We investigated the associations of plasma lipid and lipoprotein concentrations with the total plasma concentrations of ceramides and sphingoid bases, sphingosine, and dihydrosphingosine, and their 1-phosphates. We also determined the distribution of these sphingolipids between the pool localized to plasma lipoproteins compared to that found in the remaining plasma proteins mainly bound to albumin. We determined that S1P concentration in plasma is highly variable and determined further that S1P in plasma was nonuniformly distributed between the plasma lipoprotein and plasma protein pools of S1P in the six subjects investigated. We determined further that increasing concentrations of S1P in plasma were localized primarily to the plasma protein-containing fraction (Figures versus ) in subjects exhibiting a broad range of plasma lipid concentrations ().
The results of our studies in these select subjects confirmed and extended those reporting previously [12
] that S1P concentrations in the lipoprotein-containing fraction of plasma were positively correlated with HDL cholesterol concentrations (). Because HDL is the primary lipoprotein which transports S1P, and because HDL-C concentration varied widely in the six subjects (), we also normalized the levels of S1P in the lipoprotein-containing fraction to the HDL-C concentration in each subject (). We determined that this “normalized” concentration of S1P transported in the lipoprotein-containing fraction was positively correlated to the total S1P concentration in plasma, which suggests that the S1P content of HDL is variable and that S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. In fact, we have demonstrated previously that per particle, the larger VLDL particle contains the highest content of S1P, and the smallest lipoprotein particle, HDL3, contains higher S1P levels than LDL particles [9
]. Our current data may allow subsequent research on mechanisms that mediate binding of the different S1P carriers to S1P receptors, knowing that only 5% of HDL particles carry S1P molecules [21
Recent studies have reported that HDL-associated S1P is bound specifically to apolipoprotein M (apoM) and, furthermore, is transported selectively in apoM-containing HDL particles [22
]. We did not measure apoM concentration or distribution in this select subject set and thus cannot infer if differences in apoM metabolism between the subjects were associated with the observed differences in S1P metabolism. Alternatively, we [9
] and others [8
] have determined that S1P levels in HDL are significantly higher in the smaller sized HDL3 subfraction compared to S1P levels in HDL2. The smaller HDL3 particles were shown also to be enriched in apoM [23
]. Despite the very strong evidence from apoM knockout and apoM-transgenic mice that apoM determines S1P concentrations in plasma and HDL [22
], there was no statistically significant correlation between S1P and apoM concentrations in human patients with different monogenic disorders of HDL metabolism [23
]. Thus, the relative concentrations of HDL subfractions may have influenced the distribution of S1P in the individual subjects.
More recently, the crystal structure of a main S1P G protein-coupled receptor, S1P1, was revealed, and it was found that extracellular access to the binding pocket of this receptor is occluded by the aminoterminus and extracellular loops of the receptor [24
]. Interestingly, access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor [24
]. In the context of previous findings, we postulate that effects of HDL-bound S1P could be related to (1) HDL particle size and the ability to modulate cell lipid rafts and comprised receptors, (2) S1P content of HDL particles being higher in the smaller HDL particles, and (3) HDL role as scavenger/reservoir for biologically active lipids including S1P.
A confounding factor in recent studies addressing the cardioprotective role of S1P levels in HDL is the assumption that 30% of S1P is recovered in the lipoprotein-deficient serum [25
]. As shown in , S1P distribution in the plasma protein-containing fraction approximated 30% in only three of the six subjects studied. Thus, even in this limited number of subjects, S1P distribution in 50% of the subjects does not support this often cited assumption. clearly shows that there is no significant association between total plasma S1P and S1P concentration in plasma lipoprotein-containing fraction, which suggests a crucial pathophysiological role of the S1P bound to plasma proteins. Furthermore, the procedure in which apolipoprotein B-depleted plasma is prepared and S1P concentration is subsequently determined in the “HDL-containing” fraction as performed in two recent studies [21
] also does not discriminate between HDL- and albumin-associated S1P. Thus, when this methodology is employed, assumptions regarding S1P distribution in plasma may result in erroneous estimates.
We have shown previously that the secretion of plasminogen activator inhibitor-1 (PAI-1) from cultured adipocytes is significantly increased when the cells were incubated with increasing S1P concentration in the media regardless of whether the S1P is bound to HDL or transported by albumin [11
]. Previous studies have determined that triglyceride level was independently related to plasma PAI-1 activity level in both subjects with hypertriglyceridemia and in age-matched normotriglyceridemic subjects [27
]. The factor(s) contributing to this phenomenon are still unclear. In this limited study, plasma S1P concentrations were increased in the two subjects exhibiting elevated plasma triglyceride levels. Clearly, additional studies are warranted in this area.
In summary, we determined that S1P concentration in plasma is highly variable, S1P in plasma is nonuniformly distributed between the plasma lipoprotein and plasma protein pools of S1P and increasing concentrations of S1P in plasma are localized primarily to the plasma protein-containing fraction. We also determined that the S1P content of HDL, the major lipoprotein carrier of S1P, is variable and that S1P transport in lipoproteins is not limited by the concentration of HDL-C. The data further show that there is no significant association between total plasma S1P and S1P concentration in plasma lipoprotein-containing fraction, which suggests a crucial pathophysiological role of the S1P bound to plasma proteins.