Total Urinary Hydroxyproline Research Articles

Urinary collagen peptides: Source of markers for bone metabolic processes in kidney transplant recipients.

Kidney transplant recipients (KTRs) are at an increased risk of fractures. Total urinary hydroxyproline excretion served as marker for bone resorption (BR) but was replaced by β-CrossLaps (CTX), a C-terminal collagen α-1(I) chain (COL1A1) telopeptide. We investigated the low-molecular-weight urinary proteome for peptides associated with changes in bone metabolism after kidney transplantation. Clinical and laboratory data including serum levels of CTX in 96 KTR from two nephrology centers were correlated with signal intensities of urinary peptides identified by capillary electrophoresis mass spectrometry. Eighty-two urinary peptides were significantly correlated with serum CTX levels. COL1A1 was the predominant peptide source. Oral bisphosphonates were administered for decreased bone density in an independent group of 11 KTR and their effect was evaluated on the aforementioned peptides. Study of the peptides cleavage sites revealed a signature of Cathepsin K and MMP9. Seventeen of these peptides were significantly associated with bisphosphonate treatment, all showing a marked reduction in their excretion levels compared to baseline. This study provides strong evidence for the presence of collagen peptides in the urine of KTR that are associated with BR and that are sensitive to bisphosphonate treatment. Their assessment might become a valuable tool to monitor bone status in KTR.

A STUDY ON STATUS OF URINARY HYDROXYPROLINE IN POST MENOPAUSAL WOMEN

Introduction: WHO and the Stages of Reproductive Aging Workshop have defined menopausal transition as the time of an increase in follicle-stimulating hormone and either increased variability in menstrual cycle length, two skipped menstrual cycles with 60 days or more of amenorrhea, or both. It concludes with the final menstrual period. Post-menopause begins at that time, although it is not recognized until after 12 months of amenorrhea. During menopause, women face various physiological, psychological, and biochemical changes. Laboratory medicine has given a new background to overcome the clinicianÂÂs diagnostic dilemma. Hydroxyproline is mainly found in collagen and accounts for 13% of total amino acid content and derived from proline by post-translational hydroxylation. Hydroxyproline is derived from another amino acid such as proline. Direct urinary assay of hydroxyproline to measure bone resorption have clinical applications as part of screening programs to assess the risk of osteoporotic fractures. Method: A total of seventy patients with regular medical follow-up records, The Patients were pre and post-menopausal women (35 each) recruited for this study. Patient details like body mass index, education, smoking, alcohol intake, dietary habits, and family history were considered before selecting the patients. Analytical Methods: Urinary Hydroxyproline and Urinary creatinine was estimated by Modified Neumann et al and Spectrophotometric JaffeÂÂs reaction respectively. Result: The study population consisted of 70 participants of premenopausal (n=35) and postmenopausal women (n=35), mean age of 38.11 ± 4.3 and 54.40 ± 4.6 respectively. The bone mineralization marker urinary total hydroxyproline was quantified in pre and post-menopausal women, which is 80.3 ± 75mg/L and 136 ± 103mg/L respectively. The urinary creatinine level in pre and post-menopausal women was 53.7 ± 14.2mg/dL and 37.0 ± 27.3 respectively. The hydroxyproline: creatinine ratio (HCR) was 41% to 69% against the normal reference interval in pre and post-menopausal women. Conclusion: The obtained normative data for the premenopausal woman population would be a new reference range in Indian sub-population or otherwise general population normative reference range commonly being used as a reference interval in all kind of pathophysiological disorders. Hence, the derived parameter confirmed that HCR is the most prognostic significant diagnostic marker in pre and post-menopausal patients.

