Determination Of Hydroxyproline Research Articles

Determination of hydroxyproline in liver tissue by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry

A method for the determination of hydroxyproline (Hyp) in liver tissue of mice by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HILIC-HRMS) was developed. The liver tissue samples of normal mice and liver fibrosis mice induced by carbon tetrachloride were hydrolyzed by concentrated hydrochloric acid. After filtrated and diluted by solution, the diluent was separated on an Hypersil GOLD HILIC column (100 mm×2.1 mm, 3 μm). Water-acetonitrile (28:72, v/v)were used as the mobile phases with isocratic elution. Finally, the target analytes were detected in positive model by HRMS equipped with an electrospray ionization source. The linear range of hydroxyproline was from 0.78 to 100.00 μg/L with the correlation coefficient (R2) of 0.9983. The limit of quantification was 0.78 μg/L. By detecting the spiked samples, the recoveries were in the range of 97.4%-100.9% with the relative standard deviations (RSDs) between 1.4% and 2.0%. In addition, comparison of the measurement results by this method and the chloramine T method was proceeded. It was found that the linear correlation between the two methods was very good, and the Pearson correlation coefficient was 0.927. And this method had simpler operation procedure and higher accuracy than chloramine T method. This method can be used for the quick determination of hydroxyproline in liver tissue samples.

Development of a Fluorescent Labeling Reagent for Determination of Proline and Hydroxyproline in Myeloma Patient Plasma by HPLC-FLD.

A fluorescent derivatization reagent, 3,6-dimethoxy-9-phenyl-9H-carbazole-1-sulfonyl chloride (DPCS-Cl), previously designed, synthesized and developed for amino acids labeling, is here employed. It was used as a pre-column derivatization reagent for the determination of proline (Pro) and hydroxyproline (Hyp) with high-performance liquid chromatography. Both of the analytes were labeled with DPCS-Cl at 60°C for 30 min. The optimized concentration of DPCS-Cl was 25 µg mL-1 and the molar ratio of analytes to DPCS-Cl was 1:6. Excitation wavelength of 318 nm and emission wavelength of 440 nm were selected for the fluorescence detection. For Pro and Hyp, the linear correlation coefficients were all >0.999; the linear ranges of calibration curve were all 5.0 nmol L-1 to 5.0 μmol L-1; the detection limits were 20.0 and 10.0 fmol, respectively; the intra-day and inter-day relative standard deviations were 2.6-3.3% and 3.5-4.6%, respectively. This reagent was applied to the determination of Pro and Hyp in plasma of myeloma patients with its merits of high efficiency of derivatization, stability of the derivatives and high sensitivity.

Suppression of Phosphatidylinositol 3-Kinase/Akt Signaling Attenuates Hypoxia-Induced Pulmonary Hypertension Through the Downregulation of Lysyl Oxidase.

Lysyl oxidase (LOX) is a copper-dependent enzyme that catalyzes covalent cross-linking of collagen. In response to hypoxia, phosphatidylinositol 3-kinase (PI3K) pathway is activated and contributes to pulmonary arterial hypertension (PAH). However, potential role of LOX in hypoxia-induced PAH is poorly understood. In this study, we explored the mechanism responsible for the development of hypoxia-induced PAH. Potent inhibitors of PI3K/Akt and LOX, wortmannin and β-aminopropionitrile (β-APN), were administrated in rat model of hypoxia-induced PAH. The cross-linking of collagen was assessed by the determination of hydroxyproline. LOX, LOXL-1, LOXL-2, LOXL-3, LOXL-4, Akt, and phospho-Akt expression was detected by real-time polymerase chain reaction and western blot analysis. We observed that collagen cross-linking and LOX activity were elevated in hypoxia-exposed rat lung tissue, but these effects were reversed by β-APN and wortmannin. In addition, exposure to hypoxia enhanced mRNA and protein expression and activity of LOX and LOXL-1 in a PI3K/Akt-dependent manner and induced the development of PAH. After the administration of wortmannin, the upregulation of LOX and cross-linking of collagen were significantly reversed in hypoxia-exposed rat pulmonary artery tissue. Taken together, the present study demonstrated that the upregulation of LOX expression and collagen cross-linking is PI3K/Akt dependent in rat with hypoxia-induced PAH. Suppression of PI3K/Akt pathway may alleviate hypoxia-induced PAH through the downregulation of LOX.

