Vitamin B6, Pyridoxal and Pyridoxine in Cell Culture

Importance and uses of the B₆ vitamins, pyridoxal and pyridoxine, in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures

Pyridoxal and Pyridoxine, a Serum-Free Medium Supplements, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:

Vitamin B_6, in its various forms, is an essential additive to cell culture. It is a component of the basal medium formulation. Vitamin B₆ has traditionally been added to cell culture media formulations as the aldehyde, pyridoxal; the alcohol, pyridoxine; or both the aldehyde and alcohol. One exception to this is BGJb Medium, Fitton-Jackson Modification which contains pyridoxal-5-phosphate. The form of Vitamin B₆ in basal media is important because the aldehydes, pyridoxal and pyridoxal phosphate, are chemically reactive with primary amines. Any media which contains pyridoxal or pyridoxal phosphate is chemically unstable. Chemical instability is especially important in serum-free or protein-free media used for commercial applications, such as biomanufacturing of heterologous proteins and tissue engineering.

The following media contain pyridoxal alone or in combination with pyridoxine:

Media containing pyridoxal alone include: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; Fischer's Medium; Glascow Modified Eagle's Medium (GMEM); Iscove's Modified Dulbecco's Medium (IMDM); Minimum Essential Medium Eagle (EMEM); Swim's S-77 Medium; Williams Medium E and various proprietary media. The original Dulbecco's Modified Eagle's Medium (DMEM) also contains pyridoxal, but several cell culture media manufacturers offer DMEM with pyridoxine substituted for pyridoxal.

Media that contain both pyridoxal and pyridoxine include: CMRL-1066 Medium; DMEM/Ham's Nutrient Mixture F-12 (50:50); H-Y Medium (Hybri-Max^®); McCoy's 5A Modified Medium; Medium 199; and NCTC Medium.

Cell culture media that contain pyridoxine alone include: F-12 Coon's Modification; L-15 Medium; MCDB Media series; Nutrient Mixture, Ham's F-10; Nutrient Mixture, Ham's F-12; Nutrient Mixture, Ham's F-12, Kaighn's Modification (F12K); RPMI-1640; Serum-Free/Protein Free Hybridoma Medium; Waymouth Medium MB; and various proprietary media. These media benefit from improved stability, but whether a specific cell can utilize pyridoxine in culture needs to be determined.

The chemistry and bioavailability of Vitamin B₆ in cell culture plays an important role in the stability and utility of media used for biomanufacturing of heterologous proteins and tissue engineering. For a more complete discussion of Vitamin B₆ as a cell culture component, visit our Media Expert.

Primary Functions of Pyridoxal and Pyridoxine in Cell Culture Systems:

Vitamin B₆ is involved in a wide range of biochemical reactions that affect cell processes:

• amino acid trans-sulfuration pathway that degrades homocysteine to taurine and hypotaurine;
• fatty acid metabolism;
• neurotransmitter synthesis;
• immune function;
• glyconeogenesis;
• folate metabolism;
• synthesis of coenzyme Q, and
• heme synthesis.

Pyridoxal phoshate (PLP)

Pyridoxal phoshate (PLP), an adehyde, is the active coenzyme in almost all B₆ containing enzymes. The broad spectrum of biochemical reactions catalyzed by PLP-enzymes results from the formation of Schiff bases between the vitamin aldehyde and the substrate primary amines. Since all amino acids contain at least one primary amine, the Schiff base mechanism of action makes PLP-enzymes important in their metabolism.

The following are some reactions mediated by the Schiff base PLP-enzymes.

• Racemization, the inter-conversion of L and D forms of amino acids;
• Transamination: the transfer of amino groups between amino acids and alpha-keto acids;
• Elimination reactions involving amino acid side-chains; decarboxylations; dehydrations; desulfurations; and de-aldolations.

Chemical Attributes of Pyridoxal and Pyridoxine that make it a Useful Serum-Free Medium Supplement:

Vitamin B6 Forms:

The vitamin B₆ family includes pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated counterparts; pyridoxine phosphate (PNP), pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP). The vitamin is not synthesized in animal cells. However, mammalian cells can interconvert it. A series of phosphorylase, oxidase, and kinase enzymes effect the in vivo inter-conversion of pyridoxine and its derivatives.

In vivo, B₆ compounds exist primarily as phosphorylated derivatives. In vitro, B₆ compounds are provided in the media formulations as either pyridoxal or pyridoxine. The addition of serum or albumin to cell cultures may also provide pyridoxal and pyridoxal phosphate.

Pyridoxine (Pyridoxol) (PN):

• Pyridoxine-HCl, C₈H₁₁NO₃-HCl_, molecular weight = 205.64, water-soluble;
• It does not form a Schiff base and is relatively stable in culture media;
• Cells do not use pyridoxine as an enzyme co-factor. They must convert pyridoxine into pyridoxal phosphate. This requires cells to have active pyridoxine kinase (EC 2.7.1.35) and pyridoxamine-phosphate oxidase (EC 1.4.3.5);
• Pyridoxine may function as an antioxidant in cell culture.

Pyridoxal (PL):

• Pyridoxal, C₈H₉NO ₃-HCl, molecular weight = 203.64, water soluble;
• This aldehyde form reacts with primary amines, such as amino acids, to form Schiff bases;
• Pyridoxal Schiff bases non-enzymatically degrade amino acids (and other primary amines) in cell culture media:
• Pyridoxal can form a relatively stable Schiff base with a lysyl residue of albumin;
• Cells do not use pyridoxal directly as an enzyme co-factor. They must convert pyridoxal into pyridoxal phosphate. This requires cells to have an active pyridoxal kinase.

Pyridoxal-5’-phosphate, (PLP):

• This is the active coenzyme form in animal B₆ enzymes, water-soluble;
• This aldehyde form reacts with primary amines, such as amino acids, to form Schiff bases;
• PLP Schiff bases can degrade amino acids non-enzymatically in cell culture media;
• PLP is not added directly to cell culture media. However, it binds to albumin and may be fortuitously added to culture media that are supplemented with either serum or albumin. Pyridoxal phosphate can form a relatively stable Schiff base with a lysyl residue of albumin;
• PLP does not cross cell membranes. It is dephosphorylated at the cell surface, transported into the cell, and then rephosphorylated.

Schiff Base In Vitro Chemistry:

The aldehyde forms of vitamin B₆, PL and PLP, can non-enzymatically break down primary amine containing molecules, such as amino acids, and may generate toxic by-products. This non-enzymatic degradation of amino acids is enhanced by transition metals that are chelated by the Schiff base.

Similar to the PLP-enzyme mediated reactions, pyridoxal:metal:amino acid Schiff base complexes participate in several types of non-enzymatic reactions:

• Racemizations interconvert L and D amino acids;
• Transaminations transfer amino groups between amino acids and alpha-keto acids;
• Decarboxylations remove CO₂ from amino acids;
• α,β-eliminations can dehydrate, de-aldolate, or desulfurate amino acids;
• β,γ-eliminaitons can dehydrate, de-aldolate, or desulfurate amino acids.

In addition to the loss of amino acids due to these reactions, toxic by-products are often formed.

Vitamin B6 Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures.