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Urinary Calcium

As the mineralised component of bone is essentially a calcium salt, the most obvious marker of bone resorption is the estimation of urinary calcium excretion. This assay is cheap and simple to perform, being available in all clinical laboratories. However twenty-four hour urine collections on a free diet provide limited diagnostic information. While it has been suggested that in a steady state situation urinary calcium excretion reflects intestinal calcium absorption rather than bone resorption (Eriksen et al., 1995), Nordin and Polley (1987), found twenty-four hour urinary calcium excretions not to be related to dietary intake in normal postmenopausal women.

What is undisputed is that in a fasting state, calcium excreted in the urine represents the obligatory calcium loss of the individual. Although dependent on the degree of tubular resorption, calcium excretion is also a function of the filtered calcium load. In fasting individuals the assumption was made that calcium derived from bone resorption is the primary source of that load. This led to the widespread use of fasting urinary calcium (normalised for creatinine concentration to correct for body mass) as a marker of resorptive activity. A diurnal variation of bone turnover, with increased nocturnal activity, is suggested by biochemical markers of both bone formation and resorption (Hassager et a l, 1992a; McLaren et at., 1993; Nielsen et a i, 1990a; Nielsen, 1991). However evidence suggests that the fasting calcium load is derived not only from resorbed bone mineral, but the PTH-mediated exchange of calcium ions which occurs at the bone-blood interface (Reeve et a l, 1990). This exchange does not represent a net calcium loss, and even when the exchange rate is high due to the nocturnal peak of PTH concentrations, it is not reflective of resorptive activity. Fasting urine calciumicreatinine ratios (Ca/Cr) therefore lack specificity as a marker of bone resorption.

Raised fasting urinary calciumicreatinine ratios occur in post-menopausal osteoporosis, hyperparathyroidism, and Paget* s disease. Decreased values are found in hypocalcaemic states such as osteomalacia and hypoparathyroidism as well as renal failure (Morris et al.,

1993). However the intra-individual variations for fasting urine calciumicreatinine ratios are very large. The coefficient of variation of two consecutive collections has been demonstrated to be approximately 40% in osteoporotic women. Consequently the difference required for values to show a significant change (the critical difference) is very large and limits the use of Ca/Cr for monitoring bone resorption (Vasikaran et a l, 1994). Similarly the interpretation of individual clinical results using population based reference ranges is limited by a large inter-individual variation (Morris et a l, 1993).

It should be concluded that although cheap and easy to perform, Ca/Cr values are not a reliable indicator of bone resorptive activity.

Urinary Phgsphatc

Even in the fasting state only a small proportion of urinary phosphate excretion is reflective of bone resorption (Nordin et û/., 1976). Furthermore urinary phosphate excretion is poorly reproducible, especially in post-menopausal women (Morris et al.,

1990). Analysis of 24-hour urinary phosphate excretion is therefore of no great diagnostic value in the assessment of bone turnover.

Hvdroxyproline

The mineral component of bone is deposited within an organic matrix, the major constituent of which is collagen. An extremely high proportion of glycine, which appears nearly every third residue, gives this protein a remarkably regular amino acid sequence. Collagen also contains two amino acids rarely observed in other proteins; hydroxyproline and hydroxylysine. Proline and lysine are the sole precursors of collagenous hydroxyproline and hydroxylysine, dietary sources of the hydroxylated residues having no role in the final composition of collagen. These post-translational hydroxylations occur only during the synthesis of mature collagen fibrils. The free amino acids are not suitable substrates. Following resorption of bone and breakdown of collagen, these amino acids are not re-utilised for collagen synthesis (Prockop et al.y

1979). These compounds, especially the more abundant hydroxyproline, have found a potential role as markers of bone resorption.

Eighty to ninety percent of hydroxyproline released during bone resorption circulates as free amino acid (Kivirikko, 1983), which is almost entirely reabsorbed from the renal ultra-filtrate. Oxidation and enzymatic degradation to carbon dioxide and urea occurs in the liver (Kivirikko, 1983). Hydroxyproline peptides, however, are not subject to renal tubular reabsorption and are excreted into the urine. Consequently urine contains three forms of hydroxyproline, in total comprising only 10-20% of the hydroxyproline released from bone during its resorption.