Determination of Hydroxyproline in Collagen Biomaterials for Medical Use and Validation of Method

Determination of Hydroxyproline in Collagen Biomaterials for Medical Use and Validation of Method

Improvement of the liver pathology by the aqueous extract and the n-butanol fraction of Sida pilosa Retz in Schistosoma mansoni-infected mice

Ethnopharmacological relevanceSida pilosa Retz (Malvaceae) is a plant used in Africa for the treatment of intestinal helminthiasis, lower abdominal pains and dysmenorrhea. Aim of the studyIn order to determine the potential use of S. pilosa in the treatment of schistosomiasis mansoni, we evaluated the schistosomicidal, antioxidant and anti-fibrotic properties of the aqueous extract and the n-butanol fraction of its aerial parts. Material and methodsS. pilosa aqueous extract (SpAE) at 100, 200 and 400mg/kg and n-butanol fraction (SpBF) at 50, 100 and 200mg/kg were administered per os to Schistosoma mansoni-infected mice for 4 weeks. Praziquantel (100mg/kg×5 days) was used as reference drug. After sacrifice, worm burden and egg count, transaminases and proteins levels were evaluated. Malondialdehyde (MDA), lipid hydroperoxydes (LOOH), catalase (CAT), superoxide dismutase (SOD), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) were also measured. The anti-fibrotic effect of the plant was evaluated by the determination of hydroxyproline and γ-interferon (IFN-γ). ResultsThe treatment of S. mansoni-infected mice by SpAE or SpBF resulted in a moderate reduction of worm burden and egg load in the liver and intestine. Both SpAE and SpBF significantly reversed the increasing liver proteins, MDA, LOOH and CAT levels induced by the infection. Moreover, SOD activity was improved by SpAE and SpBF. Schistosomiasis mansoni considerably increased the EPO (p<0.001) and MPO activities (p<0.001). SpAE treatment significantly reduced EPO and MPO activities at all doses. SpBF failed to reduce the increasing MPO and decreased EPO only at the highest dose. S. mansoni-infection induced an increase in hydroxyproline content (p<0.001) and a decrease in IFN-γ level (p<0.001). Both SpAE and SpBF significantly reduced hepatic hydroxyproline content, while only SpAE (p<0.05) improved IFN-γ level. ConclusionThese results suggest that the liver pathology in schistosomiasis mansoni is improved by S. pilosa aqueous extract, which disclosed a moderate schistosomicidal, but strong antioxidant and anti-fibrotic activities. The n-butanol fraction was however less active than the aqueous extract.

Development of a Reference Measurement Procedure and Certified Reference Material for the Determination of Hydroxyproline in Meat

The concentration of trans-4-hydroxy-l-proline has been traditionally employed as an estimate of collagen content in meat to assess meat quality. Conventional methods use chemical derivatization followed by UV–visible detection. However, complexity of the matrix may cause interferences due to lack of specificity and consequently bias quantification. On the contrary, liquid chromatography (LC) separation coupled to mass spectrometry (MS) detection overcomes these limitations, giving more accurate results. This study describes the development and validation of an exact matching-isotopic dilution mass spectrometry method for the determination of hydroxyproline in meat hydrolysates in the certification of a candidate meat reference material (CRM LGC7154). The method is not biased by changes in method conditions or by matrix load. Hydroxyproline concentrations and relative uncertainties for quality control samples revealed good agreement with the certified values. A mean hydroxyproline concentration of 1.78 ± 0.28 g/kg (k = 2) was found for the CRM LGC 7154 material.

Scientific Assessment Of The Anti-Inflammatory And Wound Healing Potential Of Campanula Lyrata Subsp. Lyrata, A Turkish Folk Remedy

The aim of the present study is to evaluate the anti-inflammatory and wound healing activities of the extracts prepared from the aerial parts of Campanula lyrata Lam. subsp. lyrata (Campanulaceae) by using in vivo methods in order to confirm the traditional utilization. n-Hexane, diethyl ether, ethyl acetate (EtOAc), methanol (MeOH) and aqueous extracts were separately prepared from the air-dried and powdered plant materials. Carrageenan-, and serotonin- induced hind paw edema, 12-Otetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and acetic acid-induced increase in capillary permeability models were employed in mice for the anti-inflammatory activity assessment. Wound-healing activity was investigated by using incision and excision wound models along with hydroxyproline determination and histopathological analyses. MeOH extract displayed significant antiinflammatory effect in the carrageenan- and serotonin- induced hind paw edema model and in acetic acidinduced increase in capillary permeability model with the values of 25.3, 27.8 and 31.8%, respectively. MeOH extract was also found to have significant wound healing potential in the incision and excision wound models with the values of 26.9 and 39.6%, respectively. MeOH extract ointment treated group tissues also showed enhaced hydroxyproline content. The present study confirms the anti-inflammatory

