Sphingolipidome quantification by liquid chromatography- high resolution mass spectrometry: whole blood vs. plasma

Plasma and serum are the most widely used blood-derived biofluids for metabolomics and lipidomics assays, but the isolation of these products from blood may introduce additional bias as indicated by the fact that many analytes that are present at high concentrations in blood cells cannot be measured and evaluated in those samples. Of particular concern, variable hemolysis during the pre-processing of blood products could compromise accurate and reproducible quantification. Compared with plasma or serum, whole blood may be a better alternative due to simplicity of processing. In this study, we provide a comprehensive method for quantification of the whole blood sphingolipidome and the concentrations were compared with those from plasma. Combining a single-phase extraction method with liquid-chromatography high resolution mass spectrometry (R=120, 000), assisted by alkaline hydrolysis, we were able to identify and simultaneously quantify more than 150 sphingolipids. Furthermore, most of sphingolipids remained stable after a freeze/thaw cycle. Whole blood contained a higher concentration of most sphingolipids than corresponding plasma. Moreover, individual variations in the levels of sphingolipids were lower for whole blood than plasma. These findings demonstrate that whole blood could be a better alternative to plasma, and potentially guide the evaluation of sphinglipidome for biomarker discovery.


Introduction
Blood consists of different components, including red blood cells, white blood cells, platelets, and plasma. In conventional metabolomics studies, plasma or serum derived from whole blood is used for the analysis, which could underestimate a lot of metabolic pathways, such as glycolysis, coenzymes, antioxidants, and purine metabolism. Those metabolic intermediates are usually present at high concentrations in blood cells but are not detected (or are very low) in plasma [1,2]. Increasing evidence demonstrated that whole blood metabolomics could provide more useful metabolites information in delineating metabolic effects related to aging, disease, nutrition, and environmental stressors than plasma metabolomics [3][4][5]. One notable example of the use of whole blood is the quantification of Acetyl-CoA, as its detection was impossible in serum or plasma [6] together with a high-throughput method for quantitative detection of eicosanoids in human whole blood and fatty acids [3,7]. Plasma "omics" measurements have a large degree of variability from plasma isolation, so freezing the whole blood as soon as was drawn could improve this variability.
To our knowledge, limited studies used whole blood for lipidomics analysis using liquid-chromatography high resolution mass spectrometry (LC-HRMS) [8][9][10][11][12] but there are several reports using 1 H-NMR, which has by far less sensitivity when compared with LC-MS. In order to assess the advantage of using whole blood for lipidomics analysis, the lipid composition should be elucidated and compared with plasma. Marasca et al. [8] have chosen 15 lipids species from most of lipids classes and compared their profiles o from whole blood, dry blood spots (DBS) or volumetric absorptive microsampling (VAMS).
The whole blood lipidomics analysis yields a very rich data set so we focused our initial study on sphingolipids. They are one of the major classes of eukaryotic lipids that are forming an outer leaflet of the plasma membrane lipid bilayer, and a lot of bioactive sphingolipids, such as ceramides (Cer), sphingosine-1-P, and glycosphingolipids, are supposed to be involved in many aspects of cellular functions, including cell proliferation, cell differentiation, apoptosis, cell cycle arrest, senescence, autophagy, cytoskeleton rearrangement, necrosis, inflammation, neurodegeneration, and cancer cell migration and invasion [13][14][15][16].Their synthesis and degradation processes [17][18][19] have different intracellular compartmentalization, including the endoplasmic reticulum (ER), Golgi apparatus and lysosomes.
To potentially guide the evaluation of sphinglipidome in blood for biomarker discovery, we developed a comprehensive method for quantification of sphingolipids in whole blood using single phase extraction method combined with high resolution mass spectrometry. We provided robust annotations and quantification assisted by alkaline hydrolysis, parallel reaction monitoring (PRM)/ data-independent acquisition (DIA), and appropriate internal standard normalization. One freeze/thaw cycle had no significant effect of sphingolipids, which will improve the option to use stored whole blood for sphingolipid analysis. More importantly, we compared the sphingolipidome of whole blood and paired plasma, showing that whole blood contained a higher concertation of most sphingolipids than the paired plasma, making the analysis accessible to less sensitive methods. Reducing the processing steps of the whole blood during plasma or serum isolation by directly freezing the whole blood at -80 °C may provide a more robust workflow for large lipidomics studies and allow for a more complete picture of the whole metabolism.

