Introduction

The term bone markers (BM) covers a broad range of different markers (Table 1) which are being used with increasing frequency both as part of standard clinical care for patients with osteoporosis but also in various areas of research far beyond bone health. Based on the work by a working group under the auspices of the International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine [1] the resorption marker Carboxy-terminal Cross-Linked Telopeptide of Type I Collagen (CTX) and formation marker Pro-collagen Type I N-terminal Propeptide (PINP) have been recommended for use in observational and intervention studies and thus for monitoring anti-osteoporotic treatment in adults. Like PINP, pro-collagen Type I C-terminal Propeptide (PICP) is also a marker of bone matrix formation. Another bone formation marker, bone-specific alkaline phosphatase (bone ALP) is being used in relation to Paget’s disease and bone cancer or metastases to the bones [2,3,4,5,6]. Osteocalcin (OC), previously regarded as a bone formation marker, but it is rather a marker of bone turnover, is suggested to have several endocrine effects and to be involved in energy and glucose metabolism [7, 8], whereas osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) in addition to being the most important regulators of osteoclast formation and function are involved in the pathogenesis of vascular calcification and amplification of inflammation [9, 10]. Tartrate-resistant acid phosphatase exists in two isoforms where type 5b (TRAP-5b) is almost exclusively produced by osteoclasts and reflects osteoclast numbers [11]. The calcio- and phosphotropic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) are involved in and part of the regulation of calcium metabolism and thus bone turnover. PTH regulates calcium levels through several mechanisms including (a) stimulating reabsorption of calcium in the proximal tubuli in the kidneys, (b) increasing synthesis of calcitriol by activating renal 1-alpha-hydroxylation of calcidiol, with calcitriol subsequently increasing calcium absorption from the intestines, and (c) stimulating the osteoclasts to resorb bone and release calcium. FGF23 is a hormone that lowers phosphate levels through (a) downregulating surface expression of sodium-dependent phosphate transporters in the proximal tubules in the kidneys thereby increasing phosphate excretion, (b) reducing circulating levels of active vitamin D by two mechanisms; inhibiting 1-alpha-hydroxylase in the kidneys (important for conversion of 25-hydroxyvitamin D to the active 1,25-dihydroxyvitamin D), and by increasing the expression of 24-hydroxylase, which converts 1,25-dihydroxyvitamin D into its inactive metabolites, and finally, (c) inhibiting the secretion of PTH from the parathyroid glands [12].

Table 1 Systematic overview of the BM mentioned in the review
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Together this illustrates the diversity and applicability of BM circulating in the peripheral blood which combined with their easy availability through a standard blood sample make them a desirable tool for monitoring biological processes involved in regulation of bone metabolism. However, to explore the full potential of BM in research, diagnostics, and treatment monitoring, it is important to know as much as possible about the causes of preanalytical variation including the daily rhythmicity. The circadian rhythm is the endogenous, repetitive fluctuation of a marker over a 24-h time span, which can be simplified as a cosine rhythm which is schematically illustrated in Fig. 1 along with common terms used to describe these variations. In contrast, a diurnal variation is a variation primarily caused by extrinsic systems and exposures.

Fig. 1
figure 1

Schematic illustration of circadian rhythm and commonly used terms. Midline-estimating statistic of rhythm (MESOR)

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The circadian rhythm is regulated by core clock genes which regulate differentiation and proliferation of osteoblasts and osteoclasts. Thus, bone remodeling is likely to be regulated by the circadian system and studies have shown that disruption of the circadian rhythm can result in dysregulation of bone remodeling [13]. Consequently, circulating markers of bone remodeling may be affected by the circadian system.

The circadian rhythm of BM can potentially be regulated and influenced by non-modifiable factors such as age, menopause, and ethnicity, by hormonal factors and sleep or by more controllable factors such as physical activity, intake of food and light. Light is known to be the strongest regulator of the circadian rhythmicity of many genes and systems when controlled by the Suprachiasmatic nucleus. Circadian disruption of peripheral oscillation is not sufficiently investigated, but sleep disruption is shown to be a significant participant in changes in phase amplitude and width of the period of expression in rhythmic gene expression [14]].

