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PubMed1975

A study of 14C-proline and 14C-hydroxyproline incorporation in different homogenate fractions of the hepatopancreas of the snail, Helix pomatia L.

The fate of 14C in snails given 14C-proline and 14C-hydroxyproline has been examined by radiometric analyses of hepatopancreas homogenates. Free 14C-proline and 14C-hydroxyproline in the structural protein of three different hepatopancreas homogenate fractions: (a) granules, (b) small calcium spherites, and (c) large calcium sperites. The presence of hydroxyproline in granules and calcium spherites gives reason to believe that a fibrillar protein related to the collagen-type might occur within these structures. The 14C-proline and 14C-hydroxyproline incorporation test measured total radioactivity of each homogenate fraction, expressed as net counts per minute (cpm) per mg of dry weight of each fraction. The radioactivity of the granule and small calcium spherite fractions was about 6-8 times greater than that of the large calcium spherite fraction.

Animals↗
PubMed1989

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures.

We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C18 column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was less than 8%. [3H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [3H]proline and "chased" in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24-48 hour "chase" period increased the release of acid-soluble [3H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.

Animals↗
PubMed1999

Age-related changes in body composition, hydroxyproline, and creatinine excretion in normal women.

We have made a cross-sectional study of relationships among age, whole body bone mineral content (WBBMC), and non-bone lean body mass (NBLBM) measured by dual energy X-ray absorptiometry (DXA), and daily excretion of hydroxyproline (OHP) and creatinine (Cr) in a group of normal women. WBBMC fell with age from the 6th decade, whereas NBLBM was almost constant. Creatinine excretion fell with age from the 5th decade until the 9th, to a much greater degree than NBLBM, reaching a nadir in the 8th decade. Daily excretion of hydroxyproline showed a peak in the 6th decade and fell moderately thereafter. The greater fall of creatinine compared with hydroxyproline resulted in rising OHP/Cr ratios with advancing age, in contrast to the pattern of hydroxyproline excretion. The use of creatinine as a correction for urine dilution or for lean body mass (LBM) in assays for markers of bone turnover must therefore be viewed with caution.

Absorptiometry, Photon↗
PubMed2004

Relationships between tensile strength, ascorbic acid, hydroxyproline, and zinc levels of rabbit full-thickness incision wound healing.

PURPOSE: This work was carried out to follow up the healing of full-thickness incision wounds opened on the back skin of rabbits in order to gain insight into the periodical correlation among such factors as ascorbic acid, collagen (hydroxyproline), the zinc content, and tensile strength of wound tissue. The need to provide vitamin C or zinc supplements after such wound incisions is also discussed. METHODS: Full-thickness incision wounds and the ascorbic acid and hydroxyproline levels were measured in 24 rabbits by spectrophotometric methods on day 0 and on the 3rd, 5th, 7th, and 15th days after operation. The tensile strength was measured by a polygraph using a force displacement transducer. The zinc levels of the wounds were measured by atomic absorption spectrophotometry. RESULTS: The zinc and hydroxyproline levels reached the peak levels on the 5th day, but the tensile strength of wound increased sharply on the 7th day after wounding while the zinc levels did not change. These results indicated that in the first 7 days of wound healing, high levels of ascorbic acid, hydroxyproline, and zinc cumulation occurred in the wound tissue, and the tensile strength reached its highest level on the 15th day without any supplementation. CONCLUSION: The supplementation of zinc and/or ascorbic acid should therefore be given just at the beginning of the wounding period, especially if there is deficiency of these nutrients.

Animals↗
PubMed1992

Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.

Chromatography, High Pressure Liquid↗
PubMed1978

Gas chromatographic determination of hydroxyproline in urine hydrolysates.

A simple and specific method for the determination of hydroxyproline in urine hydrolysates has been described. Hydroxyproline was converted into its N-isobutyloxycarbonyl methyl ester derivative without elaborate cleanup, which was analyzed by gas chromatography. Hydroxyproline was clearly separated from other urinary constituents on a 0.60% FFAP on dimethyldichlorosilane-treated Gas-Chrom P column. Kainic acid was used as the most convenient internal standard available. The relative standard deviations of peak height ratios were 1.15--2.51% at the 10--150 microgram levels. Percent recoveries of hydroxyproline added to urine hydrolysates ranged from 98.8 to 107.3%.

Adult↗
PubMed1980

An automatic technique for the routine fractionation of the urinary hydroxyproline containing peptides.

A fully automatic method permits fractionation in a Biogel P2 column of the hydroxyproline-containing peptides into two fractions. The alkaline hydrolysis and the colorimetric evaluation of the liberated hydroxyproline are also completely automatic and allow calculation of the percentages of the two fractions. The first one, termed F1 fraction, contains the peptides of molecular weight larger than 1500, while the other, termed F2, contains the smaller peptides. The method was used for 223 assays. The F1 fraction is decreased in cases of Paget's bone disease. It is increased in cases of metastatic cancer of bone. Statistical analysis of the data demonstrates that this techique greatly improves the certainty of diagnosis when coupled to the assay of total hydroxyproline. When both total urinary hydroxyproline and the F1 fraction percentage are increased over threshold values of 485 mumol per 24 h and 28.4% respectively, the probability of the presence of a bone metastasis is 100%.

