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|Número de publicación
|Tipo de publicación
|Número de solicitud
|Fecha de publicación
|4 Ene 2000
|Fecha de presentación
|4 Sep 1997
|Fecha de prioridad
|6 Nov 1987
|Número de publicación
|David R. Eyre
|Cesionario original
|Washington Research Foundation
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|Citas de patentes (11), Otras citas (40), Citada por (8), Clasificaciones (21), Eventos legales (6)
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet
(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- |(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- | (Gly)-Ile-Hyl--Ser-Gly-Val-Ala-Val-Gly-Gly-Ser-Gly-Val-Ala-Val-Gly-Gly-Gly-His-Arg
(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- |(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- | (Gly)-Ile-Hyl--Ser-Gly-Val-Ala-Val-Gly-Gly-Ser-Gly-Val-Ala-Val-Gly-Gly-Gly-His-Arg
(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- |(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- | (Gly)-Ile-Hyl--Ser-Gly-Val-Ala-Val-Gly-Gly-Ser-Gly-Val-Ala-Val-Gly-Gly-Gly-His-Arg
(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- |(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl- | (Gly)-Ile-Hyl--Ser-Gly-Val-Ala-Val-Gly-Gly-Ser-Gly-Val-Ala-Val-Gly-Gly-Gly-His-Arg
This invention was made with government support under one or more of grants AM 26489, AR 37318, AM 30774, and AR 36794 awarded by the National Institutes of Health. The government has certain rights in the invention.
This is a continuation-in-part of provisional application Ser. No. 60/026,949 filed Sep. 6, 1996, and of application Ser. No. 08/484,833 filed Jun. 7, 1995, now U.S. Pat. No. 5,702,909, which is a continuation-in-part of application Ser. No. 07/823,270, filed Jan. 16, 1992, now U.S. Pat. No. 5,532,169, which is a divisional of application Ser. No. 07/444,881, filed Dec. 1, 1989, now U.S. Pat. No. 5,140,103, which is a continuation-in-part of application Ser. No. 07/118,234, filed Nov. 6, 1987, now U.S. Pat. No. 4,973,666.
The present invention relates to methods for detecting and monitoring collagen degradation in vivo. More specifically, it relates to methods for quantitating cross-linked telopeptides produced in vivo upon degradation of type III collagen.
Three known classes of collagens have been described to date. The Class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils. These collagens are all synthesized as procollagen molecules, made up of N-terminal and C-terminal propeptides, which are attached to the core collagen molecule. After removal of the propeptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecule consists largely of a triple-helical domain having terminal telopeptide sequences which are nontriple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extracellularly.
The present invention relates to methods of detecting collagen degradation based on assaying for particular cross-linked telopeptides produced in vivo upon collagen degradation. In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen. For example, bone turnover associated with Paget's disease has been monitored by measuring small peptides containing hydroxyproline, which are excreted in the urine following degradation of bone collagen. Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5, 255-262 (1981); and Singer, F. R., et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V. and Kane, S. M.), 489-575 (1978), Academic Press, New York.
Other researchers have measured the cross-linking compound pyridinoline in urine as an index of collagen degradation in joint disease. See, for background and for example, Wu and Eyre, Biochemistry, 23:1850 (1984); Black et al., Annals of the Rheumatic Diseases, 48:641-644 (1989); Robins et al.; Annals of the Rheumatic Diseases, 45:969-973 (1986); and Seibel et al., The Journal of Rheumatology, 16:964 (1989). In contrast to the present invention, some prior researchers have hydrolyzed peptides from body fluids and then looked for the presence of individual hydroxypyridinium residues. None of these researchers has reported measuring a telopeptide containing a cross-link that is naturally produced in vivo upon collagen degradation, as in the present invention.
U.K. Patent application GB 2,205,643 reports that the degradation of type III collagen in the body is quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid.
There are a number of reports indicating that collagen degradation can be measured by quantitating certain procollagen peptides. The present invention involves telopeptides rather than propeptides, the two being distinguished by their location in the collagen molecule and the timing of their cleavage in vivo. See U.S. Pat. No. 4,504,587; U.S. Pat. No. 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984); Niemela, Clin. Chem., 31/8:1301-1304 (1985); and Rohde et al., European Journal of Clinical Investigation, 9:451-459 (1979).
U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomer or antigenic fragments thereof in a synovial fluid sample. This patent does not relate to detecting cross-linked telopeptides derived from degraded collagen.
Dodge, G. R., and A. R. Poole, Clin. Invest., 83:647-661 (1989) discloses methods for analyzing type II collagen degradation utilizing a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. The peptides involved are not cross-linked telopeptides as in the present invention.
Amino acid sequences of human type III collagen, human proα1(III) collagen, and the entire preproα1(III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers. See Loidl et al., Nucleic Acids Research 12:9383-9394 (1984); Sangiorgi et al., Nucleic Acids Research, 13:2207-2225 (1985); Baldwin et al., Biochem. J., 262:521-528 (1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989). None of these references specifies the structures of particular telopeptide degradation products that could be measured to determine the amount of degraded fibrillar collagen in vivo.
In spite of the above-described background information, there remains a need for effective and simple assays for determining collagen degradation in vivo. Such assays could be used to detect and monitor disease states in humans, such as osteoarthritis (type II collagen degradation), and various inflammatory disorders, such as vasculitis syndromes (type III collagen degradation).
Assays for type I collagen degradation, described in the parent application, U.S. Pat. No. 4,973,666, can be utilized to detect and assess bone resorption in vivo. Detection of bone resorption may be a factor of interest in monitoring and detecting diseases such as osteoporosis. Osteoporosis is the most common bone disease in man. Primary osteoporosis, with increased susceptibility to fractures, results from a progressive net loss of skeletal bone mass. It is estimated to affect 15-20 million individuals in the United States. Its basis is an age-dependent imbalance in bone remodeling, i.e., in the rates of synthesis and degradation of bone tissue.
About 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Twelve to 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $7 billion annually (Barnes, O. M., Science, 236:914 (1987)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, no method is available for measuring bone resorption rates in patients or normal subjects. A major difficulty in monitoring the disease is the lack of a specific assay for measuring bone resorption rates.
Methods for assessing bone mass often rely on measuring whole-body calcium by neutron activation analysis or mineral mass in a given bone by photon absorption techniques. These measurements can give only long-term impressions of whether bone mass is decreasing. Measuring calcium balances by comparing intake with output is tedious, unreliable and can only indirectly appraise whether bone mineral is being lost over the long term. Other methods currently available for assessing decreased bone mass and altered bone metabolism include quantitative scanning radiometry at selected bone locations (wrist, calcaneus, etc.) and histomorphometry of iliac crest biopsies. The former provides a crude measure of the bone mineral content at a specific site in a single bone. Histomorphometry gives a semi-quantitative assessment of the balance between newly deposited bone seams and resorbing surfaces.
A urinary assay for the whole-body output of degraded bone in 24 hours would be much more useful. Mineral studies (e.g., calcium balance) cannot do this reliably or easily. Since bone resorption involves degradation of the mineral and the organic matrix, a specific biochemical marker for newly degraded bone products in body fluids would be the ideal index. Several potential organic indices have been tested. For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget's disease, a metabolic bone disorder in which bone turnover is greatly increased, as pointed out above. For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation. Singer, F. R., et al. (1978), cited hereinabove.
U.S. Pat. No. 3,600,132 discloses a process for determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. In particular, this inventor notes that in pathologic conditions such as Paget's disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism, increased collagen anabolism or catabolism as measured by hydroxyproline content in biological fluids can be determined. This inventor measures hydroxyproline by oxidizing it to a pyrrole compound with hydrogen peroxide and N-chloro-p-toluenesulphonamide followed by colorimetric determination in p-dimethyl-amino-benzaldehyde.
In the case of Paget's disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline. Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolized in the liver and never appears in the urine. Kiviriko, K. I. Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48: 1 (1969).
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally nonspecific markers of bone resorption. Krane, S. M. and Simon, L. S. Develop. Biochem., 22:185 (1981).
In addition to amino acids unique to collagen, various non-collagenous proteins of bone matrix such as osteocalcin, or their breakdown products, have formed the basis of immunoassays aimed at measuring bone metabolism. Price, P. A. et al. J. Clin. Invest., 66: 878 (1980), and Gundberg, C. M. et al., Meth. Enzymol., 107:516 (1984). However, it is now clear that bone-derived noncollagenous proteins, though potentially a useful index of bone metabolic activity are unlikely, on their own, to provide quantitative measures of bone resorption. The concentration in serum of osteocalcin, for example, fluctuates quite widely both normally and in metabolic bone disease. Its concentration is elevated in states of high skeletal turnover but it is unclear whether this results from increased synthesis or degradation of bone. Krane, S. M., et al., Develop. Biochem., 22:185 (1981), Price, P. A. et al., J. Clin. Invest., 66:878 (1980); and Gundberg, C. M. et al., Meth. Enzymol., 107:516 (1984).
The polymers of most genetic types of vertebrate collagen require the formation of aldehyde-mediated cross-links for normal function. Collagen aldehydes are derived from a few specific lysine or hydroxylysine side-chains by the action of lysyl oxidase. Various di-, tri- and tetrafunctional cross-linking amino acids are formed by the spontaneous intra- and intermolecular reactions of these aldehydes within the newly formed collagen polymers; the type of cross-linking residue varies specifically with tissue type (see Eyre, D. R. et al., Ann. Rev. Biochem., 53:717-748 (1984)).
Two basic pathways of cross-linking can be differentiated for the banded (67 nm repeat) fibrillar collagens, one based on lysine aldehydes, the other on hydroxylysine aldehydes. The lysine aldehyde pathway dominates in adult skin, cornea, sclera, and rat tail tendon and also frequently occurs in other soft connective tissues. The hydroxylysine aldehyde pathway dominates in bone, cartilage, ligament, most tendons and most internal connective tissues of the body, Eyre, D. R. et al. (1974) vida supra. The operating pathway is governed by whether lysine residues are hydroxylated in the telopeptide sites where aldehyde residues will later be formed by lysyl oxidase (Barnes, M. J. et al., Biochem. J., 139:461 (1974)).
