Here we present a relative simple model for TG redistribution by CETP among lipoproteins in human plasma. Our model is based on the model of Potter et al. [1], which itself is based on the work of Morton [12, 13]. We relied on the data on TG composition of lipoproteins in plasma stored for one hour at 37 °C of n = 91 samples compared to baseline to calculate the CETP-mediated TG-redistribution among lipoproteins.
A slightly different version of the model presented here was already introduced in our group’s published article [11]. The main difference from that model is that in the present work, we are relaxing the assumption that exchanges are equimolar. In our earlier model, only the means of TG in LDL (n = 27, normolipidemic) were used for model calibration. However, in this study, we not only acquired more data from both healthy and pathologic lipoprotein profiles to test the model, we also expanded our model to include VLDL and HDL. Moreover, we do not only compare measured and estimated medians but also considered corresponding correlations.
The model’s structure
Potter’s model describes an equimolar shuttle exchange of TG and CE by CETP between lipoproteins on the molecular level. Hence, on a formal basis, CETP is discriminated into a free and a lipoprotein-bound fraction. Correspondingly, lipoproteins are discriminated into free and CETP-bound. The model presented here simplifies and generalises this model. Our model is independent of the exact biochemical mode of CETP action (shuttle or ternary complex) - an obvious advantage, since the mode of action has still not been elucidated. Our generalised model is compatible with both the shuttle and the ternary complex models. Although differing on the molecular level, both approaches result in the same dynamics on the macroscopic level (by the law of large numbers).
As we point out later, our data do not support equimolar change in all fractions, thus we model the TG change only. However, at least in VLDL, our model seems to predict the CE flux to a strong degree. Furthermore, we are not modelling free and bound lipoproteins and CETP-molecules explicitly. The ‘CETP binding to lipoprotein’-reaction is disregarded, as the dynamics of those reactions are only relevant on a mechanistic level.
Note that there are several mathematical model approaches in the field of human lipid metabolism dealing with for example topics like oxidized lipoproteins [14], LDL endocytosis [15] or the cholesterol biosynthesis pathway [16].
Comparison to other models
There are other models besides Potter’s describing in vivo CETP dynamics. Figure 7 summarises two thereof. The model presented by Lu et al. [17] describes the flux of CE from HDL to VLDL as well as CE’s flux from LDL to HDL and vice-versa. In Lu’s model the fluxes are simple first-grade reactions\( {R}_i:C{E}_{donor}\overset{k_i}{\to }C{E}_{acceptor} \) (i = 1, 2, 3) with three different corresponding reaction rates. Applying our own data to both models, the observed ΔCE correlates much weaker to Lu’s predicted ΔCE in VLDL than to our predicted ΔCE: R = 0.315, p = 0.002 vs. R = 0.705, p = 6.4E− 15. As we are not assuming equimolar exchange and just model TG, we cannot compare Lu’s model directly to ours. However, considering the LCAT-inhibited data from experiment 2, we detected no correlation between the CE enrichment in HDL and CE mass in LDL (or VLDL).
Hübner’s model [18] considers TG and cholesterol (CH), which corresponds to FC + CE. A shuttle transport is modeled in which CETP is either free or TG- or CH-loaded. Let us replace CH by CE to better compare their model to ours. Here, five rates are used for five reactions (Fig. 7). Their model neglects CETP-mediated transport of TG out of HDL, and CE out of ApoB-containing particles. However, their model somehow makes use of the ratio of TG to (CE + TG) associated to CETP. Comparing the strength of correlation between Hübner’s model and our model, ours appears to be superior with regard to HDL-ΔTG, LDL-ΔTG, and slightly better for VLDL-ΔTG (R = 0.515, R = 0.563, R = 0.544 vs our model’s: R = 0.683, R = 0.753, R = 0.562, respectively).
Comparison between the CETP model of Lu et al. [17], Hübner et al. [18] and ours presented. Lu’s model uses first-order reactions; Hübner’s model uses 2 second-order and 3 first-order reactions. The model presented here assumes a steady state and uses one reaction rate for CETP. Due to CETP’s diffusion-like property, the amount of TG entering and leaving a lipoprotein depends on the surface and the TG and CE load of the lipoprotein and all other lipoproteins.
