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ISSN : 2153-2435
Pharmaceutica Analytica Acta

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Analysis of Free Fatty Acids in Blood of Healthy Person and that of Hepatitis Patient

Hideharu Shintani*

University of Chuo, School of Engineering, 1-13-27, Kasuga, Bunkyo, Tokyo 112-8551, Japan

*Corresponding Author:
Hideharu Shintani
University of Chuo, School of Engineering
1-13-27, Kasuga, Bunkyo, Tokyo 112-8551, Japan
Tel: +81425922336
E-mail: [email protected]

Received date: March 18, 2013; Accepted date: April 20, 2013; Published date: April 24, 2013

Citation: Shintani H (2013) Analysis of Free Fatty Acids in Blood of Healthy Person and that of Hepatitis Patient. Pharmaceut Anal Acta 4:222. doi: 10.4172/2153-2435.1000222

Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Keywords

Polyunsaturated fatty acids (PUFA) are highly susceptible to oxidation and their levels can rapidly decrease under oxidative stress [1]. A concomitant increase in monounsaturated fatty acids such as oleic and palmtoleic acids is often observed probably as a result of the activation of Δ9-desaturase [1]. Analysis of fatty acid profiles is therefore of importance. Here we describe the analysis of plasma free fatty acids (FFA) only, because the measurement of fatty acids in neutral lipids, phospholipids, and total lipids has already been well documented. Plasma FFA can, furthermore, be produced by oxidatively damaged tissues because hydrolysis of phospholipids is stimulated by oxidative stress [2].

Experimental

• Mix plasma (50 μL) with methanol (200 μL) containing margaric acid (12.5 μM; internal standard) and centrifuge at 12,000 rpm for 3 min.

• Dry the supernatant (50 μL) under a stream of N2 and mix the residue with a solution of monodansylcadaverine in N,N-dimethylformamide (2 mg/mL, 50 μL) and with diethyl phosphorocyanidate (1 μL).

• Stand for 20 min at room temperature in the dark.

• Analyse aliquots (5 μL) by HPLC with fluorescence detection. Column: 3.3 cm×4.6 mm i.d., 3 μm, octadecylsilyl (Supelco) and 25 cm×4.6 mm i.d., 5 μm, pKb-100 (Supelco) in series. Mobile phase: 17.5:65.0:17.5 (v/v) acetonitrile-methanol-water. Flow rate: 1.5 ml/mL. Column oven: 40°C. Excitation: 320 nm. Emission: 520 nm.

Calculation

Concentrations of plasma FFA can be calculated by use of the following equation.

[Plasma FFA] in μM = (50 × peak area for FFA)/(peak area for marganc ac1d).

Discussion

Only FFA in blood of healthy person and that of hepatitis patient can be measured and compared because fatty acids in phospholipids and neutral lipids are not derivatized (Figure 1).

pharmaceutica-analytica-acta-human-plasma-FFA

Figure 1: Typical HPLC chromatograms of derivatized human plasma FFA from a normal subject and from a patient with hepatitis. There is a significant difference between the content pf PUFA and monounsaturated fatty acids between the normal and the hepatitis patient.

References

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