Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS

Abstract

Background: Intracellular metabolites such as CoA thioesters are modulated in a number of clinical settings. Their accurate measurement from surrogate tissues such as platelets may provide additional information to current serum and urinary biomarkers.

Methods: Freshly isolated platelets from healthy volunteers were treated with rotenone, propionate or isotopically labeled metabolic tracers. Using a recently developed LC-MS-based methodology, absolute changes in short-chain acyl-CoA thioesters were monitored, as well as relative metabolic labeling using isotopomer distribution analysis.

Results: Consistent with in vitro experiments, isolated platelets treated with rotenone showed decreased intracellular succinyl-CoA and increased β-hydroxybutyryl-CoA, while propionate treatment resulted in increased propionyl-CoA. In addition, isotopomers of the CoAs were readily detected in platelets treated with the [(13)C]- or [(13)C(15)N]-labeled metabolic precursors.

Conclusion: Here, we show that human platelets can provide a powerful ex vivo challenge platform with potential clinical diagnostic and biomarker discovery applications.

Figure 1. Four different ex vivo challenges schemes in isolated human platelets

Platelets are purified from whole blood using differential centrifugation and treated with either (A) a toxin, or (B) a metabolic substrate, followed by absolute quantitative analysis using labeled IS. Metabolic analysis using stable-isotope tracers applied either (C) after or (D) before platelet isolation. MID: Mass isotopomer distribution; PRP: Platelet-rich plasma; SILEC: Stable-isotope labeling with essential nutrients in cell culture.

Figure 2. Representative LC–SRM/MS chromatogram of short-chain acyl-CoA thioesters extracted from untreated platelets

BHB-CoA: β-hydroxybutyryl-CoA; CoASH: Reduced CoA; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.

Figure 3. Dose-dependent effect of rotenone on succinyl-CoA and β-hydroxybutyryl-CoA in isolated human platelets

Platelets from one healthy subject were isolated, washed and resuspended in Tyrode's solution containing an increasing concentration of rotenone for 30 min. Following incubation, platelets were pelleted and processed for short-chain acyl-CoA concentrations. A small aliquot was taken after resuspension to measure platelet concentration. (A) and (B) demonstrate intracellular platelet BHB-CoA and succinyl-CoA levels, respectively; (C) demonstrates the ratio of BHB-CoA to succinyl-CoA. BHB-CoA: β-hydroxybutyryl-CoA.

Figure 4. Effect of propionate treatment on selected CoA-thioesters in isolated human platelets

Platelets from a healthy subject were isolated, washed and resuspended in either Tyrode's solution or Tyrode's solution containing 10 mM propionate for 3 min. Following incubation, platelets were pelleted and processed for short chain acyl-CoA concentrations using stable-isotope labeling by essential nutrients in cell culture methodology. A small aliquot was taken after resuspension to measure platelet concentration. Error bars show SEMs for triplicate determinations. *p < 0.005.

Figure 5. Relative isotopic labeling of pantetheine backbone of various CoA derivatives

Isolated platelets were treated for 1 h with [¹³C₃ ¹⁵N₁]-pantothenate, which was incorporated into intracellular CoASH and acyl-CoA species using a series of five enzymatic steps. Short-chain acyl-CoA thioesters were then extracted from platelets and analyzed for relative isotopic labeling by determining the relative concentration of labeled (M+4) to unlabeled (M) isotopomer. Error bars show SEMs for triplicate determinations. BHB-CoA: β-hydroxybutyryl-CoA; CoASH: Reduced CoA; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.

Figure 6. Mass isotopomer distribution of selected CoA thioesters extracted from freshly isolated human platelets treated with Tyrode's solution containing no isotopic tracers (unlabeled), [1-¹³C₁]-propionate (10 mM) or [1,2-¹³C₂]-acetate (10 mM)

Following incubation, isotopic distribution of selected CoA species was determined using mass isotopomer distribution analysis. Error bars show SEMs for triplicate determinations. BHB-CoA: β-hydroxybutyryl-CoA; CoASH: Reduced CoA; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.

Figure 7. Mass isotopomer distribution of selected CoA thioesters extracted from isolated human platelets treated in whole blood with no isotopic tracer (unlabeled), [U-¹³C₆]-glucose (1 mg/ml) or [U-¹³C₁₆]-palmitate (100 μM)

Following incubation, isotopic distribution of selected CoA species was determined using mass isotopomer distribution analysis. Error bars show SEMs for triplicate determinations. BHB-CoA: β-hydroxybutyryl-CoA; CoASH: Reduced CoA; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.