Single phase extraction combined with high resolution mass spectrometry provides accurate quantification of whole blood sphingolipidome
Due to the lack of tested methods for whole blood lipidomics, we selected several extraction protocols that were reported for plasma or serum (MTBE two phase, MTBE single phase, butanol single phase) and compared the fortified extraction efficacies of different sphingolipid internal standards. All three methods showed good recoveries for ceramides, ceramide-1-P, sphingomyelins, and hex-ceramides ( Figure 1A). However, the two-phase MTBE methods showed poor recoveries for sphingosine, sphinganine and their phosphate forms. The modified MTBE method enhanced the recovery of sphingosine-1-P and sphinganine-1-P but showed little improvement for sphingosine and sphinganine. Compared with the MTBE method, the single-phase (SP) butanol method, which was recently developed for plasma and serum lipidomics, exhibited good recoveries for all the sphingolipids ( Figure 1A, red bars). Therefore, the single-phase butanol extraction method was used for sample preparation of the whole blood sphingolipidome.
To accurately quantify the sphingolipids, we set the resolution of MS1 as high as 120,000@ m/z 200. This extreme high resolution can achieve a good accuracy with a mass error of less than 1ppm for most of sphingolipids ( Figure 1B). Even for the GM3(d34:1), with m/z higher than 1000, the resolution at m/z 1153.7217 was 53,202 with mass accuracy around 1ppm ( Figure S1). We also acquired MS2 fragmentations using data dependent acquisition (DDA) in both positive and negative mode (separately). The positive mode provided the polar lipid groups, such as SPH(d18:0) ( Figure 1C), while the negative mode provided additional information about fatty acid chain length ( Figure 1D). Combining the MS1 precursor with the MS2 fragments identification, we were able to positively identify all sphingolipid classes and typical spectra for each class in both ionization modes are included in the Supporting Information Figures S2-S6.

Alkaline hydrolysis improves the annotation of sphingolipids
Using DDA method, only the top 20 mass were selected for collision induced dissociation to acquire the MS2 fragment. For the low abundance metabolites or the ones masked by high abundant phospholipids was difficult to acquire their MS2 information. Alkaline hydrolysis was previously used to eliminate abundant phospholipids and to facilitate the annotation and quantification of sphingolipids [20,21]. However, comprehensive evaluation of stability of sphingolipids under alkaline condition was rarely performed. We fortified the extraction solvent with different sphingolipid internal standards and compared the peak intensity before and after alkaline hydrolysis (0.1 M KOH at 37℃ for 45 min). Consistent with previous results, most of sphingolipids, including ceramides, ceramide-1-P, hex-ceramides, and sphingomyelins remained stable after alkaline hydrolysis [22] (Figure 2A). Sphinganine and sphingosine levels significantly decreased after alkaline treatment, which was also found in other solvent systems, such as CH2Cl2/methanol (data not shown) and are worth investigating more. However, due to the presence of their corresponding internal standards, this should not be a major problem.
Alkaline hydrolysis facilitates the annotation of sphingolipids in two different ways, similar to the case of plasma analysis [22]. Firstly, it improves the annotation of sphingolipids by allowing for acquiring more MS2 fragmentation. For examples, Cer(m18:0/24:0) ( Figure 2B) showed similar intensity before (black peak) and after alkaline treatment (red peak), but only the alkaline treated samples showed the corresponding MS2 spectrum, which was annotated by LipidSearch with their Product search strategy. Secondly, the alkaline treatment hydrolyzed most of the phosphocholines (one of the best ionizing lipids that elute at similar retention times with most sphingolipids), so more sphingomyelins could be annotated based on their specific polar head fragment m/z 184.0732. Even combining this strategy with the HRMS, some of the fatty acid chains could not be completely annotated.

