Selective measurement of lipoprotein lipase and hepatic triglyceride lipase in heparinized plasma from horses.

The research article presents a study which identified and measured two lipolytic enzymes, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL), in heparinized plasma from horses using affinity chromatography. Using these results, the researchers developed a selective assay for direct measurement of these enzyme activities.

Identification and Purification of Enzymes

• The research utilized affinity chromatography on heparin sepharose to identify two lipolytic enzymes in heparinized plasma from horses.
• The HTGL enzyme displayed resistance to inactivation by high concentrations of NaCl and did not require additional serum for activity.
• The LPL enzyme, on the other hand, was inactivated with NaCl concentrations above 0.2M and required additional serum as a source of the apolipoprotein C-II activator.
• The enzymes were purified 347-fold for HTGL and 442-fold for LPL, producing respective yields of 54% and 58%.

Creating the Optimal Conditions and Developing the Assay

• Once the enzymes were purified, they were utilized to create incubation conditions that were optimally effective for each enzyme in vitro.
• The team then developed a selective assay for direct measurement of LPL and HTGL activities in heparinized plasma.
• A key facet of this assay was utilizing the almost complete inactivation of LPL when serum cofactor was excluded at an NaCl concentration optimal for HTGL activity.
• To measure LPL, the researches had to incubate the plasma with sodium dodecyl sulfate to inhibit HTGL activity, as HTGL maintained two thirds of its activity at the NaCl concentration required for LPL activity.

Measurement and Analysis of Enzyme Activities

• The activity of each enzyme was measured in heparinized plasma from 12 Shetland ponies.
• The mean activity for LPL was found to be 3.22 +/- 1.04 μmol of fatty acids/ml of heparinized plasma/hour, while HTGL activity was slightly higher on average at 4.9 +/- 1.56 μmol of FA/ml/hour.
• The research team assessed their assay’s performance by conducting replicate analysis on enzyme pools with high and low activities.