Changes in biochemical markers in blood and urine in case of malunion and nonunion after fracture of long bones

Background: Biochemical markers provide a dynamic view of the remodeling process of bone. Aims: To monitor biochemical markers in long bone fractures as they reflect the actual status of fracture healing. Materials and Methods: The present study after taking Institutional ethical clearance and informed consent of the subjects. Patients with normal fracture union served as controls (50); Patients with impaired fracture union served as cases. They were further divided into two groups: Fracture nonunion (20) and fracture malunion (30). Serum calcium, serum alkaline phosphatase (ALP), urinary calcium to creatinine ratio, and urinary total and free hydroxyproline were measured. Results: Serum ALP, serum calcium, urinary total and free hydroxyproline, and urinary calcium to creatinine ratio were increased in all patients after fracture. Serum ALP, urinary total and free hydroxyproline decreased after treatment in nonunion patients. Serum ALP reached to lower level of reference interval in this group. But urinary total and free hydroxyproline decreased to less than normal level. Serum calcium increased up to 2 weeks following treatment and then reduced to normal level within 1-month in fracture normal union and malunion groups. Serum calcium reduced at a very slow rate after treatment in nonunion patients and remained within reference interval. Significant differences were found between normal union and nonunion groups in case of serum ALP, urinary total and free hydroxyproline levels after treatment. Significant differences were found between normal union and nonunion groups in serum calcium level after treatment on day 7 and 14. Conclusions: Serial monitoring of biochemical markers of bone turnover reflect the actual status of bone resorption and bone formation, respectively. Thus, they can be used as an adjunct to clinical and radiological evidence of fracture healing.

Role of common biochemical markers for the assessment of fracture union.

The progress of fracture union requires close monitoring. Whereas, clinical examination and radiographic studies assess the outcome, biochemical markers like serum alkaline phosphatase and urinary hydroxyproline reflect the actual status of bone resorption and bone formation over a short time frame. 36 patients of long bone fracture were randomly allocated for the study. When the patient reported to the Department of Orthopedics after fracture, serum and urinary samples were collected and X-ray of the affected part were taken. Subsequent samples were collected and X-ray taken just after management (either operative or conservative), after 3rd, 5th, 8th and 12th week, respectively after onset of fracture. According to the course of callus formation the patients were divided into two groups that progressed to proper union or malunion. The levels of serum alkaline phosphatase, urinary total and free hydroxyproline levels were measured and statistically analysed and compared. A statistically significant positive correlation between total urinary hydroxyproline excretion and serum alkaline phosphatase indicate progress towards satisfactory union. Thus, serial monitoring of biochemical markers of bone turnover can be used as an adjunct to clinical and radiological evidence of fracture healing.

Could bone tissue be a target for luteinizing hormone/chorionic gonadotropin?

Ovariectomy (OVX) and Zoladex administration to adult rats gave conflicting results with respect to the excretion of total urinary hydroxyproline (OH-Pro), a valuable indicator of bone collagen catabolism. Whereas OVX culminated in early (1 week) increases in OH-Pro, the use of Zoladex actually lowered OH-Pro and showed no sign of increasing over controls for a 2-month period. Since both OVX and Zoladex produce a state of estrogen deficiency we reasoned that the differential effects of the two procedures on OH-Pro were attributed to LH status. Receptors for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) have been identified in many, non-gonadal, estrogen sensitive sites and although bone is receptive to estrogen what effects LH/hCG might have upon bone metabolism have received scant attention. Treatment of osteoblasts in culture with a urinary derived formulation of hCG resulted in increased alkaline phosphatase (ALP) activity, raised matrix mettaloproteinase-2 (MMP-2) levels and increased expression of type I collagen. Further studies, using murine calvaria, supported a bone-resorbing effect of hCG. Taken together our initial findings suggested that raised hCG and/or LH might lead to an overall increase in bone matrix turnover as reported for puberty, pregnancy and the menopause. However, when the urinary derived preparation of hCG was replaced with recombinant hormone no changes in osteoblast activity were found implying the presence of contaminating agents in the urine derived hCG. Herein we describe that epidermal growth factor (EGF) could account for the changes observed for urinary derived hCG in osteoblast cultures and that the effects of LH/hCG on bone tissue are probably indirect.