Freeze/thaw cycle will not influence the stability of sphingolipids
One disadvantage that could restrict the potential widespread application of whole blood in clinical metabolomics or lipidomics is the stability of metabolites in whole blood, which may be impacted by the enzymatic activity induced by the cell lysis during freeze/thaw cycle. Previous studies have reported some metabolites such acetyl-CoA may encounter dramatic loss during the thawing process [6], however, no data about the stability of sphingolipids in whole blood have been reported so far. Most of sphingolipids have been reported stable in plasma or serum at low temperatures [26]. One study reported that ceramides could be stable at room temperature for 8h [27]. To test the freeze/thaw stability of sphingolipids in whole blood samples, sphingolipids extracted from fresh collected whole blood (within 1 h after blood draw) were compared with the sphingolipid levels after freeze (at -80°C) or freeze and thaw ( Figure 5A). Surprisingly, the major sphingolipids, including SPHP, ceramides, hex-ceramides, and sphingomyelins showed no significant decrease after freeze or the freeze/thaw cycle ( Figure 5B). These results demonstrated that whole blood could be directly used for the sphingolipidome analysis. o Figure 5. Freeze/thaw cycle does not influence the intensity of sphingolipids in whole blood. (A) Schematic diagram of freeze/thaw experiment for sphingolipid stability in whole blood. (B) Comparison of the peak intensities of major sphingolipids in fresh blood and freeze/thawed blood.

Whole blood has a richer abundance of sphingolipids compared with paired plasma
The sphingolipidome of 10 whole blood samples and 10 paired plasma samples from healthy donors was performed based on the butanol single-phase extraction coupled with the LC-HRMS method developed for the analysis of the whole blood sphingolipids. Compared with plasma, whole blood contained higher levels of ceramides ( Figure 6A). The total concentration of ceramides in whole blood is nearly two-fold of that in plasma ( Figure 6B). The gangliosides GD3 and GM1 were higher in whole blood, while gangliosides GM3 were higher in plasma ( Figure 6A), which led to no significant difference of total gangliosides between whole blood and paired plasma ( Figure 6B). For glycosylceramides, Hex1Cer (glucosylceramide and galactosylceramide) were lower in whole blood than paired plasma, while Hex2Cer and Hex3Cer were dramatically higher in whole blood, especially for Hex2(3)Cer(d18:1/26:0) and Hex2(3)Cer(d18:1/26:0) that were barely detected in plasma samples ( Figure 6A and 6B). Sphingomyelins were the most abundant sphingolipids either in whole blood or plasma, but the whole blood has a much higher level of sphingomyelins ( Figure 6A and 6B). SPH(P) especially sphingosine-1-P are important small bioactive molecules, and both SPH an SPHP were 2-3 times higher in whole blood than paired plasma ( Figure 6A and 6B). Ceramide-1-P, another class of bioactive sphingolipids, also showed higher levels in whole blood, and not all plasma samples had detectable levels of ceramide-1-P using our LC-HRMS method. In conclusion, except for Hex1Cer and gangliosides, whole blood has a higher concentration of sphingolipids, including ceramide, CerP, Hex2Cer, Hex3Cer, SM, SPH, and SPHP. The lower abundance of the two classes would still allow for easy quantification (low µM levels) but for the sub-µM sphingolipids, any increase in abundance would help in the ease of quantification. After determining the corresponding internal standards, whole blood volume and freeze/thaw stability, we will investigate in further studies the contribution of each component of whole blood (red blood cells and white blood cells) to total blood sphingolipidome. Platelets sphingolipids will be determined by the difference. o Figure 6. Whole blood (WB) and paired plasma sphingolipidome. The heatmap showed the comparison of major sphingolipids, including Cer, Ganglioside, Hex1Cer, Hex2Cer, Hex3Cer, SM and SPH(P) between WB and paired plasma from healthy donators (A). The total intensity of respective sphingolipid class in WB and paired plasma (B).
Apart from containing higher abundant sphingolipids, whole blood also showed advantages in the individual variations of sphingolipid levels in 10 paired samples. For most of sphingolipids, except for HexCer, lower values (RSD) of individual variations were observed for whole blood in comparison with plasma ( Figure 6C). Specifically, some lower levels of sphingolipids, such as sphinganine, Cer(d18:0/24:2), Cer(d18:0/26:2), Cer(d18:1/26:2) showed much lower RSDs in whole blood compared with plasma, which may be due to the increased intensity and the ease of pre-process of whole blood.
One aspect that needs to be considered when using the volume of whole blood for analysis is the hematocrit-it measures the volume of red blood cells (RBC) compared to the total blood volume (red blood cells, white blood cells, platelets and plasma). The normal hematocrit for men is 40-54% and for women it is 36 to 48%. Correction for the hematocrit bias in dried blood spot was detailed before [28] and the hematocrit levels can be easily obtain from the hemoglobin levels using formula HCT=2.941*hemoglobin conc g/dL [29]. Hemoglobin can be easily measured using the Darbkin reagent and UV detection, using only 10 µL of frozen whole blood [30]. Using the hemoglobin measurements would be easy to calculate the sphingolipids levels in erythrocytes as was shown before for folates [31].