Several studies examining the circadian variation of BM have been published as well as studies examining endogenous and exogenous factors influencing the rhythmicity. While recommendations to standardize preanalytical conditions exist for CTX and PINP [15], no recommendations exist for the large majority of markers of bone turnover and metabolism.

Therefore, the aim of this systematic review is to investigate how BM are subject to circadian variation and which factors influence this variation including both non-modifiable factors as age and sex, hormonal factors, and other exogenous factors as physical activity, intake of food and liquid. Furthermore, the aim was to summarize these findings in recommendations to standardize patient preparation in relation to circadian variation to maximize the validity and reduce the variability in measuring BM and to uncover areas where further studies are warranted.

Materials and Methods

A systematic search of PubMed https://www.ncbi.nlm.nih.gov/pubmed was done on the 25th November 2019 with the following Medical Subject Headings (MeSH) terms: (("Circadian Rhythm"[Mesh]) OR ("Chronobiology Phenomena"[Mesh]) OR ("Melatonin"[Mesh])) AND (("procollagen Type I N-terminal peptide" [Supplementary Concept]) OR ("collagen type I trimeric cross-linked peptide" [Supplementary Concept]) OR ("RANK Ligand"[Mesh]) OR ("Osteoprotegerin"[Mesh]) OR ("Alkaline Phosphatase"[Mesh]) OR ("Tartrate-Resistant Acid Phosphatase"[Mesh]) OR ("fibroblast growth factor 23"[Mesh]) OR ("Parathyroid Hormone"[Mesh]) OR ("Osteopontin"[Mesh])) AND (("Bone Remodeling"[Mesh]) OR ("Osteogenesis"[Mesh])). This resulted in a total of 68 papers which were screened for relevance based on the following inclusion and exclusion criteria.

Inclusion criteria:

  • Studies investigating circadian rhythm of any of the following markers of bone turnover: PINP, CTX, Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), sclerostin, bone-specific alkaline phosphatase (bone ALP), Tartrate-Resistant Acid Phosphatase (TRAP-5b), fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH), osteopontin or previously measured equivalents

  • Studies conducted in humans, age ≥ 18 years

  • Biomarkers must be sampled ≥ 3 times over a 24-h period, with a minimum interval of 6 h between at least 2 of the samples

  • The primary aim of the studies being BM measures on blood sampling on arterial or venous blood

Exclusion criteria:

  • Studies only measuring urinary excretion of BM or only markers of calcium metabolism

  • Papers in other language than English

  • In vivo or in vitro studies

  • Case-reports, opinion pieces, and reviews

There exists a range of studies only measuring BM by urinary excretion, however, since urine markers are generally not used in clinical practice or in clinical trials today, they were excluded. This resulted in 45 eligible papers included in the review. The reference lists of these papers were subsequently evaluated for further relevant literature whereof an additional 38 papers were identified. Following a second round of screening and full-text assessment this resulted in a total of 40 papers included in the review. The search was repeated on the 3rd May 2021 and 1 additional paper was found eligible for inclusion together with an additional 4 papers included during the review process, resulting in a total of 45 papers included in the review. The screening process is illustrated in Fig. 2 adapted from the PRISMA statement [16]. The screening process and the evaluation of the quality of the included papers were done by the authors SS Diemar and SS Dahl. In case of disagreement all authors were included in the decision.

Fig. 2
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The literature screening process

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Results

The results of the literature search and subsequent screening are summarized in Table 2, where the studies are classified based on primary research objective (Table 3).

Table 2 Systematic classification of the studies studying circadian rhythm of bone turnover markers
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Table 3 Summary of characteristics of circadian rhythms for the individual bone turnover markers
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Studies on the Existence of a Circadian Rhythm of BM and the Influence of Non-modifiable Factors, e.g., Age, Sex, Menopause, and Ethnicity