Hyp levels in adults submitted to dynamic stretch in liquid medium and ground medium

Collagen catabolism in the locomotion system implies liberation of an increased hydroxyproline (HYP) level in winary excretion, which in turn constitutes a biochemical marker indicating damage in the conjunctive tissue. The objective of the present study was to verify the locomotion systems collagen catabolism degree through the HYP level excreted in 10 (ten) non – athletes adults men’s wire, with age group between 24 to 45, submitted to dynamic stretch in liquid medium or ground medium. Four movements limited by both muscular elasticity and joint mobility were selected for dynamic stretch performance and three urine sample collections were used in laboratory. As a methodological procedure sample collection was carried out for a two hour period (2:00) post hydration, and taken to the laboratory within 3 (three) hours. The HPLC – system liquid chromatographic determination of total hydroxyproline in urine – was used with reference value of 15 – 43 mg/d (Cherneeky et al., 1993). The fi rst collection was carried out 24 hours before the dynamic stretch to verify the basal level of HYP as sample group reference of volunteers. The second and the third collections were carried out 24 hours after the dynamic stretch was accomplished in both means. The results found for HYP levels (for p= 0.05) were for the basal level 39.07 + 15.27; for the levels 24 hours post – ground training were 71.28 + 12.15 and for the levels 24 hours post-water training were 66.74 + 10.31. From the results it is concluded that there is difference between the basal instants and the 24 hour post – training, indicating therefore the occurrence of damage of the conjunctive tissue. The direct analysis among the environments didn’t present a signifi cant difference (for p < 0.05). In spite of the occurrence of damage in both means with the results obtains, it isn’t possible to conclude which mean can prevail for the training with lesser risk of damage.

Determination of hydroxyproline in rat urine by high-performance liquid chromatography with electrogenerated chemiluminescence detection using tris(2,2′-bipyridyl) ruthenium(II)

A high-performance liquid chromatographic (HPLC) method for the determination of total urinary hydroxyproline (Hyp) in rat was developed and validated. Urine samples hydrolyzed with concentrated hydrochloric acid were filtered using a disposable centrifugal filter after the sample solutions had been adjusted to a slightly acidic condition by a buffer solution. Hyp in the fraction could be separated by column-switching HPLC and detected with electrogenerated chemiluminescence detection using tris(2,2′-bipyridyl) ruthenium(II) within an analysis time of 25 min. The calibration curve gave good linearity in the concentration range of 30–1500 nmol/ml, and the lower limit of quantification was 30 nmol/ml. The recovery of Hyp from urine samples was more than 96%. Precision and accuracy including within- and between-run values were less than 6 and ±5%, respectively, in the assay range. We concluded that the present method could be used to measure Hyp in rat urine.

INCIDENCE OF SKELETAL COMPLICATIONS IN PATIENTS WITH BONE METASTATIC PROSTATE CANCER AND HORMONE REFRACTORY DISEASE: PREDICTIVE ROLE OF BONE RESORPTION AND FORMATION MARKERS EVALUATED AT BASELINE

We evaluated the incidence of skeletal complications in patients with bone metastatic prostate cancer and hormone refractory disease. We also assessed the predictive role of bone turnover markers determined at baseline. A total of 112 patients were consecutively enrolled in our study from July 1990 to July 1998 and followed until death or the last followup. Bone pain, disease extent in bone, serum prostate specific antigen, hemoglobin, and a panel of bone formation and resorption markers were assessed at baseline before any second line treatment. Skeletal complications in 34 patients (30.3%, estimated yearly incidence 12.3%) involved vertebral deformity or collapse requiring spinal orthosis in 20 (17.9%), spinal cord compression in 7 (6.2%), pathological bone fracture in 10 (8.9%), symptomatic hypercalcemia in 1 (0.9%) and symptomatic hypocalcemia in 1 (0.9%). Median time to the evidence of the initial skeletal complication was 9.5 months. These adverse events did not influence overall survival. At baseline patients with eventual skeletal complications had greater bone pain (p = 0.02), a heavier tumor load in bone (p = 0.005), lower performance status (p = 0.05), and higher serum alkaline phosphatase (p <0.02) and urinary deoxypyridoline (p <0.05) than their counterparts. Multivariate analysis revealed that only urinary deoxypyridinoline was independently associated with the onset of these events (p <0.02). The scatterplot of urinary deoxypyridinoline values in patients with and without skeletal complications enabled us to detect a cutoff of 38 pM./mM. for predicting 51% of skeletal events with only an 8% false-positive rate. Skeletal complications are common in patients with prostate cancer and hormone refractory disease. Bone loss is the major cause of onset. Baseline deoxypyridinoline at the cutoff point noted had moderate sensitivity but high specificity for predicting these adverse skeletal events.

