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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

Representative Results

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…

Discussion

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 β…

Materials

Reagent
Sodium Chloride (NaCl)	Sigma-Aldrich	746398
Sodium Bicarbonate (NaHCO3)	Sigma-Aldrich	S5761
Calcium Chloride Dihydrate (CaCl2 * H2O)	Sigma-Aldrich	223506
Potassium Chloride (KCl)	Sigma-Aldrich	P9541
Magnesium Chloride (MgCl2)	Sigma-Aldrich	208337
Glucose	Sigma-Aldrich	G8270
13C6-Glucose	Sigma-Aldrich	389374
Palmitic acid	Cayman	10006627
13C16-Palmitic Acid	Sigma-Aldrich	605573
Rotenone	Sigma-Aldrich	R8875
Trichloro Acetic Acid	Sigma-Aldrich	T6399
5-Sulfosalicylic Acid	Sigma-Aldrich	390275
Acetonitirle	Fischer Scientific	A996-4	(optima)
Water (H2O)	Fischer Scientific	W7-4	(optima)
Formic acid	Fischer Scientific	85171	(optima)
Dimethyl Sulfoxide	Sigma-Aldrich	472301
Ethanol	Fischer Scientific	04-355-222
Methanol	Fischer Scientific	A454-4	(optima)
Ammonium Acetate	Fischer Scientific	A639-500
2 mL Eppendorf Tubes	BioExpress	C-3229-1
LC vials (plastic)	Waters	186002640
10 mL Glass Centrifuge Tubes	Kimble Chase	73785-10
Oasis Solid Phase Extraxtion (SPE) Columns	Waters	WAT094225
Pastuer Pipets	Fischer Scientific	13-678-200
Name	Company	Catalog Number	Comments
Equipment
CO2 Water-Jacketed Incubator	Nuaire	AutoFlow NU-8500
Triple Quadropole Mass Spectrometer	Thermo Scientific	Finnigan TSQ Quantum
HPLC	Thermo Scientific	Dionex Ultimate 3000
Source	Thermo Scientific	HESI II
HPLC Column	Phenomenex	Luna C18	3 μm particle size, 200 mm x 2 mm