The chemical structure(s) of the mature cross-linking amino acids on the lysine aldehyde pathway are unknown, but hydroxypyridinium residues have been identified as mature products on the hydroxylysine aldehyde route. On both pathways and in most tissues the intermediate, borohydride-reducible cross-linking residues disappear as the newly formed collagen matures, suggesting that they are relatively short-lived intermediates (Bailey, A. J. et al., FEBS Lett., 16:86 (1971)). Exceptions are bone and dentin, where the reducible residues persist in appreciable concentration throughout life, in part apparently because the rapid mineralization of the newly made collagen fibrils inhibits further spontaneous cross-linking interactions (Eyre, D. R., In: The Chemistry and Biology of Mineralized Connective Tissues, (Veis, A. ed.) pp. 51-55 (1981), Elsevier, New York, and Walters, C. et al., Calc. Tiss Intl., 35:401-405 (1983)).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified (Formula I and II). Both compounds are naturally fluorescent, with the same characteristic excitation and emission spectra (Fujimoto, D. et al. Biochem. Biophys. Res. Commun., 76:1124 (1977), and Eyre, D. R., Develop. Biochem., 22:50 1981)). These amino acids can be resolved and assayed directly in tissue hydrolysates with good sensitivity using reverse phase HPLC and fluorescence detection. Eyre, D. R. et al., Analyte. Biochem., 137:380-388 (1984). It should be noted that the present invention involves quantitating particular peptides rather than amino acids. ##STR1##
In growing animals, it has been reported that these mature cross-links may be concentrated more in an unmineralized fraction of bone collagen than in the mineralized collagen (Banes, A. J., et al., Biochem. Biophys. Res. Commun., 113:1975 (1983). However, other studies on young bovine or adult human bone do not support this concept, Eyre, D. R., In: The Chemistry and Biology of Mineralized Tissues (Butler, W. T. ed.) p. 105 (1985), Ebsco Media Inc., Birmingham, Ala.
The presence of collagen hydroxypyridinium cross-links in human urine was first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. and Boucek, R. J., Biochem J., 197:759-762 (1981)) using lengthy isolation procedures for peptides and conventional amino acid analysis. At that time, they were aware only of the HP form of the cross-link. Robins (Robins, S. P., Biochem J., 207:617-620 (1982) has reported an enzyme-linked immunoassay to measure HP in urine, having raised polyclonal antibodies to the free amino acid conjugated to bovine serum albumin. This assay is intended to provide an index for monitoring increased joint destruction that occurs with arthritic diseases and is based, according to Robins, on the finding that pyridinoline is much more prevalent in cartilage than in bone collagen.
In more recent work involving enzyme-linked immunoassay, Robins reports that lysyl pyridinoline is unreactive toward antiserum to pyridinoline covalently linked to bovine serum albumin (Robins et al., Ann. Rheum. Diseases, 45:969-973 (1986)). Robins' urinary index for cartilage destruction is based on the discovery that hydroxylysyl pyridinoline, derived primarily from cartilage, is found in urine at concentrations proportional to the rate of joint cartilage resorption (i.e., degradation). In principle, this index could be used to measure whole body cartilage loss; however, no information on bone resorption would be available.
A need therefore exists for a method that allows the measurement of whole-body bone resorption rates in humans. The most useful such method would be one that could be applied to body fluids, especially urine. The method should be sensitive, i.e., quantifiable down to 1 picomole and rapidly measure 24-hour bone resorption rates so that the progress of various therapies (e.g., estrogen) can be assessed.
The present invention is based on the discovery of the presence of particular cross-linked telopeptides in body fluids of patients and normal human subjects. These telopeptides are produced in vivo during collagen degradation and remodeling. The term "telopeptides" is used in a broad sense herein to mean cross-linked peptides having sequences that are associated with the telopeptide region of, e.g., type II and type III collagens and which may have cross-linked to them a residue or peptide associated with the collagen triple-helical domain. Generally, the telopeptides disclosed herein will have fewer amino acid residues than the entire telopeptide domains of type II and type III collagens. Typically, the telopeptides of the present invention will comprise two peptides linked by a pyridinium cross-link and further linked by a pyridinium cross-link to a residue or peptide of the collagen triple-helical domain. Having disclosed the structures of these telopeptides herein, it will be appreciated by one of ordinary skill in the art that they may also be produced other than in vivo, e.g., synthetically. These peptides will generally be provided in purified form, e.g., substantially free of impurities, particularly other peptides.
The present invention also relates to methods for determining in vivo degradation of type II and type III collagens. The methods involve quantitating in a body fluid the concentration of particular telopeptides that have a 3-hydroxypyridinium cross-link and that are derived from collagen degradation. The methods disclosed in the present invention are analogous to those previously disclosed in U.S. Pat. No. 4,973,666, for determining the absolute rate of bone resorption in vivo. Those methods involved quantitating in a body fluid the concentration of telopeptides having a 3-hydroxypyridinium cross-link derived from bone collagen resorption.
In a representative assay, the patient's body fluid is contacted with an immunological binding partner specific to a telopeptide having a 3-hydroxypyridinium cross-link derived from type II or type III collagen. The body fluid may be used as is or purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.
Other representative embodiments of quantitating the concentration of peptide fragments having a 3-hydroxypyridinium cross-link in a body fluid include electrochemical titration, natural fluorescence spectroscopy, and ultraviolet absorbance. Electrochemical titration may be conducted directly upon a body fluid without further purification. However, when this is not possible due to excessive quantities of contaminating substances, the body fluid is first purified prior to the electrochemical titration step. Suitable methods for purification prior to electrochemical detection include dialysis, ion exchange chromatography, alumina chromatography, molecular sieve chromatography, hydroxyapatite chromatography and ion exchange absorption and elution.
Fluorometric measurement of a body fluid containing a 3-hydroxypyridinium cross-link is an alternative way of quantitating collagen degradation (and, hence, bone resorption, if type I peptides are quantitated). The fluorometric assay can be conducted directly on a body fluid without further purification. However, for certain body fluids, particularly urine, it is preferred that purification of the body fluid be conducted prior to the fluorometric assay. This purification step consists of dialyzing an aliquot of a body fluid such as urine against an aqueous solution thereby producing partially purified peptide fragments retained within the nondiffusate (retentate). The nondiffusate is then lyophilized, dissolved in an ion pairing solution and adsorbed onto an affinity chromatography column. The chromatography column is washed with a volume of ion pairing solution and, thereafter, the peptide fragments are eluted from the column with an eluting solution. These purified peptide fragments may then be hydrolyzed and the hydrolysate resolved chromatographically. Chromatographic resolution may be conducted by either high-performance liquid chromatography or microbore high performance liquid chromatography.
The invention includes peptides having structures identical to peptides derived from collagen degradation, substantially free from other human peptides, which may be obtained from a body fluid. The peptides contain at least one 3-hydroxypyridinium cross-link, in particular, a lysyl pyridinoline cross-link or a hydroxylysyl pyridinoline cross-link, and are derived from the telopeptide region of type II or type III collagen linked to one or more residues from a triple-helical domain, typically by the action of endogenous proteases and/or peptidases.
The structures of the type II and type III telopeptides are disclosed below. Information on the type I telopeptides, originally presented in U.S. Ser. No. 118,234, is also included.
Structure of Cross-Linked Telopeptides Derived from Type I Collagen
A specific telopeptide having a 3-hydroxypyridinium cross-link derived from the N-terminal (amino-terminal) telopeptide domain of bone type I collagen has the following amino acid sequence:
FORMULA III Asp-Glu K-Ser-Thr-Gly-Gly |Gln-Tyr-Asp-Gly K-Gly-Val-Gly | Kwhere K is hydroxylysyl pyridinoline or lysyl | K | Kpyridinoline, and Gln is glutamine or
K is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamine or a pyrrolidine carboxylic acid.
The invention also encompasses a peptide containing at least one 3-hydroxypyridinium cross-link derived from the C-terminal (carboxy-terminal) telopeptide domain of bone type I collagen. These C-terminal telopeptide sequences are cross-linked with either lysyl pyridinoline or hydroxylysyl pyridinoline. An example of such a peptide sequence is represented by the formula:
FORMULA IVAsp-Gly-Gln-Hyp-Gly-Ala | Hyp-Glu-Gly-Lys | Gly-Asp-Ala-Gly-Ala-K-Gly-Asp | Glu-K-Ala-His-Asp-Gly-Gly-Arg | Glu-K-Ala-His-Asp-Gly-Gly-Argwhere K | K | K
is hydroxylysyl or lysyl pyridinoline.
Since the filing of U.S. Ser. No. 118,234, the inventor has discovered evidence of two additional type I collagen telopeptides in body fluids, having the following structures:
FORMULA VGly-Glu-Hyp | Gly-Asp-Ala-Gly-Ala-K-Gly-Asp | Glu-K-Ala-His-Asp-Gly-Gly-Arg | Glu-K-Ala-His-Asp-Gly-Gly-ArgandFORMULA VI K |Glu K-Ala-His-Asp-Gly-Gly-Arg |Glu K-Ala-His-Asp-Gly-Gly-Arg
These telopeptides may also be quantitated in body fluids in accordance with the present invention.
Structure of a Cross-Linked Telopeptide Derived from Type II Collagen
A specific telopeptide having a hydroxylysyl pyridinoline cross-link derived from the C-terminal telopeptide domain of type II collagen has the following amino acid sequence (referred to hereinbelow as the core peptide structure):
FORMULA VII 17C Glu-Hyl-Gly-Pro-Asp α1(II)C-telopeptide | Glu-Hyl-Gly-Pro-Asp α1(II)C-telopeptide |Gly-Val-Hyl α1(II) helical domain 87
wherein the cross-linking residue depicted as Hyl-Hyl-Hyl is hydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium residue present in mature collagen fibrils of various tissues.
Additional cross-linked peptides that embody type II collagen N-telopeptides can be isolated from human body fluid. Peptides having the following N-terminal cross-linked structures were identified:
FORMULA XVI(Gly)-(Gly)-Phe-Asp-Glu K-Ala-Gly-Gly-(Ala)-(Gln)-(Leu) α1(II)N-telopeptide |(Gly)-(Gly)-Phe-Asp-Glu K-Ala-Gly-Gly-(Ala)-(Gln)-(Leu) α1(II)N-telopeotide | Kwhere K is hydroxylysyl pyridinoline (HP) or lysyl | K | Kpyridinoline (LP), and the parentheses indicate
optional amino acid residues. Preferred isolates for monitoring type II collagen degradation have α(II)N-telopeptide amino terminus sequence(s) selected from among Gly-Gly-Phe-Asp- and Phe-Asp-.
Structure of Cross-Linked Telopeptides Derived from Type III Collagen
By analogy to the above disclosure, cross-linked peptides that are derived from proteolysis of human type III collagen may be present in body fluids. These peptides have a core structure embodied in the following parent structures:
FORMULA VIIIGln-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly α1(III)N-telopeptide |Gln-Tys-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly α1(III)N-telopeptide | Gly-Ala-Ala-Gly-Ile-Hyl-Gly-His-Arg α1(III) helical domain 939andFORMULA IXGly-Ile-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-Ala α1(III)C-telopeptide |Gly-Ile-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-Ala α1(III)C-telopeptide |Gly-Phe-Pro-Gly-Met-Hyl-Gly-His-Arg α1(III) helical domain 96where K is hydroxylysyl or lysyl pyridinoline, and Gln is glutamin or | K | Kpyrroldine carboxylic acid.