Comparison of biochemical markers of bone remodelling in the assessment of the effects of alendronate on bone in postmenopausal osteoporosis

The effects of alendronate treatment on biochemical markers of bone remodelling and bone mineral density (BMD) were studied in 30 Caucasian women (postmenopausal for at least 3 years, age 42–76 years, with BMD of the lumbar spine at least 2 S.D. below the mean for mature, premenopausal women). The patients were randomly assigned to receive alendronate (10 mg/day) or placebo for 12 months (double blind). The study was subsequently extended to a second year of open alendronate treatment. The treatment with alendronate resulted in a significant and progressive increase in BMD of the lumbar spine and femoral neck. Under the treatment, the maximal decrease of biochemical markers of bone remodelling (osteocalcin in plasma, bone-specific alkaline phosphatase, N-terminal propeptide of type I procollagen and C-terminal telopeptide of type I collagen in serum, and cross-linked amino-terminal N-telopeptide and total hydroxyproline in urine) was observed at 6 months with no further change during the 2-year period. There were no significant differences in discriminating between patients treated for 1 year with alendronate or placebo using either the percentage change in spine BMD at month 12, or a single measurement of the marker at month 6, or log (percent of baseline at month 6 of value of the marker). In this respect, the power of all the biochemical markers were comparable. The markers are a valuable adjunct to the measurements of BMD, especially in the patients not showing an increase of 3% or more at the lumbar spine BMD after 1 year of treatment.

Simultaneous determination of serum and urinary hydroxyproline and proline by liquid chromatography using two fluorescent labeling reagents

A method for the simultaneous determination of free serum and total urinary hydroxyproline (Hyp) and proline (Pro) concentrations by liquid chromatography (LC) using two fluorescent labeling reagents was developed. Serum Hyp and Pro, after treatment with o-phthalaldehyde, were derivatized with 4-(2-phthalimidinyl)phenylsulfonyl chloride (Phisyl-Cl). Imino acids in hydrolysed urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride (DPS-Cl) after treatment with o-phthalaldehyde and cleanup on a Bond Elut C 18 column. The two reaction mixtures were combined and mixed with dichloromethane to extract the excess of the reagents, and then the aqueous layer (10 μl) was subjected to LC. The Phisyl and DPS derivatives of imino acids were separated on a reversed-phase column by gradient elution with phosphate buffer (5 mM, pH 2.5) and acetonitrile at 35°C and detected by fluorescence measurement at 300 nm (excitation) and 400 nm (emission). The detection limits ( s: n=3) for Hyp and Pro were 30 and 40 fmol/injection, respectively, for Phisyl derivatives and 5 fmol/injection for both DPS derivatives. The within-day and day-to-day relative standard deviations for Hyp and Pro in serum and urine were <2.8%. The recoveries of Hyp and Pro added to serum and urine were 97.6–103.6%.

Supplemented gamma-linolenic acid and eicosapentaenoic acid influence bone status in young male rats: Effects on free urinary collagen crosslinks, total urinary hydroxyproline, and bone calcium content

The effect of different ratios of the prostaglandin precursors gamma-linolenic (GLA) and eicosapentaenoic (EPA) acids on bone status in growing rats measured as a function of free urinary pyridinium crosslinks and hydroxyproline levels was investigated. Male Sprague-Dawley rats were weaned onto an essential fatty acid deficient diet and from their fifth week, different groups of rats received a balanced, semisynthetic diet, supplemented with different ratios of GLA:EPA supplied as a mixture of evening primrose oil (EPO) and fish oil (FO). Controls were supplemented with linoleic (LA; sunflower oil) and alpha-linolenic (ALA; linseed oil) acids (3:1) or a commercially available rat chow. Animals were terminated at 84 days and femur length, ash weight, calcium content, free urinary pyridinium crosslinks (Pyd and Dpyd), total hydroxyproline (Hyp), and creatinine levels measured. Free urinary Pyd and Dpyd are good indicators of bone status and they correlated well with Hyp. Pyd and Dpyd excretion were significantly decreased in the higher GLA:EPA dietary groups and correlated well (r = 0.7) with Hyp levels. Concomitantly, bone calcium content increased significantly in the same dietary groups. These results suggest that diet supplementation with relatively high GLA:EPA ratios are more effective in inhibiting bone resorption than LA:ALA.

