Total Urinary Hydroxyproline Research Articles

Simple, optimized liquid-chromatographic method for measuring total hydroxyproline in urine evaluated

We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.

Hypophosphatemic rickets with hypocalciuria following long-term treatment with aluminum-containing antacid

We present what we believe is the first case of rickets following prolonged treatment with aluminum containing antacids that bind phosphate, in an 18-year-old mentally retarded boy with cerebral palsy and spastic quadriplegia. As expected, serum calcitriol was increased, and urinary phosphate excretion was very low. However, in contrast to all published cases of antacid induced hypophosphatemic osteomalacia in adults, despite a substantial increase in bone resorption reflected by urinary total hydroxyproline excretion, urinary calcium excretion was low rather than high, and significant hypocalcemia occurred after antacids were ceased and a phosphate salt administered. We suggest that the skeleton was so under-mineralized because of growth during prolonged phosphate deficiency, possibly augmented by anticonvulsant administration and immobilization, that increased bone resorption did not release enough calcium to cause hypercalciuria, or to prevent hypocalcemia during resumption of normal mineralization.

Assessment of bone turnover in the dry period of dairy cows by measurement of plasma bone GLA protein, total plasma alkaline phosphatase activity and urinary hydroxyproline

Plasma osteocalcin, or plasma bone GLA protein (BGP), total plasma alkaline phosphatase activity and urinary hydroxyproline excretion of twenty-four pregnant dairy cows (thirteen in their first or second pregnancy, i.e. low parity, and eleven in their third or more pregnancies, i.e. high parity) were measured from 7 weeks before parturition until 1 week after parturition. Seven weeks before parturition the cows' ration was changed to one containing either 0.22% magnesium (low magnesium, LMg) or 0.82% magnesium (high magnesium, HMg) in the dry matter, and the potassium content of both rations was increased to approximately 4.1% in the dry matter to reduce the absorption of magnesium. Plasma BGP levels decreased significantly (P less than 0.01) as parturition approached while total plasma alkaline phosphatase activity and urinary hydroxyproline excretion did not. Magnesium supply and parity had no significant effect on this decrease. The overall plasma concentration of BGP, total plasma alkaline phosphatase activity and the urinary hydroxyproline/creatinine ratio in the prepartum period were affected by parity (P less than 0.05) with higher values in the lower parity cows. A significant positive correlation (r = 0.58, P less than 0.01) was found in all cows between plasma BGP level at parturition and the percentage of the bone surface covered with osteoblasts; however, plasma BGP was not correlated either with other histomorphometric variables or with total alkaline phosphatase activity during this time.

Increased Monocyte Interleukin-1 Activity and Decreased Vertebral Bone Density in Patients with Fasting Idiopathic Hypercalciuria

Idiopathic hypercalciuria (IH) is a heterogeneous disorder frequently observed in patients with nephrolithiasis. At one extreme of its clinical spectrum is fasting hypercalciuria (FH), a condition characterized by increased bone resorption and turnover. In previous studies we have shown that monocytes from patients with high turnover osteoporosis and from women in early postmenopause elaborate increased amounts of interleukin-1 (IL-1), a cytokine that stimulates bone resorption in vitro and in vivo. Since IL-1 could also mediate the resorptive mechanism of FH and cause a clinically significant bone loss, we have studied the relationship of IH, vertebral mineral density, bone turnover, and monocyte IL-1 activity in 47 patients with absorptive hypercalciuria (AH), 23 with FH, and 38 nonhypercalciuric subjects with recurrent nephrolithiasis (controls). Vertebral mineral density, as measured by quantitative computer tomography, was decreased in each of the three patient groups, but was significantly lower in FH patients than in AH patients or control subjects. Twenty-four-hour total urinary hydroxyproline excretion was increased in FH patients compared to that in AH patients or controls, but blood levels of osteocalcin were not. Monocytes from FH subjects yielded significantly more IL-1 (alpha + beta) activity than those from AH patients or controls; levels of IL-1 activity in monocytes of AH and control patients were similar. In IH subjects, significant correlations were found between IL-1 and hydroxyproline (r = 0.70; P less than 0.0001), IL-1 and quantitative computer tomography values (r = -0.49; P less than 0.005), and IL-1 and urinary calcium (r = -0.36; P less than 0.05). Serum PTH levels were within normal limits in all subjects and were similar in the three study groups, 1,25-Dihydroxyvitamin D3 levels, although higher in IH patients than in controls, were not significantly different in FH and AH subjects. Increased IL-1 activity and decreased vertebral mineral density are features of a subset of patients with IH. Although a cause-effect relationship remains to be established, increased monocytic IL-1 activity, rather than elevated PTH or 1,25-dihydroxyvitamin D3 levels, could underlie the resorptive component of FH.