Cross-linked α1(III) N-telopeptides embodying the Formula VIII core structure found in human urine (and other body fluids) terminate in the C-terminal sequence shown above:
where the parentheses indicate optonal amino acid residues, and where the cross-link to the hydroxylysine shown (or lysine) can be a pyridinoline or other chemical form of cross-link.
A cross-linked peptide derived from type III collagen in body fluids has the core structure:
FORMULA X Asp-Val-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly | Asp-Val-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly |(Gly)-Ile-Hyl-Gly-His-Arg
that is derived from two α1(III)N-telopeptide domains linked to an hydroxylysyl pyridinoline residue (Hyl-Hyl-Hyl).
A possible fragment of the collagen type III C-telopeptide cross-linking domain, based on the collagen types I and II peptides observed in urine, has the core structure:
FORMULA XIGlu-Hyl-Ala-Gly-Gly-Phe|Glu-Hyl-Ala-Gly-Gly-Phe| Hyl
Smaller and larger versions (differing by one to three or more amino acids on each component chain) of these two peptides corresponding to the parent sequences shown above (FORMULAE VIII and IX, particularly on the N-terminus or C-terminus thereof, respectively) may also be present and measurable in body fluids. Analogous smaller and larger versions of each of the peptides disclosed herein form part of the present invention as well.
The invention includes fused cell hybrids (hybridomas) that produce monoclonal antibodies specific for the above-described collagen peptides having 3-hydroxypyridinium cross-links. The subject antibodies preferably bind not only to the disclosed 3-hydroxypyridinium cross-linked structure, but also to related structures having identical amino acid component chains except for having other chemical forms of lysine-derived cross-links, e.g., pyrroles, ketoamines and others, particularly derived from lysyl oxidase-generated aldehydes of the telopeptide lysine or hydroxylysine.
The invention further includes monoclonal antibodies produced by the fused cell hybrids, and those antibodies (as well as binding fragments thereof, e.g. Fab) coupled to a detectable marker. Examples of detectable markers include enzymes, chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic metals, spin labels and radioisotopes.
The invention also includes test kits useful for quantitating the amount of peptides having 3-hydroxypyridinium cross-links derived from collagen degradation in a body fluid. The kits may comprise a monoclonal antibody specific for a peptide derived from degraded collagen and containing a 3-hydroxypyridinium or other form of cross-link. The monoclonal antibodies of the test kits may be coupled to detectable markers such as described above.
FIG. 1 is a depiction of type II collagen and a proposal for the source of telopeptides. It is not established whether the two telopeptides shown come from one collagen molecule as depicted in FIG. 1 or from two collagen molecules.
FIG. 2 shows relative fluorescence (297 nm excitation; 390 nm emission) versus fraction number (4 ml), obtained during molecular sieve chromatographic purification of cross-linked telopeptides. Cross-linked type II collagen telopeptides are contained in the fractions designated II.
FIG. 3A shows relative fluorescence (330 nm excitation, 390 nm emission) versus elution time of fractions during ion exchange HPLC (DEAE-5PW). Cross-linked type II collagen telopeptides are contained in the fraction designated IV.
FIG. 3B shows absorbance (220 nm) versus elution time in minutes for the same chromatogram.
FIG. 4A shows relative fluorescence (297 nm excitation, 390 nm emission) versus elution time of fractions during reverse phase HPLC. Cross-linked type II collagen telopeptides are eluted as indicated. The fractions indicated by the bar (-) show evidence by sequence and composition analysis of the peptides indicated that retain or have lost the gly (G) and pro (P) residues.
FIG. 4B shows absorbance (220 nm) as a function of elution time during reverse phase HPLC.
FIG. 5 compares the concentration of HP and LP in both cortical and cancellous human bone with age.
FIG. 6 depicts the cross-link molar ratios of HP to LP as a function of age.
FIG. 7A shows relative fluorescence (297 nm excitation, >370 nm emission) as a function of elution volume during reverse phase HPLC separation of cross-linked type I collagen N-telopeptides.
FIG. 7B shows relative fluorescence (297 nm excitation, >370 nm emission) versus elution volume during reverse phase HPLC separation of cross-linked type I collagen C-telopeptides.
FIG. 8A shows relative fluorescence (297 nm excitation, >380 nm emission) as a function of elution time for the cross-linked type I collagen telopeptides.
FIG. 8B shows relative fluorescence (297 nm excitation, >380 nm emission) as a function of elution time for the cross-linked type I collagen telopeptides.
FIG. 9 shows relative fluorescence (330 nm excitation, 390 nm emission; bold upper trace) and UV absorbance of peptides (lower trace) versus elution time during ion-exchange HPLC (DEAE-5PW). Cross-linked type II collagen N-telopeptides are contained in the fraction designated II. For comparison, cross-linked type I collagen C-telopeptides are contained in the fraction designated I.
The core peptide structure of the type II collagen peptides may be found in body fluids as a component of larger peptides that bear additional amino acids or amino acid sequences on one or more ends of the three peptide sequences joined by the HP residue. FIG. 1 shows how type II collagen telopeptides, which are linked to a triple-helical sequence, may be produced in vivo from a human source using the proteolytic enzymes pepsin and trypsin. Smaller fragments that have lost amino acids from the core peptide structure, particularly from the helical sequence, may also occur in body fluids. Generally, additions or deletions of amino acids from the core peptide structure will involve from 1 to about 3 amino acids. Additional amino acids will generally be determined by the type II collagen telopeptide sequence that occurs naturally in vivo. As examples, peptides having the following structure:
FORMULA XII Glu-Hyl-Gly-Pro-Asp-Pro-Leu | Glu-Hyl-Gly-Pro-Asp |Gly-Val-HylandFORMULA XIII Glu-Hyl-Gly-Pro-Asp-Pro | Glu-Hyl-Gly-Pro-Asp |Gly-Val-Hyl
can be isolated chromatographically from urine, and another of structure:
FORMULA XIVGlu-Hyl-Gly-Pro-Asp|Glu-Hyl-Gly-Pro-Asp|Val-Hyl
may also be isolated. In addition, glycosylated variants of the core structure and its larger and smaller variants may occur in which a galactose residue or a glucosyl galactose residue are attached to the side chain hydroxyl group of the HP cross-linking residue. Each peak in the graph shown in FIGS. 4A and 4B may correspond to a cross-linked fragment of particular structure that may be quantitated for purposes of the present invention.
These structures are consistent with their site of origin in human type II collagen fibrils at a molecular cross-linking site formed between two α1(II) C-telopeptides and residue 87 of a triple-helical domain, the known sequences about which are:
FORMULA XV 17CGly-Leu-Gly-Pro-Arg-Glu-Hyl-Gly-Pro-Asp-Pro-Leu Human α1(II)C |Gly-Leu-Gly-Pro-Arg-Glu-Hyl-Gly-Pro-Asp-Pro-Leu Human α1(II)C | Gly-Leu-Pro-Gly-Val-Hyl-Gly-His-Arg Human α1(II)H 87
Cross-linked peptides that embody type II collagen N-telopeptides can also be isolated from human body fluids. As a first step, molecular sieve fractions equivalent to pools I and II of FIG. 2 (as described below) are prepared. By further chromatography on a DEAE-HPLC column (DEAE-5PW, Bio-Rad Labs) eluted with a NaCl gradient, a late eluting fraction (pool II, FIG. 9) reveals peptides having the N-terminal sequences Gly-Gly-Phe-Asp-Glu-X-Ala-Gly-Gly or Phe-Asp-Glu-X-Ala-Gly-Gly (where X is a blank sequencing cycle), which are component sequences of cross-linked structures of formula:
FORMULA XVI (Gly)-(Gly)-Phe-Asp-Glu-K-Ala-Gly-Gly-(Ala)-(Gln)-(Leu) | (Gly)-(Gly)-Phe-Asp-Glu-K-Ala-Gly-Gly-(Ala)-(Gln)-(Leu) | Kwhere K is hydroxylsyl pyridinoline (HP) or lysyl pyridinoline (LP), andthe | K | K
parentheses indicate optional amino acid residues. An LP/HP ratio of <1/20 was observed.
The structures are consistent with their site of origin in human type II collagen fibrils at a molecular cross-linking site formed between two α1(II)N-telopeptides and residue 930 of a triple-helical domain, the known sequences about which are
FORMULA XVII 9NGln-Met-Ala-Gly-Gly-Phe-Asp-Glu-K-Ala-Gly-Gly-Ala-Gln-Leu Human α1(II)N |Gln-Met-Ala-Gly-Gly-Phe-Asp-Glu-K-Ala-Gly-Gly-Ala-Gln-Leu Human α1(II)N | Gly-Leu-K-Gly-His-Arg-Gly Human α1(II)H 930
The isolated peptide fragments represent the products of proteolytic degradation of type II collagen fibrils within the body. The core structures containing the HP residue are relatively resistant to further proteolysis and provide a quantitative measure of the amount of type II collagen, and hence cartilage, degraded.
Collagen type II is present in hyaline cartilage of joints in the adult skeleton. Quantitation of the collagen type II telopeptides in a body fluid, for example by way of a monoclonal antibody that recognizes an epitope in the peptide structure, would provide a quantitative measure of whole-body cartilage destruction or remodeling. In a preferred embodiment, the present invention involves an assay for cartilage tissue degradation in humans based on quantifying the urinary excretion rate of at least one member of this family of telopeptides. Such an assay could be used, for example, to:
(1) screen adult human subjects for those individuals having abnormally high rates of cartilage destruction as an early diagnostic indicator of osteoarthritis;
(2) monitor the effects of potential antiarthritic drugs on cartilage metabolism in osteoarthritic and rheumatoid arthritic patients; or
(3) monitor the progress of degenerative joint disease in patients with osteoarthritis and rheumatoid arthritis and their responses to various therapeutic interventions.
Osteoarthritis is a degenerative disease of the articulating cartilages of joints. In its early stages it is largely non-inflammatory (i.e. distinct from rheumatoid arthritis). It is not a single disease but represents the later stages of joint failure that may result from various factors (e.g. genetic predisposition, mechanical overusage, joint malformation or a prior injury, etc.). Destruction of joint articular cartilage is the central progressive feature of osteoarthritis. The incidence of osteoarthritis, based on radiographic surveys, ranges from 4% in the 18-24 year age group to 85% in the 75-79 year age group. At present the disease can only be diagnosed by pain and radiographic or other imaging signs of advanced cartilage erosion.