1. Free amino-acid that has escaped reabsorption (around 5% of total urinary hydroxyproline).

2. Peptides of less than approximately 5 kD (85%).

The Specificity of Hydroxyproline as a MarkerolBone Resorption

Non-dialysable hydroxyproline, is derived from the degradation of the N-terminal fragment of the procoUagen propeptide. ProcoUagen processing is a prerequisite step in coUagen fibrU formation and so bone matrix production, and therefore this fraction of hydroxyproline is actuaUy indicative of bone formation, although it lacks the specificity to be used as a marker of this process (Haddad e t a l y 1970). However it is clear that despite their clinical use, total urinary hydroxyproline concentrations are not specific for bone resorption. Nor is aU urinary hydroxyproline derived from bone. The C lq fraction of complement contains significant amounts of hydroxyproUne, as do other coUagen containing tissues such as skin, tendon, cartilage, e t c . (Deacon et aly 1987). HydroxyproUne is absorbed from dietary gelatine, raising concentrations in the urine (ColweU e t a l y 1990). A number of attempts to overcome dietary influences have been made. The use of a coUagen-free diet is effective (Gasser e t a l y 1979), but laborious to achieve and compliance difficult to monitor. A fast of nine hours removes dietary influences (Hodgkinson & Thompson, 1982), allowing fasting hydroxyproline concentrations to be used once corrected for creatinine concentration. WhUe no seasonal variations in hydroxyproline excretion are found (Vanderschueren e t a l y 1991), diurnal variations do occur, peaking between 00:00h and 08:00h with a trough between 12:00h and 20:00h (Mautalen, 1970). Fasting urine collections are more susceptible to the diurnal variations which 24 hour coUections eliminate (EasteU et a l y 1988). Fasting coUections at standardised times minimise both dietary and diurnal variation. Intra­ individual variations in such samples are lower than in 24 hour coUections, giving a more reliable indication of the hydroxyproline:creatinine ratio (Morris e ta/., 1990; Podenphant e t a l y 1986). Some workers suggest that a fasting second morning void urine is adequate for reUable estimation. (Wilson e t a l . y 1984).

While Deacon et a l (1987), found hydroxyproline excretion to be a good indicator of radioisotope measured bone resorption, Delmas (1990), reported poor correlation with resorption as assessed by calcium kinetics and bone histomorphometry . As such hydroxyproline wiU eventuaUy be replaced by more specific assays of bone resorption. The CUnical Use of HvdroxvoroUne

In common with most bone markers, apart from a peak during puberty, elevated hydroxyproUne concentrations in infancy decline in adulthood (Charles et a l y 1985; Stepan et a l y 1985). WhUe population based reference ranges for hydroxyproline are greatly limited by large inter-individual biological variation (Morris et a l y 1990), cross- sectional studies indicate increased excretion in females in their sixth decade, presumably due to increased post-menopausal bone loss (Hyldstrup et a l y 1984). Some workers indicate levels rise by up to 50% within one year of the menopause (Nordin & PoUey,

1987), and by a further 20% in post-menopausal women diagnosed as osteoporotic. Although hydroxyproline excretion in osteoporotic women rises, the scatter of values increases, reflecting the heterogeneity of bone turnover within this group. In such circumstances population based reference ranges become even less reliable.

Hydroxyproline has been used to monitor treatment in osteoporosis. Short term oestrogen therapy and bisphosphonate infusion both produce a significant decline in hydroxyprolinexreatinine ratios (Aloia et a l y 1991; Mallmin et a l . , 1991; Thiebaud et a l y

1994), . In early post-menopausal women, Podenphant et a l (1986) found fasting hydroxyprolinexreatinine ratios were decreased on anti-resorptive treatment, a change not detected with 24 hour samples. Hydroxyproline excretion correlates well with forearm bone mineral content (BMC) in both adult men and women (Hyldstrup et aly 1984). U rinary hydroxyproline concentrations are significantly increased in hyperparathyroidism, correlating well with raised levels of osteocalcin (Hyldstrup et a l y

1984). Significantly elevated hydroxyprolinexreatinine ratios (which decrease with treatment), reflect increased bone turnover in Paget's disease (Russell, 1984; Torres et

a l y 1991b). Correlation with alkaline phosphatase levels in these subjects is high, and either marker may be used to assess disease activity and response to therapy.

