Pyruvate Kinase Deficiency (PKD)

Pyruvate Kinase Deficiency (PKD) is an one of several intrinsic hemolytic anemias and is a genetic condition that you are born with. These are called congenital hemolytic anemias. There are many types of congenital hemolytic anemias. The intrinsic hemolytic anemias include: sickle cell anemia, hereditary spherocytosis, pyruvate kinase deficiency and G6PD deficiency.

3D structure of pyruvate kinase

Pyruvate kinase is the enzyme that catalyzes the final step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP.¹ Pyruvate kinase is present in four distinct, tissue-specific isozymes in animals, each consisting of particular kinetic properties necessary to accommodate the variations in metabolic requirements of diverse tissues.

Genetic defects of this enzyme (pyruvate kinase) cause the disease known as pyruvate kinase deficiency. In this condition, a lack of pyruvate kinase slows down the process of glycolysis. This effect is especially devastating in cells that lack mitochondria, because these cells must use anaerobic glycolysis as their sole source of energy because the TCA cycle is not available. For example, red blood cells, which in a state of pyruvate kinase deficiency, rapidly become deficient in ATP and can undergo hemolysis. Therefore, pyruvate kinase deficiency can cause chronic nonspherocytic hemolytic anemia (CNSHA).²

Tetrahydrobiopterin Deficiency

Tetrahydrobiopterin Deficiency is a rare disorder characterized by a shortage (deficiency) of a molecule called tetrahydrobiopterin or BH4. This condition alters the levels of several substances in the body, including phenylalanine. Phenylalanine is a building block of proteins (an amino acid) that is obtained through the diet. It is found in foods that contain protein and in some artificial sweeteners. High levels of phenylalanine are present from early infancy in people with untreated tetrahydrobiopterin deficiency. This condition also alters the levels of chemicals called neurotransmitters, which transmit signals between nerve cells in the brain.

Tetrahydrobiopterin deficiency can be caused by mutations in one of several genes, including GCH1, PCBD1, PTS, and QDPR. These genes provide instructions for making enzymes that help produce and recycle tetrahydrobiopterin in the body. Tetrahydrobiopterin normally helps process several amino acids, including phenylalanine. It is also involved in the production of neurotransmitters.

If one of the enzymes fails to function correctly because of a gene mutation, little or no tetrahydrobiopterin is available to help process phenylalanine. As a result, phenylalanine can build up in the blood and other tissues. Because nerve cells in the brain are particularly sensitive to phenylalanine levels, excessive amounts of this substance can cause brain damage. Tetrahydrobiopterin deficiency can also alter the levels of certain neurotransmitters, which disrupts normal brain function. These abnormalities underlie the intellectual disability and other characteristic features of the condition.

Phenylalanine is found to function as a competitive inhibitor of pyruvate kinase in the brain. Although the degree of phenylalanine inhibitory activity is similar in both fetal and adult cells, the enzymes in the fetal brain cells are significantly more vulnerable to inhibition than those in adult brain cells. A study of PKM2 in babies with the genetic brain disease phenylketonurics (PKU), showed elevated levels of phenylalanine and decreased effectiveness of PKM2. This inhibitory mechanism provides insight into the role of pyruvate kinase in brain cell damage.3 4

Tetrahydrobiopterin (BH4, THB), also known as sapropterin, is a cofactor of the three aromatic amino acid hydroxylase enzymes,5 used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin (5-hydroxytryptamine, 5-HT), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and is a cofactor for the production of nitric oxide (NO) by the nitric oxide synthases.6 Chemically, its structure is that of a reduced pteridine derivative.

Tetrahydrobiopterin (BH4) has three roles:

Tetrahydrobiopterin is biosynthesized from guanosine triphosphate (GTP) by three chemical reactions mediated by the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR).[8]

BH4 can be oxidized by one or two electron reactions, to generate BH4 or BH3 radical and BH2, respectively. Research shows that ascorbic acid (also known as ascorbate or vitamin C) can reduce BH3 radical into BH4,[9] preventing the BH3 radical from reacting with other free radicals (superoxide and peroxynitrite specifically). Without this recycling process, uncoupling of the endothelial nitric oxide synthase (eNOS) enzyme and reduced bioavailability of the vasodilator nitric oxide occur, creating a form of endothelial dysfunction.[10] Ascorbic acid is oxidized to dehydroascorbic acid during this process, although it can be recycled back to ascorbic acid.

Folic Acid (Folate) and its metabolites seem to be particularly important in the recycling of BH4 and NOS coupling.[11]

MTHFR 1298 and the BH4 Cycle

The MTHFR A1298C mutation appears to disrupt the function of the BH4 Cycle. That is the Recycling of BH2 into BH4 [Tetrahydrobiopterin]. Tetrahydrobiopterin [BH4] is absolutely critical for many functions within the body including but not limited to:

  1. BH4 assists with the breakdown of PHENYLALANINE
  2. Helps as a cofactor for the formation NEUROTRANSMITTERS:
    • a. Serotonin
    • b. Melatonin
    • c. Dopamine
    • d. Norepinephrine (noradrenaline)
    • e. Epinephrine (adrenaline)
  3. cofactor to produce Nitric Oxide (NO)
  4. BH4 assists with the breakdown of AMMONIA.