The assays disclosed above may be used to detect early evidence of accelerated cartilage degradation in mildly symptomatic patients, to monitor disease progress in more advanced patients, and as a means of monitoring the effects of drugs or other therapies.
In normal young adults (with mature skeletons) there is probably very little degradation of cartilage collagen. A test that could measure fragments of cartilage collagen in the urine (and in the blood and joint fluid) would be very useful for judging the "health" of cartilage in the whole body and in individual joints. The type II collagen-specific peptide assays described above will accomplish this. In the long term, such an assay could become a routine diagnostic screen for spotting those individuals whose joints are wearing away. They could be targeted early on for preventative therapy, for example, by the next generation of so-called chondroprotective drugs now being evaluated by the major pharmaceutical companies who are all actively seeking better agents to treat osteoarthritis.
Other diseases in which joint cartilage is destroyed include: rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, the low back pain syndrome, and other infectious forms of arthritis. The type II collagen-specific assays described herein could be used to diagnose and monitor these diseases and evaluate their response to therapy, as disclosed above in connection with osteoarthritis.
Type III Collagen Telopeptides
As pointed out above, human type III collagen telopeptides that may be present in body fluids are expected to have a core structure embodied in the following parent structures:
FORMULA VIII(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly α1(III)N-telopeptide |(Gln)-(Tyr)-(Ser)-(Tyr)-(Asp)-(Val)-Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly α1(III)N-telopeptide | (Gly)-Ile-Hyl-Gly-His-Arg α1(III) helical domain 939andFORMULA IX Gly-Ile-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-Ala α1(III)C-telopeptide | Gly-Ile-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-Ala α1(III)C-telopeptide | Gly-Phe-Pro-Gly-Met-Hyl-Gly-His-Arg α1(III) helical domain 96wherein Hyl | Hyl | Hyl
is hydroxylysyl pyridinoline.
With respect to the type III collagen N-telopeptide metabolites, the subject antibodies preferably bind to:
Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly| Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly| Hylor Hyl-Ser-Gly-Val-Ala-Val-Gly-Gly| Hyl| Hyl
By analogy to the type II peptides, the type III collagen peptides may occur in glycosylated forms of the core structure. For example, galactose residues or glucosylgalactose residues may be attached to the core structure, e.g., by way of hydroxyl groups.
The cross-linking residue of the type III collagen peptides is depicted as a 3-hydroxypyridinium residue, hydroxylysyl pyridinoline. The type II telopeptide structures in urine and cartilage have been found to primarily have hydroxylysyl pyridinoline cross-linking residues. Type III collagen in tissue (e.g., aorta) contains primarily hydroxylysyl pyridinoline.
Type III collagen is present in many connective tissues in association with type I collagen. It is especially concentrated in vascular walls, in the skin and in, for example, the synovial membranes of joints where its accelerated turnover might be observed in inflammatory joint diseases such as rheumatoid arthritis.
A specific assay for type III collagen degradation by quantitating cross-linked type III collagen peptides as disclosed above, can be used for detecting, diagnosing, and monitoring various inflammatory disorders, possibly with particular application to the vasculitis syndromes. In conjunction with assays for measuring bone type I and cartilage type II collagen degradation rates, such an assay could be used as a differential diagnostic tool for patients with various degenerative and inflammatory disorders that result in connective tissue destruction or pathological processes.
Isolation of Type II and Type III Collagen Telopeptides
General Procedure
Urine is collected form a normal adolescent during a rapid phase of skeletal growth. Using a sequence of chromatographic steps that include but are not limited to, adsorption on selective cartridges of a hydrophobic interaction support and an ion-exchange support and molecular sieve, ion-exchange and reverse-phase HPLC column chromatography steps, individual peptides are isolated. The cross-linked peptides containing HP (and LP) residues are detected during column chromatography by their natural fluorescence (Ex max 297 nm<pH 4, Ex max 330 nm, >pH 6; Em max 390 nm). An exemplary isolation procedure is provided in the Example below.
Specific Example
Fresh urine (at 4° C.) diluted 5 times with water and adjusted to 2% (v/v) trifluoroacetic acid, passed through a C-18 hydrophobic binding cartridge (Waters C-18 Sep-pak prewetted with 80% (v/v) acetonitrile then washed with water). Retained peptides were washed with water then eluted with 3 ml of 20% (v/v) acetonitrile, and this eluent was adjusted to 0.05 M NH4 HCO3, 10% (v/v) acetonitrile by addition of an equal volume of 0.1 M NH4 HCO3. This solution was passed through a QMA-Sep-pak (Waters), which was washed with 10 ml of 0.1M NaCl, 20% (v/v) acetonitrile followed by 10 ml of water and the peptides were then eluted with 3 ml of 1% (v/v) trifluoroacetic acid and dried by Speed-Vac (Savant).
Peptides were fractionated in three chromatographic steps. The first step was molecular sieve chromatography on a column of Bio-Gel P-10 (Bio Rad Labs, 2.5 cm×90 cm) eluted by 10% (v/v) acetic acid, monitoring the effluent for HP fluorescence as shown in FIG. 2. In FIG. 2, the Y-axis is the relative fluorescence emission at 390 nm (297 nm excitation), and the X-axis is the fraction number. The fraction size was 4 ml. The fractions indicated as II (and to a lesser extent I) are enriched in the cross-linked collagen type II telopeptides. The cross-linked collagen type I telopeptides are contained in the fractions indicated as III and IV. Fractions spanning pool II (enriched in the type II collagen cross-linked peptides) were combined, freeze-dried and fractionated by ion-exchange column chromatography on a DEAE-HPLC column (TSK-DEAE-5PW, 7.5 mm×7.5 mm, Bio-Rad Labs), equilibrated with 0.02 M Tris/HCl, 10% (v/v) acetonitrile, pH 7.5 and eluted with a gradient of 0-0.5M NaCl in the same buffer, as shown in FIG. 3.
FIG. 3A plots relative fluorescence emission at 390 nm (330 nm excitation) versus elution time. The cross-linked collagen type II telopeptides (C-telopeptides) are found primarily in the segment indicated as IV. FIG. 3B plots absorbance at 220 nm as a function of elution time in minutes. Pool IV contains the type II collagen cross-linked peptides. Individual peptides were then resolved from pool IV by reverse phase HPLC on a C-18 column (Aquapore RP-300, 25 cm×4.6 mm, Brownlee Labs), eluting with a gradient of 0-30% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. FIG. 4A shows a plot of relative fluorescence intensity at 390 nm (297 nm excitation) as a function of elution time. The peaks associated with particular peptides are indicated in FIG. 4A. FIG. 4B shows the relative absorbance at 220 nm as a function of time.
FIG. 9 shows an equivalent chromatogram on DEAE-HPLC of a pool from another P-10 run (peak I/II) from which the collagen type II cross-linked N-telopeptides were isolated. Pool II in this chromatogram (FIG. 9) is equivalent in elution position to pool V shown in FIG. 3A.
Cross-linked peptide fragments of type III collagen containing HP cross-linking residues may be isolated by a similar combination of steps from the urine of normal growing subjects or, for example, from the urine of patients with inflammatory disorders of the vasculature.
Type I Collagen Telopeptides
This aspect of the invention is based on the discovery that both lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptide fragments (i.e., telopeptides, as used herein) derived from reabsorbed bone collagen are excreted in the urine without being metabolized. The invention is also based on the discovery that no other connective tissues contain significant levels of LP and that the ratio of HP to LP in mature bone collagen remains relatively constant over a person's lifetime.
FIG. 5 compares the concentration of HP and LP in both cortical and cancellous human bone with age. It is observed that the concentration of HP plus LP cross-links in bone collagen reaches a maximum by age 10 to 15 years and remains reasonably constant throughout adult life. Furthermore, the ratio of HP to LP, shown in FIG. 6, shows little change throughout life, remaining constant at about 3.5 to 1. These baseline data demonstrate that the 3-hydroxypyridinium cross-links in bone collagen remains relatively constant and therefore that body fluids derived from bone collagen degradation will contain 3-hydroxypyridinium cross-linked peptide fragments at concentrations proportional to the absolute rate of bone resorption.
Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen, the method for determining the absolute rate of bone resorption, in its simplest form, is based on quantitating the concentration of peptide fragments containing 3-hydroxypyridinium cross-links and preferably lysyl pyridinoline (LP) cross-links in a body fluid.
As used in this description and in the appended claims with respect to type I, II, or III telopeptides, by "quantitating" is meant measuring by any suitable means, including but not limited to spectrophotometric, gravimetric, volumetric, coulometric, immunometric, potentiometric, or amperometric means the concentration of peptide fragments containing 3-hydroxypyridinium cross-links in an aliquot of a body fluid. Suitable body fluids include urine, serum, and synovial fluid. The preferred body fluid is urine.
Since the concentration of urinary peptides will decrease as the volume of urine increases, it is further preferred that when urine is the body fluid selected, the aliquot assayed be from a combined pool of urine collected over a fixed period of time, for example, 24 hours. In this way, the absolute rate of bone resorption or collagen degradation is calculated for a 24 hour period. Alternatively, urinary peptides may be measured as a ratio relative to a marker substance found in urine such as creatinine. In this way the urinary index of collagen degradation and bone resorption would remain independent of urine volume.
In one embodiment of the present invention, monoclonal or polyclonal antibodies are produced which are specific to the peptide fragments containing lysyl pyridinoline cross-links found in a body fluid such as urine. Type I telopeptide fragments may be isolated from a body fluid of any patient, however, it is preferred that these peptides are isolated from patients with Paget's disease or from rapidly growing adolescents, due to their high concentration of type I peptide fragments. Type II and type III telopeptides may be isolated from a body fluid of any patient but may be more easily obtained from patients suffering from diseases involving type II or type III collagen degradation or from rapidly growing adolescents.
Isolation of Type I Collagen Telopeptides
Urine from patients with active Paget's disease is dialyzed in reduced porosity dialysis tubing (<3,500 mol. wt. cut off Spectropore) at 4° C. for 48 h to remove bulk solutes. Under these conditions the peptides of interest are largely retained. The freeze-dried non-diffusate is then eluted (200 mg aliquots) from a column (90 cm×2.5 cm) of Bio-Gel P2 (200-400 mesh) in 10% acetic acid at room temperature. A region of effluent that combines the cross-linked peptides is defined by measuring the fluorescence of collected fractions at 297 nm excitation/395 nm emission, and this pool is freeze-dried. Further resolution of this material is obtained on a column of Bio-Gel P-4 (200-400 mesh, 90 cm×2.5 cm) eluted in 10% acetic acid.