The Measurement of Urinarv Hvdroxvproline

The assay for hydroxyproline is well characterised, having been first described in 1933 (Lang, 1933). Acid hydrolysis of urine at high temperature or with a cation exchange resin releases peptide-bound hydroxyproline (Goverde & Veenkamp, 1972). The hydrolysate is then oxidised with chloramine T to form a pyrrole. After removal of interfering substances, colorimetric quantification of the pyrrole is achieved by reaction with Ehrlich’s reagent The method has been successfully applied to the continuous flow analyser (Blumenkrantz & Asboe-Hansen, 1974) and the centrifugal autoanalyser (Ho & Pang, 1989). Suppression of colour development may occur in urine samples but not aqueous standards, resulting in an underestimation in sample values. This may be avoided by decolouring the urine with activated charcoal or by the use of an internal standard (Buttery et a l y 1991). Increased analytical specificity may be conferred by high performance liquid chromatography, a process that with the appropriate equipment may be semi-automated (Dawson et a l , 1988; Paroni et a l y 1992).

Hydroxylysine Glycosides

Lysine, the other collagen amino acid which is rarely found in other tissues is also post- translationally hydroxylated. As with hydroxyproline, hydroxylysine is not re-utilised after bone resorption for new collagen synthesis. In mature collagen molecules, hydroxylysine is glycosylated, the degree of glycosylation differing according to the collagen type. The monoglycosylated form, galactosyl hydroxylysine (GalH) is most

predominant in bone, while the major form in skin collagen is the diglycosylated species, glucosyl-galactosyl hydroxylysine (GGalH). The ratio of GGalHiGalH, is 1.61 in human adult skin but only 0.15 in bone. This compares favourably with the ratio of hydroxyproline present in osseous and non-osseous collagen (Segrest, 1982). GalH may therefore be regarded as a more speciHc index of bone resorption than hydroxyproline. GalHxreatinine ratios have been found to be significantly negatively correlated with bone mineral content, and Moro et a l (1988) suggested a possible role for this marker in the identification of osteoporotic women. Indeed it has also been suggested that GalH is as effective a marker of bone resorption as urinary pyridinoline (Bettica et a/., 1992).

Analysis of GalH is currently only available using reverse phase HPLC with fluorescence detection. Such methodology requires specialised equipment, is labour intensive, technically demanding, and therefore relatively slow and expensive. Consequently the use of this marker has been limited. Evaluation against reference methods is required to assess its possible usefulness as a biochemical marker of bone resorption, while the development of a specific immunoassay would widen the availability of GalH measurements.

Collagen Crosslink Macromolccules Pyridinium Crosslinks: Formation and Structure

Aggregation of individual collagen molecules in the extra-cellular space leads to the formation of macromolecular collagen fibrils which are initially stabilised by both electrostatic and hydrophobic forces. A time-dependent maturation then occurs due to the formation of covalent crosslinks within and between the collagen molecules. Precursor lysine and hydroxylysine aldehydes produce separate cross-linking pathways (Eyre et al.,

1984). The initial products of these pathways, the borohydride-reducible keto-amines, disappear from skeletal connective tissues with age, undergoing conversion to mature non-reducible intermolecular cross-linking compounds (Eyre et a l, 1988). Mature crosslinks confer stability and high tensile strength to fibrous collagen and hence are vital to the structural integrity of the skeleton (Eyre & Oguchi, 1980). These molecules, lysylpyridinoline and hydroxylysylpyridinoline have become more commonly known as deoxypyridinoline (DPyr) and pyridinoline (Pyr) respectively. While DPyr is formed exclusively in the mineralised coUagens of dentine and bone, Pyr is widely distributed among many tissue types, especially cartilage.(Boucek, 1981; Eyre & Oguchi, 1980, Eyre et aly 1988). Both crosslink species are absent from the skin, as post-translational glycosylation of skin collagen differs from other coUagens (PinneU et aly 1971). Pyr and DPyr occur in a molar ratio of 3.5:1 in mature bone (Eyre et a l, 1988). During bone resorption these compounds are released and without being further metabolised, excreted

