• BMB Reports
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• v.36 no.2
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• pp.154-158
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• 2003

Collagen is a family of proteins which consists of several genetically distinct molecular species and is intimately involved in tissue organization, function, differentiation and development. The purpose of this study was to investigate the concentration of different hydroxyproline (Hyp) fractions viz., total, free, peptide-bound, protein-bound, soluble- and insoluble-collagen hydroxyproline (Hyp) in various bovine tissues. Results showed that liver had the highest concentration of free Hyp followed by kidney, brain, spleen, lungs, muscle and heart. Liver also had the highest concentration of peptide-bound collagen Hyp followed by kidney, heart, spleen, lungs, brain and muscle. The concentration of protein-bound collagen Hyp was highest in the liver, followed by kidney, spleen, lungs, muscle, brain and heart. Total Hyp was highest in the liver, followed by kidney, spleen, brain, heart, muscle and lungs. Liver also had significantly high concentration of collagen as compared to other tissues examined (P<0.001). Spleen had the significantly higher concentration of soluble-collagen Hyp when compared to other tissues (P<0.001). This was followed by heart, muscle, lungs, brain, kidney and liver. Heart had the highest concentration of insoluble-collagen Hyp followed by lungs, kidney, liver, muscle, spleen and brain. The variation among the insoluble-collagen Hyp concentration of heart and muscle, spleen and brain was significant (P<0.001). We speculate that these differences could be due to the variation in turn over of rate of collagen metabolism in this species.

• Food Science of Animal Resources
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• v.26 no.3
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• pp.362-367
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• 2006

This study was conducted to evaluate the feasibility of determination of hydroxyproline (Hyp) contents in pork meat for the quality evaluation of meat products. The relationship between the Hyp contents and pork grade was not high in porcine semitendinosus muscle. Whereas, Hyp contents of pork fresh ham and hamburger patties prepared with them were significantly increased as the decrease of pork grade. These results showed that the determination of Hyp contents might be a useful method for the quality evaluation of meat products.

• Journal of Preventive Medicine and Public Health
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• v.44 no.1
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• pp.9-13
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• 2011

Objectives: Although increased reactive oxygen species (ROS) is caused by stress accelerates collagen degradation, there was no data on the relationship between stress and urinary hydroxyproline (Hyp) and proline (Pro), a good marker of collagen degradation. The purpose of this study was to evaluate the relationship between depression, anxiety, and stress (DAS) and concentrations of urinary Hyp and Pro. Methods: 97 hospital employees aged 20 to 58 were asked to fill out comprehensive self-administrated questionnaires containing information about their medical history, lifestyle, length of the work year, shit-work and DAS. Depression Anxiety Stress Scale (DASS) was applied to evaluate chronic mental disorders. Urine samples were analyzed using High Performance Liquid Chromatography (HPLC) with double derivatization for the assay of hydroxyproline and proline. Results: The mean value of Hyp and Pro concenturation in all subjects was $194.1{\pm}113.4\;{\mu}mol/g$ and $568.2{\pm}310.7\;{\mu}mol/g$. DASS values and urinary Pro concentrations were differentiated by sex (female > male, p < 0.05) and type of job (nurse > others, p < 0.05). In the stepwise multiple linear regressions, urinary Hyp and Pro concentrations were influenced by stress (Adjusted $r^2$ = 0.051) and anxiety and job (Adjusted $r^2$ = 0.199), respectively. Conclusions: We found that stress and anxiety were correlated with urinary Hyp and Pro concentrations. To identifying a definite correlation, further study in large populations will be needed.

• Korean Journal of Food Science and Technology
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• v.40 no.4
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• pp.436-442
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• 2008

Hydroxyproline and Pro-Hyp dipeptide are the digestive products of collagen hydrolysate called collagen peptide. Some suggested that collagen peptides could improve aged or damaged skins, however, the effects of collagen peptides on the skin have not been known. In this study, we investigated the effects of digestive products of collagen peptides, hydroxyproline and Pro-Hyp dipeptide on skin quality using the UV-damaged dorsal skin of hairless mouse as a model system. Female SKH hairless mice were pre-irradiated with UV for 7 weeks, and then hydroxyproline, Pro-Hyp dipeptide were orally administered for 7 weeks with UV irradiation. Wrinkle formation (by replica image), skin elasticity, barrier status (by TEWL, transepidermal water loss), epidermis thickness, and biophysical changes in the stratum comeum (by hematoxylin & eosin staining) were examined. With the oral peptide treatment, effects such as skin barrier maintenance, anti-skin thickening, and recovery of the stratum corneum were observed. These results indicate that oral intake of collagen peptides may have beneficial effects on damaged skin cells.