Urinary total hydroxyproline measured by HPLC: comparison of spot and timed urine collections

Urinary total hydroxyproline measured by HPLC: comparison of spot and timed urine collections

Environmental cadmium exposure in the past and urinary levels of hydroxylysine glycosides.

In our previous report (Hayakawa et al., 1987), it was suggested that there occurred some changes in the collagen metabolism among the inhabitants in a cadmium polluted area. The urinary total hydroxyproline (THP) excretion level of female inhabitants in the cadmium polluted area was significantly higher than that of the inhabitants in nonpolluted area, and a positive correlation was observed among the urinary levels of cadmium, T-HP and β2-microglobulin, though the correlation was low. However, it is known that urinary level of T-HP is influenced by the turnover of several tissue collagens other than bone. Therefore, for the confirmation of the above estimation, it is necessary to re-examine the results using the index of bone collagen turnover, which is known to be more reliable. Segrest and Cunningham (1970) pointed out that two hydroxylysine glycosides, 2-O-α-D-glucopyranosyl-O-β-D-galactopyranosyl.5- hydroxylysine (GGH) and O-β-galactopyranosyl-5-hydroxylysine (GH), are the endogenously derived collagen metabolites, not influenced by normal diet. The measurement of urinary GGH and GH levels provides a more quantitative indication in estimating the rate of collagen turnover because the amounts of GGH and GH differ according to the types of collagen and the kinds of tissue. In the present study, the relation between cadmium exposure and bone collagen metabolism reported in our previous study was further evaluated through the measurement of urinary GGH and GH levels in male inhabitants in the area where our previous survey was performed.

Automated analysis of urinary hydroxyproline

4-Hydroxyproline released during collagen breakdown is not reutilised and its excretion in urine therefore reflects collagen breakdown [l]. Excretion of hydroxyproline is increased during accelerated collagen turnover such as growth [2,3], bone diseases [1,4] and tumors [5,6]. Almost all endogenous 4-hydroxyproline in man is confined to collagen, where it constitutes about 14% of the total amino acid content [7]. Quantitation of urinary 4-hydroxyproline is therefore helpful in the diagnosis and management of a variety of diseases [1,7]. Over 90% of the hydroxyproline excreted in urine is peptide bound [l]. Q uantitation of total urinary hydroxyproline therefore involves acid hydrolysis of the hydroxyproline-containing peptides. We have previously described a HPLC method for analysis of hydroxyproline in urine [8]. In this study we have modified the HPLC method to improve the accuracy of hydroxyproline measurement. The conventional spectrophotometric method by Ehrlich’s reaction after chloramine T oxidation to pyrrole [9] was also adapted to a Cobas Bio Centrifugal Analyzer for quick and automated analysis of large number of specimens.

Clinical value of the measurement of bone remodelling markers in primary hyperparathyroidism.

This study was performed in order to evaluate the clinical usefulness of the measurement of total serum alkaline phosphatase activity (AP), serum osteocalcin (BGP) and urinary hydroxyproline (OHPr) in assessing bone remodelling in primary hyperparathyroidism. Thirty-two patients with primary hyperparathyroidism were included in the study. No statistically significant differences were observed between the mean values of Z-scores obtained for each marker. Furthermore, an inverse correlation was found between percentage of bone mineral content at the distal radius and both BGP (r = -0.57; p less than 0.05) and AP (r = -0.49; p less than 0.05). The results obtained demonstrate that, contrary to other metabolic bone diseases (e.g. Paget's disease of bone), all three markers examined may be used in clinical practice to evaluate the entity of skeletal turnover in primary hyperparathyroidism.

Effects of dietary NaCl supplementation on bone synthesis of hydroxyproline, urinary hydroxyproline excretion and bone 45Ca uptake in the rat.

High sodium chloride intakes are regarded as a risk factor for osteoporosis because they increase the obligatory urinary calcium loss and stimulate parathyroid activity. Sodium chloride loads induce osteopenia in the rat. The effect could be due to a decrease in bone formation or a rise in bone resorption. Two experiments were undertaken to study the effects of dietary NaCl supplementation on 3H-hydroxyproline synthesis and 45Ca uptake in femoral bone. Salt-treated rats excreted 1.7 times more total urinary hydroxyproline (P less than 0.001) and 2.1 times more recently labelled 3H-hydroxyproline than controls (P less than 0.02) but they did not accumulate less 3H-hydroxyproline or less 45Ca in their bones than controls. These results indicate that salt-mediated osteopenia is due to an increase in bone resorption, rather than to a decrease in bone formation.

