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The postabsorptive hydroxyproline in the long-term evaluation of patients with breast cancer.

The postabsorptive urinary hydroxyproline excretion test (Spot-HYPRO) was evaluated for its usefulness in reflecting the presence or absence of bone metastasis in 75 women with breast cancer. A comparison was made between the Spot-HYPRO values and bone disease, as documented by bone scanning supported by skeletal x-rays. Breast cancer patients with skeletal metastasis had 3-4-fold elevations in Spot-HYPRO above the control values (P less than 0.001). Mild elevations were noted in breast cancer patients without skeletal metastasis (P less than 0.025). Thirty patients received serial Spot-HYPRO and bone scans for 6 to 48 months (average, 24 months). There was a 90% correlation between changes in Spot-HYPRO and simultaneous changes on bone scan. Elevations in Spot-HYPRO preceded changes found on bone scan by an average of 3 months. The authors conclude that the Spot-HYPRO is a simple, convenient, and accurate method of documenting and following bone metastasis in patients with breast cancer.

Adult↗

A rational postoperative follow-up with carcinoembryonic antigen, tissue polypeptide antigen, and urinary hydroxyproline in breast cancer patients.

Breast cancer patients (n = 224) aged 28 to 81 were postoperatively followed up with serial determinations of carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), and urinary hydroxyproline (OHP). The clinical usefulness of these tumor markers to diagnose and monitor distant metastases was compared with that of the imaging techniques commonly used to monitor breast cancer patients (bone scanning [BS], liver echography [LE], chest radiograph, and skeletal radiograph). So far, 23 patients withdrew from the study, and distant metastases occurred in 33 patients. In 91% of the metastatic patients, constant elevation or progressive increase in serum CEA and/or TPA levels were the first pathologic findings of the relapse. Of the remaining 168 nonrelapsed patients, 122 were followed up longer than 24 months (43 +/- 17 months; mean +/- SD). In these 122 patients the false-positive results of CEA, TPA, and OHP were 0.8%, 2.4%, and 0%, respectively, when used simultaneously with clinical examination and the common laboratory examinations. BS and LE are the only imaging techniques that showed such a high sensitivity to be suitable in the postoperative follow-up of breast cancer patients. Nevertheless, because BS has a low specificity and is not harmless, it should be performed at longer intervals than tumor markers. Eventually, in the relapsed patients, TPA and OHP well reflected the response to treatment better than CEA and prevented useless radiologic examinations.

Adult↗

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius).

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.

Animals↗

Concentration of hydroxyproline in blood: a biological marker in occupational exposure to asbestos and its relationship with Pi*Z and Pi*S polymorphism in the alpha-1 antitrypsin gene.

BACKGROUND: Hydroxyproline (OHP) is one of the most abundant amino acids in collagen and, in general, it provides a good measure of overall collagen catabolism. METHODS: Asbestos workers suffering from asbestosis (cases n = 85); asbestos exposed workers without asbestosis (exposed controls, EC, n = 86), and non-exposed population (non-exposed controls, NEC, n = 122) were studied. The concentration of free OHP in whole blood was measured following the Pico-Tag procedure. RESULTS: Concentration of OHP in blood was significantly different in the three groups studied (P < 0.001), being higher in cases (19.8 +/- 14.7 micromol/L) than in EC (16 +/- 12.4) and NEC (13.5 +/- 6.7). When all individuals were grouped and stratified by the Pi*S and Pi*Z polymorphisms in the alpha-1-antitrypsin gene, the highest OHP levels were detected in the Pi*S homozygotes, one of the asbestosis-at risk-genotypes (Pi*S homozygotes, x = 24.5 +/- 11.7; Pi*S heterozygotes, x = 16.6 +/- 10.0; wild type, wt, x = 15.9 +/- 11.8). CONCLUSIONS: Blood OHP concentration could be used for monitoring human exposure to asbestos, either as a marker for occupational monitoring or as an additional clinical parameter in diagnostic exploration of asbestosis.

Alleles↗

A rapid capillary electrophoresis with electrochemiluminescence method for the assay of human urinary proline and hydroxyproline.

A novel simultaneous determination method for free and total proline (Pro) and hydroxyproline (Hyp) in human urine was developed, based on capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection, using tris-(2,2'-bipyridyl) ruthenium(II). Experimental conditions, such as the Ru(bpy)(3)2+ concentration, detection potentials, buffer concentration and pH in CE or in the ECL cell, injection voltage and time were investigated in detail. Under optimized conditions, the linear range, detection limit and sample recoveries for the method were 0.01-2 mmol/L (correlation coefficient, 0.9970), 4 micromol/L and 96.4-101.2% in human urine, respectively. The results show that the method has potential applications in monitoring the level of Pro and Hyp in body fluids from patients with bone disease, tumours or chronic uraemia.