A handful of studies have documented the existence of a circadian variation of various BM in healthy individuals and evaluated the effect of age, sex, and ethnicity. It is a general finding that the bone formation markers OC, PINP, and bone ALP exhibit a circadian variation with a nighttime or early morning peak and daytime nadir, which is unaffected by sex, age, and ethnicity [17,18,19,20,21,22]. For Pro-collagen PICP this resulted in 20–25% higher values at night than in the afternoon [18]. PINP also exhibits circadian rhythm with a nighttime or early morning peak between 5 and 10% above the 24-h mean and a noon nadir around 5–10% below 24-h mean [21]. Similar slight changes in PINP were found by Ahmad et al. showing a Midline-Estimating Statistics Of Rhythm (MESOR) of 63.82 µg/l and an amplitude of 5.97 µg/l [23]. OC had nadir around noon which was 30% less than the peak between 4 and 8 in the morning [17]. Cosine curves of OC found the same circadian rhythm with MESOR of 10.15 µg/l and amplitude of 0.69 µg/l [19]. Similar is true for bone ALP but with a magnitude of circadian variation between 10 and 20% of the 24-h mean [20]. One study found a visual but not significant circadian variation of PINP, which was believed to reflect either the absence or small amplitude of circadian changes in PINP [24]. The studies on bone resorption markers CTX and carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) also found a clear circadian rhythm with nighttime or early morning peak which appears to be unaffected by ethnicity [18, 19, 21, 24, 25]. For CTX the 24-h MESOR for young participants was 890 ± 100 ng/l with an amplitude of 250 ± 50 ng/l and a nadir at 13.30 [24]. There are marked differences in the 24-h mean concentrations of CTX where post-menopausal women had higher levels than elderly men whom again had higher levels than pre-menopausal woman; however, these differences do not affect the circadian rhythmicity [25]. For PTH, involved in calcium metabolism and a promotor of bone resorption, there exists a biphasic circadian rhythm with two peaks, one early morning and one late evening [26,27,28]; however, interestingly the circadian rhythm of PTH is not a mediator of the circadian rhythm of other BM [29]. There appeared to be no effect of ethnicity on the circadian rhythm of PTH but a clear effect of age, with elderly men and postmenopausal women having the highest MESOR of 5.38 ± 0.1 pmol/l and 5.83 ± 0.06 pmol/l, respectively [20, 25, 30]. Likewise, was there a sex difference with men having an earlier and greater increase at night than women [31]. Very limited data exists on the osteocyte inter-osteoblast-osteoclast regulatory markers; however, one study showed a circadian rhythm for FGF23 with a variable night to late morning peak with a MESOR of 37.32 ± 1.20 pg/ml and an amplitude of 3.31 ± 1.20 pg/mL [24] while another study showed that intact FGF23 had a more pronounced circadian variation than the C-terminal FGF23 fragment [32]. For markers such as RANKL and sclerostin there was found no circadian rhythm [19, 24]; however, for OPG the results are less clear as a study found a circadian rhythm of OPG with daytime increase and nighttime decrease with MESOR in premenopausal women of 3.66 pmol/L and an amplitude of 0.19 pmol/l [25], whereas another study by Tarquini et al. found no 24-h circadian rhythm of OPG but a 12-h component with peaks around noon and midnight [33]. Only one study found circadian rhythm in OPG; however, this was with a daytime increase and nighttime nadir. Furthermore, the 24-h mean concentration was highest in post-menopausal women followed by elderly men and lowest in pre-menopausal women [25].

Influence of Hormonal Factors on the Circadian Rhythm of BM

Another interesting area of research has been whether the circadian rhythm of BM is influenced or regulated by other hormonal factors. In our search, we found studies investigating hyperparathyroidism, prolactin, cortisol levels, growth hormone (GH), and sleep–wake cycle in relation to BM.

Hyperparathyroidism has a clear effect on the circadian rhythm of PTH by abolishing the nighttime peak and the correlation with levels of calcium and phosphorus, which interestingly is restored post-surgery suggesting that changes in calcium and phosphorus cannot fully be explained by the circadian rhythm of PTH [34, 35]. Another interesting correlation exists between PTH and prolactin, where a strong temporal correlation exists with changes in PTH occurring around 2 h prior to similar changes in prolactin; however, there appears to be no evidence of a coupling between the two but it seems more likely that both hormones are controlled by the same neuroendocrine control mechanisms [36].