Correlation between Vitamin C Deficiency and Hydroxyproline Amino Acid in Young Children of Northern Part in Palestine

Vitamin C is a water soluble vitamin found in many natural sources, citric fruits, vegetables, particularly as antioxidants and as a factor to catabolism and metabolism. The aim of this article is to correlate the level of vitamin C and the hydroxyproline level in urine samples among young palestinian children from age 5 - 14 years. Materials and Methods: Urine samples of 34 individuals of both sexes, their ages ranged from 5 - 14 years were collected and analyzed for determination of Urinary Hydroxyproline by using Modified Neuman and Logan Method. The result of this study shows a significant correlation between Vitamin C and Hydroxyproline at level 0.01. The conclusion of this study was that low vitamin C intake was found among young palestinian children from age 5 - 14 years, Vitamin C should be supplemented in the drinks taken by young palestinian children from age 5 - 14 years as well as further research and investigation with large samples required to include all children from middle and southern Palestine.

Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque.

Transforming growth factor-β1 (TGF-β1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

Resveratrol ameliorates lipopolysaccharide-induced epithelial mesenchymal transition and pulmonary fibrosis through suppression of oxidative stress and transforming growth factor-β1 signaling

Fibrotic changes seem to be responsible for the high mortality rate observed in patients with acute respiratory distress syndrome (ARDS). The present study aimed to determine whether resveratrol, a natural antioxidant polyphenol, had anti-fibrotic effects in the murine model of lipopolysaccharide (LPS)-induced pulmonary fibrosis. Fibrosis was assessed by determination of collagen deposition, hydroxyproline and type I collagen levels in lung tissues. Development of epithelial-mesenchymal transition (EMT) was identified by the loss of E-cadherin accompanying by the acquisition of α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, levels of phosphorylated Smad2/Smad3 and Smad4, malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, and catalase (CAT) activity in lung tissues were determined. LPS increased collagen deposition, hydroxyproline and type I collagen contents, and meanwhile induced EMT process, stimulated TGF-β1 production and Smad activation in lung tissues on day 21 to day 28 after LPS administration. In addition, LPS treatment resulted in a rapid induction of oxidative stress as evidenced by increase of MDA and decreases of T-AOC, CAT and SOD activities as early as 7 days after LPS treatment, which was persistent for at least 4 weeks. In contrast, resveratrol treatment attenuated LPS-induced EMT and pulmonary fibrosis, meanwhile it suppressed LPS-induced oxidative stress, TGF-β1 production and activation of Smad signaling pathway. Resveratrol may ameliorate LPS-induced EMT and pulmonary fibrosis through suppression of oxidative stress and TGF-β1/Smad signaling pathway. Application of antioxidants may represent a useful adjuvant pharmacologic approach to reduce ARDS-associated pulmonary fibrosis.

Extraction and characterization of collagen from rabbit skin: partial characterization

Extraction and characterization of collagen were carried out in rabbit skins as a new alternative for collagen type I. Acetic acid and pepsin were for the extraction of soluble and insoluble collagen, respectively. The enzymatic treatment yielded higher amount of collagen (71%). The average pH value was 6.3, no matter what the method of extraction was. Denaturation temperature of collagen was found at 36°C approximately in two different techniques: Rheometer and Differential Scanning Calorimetry (DSC). Despite the amount of collagen in solution was low, its viscosity was high because of the hydrodynamic behaviour of collagen molecules. Sodium dodecyl sulphate polyacrylamide gel electrophoresis results showed three different bands that reflected two alpha-chains and one beta-chain with molecular weights of 102, 118 and 220 kDa, respectively. Determination of hydroxyproline gave evidence that the extracted material was collagen. It was concluded that rabbit skin could be an alternative source for the extraction of collagen.

Protective Effects of Norursodeoxycholic Acid Versus Ursodeoxycholic Acid on Thioacetamide-induced Rat Liver Fibrosis