Buffers↗

O-acylation of hydroxyproline residues: effect on peptide-bond isomerization and collagen stability.

In collagen, strands of the sequence XaaYaaGly form a triple-helical structure. The Yaa residue is often (2S,4R)-4-hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O-acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cgamma atom: AcHyp[C(O)CF3]OMe congruent with Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe congruent with Achyp[C(O)CH3]OMe > or = AcHypOMe congruent with AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)(10), which is O-acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)(10) > {ProHyp[C(O)CH3]Gly}(10) >> (ProProGly)(10). Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group.

Acylation↗

Distribution of plasma-bound hydroxyproline in breast cancer, benign breast disease and healthy females.

In normal adult females, patients with benign breast disease and patients with breast cancer, hydroxyproline (OHPro) was found in the plasma attached to the protein fractions which were separated by Sephadex gel filtration. In addition, a distinct moiety, probably a protein sub-unit, containing substantial amounts of this imino acid, occurred in between gel filtration fractions III and IV. Quantitative differences in the OHPro content of these various fractions were observed in the groups of patients studied. Significant elevations in the peptide-bound OHPro (fraction IV) were found only in patients with breast cancer and the level appeared to correlate with the degree of advancement of the disease. Similar findings relate to the OHPro content of the sub-protein moiety which is found between gel filtration fractions III and IV. A reduced level of high molecular weight hyproprotein (fraction I) was found in the breast cancer group.

Breast Diseases↗

Determination of free and total proline and hydroxyproline in plasma and tissue samples by gas chromatography with flame photometric detection.

A selective and sensitive method for the determination of free and total proline (Pro) and 4-hydroxyproline (Hyp) by gas chromatography (GC) was developed. For free Pro and Hyp analysis, plasma and tissue homogenate were extracted with methanol. For total Pro and Hyp analysis, these samples were hydrolysed in 6 M HCl. After removal of primary amino compounds by the reaction with o-phthaldialdehyde, Pro and Hyp in methanol extract and acid hydrolysate were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. This method was successfully applied to small samples without prior clean-up, and Pro and Hyp in these samples could be analysed without any influence from coexisting substances. Overall recoveries of Pro and Hyp added to plasma and tissue samples were 92-106%. The analytical results of free and total Pro and Hyp in human plasma and mouse tissue samples are presented.

Adult↗

On the mode of action of hydroxyproline in Drosophila development.

Hydroxyproline (OHP) fed to Drosophila larvae is known to impair the ability of pupae to evert cephalic structures and leg and wing discs. In order to gain insight into the metabolic basis of OHP action we have investigated the effects of combined feeding of OHP and several compounds. Proline, glucosamine HCl, and N-acetyl glucosamine considerably reduced the extent of morphological defects of OHP, while alanine and sodium glutamate were less effective. Glutamine and glutathione did not reduce the effects of OHP. The results are consistent with the hypothesis that OHP may indirectly interfere with cuticle synthesis.

Acetylglucosamine↗

Laser photobioactivation mechanisms: in vitro studies using ascorbic acid uptake and hydroxyproline formation as biochemical markers of irradiation response.

Clinical investigations of laser photobioactivation, or biostimulation, might be differently designed and more fruitful if knowledge of basic biochemical mechanisms were better understood. In this investigation, biochemical events identified as responses to 904 nm irradiation included increased ascorbic acid uptake by fibroblasts. These cells also showed increased hydroxyproline formation, and this was increased several-fold by the addition of proline to the medium. Maximum biochemical responses were observed at a pulse frequency of 67 Hz and a pulse width of 150 nsec with an energy density of approximately 7 mJ/cm2 per exposure. Elements in the mitochondrial cytochrome system are proposed as the radiation absorbing chromophore(s). Hypothetically, the energy generated is linked to ascorbic acid uptake, which in turn stimulates collagen synthesis.

Ascorbic Acid↗

Use of urinary hydroxyproline excretion as a tumor marker in diagnosis and follow-up of prostatic carcinoma.

Urinary hydroxyproline/creatinine ratio was measured in 49 patients of prostatic carcinoma and 13 patients with benign prostatic hypertrophy, the latter group serving as controls. The results show that it is a very sensitive indicator of osseous metastases in prostatic carcinoma. The ratio was also measured in 18 patients of prostatic carcinoma with bony metastases before commencement of treatment and during treatment. The results show that the elevated pretreatment values were significantly reduced in those who responded to therapy whereas in nonresponders, the values remained high. Changes in urinary hydroxyproline appeared to reflect the nature of response to treatment better than other parameters.