Studies on cortisol showed that circadian variation of CTX or OC are not associated with serum cortisol levels as patients with no endogenous cortisol production had exactly the same circadian variation in BM as participants with normal serum cortisol levels [22]. In contrast, exogenous glucocorticoids inhibit the nighttime rise in OC, and the inhibition is dose-dependent so that lower doses of glucocorticoids result in shorter duration of inhibition [37, 38]. Interestingly this effect of cortisol appears only to apply to OC, as a similar study, found no effect on PINP of inhibiting morning peak of cortisol [39]. These findings are further supported by a study of patients on hydrocortisone substitution, receiving 4 equal hydrocortisone substitution doses a day. These patients displayed a normal circadian rhythm of PINP but with no afternoon nadir for OC [26]. Another hormone of relevance to the circadian rhythm of BM is GH. Patients with GH deficiency show circadian rhythm for PINP and CTX but with a lower MESOR (Fig. 1) [23] and in another study on patients with active acromegaly were the 24-h mean for PINP and CTX higher compared to matched controls [36]. For PTH the circadian rhythm was unaffected by the hydrocortisone substitution, but a higher MESOR for PTH was found in patients with GH deficiency but also in patients with acromegaly [23, 26, 40]. Although, GH clearly affects the mean levels of markers, no effect is seen on the circadian rhythmicity, which remains intact. Another important endogenous factor in close relation to GH is the sleep–wake cycle, which was investigated in a study where participants were kept awake for one night. The study found that sleep deprivation did not affect the circadian rhythm of OC [41], similar is found in two studies by Swanson et al. where more than 3 weeks of forced desynchronization of sleep did not affect the circadian rhythm of CTX, PINP OC, and sclerostin. However, the study found that for men the level of PINP decreased following the intervention, whereas both PINP and OC decreased for women. Levels of sclerostin increased in young men only and no change was found in levels of CTX, whereas CTX increased in young women [42, 43].

Influence of Other Exogenous Factors on the Circadian Rhythm of BM

Exogenous factors such as physical activity or intake of food and liquid can have a similar impact on the circadian rhythm of BM. Studying the beneficial effect of weightbearing exercise on bone health is a topic of obvious interest and in a study of five days of bedrest in healthy participants there was found no effects of the intervention on the circadian variation of OC or PINP. However, OC levels increased, whereas PINP levels decreased continuously during the five days study period, however, with no change in the circadian pattern [44].

Other exogenous factors that could be of great influence is the daily intake of food and liquid as well as the effects of fasting as the nocturnal increase in BM could potentially be an effect of fasting. Consequently, there are studies investigating the effects of fasting on the circadian rhythm of OC and found no effect hereof whereas fasting decreased levels of PTH and abolished or diminished the circadian rhythm and night time peak [45, 46]. Another study investigated whether fasting, oral glucose tolerance test (OGTT), or intravenous glucose tolerance test (IVGTT) affected the circadian rhythm of OC or CTX compared to normal diet. As in the previous study OC was not affected by any of the interventions. In contrast, the circadian rhythm of CTX was greatly affected by fasting, OGTT, and IVGTT compared to normal diet. Fasting markedly affected the circadian rhythm of CTX by reducing the amplitudes of both the morning decrease and the nighttime increase to less than 10%. The OGTT induced the same morning decrease as seen in normal diet but the duration was significantly shorter, and the amplitude of the nighttime increase was also reduced and similar to that of fasting. The IVGTT also reduced the amplitudes of CTX both the morning decrease and the nighttime increase in a manner similar to fasting [47]. The effects of fasting on the circadian rhythm of CTX was confirmed in another similar study [22]. Finally, the effect of light exposure was evaluated in patients with blindness. No differences in circadian rhythm of CTX or OC could be detected between blind and patients with normal vision indicating the lack of effect of light on the diurnal variation of BM [22].

Influence of Osteoporosis and Anti-Osteoporotic Treatment on Circadian Rhythm of BM

Osteoporosis is often related to a state of increased bone turnover leading to bone loss [48] and the BM are frequently used to monitor the effects of both anti-resorptive and anabolic treatment. Consequently, the circadian rhythm of bone turnover is highly important as to whether they are preserved in osteoporosis, could be a potential treatment target or be affected by the treatments.