The Short-Term Effects of Conjugated Estrogen on Bone Turnover in Older Women

Estrogen replacement therapy (ERT) prevents bone loss and fracture in early postmenopausal women, but its benefit for women over 70 yr of age has not been determined. We have examined the effect of a short course of ERT on biochemical markers of bone turnover in older women. Eleven women (mean age, 77 yr) were given conjugated estrogen (Premarin; 0.625 mg/day) for 6 weeks. Biochemical markers were measured on serum and urine collected at baseline (two samples), after 5 and 6 weeks of ERT, and 5 and 6 weeks post-ERT. Markers of bone formation were osteocalcin, bone alkaline phosphatase, and type I procollagen peptide. Markers of bone resorption were total urinary hydroxyproline, total and free pyridinoline and deoxypyridinoline cross-links, type I collagen cross-linked N-telopeptides, and serum C-terminal cross-linked telopeptide. Data were analyzed by repeated measures multivariate analysis of variance to estimate the overall effect of ERT on the biochemical markers. Markers of bone resorption decreased d...

A simple and automated HPLC method for determination of total hydroxyproline in urine. Comparison with excretion of pyridinolines

An HPLLC method for measuring total hydroxyproline in human urine was validated. Hydroxyproline derivatization was achieved with 9-fluorenylmethyl chloroformate after blocking primary amino acids with orthophthaldialdehyde. The derivatives (hydroxyproline and internal standard) were separated by reversed phase high-performance liquid chromatography and detected by absorbance at 254 nm. Duplicate measurements of hydroxyproline have a coefficient of variation of 3.9% and the recovery in spiked urine samples is between 99.5 and 100.8%. We have compared the HPLC procedure with a commercial colorimetric assay. Analytical criteria of these methods are identical. Regression analysis, involving 50 samples, shows an excellent correlation between hydroxyproline chromatographic ( y) and colorimetric ( x) procedures: y = 0.989 x + 2.99 ( r = 0.976). Hydroxyproline excretion was determined in urine samples from 76 women more than 5 years post-menopause. The mean hydroxyproline/creatinine ratio in this group was19.3 ± 5.6 μmol/mmol (range 10.6–34.7). Finally, we compared in the same urinary samples hydroxyproline excretion with pyridinoline excretion (hydroxylysylpyridinoline and lysylpyridinoline), a new marker of bone resorption. The values show a significant correlation, with r = 0.417 for hydroxylysylpyridinoline and r = 0.443 for lysylpyridinoline.

The short-term effects of conjugated estrogen on bone turnover in older women.

Estrogen replacement therapy (ERT) prevents bone loss and fracture in early postmenopausal women, but its benefit for women over 70 yr of age has not been determined. We have examined the effect of a short course of ERT on biochemical markers of bone turnover in older women. Eleven women (mean age, 77 yr) were given conjugated estrogen (Premarin; 0.625 mg/day) for 6 weeks. Biochemical markers were measured on serum and urine collected at baseline (two samples), after 5 and 6 weeks of ERT, and 5 and 6 weeks post-ERT. Markers of bone formation were osteocalcin, bone alkaline phosphatase, and type I procollagen peptide. Markers of bone resorption were total urinary hydroxyproline, total and free pyridinoline and deoxypyridinoline cross-links, type I collagen cross-linked N-telopeptides, and serum C-terminal cross-linked telopeptide. Data were analyzed by repeated measures multivariate analysis of variance to estimate the overall effect of ERT on the biochemical markers. Markers of bone resorption decreased during ERT and returned to baseline after ERT (P < 0.05). Markers of bone formation declined less during ERT and continued to decline after ERT (P < 0.05). We conclude that ERT reduces bone turnover in older women and that markers of bone turnover may be useful in assessing the response to treatment in this age group.

