柱前衍生高效液相色谱法对动物组织中胶原蛋白的测定

动物组织中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用邻苯二甲醛（OPA）与其中的一级氨基酸衍生,再用9-芴基甲氧基羰酰氯（FMOC）与其中的羟脯氨酸衍生,用反相高效液相色谱法测定羟脯氨酸含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量计算胶原蛋白含量。在0.01-50 mg.L^-1范围内,羟脯氨酸的峰面积和质量浓度之间的相关系数为0.9993,保留时间和峰面积的相对标准偏差分别为0.30%和2.9%。该法选用FMOC与氨基酸衍生产物的特征波长检测,能够完全屏蔽一级氨基酸衍生物的干扰,可快速、准确、高效地测定羟脯氨酸及胶原蛋白的含量。     

Hydroxyproline Measurement by High Performance Liquid Chromatography: An Improved Method of Derivatization

Abstract

Hydroxyproline is a postsynthetic derivative of proline which is commonly used to estimate the collagen content of tissues. Values (weight %) of hydroxyproline range from ten to twenty percent in most collagen types, with Type I having 11.4 percent hydroxyproline. This fact is useful for estimating the collagen content in various acid hydrolyzed tissue samples. In this paper, the authors describe a technique which produces linear hydrolysis of collagen coupled with a sensitive ultraviolet detection scheme for the 9-Fluorenylmethoxycarbonyl Chloride(FMOC-Cl) derivative of hydroxyproline. The separation itself employs reverse phase chromatography with a sensitivity of approximately 0.12 nmoles of hydroxyproline or 0.14 μg Type I collagen.     

雄黄染毒后大鼠脑组织中氨基酸类神经递质含量的变化

研究了雄黄对大鼠脑组织氨基酸类神经递质含量的影响.将32只Wistar大鼠随机分为对照组(0.5%CMC-Na)以及低剂量(0.3g/kg)、中剂量(0.9g/kg)、高剂量(2.7g/kg)雄黄染毒组,通过连续灌胃给予雄黄混悬液两周.采用高效液相色谱-柱前衍生化法测定了大鼠脑组织中氨基酸类神经递质含量的变化.结果表明,与对照组比较,低剂量组大鼠脑组织中丝氨酸、甘氨酸和γ-氨基丁酸含量明显增加.中、高剂量组大鼠脑组织中同型半胱氨酸、谷氨酰胺、丝氨酸和天冬氨酸含量明显比对照组的低.总体而言,雄黄可对大鼠脑组织氨基酸类神经递质产生影响,氨基酸类神经递质可能是雄黄毒性作用的靶点之一.     

Microderivatization of 4-[18O]hydroxyproline and quantitation with a benchtop mass spectrometer

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [18O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [18O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph-mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of 18O incorporation.     

Identification of collagen types in tissues using HPLC-MS/MS

A method for the determination and quantification of collagen types I-V in rat tissues has been developed. This method is based on collagen fragmentation by cyanogen bromide followed by trypsin digestion. After that, HPLC-MS/MS (HPLC coupled to an IT mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Specific peptides for each collagen type were selected. According to online databases, these peptides are present in human, bovine, and rat collagens. As a result, this method can be potentially applied to other species' tissues as well, such as human tissues, and provides a universal and simple method of quantifying collagen types. The applicability of this method for analyzing collagen types was demonstrated on rat tissues (skin, tendon, and aorta).     

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice.

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.     

Surprisingly high stability of collagen ABC heterotrimer: evaluation of side chain charge pairs

Type I collagen is a major component of skin, tendon, and ligament and forms more than 90% of bone mass. It is an AAB heterotrimer assembled from two identical alpha1 and one alpha2 chains. However, the majority of studies on the effects of amino acid substitution on triple helix stability have been performed on collagen homotrimeric helices. In a homotrimer, it is impossible to determine whether the contribution to stability is from the polyproline II helix propensity of the amino acids or from interhelix amino acid interactions. The presence of amino acids in all three chains further exaggerates their contribution. In contrast, in a heterotrimer, the individual chains may be tailored in order to have the substitution in one, two, or all three chains. Therefore, a heterotrimer can divulge specific information about any interaction based upon the substitutions in individual chains. In this paper, we evaluate the contribution of electrostatic interactions between side chain charge pairs on the stability of heterotrimers. We synthesize and analyze the stability of four AAB and four ABC heterotrimers including a surprisingly stable ABC heterotrimer composed of (DOG)10, (PKG)10, and (POG)10 chains (O = hydroxyproline). This heterotrimer has a stability comparable to that of a (POG)10 homotrimer even though D and K occur 20 times in the heterotrimeric helix and have been previously shown to significantly destabilize the triple helix compared to the P and O imino acids. These results show that the stability of heterotrimers cannot be directly determined from the analysis of charge pairs in homotrimers. Because collagen heterotrimers can be designed to have substitution in one, two, or three chains, it gives us the ability to decode cross-strand interactions in collagen in a similar fashion to alpha-helical coiled-coil interactions and DNA duplex hydrogen bonding.     