Comparing the Hübner and Lu models to ours presented here, note that ours is less complex, as it only requires one reaction rate instead of 5 or 3, respectively. Hence, unlike their models, which describe each flux from one fraction to another with their own reaction, our newly derived model represents a rather holistic model.
Concept of isotransfer point (ITP)
Figure 6a displays the medians of the ITP in the ‘normal’ and the ‘high TG’ group. Intuitively speaking, the point is lower in the ‘normal’-group. Based on our model, the ITP ‘CETPTG’ is the mean of all fraction’s TG/(TG + CE) ratios weighted by the corresponding surface. As clarified in Fig. 6 the greater the distance between the TG/(TG + CE) ratio of a lipoprotein to the ITP, the higher is the net flux of TG into/out of this fraction (if the ratio is smaller/greater than the ITP). Thus we expect LDL and HDL to have a higher TG turnover in the ‘high TG’ situation.
The ITP may be changed due to lipid-lowering medication or during the postprandial state. This change may also exert a significant influence on HDL metabolism. The TG/(CE + TG) ratio in IDL in the ‘high TG’ group is surprisingly low. This might be caused by a prolonged retention time in plasma, which may be characteristic in the hypertriglyceridemic state. The clinical significance of the shift of the ITP from LDL to IDL in hyperlipidemia remains unclear. However, the atherogenity of certain lipoproteins (e.g. IDL) may be altered if its metabolic function (CETP-mediated TG transfer) changes.
Data
Our in vitro data mirror the corresponding in vivo redistribution of lipids to a fairly good degree, as data from Liu et al. [19] as well as our group’s experiments have revealed evidence that the net change in TG in lipoprotein fractions by CETP action during plasma storage at 37 °C is linear for at least four hours.
Further data from experiment 2 suggest that CETP is the only factor responsible for TG redistribution among lipoprotein subfractions. CETP inhibition suggests that the esterification rate on non-HDL is very low or even non-existent. However, the data may be too noisy to make precise claims.
In contrast to other methods measuring CETP-activity, the data we used to estimate the CETP-mediated TG-change enables the estimation of TG in all fractions, namely VLDL, IDL, LDL and HDL, simultaneously. The data cover a broad physiological range of TG changes mediated by CETP, as individuals with healthy and various pathologic lipid profiles have been considered.
Very probably CETP activity differs among individuals. However, as the first step, our model estimates the CETP-mediated relative redistribution of TG among the lipoproteins, which is independent of the CETP reaction rate, given only the TG, CE and ApoB (or ApoA1) concentration of VLDL, IDL, LDL and HDL. Based on this estimation, absolute fluxes may be inferred assuming a fixed reaction rate in a second step, if no additional information is given.
Model fit
Despite the noise of TG change data in lipoprotein fractions, the predicted TG fluxes correlate well with the observed corresponding changes (Fig. 2). While HDL and LDL fit well, VLDL and especially IDL appear more problematic. This could be mainly caused by relatively greater measurement imprecision, but also by a shift of ApoB from IDL to VLDL or vice-versa during the one hour storage at 37 °C – as our group already observed [11]. Considering the R2 values between modelled and measured TG changes in VLDL, LDL and HDL (Fig. 2), mind that on the one hand our rather imprecise method of measurement may strongly lower the ‘true’ R2 value and on the other hand that our prediction is superior compared to the other models mentioned above, as well as to possible linear correlations mentioned in the results part (Table 2).
Equimolarity
Assuming an equimolar lipid-exchange it is expected that the amount of TG entering/leaving a lipoprotein fraction equals the amount of CE leaving/entering it if LCAT inhibition is given. Considering ΔCE and ΔTG in experiment 2 (Fig. 1) we noted a significant increase in the TG-median of 11.3 μmol/L and 19.2 μmol/L in LDL and HDL under LCAT inhibition. Surprisingly no corresponding loss of CE (0 and − 5.2 μmol/L, respectively) was observed. However, due to the small sample size and measurement imprecision, we cannot make a solid statement on this point.
