Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources
Martina Lioi
;
Sara Tengattini
;
Roberto Gotti
, et al.
J. Chromatogr. A,2024,464771.
DOI:
10.1016/j.chroma.2024.464771
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Abstract: During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers.
The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase.
The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).
Keywords:
Collagen ;
Amino acid analysis ;
Hydroxyproline isomers ;
Recombinant collagen ;
Reverse phase chromatography ;
Mass spectrometry
Purchased from AmBeed:
147-85-3 ;
73-32-5 ;
61-90-5 ;
51-35-4
Downregulation of hepatic fat accumulation, inflammation and fibrosis by nerolidol in purpose built western-diet-induced multiple-hit pathogenesis of NASH animal model
Usman Sabir
;
Hafiz Muhammad Irfan
;
Alamgeer
, et al.
Biomed. Pharmacother.,2022,150,112956.
DOI:
10.1016/j.biopha.2022.112956
PubMed ID:
35447548
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Abstract: Western diet style (fast food), which includes fatty frozen junk food, lard, processed meats, whole-fat dairy foods, cream, mayonnaise, butter, snacks, and fructose, is a primary etiological determinant for developing nonalcoholic steatohepatitis (NASH) worldwide. Here the primary focus is to see the impact of naturally identified essential oil on disease mechanisms developed in an animal model using the same ingredients. Currently, symptomatic therapies are recommended for the management of NASH due to non-availability of specific treatments. Therefore, the present study was designed to evaluate the potential anti-NASH effect of nerolidol in a rat model fed with a purpose-built diet. The diet substantially induced insulin resistance, hepatic steatosis, dyslipidemia, and elevation of liver enzymes in the experimental animals. The levels of liver oxidative stress markers, nitrites (NO2–), serum pro-inflammatory cytokine (TNF-α) and hepatic collagen were increased in disease control rats. Nerolidol oral treatment in ascending dose order of 250 and 500 mg/kg substantially reduced the steatosis (macrovesicular and microvesicular), degeneration of hepatocytes, and inflammatory cells infiltration. The amounts of circulatory TNF-α and tissue collagen were also reduced at 500 mg/kg dose of nerolidol, expressing its anti-fibrotic effect. The current study described the multiple-hit pathophysiology of NASH as enhanced steatosis, pro-inflammatory markers, and oxidative stress in rats, which resulted in the development of vicious insulin resistance. Nerolidol treatment significantly reduced hepatic lipid accumulation and halted disease progression induced by a hypercaloric diet.
Keywords:
Western diet ;
Non-alcoholic steatohepatitis ;
Insulin resistance ;
Inflammation ;
Nerolidol
Purchased from AmBeed:
100-10-7 ;
53-59-8 ;
51-35-4
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