Comparison of Serum Osteocalcin with Total and Bone Specific Alkaline Phosphatase and Urinary Hydroxyproline: Creatinine Ratio in Patients with Paget's Disease of Bone

Osteocalcin or bone Gla protein (BGP) is secreted by osteoblasts, and its serum concentration is elevated in a number of conditions with high bone turnover. A comparison of serum osteocalcin with total (TALP) and bone specific alkaline phosphatase (BALP) and urinary hydroxyproline/creatinine (OHP/Cr ratio) was performed in 13 patients with active Paget's disease of bone. BGP did not correlate significantly with either BALP or TALP, but did show a significant correlation with OHP/Cr ratio (r = 0.76; P < 0.01). BGP does not appear to be as sensitive a marker as BALP or TALP in Paget's disease.

Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption.

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an 'uncoupling index' by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5; n = 63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6; n = 8.9 ng/ml (SD 2.0; p < 0.01). The variance of serum osteocalcin levels in the two groups was similar. Compared with this 11% increase in the biochemical marker for bone formation, the markers of bone resorption, total urinary deoxypyridinoline (bone-specific), pyridinoline and hydroxyproline were increased by 40% (p < 0.0001), 61% (p < 0.0001) and 25% (p < 0.01), respectively. Furthermore, these biochemical markers of bone resorption had greater variance in women in type I osteoporosis than in the normal postmenopausal women (p < 0.01). The urinary excretion of the free crosslinks deoxypyridinoline, pyridinoline and glycosylated pyridinoline were increased by 26% (p < 0.001), 17% (p < 0.01) and 13% (NS) respectively. An 'uncoupling index' was calculated for the difference between urinary deoxypyridinoline and serum osteocalcin using the results from the normal women and expressed as z-scores. We conclude that the pyridinium crosslinks of collagen enable better discrimination between normal and osteoporotic women than does hydroxyproline.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic idiopathic hyperphosphatasemia: Case report

Chronic idiopathic hyperphosphatasemia, or juvenile Paget disease is a very rare syndrome that is characterized by fragile bones, bowing deformities, shortness of stature, large head, premature loss of teeth, radiographic evidence of expanded osteoporotic long bones with coarse trabeculations, and widened bones of the skull. Increased levels of serum alkaline phosphatase and increased levels of urinary total hydroxyproline are notable. We present a case of juvenile Paget disease that was associated with a history of precocious puberty. The patient had odontogenic osteomyelitis of the mandible that was treated by drainage, surgical debridement and antibiotic therapy.

Selective and sensitive determination of urinary total proline and hydroxyproline by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of total urinary proline (Pro) and hydroxyproline (Hyp) by gas chromatography (GC) was developed. After acid hydrolysis of urine, primary amino compounds were eliminated by reaction with o-phthaldialdehyde. Subsequently, Pro and Hyp were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. The derivatives were volatile and stable, giving single and symmetrical peaks. The detection limits for Pro and Hyp were 0.1 and 0.2 pmol injected, respectively. 3,4-Dehydroproline was used as an internal standard. The calibration curves for Pro and Hyp in the range 0.4–10 nmol were linear and sufficiently reproducible for quantitative determination. Overall recoveries of Pro and Hyp added to urine samples ranged from 93% to 103%. By using this method, Pro and Hyp in a small urine sample could be accurately and precisely determined without any influence from other constituent substances.

Total Urinary Hydroxyproline Determined with Rapid and Simple High-Performance Liquid Chromatography

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).

Paget's disease of bone

Paget's disease of bone is a common disorder of older patients that may sometimes cause a variety of signs and symptoms referable to the skeleton. An intrinsic bone-remodeling abnormality in this disease may lead to bone deformity, pain, and various arthritic or neurologic complications. Characteristic radiographic changes can be seen, and typically the serum alkaline phosphatase level and total urinary hydroxyproline excretion are elevated. Current therapies that interfere with excess osteoclast activity are helpful in many cases, but treatment choices and expected responses must be considered in the context of the individual patient.