• Journal of Preventive Medicine and Public Health
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• v.35 no.4
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• pp.282-286
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• 2002

Objectives : The level of 4-hydroxyproline (4-Hyp) in human urine was measured using high performance liquid chromatography (HPLC) with a fluorescence detector. This method is useful for medical examinations and investigating the radicals induced by physical, chemical, mental stresses. This method is superior to many published several methods in terms of its low cost and ability to analyze many samples. Methods : The urine from workers in a tire manufacturing company (22 male pre- and post-shift workers) and 18 office-workers as controls were analyzed. Data concerning age, the cumulative drinking amount and the cumulative smoking amount was collected with a questionnaire. The optimum applied amount of dansyl-Cl, the optimum reaction temperature and time, the recoveries and the optimum pH of the eluent and buffer were determined.4-Hyp from human urine was derivatized with dansyl-Cl (dimethylamino-naphthalene-1-sulfonyl chloride) after removing the a-amino acid by a treatment with phthalic dicarboxaldehyde (OPA) and cleaned with Bond Elut C18 column. The 4-Hyp derivatives were separated on a reversed phase column by gradient elution with a phosphate buffer (5 mmol, pH 8.0) and acetonitrile, and detected by fluorescence measurements at 340 nm (excitation) and 538 nm (emission). Results : The detection limit for the urinary free 4-Hyp was $0.364{\mu}mol/l$. The recovery rate of 4-Hyp was 99.7%, and the effective pH of the phosphate buffer and borate buffer were 3.0 and 8.0, respectively. From statistical analysis, age, drinking and smoking did not affect the urinary free 4-Hyp in both the controls and workers. The range of urinary 4-Hyp in the controls, pre-shift, and post-shift workers were 0.33-16.44, N.D-49.06, and $0.32-56.27{\mu}mol/l$. From the pared-sample t-test, the urinary 4-Hyp levels in post-shift workers ($11.82{\pm}6.73\;nmmol/mg\;Cre$) were 2-fold higher than in pre-shift workers ($5.36{\pm}5.53\;nmol;/mg\;Cre$) and controls ($4.91{\pm}4.89\;nmol;/mg\;Cre$). Conclusions : This method was developed with high sensitivity, accuracy, and precision. The present method was effectively applied to analyze the urinary free 4-Hyp in both controls and workers.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.135-140
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• 2004

Anti-skin aging agent could be have an inhibition effect of ROS production as well as fragmentation and change of collagen cross linkage in collagen molecule. For the monitoring of lipid peroxidation and collagen degradation, the skin of young and old rats were incised and observed 7 days. In the result, the wound closure was observed in the skin from 10 of 11 young rats and in 8 of 11 old rats. And the longer wound length but shorter wound closure, weaker collagen density and thicker epidermis were observed in old rats than in young rats. The level of hydroxyproline as a parameter of collagen synthesis and MDA as a parameter of lipid peroxidation was lower in old group than in young group. The cyst and lacuna between collagen bundle and fibroblast were observed in old rats in contrast to young rats. So that we propose that MDA and hydroxyproline could be used for monitoring of anti-skin aging agent.

• Biomolecules & Therapeutics
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• v.15 no.4
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• Nutrition Research and Practice
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• v.13 no.6
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• pp.480-487
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• 2019

BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of osteoporosis. We speculated that chicken legs, a food consumed in many regions of the world, may also contain such ingredients. In this study, we aim to (i) evaluate the effect of chicken leg extract (CLE) on the promotion of cartilage matrix production and (ii) identify the active ingredient in CLE that contributes to this function. MATERIALS/METHODS: Artificial CLE digest was prepared, and the acid mucopolysaccharide production-promoting activity of the CLE digest was evaluated by alcian blue staining of ATDC5 cells. CLE was orally administered to rabbits with burr holes in the knee joint of the femur, and the degree of regeneration of cartilage matrix was evaluated. Furthermore, we investigated orally administered CLE-derived peptides in human plasma using LC-MS. From measuring the acid mucopolysaccharide production-promotion activity of these peptides, a molecule considered to be an active ingredient in the CLE digest was identified. RESULTS: CLE digest promoted acid mucopolysaccharide production and facilitated regeneration of cartilage matrix in in vitro and in vivo experiments. Four peptides including phenylalanyl-hydroxyproline (Phe-Hyp) were detected as CLE-derived peptides in human plasma. The effect of CLE was inferred to be due to Phe-Hyp, which was confirmed to be present in the CLE digest. CONCLUSIONS: It was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for preventing or treating OA. Furthermore, only Phe-Hyp was confirmed as the active compound in the CLE digest, suggesting that the activity of CLE was due to Phe-Hyp.

• Toxicological Research
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• v.25 no.2
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• pp.71-78
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• 2009

We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and $CCl_4$-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of $CCl_4$-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen ${\alpha}1$(III) and $\beta$-actin mRNA was observed by RTPCR. The histological change in liver tissue was also observed by Masson's trichrome and H&E staining. Correlation analysis was carried by Spearman's rho method. All biochemical parameters except total-bilirubin were significantly higher in the $CCl_4$-thiopental sod. treated group than that of the normal group (p < 0.01). In the $CCl_4$-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p < 0.001) respectively. The activity of SOD in the $CCl_4$-thiopental sod. treated group is decreased significantly by 44.8% (p < 0.001). And collagen ${\alpha}1$(III) mRNA was more expressed in the $CCl_4$-thiopental sod. treated group than that of the normal group. However, the expression of $\beta$-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = -0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = -0.40, n = 25) in the two groups. Elevated levels of MDA in $CCl_4$-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that (1) lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of $CCl_4$-thiopental sod. during a short terms. And (2) the intoxication of $CCl_4$-thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and deposition) for test of antioxidant and antifibrotic agent.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.154.1-154.1
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• 2003

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase which plays a crucial role in the synthesis of all collagens, because the 4-hydroxyproline residues are essential for the folding of the newly synthesized collagen polypeptide chains into triple-heical molecules. Considering the role of collagen and its significance in many clinically important diseases such as liver cirrhosis, a great deal of attention has been directed toward the development of an assay at cell-based system. (omitted)