Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
Uploaded: 2016-04-04T15:00:00
Description: Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

We acknowledge the support of NIH grants P30ES013508 and T32ES019851.

Ethics Statement: All protocols concerning the treatment of human samples follow the guidelines of The University of Pennsylvania&#39;s human research ethics committee.
1. Preparation of Buffers and 100x Stock Solutions
<ol>
	<li>Prepare 1 L of base Tyrode&#39;s buffer. Combine 8.123 g NaCl, 1.428 g NaHCO3, 0.466 g CaCl2&#8729;2 H2O, 0.224 g KCl, and 0.095 g MgCl2. Adjust total volume to 1 L with ddH2O. Filter sterilize base Tyrode&#39;s...

<ol>
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Here we have shown the utility of isolated platelets as a platform for studying perturbed mitochondrial metabolism. Specifically, we have characterized metabolic adaptation in response to complex I inhibition by rotenone.
The present study has extended previously reported findings on the role of complex I inhibition by rotenone in cell lines to human platelets. Importantly, this has revealed that rotenone also inhibited platelet succinyl-CoA formation, stimulated an increase in platelet &#946;...

Dysfunctional mitochondrial metabolism has been implicated in a wide range of diseases including neurodegeneration, cancer, and cardiovascular disease 30. As such, great effort has been placed on characterizing metabolic defects that contribute to disease pathogenesis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard for quantification of analytes from complex biological matrices and is often employed for metabolic studies 8. However, as is often the case with biomedical studies, attaining an accessible and well-defined model relevant to human disease is a challenge.
Many studies employ transformed cellular models for probing the impact of xenobiotics or genetic abnormalities on cellular metabolism 7,9. The metabolic reprogramming that occurs in cancer cells can introduce confounding factors 21 and are therefore not ideal. These issues can be circumvented with primary cell models, although obtaining sufficient biomass for metabolic analyses can be challenging. Furthermore, the impact of high amounts of antibiotics used in culture has been highlighted as potentially confounding mitochondrial studies 16.
Human platelets afford the opportunity to utilize a primary cell model with sufficient mitochondrial content for metabolic studies 5,22,27,32. First, platelets can be easily acquired, through blood draws from individual donors, or in large volumes from blood banks, and therefore provide a model in which external factors can be readily controlled. Secondly, due to their small size, platelets can be easily isolated from other blood components with minimal preparatory work in even minimally equipped laboratories 5. Of note, platelets do not contain nuclei and can therefore be used to study alterations to metabolism independently of transcriptional regulation. Here we show that in addition to relative quantification of acyl-coenzyme A (CoA) thioesters, the isolated platelet system can be used to examine carbon metabolism. Specifically, we report the use of metabolic labeling with stable isotope (non-radioactive) labeled [13C6]-glucose and [13C16]-palmitate to probe incorporation of the [13C]-label into the important metabolite acetyl-CoA via glycolysis or fatty acid oxidation. This provides a powerful, generalizable, and versatile platform due to the extensive involvement of acyl-CoA species in biochemical pathways 13,24 and the tractability of this system to testing other variables, such as inhibition of complex I with rotenone 3,33. In addition to information provided in the Protocol below, an extensive description of the methods used for isotope labeling and for the LC-MS-based analyses can be found in Basu and Blair 4.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

To demonstrate the utility of this methodology we have reproduced the generalizability of previously described compensatory metabolic adaptation resulting from exposure to rotenone. This finding was previously identified in cell culture models and this investigation was aimed to test if this metabolic shift also occurs in platelets, which are anuclear and not prone to the same experimental artifacts as cell culture. This work was performed with 6-day-old platelets from the Penn Trauma Cen...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

The overall goal of this procedure is to determine how either a xenobiotic treatment or a disease state alters cellular metabolism. This method has the potential to reveal how a disease state or an applied toxin affects cellular metabolism. And once this is known, our method has the ability to reveal whether some kind of intervention can correct the defect.The main advantage of this technique is it doesn't rely on transformed cell lines, so it's more likely to provide answers that are directly relevant to human health. To begin this procedure prepare a 10 millimolar stock solution of rotenone in dimethyl sulfoxide. Perform a serial dilution of the 10 millimolar rotenone stock solution to obtain solutions with rotenone concentrations ranging from one nanomolar to 100 micromolar.Next, add 50 microliters of each stock solution to five milliliters of a previously-prepared enriched Tyrode's buffer solution to prepare solutions with rotenone concentrations ranging from 10 picomolar to one micromolar. Add 50 microliters of DMSO to one of the buffer solution samples to serve as a vehicle control. To isolate platelets from whole blood, centrifuge whole blood samples at 175 x g for 15 minutes with no breaks.When finished, transfer one milliliter of the upper platelet-rich plasma layer to a 1.5 milliliter microcentrifuge tube. When transferring the platelet-rich plasma, be careful not to disturb the buffy coat so the platelet pellet is not contaminated by lymphocytes. Centrifuge the platelet-rich plasma layer at 400 x g for five minutes.Then, aspirate the supernatent. Using no fewer than three biological replicates for each condition, resuspend the platelet pellet in one milliliter of each previously-prepared rotenone solution. Following this, incubate the resuspended platelets in a water-jacketed carbon dioxide incubator set to 95%humidity and 5%carbon dioxide at 37 degrees Celsius for one hour.At this point, pellet the platelets by centrifuging the samples at 3000 x g for three minutes. After aspirating the supernatent, add the appropriate stable isotope-labelled internal standard. Then, resuspend the platelets in 750 microliters of ice-cold 10%trichloroacetic acid.Pulse sonicate each sample 30 times with 0.5 second pulses. Following centrifugation, affix C18 solid phase extraction columns to a vacuum manifold. Condition the columns with one milliliter of methanol.Then, equilibrate the columns with one milliliter of double-distilled water. Run the sample-derived supernatent through the columns. When finished, wash the columns with one milliliter of water.Next, load 10 milliliter glass centrifuge tubes into the vacuum manifold to collect the elution fractions. Elute the columns with one milliliter of 25 millimolar ammonium acetate and methanol. After drying the eluate under nitrogen gas, resuspend the dried residues in 50 microliters of 5%5-sulfosalicyclic acid and transfer the samples to HPLC files for analysis.As previously reported in SH-SY5Y cells, changes in relative levels of aceyl CoA thioesters in response to rotenone are hypothesized to result from the inhibition of complex one. Specifically, there is a dose-dependent decrease in succinyl CoA with a simultaneous increase in beta-Hydroxybutyryl-CoA, while acetyl CoA levels were unchanged. The analysis of human platelets treated with rotenone reveals highly consistent results and provides a unique set of experimental data.It is important to note that this observation underscores the mitochondrial dependence of the response to rotenone because platelets lack nuclei. Following this procedure, other methods like metabolic tracing can be performed in order to answer additional questions, such as:How does this treatment or disease state affect substrate utilization? If we identify changes in the levels of some metabolite, why is it changing?Which steps in the pathway are affected, and in which way? After watching this video, you should have a good understanding of how to isolate platelets from whole blood, perform the controlled ex-vivo challenge and extract the aceyl CoA thioesters for LCMS anaylsis.

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