Two contiguous fraction pools are defined by monitoring the fluorescence of the eluant above. The earlier fraction is enriched in peptide fragments having two amino acid sequences that derive from the C-terminal telopeptide domain of the α1(I) chain of bone type I collagen linked to a third sequence derived from the triple-helical body of bone type I collagen. These three peptide sequences are cross-linked with 3-hydroxypyridinium. The overlapping later fraction is enriched in peptide fragments having an amino acid sequence that is derived from the N-terminal telopeptide domain of bone type I collagen linked through a 3-hydroxypyridinium cross-links.
Individual peptides are then resolved from each of the two fractions obtained above by ion-exchange HPLC on a TSK DEAE-5-PW column (Bio Rad 7.5 cm×7.5 mm) eluting with a gradient of NaCl (0-0.2M) in 0.02M Tris-HCl, pH 7.5 containing 10% (v/v) acetonitrile. The N-terminal telopeptide-based and C-terminal telopeptide-based cross-linked peptides elute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15M NaCl. The C-terminal telopeptide-based cross-linked peptides elute first as a series of fluorescent peaks, and the major and minor N-terminal telopeptide-based cross-linked peptides elute towards the end of the gradient as characteristic peaks. Each of these is collected, freeze-dried and chromatographed on a C-18 reverse phase HPLC column (Vydac 218TP54, 25 cm×4.6 mm) eluted with a gradient (0-10%) of acetonitrile: N-propanol (3:1 v/v) in 0.01M trifluoroacetic acid. About 100-500 μg of individual peptide fragments containing 3-hydroxypyridinium cross-links can be isolated by this procedure from a single 24 h collection of Paget's urine.
Amino acid compositions of the major isolated peptides confirmed purity and molecular sizes by the whole number stoichiometry of recovered amino acids. N-terminal sequence analysis by Edman degradation confirmed the basic core structures corresponding to the sequences of the known cross-linking sites in type I collagen and from the matching amino acid compositions. The N-terminal telopeptide sequence of the α2(I) chain was blocked from sequencing analysis due presumably to the known cyclization of the N-terminal glutamine to pyrrolidone carboxylic acid.
A typical elution profile of N-terminal telopeptides obtained by the above procedure is shown in FIG. 7A. The major peptide fragment obtained has an amino acid composition: (Asx)2 (Glx)2 (Gly)5 Val-Tyr-Ser-Thr, where Asx is the amino acid Asp or Asn and Glx is the amino acid Gln or Glu. The sequence of this peptide is represented by Formula III below.
The C-terminal telopeptide-based cross-linked peptides resolved by reverse phase HPLC as described above are shown in FIG. 7B. As can be seen from this figure, these peptides are further resolved into a series of C-terminal telopeptides each containing the 3-hydroxypyridinium cross-links. The major peptide, shown in FIG. 7B, was analyzed as described above and was found to have the amino acid composition: (Asp)5 (Glu)4 (Gly)10 (His)2 (Arg)2 (Hyp)2 (Ala)5. The sequence of this peptide is represented by Formula IV below. It is believed that the other C-terminal telopeptide-based cross-linked peptides appearing as minor peaks in FIG. 7B represent additions and deletions of amino acids to the structure shown in Formula IV. Any of the peptides contained within these minor peaks are suitable for use as immunogens as described below.
FORMULA III Asp-Glu-K-Ser-Thr-Gly-Gly |Gln-Tyr-Asp-Gly-K-Gly-Val-Gly | KFORMULA IVAsp-Gly-Gln-Hyp-Gly-Ala | Hyp-Glu-Gly-Lys | Gly-Asp-Ala-Gly-Ala-K-Gly-Asp | Glu-K-Ala-His-Asp-Gly-Gly-Arg | Glu-K-Ala-His-Asp-Gly-Gly-ArgFORMULA V Hyp-Glu-Gly | Gly-Asp-Ala-Gly-Ala-K-Gly-Asp | Glu-K-Ala-His-Asp-Gly-Gly-Arg | Glu-K-Ala-His-Asp-Gly-Gly-ArgandFORMULA VI K | Glu-K-Ala-His-Asp-Gly-Gly-Arg | Glu-K-Ala-His-Asp-Gly-Gly-Argwhere K represents the HP or LP cross-links and Gln represents glutamineor | pyrrolidone carboxylic acid. K | K
Equivalents of the peptides represented by the above structures include those cases where there is some variation in the peptide structure. Examples of such variation include 1-3 amino acid additions to the N and C termini as well as 1-3 terminal amino acid deletions. Smaller peptide fragments of the molecule represented by Formula IV derived from bone resorption are especially evident in urine. These are found in the minor peaks of the C-terminal telopeptide fraction seen in FIG. 7B and can be identified by amino acid composition and sequence analysis.
A. Immunological Procedure for Quantitating Peptides
Immunological binding partners capable of specifically binding to peptide fragments derived from bone collagen obtained from a physiological fluid can be prepared by methods well known in the art. The preferred method for isolating these peptide fragments is described above. By immunological binding partners as used herein is meant antibodies and antibody fragments capable of binding to a telopeptide.
Both monoclonal and polyclonal antibodies specifically binding the peptides disclosed herein and their equivalents are prepared by methods known in the art. For example, Campbell, A. M. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13 (1986). Elsevier, herein incorporated by reference. It is possible to produce antibodies to the above peptides or their equivalents as isolated. However, because the molecular weights of these peptide fragments are generally less than 5,000, it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.
It is well known in the art that the orientation of the hapten, as it is bound to the carrier protein, is of critical importance to the specificity of the antiserum. Furthermore, not all hapten-protein conjugates are equally successful immunogens. The selection of a protocol for binding the particular hapten to the carrier protein therefore depends on the amino acid sequence of the urinary peptide fragments selected. For example, if the peptide represented by Formula III is selected, a preferred protocol involves coupling this hapten to keyhole limpet hemocyanin (KLH), or other suitable carrier, with glutaraldehyde. An alternative protocol is to couple the peptides to KLH with a carbodiimide. These protocols help to ensure that the preferred epitope, namely Tyr and a 3-hydroxy-pyridinium cross-link, are presented to the primed vertebrate antibody producing cells (e.g., B lymphocytes).
Other peptides, depending on the source, may require different binding protocols. Accordingly, a number of binding agents may be suitably employed. These include, but are not limited to, carbodiimides, glutaraldehyde, mixed anhydrides, as well as both homobifunctional and heterobifunctional reagents (see for example the Pierce 1986-87 catalog, Pierce Chemical Co., Rockford, Ill.). Preferred binding agents include carbodiimides and heterobifunctional reagents such as m-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).
Methods for binding the hapten to the carrier molecule are known in the art. See for example, Chard, T., Laboratory Techniques in Biochemistry Molecular Biology, Vol. 6 (1987) Partz Elsevier, N. Y., herein incorporated by reference.
Either monoclonal or polyclonal antibodies to the hapten-carrier molecule immunogen can be produced. However, it is preferred that monoclonal antibodies (MAb) be prepared. For this reason it is preferred that immunization be carried out in the mouse. Immunization protocols for the mouse usually include an adjuvant. Examples of suitable protocols are described by Chard, T. (1987) vida supra. Spleen cells from the immunized mouse are harvested and homogenized and thereafter fused with cancer cells in the presence of polyethylene glycol to produce a fused cell hybrid which produces monoclonal antibodies specific to peptide fragments derived from collagen. Examples of such peptides are represented by the formulas given above. Suitable cancer cells include myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of this procedure, including screening protocols, protocols for growing selected hybrid cells and harvesting monoclonal antibodies produced by the selected hybrid cells are provided in Galfre, G. and Milstein, C., Meth. Enzymol., 73:1 (1981). A preferred preliminary screening protocol involves the use of peptide fragments derived from bone collagen resorption and containing 3-hydroxypyridinium cross-links in a solid phase radioimmunoassay.
The monoclonal antibodies or other immunological binding partners used in connection with the present are preferably specific for a particular type of collagen telopeptide. For example, assays for the type II or type III collagen degradation telopeptides should preferably be able to distinguish between the type I, type II, and type III peptides. However, in some cases, such selectivity will not be necessary, for example, if it is known that a patient is not suffering degradation of one type of collagen but is suspected of suffering degradation from the assayed type of collagen. Because of the differences in amino acid sequences between the type I, type II, and type III families of telopeptides, cross-reactivity should not occur to a significant degree. Indeed, hybridomas can be selected for during the screening of splenocyte fusion clones that produce monoclonal antibodies specific for the cross-linked telopeptide of interest (and lack affinity for those of the other two collagen types). Based on the differences in sequence of the isolated peptide structures, such specificity is entirely feasible. Peptide fragments of the parent types I, II, and III collagens, suitable for such hybridoma screening, can be prepared from human bone, cartilage and other tissues and used to screen clones from mice immunized appropriately with the individual cross-linked peptide antigens isolated from body fluid.
Immunological binding partners, especially monoclonal antibodies, produced by the above procedures, or equivalent procedures, are employed in various immunometric assays to quantitate the concentration of the peptides having 3-hydroxypyridinium cross-links described above. These immunometric assays comprise a monoclonal antibody or antibody fragment coupled to a detectable marker. Examples of suitable detectable markers include but are not limited to: enzymes, coenzymes, enzyme inhibitors, chromophores, fluorophores, chemiluminescent materials, paramagnetic metals, spin labels, and radionuclides. Examples of standard immunometric methods suitable for quantitating the telopeptides include, but are not limited to, enzyme linked immunosorbent assay (ELISA) (Ingvall, E., Meth. Enzymol., 70 (1981)), radio-immunoassay (RIA), and "sandwich" immunoradiometric assay (IRMA).
In its simplest form, these immunometric methods can be used to determine the absolute rate of bone resorption or collagen degradation by simply contacting a body fluid with the immunological binding partner specific to a collagen telopeptide having a 3-hydroxypyridinium cross-link.
It is preferred that the immunometric assays described above be conducted directly on untreated body fluids (e.g. urine, blood, serum, or synovial fluid). Occasionally, however, contaminating substances may interfere with the assay necessitating partial purification of the body fluid. Partial purification procedures include, but are not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography and combinations thereof.
Test kits, suitable for use in accordance with the present invention, contain monoclonal antibodies prepared as described above that specifically bind to peptide fragments having 3-hydroxypyridinium cross-links derived from collagen degradation found in a body fluid. It is preferred that the monoclonal antibodies of this test kit be coupled to a detectable marker of the type described above. Test kits containing a panel of two or more immunological binding partners are also contemplated. Each immunological binding partner in such a test kit will preferably not cross-react substantially with another type of telopeptide. For example, an immunological binding partner that binds specifically with a type II collagen telopeptide should preferably not cross-react with either a type I or type III collagen telopeptide. A small degree (e.g. 5-10%) of cross-reactivity may be tolerable.