in the urine, mostly in their free form (see Table 1.4) (Gunja-Smith and Boucek, 1981; Fujimoto e t al . y 1983). As pyridinium crosslinks are formed exclusively in mature collagen, their presence in urine relates only to degradation of collagen established in the extra cellular matrix, and not that protein which has been synthesised without being incorporated into collagen fibrils. Furthermore, pyridinoline compounds are not signifîcantly absorbed from the diet, adding to their specificity as markers of mature collagen breakdown (Colwell e t a L y 1990).

Crosslink Species In Urine Composition (% )

Free 38

Glycosylated forms and peptides of 550 -1000 kD 40

Peptides 1000 - 3500 of kD 15

Peptides 3500 kD and over 7

Table 1.4: Distribution of pyridinium crosslink forms in normal adult human urine. Species of Pyr isolated by diiysis and measured by HPLC according to Seyedin et al.

(1993).

The Measurement of Pyridinium Crosslinking Species

Pyridinoline and Deoxypyridinoline

Reference methods for analysis of mature crosslinks utilise HPLC (Black et aly 1988; Fujimoto et al . y 1983; Gunja-Smith & Boucek, 1981; James et a l y 1990). With minor modifications, the majority of these techniques have common steps of sample hydrolysis, pre-fractionation by partition chromatography, separation by HPLC and fluorescence detection. Such techniques contributed a great deal to the early understanding of crosslink excretion and metabolism (e.g. the presence in urine of both free and peptide- bound forms) (Fujimoto et a l y 1983). Despite the introduction of an internal standard and consequent improvements in the precision of this assay (Calabresi et al . y 1994), HPLC methods are limited by their technically laborious, time consuming nature and the requirement for expensive specialised equipment

The introduction of immunoassays has increased the availability of collagen crosslink measurements. While the first such assay was described in 1982 (Robins, 1982a), only in the last few years have commercial assays been produced. The oldest, ‘Pyrilinks’ (Metra Biosystems Ltd, Oxford, UK) measures free Pyr and DPyr in urine with 100% cross-reactivity (Seyedin et a l y 1993). Recognition of glycosylated and peptide bound forms of Pyr is minimal. While the Pyrilinks assay remains available for measurement of general collagen turnover with a possible role in the assessment of rheumatoid arthritis (Astbury et a l y 1994; Seibel et a l y 1989), it has been superseded by a similar enzyme immunoassay (‘Pyrilinks-D’ Metra Biosystems Ltd, Oxford, UK). This assay measures

free DPyr and is therefore more specific for bone resorption (Robins et al. 1994a). The monoclonal antibody used exhibits no significant interaction with peptide-bound crosslinks and less than 1% cross-reactivity with free Pyr. Both immunoassays exhibit excellent correlation with appropriate HPLC measurements (Robins et al.y 1982; Seyedin

et al.y 1993; Robins et aly 1994a). A further commercial immunoassay for DPyr (Nichols Institute Diagnostics, Essex, UK.) utilises polyclonal antibodies to measure total DPyr by radioimmunoassay. While this assay claims to be suitable for the analysis of DPyr in serum, virtually no published data is available.

Type I Collagen Telopeptides

Type I collagen accounts for more than 90% of the organic matrix of bone (Ayad et aly

1994). As pyridinoline residues are primarily excreted as peptides, a reproducible fraction of cross-linked type I collagen derived pyridinolines would act as a quantitative measure of systemic bone resorption. Within bone type I collagen two pyridinoline forming sites occur; N-telopeptide to helix and C-telopeptide to helix (Eyre et a l y 1984). While the C-telopeptide structure is common to all tissues in which collagen is cross- linked, the N-telopeptide interactions are characteristic of those occurring in bone type I collagen (Hanson, et a l 1992). Urinary pyridinoline cross-linked N-telopeptides (NTx) are therefore postulated to be a specific index of bone collagen resorption. The commercial version of this assay ('Osteomark', Lifescreen Ltd, Watford, UK) utilises monoclonal antibody technology in an enzyme-linked immunoassay. NTx has been found to be sensitive marker of bone resorption, correlating significantly with other markers of bone resorption in healthy individuals (Rosen et aly 1994). In early post­ menopausal women, NTx excretion correlates positively with urinary DPyr and serum osteocalcin, and negatively with spinal bone mineral density (Gertz et a l y 1994). Excretion of NTx alters more dramatically and for a longer duration than that of Pyr or hydroxyproline in bisphosphonate or thyroxine-induced changes of bone resorption in healthy subjects (Rosen et a l y 1994).