For PTH the nocturnal rise was preserved for premenopausal women and for postmenopausal women without osteoporosis, whereas this rise was absent in postmenopausal women with osteoporosis. Establishing the importance of PTH for normal bone health raises the question of whether the difference is causative or a response to pathology [49]. Two studies investigated whether calcium supplements can affect the circadian rhythm of PTH and found that evening doses but not morning doses of calcium inhibit the nocturnal rise in PTH. It was a general effect that the participants on highest doses of calcium had the lowest levels of PTH and in general there was found no effect of age [50, 51].

A study investigated the effects of administering calcitriol either p.o. or i.v. on circadian rhythm of OC and found that following administration of calcitriol in the morning, the decrease of OC was eliminated and substituted by a rapid increase that was sustained for more than 24 h [52]. No difference was detected between administering calcitriol as p.o. or i.v.

In terms of anti-osteoporotic treatment various drugs have been investigated, including the anti-resorptive bisphosphonates, alendronate, and clodronate. Alendronate treatment for 12–15 months did increase the nocturnal PTH secretion but did not affect daytime levels, nor did the treatment alter the circadian rhythm of OC but it reduced the amplitude of the circadian rhythm of Type I collagen cross-linked N-telopeptides (NTX) in serum in women with femoral neck osteoporosis. In general the alendronate-treated women had lower levels of the two BM than controls [53, 54]. Clodronate treatment for 4 weeks had no effect on the circadian rhythm of PICP or ICTP in healthy women nor was there any difference in the serum levels of the two markers, which is surprising since this is a known effect of antiresorptive treatment. However, this could be due to the short duration of treatment that the study was done in healthy women or a combination of the two. Clodronate however affected PTH and increased area under the curve, which could suggest an effect, however, only detectable in the calcium metabolism after 4 weeks [55].

The effects of timing of the anabolic anti-osteoporotic drug, teriparatide, which is the active fragment of PTH (PTH1-34), has been investigated in women with osteoporosis. In general, it was found that CTX showed circadian rhythm during teriparatide treatment while PINP was not affected by the time of day of dosing. The circadian rhythm of CTX was preserved with evening doses of teriparatide while the morning dose inhibited this rhythmicity. Furthermore, had the 12-h response curve of CTX a greater daily mean value following evening dose compared to morning dose. For PINP, the amplitudes were smaller than for CTX, but with no difference in the overall 24-h mean [56].

Calcitonin has been investigated for optimal drug delivery and effects on circadian rhythm of OC in healthy women. The study found no circadian rhythm of OC and there was no effect of calcitonin in general, nor on the time of administration [57]. Another trial with calcitonin in healthy postmenopausal women found a clear circadian rhythm for CTX with lower peaks following evening administration of calcitonin compared to morning and pre-dinner dose. Likewise was the maximum difference from placebo seen after evening dose [58].

Cathepsin K-inhibitors are a group of drugs aimed at inhibiting the osteoclast enzyme cathepsin K which primary function is to cleave collagen and elastin. The cathepsin K-inhibitor, ONO-5334, has been shown in healthy women to reduce 24-h mean serum CTX when administered both morning and evening; however, the reduction was most consistently with > 60% reduction over 24 h following morning dose [59]. There are currently no cathepsin K-inhibitors marketed for clinical use to treat osteoporosis.

Influence on Chronic Kidney Disease on Circadian Variation of BM

In patients with chronic kidney disease (CKD), the circadian rhythm of various functions such as blood pressure regulation, protein excretion in the urine, and plasma sodium are affected. This is highly important as CKD severely affects bone turnover and could therefore also affect the circadian variation of BM. Moreover, several BM (e.g., CTX, PINP (monomers only), and OC) are excreted through the kidneys and reduced kidney function will therefore lead to accumulation of the BM in the circulation. For PINP this only applies for assays measuring both trimers and monomers, while assays measuring only the “intact” PINP molecule (the trimer) will most likely not result in increased PINP values in CKD patients. However, very few studies are published on whether renal failure affects the circadian variation of BM. A recent study demonstrated that patients with stage IV CKD had unaltered circadian variation of both PTH and FGF23 as compared to healthy controls, though there was a disturbance in the circadian variation of 1,25-dihydroxyvitamin D [60]. Similarly, another study showed that patients with end-stage renal disease in hemodialysis have preserved circadian rhythm of PTH [61].

