determine whether there are diurnal fluctuations in the
concentration of CTx in serum; if this proves true, it is important to
specify the time of blood sampling.
Six healthy 24- to 27-year-old men (weight, 74–85 kg) were studied over a single 24-h period. Blood was sampled every 60 min from an intravenous catheter placed in each subject between 0800 and 0830. Meals were offered at 1000, 1400, and 1930. Activity was restricted to small movement within and around the study room, and lights were turned out between 0000 and 0700. Blood was centrifuged at 3000g for 10 min, and serum was separated and stored at -20 °C until assay.
CTx was measured by a one-step enzyme-linked immunosorbent assay
(Osteometer), which is based on one highly specific monoclonal antibody
against a ß-aspartate isomerized form of the epitope EKAHD-ß-GGR
derived from the C-terminal telopeptide region of the type I collagen
1-chain. According to the manufacturer, the detection limit of the
assay is 92 pmol/L, the intra- and interassay CVs are 4.9% (mean, 3500
pmol/L) and 6.6% (mean, 3501 pmol/L).
Hormone concentrations are presented as median values and individual secretion profiles. The 24-h concentration profiles of CTx were tested for diurnal variation by cosinor rhythmometry, a computer program provided by Dr. W. Burr (Clinic of Epileptology, University of Bonn) as described previously (9). A significant fit of the curve was defined when the possibility of the data representing a horizontal line rather than a cosine curve was <5%. The acrophases, which were not necessarily the detected maximal concentrations, represented the time of occurrence of the maximal values of the best-fitting pattern; the amplitude was defined as 50% of the difference between the acrophase and the nadir concentrations. A value <0.05 was considered significant.
Individual serum CTx concentration profiles over 24 h in six
healthy male volunteers are illustrated in Fig. 1
. The highest concentrations were observed at night between 0130
and 0430, with peak values of 3948–9945 pmol/L; the lowest values were
measured between 1100 and 1500 (443–2356 pmol/L). The concentrations
fell quickly in the morning between 0800 and 1100 and came to a nadir
between 1100 and 1500. After the nadir, they increased slowly to the
initial concentrations between 0130 and 0430 at night. The peak was
66% greater than the 24-h mean, and the nadir was 60% below the 24-h
mean. A significant (P <0.01) diurnal secretion in all
six volunteers was validated by cosinor rhythmometry (Table 1
).
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The diurnal variation of markers of the bone mineral metabolism needs to be taken into account when interpreting the results of biochemical marker measurements. Most markers tend to be higher at night. This is most characteristic for the bone resorption markers.
A new direct immunoassay using antibodies that recognize serum type I C-telopeptide breakdown products, CrossLaps (CTx), is now available. CTx is a very important marker for the follow-up treatment of osteoporosis. A recent study showed that serum and urine CTx concentrations decrease significantly among women treated with clinically relevant doses of different antiresorptive agents. Furthermore, the annual percentage of change in bone mineral density correlated with the measured changes in CTx concentration (10). Unfortunately, the investigators did not take into account the timing of blood sampling.
The present investigation demonstrated a significant circadian rhythm of this bone resorption marker in serum with a peak value between 0130 and 0430 at night and a nadir between 1100 and 1500 during the day. The amplitude of variation is ~60-66% of the mean value. This finding is in agreement with a recent study that measured urinary CTx during 24 h in seven separate collections. A significant variation with a peak at night and a nadir at 1700 h was found (1).
The pattern of the circadian variation of CTx in serum was similar to those of other metabolic bone markers. It followed the rhythmicity of osteocalcin (2) and NTx (4) in serum. Urinary excretion of creatinine-corrected pyridinoline, deoxypyridinoline, and NTx showed a diurnal rhythm, with a peak between 0400 and 0700 and a nadir between 1300 and 1600. Their excretion at night was ~50% higher than during the day (11)(12). It should be noted that most patients usually are older than our subjects, and one study found that urinary NTx values in men of 20–30 years were higher than at subsequent ages (13). But another study also showed a circadian rhythm of bone mineral turnover in elderly men and women (3). Therefore, we did not expect that the course of CTx secretion is very different between younger and older subjects.
In conclusion, we found that CTx in serum demonstrated a marked circadian rhythm in all subjects. The peak value, which on an average is 66% higher than the mean value, highlights the importance of the timing of sample collection for appropriate interpretation of therapeutic response.
References
1-chain of type I collagen. Application in an enzyme immunoassay and comparison to CrossLapsTM ELISA. Scand J Clin Lab Investig 1997;57:73-83.
[Web of Science][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  F. R. SINGER and D. R. EYRE Using biochemical markers of bone turnover in clinical practice Cleveland Clinic Journal of Medicine, October 1, 2008; 75(10): 739 - 750. [Abstract] [Full Text] [PDF]
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 D. B. Henriksen The Gut Feeling of Bone Remodeling IBMS BoneKEy, November 1, 2005; 2(11): 16 - 23. [Full Text] [PDF]
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 S. Christgau Circadian Variation in Serum CrossLaps Concentration Is Reduced in Fasting Individuals Clin. Chem., March 1, 2000; 46(3): 431 - 431. [Full Text] [PDF]
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