Hydroxyproline quantification for the estimation of collagen in tissue using multiple reaction monitoring mass spectrometry

Collagens are highly abundant mammalian proteins that contain a high content of hydroxylated amino acids such as hydroxyproline. We have exploited the high hydroxyproline content of collagen and developed a method for hydroxyproline quantification as a measure of collagen content in muscle samples. The novel method utilizes a highly selective and sensitive method of multiple reaction monitoring (MRM) by mass spectrometry. The analytical method is simple, rapid (5min), convenient (no derivatization), precise (<17% RSD), accurate (90-108%), sensitive (4.88nmol/L) and linear (R(2)>0.999) over three orders of magnitude (5-5000nmol/L).     

Determination of 4‐hydroxyproline‐2‐epimerase activity by capillary electrophoresis: A stereoselective platform for inhibitor screening of amino acid isomerases

Isomerases involved in the metabolism of D /L ‐amino acids represent promising therapeutic targets for treatment of disease. Herein, we report a tunable platform for the assessment of enzymatic kinetics involving amino acid isomerization by CE that offers improved selectivity and sensitivity over traditional methods. Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4‐hydroxyproline‐2‐epimerase as a model system. In this work, pyrrole 2‐carboxylic acid was found to be a selective inhibitor of 4‐hydroxyproline‐2‐epimerase with a half‐maximal inhibition concentration of (2.3±0.1) mM. Reliable methods for unambiguous characterization of amino acid isomerases are required for the screening of novel inhibitors with epimerase and/or racemase activity.     

Solid-phase synthesis and structural characterization of highly substituted hydroxyproline-based 2,5-diketopiperazines

Two general solid-phase methods for the synthesis of a new class of 2,5-diketopiperazines (DKPs) containing the trans-4-hydroxy-L-proline amino acid residue (Hyp) have been developed. An N-protected hydroxyproline methyl ester was linked through the hydroxyl function to the Ellman resin. The synthesis procedures were conceived to enable a sequence of Hyp alkylation, Hyp N-acylation, cyclization, and amide bond alkylation. Up to three different centers of molecular diversity were introduced around the DKP scaffold. Highly functionalized bicyclic compounds were obtained in good yield and purity. The alkylation of hydroxyproline alpha CH was performed without control of the diastereoselectivity. During the final alkylation of the backbone, amide bond epimerization at the alpha-carbon atoms of the two amino acid residues was observed. The structures of representative DKPs were elucidated with multidimensional NMR experiments. The described reaction pathways can be applied to the identification of heterocyclic molecule inhibitors to diverse enzyme targets.     

Kinetics of biomethanation of solid tannery waste and the concept of interactive metabolic control

Anaerobic digestion of calf skin collagenous waste was optimized for a batch process based on accelerated maximal methane yield per gram of input volatile solid. A kinetic analysis with respect to changes in the levels of volatile solid, collagen, amino sugars, amino acids, hydroxyproline, ammonium ions, and volatile fatty acid were followed for a period of 80 d. Distinct metabolic phases included an initial high rate collagenolysis for 4 d, with 50% degradation and was followed by an acidogenic phase between 4–12 d with voltatile fatty acids levels increasing to 215 mmol/L. Subsequently methanogenesis ensued and was maximal between 12–24 d when volatile fatty acids attained steady state levels. During the period of 80 d, the overall decrease in volatile solid level was 65%, whereas the collagen level declined by 85% with 0.45 L of methane yield/g of volatile solid degraded. Based on the levels of various metabolites detected, the concept of interactive metabolic control earlier proposed has been validated.     

The effect of NDGA-modified etchant on the enzymatic degradation resistance and mechanical properties of collagen matrix

Bio-modified etchant can significantly improve the biostability of demineralized dentin collagen matrix, which validates the concept of etch-andcrosslink in dentin bonding.     

Determination of proline and hydroxyproline in wheat flour by spectrophotometry and measurement of radioactivity of proline-14C and hydroxyproline-14C

Proline and hydroxyproline are two amino acids which due to their analogous chemical structures give similar reactions and often appear together. In view of the high percentage of proline in wheat gluten we have investigated the presence of hydroxyproline in wheat flour. We have developed a method to separate the two amino acids after separation from other interfering amino acids which are present in flour. The method of separation utilizes an ion-exchange column/Dowex 50 X-8, 100–200 resin/. The separated amino acids are determined either by spectrophotometry or isotope dilution /¹⁴C/ The latter method is more rapid and more sensitive than the spectrophotometric method.     

