Ducci M, Pacchini S, Niccolini A, Gazzano A, Cerri D, Gadea J, Bobowiec R, Sighieri C, Martelli F
Department of Anatomy, Biochemistry and Veterinary Physiology, Faculty of Veterinary Medicine, University of Pisa, 2 Viale delle Piagge, 56124 Pisa, Italy.
Pol J Vet Sci. 2006;9(3):159-63.
The present study deals with the application of high-performance-liquid-chromatography (HPLC) method for a quantitative detection of carnosine, anserine, L-histidine and 3-methyl-L-histidine in biological material with o-phthaldialdehyde (OPA) post column derivatisation at the constant temperature of 50 degrees C. For this purpose, some mobile-phases were prepared with scalar acetonitrile concentrations. A complete separation of all molecules, particularly for carnosine and 3-methyl-L-histidine, was obtained with a solution of acetonitrile and 6mM hydrochloric acid with 0.48 M sodium chloride (5%:95% v/v). Post column derivatisation reaction at temperature of 50'C permitted to obtain an increase in sensibility of all molecules. This method has been utilised for detection of histidine dipeptides in boar spermatozoa and in sheep milk. Concentrations (mean +/- S.E. nmol/10(9) spermatozoa) of carnosine (0.96 +/- 0.14) and anserine (0.83 +/- 0.18) in boar spermatozoa were significantly lower than those of L-histidine (52.85 +/- 4.86) and 3-methyl-L-histidine (83.07 +/- 7.1). Positive correlation was found between carnosine and anserine contents (r = 0.740; p < 0.01) and between L-histidine and 3-methyl-L-histidine (r = 0.657; p < 0.01). All histidine dipeptides studied were also present in 40 samples of sheep milk. In a case of samples without unit-forming colonies (UFC) of Staphylococcus coagulase-positive, carnosine concentrations (9.17 +/- 0.89 nmol/ml) were higher than anserine (0.51 +/- 0.02 nmol/ml) and both were significantly lower in respect to L-histidine (49.51 +/- 6.48 nmol/ml) and 3-metyl-L-histidine (81.21 +/- 6.82 nmol/ml). A negative correlation was observed between carnosine milk levels (r = -0.773; p < 0.01) and UFC/ml of Staphylococcus coagulase-positive. In conclusion this very simple and fast method can be used to detect histidine dipeptides in biological compartments where their concentrations are very low.
本研究探讨了高效液相色谱（HPLC）法在生物材料中定量检测肌肽、鹅肌肽、L-组氨酸和3-甲基-L-组氨酸的应用，采用邻苯二甲醛（OPA）柱后衍生化，温度恒定为50摄氏度。为此，制备了不同乙腈浓度梯度的流动相。采用乙腈与6mM盐酸及0.48M氯化钠的溶液（5%:95% v/v）可实现所有分子的完全分离，尤其是肌肽和3-甲基-L-组氨酸。50摄氏度的柱后衍生化反应可提高所有分子的检测灵敏度。该方法已用于检测公猪精子和羊奶中的组氨酸二肽。公猪精子中肌肽（0.96 +/- 0.14）和鹅肌肽（0.83 +/- 0.18）的浓度（平均 +/- 标准误，nmol/10(9)精子）显著低于L-组氨酸（52.85 +/- 4.86）和3-甲基-L-组氨酸（83.07 +/- 7.1）。肌肽和鹅肌肽含量之间呈正相关（r = 0.740；p < 0.01），L-组氨酸和3-甲基-L-组氨酸之间也呈正相关（r = 0.657；p < 0.01）。所研究的所有组氨酸二肽在40份羊奶样品中也均有存在。在无凝固酶阳性葡萄球菌单位形成菌落（UFC）的样品中，肌肽浓度（9.17 +/- 0.89 nmol/ml）高于鹅肌肽（0.51 +/- 0.02 nmol/ml），且两者均显著低于L-组氨酸（49.51 +/- 6.48 nmol/ml）和3-甲基-L-组氨酸（81.21 +/- 6.82 nmol/ml）。观察到肌肽在羊奶中的水平与凝固酶阳性葡萄球菌UFC/ml之间呈负相关（r = -0.773；p < 0.01）。总之，这种非常简单快速的方法可用于检测生物样本中浓度极低的组氨酸二肽。
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