What is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)?

From the Guidelines

HEPES is a crucial biological buffer that maintains stable pH levels in various laboratory solutions, including cell culture media, and is particularly valuable for its ability to maintain pH stability even when carbon dioxide concentrations change. According to the most recent study 1, HEPES is used in the incubation of patient samples, specifically in the presence of buffer and low- (therapeutic) and supratherapeutic concentrations of heparin. The study highlights the importance of sufficient final concentrations of calcium in the buffer to enable the generation of a procoagulant platelet response. Key characteristics of HEPES include:

• Effective pH range of 6.8-8.2, making it ideal for cell culture applications
• Non-toxic to most cell types at standard concentrations (typically 10-25 mM)
• Does not readily cross cell membranes
• Can generate free radicals when exposed to strong light and may interfere with some biochemical assays
• Often used as a supplementary buffer alongside bicarbonate systems to provide additional pH stability The use of HEPES in laboratory settings is supported by multiple studies, including 2 and 3, which demonstrate its effectiveness in maintaining pH stability in various applications. However, it is essential to use HEPES cautiously in certain applications, as it can generate free radicals when exposed to strong light and may interfere with some biochemical assays. In terms of specific applications, HEPES is commonly used in cell culture work, particularly when cultures must be removed from CO2 incubators for extended periods. Overall, HEPES is a valuable tool in laboratory settings, providing stable pH levels and supporting various biological applications.

From the Research

Properties and Effects of HEPES

• HEPES (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid) is a buffer that has advantages over other buffers commonly used in radiopharmaceutical preparation, as it exhibits significantly lower complexation tendency with metal ions 4.
• The use of HEPES buffer for radiolabeling reactions has been underrated due to the non-availability of sufficient toxicity data, but studies have shown that it may be safe for clinical applications 4.
• HEPES can affect blood clotting tests, with shorter clotting times found in the presence of 50 mM HEPES buffer than of 50mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin 5.

Physiological and Cellular Effects

• HEPES buffer has been proposed as a calibrator for pH measurement in human blood, with a physiological HEPES buffer (PHB) defined operationally with respect to the glass electrode and to phosphate buffer 6.
• HEPES can activate a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells, which can alter lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling 7.
• The addition of HEPES to cell growth and incubation media can significantly alter the uptake and transport of P-gp substrates, and can also stimulate the production of ATP 8.

Safety and Toxicity

• Studies have shown that HEPES is relatively non-toxic, with no mortality observed up to 2000 mg/kg doses of HEPES in female Sprague Dawley rats, and the doses beyond 300 mg/kg resulted in few temporary adverse effects that disappeared within 4-5 days of dosing 4.
• The amount of HEPES to be administered during clinical intervention is usually much lower than the maximum tolerated dose (MTD) determined in rat models, suggesting that the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe 4.