Name: differential effects of lipid-lowering drugs in modulating morphology of cholesterol particles
Uploaded: 2017-11-10T15:00:00
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Description: Watch this Scientific Journal Video about Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles at JoVE.com

This work was funded by a Plaxgen Research grant awarded to SM (PLX-1008). We thank Palo Alto Medical Research Foundation Research Institute for collecting serum samples from atherosclerosis subjects under IRB approval.

1. Preparation of Fluorescent-labeled Cholesterol and Lipid-lowering Drugs
NOTE: Fluorescent-labeled cholesterol aggregates and statins were prepared as described in our previous article21. Please see Table of Materials for details of reagents, imaging flow cytometer, chemistry analyzers, and data analysis software used in this study.
<ol>
	<li>Solubilize fluorescent-labeled lyophilized cholesterol (1 mg, Ex/Em = 495 nm/507 nm) powder in 1 mL of 100% alcohol.</li>
	<li>Centrifuge the sample for 3 min at 2,040 x g and use the supernatants containing soluble fluorescence-labeled cholesterol aggregates in the plaque array assay.</li>
	<li>For the preparation of drugs, individually solubilize the powders (2 mg) of simvastatin, lovastatin, atorvastatin, ezetimibe, and omega-3 fatty acid in 1 ml of 100% alcohol.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the assay.</li>
	<li>Similarly, solubilize individually the powders (2 mg) of fluvastatin, rosuvastatin, niacin, and fibrate in 1 mL of deionized water to make a stock solution of 2 mg/mL.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the plaque array assay.</li>
</ol>
2. Plaque Array Assay for Examining In Vitro Cholesterol Particles Formation
<ol>
	<li>Use chemistry analyzer-1 (see Table of Materials) to set up the plaque array assay for cholesterol particles formation. Perform the assay in a round bottom, low protein binding 96-well plate using the reagents prepared in Section 1. Maintain the final reaction volume in each well at 200 &#181;L and perform all assays in triplicate.
	<ol>
		<li>First, load 194.5 &#181;L of phosphate buffered saline (PBS) to each well.</li>
		<li>To each well, add 2.5 &#181;L (5 &#181;g) of each lipid-lowering drug solution and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate to uniformly mix the drug into solution. No drug is added to the negative control samples.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescent-labeled cholesterol aggregate solution to each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
		<li>Incubate the plate on a lab shaker for 2 h set at 37 &#176;C and 200 rpm.</li>
		<li>After incubation, acquire the images of particles by imaging flow cytometry.</li>
	</ol>
	</li>
</ol>
3. Capturing Images of Cholesterol Particles Using Imaging Flow Cytometry
<ol>
	<li>Open the data acquisition template to load the correct instrument settings. Click &#34;File&#34;, then select &#34;Open Template&#8230;&#34; and select the template file.</li>
	<li>Click &#34;Flush, Lock, Load&#34; to prepare the instrument for sample loading.</li>
	<li>When the &#34;Load Sample&#34; dialogue box opens, click &#34;OK&#34; to load 50 &#181;L of the samples from each well prepared in Section 2 into the imaging flow cytometer.</li>
	<li>Click &#34;Run|Setup&#34; to view the particles in the imaging area in real time.</li>
	<li>When the particles in the imaging area are in good focus, click &#34;Run|Acquire&#34; to acquire images of each object simultaneously in a high-throughput manner for dark field (side scatter/SSC), bright field (BF), green fluorescence, and yellow fluorescence.</li>
	<li>Repeat steps 3.2 - 3.5 to acquire 5,000 - 10,000 particles from each sample.</li>
	<li>Analyze all raw image files using the image analysis software for determining object fluorescence intensity and morphological variations.
	<ol>
		<li>Click the &#34;Tools&#34; button at the top of the image analysis software, then select &#34;Batch Data Files&#34; from the drop-down menu, which will open the &#34;Batches&#34; window.</li>
		<li>In the &#34;Batches&#34; window, click the &#34;Add Batch&#34; button, which will open the &#34;Define a Batch&#34; window.</li>
		<li>In the &#34;Define a Batch&#34; window, click the &#34;Add Files&#34; button and select the raw image files, click the &#34;Template&#34; button and select the appropriate data analysis template file, then select &#34;OK&#34; and &#34;Submit Batches&#34; to begin processing and analysis of the raw image files.</li>
	</ol>
	</li>
</ol>
4. Plaque Array Assay Based Evaluation of Lipid-lowering Drugs Effect on Purified Cholesterol Particles
<ol>
	<li>As described in step 2.1, perform the cholesterol particles formation assay using purified VLDL, LDL, and HDL lipoproteins/particles. Perform the plaque array assay in a 96-well plate.
	<ol>
		<li>First, load all wells with PBS; use a 200 &#181;L final volume for each well.</li>
	</ol>
	</li>
	<li>For negative control, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually in each well without drugs and shake the plate for 30 s.
	<ol>
		<li>For examining the lipid-lowering drug effect, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually to each well and shake the plate for 30 s.</li>