Influence of other Diseases on Circadian Rhythm of BM

The search resulted in only one study investigating the effects of diseases other than osteoporosis or chronic kidney disease on the circadian rhythm of BM. In a study on women with breast cancer and lytic bone disease a normal synchronization of the circadian variation in OC, CTX, and bone ALP was found. This normal rhythmicity was not altered or affected with increasing tumor load [62].

Discussion

From this literature review it is evident that some BM exhibit a clear circadian variation. For resorption markers such as ICTP and CTX there is a marked circadian rhythmicity with nighttime or early morning peak [18, 19, 21, 24]. Thus ICTP had a 20–25% higher night mean than in the afternoon [18] with CTX having even higher differences (40–50%) between nighttime and afternoon levels [19]. A similar circadian rhythmicity is true for the formation markers OC, bone ALP, PICP, and PINP also with a nighttime peak [17,18,19,20,21]. The oscillations were similar across the formation markers with PICP and PINP varying between 20–25% and 10–20%, respectively, across the day. OC varied 30% and bone ALP varied 10–20% across the day [17, 18, 20]. Thus, there appear to be similar oscillations between formation and resorption markers; however, it should be noted that several of these studies are non-fasting and only a few with standardized meals, which could limit the generalizability. However, the changes in PINP appear to be less pronounced and the rhythmicity with less amplitude and fluctuation, which could explain why not all studies find a circadian rhythm of PINP [24]. PTH was found to have a biphasic circadian pattern with two peaks, one early morning and one late evening [21, 25, 27]. As for regulatory BM as RANKL and sclerostin, there appears to be no circadian rhythm [19, 24], only FGF23 showed a moderate circadian rhythmicity with a late morning peak [24]. The result on OPG are less clear as a study found a circadian rhythm of OPG with daytime increase and nighttime decease, whereas another study found a 12-h component with peaks around noon and midnight [25, 33]. However, it should be noted that all these studies are done in relatively few participants under varying conditions suggesting that further research in these regulatory BM is needed.

Although it is generally known that levels of BM are affected by sex, age, and menopausal status, none of these factors influenced the circadian rhythm of markers such as CTX, OC, or bone ALP [20, 25] nor was there any effect on circadian rhythm of ethnicity [21]. Neither was PTH levels affected by ethnicity but a sex difference was found with an earlier and greater increase at night in men suggesting that markers of bone turnover and markers of calcium metabolism differ on some parameters [30, 31]. This is further supported by a study by Ledger et al. where suppression of PTH with calcium infusion had no effect on the circadian patterns of bone resorption markers [29].

What these studies further illustrate is that the term BM covers a broad range of different markers reflecting biologically diverse processes in bone modeling and remodeling. This is evident from the studies on the effects of cortisol on circadian rhythm of OC and PICP. Although both are markers of formation, there were no effects of cortisol on the circadian rhythm of PICP, whereas the circadian rhythmicity and peak of OC was strongly inhibited by increases in cortisol levels, and this even dose-dependent [26, 37,38,39]. This difference might be explained by the different biology of the markers as PICP is a marker reflecting osteoblast activity through collagen synthesis and matrix deposition, whereas OC is a bone turnover marker involved in the mineralization process but which also exhibits several endocrine functions on stress response and energy metabolism, especially in relation to glucose metabolism [7, 8, 63]. For PTH there is a clear effect of primary hyperparathyroidism which abolishes the circadian rhythm of PTH [34, 35] but it appears that there is no relation to circadian rhythm of cortisol nor an effect of abolishing the morning peak [26]. Although not affecting the circadian rhythm of PTH there appears to be an association with levels of GH as both GH deficiency and acromegaly increased levels of PTH, which could suggest that PTH and calcium metabolism are sensitive to changes in GH and that both too high and too low levels could result in increased bone turnover and resorption [20, 36]. GH also have effects on other bone turnover markers and although not affecting the circadian rhythm of CTX, PINP and OC GH deficiency lowered MESOR of both CTX and PINP while active acromegaly increased the 24-h mean of CTX and PINP [23, 40]. GH was found to have no effect on OC [41].