Bone Neoplasms↗

Estimation of creatine phosphokinase and hydroxyproline in the developing limb: its use in evaluating the effect of teratogens on myogenesis and chondrogenesis.

Forelimbs of mouse fetuses were examined for tissue-specific, drug-induced alterations in their biochemical composition. The activity of the enzyme creatine phospholinase (CPK; to estimate myogenesis) and the content of hydroxyproline (HP; to estimate chondrogenesis) were compared in homogenates of control and treated mouse-fetus forelimbs on day 14 of gestation. In addition, the content of DNA, RNA, and protein was also measured. Injection of 6-aminonicotinamide (6-AN) (15 mg/kg) on day 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a significant reduction in day 14 forelimb HP and RNA content, without altering CPK, DNA, or protein; thus, the HP:CPK ratio was decreased. These results indicated that 1) 6-AN nonspecifically retards growth and cyto-differentiation in limbs; 2) retinoic acid inhibits synthesis of collagen and RNA; 3) retinoic acid has a differential effect upon chondrogenic and myogenic tissues of the limb, and 4) the comparison of HP content and CPK activity in tissue homogenates is an acceptable method of evaluating teratogenic compounds for selective effects.

6-Aminonicotinamide↗

4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent for determination of amino acids in high-performance liquid chromatography and its application for determination of urinary free hydroxyproline.

A highly sensitive fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl), for determination of amino acids by HPLC has been developed. DMS-Cl reacted with amino acids in the basic medium to produce the corresponding fluorescent sulfonamides (excitation and emission wavelength: 318 and 406 nm in aqueous acetonitrile, respectively). When the reactivity of DMS-Cl was investigated by means of reversed-phase HPLC using hydroxyproline (Hyp) and alanine (Ala) as model compounds, the reaction of Hyp was completed within 5 min at 25 degrees C and that of Ala within 15 min at 70 degrees C. The efficiency of conversion of Hyp into the fluorescent derivative was about 100%. The detection limits (signal-to-noise ratio = 3) of almost all amino acids were less than 5 fmol/injection. When the concentration of urinary free Hyp was measured with HPLC using DMS-Cl, the concentration (mean +/- SD, n = 10) was 3.8 +/- 2.76 microM (2.7 +/- 1.71 nmol/mg creatinine).

Amino Acids↗

Probing the bradykinin receptor: mapping the geometric topography using ethers of hydroxyproline in novel peptides.

Five decapeptides were prepared, each having the generic primary sequence D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-X7 -Y8-Arg9. A C-terminal beta-turn was anticipated when X was an alkyl ether of D-4-hydroxyproline in either the cis or trans geometric state and Y was either a Tic or Oic residue. Whereas cis ethers have only very weak receptor affinities, the trans ethers are significantly more potent in binding to guinea pig smooth muscle having Ki values as low as 0.16 nM. Notably, these peptides do not contain a D-aromatic amino acid at position 7 of the primary sequence.

Amino Acid Sequence↗

Mapping of the loci involved in the catabolic oxidation of L-hydroxyproline in Pseudomonas aeruginosa PAO.

Genes specifying the oxidative utilization of hydroxyproline (Hyp) in P. aeruginosa PAO were located on the chromosome, around 19th minute by conjugation experiments. A map order of his-68-his-07-Hyp was assigned. Confirmation of this gene order was also demonstrated by transductional mapping studies. All the genes determining the enzymes of Hyp dissimiliatory pathway were closely linked.

Amino Acid Isomerases↗

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related species.

The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 bp region immediately upstream of the TATA box is highly conserved in the Zea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5' region, which is common to the four genes and is probably essential for their expression. The 3' end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.

Amino Acid Sequence↗

[Hydroxyproline concentration in normal and pathologic altered placentae of the second part of pregnancy (author's transl)].

By using microanalytic Hydroxyproline (HP)-determination method (Woessner and Stegemann) the content of collagen in 50 normal and pathologicaltered placentae of the second part of pregnancy was measured. A positive linear correlation is demonstrated between HP-concentration and age of pregnancy in normal placentae. Most of the pathologic altered placentae have a lower content of collagen. HP-content measured in main stem and terminal villi of further 35 placentae showed the essential influence of the main stem villi on total placental collagen concentration. In the second part of gestation the HP-concentrations of the main stem villi rise on a factor about 5-6, in the terminal villi they remain constant. The values measured within both groups of villi show a great dispersion. The quantitative results were compared with the histology of the placentae.

Collagen↗