Effects of norursodeoxycholic acid (norUDCA) and ursodeoxycholic acid (UDCA) on liver fibrosis progression and liver fibrosis reversal in thioacetamide (TAA)-treated rats were studied. Advanced liver fibrosis was induced by TAA treatment (200mg/kg, i.p.) for 12 weeks. In the second experiment resolution of liver fibrosis was assessed after 8 weeks of TAA withdrawal. During 8 last weeks of each trial, fibrotic rats were daily administered with UDCA (80mg/kg) and norUDCA (equimolar to 80mg/kg of UDCA) by oral gavage. Liver fibrosis was assessed by Sirius red staining, liver hydroxyproline and serum fibrosis markers determination. The TAA treatment resulted in advanced fibrosis and increase in liver hydroxyproline content and serum fibrosis markers. These signs of fibrosis were less pronounced in rats after TAA withdrawal. Treatment with of norUDCA significantly decreased the total and relative liver hydroxyproline contents in rats with fibrosis reversal, whereas UDCA did not change these parameters. Both compounds decreased serum TGFβ and type IV collagen contents, whereas other serum markers did not differ from the placebo group. In the fibrosis progression model the square of connective tissue was decreased by norUDCA. Serum type IV collagen and procollagen III-NT contents in these experiments were lowered by both UDCA and norUDCA, whereas rest of serum fibrosis markers were diminished only by norUDCA. Both norUDCA and UDCA showed therapeutic and prophylactic antifibrotic effect in rats with TAA-induced liver fibrosis. For most of tested parameters norUDCA was more effective than UDCA, especially in the experiment with liver fibrosis regression.

Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes

Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV(+) F344 rats were transplanted into DPPIV(-) rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV(-) F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1(+), AFP(+), CD133(+), Sox-9(+), FoxJ1(+)) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor β, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells. This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Administration of Bleomycin via the Oropharyngeal Aspiration Route Leads to Sustained Lung Fibrosis in Mice and Rats as Quantified by UTE-MRI and Histology

Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats.

Hepatic apoptosis can modulate liver fibrosis through TIMP1 pathway

Apoptotic injury participates in hepatic fibrosis, but the molecular mechanisms are not well understood. The present study aimed to investigate the role of inducible TIMP1 in the pathogenesis of hepatic apoptosis-fibrosis. Apoptosis was induced with GCDC, LPS, and alcohol in precision-cut liver slices or bile duct ligation (BDL) in rats, as reflected by caspase-3 activity, TUNEL assay, and apoptosis-related gene profiles. The hepatic fibrosis was detected with Picrosirius staining, hydroxyproline determination, and expression profiling of fibrosis-related genes. Levels of TIMP1 were upregulated by the hepatic apoptosis, but downregulated by caspase inhibitor. The inducible TIMP1 was apoptosis-dependent. Once TIMP1 was inhibited with treatment of TIMP1-siRNA, the fibrotic response was reduced as demonstrated by hydroxyproline assay. In addition, the expression of fibrosis-related genes aSMA, CTGF, and TGFb2r were down-regulated subsequent to the treatment of TIMP1-siRNA. TIMP1 could mediate the expression of fibrosis-related genes. TIMP1 was transcriptionally regulated by nuclear factor c-Jun as demonstrated by EMSA and ChIP assay. The treatment of c-Jun siRNA could significantly decrease the expression of TIMP1 induced by alcohol, GCDC, or LPS treatment. Hepatic apoptosis induces the expression of TIMP1. Inducible TIMP1 can modulate the expression of fibrosis-related genes in liver. TIMP1 pathway is a potential target for therapeutic intervention of fibrotic liver diseases.

Targeted Gene Transfer of Hepatocyte Growth Factor to Alveolar Type II Epithelial Cells Reduces Lung Fibrosis in Rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

Laser therapy of muscle injuries

Low-level lasers are used in general therapy and healing process due to their good photo-bio-stimulation effects. In this paper, the effects of diode laser and Nd:YAG laser on the healing process of practically managed skeletal muscle trauma has been successfully studied. Standard impact trauma was induced by using a specially designed mechanical device. The impacted muscle was left for 3 days for complete development of blunt trauma. After that it was irradiated by five laser sessions for 5 days. Two types of lasers were used; 785-nm diode laser and 1.064-nm Nd:YAG laser, both in continuous and pulsed modes. A special electronic circuit was designed and implemented to modulate the diode laser for this purpose. Tissue samples of crushed skeletal muscle have been dissected from the injured irradiated muscle then bio-chemically analyzed for the regeneration of contractile and collagenous proteins using Lowry assay for protein determination and Reddy and Enwemeka assay for hydroxyproline determination. The results showed that both lasers stimulate the regeneration capability of traumatized skeletal muscle. The diode laser in CW and pulsed modes showed better results than the Nd:YAG in accelerating the preservation of the normal tissue content of collagenous and contractile proteins beside controlling the regeneration of non-functional fibrous tissue. This study proved that the healing achieved by the laser treatment was faster than the control group by 15-20 days.