		<li>For wells added with 4 &#181;g VLDL, LDL, or HDL, add 2.5 &#181;L (5 &#181;g) ezetimibe, lovastatin, simvastatin, or niacin solution and shake the plate for 30 s by placing it in chemistry analyzer-1.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescence-labeled cholesterol aggregate solution to all wells and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples using imaging flow cytometry following steps 3.1 - 3.5.</li>
	<li>Analyze raw image files using image analysis software as described in step 3.7.</li>
	<li>In the analysis template, use the following gating scheme for identification of cholesterol particles subpopulations.
	<ol>
		<li>On a plot of green channel saturated pixel count (x-axis) and dark field channel saturated pixel count (y-axis), reject objects with one or more saturated pixels in either channel.</li>
		<li>Plot the objects/particles without saturated pixels using green channel intensity (x-axis) and SSC intensity (y-axis): objects fall into one of three regions (VLDL, LDL, HDL) based on their green channel and SSC intensities. 
		NOTE: These gates were created based on data generated from control experiments carried out using purified VLDL, LDL, and HDL particles/proteins without the drug.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect for each sample, calculate the total lipoprotein particles concentrations, as well as the percentage of each subpopulation (VLDL, LDL, HDL) as described in step 6.5.
	<ol>
		<li>Calculate the particle concentrations (particles/mL) using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/56596/56596eq1.jpg" /> 
		NOTE: The VLDL, LDL, and HDL gating regions in the fluorescence dot-plot detect ~ 80% of their respective subpopulations, and the remaining ~ 20% of the particles overlap with other gating regions.</li>
	</ol>
	</li>
</ol>
5. Measurement of Lipid Concentrations in the Serum Samples
<ol>
	<li>For determining concentrations of LDL, HDL, cholesterol, and triglycerides by chemistry analyzer-2, use serum samples obtained from age-matched normal and dyslipidemia subjects.</li>
	<li>Define abnormal concentration of LDL, HDL, cholesterol, and triglycerides based on their concentration identified beyond the upper and lower reference values in serum samples.</li>
	<li>Follow the normal reference value recommended by the reagent manufacturer: cholesterol (4 - 200 mg/dL), HDL (3.0 - 65 mg/dL), LDL (4 - 100 mg/dL), and triglycerides (9 - 200 mg/dL).</li>
	<li>Identify serum samples that have an abnormal value as either below the lower reference value or above the higher reference value for cholesterol and triglycerides, and use them for screening in the presence and absence of lipid-lowering drugs.</li>
</ol>
6. Analyzing the Effect of Drugs on Cholesterol Particles Formation in Serum Samples
<ol>
	<li>Use the chemistry analyzer-1 to set up the plaque array assay for cholesterol particles formation in serum samples. Perform the assay in a round bottom, low protein binding 96-well plate. Use a final reaction volume of 200 &#181;L/well and perform all assays in triplicate.</li>
	<li>Prepare the plate by loading reagents in a stepwise manner.
	<ol>
		<li>Prepare the control wells.</li>
		<li>Load 193 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on chemistry analyzer reaction plate.</li>
		<li>Add 2 &#181;L (2 &#181;g) of fluorescence-labeled cholesterol aggregate solution into each well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Prepare the wells with drugs.
	<ol>
		<li>Load 191 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L ezetimibe, lovastatin, simvastatin or niacin solution (4 &#181;g) to all wells except the negative control wells. Add only one drug per well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L of fluorescence-labeled cholesterol aggregate solution (2 &#181;g) into each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Load drugs only after all wells (control and drug-treated) have been loaded with their respective serum samples, and add fluorescence-labeled cholesterol at the end of this step.</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples by imaging flow cytometer following settings described in steps 3.1 - 3.6.</li>
	<li>Use the image analyzing software for batch processing all the image files using the same template as described in steps 3.7.
	<ol>
		<li>Perform the data analysis by drawing gates on the plot as described in steps 4.5 and 4.6.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect/response (low, moderate, and high) for each sample, calculate the total lipoprotein particle concentration, as well as the percentage of the total particles comprising of VLDL, LDL, and HDL subpopulations.</li>
	<li>Plot the VLDL, LDL, and HDL populations on a histogram of the object&#39;s bright field image, width subtracted from length (length - width), and gate into either globular or linear regions: objects with (length - width) &#8804;2&#160;&#181;m fall into the globular region for calculating the percentage of globular and linear shaped particles.</li>
	<li>Compare the percentage of globular and linear shaped LDL and HDL cholesterol particles identified for each serum sample incubated with and without each drug. Among triplicate values obtained for each serum sample, accept the percentage of globular and linear shaped LDL and HDL particles that fall within SD &#177;2 range for determination of the drug effect.</li>
</ol>