B. Electrochemical Procedure for Assaying for Peptides
An alternative procedure for assaying for the above-described peptides consists of measuring a physical property of the peptides having 3-hydroxypyridinium cross-links. One such physical property relies upon electrochemical detection. This method consists of injecting an aliquot of a body fluid, such as urine, into an electrochemical detector poised at a redox potential suitable for detection of peptides containing the 3-hydroxypyridinium ring. The 3-hydroxypyridinium ring, being a phenol, is subject to reversible oxidation and therefore the electrochemical detector (e.g., Model 5100A Coulochem sold by Esa 46 Wiggins Ave., Bedford, Mass.) is a highly desirable instrument for quantitating the concentration of the present peptides. Two basic forms of electrochemical detector are currently available: amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc., Bedford, Mass. 01730). Both are suitable for use in accordance with the present invention, however, the latter system is inherently more sensitive and therefore preferred since complete oxidation or reduction of the analyzed molecule in the column effluent is achieved. In addition, screening or guard electrodes can be placed "upstream" from the analytical electrode to selectively oxidize or reduce interfering substances thereby greatly improving selectivity. Essentially, the voltage of the analytical electrode is tuned to the redox potential of the sample molecule, and one or more pretreatment cells are set to destroy interferents in the sample.
In a preferred assay method, a standard current/voltage curve is established for standard peptides containing lysyl pyridinoline or hydroxylysyl pyridinoline in order to determine the proper voltage to set for optimal sensitivity. This voltage is then modified depending upon the body fluid, to minimize interference from contaminants and optimize sensitivity. Electrochemical detectors, and the optimum conditions for their use are known to those skilled in the art. Complex mixtures of body fluids can often be directly analyzed with the electrochemical detector without interference. Accordingly, for most patients no pretreatment of the body fluid is necessary. In some cases however, interfering compounds may reduce the reliability of the measurements. In such cases, pretreatment of the body fluid (e.g., urine) may be necessary.
Accordingly, in an alternative embodiment of the invention, a body fluid is first purified prior to electrochemically titrating the purified peptide fragments. The purification step may be conducted in a variety of ways including but not limited to dialysis, ion exchange chromatography, alumina chromatography, hydroxyapatite chromatography, molecular sieve chromatography, or combinations thereof. In a preferred purification protocol, a measured aliquot (25 ml) of a 24 hour urine sample is dialyzed in reduced porosity dialysis tubing to remove the bulk of contaminating fluorescent solutes. The non-diffusate is then lyophilized, redissolved in 1% heptafluorobutyric acid (HFBA), an ion pairing solution, and the peptides adsorbed on a Waters Sep-Pak C-18 cartridge. This cartridge is then washed with 5 ml of 1% HFBA, and then eluted with 3 ml of 50% methanol in 1% HFBA.
Another preferred method of purification consists of adsorbing a measured aliquot of urine onto an ion-exchange adsorption filter and eluting the adsorption filter with a buffered eluting solution. The eluate fractions containing peptide fragments having 3-hydroxypyridinium cross-links are then collected to be assayed.
Still another preferred method of purification employs molecular sieve chromatography. For example, an aliquot of urine is applied to a Bio-Gel P2 or Sephadex G-20 column and the fraction eluting in the 1000-5000 Dalton range is collected. It will be obvious to those skilled in the art that a combination of the above methods may be used to purify or partially purify urine or other body fluids in order to isolate the peptide fragments having 3-hydroxypyridinium cross-links. The purified or partially purified peptide fragments obtained by the above procedures may be subjected to additional purification procedures, further processed or assayed directly in the partially purified state. Additional purification procedures include resolving partially purified peptide fragments employing high performance liquid chromatography (HPLC) or microbore HPLC when increased sensitivity is desired. These peptides may then be quantitated by electrochemical titration.
A preferred electrochemical titration protocol consists of tuning the redox potential of the detecting cell of the electrochemical detector (Coulochem Model 5100 A) for maximum signal with pure HP. The detector is then used to monitor the effluent from a C-18 HPLC column used to resolve the partially purified peptides.
C. Fluorometric Procedure for Quantitating Peptides
An alternative preferred method for quantitating the concentration of peptides having 3-hydroxypyridinium cross-links as described herein is to measure the characteristic natural fluorescence of these peptides. For those body fluids containing few naturally occurring fluorescent materials other than the 3-hydroxypyridinium cross-links, fluorometric assay may be conducted directly without further purification of the body fluid. In this case, the peptides are resolved by HPLC and the natural fluorescence of the HP and LP amino acid residues is measured at 395 nm upon excitation at 297 nm, essentially as described by Eyre, D. R., et al., Analyte. Biochem. 137:380 (1984), herein incorporated by reference.
It is preferred, in accordance with the present invention, that the fluorometric assay be conducted on urine. Urine, however, usually contains substantial amounts of naturally occurring fluorescent contaminants that must be removed prior to conducting the fluorometric assay. Accordingly, urine samples are first partially purified as described above for electrochemical detection. This partially purified urine sample can then be fluorometrically assayed as described above. Alternatively, the HP and LP cross-linked peptides in the partially purified urine samples or other body fluids can be hydrolyzed in 6M HCl at about 108° C. for approximately 24 hours as described by Eyre, et al. (1984) vida supra. This process hydrolyzes the amino acids connected to the lysine precursors of "tripeptide" HP and LP cross-links, producing the free HP and LP amino acids represented by Formulae I and 11. These small "tripeptides" are then resolved by the techniques described above, preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm, Ex 390 nm).
Optionally, the body fluid (preferably urine) is passed directly through a C-18 reverse phase affinity cartridge after adding acetonitrile/methanol 5 to 10% V/V. The non-retentate is adjusted to 0.05-0.10M with a cationic ion-pairing agent such as tetrabutyl ammonium hydroxide and passed through a second C-18 reverse phase cartridge. The washed retentate, containing fluorescent peptides, from this second cartridge is eluted with acetonitrile:water (or methanol:water), dried and fluorescent peptides are analyzed by reverse phase HPLC or microbore HPLC using an anionic ion-pairing agent such as 0.01M trifluoroacetic acid in the eluant.
FIG. 8A displays the elution profile resolved by reverse phase HPLC of natural fluorescence for a hydrolysate of peptide fragments from normal human urine. Measurement of the integrated area within the envelope of a given component is used to determine the concentration of that component within the sample. The ratio of HP:LP found in normal human urine and urine from patients having Paget's disease, FIG. 8B, are both approximately 4.5:1. This is slightly higher than the 4:1 ratio found in bone itself (Eyre, et al., 1984). The higher ratio found in urine indicates that a portion of the HP fraction in urine may come from sources other than bone, such as the diet, or other sources of collagen degradation, i.e., cartilage catabolism. It is for this reason that it is preferred that LP which derives only from bone be used to provide an absolute index of bone resorption. However, in the absence of excessive cartilage degradation such as in rheumatoid arthritis or in cases where bone is rapidly being absorbed, HP or a combination of HP plus LP may be used as an index of bone resorption.
While the invention has been described in conjunction with preferred embodiments, one of ordinary skill after reading the foregoing specification will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.
|Patente citada
|Fecha de presentación
|Fecha de publicación
|13 Abr 1979
|26 Ene 1982
|Radioimmunological determination of procollagen (type III) and procollagen peptide (type III)
|11 Mar 1983
|12 Mar 1985
|Hoechst Aktiengesellschaft
|Procedure for immunologic determination of procollagen peptide (type III) and procollagen peptide col 1 (type III) together and a process for preparing anti-procollagen peptide col 1 (type III) serum
|2 May 1988
|30 Ago 1994
|Orion-Yhtyma Oy
|Type III collagen degradation assay
|Título no disponible
|10 Mar 1983
|21 Sep 1983
|Hoechst Aktiengesellschaft
|Highly specific antiserum against Procollagen-peptide Col 1 (Type III),and Procollagen-peptide (Type III), its preparation and use thereof.
|27 Abr 1988
|9 Nov 1988
|Hoechst Aktiengesellschaft
|Monoclonal antibody for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids
|27 Abr 1988
|11 Ene 1989
|Hoechst Aktiengesellschaft
|Method for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids and reagents used therefor
|17 Ago 1988
|22 Feb 1989
|Orion-Yhtymä Oy
|Antibody to propeptide of procollagen type III, and assay method using it
|19 Abr 1989
|2 Nov 1989
|Hoechst Aktiengesellschaft
|Monoclonal antibody for the selective immune determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids
|Título no disponible
|2 May 1988
|17 Nov 1988
|Farmos-Yhtymä Oy
|Type iii collagen degradation assay
|Ala Kokko, L. et al., Structure of cDNA clones coding for the entire prepro 1(III) chain of human type III precollagen, Biochem. J. 260:509 516 (1989).
|Ala-Kokko, L. et al., "Structure of cDNA clones coding for the entire preproα1(III) chain of human type III precollagen," Biochem. J. 260:509-516 (1989).
|Eyre, D.R. et al., "Cross-Linked Telopeptides From Collagen Types I, II, and III in Human Urine," ASBMR Meeting, Sep. 1996.
|Eyre, D.R. et al., Cross Linked Telopeptides From Collagen Types I, II, and III in Human Urine, ASBMR Meeting, Sep. 1996.
|Eyre, D.R., "Cross-Linking in Collagen and Elastin," Ann. Rev. Biochem. 53:717-748 (1984).
|Eyre, D.R., Cross Linking in Collagen and Elastin, Ann. Rev. Biochem . 53:717 748 (1984).
|Gosslau, B. and Barrach, H. J., Enzyme Linked Immunosorbent Microassay for Quantification of Specific Antibodies to Collagen Type I, II, III, Immunol. Meth . 29:71 77 (1979).
|Gosslau, B. and Barrach, H.-J., "Enzyme-Linked Immunosorbent Microassay for Quantification of Specific Antibodies to Collagen Type I, II, III," Immunol. Meth. 29:71-77 (1979).
|Hanson, D.A. and Eyre, D.R., "Molecular Site Specificity of Pyridinoline and Pyrrole Cross-links in Type I Collagen of Human Bone," J. Biol. Chem. 271(43):26508-26516 (1996).
|Hanson, D.A. and Eyre, D.R., Molecular Site Specificity of Pyridinoline and Pyrrole Cross links in Type I Collagen of Human Bone, J. Biol. Chem . 271(43):26508 26516 (1996).
|Hanson, D.A. et al., "A Specific Immunoassay for Monitoring Human Bone Resorption: Quantitation of Type I Collagen Cross-linked N-Telopeptides in Urine," J. Bone and Min. Res. 7(11):1251-1258 (1992).