Adolescent↗
PubMed1987

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 mumol/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.

Aged↗
PubMed1989

Selective determination of hydroxyproline in urine by high-performance liquid chromatography using precolumn derivatization.

A method to measure total hydroxyproline in human urine was developed. Primary amino acids were derivatized with ortho-phthaldialdehyde, followed by derivatization of imino acids with 9-fluorenylmethyl chloroformate. The fluorescent 9-fluorenylmethyl chloroformate derivatives were separated by reversed phase high-performance liquid chromatography. 3,4-Dehydroproline was used as internal standard. Calibration curves for hydroxyproline and internal standard were linear from 1 to 200 pmol injected. Both within- and between-run precision were below 3.2%. Analytical recovery of hydroxyproline added to urine samples was 99.2 +/- 2.6%. Values for excreted hydroxyproline were determined by analysis of urine samples from adult volunteers. The hydroxyproline/creatinine ratio was found to be 15.8 +/- 4.6 mmol/mol (range 7.0-27.3) with no significant sex-related difference.

Chromatography, High Pressure Liquid↗
PubMed1993

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride.

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20-200 pmol, with equations y = 1.10x - 0.80 (r = 0.999) and y = 1.12x - 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 +/- 0.04 and 2.03 +/- 0.04 nmol/micrograms, respectively.

Animals↗
PubMed1993

Gas chromatography-mass spectrometry determination of 18O2 in 18O-labelled 4-hydroxyproline for measurement of collagen synthesis and intracellular degradation.

The use of gas chromatography-mass spectrometry (GC-MS) and 18O2, a stable isotope which is incorporated into collagen during the post-translational conversion of proline to hydroxyproline, offers the potential advantages of high levels of sensitivity and specificity as compared to other techniques for measuring rates of collagen synthesis and degradation in vitro and vivo. Trifluoracetylation and methanol esterification of hydroxyproline yields two derivatives of hydroxyproline: N,O-trifluoroacetyl methyl 4-hydroxy-L-proline (N,O-TFA-Hyp) and N-trifluoroacetyl methyl 4-hydroxy-L-proline (N-TFA-Hyp). In the past, N-TFA-Hyp, which yields the 16O/18O-containing m/z 182/184 ion pair [M-COOH3]+ when analyzed by electron impact ionization GC-MS, has been proposed for analysis of 18O-enriched collagen. Although N,O-TFA-Hyp can be converted to N-TFA-Hyp by solvolysis, we find that this leads to degradation of the chromatography in GC-MS and demonstrate here that this extra chemical step is unnecessary if the m/z 278/280 ion pair (representing the [M-COOCH3]+. fragment) is measured by selected ion monitoring. By labelling fibroblasts in culture with 18O2, a sample of isotope-enriched collagen was obtained which was used to calibrate the GC-MS over the range 0.5-49% atom percent enrichment (APE). The greater sensitivity of 18O2 versus [15N]proline for labelling newly synthesized collagen was demonstrated by the finding of a ten-fold higher enrichment in the former isotope when administered to cell cultures at the same precursor APE. Thus, the approach described herein permits the determination of total hydroxyproline and APE on the same sample avoiding additional processing steps while maintaining the quality of chromatography and the sensitivity of detection. Measurement of absolute rates of both collagen synthesis and intracellular degradation of newly synthesized collagen in cell cultures is thus possible. Preliminary results comparing collagen metabolism in pairs of fibroblasts from hypertrophic scars and normal skin in post-burn patients are presented.

Cicatrix↗
PubMed2004

Quantitation of hydroxyproline in bone by gas chromatography-mass spectrometry.

A validated gas chromatography (GC)-mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC-MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 micromol/l bone hydrolysate.

Animals↗
PubMed2006

Effects of sparfloxacin on CNS functions and urinary hydroxyproline in mice.

Sparfloxacin is a widely prescribed drug for various infections. In the present study, sparfloxacin, at two doses, 25 and 50 mg kg(-1) was screened on seven neurological and neurobehavioural parameters in mice. In addition, a chronic study was performed to measure the grip strength of the animal to simulate tendon damage and corresponding hydroxyproline levels were determined in mice urine. Increasing current electroshock test (ICES), pentylenetetrazole (PTZ)-induced seizures, forced swim test (FST), passive avoidance response (PAR), spontaneous alternation behaviour (SAB) and elevated plus maze (EPM) were used for studying the acute effects of sparfloxacin while ICES, grip strength test (GST) and urinary hydroxyproline estimations were done for studying its chronic effects. The results indicate a proconvulsive potential of sparfloxacin on the ICES test (when administered for 21 days) and on PTZ when administered for 4 days. Further sparfloxacin (50 mg kg(-1)x4 days) exhibited anxiety like effects on EPM while mood and memory remained unaffected. Sparfloxacin 50 mg kg(-1) reduced the grip strength of mice after 2 weeks while both 25 and 50 mg kg(-1) reduced the same after 3 weeks. On urinary hydroxyproline, sparfloxacin 25 mg kg(-1) significantly increased the levels after 3 weeks of treatment. At 50 mg kg(-1), an elevation was evident from the first week onwards. In conclusion, sparfloxacin produced proconvulsant and anxiogenic effects in mice and was devoid of effects on mood and memory. On chronic treatment, it reduced grip strength and caused an elevation in urinary hydroxyproline levels.