Environmentally friendly hide unhairing: enzymatic hide processing for the replacement of sodium sulfide and delimig

In this work is described the use of an enzyme preparation produced by a new strain of Bacillus subtilis for hide unhairing aiming at completely replace the chemical process, which uses toxic sulfides and lime. The whole process was designed to be fast and cheap, by applying the crude enzymatic extracts of B. subtilis BLBc 11 cultures on the hides, with good results obtained in just 6 h of process. The treated hides were evaluated for their quality by optical microscopy and analyzes of inter-fibrillary proteins (proteoglycans and glycosaminoglicans). Undesired damage to the hide grain was evaluated by the determination of free hydroxyproline in the process liquor. Furthermore, the environmental impact of chemical and enzymatic treatments were compared, and the processes were evaluated by analyzing the chemical oxygen demand (COD), biological oxygen demand (BOD), total nitrogen, and sulfides in the residual wastewater, as the proprieties of the finished leather. COD reductions were of 42%, 100%, and 82% compared with the chemical process for the unhairing, deliming, and bating steps, respectively. The enzymatic unhairing process showed excellent results, with time reduction for unhairing of more than 50%, without the need for deliming, suggesting that this enzymatic preparation could be used to replace the chemical process.

Rapid and sensitive determination of hydroxyproline in dairy products using micellar electrokinetic chromatography with laser-induced fluorescence detection

Many reports have focused on the determination of hydroxyproline (Hyp) in blood plasma, urine sample, meat and meat products, however, there are few concerned with the Hyp assay in dairy products for food quality assurance up to now. In this paper, we described a sensitive and automated approach for the determination of Hyp in milk powder, liquid milk, milk drink and soymilk powder samples by micellar electrokinetic chromatography (MEKC) based on in-capillary derivatization for the first time. Under the optimal conditions, derivatization and separation procedure could be completed within 7min and the detection limit for Hyp was 1.6±0.5ngmL−1. Comparing with the existing alternatives, the present method exhibited some relevant advantages, including full automation, satisfactory sensitivity, and short analysis time for Hyp assay in dairy products.

Prenatal nicotine exposure induces gender-associated left ventricular-arterial uncoupling in adult offspring.

Alterations in vascular or myocardial structure and function have been demonstrated in offspring subjected to prenatal nicotine exposure (PNE), however, limited data are available on how these changes interact. The present study assessed the hypothesis that prenatal nicotine exposure induced gender-specific alterations of left ventricular-arterial coupling indices in adult offspring. Female Sprague-Dawley rats were exposed to either nicotine (8 mg/kg/day) or saline via subcutaneous osmotic mini-pumps throughout gestation. Male and female offspring, aged 12 months, underwent non-invasive echocardiography and invasive left ventricular cannulation. Left ventricular-arterial coupling was analysed as the ratio of effective arterial elastance (Ea) to ventricular end-systolic elastance (Ees). The left ventricular myocardium and aorta were stained with hematoxylin and eosin and the myocardial cell cross-sectional area was calculated. Simultaneously, the ratio of medium thickness to internal diameter in the aorta and mesenteric artery was determined. The fibrosis component of left ventricle myocardium was analyzed by Sirius-red staining and further confirmed by hydroxyproline determination. The elastic properties of the aortic wall were analyzed by van Gieson staining. PNE caused significant increases in pulse pressure (56.36 ± 7.41 vs. 50.16 ± 4.94 mmHg; P<0.05) and left ventricular meridional wall stress (78.25 ± 9.12 vs. 69.64 ± 7.58 kdyne/cm(2); P<0.05) in male offspring compared with the control. Conversely, no similar effect was observed in female offspring. An elevated augmentation index was noted in male and female pups. Additionally, Ea/Ees was reduced in PNE males compared with control males, due to a disproportionate increase in Ees vs. Ea whereas in females, Ea/Ees did not differ significantly due to tandem increase in Ea and Ees. In addition, collagen cross-linking was markedly higher in male offspring, whereas it was unaltered in females compared with their respective controls. Fragmentation of the elastic network in the aorta and the increased ratio of medial thickness to internal diameter in the mesenteric artery were more evident in male offspring when compared with female offspring. PNE caused combined ventricular-arterial stiffening in male and female offspring, with lower Ea/Ees in males, while Ea/Ees was preserved in females. Enhanced collagen cross-linking in the myocardium, underdeveloped elastic fibers in the aorta and remodeled resistance vessels were associated with pathological ventricular arterial mismatching. The results of the present study indicated that male offspring were more susceptible to the development of ventricular and arterial dysfunction in response to PNE compared with female offspring.