|Hanson, D.A. et al., A Specific Immunoassay for Monitoring Human Bone Resorption: Quantitation of Type I Collagen Cross linked N Telopeptides in Urine, J. Bone and Min. Res . 7(11):1251 1258 (1992).
|Hasselblach, H. et al., "Procollagen Type III Aminoterminal Peptide in Serum in Idiopathic Myelofibrosis and Allied Conditions: Relation to Disease Activity and Effect of Chemotherapy," Am. J. Hematol. 33:16-26 (1990).
|Hasselblach, H. et al., Procollagen Type III Aminoterminal Peptide in Serum in Idiopathic Myelofibrosis and Allied Conditions: Relation to Disease Activity and Effect of Chemotherapy, Am. J. Hematol . 33:16 26 (1990).
|Horslev Petersen, K., Circulating extracellular matrix components as markers for connective tissue response to inflammation, Danish Med. Bul . 37(4):308 329 (1990).
|Horslev-Petersen, K., "Circulating extracellular matrix components as markers for connective tissue response to inflammation," Danish Med. Bul. 37(4):308-329 (1990).
|Kuhn, K., "The Classical Collagens: Types I, II, and III," In Structure and Function of Collagen Types, Academic Press, Inc., pp. 1-42 (1987).
|Kuhn, K., The Classical Collagens: Types I, II, and III, In Structure and Function of Collagen Types , Academic Press, Inc., pp. 1 42 (1987).
|Lane, J.M. et al., "Immunofluorescent Localization of Structural Collagen Types in Endochondral Fracture Healing," Orthopaedic Transactions VI(2):236 (1982).
|Lane, J.M. et al., Immunofluorescent Localization of Structural Collagen Types in Endochondral Fracture Healing, Orthopaedic Transactions VI(2):236 (1982).
|Loid, H.R. et al., "Molecular cloning and carboxyl-propeptide analysis of human type III procollagen," Nucleic Acids Research 12(24):9383-9394 (1984).
|Loid, H.R. et al., Molecular cloning and carboxyl propeptide analysis of human type III procollagen, Nucleic Acids Research 12(24):9383 9394 (1984).
|Macarak, E.J. et al., "Production and characterization of a monoclonal antibody to human type III collagen," J. Histochem, Cytochem. 34(8):1003-1011 (1986), Chem. Abstracts, 105:95762y (1986).
|Macarak, E.J. et al., Production and characterization of a monoclonal antibody to human type III collagen, J. Histochem, Cytochem . 34(8):1003 1011 (1986), Chem. Abstracts , 105:95762y (1986).
|Myllyla, R. et al., "Markers of collagen metabolism in sera of patients with various rheumatic diseases," Clinica Chimica Acta 183:243-252 (1989).
|Myllyla, R. et al., Markers of collagen metabolism in sera of patients with various rheumatic diseases, Clinica Chimica Acta 183:243 252 (1989).
|Niemela, O. et al., "Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum," Clinica Chimica Acta 124:39-44 (1982).
|Niemela, O. et al., Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum, Clinica Chimica Acta 124:39 44 (1982).
|Niemela, O., "Radioimmunoassays for Type III Procollagen Amino-Terminal Pepetides in Humans," Clin. Chem. 31(8):1301-1304 (1985).
|Niemela, O., Radioimmunoassays for Type III Procollagen Amino Terminal Pepetides in Humans, Clin. Chem . 31(8):1301 1304 (1985).
|Otter, A. et a l., "Conformational analysis of the type II and III collagen α-1 chain N-telopeptides by proton-NMR spectroscopy and restrained molecular mechanics calculations," Biopolymers 33(9):1443-1459 (1993), Chem. Abstracts, 199:264860a (1993).
|Otter, A. et a l., Conformational analysis of the type II and III collagen 1 chain N telopeptides by proton NMR spectroscopy and restrained molecular mechanics calculations, Biopolymers 33(9):1443 1459 (1993), Chem. Abstracts , 199:264860a (1993).
|Pierard, D. et al., "Radioimmunoassay for the Amino-Termianl Sequences of Type III Procollagen in Human Body Fluids Measuring Fragmented Precursor Sequences," Analytical Biochem. 141:127-136 (1984).
|Pierard, D. et al., Radioimmunoassay for the Amino Termianl Sequences of Type III Procollagen in Human Body Fluids Measuring Fragmented Precursor Sequences, Analytical Biochem . 141:127 136 (1984).
|Rohde, H. et al., "Radioimmunoassay for type III procollagen peptide and its application to human liver disease," Euro. J. Clin. Invest. 9:451-459 (1979).
|Rohde, H. et al., Radioimmunoassay for type III procollagen peptide and its application to human liver disease, Euro. J. Clin. Invest . 9:451 459 (1979).
|Seibel, M.J., "Komponenten extrazellularen Gewebematrix als potentielle "Marker" des Bindegewebs-, Knorpel- und Knochenmetabolismus bei Erkrankungen des Bewegungsapparates," Z. Rheumatol. 48:6-18 (1989).
|Seibel, M.J., Komponenten extrazellul a ren Gewebematrix als potentielle Marker des Bindegewebs , Knorpel und Knochenmetabolismus bei Erkrankungen des Bewegungsapparates, Z. Rheumatol . 48:6 18 (1989).
|Stoltenberg, M. et al., "Intra-articular and circulating levels of type I and III collagen markers in inflammatory and degenerative joint disease," Acta. Orthop. Scand. 66(Suppl 266):147-148 (1995).
|Stoltenberg, M. et al., Intra articular and circulating levels of type I and III collagen markers in inflammatory and degenerative joint disease, Acta. Orthop. Scand . 66(Suppl 266):147 148 (1995).
|Patente citante
|Fecha de presentación
|Fecha de publicación
|18 Jul 2005
|3 Ago 2010
|Medtronic, Inc.
|Medical devices and methods for reducing localized fibrosis
|16 Jun 2009
|21 Abr 2015
|Abraham Amir
|Monitoring skin metabolism products for evaluating burn injury
|4 Nov 2008
|7 Jun 2016
|Nordic Bioscience A/S
|Biochemical markers for CVD risk assessment
|18 Jul 2005
|9 Feb 2006
|Medtronic, Inc.
|Medical devices and methods for reducing localized fibrosis
|18 Jul 2005
|9 Feb 2006
|Medtronic, Inc.
|Methods for reducing or preventing localized fibrosis using SiRNA
|16 Ene 2009
|23 Jul 2009
|Ogle Matthew F
|Diagnostic biomarkers for vascular aneurysm
|16 Jun 2009
|23 Jun 2011
|Abraham Amir
|Monitoring skin metabolism products for evaluating burn injury
|4 Nov 2008
|21 Jul 2010
|Nordic Bioscience A/S
|Biochemical markers for cvd risk assessment
|Clasificación de EE.UU.
|435/7.1, 436/518, 435/331, 530/389.1, 435/975
|Clasificación internacional
|Clasificación cooperativa
|Clasificación europea
|24 Jun 2003
|Fee payment
Year of fee payment: 4
|8 Jun 2007
|Fee payment
Year of fee payment: 8
|10 Jun 2010
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF WASHINGTON, CENTER FOR COMMERCIALIZATION;REEL/FRAME:024505/0879
Effective date: 20100602
|8 Ago 2011
|Maintenance fee reminder mailed
|4 Ene 2012
|Lapse for failure to pay maintenance fees
|21 Feb 2012
|Expired due to failure to pay maintenance fee
Effective date: 20120104


text: Demo is out. Presentation is nice and the gameplay is a bit improved, but it still feels very similar to the last one. I hate how they don't let you do rematches so you have to go through the game menu and the slideshow if you want to play again.

subset: commoncrawl


text:
DeLAND, Fla. - DeLand police arrested a 21-year-old man on Thursday after an investigation revealed he was attempting to lure a 14-year-old boy to engage in sex acts.
Maxwell Arturo Gonzalez, of Orange City, has been charged with traveling to seduce/solicit/lure or entice a child and use a computer to seduce/solicit/lure or entice a child.
The victim's mother first alerted detectives when her older son saw an older man leaving their house on Tuesday. The mother then found out her 14-year-old son was communicating with Gonzalez about sex.
Police said Gonzalez was arrested after he went to the victim's home to engage in sex acts, where police were waiting to arrest him.
"Any circumstances when an adult interacts with a child inappropriately we take seriously," said DeLand police Sgt. Chris Estes. "It's an outrage to the community and we're working real hard on it."
Doreen Berkie of Orange City lives a few doors down from Gonzalez and said she's never felt comfortable around him.
"In one way it doesn't surprise me, and in another way it just makes me sick," she said.
Police believe there could be additional victims and urge anyone with information to contact police at 386-736-5999.
Gonzalez was booked into the Volusia County jail on $10,000 bond. According to FDLE records, Gonzalez was previously arrested in 2005 for trying to make an explosive device.
Copyright 2013 by ClickOrlando.com. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


text:
The Ambassador of Ireland to the United States will kick off the 12th Annual European Union Day with the "State of the European Union" address, Friday, February 15, at 10 am in the University of Illinois' Alice Campbell Alumni Center Ballroom.
His Excellency Michael Collins will provide his perspective on the present and future direction of the 27-member European Union during EU Day's keynote address, offering insight on timely topics such as the ongoing euro crisis and EU-U.S. relations. Ireland currently holds the rotating presidency of the Council of the European Union. Before assuming his current role, Collins served as second secretary general in the government department of the prime minister of Ireland, with responsibility for international and European Union affairs and particularly the Northern Ireland Peace Process. Prior to that, he was appointed Ireland's Ambassador to the Czech Republic and Ukraine.
This event is followed by a "Visions for a Future Europe" panel discussion with members of the Chicago Consular Corps at 2:00 PM.
This event is free and open to the public.


text:
American Heart Association http://www.americanheart.org
Heart Information Network http://www.heartinfo.org
Canadian Cardiovascular Society http://www.ccs.ca/home/index%5Fe.aspx
Canadian Family Physician http://www.cfpc.ca/cfp/
Aortic regurgitation. American Heart Association website. Available at:
Accessed on December 5, 2002.
Aortic valve disease: aortic regurgitation. The Merck Manual website. Available at:
. Accessed on December 15, 2002
Cheitlin M. Surgery for chronic aortic regurgitation: when should it be considered?
Am Fam Physician
. 2001;64(10).
Tarasoutchi F, et al. Symptoms, left ventricular function, and timing of valve replacement surgery in patients with aortic regurgitation.
Am Heart J
. 1999;138:477-485.
This content is reviewed regularly and is updated when new and relevant evidence is made available. This information is neither intended nor implied to be a substitute for professional medical advice. Always seek the advice of your physician or other qualified health provider prior to starting any new treatment or with questions regarding a medical condition.