Liquid-chromatographic determination of total hydroxyproline in urine.

In this method for quantifying 4-hydroxyproline in human urine, 50 microL of urine is hydrolyzed, derivatized with phenylisothiocyanate (PITC), and then quantified by reversed-phase HPLC with ultraviolet detection. The detection limit in urine is 373 pg of hydroxyproline per 50-microL injection. The total CVs for high- and low-concentration pools are 5.3% and 3.9%, respectively (10 runs in 10 days). The standard curve of the assay is linear over a range of 0 to 22 nmol per injection. We estimated the normal range for hydroxyproline excretion in men on an unrestricted diet to be 123-308 mumol/24 h. We also report hydroxyproline concentrations in patients with metastatic bone disease and cirrhosis of the liver.

Estimation of whole body bone resorption rate: A comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis

Estimation of whole body bone resorption rate: A comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis

Urinary hydroxyproline excretion in the myelofibrosis-osteomyelosclerosis syndrome and related diseases.

The total urinary hydroxyproline excretion was assessed in 47 patients with chronic myeloproliferative disorders. Urinary hydroxyproline excretion was normal in 16 patients with idiopathic myelofibrosis and in 5 out of 6 patients with acute myelofibrosis. In patients with osteomyelosclerosis (n = 8) values for urinary hydroxyproline excretion were higher (median 202, range 54-652) than those in idiopathic myelofibrosis (median 139, range 84-216). This difference was not significant (p greater than 0.1). Elevated values for urinary hydroxyproline excretion were found in 10 patients (1 AMF patient, 3 OMS patients and 6 patients with CML in the accelerated phase of the disease). All but 1 of these patients had been treated, or were being treated, with cytotoxic agents at the time of investigation. These findings are compatible with impaired degradation of bone marrow collagen which, together with enhanced collagen synthesis from bone marrow fibroblasts, accounts for progressive accumulation of connective tissue in the bone marrow. This process appears to be influenced by cytotoxic treatment as reflected in increased urinary hydroxyproline excretion in those patients receiving cytotoxic agents.

Estimation of whole body bone resorption rate: A comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis

In 37 female patients with primary osteoporosis, urinary hydroxyproline excretion, determined in 18 24-h consecutive complete urine collections was compared with two radioisotopic measurements of bone resorption rate measured simultaneously using 85Sr. A somewhat better fit was obtained when the kinetically determined bone resorption rate was corrected for long-term exchange processes within bone. Regression analysis showed that the intercept of the regression of hydroxyproline excretion on resorption rate, corrected or uncorrected for exchange, was significantly higher than zero at about 100 μmol/day. This is consistent with a substantial fraction of urinary hydroxyproline arising from non-bony sources. Fifteen paired studies were analysed and the results suggested that intra-individual variability in these relationships (when studies were separated by a year or more) were similar to inter-individual variability. We calculated the precision with which an estimate of bone resorption could be determined based on the calculated regressions. As a means of non-invasive quantitation of whole body bone resorption rate, the excretion rate of hydroxyproline, measured over 5 days, for example, appeared competitive with isotopic methods making no correction for exchange and relatively little worse than our exchange corrected method.

Analysis of total hydroxyproline in urine by high-performance liquid chromatography and pre-column derivatization

Analysis of total hydroxyproline in urine by high-performance liquid chromatography and pre-column derivatization

Bone resorption and hypercalciuria in calcium stoneformers

The circadian rhythm of urinary total hydroxyproline (THP) excretion was determined in matched groups of ten male idiopathic calcium stoneformers and ten normal subjects in order to determine whether enhanced resorption of bone might contribute to hypercalciuria in these patients. THP increased progressively in normal subjects in successive eight-hour urine collections from period 1 (8 am to 4 pm) to period 3 (12 midnight to 8 am), the nocturnal high level in period 3 being significantly greater than in period 1 ( P < 0.01) and in period 2 ( P < 0.05). By contrast, no significant circadian rhythm was observed in THP excretion in the stoneformers. Their THP excretion was similar to that of normal subjects in period 3, but was significantly higher than that of normal subjects in period 1 (THP/creatinine ratio(mg/mg); 0.026 ± 0.003 v 0.017 ± 0.001; P < 0.05. Indices of parathyroid hormone activity were not significantly different between stoneformers and normal subjects; mean serum 1,25(OH) 2 vitamin D levels were higher in the stoneformers than the normal subjects (44 v 37 pg/mL) but the difference was not significant ( P > 0.05). These studies suggest that increased bone turnover may contribute to hypercalciuria in these calcium stoneformers.

