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Rapid Profiling of Prostanoids in Biological Samples (X-LC)

Introduction

Inflammation is implicated in number of diseases including, hypertension, ischemic heart injury rheumatoid arthritis and atherosclerosis. Therefore, a mechanistic understanding of the inflammation process at the molecular level would contribute to the mechanism of the disease state, injury and recovery. Eicosanoids are specific biomarkers for inflammation Arachidonic acid (AA) which is an omega-6 polyunsaturated fatty acid can undergo oxidative metabolism by cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome (P450) to produce a number of these inflammatory lipid biomolecules.

The oxidation of AA by COX-2 results in a number of prostanoids which include PGE2, PGD2 and PGF2, PGJ2, prostacyclin (PGI2) and thromboxane A2 (TXA2). Prostacyclin is considered to be a potent vasodilator whereas thromboxane A2 is considered to be a potent vasoconstrictor. PGI2 and TXA2 are unstable at physiological pH and are readily converted to 6-keto PGF and 11-dehydro TXB2 respectively. Hence these are considered to be reliable for the in vivo estimation of PGI2 and TXA2 respectively. PGE2, PGD2 and PGF2 are considered to be pro inflammatory biomarkers.13, 14 -dihydro-15-keto PGE2 and 13, 14 -dihydro-15-keto PGA2 are major metabolites of PGE2 in plasma.15-deoxy PGJ2 which is the major metabolite of PGJ2 is thought to have anti inflammatory properties especially in rheumatoid arthritis. Free radical peroxidation of AA produces a series of prostaglandin like compounds known as the isoprostanes of which F2 isoprostane which are PGF2 like compounds is considered to be the most important.8-iso PGF2 also known as 8-isoprostane the most abundant form of the F2 isoprostane is considered to be an indicator of in vivo oxidative stress Figure 1 shows the understood pathways for the metabolism of arachidonic acid.

This application note describes a 6.8 min method for identifying a mixture of 9 prostanoids using Jasco XLC. Separation of these compounds using conventional HPLC typically takes around 21 minutes.

Experimental

stress measurement

Figure 1. Arachidonic acid metabolism by COX-2 and non-enzymatic pathway.

Sample Preparation

8-iso PGF2 was supplied in methanol with a stock mass concentration of 1 mg/ml and 13, 14 -dihydro-15-keto PGE2 and 13, 14 -dihydro-15-keto PGA2 were supplied in methyl acetate with a stock mass concentration of 1 mg/ml. All the other prostanoids were dissolved in 1 ml methanol such that the stock mass concentration for all was 1mg/ml.Working standards for the analytes were prepared by serial dilution using ACN.

X-LC System and Operating Conditions

Jasco X-LC System

Jasco X-LC System

 

System: Jasco X-LC
Mobile Phase A: 0.1 % Formic Acid
Mobile Phase B: ACN
Flow Rate: 0.2 ml/min
Gradient:

Time (min)B (%)
0.0 35
4.0 35
6.0 90
6.5 35
6.8 35

 

Injection volume

2µl

Stationary Phase

Column: Restek Pinnacle DB C18 1.9µm (50*2.1 mm)
Column Temperature: 25°C

UV Detection

 

Time (min)Wavelength (nm)
0.0 220
1.0 196

 

Results and Discussion

Table 1 provides a list of the compounds from the method and figure 1 shows an X-LC chromatogram of a standard mixture of prostanoids (150 ng each). The compounds are separated by a gradient elution of 0.1 % formic acid and acetonitrile on a Restek Pinnacle DB C18 1.9µm (50*2.1 mm) at a flow rate of 0.2 ml/min.

 

stress measurement

Figure 1. X-LC Chromatogram of Prostanioids from Table 1.

 

Compound NameSymbolRT (min)
6-keto-ProstaglandinF 6-keto PGF 1.25
8-isoprostane 8-iso PGF 2.25
Prostaglandin F PGF 2.88
11-dehydro Thromboxane B2 11-dehydro TXB2 3.86
Prostaglandin D2 PGD2 4.18
13,14-dihydro-15 keto Prostaglandin E2 13,14-dihydro-15 keto PGE2 5.43
13,14-dihydro-15 keto Prostaglandin A2 13,14-dihydro-15 keto PGA2 6.01
15-deoxy Prostaglandin J2 15-deoxy PGJ2 6.48

 

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About the Author

Kristen Burkhardt Kristen Burkhardt

has a Bachelor's degree in Marketing from York College of Pennsylvania. She is the current CD/Export Sales Coordinator/Marketing at JASCO Incorporated.