A study by Pedersen et al. investigated the effects of 5 days of skeletal unloading on circadian rhythm of OC and PINP. The intervention had no effect on the circadian rhythm of the markers but during bedrest levels of PINP decreased while levels of OC increased [44]. It is well documented that exercise has anabolic effects on bone and it is likely that this decrease in PINP during bedrest illustrates the inverse of this effect. The increase in OC might be explained by the increased resorption during immobility as OC is imbedded in the bone matrix and consequently released during resorption. Moreover, could endocrine changes in response to complete physical inactivity such as effects on stress hormones or glucose metabolism also influence OC levels and is an interesting topic for further research. In the light of our knowledge of the effects of OC on glucose metabolism it is interesting that studies investigating the effects of fasting, normal diet, OGTT, and IVGTT had no effect on circadian rhythm of OC [45, 47], suggesting that there is still several aspects of OC that currently are not fully known or understood. However, while no effects were seen on OC, the circadian rhythms of CTX and PTH were markedly diminished during fasting compared to normal diet [45, 47]. For CTX OGTT resulted in a decrease similar to normal diet but with a shorter duration and reduced nocturnal increase, while IVGTT showed similar pattern as fasting but with slightly larger oscillations [47]. These differences probably illustrate the influence of incretin hormones, e.g., gastro inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) on the circadian variation of CTX and bone metabolism [64] and raise the question of whether food intake in it-self affects circadian rhythm or is a surrogate marker [65]. Likewise, is PTH suppressed by fasting and the nighttime peak diminished, but the changes that occur during fasting however indicates that other factors than calcium contribute to the circadian rhythm of PTH [46].

The marked circadian rhythm of several of the BM makes the topic of interest both in the context of effects of osteoporosis on this circadian pattern but also in the context of timing anti-osteoporotic treatment to maximize the beneficial effects of treatment. PTH appears to be the only marker where osteoporosis alters the circadian rhythm [49], though not all studies find this association [66]. Otherwise it is a general finding that patients with osteoporosis have preserved the circadian rhythm of BM, despite the general increase in levels of turnover markers. In terms of supplements and treatment, the idea of suppressing PTH induced bone resorption by timing calcium supplements is adjacent and it is clear from the literature that patients on high levels of supplements have lower levels of PTH and that if the supplement is taken in the evening is suppresses the night time rise in PTH [50, 51]. However, there is evidence to suggest that PTH is not the only mediator of the night time increase in bone turnover [29]. As for anti-osteoporotic treatment the anti-resorptive treatment with bisphosphonates had no effect on the circadian rhythm of OC but reduced the amplitude of NTX, which makes sense as the anti-resorptive treatment targets the osteoclastic bone resorption. Interestingly, not all studies found an effect on resorption markers; however, there are marked differences in the studies as one showing an effect was done in women with femoral osteoporosis following 12–15 months alendronate treatment, whereas the other was done in healthy participants following 4 weeks of clodronate treatment. It is likely that a few weeks treatment is not enough to detect effects of clodronate and that healthy participants do not respond in the same way to treatment as osteoporosis patients [53,54,55]. Another drug also targeting the resorption, although not marketed, is the cathepsin K-inhibitor ONO-5334, which reduced CTX levels but equally following both morning and evening dose [59]. Together this indicates that anti-resorptive treatments affect the levels of markers but to a lesser extent interfere with circadian rhythmicity of BM. Other drugs such as teriparatide and calcitonin had more profound effects on the circadian rhythm of CTX that was dependent upon timing of dosage, with the most effective dosages inhibiting the nocturnal or early morning rise [56,57,58]. Vitamin D is essential for bone health, which if administered in its active form, calcitriol, eliminates the morning decrease in levels of OC, an effect sustained for more than 24 h [52].

