Blood microsampling is widely used to collect samples for dried blood spot (DBS) testing in newborn screening programmes, metabolic disease monitoring, and therapeutic drug monitoring. In a recent study from Greece, Capitainer’s quantitative DBS device Capitainer®B was used to develop a detection protocol for ceramides, a class of lipids that are emerging as important biomarkers for cardiovascular diseases, type 2 diabetes, and insulin resistance. The study provides proof-of-concept that DBS microsampling can be used to analyse ceramide biomarkers of cardiovascular disease, and highlights advantages including accuracy, minimal invasiveness, and possibility for at-home sampling.
Blood microsampling typically involves pricking a fingertip (or heel in newborns) and collecting a few drops of blood in the microliter volume range. Because microsampling is much less invasive than the venous blood draws used in many standard diagnostic and medical screening procedures, it is an ideal option for elderly individuals, children and individuals who require frequent testing. Dried blood spot (DBS) testing, which is a microsampling method based on dried spots of whole blood, has become the preferred sampling method in newborn screening (NBS) programmes throughout the world, and it is also widely used to monitor patients with inherited metabolic disorders (1,2).
Recent studies have shown that, when used quantitatively, DBS microsampling has the potential to aid patient-centric approaches to diagnostics and disease monitoring beyond the applications mentioned above. For instance, quantitative DBS was recently demonstrated to greatly increase access to COVID-19 diagnostics in rural Sweden (3), while another recent study from Sweden found that DBS testing has the potential to save resources while improving compliance, to increase the safety of lithium treatment in psychiatric disorders (4).
Ceramide biomarkers – reliable indicators of diverse disease states
Ceramides are a class of waxy lipid molecules known as sphingolipids, which are abundant within the lipid-rich cell membrane of eukaryotic cells. Beyond their structural purpose in the cell membrane, they also play key roles in cell signalling, cell growth arrest and apoptosis, proliferation, differentiation, senescence, adhesion, migration, inflammation, and angiogenesis.
Although the underlying mechanisms are not completely understood, ceramides and their downstream metabolites have been associated with several disease states including cancer, neurodegeneration, diabetes, infectious disease, obesity, and inflammation. A growing body of evidence indicates that certain circulating ceramides can be used to predict atherosclerotic cardiovascular disease (ACVD) risk, and may even more predictive than low-density (LDL), the so-called ‘bad’ cholesterol that is commonly used as a biomarker for risk of heart disease and stroke (5, and references within).
Ceramides are found within lipoprotein complexes and are produced by various cell and tissue types, including the liver and platelets, with the latter potentially linking ceramide signalling to atherosclerosis progression and pathology. In mice and rats, pharmacological inhibition or depletion of the enzymes that drive de novo ceramide biosynthesis prevents the development of diabetes, atherosclerosis, hypertension, and heart failure (6, and references within). As a result of such findings, ceramides have attracted attention not only as potential therapeutic targets but also as biomarkers for many of the diseases mentioned above.
How are ceramides analysed?
The use of ceramides as biomarkers for any disease state requires that accurate, sensitive, specific, and reproducible assays are available to detect and determine individual ceramide concentrations in the relevant bodily fluid. While ceramide analysis is not currently widespread in diagnostics, several approaches have been explored to measure ceramides in blood, plasma, and serum, such as:
- Antibody-based methods. Here, monoclonal antibodies with specificity for a given ceramide are used in an immunoassay setup, e.g., immunohistochemistry, ELISA, western blotting.
- Gas chromatography-mass spectrometry (GC–MS). This method combines the principle of GC, i.e., that a mixture will separate into individual substances (in this case ceramides) when heated, with the detection capacity of MS. The heated gases are carried through a column containing an inert gas, e.g., helium, and as the separated ceramides emerge from the column opening, they flow into the MS where they are detected based on their size.
- Liquid chromatography–tandem mass spectrometry (LC/MS), e.g., HPLC, LC-ESI-MS/MS, HPLC-MSMS-MRM. This approach is somewhat like GC-MS, in that it combines a separation method with a detection method. Here, compounds in a mixture are separated based on their affinity to a chromatography column (the material in the chosen column depends on the target ceramides), and then detected in a MS based on size.
Growing interest in quantifying ceramide biomarkers for diagnostic purposes
Although not discussed in detail here, the methods described above collectively suffer certain limitations, including time- and labour-intensiveness in sample preparation, unsuitability for high-throughput analysis, and lack of discriminatory power due to non-specific antibody binding. However, with increasing focus on ceramides as biomarkers and therapeutic targets, much progress has been made in recent years to develop new methods that overcome some of those limitations.