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Dr. Madasamy received grant support from Plaxgen, Inc and has a competing financial interest. The other authors have no competing financial interests to disclose.

In general, the distribution and functional properties of VLDL, LDL, and HDL cholesterol particles in the blood circulation are mainly determined by metabolic, genetic, epidemiological,&#160;cellular, and plasma factors22,23. In the present study, examining the effects of lipid-modifying drugs in the buffer revealed that highly lipophilic drugs such as ezetimibe, lovastatin, simvastatin, and atorvastatin induced a higher level complexity on the morphology of cholesterol particles compared to the lower level effect observed with highly hydrophilic rosuvastatin and fluvastatin drugs. These results are in good agreement with our previous study describing a non-enzymatic mechanism based effect of statins in modulating LDL and HDL cholesterol particles formation in the buffer and serum samples21. Accordingly, the results from present study revealed a non-enzymatic mechanism of action of ezetimibe, niacin, fibrate, and omega-3 fatty acid drugs that may play a direct role in modulating cholesterol particles formation.It is possible that the interactions between drugs and cholesterol aggregates leads to the assembly of large size cholesterol particles that are 2 - 60 &#181;m2, exhibiting globular and linear strand morphologies.
Besides, the results obtained using purified lipoprotein particles suggest interactions between cholesterol aggregates and plasma factors including VLDL, LDL, and HDL proteins that may alter the compositions and morphological properties of the cholesterol particles. The drug treatment results involving purified lipoprotein particles indicated a higher level drug effect on VLDL particles formation compared to their effect observed on LDL cholesterol particles formation. The lovastatin, simvastatin, and ezetimibe drugs were used as pro-drugs and their doses in the assays may be higher than the physiological concentrations.
Interestingly, screening of serum samples showed variations of drug effect on altering the profiles of VLDL, LDL, and HDL cholesterol particles formation, particularly their effect on the formations of linear shaped LDL and HDL particles. These drugs induced reduction on linear shaped LDL and HDL cholesterol particles formation in both dyslipidemia and age-matched normal serum samples. The drug effects observed on reducing linear shaped particles formation was higher in simvastatin, ezetimibe, lovastatin, and niacin. The identification of cholesterol particles with globular and linear strand morphologies in the normal and dyslipidemia serum samples suggests that particles with similar morphologies may form in in vivo conditions. Previous studies have identified the presence of disc and needle-like cholesterol crystals in the atherosclerotic plaques of human and ApoE-/- and LDLR-/- mice models24,25,26,27,28.
The HDL particles circulating in the blood exist as a heterogeneous mixture and the level of small and large size HDL particles along with functional activity are important factors to exert their cardio-protective effect via the reverse cholesterol transport pathway29,30. Recent studies have highlighted the importance of identifying HDL cholesterol particle subfractions for elucidating their role in multiple biological functions such as cholesterol efflux, anti-inflammation, anti-thrombotic, and anti-oxidative31. In addition, a number of studies have reported the effect of lipid-lowering therapy in increasing a low to moderate level of HDL in the plasma1,5,21. Accordingly, the results from this study provide new insights on morphological features of cholesterol particles. Notably, the detection of a higher level of linear shaped HDL cholesterol particles in the serum samples of dyslipidemia subjects suggests that they may be the reliable biomarker for both diagnosis and evaluating effects of lipid-modifying drugs in patients. However, further investigation is required using large clinical samples to better understand the cholesterol particles with distinct morphologies and their association to CVD.
In the plaque array assay for examining the drug effect on assembly of cholesterol particles, we used 2 &#181;g of fluorescence labeled cholesterol aggregates and 5 &#181;gof each drug because: (1) drugs competitively bind to both fluorescence labeled cholesterol and endogenous lipids present in the serum samples; (2) from each sample, we acquired 5,000 to 10,000 cholesterol particles that are assembled into large sizes and shapes ranging from ~2 - 60 &#181;2; (3) we observed a wide variations of drug response among serum samples incubated with the drugs (doses 300 ng to 5 &#181;g) and ~1 - 5% of them incubated with high doses showed no detectable changes in the profile of cholesterol particles formation; and (4) the interaction between cholesterol aggregates and lipid-lowering drugs is mediated by a non-enzymatic process. Hence, the concentrations of the reagents used in the assay may be higher than their physiological level.
In conclusion, we have successfully demonstrated the advantages of an in vitro imaging method described in this study for determining the effect of a broad spectrum of lipid-lowering drugs on modulating morphology and composition of cholesterol particles. The approach of visualizing and quantifying the morphology of lipid particles by employing a constellation of image analysis algorithms may help both the diagnosis of atherosclerosis and to evaluate outcomes of lipid-lowering therapy in patients.