While many human studies exist on factors affecting the circadian variation on BM, only few studies have directly investigated the effect of factors on the circadian rhythm in humans. The question is then whether patients with disrupted circadian rhythms have increased or altered bone turnover and whether these patients are more prone to osteoporosis than patients with preserved circadian rhythm. The studies by Swanson et al. with 3 weeks of forced desynchronization of sleep found no effect on circadian rhythm of BM but found lower levels of PINP and for women also lower levels of OC. For men CTX levels remained unchanged while they increased for young women [42, 43]. If this margin between formation and resorption persists it might potentially result in bone loss suggesting that disrupted sleep could result in bone loss but not by interfering with the circadian rhythm. However, no clinical data have demonstrated the negative effects on bone mass and this is a topic for further research. As is the difference between age groups which could indicate that the risk of bone loss by disrupted sleep differs with age.

In general, the studies included in this review have several strengths including a thorough screening of the patients and participants with standard biochemical analyses and investigations for any diseases or medications that could affect the bone turnover. Likewise, are several of the studies cross-over designs with repeated measures using the participants as their own controls, which reduces variability. In the studies with control groups these are highly matched on several parameters to the intervention groups. However, the studies are in general small with few participants reducing the reliability and generalizability, making comparison between studies difficult (sample sizes for the individual studies can be found in Table 2). This is further challenged by a high degree of variation in the set-ups including time of initiating the investigation, blood sampling frequency, circumstances as activity levels, and fasting vs non-fasting. Furthermore, it can require influencing the circadian rhythm for a certain amount of time before changes occur and several of the studies have no or only short adjustment-phases prior to the experiment which could explain some of the studies where no effects are found. There is also great variation in which and how many markers are measured, rendering more or less adequate and complete insight in the complexity of bone turnover, and rendering it difficult to compare as the biological diversity between markers is clear and profound. Likewise, are several different methods of analyses and various assays used which also reduces generalizability. Unfortunately, there is currently no agreement on standardization of BM assay, and systematic bias has been shown between assays for PINP and CTX, respectively [67,68,69]. Furthermore, are several of the studies of older date, and while this is not a disadvantage in itself, it is reflected in the measured markers which represent what was used at the current time, which means that the circadian variation for several newer markers such as sclerostin, uncarboxylated OC, FGF23, and klotho are virtually uninvestigated. Likewise, are aspects as light and melatonin levels areas of interest for future research on circadian rhythm of BM. In general, does our current knowledge rest on few studies which could be repeated and expanded with new aspects, e.g., the effects of fasting and OGTT could be repeated and expanded with effects of insulin- or glucose-clamps, or the effects of cortisol could be studied on patients with increased risk of osteoporosis and use of high-dose steroids as patients with rheumatoid arthritis or chronic obstructive pulmonary disease.

With the increasing use of BM not only in research but also in clinical practice there is great need for increasing our knowledge on factors known to influence these markers so that a high degree of standardization can be implemented when measuring BM. There are several aspects of standardization including patient preparation, handling of blood samples, and choice of assay. The aspect of circadian rhythmicity is an important contributor to biological and pre-analytical variation. As illustrated in this review there exists a circadian variation of several BM and consequently should time of day for blood sampling for BM measurements be taken into account when preparing the patient. The rhythmicity appears not to be affected by age, sex, or menopausal status although all these factors are known to influence the overall levels of the markers. The exogenous factors which appear to have the greatest impact on the circadian rhythm is fasting which reduced variation. Consequently, are standardization of timing of sampling and fasting of great importance as part of patient preparation in the light of the circadian variation of BM.

Conclusion

In general, we conclude that there exists a circadian variation for several BM including PINP, CTX, OC and bone ALP and PTH. This rhythmicity appears to be associated with hormonal axes such as cortisol and GH but differs depending on the BM in question. In contrast, it is not affected by non-modifiable factors such as age, sex, or menopausal status for women. Of exogenous factors is fasting perhaps the most striking, however, sleep–wake cycle could be an interesting influencer. Consequently, we recommend that in terms of circadian rhythmicity of BM the blood sampling should be done in the morning and if measurements are repeated then preferably at the same time of day. Furthermore, should the patients be fasting overnight to minimize amplitudes in the rhythmicity, which would reduce variation and increase generalizability of measuring BM. Moreover, is it clear from current research that the term BM covers a broad range of biologically different markers where several aspects are in need of further research including several of the less well-described regulators of the circadian rhythm in bone turnover.