Last year, a team of researchers in China and the UK developed a simple and fast method to quantify 37 sphingolipids from 8 different lipid classes in human plasma and evaluated their role in insulin resistance, with comparable findings to previous studies (7). In 2021, a research group in Greece reported a new UHPLC-MS/MS method to rapidly determine four ceramides that are known to be predictive and prognostic biomarkers of atherosclerotic cardiovascular disease (Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0 and Cer d18:1/24:1) in human serum, and evaluated the method’s clinical utility by measuring serum ceramides in patients with coronary artery disease (8).
Proof-of-concept: DBS microsampling can be used to analyse ceramide biomarkers of atherosclerotic cardiovascular disease
The recent attempts to develop rapid detection methods for ceramides were based on sample volumes of at least 50 μL (6,7). Recognising the advantages of microsampling and building upon their previous experiences, the research group in Greece, mentioned above, sought to re-develop their protocol using dried blood spots from 10 μL whole blood collected using our quantitative DBS device Capitainer®B. Their workflow involved collecting blood samples using fingerprick and Capitainer®B (or venous blood draw for comparison), extracting the four ceramides of interest from the DBS sample disc, followed by analysis via RPLC-MS/MS. Isotope-labelled solutions of the four ceramides of interest were included as internal standard controls, while working standard mixture solutions were used as calibration controls (9).
Overall, the researchers detected similar levels of ceramides in whole blood collected by venous blood draw and in DBS samples, which is encouraging given that the ceramides of interest remain stable at room temperature, and the major advantages of DBS microsampling for at-home or remote testing. Their findings, published last month in Analytica Chimica Acta, estimated the intra and inter-day accuracy of the assay to be between 87.6% – 113% and 90.6% -113%, respectively, while intra- and inter-day precision were calculated from 0.2% to 9.9% %RSD and 0.1% – 8.0% %RSD, respectively. The authors attribute the observed accuracy and precise quantitation to the precise sampling volume made possible with Capitainer®B.
Upon sampling ten healthy individuals with Capitainer®B, the researchers found that circulating ceramides were more abundant in whole blood taken from the fingertip as compared to reported values in plasma or serum. To the best of our knowledge, this study is the first to demonstrate that quantitative DBS sampling can be used to detect clinically relevant ceramide biomarkers (9).
Researchers acknowledge value of quantitative DBS microsampling in health and disease surveillance
The authors note that further studies are warranted to yield more rigorous reference ranges before ceramides can be used in diagnostics, but their findings so far demonstrate the potential of DBS microsampling to monitor an increasingly important set of biomarkers with relevance in diverse disease areas. Finally, the researchers highlight advantages including access in remote/distant areas for high-risk patients and acknowledge the value of volumetric microsampling devices in health and disease surveillance.
References
- Moat, S. J., George, R. S., & Carling, R. S. Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders. International Journal of Neonatal Screening 2020. 6(26), doi: 10.3390/ijns6020026.
- Pitt, J. J. Newborn Screening. 2010. Clin. Biochem. Rev. 31(2):57-68.
- Byström JW, Vikström L, Rosendal E, et al. At-home sampling to meet geographical challenges for serological assessment of SARS-CoV-2 exposure in a rural region of northern Sweden, March to May 2021: a retrospective cohort study. Euro Surveill. 2023. 28(13):2200432. doi: 10.2807/1560-7917.ES.2023.28.13.2200432.
- Wikström F, Olsson C, Palm B, et al. Determination of lithium concentration in capillary blood using volumetric dried blood spots. J Pharm Biomed Anal. 2023. 1;227:115269. doi: 10.1016/j.jpba.2023.115269.
- McGurk KA, Keavney BD, Nicolaou A. Circulating ceramides as biomarkers of cardiovascular disease: Evidence from phenotypic and genomic studies. Atherosclerosis. 2021 Jun;327:18-30. doi: 10.1016/j.atherosclerosis.2021.04.021.
- Choi RH, Tatum SM, Symons JD, Summers SA, Holland WL. Ceramides and other sphingolipids as drivers of cardiovascular disease. Nat Rev Cardiol. 2021 Oct;18(10):701-711. doi: 10.1038/s41569-021-00536-1.
- Xu Y, Li H, Han Y, Wang T, et al. A simple and rapid method for extraction and measurement of circulating sphingolipids using LC-MS/MS: a targeted lipidomic analysis. Anal Bioanal Chem. 2022 Mar;414(6):2041-2054. doi: 10.1007/s00216-021-03853-z.
- Begou OA, Deda O, Karagiannidis E, et al. Development and validation of a RPLC-MS/MS method for the quantification of ceramides in human serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 15;1175:122734. doi: 10.1016/j.jchromb.2021.122734.
- Meikopoulos T, Begou O, Theodoridis G, Gika H. Ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS. Anal Chim Acta. 2023 May 15;1255:341131. doi: 10.1016/j.aca.2023.341131.