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text:
Part of the reason was Washington's defense, ranked first in the Pac-12 in pass defense as well as total defense.
When Stanford (3-0 in the Pac-12, 5-0 overall) travels to Salt Lake City to take on Utah (0-2, 3-2) on Saturday (3 p.m.), Hogan will be going up against the conference's 10th-ranked defense.
"There's a pit in his stomach," Cardinal coach David Shaw said. "He can't wait to play the next game. He's pushing himself to be as good as he can be."
In terms of his experience, it's as though Hogan were nearing the end of his first full season as a quarterback. It's been a high-profile experience to be sure, which includes sparkling efforts in winning the Pac-12 championship, the Rose Bowl and leading the charge against Oregon.
"He's going to go through a couple more growing pains," Shaw said. "He's not a finished product. He's going to have a couple more outstanding games and he will have a couple more rough games. I see a guy who is not satisfied."
Hogan has thrown an interception in each of his past four games and he also has 11 touchdown passes. He's ranked 10th in the conference in passing yardage per game, but he's also ranked fourth in passing efficiency.
There's plenty of upside with Hogan, who can still direct a game with the best of them. That's another quality Shaw admires.
"Washington did a great job of pass coverage," Shaw said. "Kevin missed four throws, including one on fourth down. We were terrible on third downs."
Hogan likely will rebound against the Utes, who do present a physical defensive line.
"They are hard to move and they are hard to block," Shaw said.
Utah is ranked fourth in rushing defense while Stanford is fifth in rushing offense. Watch for Hogan to dissect the Utes' secondary.
Ty Montgomery, named the Pac-12 Special Teams Player of the Week, has been effective as a wide receiver and at kickoff returns all season. He seems to be on a steady rise. He averages 76.6 receiving yards a game on just 23 receptions. He gains an average of 16.7 yards a catch, fifth in the conference.
Montgomery was the difference in the game against Washington. He returned the opening kickoff for a touchdown, raced 79 yards with another kickoff and caught a 39-yard touchdown pass.
"We know how important all three phases of the game are," Montgomery said. "Every kickoff return, I can do what I do because of the blocking."
Defensively, the Cardinal has had a few problems against the passing game, though they've already played against three of the top six passing offenses in the Pac-12.
Utah is ranked in the bottom half, though quarterback Travis Wilson averages 281.2 yards a game and has thrown 11 touchdown passes. He's also thrown nine interceptions, second most in the Pac-12. Dres Anderson ranks fifth in the conference with 102.0 receiving yards per contest.
"They have an elite receiver," Stanford linebacker A.J. Tarpley said. "Their quarterback is not afraid to throw the ball and they have a big offensive line. It will be a challenge."
Tarpley saw his playing time suddenly increase two years ago, when Shayne Skov was hurt and declared out for the season. Since then he has developed into one of the top linebackers on the West Coast.
"He's always in the right place," Shaw said. "He doesn't miss tackles and we trust him with everything. He's an NFL player."
Tarpley certainly showed he was a premiere linebacker, earning a starting spot even as Skov returned to the lineup.
"Being a senior I have changed a lot," Tarpley said. "I've grown and I feel more like a veteran."
He's also part of the punt coverage team, something that gives him a lot of pleasure. He's been part of most every special teams unit while at Stanford.
"We (the linebackers) want to be the core of special teams," Tarpley said. "We want to get out, make tackles and force turnovers. We feel special teams is a third of the game and everybody out there takes pride in that."
Stanford makes its first trip to the state of Utah since 2003, and its first trip to Salt Lake City since 1995. The Cardinal and Utah first met in 1902, in Salt lake City.
Stanford has played Utah fewer times (5) than any other Pac-12 opponent.
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text:
Gwen Stefani and Gavin Rossdale Father's Day Pictures
Gwen and Gavin Celebrate Father's Day With the Boys and A Date Night
Yesterday was a family-filled Father's Day for Gavin Rossdale, who spent time with wife Gwen Stefani and their boys, Kingston and Zuma, at her parents' home in LA. Zuma was all smiles as he ran ahead of his mom and dad on the way into his grandparents' house. Later that evening, Gwen and Gavin paired off for a date night. They were hand in hand as they strolled into a restaurant to continue the celebrating. Gavin shared his Father's Day wishes, tweeting, "Happy Father's Day everyone and thanks for all your messages." The Stefani-Rossdales were just one of the famous families spending the holiday together — see how more stars spent Father's Day.
Gwen has been spending much of her time with her boys, when she isn't in the studio with No Doubt. The release of the group's comeback album is just three months away, and Gwen will hit the stage with her band multiple times this Summer.


text: Well, with Christmas just over, I thought it was time to give one final nod to our favorite Christmas specials--any movie or TV show that you love watching, year after year, at Christmas time. I'll allow any genre, as long as there is a Christmas theme to it somehow. So Die Hard, Doctor Who Specials? Sure. True Grit? No, even if you watch it every Christmas. Standard avatar rules apply. I'll try to get the poll up on January 2nd. I'll get us started, with one of the movies I watch every year, It's a Wonderful Life.


text:
Exclusive Video: Flying the Zeppelin NT with Airship Ventures
Original, Exclusive Videos from AVweb | Reader-Submitted & Viral Videos
Pilots who signed up for sightseeing flights over San Francisco in the new Zeppelin NT kept asking the crew if they could take a turn in the front seat. Tired of having to say no, the company created a special day-long program just for pilots. AVweb's Mary Grady tried it out during a session in Long Beach last month, and here is her report.
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text:
Nicole C. Mullen
Genre: R&B
Styles: Funk, Neo-soul, Contemporary Christian
Location: Nashville, Tn.
Label: Word Records
Website: NicoleCMullen.com
Christian label: yes
Category: The Spiritual whats this?
Nicole C. Mullen is a peculiar artist. Her music is a mix of Contemporary Christian Music anthems such as the Dove award winning "On My Knees", "Redeemer" and "Come Unto Me"; African, funk, R&B raves such as "This This", "Freedom", "Under The Shadow"; and Neo-soul grooves like "Dancin' in The Rain" and "Color". Nicole C. Mullen is a highly sought after songwriter, choreographer, author and speaker.
Why is our site, which covers underground Christian music, covering an eight-time Dove award winner? Nicole C. Mullen is one of the rare "Christian" artists who can sing in front of people who know nothing about Jesus Christ and leave them wanting to know Him in a close and intimate way through her well-crafted, stylish songs.
Nicole C. Mullen's cds are as musically diverse as anyone on today's music scene. Her soft R&B-twinged voice and a relaxed soulful phrasing style is the central core that runs through every song. Some of Nicole C. Mullen's songs feature heavy African instrumentation and rhythm progressions. Many of Nicole C Mullen's songs are lavishly orchestrated anthems that are sung in churches throughout America. Experience both in this long video where she performs both "Redeemer" and "Witness".
Nicole C. Mullen recently said, "I love writing about reality from a Christian's point-of-view. I believe that we as believers are called to make an impact on our culture. We are compelled to do that," she says. "We as believers are called to give light to the world and we can't give light by hiding. We've got to let those around us know we are believers in Jesus Christ, so I do my best to do that in my songs." She expresses that philosophy in her music as well. Read the lyrics from "Blowing Kisses/Love Divine":
Walkin' down Broadway I saw a mom and her baby She came up to me And asked me for a dime I said now come on I'll take you to this diner Gave her more than food It cost me 6 o' 5
People don't need just money, people need love and time Spent a little time with the hurting honey and got myself love-a-fied Now I'm
(Chorus) Blowin' kisses to the one I love Blowin' kisses to the father above Helpin' others to feel His touch Blowin' kisses to the one I love
Well, she said their names Were Rachel and Little Nikki I said, "Hey, that's strange Cause Niki's my name too" As she turned to go She spoke the words of blessing With an angel's voice, she said "I see the Christ in you"
Nicole C Mullen uses her talents and fame to reach to the world as well. She established a mentor group for girls called "The Baby Girls Club". She and her husband, David Mullen, are youth leaders in their local church, tutor inner-city youth, and participate in Kids Across America summer camp( http://kidsacrossamerica.org/) for inner city youth. Nicole C. Mullen is also active with the International Needs Network Ghana, an organization that works to free Trokosi slaves in Ghana. Here she is creating a place for these horribly abused female Trokosi slaves in a few minutes of joyful singing
Nicole C Mullen is an important R&B artist who sings about Christ. Nicole C. Mullen is a vital Christian music artist whose songs fill churches across America every Sunday. Nicole C. Mullen is a fun recording artist who makes dancable, singable songs. Enjoy.
Download Nicole C. Mullen's entire libaray of music here
Or you can order Nicole C. Mullen cds from Amazon by clicking on the links below. I would suggest the Christams cd as a must have.
Redeemer: The Best of Nicole C. Mullen
A Dream To Believe In, Vol. II
Live From Cincinnati: Bringing It Home


text:
I was asked to be one of the five judges at the Miss LifeWalk 2011 contest, which took place Sunday night at the Round-Up. It's the first time I've judged a drag show, but the talent was evident — not only from the contestants, but from the other entertainers. Jenna Skyy gave a rousing performance (before she sat down alongside me to judge, with Dr. Bill Henderson, John Loza and Dallas Voice Ultimate Diva! Stacey McKinney), while Iona Trailer camped it up hilariously. One of the remarkable discoveries was former Voice of Pride Jason Huff, who donned a dress and gave a roaring church-revival-esque performance late in the show.
The crowd responded. Money was free-flowing for all three contestants, with Lotta Pink — who bested runners-up Heather Thomas and Mowlawn Rouge — in total money raised (before she even entered the club, she came with $7,000 in benefits for the LifeWalk). Lotta won the whole she-bang, largely as a result of having elaborate choreography (including dancing boys, pictured) during the talent portion.
Keeping everything going were co-hosts Ima Lush and Victoria Weston (who sang without lip-synching).
(Watch some reader video of Lotta Pink performing Pink's "Raise Your Glass" after the jump.)


text:
FAWN GROVE, Pa. (AP) – Eight horses have been removed from a central Pennsylvania farm and animal welfare officials say their owner will face animal neglect charges.
The horses were removed from a property near Fawn Grove after York County Humane Society officials were called to the property and said some of the animals were hundreds of pounds underweight.
The owner of the horses voluntarily relinquished their care to the York County Society for the Prevention of Cruelty to Animals.
York County SPCA executive director Melissa Smith said the horses are very thin and suffering from hoof problems, skin disease and open wounds. A ninth horse – a 4-month-old foal – was found dead.
Several local farms are fostering a few of the horses, while others went to one of two area animal rescues.
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