Animals↗
PubMed1975

Differential alkali-extraction of hemicellulose and hydroxyproline from non-delignified cell walls of lupin hypocotyls.

The extraction by alkali of hemicellulose polysaccharides and polymer hydroxyproline from non-delignified, primary cell-walls of lupin hypocotyls has been studied, using sequential extractions at 0 degrees and 18-22 degrees. 10% Aqueous potassium hydroxide at 0 degrees rapidly removed about two-thirds of the hemicellulose normally extracted in 10% KOH at 18-22 degrees and including nearly all of the hemicellulose-A. Little hydroxyproline was released at 0 degrees. When the temperature was subsequently raised to 18-22 degrees, the remaining 10% KOH-soluble hemicellulose, along with most of the hydroxyproline, was released. The monosaccharide composition of these fractions changed markedly with the time of extraction. Arabinose increased from 35% of the polysaccharide extracted during the first hour at 18-22 degrees to similar to 65% of the polysaccharide extracted between 16 and 20 h at 18-22 degrees. Hydroxyproline changed similarly as a proportion of polymer. The implications of these and other results are discussed in relation to polysaccharide and polymer extraction without prior delignification and to models of the primary cell-wall.

Arabinose↗
PubMed1978

A new method of preparation and some properties of 3-hydroxyproline.

The hydrolyzate of Delonix Regia seed extract is fractionated sequentially on Dowex 50 X 8 resin and on QAE Sephadex A 25. Purification is completed by recrystallisation from ethyl alcohol. 3-hydroxyproline is destroyed by NO2H and by chloramine T, which prevents from using most of the colorimetric reactions in use for 4-hydroxyproline. For its characterization, 3-hydroxyproline may be clearly separated from 4-hydroxyproline by TLC chromatography and by high voltage paper electrophoresis.

Chromatography↗
PubMed1986

Healing after myocardial infarction in the dog: changes in infarct hydroxyproline and topography.

Temporal changes in infarct collagen and left ventricular topography during healing after myocardial infarction were studied in 132 dogs with coronary artery ligation: 8 sham dogs and 13 with no infarction (controls) and 111 with infarction (3 at 1 day, 54 at 2 days, 25 at 7 days, 3 at 2 weeks, 9 at 4 weeks and 17 at 6 weeks). Myocardial hydroxyproline (a marker of collagen) was measured by spectrophotometry and pathologic infarct size, arteriographic occluded bed size and topography by computerized planimetry of weighed left ventricular rings. Over 6 weeks, hydroxyproline was unchanged in normal regions (average 4.20 mg/g dry weight) but increased progressively between 7 days and 6 weeks (9.94 versus 55.55 mg/g, p less than 0.001) in infarct zones. Progressive infarct contraction occurred over 6 weeks, with infarct size at 6 weeks being 40% less than at 2 days (9.7 versus 16.3% of the left ventricle, p less than 0.001), although total infarct hydroxyproline was directly related to infarct size at each time period (r = 0.73 to 0.81, p less than or equal to 0.05). Significant (p less than or equal to 0.05) left ventricular topographic changes in infarct hearts compared with control hearts included: 1) increase in cavity area (5.0 versus 3.9 cm2), endocardial circumference (8.8 versus 7.4 cm) and expansion index (infarct/normal endocardial segment length, 1.21 versus 1.02) by 7 days; and 2) decrease in thinning ratio (infarct/normal wall thickness, 0.71 versus 0.98) by 6 weeks. Also, compared with 2 day infarcts, by 6 weeks infarct area was decreased (1.8 versus 3.4 cm2) and the noninfarcted segment length increased (6.9 versus 5.4 cm). Changes in hydroxyproline and topography were similar for anterior (n = 54) and posterior (n = 57) infarcts. Thus, healing in canine infarcts is associated with cavity dilation and infarct expansion within 7 days followed by infarct contraction and thinning by 6 weeks, whereas collagen increases between 7 days and 6 weeks. Collagen deposition in expanded and thinned infarct segments explains the permanent regional shape distortion associated with ventricular aneurysms.

Animals↗
PubMed2003

Liquid chromatographic determination of hydroxyproline in tissue samples.

We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25-500 microM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08+/-0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.

Animals↗
PubMed1975

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.

Arginine↗