The determination of microgram amounts of collagen metabolites in the urine of primates inhaling silica dust

Collagen metabolites in urine are determined by means of the hydroxyproline index for collagen. A simple and reproducible method for hydrolysis is outlined obviating the need to heat-seal reaction tubes under vacuum. Six or more urine specimens can be processed simultaneously without risk of loss of specimen. In particular, specimens containing very small amounts of collagen metabolities may be determined by repeatedly evaporating 1-ml aliquots of urine before hydrolysis.     

Purification and Characterization of Pepsin-Solubilized Collagen from Skin of Sea Cucumber Holothuria parva

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α₁ chains (α₁)₃, in contrast to calf skin collagen type I with two α₁ and one α₂ chains (α₁)₂α₂ with approximately 130 kDa each. The maximum transition (T_m) and denaturation temperature (T_d) of PSC were determined to be 46.94 and 32.5 °C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.     

New protocol for compound‐specific radiocarbon analysis of archaeological bones

Rationale

For radiocarbon results to be accurate, samples must be free of contaminating carbon. Sample pre‐treatment using a high‐performance liquid chromatography (HPLC) approach has been developed at the Oxford Radiocarbon Accelerator Unit (ORAU) as an alternative to conventional methods for dating heavily contaminated bones. This approach isolates hydroxyproline from bone collagen, enabling a purified bone‐specific fraction to then be radiocarbon dated by accelerator mass spectrometry (AMS).

Methods

Using semi‐preparative chromatography and non‐carbon‐based eluents, this technique enables the separation of underivatised amino acids liberated by hydrolysis of extracted bone collagen. A particular focus has been the isolation of hydroxyproline for single‐compound AMS dating since this amino acid is one of the main contributors to the total amount of carbon in mammalian collagen. Our previous approach, involving a carbon‐free aqueous mobile phase, required a two‐step separation using two different chromatographic columns.

Results

This paper reports significant improvements that have been recently made to the method to enable faster semi‐preparative separation of hydroxyproline from bone collagen, making the method more suitable for routine radiocarbon dating of contaminated and/or poorly preserved bone samples by AMS. All steps of the procedure, from the collagen extraction to the correction of the AMS data, are described.

Conclusions

The modifications to the hardware and to the method itself have reduced significantly the time required for the preparation of each sample. This makes it easier for other radiocarbon facilities to implement and use this approach as a routine method for preparing contaminated bone samples.

     

Thermal and non-thermal effects of nonablative IR laser irradiation of the collagen of annulus fibrosus tissues

Collagen modifications in the tissues of an annulus fibrosus under moderate-intensity IR laser irradiation at various pulse durations and frequencies were studied. Changes at various levels of the ordered structural hierarchy of collagen in tissues were detected by differential scanning calorimetry, multiphoton microscopy with second harmonic generation, and cross-polarized optical coherence tomography. The obtained data confirm the photothermal and photomechanical effect of IR laser action on fibrous structures. Details of these effects (mechanical defect formation or protein denaturation) were determined, and the greatest tissue damage was demonstrated to be generated by their combined action.     

Determination of terbinafine in tissues

Terbinafine and N-demethyl terbinafine concentrations were determined simultaneously in rat tissues by a high-performance liquid chromatography method. This method involved the homogenization of tissues (except for skin) followed by a liquid-liquid extraction. Skin samples were dissolved in sodium hydroxide prior to extraction. Terbinafine and its N-demethylated metabolite were assayed using a C(18) reversed-phase column with a mobile phase of acetonitrile and water (40:60) containing ortho phosphoric acid (0.02 M) and triethylamine (0.01 M), and UV detection (at 224 nm). The standard curve for the assay (constructed using clotrimazole as internal standard) was linear over the concentration range 100-3000 ng/g in skin and 10-600 ng/g in all other tissues. The inter- and intra-day precision for both terbinafine and metabolite was between 0.2% and 16%. The limit of quantification was 10 ng/g in all tissues and 100 ng/g in skin. This assay was found to be reliable and reproducible for the determination of terbinafine and N-demethyl terbinafine concentration in all rat tissues and has been used for tissue distribution studies.     

CHANGES IN TOTAL DIFFUSE SPECTRAL REFLECTANCE OF AMINO ACIDS AND PROTEINS AFTER ULTRAVIOLET IRRADIATION

Abstract— Total diffuse reflectance spectra of air-dried surfaces of free and neutralized amino acid preparations before and after irradiation in vitro are reported. It was found that some free or neutralized amino acid surfaces underwent modification which resulted in changes in the diffuse reflectance spectra after U.V. exposure. It is suggested that these reflectance changes result from transformations in the side chains of the amino acids and that the transformations may differ from those occurring when amino acids in solution are irradiated. Histidine, cystine, hydroxyproline and some protein surfaces showed changes in reflectance of 330–400 nm light similar to those reported in skin after U.V. irradiation in viuo.