Taken together, we identified some indicators suggesting that the CETP exchange may not necessarily be equimolar. However, due to experiment 2’s low case numbers, our data do not suffice to clarify this issue.
Lipid transfer inhibitor protein (LTIP)
There is strong evidence of a protein inhibiting CETP, namely lipid transfer inhibitor protein (LTIP) or Apolipoprotein F, which mainly interacts with LDL particles [20]. We investigated whether the resulting lower availability of LDL would have a positive effect on our model’s fit by reducing s(PLDL), the surface of LDL. Potter uses a factor of 0.73 for LDL inhibition by LTIP. Such a reduction in our case leads in fact to a better correlation between predicted and observed changes in LDL and especially HDL: If s(PLDL)*0.4 is used instead of s(PLDL), the correlation is optimised. If s(PLDL) is reduced, the correlation coefficients R in HDL and LDL increase slightly, while the corresponding coefficient in VLDL decreases simultaneously. Further the estimated and observed proportion of TG enrichment between LDL and HDL tends to impair as s(PLDL) is reduced. A possible inhibitory effect of ApoF is thus not implemented in our model.
CETP mass
Estimating TG fluxes in experiment 1, we assumed all rcetp to be equal. This of course did not hold true. The parameter rcetp rather depends on CETP’s mass concentration and the ability of the particular CETP-phenotype to mediate lipid exchange.
In the literature, CETP’s plasma concentration does not differ that much depending on gender or pathologies [8, 21,22,23,24,25]. We are thus assuming that the mass is equal in experiment 1’s different subgroups. However, our model may not account for changes in rcetp due to genetic variants of CETP.
Properties of CETP exchange
Considering our model and applying it to several lipid-profiles, two issues deserve attention: First, the driving force for CETP exchange is the mass and composition of triglyceride-rich lipoproteins such as VLDL and chylomicrons, as they contain the most TG in plasma. Figure 4 illustrates this fact, as in the ‘high TG’ case, LDL and HDL both revealed the highest increase in TG mass. This issue might be an important factor, when assessing TG influence on lipid metabolism (for example the postprandial state).
Second, TG from VLDL is distributed to LDL and HDL to a similar degree in all four metabolic states investigated (Fig. 4). However, depending on their surface and lipid composition, the proportion of TG entering HDL compared to TG entering LDL may differ strongly, as both lipoprotein-species compete against each other (consider for example the ‘high LDL’ case in Fig. 4).
Let us assume that TG’s high flux to LDL or HDL is atherogenic, as (in combination with hepatic lipase) it can lead to small, dense LDL and small HDL. Following the model of Lu et al. [17], lipolysis by hepatic lipase is the main pathway causing HDL particles to shrink. Small HDL are more likely to exit the plasma. Hence, via CETP hypertriglyceridemic plasma may lead to an atherogenic lipoprotein phenotype characterized by low HDL particle concentrations and small HDL and LDL particles. Our model might thus help to quantify the causal relationship between hypertriglyceridemia and those atherogenic characteristics. Furthermore, following our model’s dynamics, a high HDL particle concentration may prevent small dense LDL particles from forming by reducing the net flux of TG to LDL in such a hypertriglyceridemic state. However, the focus of this paper making is not claims about HDL functionality by relying on its lipid-composition.
Note that only 43 normolipidemic persons out of experiment 1 were used for model calibration. Hence, the results for ‘high TG’, high LDL’ and ‘low HDL’ presented in Fig. 4 are extrapolated and demonstrate our model’s ability to predict CETP redistribution of TG based on lipid and apolipoprotein concentrations only.
When studying lipoprotein metabolism, the concepts considered are usually delipidation of ApoB containing lipoproteins and the reversed cholesterol transport mediated by HDL. However, both metabolic pathways are intimately connected by the CETP-mediated flux of TG and CE. This paper is intended to give a new perspective on lipoprotein metabolism concerning CETP-mediated fluxes of TG and CE.