Haptoglobin inhibits phospholipid transfer protein activity in hyperlipidemic human plasma

Abstract

Background: Haptoglobin is a plasma protein that scavenges haemoglobin during haemolysis. Phospholipid Transfer Protein (PLTP) transfers lipids from Low Density Lipoproteins (LDL) to High Density Lipoproteins (HDL). PLTP is involved in the pathogenesis of atherosclerosis which causes coronary artery disease, the leading cause of death in North America. It has been shown that Apolipoprotein-A1 (Apo-A1) binds and regulates PLTP activity. Haptoglobin can also bind to Apo-A1, affecting the ability of Apo-A1 to induce enzymatic activities. Thus we hypothesize that haptoglobin inhibits PLTP activity. This work tested the effect of Haptoglobin and Apo-A1 addition on PLTP activity in human plasma samples. The results will contribute to our understanding of the role of haptoglobin on modulating reverse cholesterol transport.

Results: We analyzed the PLTP activity and Apo-A1 and Haptoglobin content in six hyperlipidemic and six normolipidemic plasmas. We found that Apo-A1 levels are proportional to PLTP activity in hyperlipidemic (R2 = 0.66, p < 0.05) but not in normolipidemic human plasma. Haptoglobin levels and PLTP activity are inversely proportional in hyperlipidemic plasmas (R2 = 0.57, p > 0.05). When the PLTP activity was graphed versus the Hp/Apo-A1 ratio in hyperlipidemic plasma there was a significant correlation (R2 = 0.69, p < 0.05) suggesting that PLTP activity is affected by the combined effect of Apo-A1 and haptoglobin. When haptoglobin was added to individual hyperlipidemic plasma samples there was a dose dependent decrease in PLTP activity. In these samples we also found a negative correlation (-0.59, p < 0.05) between PLTP activity and Hp/Apo-A1. When we added an amount of haptoglobin equivalent to 100% of the basal levels, we found a 64 +/- 23% decrease (p < 0.05) in PLTP activity compared to basal PLTP activity. We tested the hypothesis that additional Apo-A1 would induce PLTP activity. Interestingly we found a dose dependent decrease in PLTP activity upon Apo-A1 addition. When both Apo-A1 and Hpt were added to the plasma samples there was no further reduction in PLTP activity suggesting that they act through a common pathway.

Conclusion: These findings suggest an inhibitory effect of Haptoglobin over PLTP activity in hyperlipidemic plasma that may contribute to the regulation of reverse cholesterol transport.

Figure 1

a. Association between PLTP activity and haptoglobin levels in hyperlipidemic plasma (R² = 0.5733, n = 6). PLTP activity after 60 min was determined as described in Materials and Methods. Haptoglobin levels were determined using a two site Human Haptoglobin ELISA kit. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment. b. Association between PLTP activity and haptoglobin levels in normolipidemic plasma (R² = 0.0007, n = 6). PLTP activity after 60 min was determined as described in Materials and Methods. Haptoglobin levels were determined using a two site Human Haptoglobin ELISA kit. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment.

Figure 2

a. Association between Apolipoprotein A1 levels and PLTP activity (60 min) in hyperlipidemic plasma. Apolipoprotein levels were determined using an EIA kit as outlined in Materials and Methods. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment. The graph depicts the mean ± standard deviation (R² = 0.6635, n = 6, p < 0.05). b. Association between Apolipoprotein A1 levels and PLTP activity (60 min) in normolipidemic plasma (R² = 0.2732, n = 6, p > 0.05). c. Association between PLTP activity (60 min) and Hp/Apo-A1 ratio in hyperlipidemic plasma (R² = 0.69, n = 6, p < 0.05).

Figure 3

a. Effect of haptoglobin addition (2, 10 and 20 μg per sample) on PLTP activity (5 min) of individual hyperlipidemic (H, n = 6) and normolipidemic (N, n = 2) plasma samples. From left to right: control, addition of 2 μg, 10 μg and 20 μg of Hp, respectively. One of two representative experiments with duplicate measurements per treatment. b. Effect of haptoglobin addition (2, 10 and 20 μg per sample) on PLTP activity (5 min) in hyperlipidemic plasma as a percentage of basal PLTP activity. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment. The graph depicts the mean ± standard deviation of each group of six hyperlipidemic plasma (n = 6, p < 0.05). c. Ibidem, except that PLTP activity was analyzed after 60 min. d. Correlation between PLTP activity and Hp/Apo-A1 ratio in hyperlipidemic plasma samples with added haptoglobin (n = 11, Correlation coefficient -0.679, p < 0.05).

Figure 4

Effect of Haptoglobin addition on PLTP activity (5 min) in hyperlipidemic plasma. The equivalent of 100% of basal haptoglobin levels was added to each plasma sample and PLTP activity was measured as described previously (n = 6, p < 0.05).

Figure 5

a. Effect of Apo-A1 addition to the rate of PLTP activity within the first 60 minutes of reaction in hyperlipidemic plasma. Two and four micrograms of Apo-A1 were added to the plasma samples and PLTP activity was measured within the first two and 60 minutes to calculate the rate of activity (nmoles product/min). One of two representative experiments. b. Effect of Apo-A1 and Haptoglobin addition to the rate of PLTP activity within the first 60 minutes of reaction in hyperlipidemic plasma. Two micrograms of Apo-A1 and/or the equivalent of 100% of basal haptoglobin were added to the plasma (Pl: plasma alone, Pl+Apo-A1: plasma plus Apo-A1, Pl+Hp: plasma plus haptoglobin and Pl+Apo-A1+Hp: plasma plus Apo-A1 plus Haptoglobin) and PLTP activity was measured within the first two and 60 minutes to calculate the rate of activity (nmoles product/min). One of three representative experiments.