October 1, 2015 (Vol. 35, No. 17)

Mats Boren, Ph.D.

Heat-Induced Enzymatic Inactivation Preserves Metabolites for Biomarker Research

In the quest for a better understanding of disease processes and improved diagnostics, the study of low molecular weight molecules, a.k.a. metabolomics, has great potential. Metabolomics involves the quantitative detection of multiple small-molecule metabolites in biological systems using the multi-informative analytical techniques of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS).

Metabolites are a diverse group of molecules, including components of the natural metabolic process (such as lipids) as well as any added synthetic pharmaceutical compounds. Metabolomics can reveal abundant information with regard to the state of a biological system and is a vital complement to other life science omics disciplines.

Several recent metabolomics studies have been conducted with the focus on identifying changes in metabolite patterns that have prognostic value for early detection, therapy monitoring, and personalized medicine for common diseases such as cancer, diabetes, metabolic disturbances, and cardiovascular disease.

Metabolites in a State of Flux

In the healthy biological state, metabolite levels are tightly controlled, and fluctuate to reflect natural changes due to such things as food intake, diurnal rhythms, and level of activity. Nevertheless, metabolites are in a constant state of flux as metabolic processes occur constantly. Molecules are converted in metabolic processes and flow through multiple enzyme-catalyzed and interconnected conversion steps. Due to their nature as enzymatic substrates in constantly ongoing cellular processes, biologically relevant levels of metabolites are challenging to measure accurately.

Enzymatic processes continue post-sampling in addition to new processes initiated by the sudden change in the cellular environment caused by the sampling itself. This problem is addressed by experimental practices such as adding inhibitors to buffers, extracting in neat organic solvents, and keeping the samples cold during extraction. Although such practices help to reduce post-sampling changes, substantial alterations can still occur from the in vivo state prior to analysis.

The rate of change can differ between individuals and tissues as well as with post-sampling handling, which makes it impossible to compensate for post-sampling alterations. The potential differences in actual measured levels and true in vivo levels due to post-sampling changes may lead to erroneous diagnosis and make it more difficult to understand the underlying metabolism.

Heat-Induced Enzyme Inactivation to Prevent Post-Sampling Metabolite Changes

Denator has developed an additive-free heat-stabilization technology that utilizes conductive heating, under controlled pressure, to rapidly and irreversibly eliminate enzymatic degradation or changes in biological samples. This preserves the quality of samples throughout the entire workflow thereby increasing the accuracy of downstream analytical results.

The company’s Stabilizor™ system can be readily combined with most sample types and standard mass spectrometric and NMR based metabolomics analysis workflows to enable analytical results that better reflect the actual metabolomics state of the sample.

Case Study I: Pharmaceuticals in Whole Blood with DBS

The rate of metabolism of pharmaceutical compounds varies between individuals, and it is important to establish the correct dose with efficacy while minimizing side effects. The use of dried blood spot (DBS) sampling is a convenient and cost-effective way to determine the pharmaceutical concentration in whole blood. Although the liver is the major site of pharmaceutical metabolism, many drugs are also metabolized in the blood.

Metabolism in the blood is a problem when using DBS sampling as drugs can be further metabolized after blood is applied to the DBS card filter paper resulting in detection of lower levels than those actually present in the blood. Once on the filter paper, metabolism is caused by enzymatic activity in the blood, which can differ between individuals, as well as by drying time. To avoid the residual ongoing metabolism during DBS card drying, Blessborn et al. (Mahidol Oxford Tropical Medicine Research Unit Mahidol University, Thailand) applied heat inactivation using the Stabilizor system for the study of pharmaceutical concentrations in whole blood with DBS.

Using a number of pharmaceutical compounds they were able to show a time-dependent degradation during drying in the standard DBS cards. With heat-based enzyme inactivation, the enzymatic degradation could be stopped and higher levels of the intact pharmaceuticals of interest were detected coinciding with lower levels of the degradation products (Figure 1).

Figure 1. Difference in recovery of four pharmaceuticals in blood samples either heat stabilized directly after application to DBS cards, or nontreated and left to dry at room temperature. Heat stabilized blood samples gave between 12 and 70% higher recovery of the intact analyte compared to standard practice DBS cards dried at room temperature. It was shown that the increased recovery was due to prevention of post-sampling degradation after spotting the sample onto the DBS card. The four compounds were tested independently and spiked into fresh human whole blood just prior to application on the DBS cards. For analysis 3.2 mm round discs were punched from the DBS filter paper and the compounds extracted and subsequently analyzed using HPLC.

Case Study II: Free Fatty Acids in Tissue

The study of free fatty acids (FFA) has  recently gained in importance from just being part of energy metabolism to also being implicated in various diseases such as metabolic disorders and cancer. This is partly a result of improvements in analytical techniques, which have enabled a more detailed analysis of FFA. Both the ability to measure and to understand their importance have increased the necessity of being able to accurately quantify FFA.

This is challenging due to the role of FFAs in energy metabolism post-sampling where FFA are enzymatically released from triglycerides in an effort to recover energy, leading to a massive increase of FFA in the sample. To investigate the release of free FFAs post-sampling and the effect of heat- induced enzyme inactivation Karlsson et al. (Karolinska Institutet, Stockholm, Sweden) tested the Stabilizor system. They showed a time-dependent increase of FFAs in brain and liver after sampling. This resulted in much higher levels of a range of FFAs in traditionally harvested snap frozen samples compared to heat stabilized samples (Figure 2).

Figure 2. Levels of free fatty acids (FFA) in brain and liver are considerably lower in heat stabilized samples compared to standard snap frozen samples. FFA are released post-sampling from triglycerides first in an effort to recover energy and later more randomly due to active lipases, leading to a massive increase of FFA in the standard snap frozen samples compared to heat stabilized samples. Rat brain and liver samples were harvested and either snap frozen directly or heat stabilized to inactivate enzymes prior to snap freezing on dry ice. FFA were extracted with series of ice-cold neat organic solvents using a modified Folch protocol and analyzed using LC-MS.


Metabolites play a prominent part in understanding and managing diseases. However,  with a systematic and standardized approach to sample handling in metabolomic analysis they could have an even bigger role. The labile post-sampling nature of many metabolites makes them difficult to study in clinical research and later to implement in clinical routine practice.

The use of heat-induced enzyme inactivation with the Stabilizor system is an efficient means to stabilize metabolite levels and prevent changes and loss of sample quality and ultimately information on the biological state. The stability of metabolites  at room temperature after heat stabilization opens possibilities for new ways of sample handling for clinical samples, which could enable a greater use of metabolic biomarkers in healthcare as well as clinical and basic research. Heat stabilization using the Stabilizor system has the potential to be part of the work flow to unleash the full potential of metabolites as a research tool and within clinical practices.

Mats Boren, Ph.D. ([email protected]), is head of development at Denator.

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