N

\ V N

< ?

W

Figure 1.6: Diagrammatic representation of inter-m olecular pyridinium crosslinking between aligned quarter-staggered collagen fibrils. C -telopeptide to helix and N- telopeptide to helix (NTx assay epitope) collagen crosslinks shown. Intra-m olecular crosslinking also occurs within each collagen fibril.

A more recent assay for products o f bone resorption measures a C-terminal telopeptide sequence of eight amino acids specific for type I collagen (‘C rosslaps’, O steom eter, Denmark) (Bonde et a i , 1994). It is likely that this epitope appears in urine in several fomis; as a free peptide, as peptides linked to Pyr and DPyr and possibly peptides linked to the as yet unidentified cross-linking m olecules of type I collagen postulated by Kuypers (Kuypers etal., 1992). It is suggested that the direction of antibodies to a linear peptide sequence will allow recognition of that sequence irrespective of the fomi in which it occurs. The conform ational dependency exhibited by the N-telopeptide crosslink (NTx) assay would then be avoided. ‘C rosslaps’ values correlate well with urinary DPyr, Pyr and hydroxyproline, while in women reaching the menopause, the increase in crosslaps excretion, like that of NTx, is greater than the increase in other markers of bone turnover (Bonde È-ra/., 1994; Gam ero era/., 1994c).

Clinical Use Of Collagen Crosslinking Compounds

Many assessments of crosslinking com pounds of mature collagen as markers of bone resorption have been made. Broadly speaking, the changes in bone resorption in a particular disease state, or a response due to treatment produce similar alterations in all of the markers discussed. W hat may differ is the m agnitude of such changes. Unless stated, the findings discussed below refer to all of this group of markers.

The excretion of collagen-crosslink macromolecules is unaffected by diet and renal function (McLaren et al., 1993; Seibel et at., 1992a). However individuals exhibit significant variation in levels of excretion (Dawson et al., 1988; Schlemmer et at., 1992). and large circadian variations have been reported (McLaren et al., 1993), As with most biochemical markers of bone turnover, markedly elevated values in children and adolescents decrease after the pubertal growth spurt (Hanson et a l, 1992). In younger adults, male and female levels do not differ (Beardsworth et a l, 1990; Uebelhart et a l,

1990). On reaching the menopause, levels in females rise two to three fold, increases which may be reversed by hormone replacement therapy (Uebelhart et al., 1991; Hassager et a l, 1992b). In vertebral osteoporosis collagen-crosslink levels correlate with histomorphometric assessment of bone turnover (Delmas et a l, 1991), and rate of bone loss as assessed by bone densitometry (Mole et a l, 1992; Robins et a l, 1990; Uebelhart et a l, 1991). Treatment of established osteoporosis with bisphosphonates (Gamero et a l, 1994a; Gertz et a l, 1994; Harris et a l, 1993; Rosen et a l, 1994), oestrogen (Uebelhart et a l, 1991), or calcitonin (Overgaard et a l, 1994), reduces the excretion of crosslinking molecules. Similarly in these markers are elevated (and may be normalised on treatment) in Paget* s Disease, primary hyperparathyroidism and osteomalacia (Delmas e ta l, 1993; Gamero e ta l, 1994c; Seibel e ta l, 1992b; Uebelhart

etal., 1990).

Cross-linking compounds of collagen are highly stable in vitro. Numerous freeze-thaw cycles and storage for several days at 4°C has little effect on sample concentration. While it has been recognised that crosslink molecules may be sensitive to ultraviolet light, (Fujimori et a l, 1985; Sakura et a l, 1982), the light transmission through undiluted