Liquid-chromatographic determination of total hydroxyproline in urine.

A reversed-phase "high-performance" liquid chromatographic assay for total hydroxyproline in urine is described. The urine samples are hydrolyzed overnight with acid, evaporated, solubilized, and derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl chloride. The derivatives are chromatographed with a solvent gradient consisting of sodium acetate buffer and acetonitrile, the effluent being monitored at 436 nm. The useful lower limit of sensitivity for quantification is 13 pmol of hydroxyproline per 5-microL injection, corresponding to 33 mumol/L of urine. Either glutamine or cysteic acid is satisfactory as the internal standard. Peak heights and the amounts of hydroxyproline applied to the column are linearly related from 13.3 to 266 pmol. Mean analytical recovery was 83%. For four different concentrations the mean within-assay CV was 9.0% and the between-assay CV 12%. The normal reference interval found for 31 healthy adults was 31 to 177 mumol/24 h per square meter of body surface. We compared results for 10 samples from patients.

High-performance liquid chromatographic analysis of free hydroxyproline and proline in blood plasma and of free and peptide-bound hydroxyproline in urine

A rapid, accurate and sensitive method for the determination of free hydroxyproline and proline in plasma and of total hydroxyproline in urine has been developed. Free imino acids and internal standard are extracted from plasma by trichloroacetic acid precipitation of protein and they are selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of the acid extract with o-phthalaldehyde. The highly fluorescent adducts of imino acids are separated on a Spherisorb ODS 2 reversed-phase column using acetonitrile—0.1 M sodium phosphate buffer, pH 7.2 (9:91, v/v) as mobile phase, followed by fluorometric detection. Total hydroxyproline determination in urine hydrolysates is carried out by reaction of the imino acid with 4-chloro-7-nitrobenzofurazan after clean-up on a Sep-Pak C 18 cartridge of the o-phthalaldehyde-treated sample, high-performance liquid chromatographic separation and fluorometric quantitation of the derivative.

Plasma somatomedin activity and urinary hydroxyproline excretion during administration of human growth hormone in children with short stature. Long-term effects and relation with short-term changes.

Growth velocity, somatomedin activity (SM-act) and total urinary hydroxyproline excretion (THP) were studied in 9 prepubertal short children on long-term human growth hormone (hGH) therapy, and compared to the short-term responses to hGH, described in the accompanying paper. Positive correlations were found between the short-term increases of either SM-act or THP and growth acceleration, but these were too weak to be used as a predictor. On a schedule of biweekly injections, pre-injection SM-act values were only slightly higher than pre-treatment values, but post-injection values were considerably higher and similar to the values obtained with comparable hGH doses in the short-term study. There was an excellent relationship of the increment of SM-act during chronic therapy over untreated values and the increases of growth velocity. During the first year on hGH therapy the mean SM-act, mean THP and growth acceleration showed strong correlations.

Plasma Somatomedin Activity and Urinary Hydroxyproline Excretion during Administration of Human Growth Hormone in Children with Short Stature

15 prepubertal children with short stature and varying peak growth hormone (GH) levels were given daily injections of increasing doses of human growth hormone (hGH) for consecutive periods of 7 days. Somatomedin activity (SM-act) and total urinary hydroxyproline excretion (THP) were determined in each period. In patients with a varying degree of GH deficiency, but without non-pituitary dependent abnormalities, there was a high correlation between basal SM-act and height velocity. Patients with catch-up growth had an unproportionally low SM-act and the Prader-Willi and transient Cushing patients had an unproportionally high one. All patients showed increases of SM-act and THP on hGH administration, but there was considerable variation of the shape of the curve and of the amplitude of the response. 3 1/2 days after the last injection, SM-act was back to basal level. There was a good correlation between weight-for-height and SM-act during the first two hGH doses, which fits the hypothesis of GH and insulin synergism on SM generation.

Total, Dialyzable, and Nondialyzable Postabsorptive Hydroxyproline

The postabsorptive urinary total (T), dialyzable (D), and nondialyzable (ND) hydroxyproline (HYPRO) tests were evaluated to determine whether the patterns of excretion varied according to the predominance of osteoblastic v osteolytic bone involvement in 58 patients with neoplastic disease. In patients with osteolytic lesions from multiple myeloma, elevated T and D levels with normal ND HYPRO values were observed, along with elevated D/ND ratios. In prostate cancer, the T, D, and ND values were all elevated and the D/ND ratio was normal. Patients with Hodgkin's disease had elevated T, D, and ND HYPRO levels, and the D/ND ratio was in the range of patients with prostate cancer. The data suggest that these collagen markers may be useful in the long-term evaluation of these neoplasms in patients.