Chemicals and reagents.
Acetonitrile

Blood and plasma collection.
Whole blood samples were collected from healthy controls (6 males, 4 females, average age 28.6) after overnight fasting into EDTA coated tubes (Catalog No. 367861, 4 mL tubes with 7.2 mg EDTA). Each tube was gently inverted (without shaking or foaming contents) five times prior to transfer of 1 mL aliquots to separate LoBind tubes. All controls are enrolled in an ongoing natural history study at the Children's Hospital of Philadelphia (IRB # 01−002609). Plasma was obtained from whole blood after centrifugation at 500×g for 20 min at 4 °C.

Butanol single phase extraction
This method was used for the final extraction protocol for the whole blood and plasma. The method was modified according to previous papers [24,32]. Briefly, 50 μL whole blood (or other volumes as indicated on graphs for the method development part) or plasma, mixed with 10 μL internal standard solution (Cer/Sph Mixture I, was used as provided, and the concertation of each of the components is indicated in Table S1, as it was found from the Avanti website), were extracted with 1 mL butanol/methanol (1:1) (v:v) with 10 mM ammonium formate. After centrifugation at 500×g for 10 min, the supernatant was collected and dried to completeness under a stream of nitrogen. 3.3.2 MTBE two phase extraction 50 μL whole blood or plasma, mixed with 10 μL internal standard solution, were extracted with 0.75 mL methanol and 2.5 mL MTBE, then 0.625 mL water was added to induce two phase separation [33,34]. After centrifugation at 500×g for 10 min, the upper organic phase was transferred into a new tube and dried under a stream of nitrogen.

MTBE single phase extraction
This method was modified according to a published protocol (with chloroform being replaced by MTBE) to achieve better result for polar lipids [35]. Briefly, 50 μL whole blood or plasma, mixed with 10 μL internal standard solution, were extracted with 0.5 mL water, 1.25 mL methanol, and 1.25 mL MTBE. Different from the MTBE two phase method, this mixture would result in a final single phase for lipidomic analysis.
Each of the extraction steps was repeated, and the combined steps were dried to completeness under a stream of nitrogen. 100 μL methanol/MTBE (1:3) (v:v) was used for the re-suspension for all of the extraction methods.

Alkaline hydrolysis
For the butanol single phase method, 100 μL 0.1M KOH was added to extraction solvent, and incubated at 37℃ for 45 min, then neutralized with 6 μL glacial acetic acid (monitored with pH paper).

Freeze/thaw cycle
The freeze/thaw cycle was performed with fresh collected whole blood samples that were processed within 30 minutes of the blood draw. Fresh blood samples were aliquoted into 100 μL. Three technical replicates were used directly for extraction with butanol single phase method; the remaining aliquots were frozen at -80℃ for at least 24 h. After 24 h, the frozen blood samples were taken out of the freezer, and three technical replicates were extracted with butanol/methanol without thawing, while other three were thawed on ice (~2h) and then processed with the same extraction method as for fresh blood.
Full scan/PRM or full scan/DIA were performed with scheduled inclusion list (Table  S2). Full scan settings were as follows: m/z 200−1500, resolution 120,000, Maximum IT 200 ms, AGC target 1e6. For PRM, spectra were acquired as follows: isolation window 1.0 m/z, resolution 15000, AGC 1e5, maximum IT 100ms. For DIA, spectra were acquired as follows: isolation window 1.0 m/z, resolution 15,000, AGC 1e5, maximum IT 100ms, loop count 20.

Lipidomics data analysis
Peak detection, identification, alignment, and quantification were performed with LipidSearch 4.2 (Thermo Fisher Scientific, USA). Peak detection was achieved by searching their target database (HCD), and identification was performed using comprehensive ID algorithms (search type, Product) with precursor tolerance = 5 ppm, product tolerance = 10 ppm, m-Score threshold = 2.0. Identified peaks were filtered with m-Score threshold (higher than 5.0) and ID quality filter (A and B and C). Adducts included +H, +NH4, +Na, and +H-H2O in positive mode and -H, +HCOO, -2H, -CH3 in negative mode. Lipid results from each sample were then aligned within a RT window of 0.1 min and quantified by detecting their precursor ions from full MS and integrating XIC's.

