WHITE PAPER

ISCHEMIA/HYPOXIA-INDUCED CASCADE OF METABOLIC EVENTS

Imagine trying to reconstruct the contents and operations of a busy factory after a cataclysmic hurricane or flood. Scientists are faced with a similar task when they attempted to reconstruct and understand the metabolism of living cells from excised postmortem or surgical samples.

Scientists who work with biospecimens know that specimens must be preserved immediately to maintain the specimen’s molecular integrity. However, this is not always possible with human biospecimens. This whitepaper discusses the consequences of this practical limitation and possible solutions to address it.

The moment a sample is removed from its primary blood supply, ischemia/hypoxia triggers an irreversible flood of events that alter cellular metabolism and cytoarchitecture.

Within seconds, the concentrations of signaling molecules, second messengers such as cAMP, and metabolites integral to energy metabolism (e.g., NAD+/NADH, ATP/ADP) begin to change. The effects of these changes bring about metabolic consequences. For example, a single molecule of cAMP can activate an enzyme cascade that yield a 10-billion-fold increase in the activity of the enzymes that breaks down glycogen into glucose. Rapid fluxes in calcium can activate other enzyme pathways and proteases with major metabolic consequences.

Within minutes of hypoxia, gene transduction factors and gene transcription are activated. Depending upon the tissue and other factors, RNA integrity deteriorates. The phosphorylation state for many proteins changes quickly. Apoptosis and eventual cell death occur. Some of these changes are highlighted in the adjacent figure.

This can be alarming if you are using human biospecimens to further your research. Many studies on metabolism using surgical, and even more so, postmortem samples are based upon a metabolic landscape that is dramatically changed from the normal in vivo situation. This problem is rarely discussed because it is omnipresent and very difficult to address with human studies. The next section discusses how this issue can be addressed currently and in the future with improved experimental design and techniques.

APPROACHES AND SOLUTIONS

There are four strategies that can be used to guide specimen collection and preservation. Implementing these strategies will help to minimize or at least account for the cellular alterations caused by ischemia/hypoxia.

1. Control what you can control. For example, there should be detailed SOPs for sample collection specifying permissible intervals between the cessation of blood flow and sample excision, preservations methods, and storage conditions. NIH’s GTex, TCGA, and CPTAC
   programs provide good examples of these along with ISBER’s and NCI’s Best Practices.
2. If you can’t control it, record it. During normal surgical practice or when using postmortem samples, it is often not possible to control as many variables as one would prefer. For example, clinical variables such as anesthesia, time on a respirator, radiation therapy, chemotherapy, pre- surgical medications, etc. have all been shown to affect sample biochemistry. These should all be annotated whenever possible.
   
   
3. Develop more rapid means of better preserving samples. With animal experiments, it is often possible to preserve samples within less than one minute of their excision using flash freezing, microwave irradiation, or various fixatives. Although, as pointed out above, even a minute’s delay in halting enzymatic activity can have enormous effects. In the clinical situation or when using postmortem samples, the situation is much worse. Some groups have tried to address this by taking biopsy samples and rapidly freezing or otherwise preserving them. This is not typically done with most surgical samples because, as it should be, patient care is the foremost priority. Nevertheless, efforts to rapidly collect samples, such as punch biopsies followed rapid freezing, will be rewarded with more physiological relevant data.
4. Measure tissue quality. There should be a good assay or more likely set of assays that assess tissue quality. For example, ABS has used a variety of measures such as sample pH, lactate concentration, oxidation-reduction status, thiol concentration, lipid peroxidation, protein degradation, and RNA integrity analyses to assess the biochemical integrity of tissue samples. Similarly, ABS is developing methods to assess the quality of formalin-fixed samples for immunohistochemical studies. With further development, these assays should greatly enhance one’s confidence in sample quality.

Ideally, all of these are used concurrently with goal of ensuring that the sample is satisfactory for its intended purpose. So how do we know if a sample is satisfactory? Thorough knowledge of analytes’ stability both in situ, after preservation, and following storage is essential. Without this information, all one is doing is measuring something, but its relationship to normal physiology or pathophysiology is ambiguous at best. For example, DNA is relatively stable whereas RNA is considerably less stable in many tissues. It is also important to utilize large numbers of samples because so many variables cannot be precisely controlled. If the approaches that are outlined above are followed, one can have some confidence in the data that are obtained.