Numerous clinical studies have demonstrated beneficial effects of the lipid-lowering drugs in reducing plasma levels of low-density lipoproteins (LDL) cholesterol and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol, which prevents both primary and secondary incidences of atherosclerosis-related adverse cardiovascular events1,2,3,4,5. Statins, a group of HMG-CoA reductase enzyme inhibitors, block endogenous cholesterol synthesis in the liver that in turn lead to lower circulating levels of LDL cholesterol in the blood6,7. Likewise, the lipid lowering effect of niacin is mediated by its direct and noncompetitive inhibition of hepatocyte diacylglycerol acyltransferase-2, a key liver enzyme involved in triglyceride synthesis8. Comparatively, ezetimibe reduces plasma level of LDL by limiting absorption of exogenous cholesterol by binding to Niemann-Pick C1-Like 1 (NPC1L1) protein located in the epithelial cells of the small intestine9. Fenofibrate, another lipid-lowering drug, substantially reduces plasma concentrations of triglycerides and also moderately decreases LDL cholesterol through the peroxisome proliferator-activated receptors pathway10. Besides, omega-3 fatty acid is reported to have the anti-atherosclerotic effect due to its ability to lower the plasma levels of LDL11.
The lipid-lowering drugs, in addition to their primary effect on lowering LDL cholesterol, have a number of beneficial pleiotropic effects including enhancing the HDL level, improving endothelial functions, reducing inflammation, and inhibiting platelet aggregations12,13,14. However, the underlying mechanism of these drugs in increasing HDL cholesterol particles and modifying their structural features are not fully understood. Since these medicines are widely prescribed to treat atherosclerosis-related cardiovascular diseases (CVDs), it is essential to further investigate their possible roles in determining morphological features and distribution of the lipid particles. The human plasma lipidome consists of approximately 600 distinct lipids and 22 different molecular types of cholesterols that are present in various sizes, shapes, densities, and compositions15,16,17. Analytical methods such as ultra-centrifugation, NMR, and gradient gel electrophoresis are used to characterize LDL and HDL particles and their subfractions18,19. However, application of these methods is limited to studies aimed at determining the effect of drugs in modulating morphology and assembly of the lipid particles. The flow cytometer based plaque array is a functional biochemical assay developed for detection and visualization of serum derived lipid and amyloid plaque particles20. The advantages of in vitro imaging method described in this study enable identification of lipid-modulating drug effects in altering morphology and distribution of cholesterol particles in buffer and serum samples.

<table>
	<tbody>
		<tr>
			<td>TopFluor fluorescent cholesterol</td>
			<td>Avanti Polar lipids</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>simvastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>lovastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>rosuvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>atorvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fluvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>ezetimibe (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Niacin</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fibrate</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>omega-3 fatty acid</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>purified VLDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified LDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified HDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human age-matched serum</td>
			<td>Dx Biosamples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human atherosclerosis serum</td>
			<td>Bioserve</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human normal serum</td>
			<td>Stanford Blood center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Total cholesterol measurment</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtitre plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triglycerides measrument</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amnis Imaging Flow cytometer</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS image analysing software</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-1, ChemWel 2902</td>
			<td>Awarness Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-2, Intergra 400</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

differential effects of lipid-lowering drugs in modulating morphology of cholesterol particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

Watch this Scientific Journal Video about Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles at JoVE.com

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

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