Data and Statistical analysis
All the data were presented with mean ± SD. Two-tailed unpaired t-test was used to compare the significant difference with P<0.05. Graphpad Prism 9.0 was used to generate the illustrations and perform statistical analysis. Heatmap was conducted using R 3.6.3 with package pheatmap [36]. The approximate concentration for each compound was calculated by multiplying the area ratio of the compound to the relevant internal standard by the concentration of the internal standard added to each sample. .

Conclusions
In this study, we developed a comprehensive and robust method for whole blood sphingolipidome profiling by combing single phase extraction, alkaline hydrolysis, appropriate internal standards correction, and ultra-high resolution (R=120,000) mass spectrometry (DDA/PRM/DIA). Our results demonstrated that freeze/thaw process will not impact the stability of the sphingolipids in whole blood, implying that whole blood could be widely used for sphingolipid research. Comparison between whole blood and paired plasma from healthy controls indicated that whole blood had higher abundance sphingolipids than plasma, and whole blood showed much smaller individual variations in sphingolipid levels than plasma. All these results indicated that whole blood could be used as an alternative to plasma to guide sphingolipid biomarker discovery in clinical research, especially given the low volume of blood that could be used. Ceramides have been reported as promising biomarkers of cardiac events [37,38] and other pathologies as well [39], but their plasma concentrations varies depending on diet, time of day and other factors [40]. The RBC have a half-life of approximatively 120 days, so RBC sphingolipid o measurements could reflect the average levels during the prior 4 months, which could be a better measure of the absolute sphingolipid levels than the fluctuating ones from plasma. Recent results suggested that sphingolipids overload in RBCs could occur during erythropoiesis [41]. Also as recently reported that differences at individual levels in plasma may favor the use of erythrocytes [42] our study investigated the best protocol to do that by using the minimum volume of whole blood and the relevant internal standards for normalization. We will move next to a case-control study of a particular disease to show the applicability of this new approach.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: The chromatogram and MS spectra of GM3(d34:1), Figure S2: Representative MS2 spectra of Cer(d18:1/24:0), Figure S3: Representative MS2 spectra of Hex1Cer(d18:1/24:0), Figure S4: Representative MS2 spectra of sphingosine-1-P in both positive (left) and negative (right) ion mode, Figure S5: Representative MS2 spectra of SM(d18:1/24:1) in both positive (left) and negative (right) ion mode, Figure S6: Representative MS2 spectra of GM3(d34:1) in both positive (left) and negative (right) ion mode, Figure S7: The MS2 product extracted chromatograms of Cer(d19:1/25:0) (lower channel, RT 16.63 min) and Cer(d18:1/26:0) (middle channel, RT 16.83 min) in data independent acquisition (DIA) mode, Figure S8: Representative MS2 spectra of CerP(d18:1/16:0) acquired from whole blood samples (left panel) or standard solution (right panel) in positive ion mode, Figure S9. Comparison of the raw peak intensity or normalized intensity (internal standard 12:0 ceramide) of ceramides, and their linear response vs different volume of blood, Figure S10: Comparison of the raw peak intensity or normalized intensity (internal standard 12:0 ceramide) of dihydroceramides, and their linear response vs different volume of blood, Figure S11: Comparison of the raw peak intensity or normalized intensity (internal standard C17 base SPHP) of SPHP, and their linear response vs different volume of blood, Figure S12: Comparison of the raw peak intensity or normalized intensity (internal standard C15 Hex1Cer-D7) of Hex1Cer, and their linear response vs different volume of blood. Figure S13: Comparison of the raw peak intensity or normalized intensity (internal standard C15 Hex1Cer-D7) of Hex2Cer, and their linear response vs different volume of blood. Figure S14: Comparison of the raw peak intensity or normalized intensity (internal standard C15 Hex1Cer-D7) of Hex3Cer, and their linear response vs different volume of blood. Figure S15: Comparison of the raw peak intensity or normalized intensity (internal standard 18:1 d7 SM) of SM, and their linear response vs different volume of blood. Figure S16: Comparison of the raw peak intensity or normalized intensity (internal standard C18:0 GM3-d5) of GM3, and their linear response vs different volume of blood, Table S1: Ceramide/Sphingoid Internal Standard Mixture I,  Funding: This research was funded by the NIH grants R21NS116315 and P30ES013508.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Children's Hospital of Philadelphia (protocol code IRB 01-002609 and date of approval 12-09-2020) Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is contained within the article or supplementary material.