BH4 regeneration is supported by methylfolate and SAMe

There is a pathway through which methyl-folate will regenerate BH4.  MTR removes the methyl group from methyl-folate as it remethylates homocysteine back in to methionine, generating raw material THF.  In the folate processing section we will talk a little more about DHF (Dihydrofolate Reductase).  DHF converts oxidized folate molecules to DHF and then in to useful THF, and this reaction is reversible.  High levels of methyl-folate will favor this reverse reaction, and in the process BH2 is recycled back to BH4.  Also, Methyl-folate looks a lot like BH4.  With respect to eNOS (endothelial nitric oxide synthase) function, methyl-folate enhances binding between eNOS and BH4, and when BH4 is in short  supply, methyl-folate can “stand in” for BH4.  Whether A1298C affects BH4 recycling or not, strong methyl-folate status will lead to strong BH4 status, a desirable state of affairs.  We often need help with respect to BH4 recycling. The enzyme DHPR recycles BH2 back in to BH4. DHPR is sensitive to toxins (mercury, lead, and particularly aluminum).  Oxidative stress (superoxide and peroxynitrite in particular) degrade BH4, and we also use up BH4 dealing with ammonia generated when the CBS pathway is unregulated.  Methyl-folate improves endothelial function and we are quite liberal with its use in the treatment of cardiovascular conditions.

Other than PKU studies, tetrahydrobiopterin has participated in clinical trials studying other approaches to solving conditions resultant from a deficiency of tetrahydrobiopterin. These include autism, ADHD, hypertension, endothelial dysfunction, and chronic kidney disease.[10][11] Experimental studies suggest that tetrahydrobiopterin regulates deficient production of nitric oxide in cardiovascular disease states, and contributes to the response to inflammation and injury, for example in pain due to nerve injury. A 2015 BioMarin-funded study of PKU patients found that those who responded to tetrahydrobiopterin also showed a reduction of ADHD symptoms.[12]


In 1997, a small pilot study was published on the efficacy of tetrahydrobiopterin (BH4) on relieving the symptoms of autism, which concluded that it “might be useful for a subgroup of children with autism” and that double-blind trials are needed, as are trials which measure outcomes over a longer period of time.[13] In 2010, Frye et al. published a paper which concluded that it was safe, and also noted that “several clinical trials have suggested that treatment with BH4 improves ASD symptomatology in some individuals.”[14]

For more information regarding methylation, please refer to “The Yasko Hypothesis of Neurodegenerative and Autoimmune Disease” and Methylation: Your Body Chemistry.

Cardiovascular disease

Since nitric oxide production is important in regulation of blood pressure and blood flow, thereby playing a significant role in cardiovascular diseases, tetrahydrobiopterin is a potential therapeutic target. In the endothelial cell lining of blood vessels, endothelial nitric oxide synthase is dependent on tetrahydrobiopterin availability.[15] Increasing tetrahydrobiopterin in endothelial cells by augmenting the levels of the biosynthetic enzyme GTPCH can maintain endothelial nitric oxide synthase function in experimental models of disease states such as diabetes,[16] atherosclerosis, and hypoxic pulmonary hypertension.[17] However, treatment of patients with existing coronary artery disease with oral tetrahydrobiopterin is limited by oxidation of tetrahydrobiopterin to the inactive form, dihydrobiopterin, with little benefit on vascular function.[18]


1 Gupta V, Bamezai RN (November 2010). “Human pyruvate kinase M2: a multifunctional protein”. Protein Science. 19 (11): 2031–44. doi:10.1002/pro.505. PMC 3005776. PMID 20857498.

2 Grace RF, Zanella A, Neufeld EJ, Morton DH, Eber S, Yaish H, Glader B (September 2015). “Erythrocyte pyruvate kinase deficiency: 2015 status report”. American Journal of Hematology. 90 (9): 825–30. doi:10.1002/ajh.24088. PMC 5053227. PMID 26087744.

3 Miller AL, Hawkins RA, Veech RL (March 1973). “Phenylketonuria: phenylalanine inhibits brain pyruvate kinase in vivo”. Science. 179 (4076): 904–6. Bibcode:1973Sci…179..904M. doi:10.1126/science.179.4076.904. PMID 4734564.

4 Weber G (August 1969). “Inhibition of human brain pyruvate kinase and hexokinase by phenylalanine and phenylpyruvate: possible relevance to phenylketonuric brain damage”. Proceedings of the National Academy of Sciences of the United States of America. 63 (4): 1365–9. Bibcode:1969PNAS…63.1365W. doi:10.1073/pnas.63.4.1365. PMC 223473. PMID 5260939.

5 Kappock, T. Joseph; Caradonna, John P. (1996). “Pterin-Dependent Amino Acid Hydroxylases”. Chemical Reviews. 96 (7): 2659–2756. doi:10.1021/CR9402034. PMID 11848840.

6 Całka, Jarosław (2006). “The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons”. Folia Histochemica et Cytobiologica. 44 (1): 3–12. PMID 16584085.

7 Kappock, T. Joseph; Caradonna, John P. (1996). “Pterin-Dependent Amino Acid Hydroxylases”. Chemical Reviews. 96 (7): 2659–2756. doi:10.1021/CR9402034. PMID 11848840.

Thöny, Beat; Auerbach, Günter; Blau, Nenad (2000). “Tetrahydrobiopterin biosynthesis, regeneration and functions”. Biochemical Journal. 347: 1–16. doi:10.1042/0264-6021:3470001. PMC 1220924. PMID 10727395.

9 Kuzkaya, N.; Weissmann, N.; Harrison, D. G.; Dikalov, S. (2003). “Interactions of Peroxynitrite, Tetrahydrobiopterin, Ascorbic Acid, and Thiols: Implications For Uncoupling Endothelial Nitric-Oxide Synthase”. Journal of Biological Chemistry. 278 (25): 22546–54. doi:10.1074/jbc.M302227200. PMID 12692136.

10 Muller-Delp, J. M. (2009). “Ascorbic acid and tetrahydrobiopterin: looking beyond nitric oxide bioavailability”. Cardiovascular Research. 84 (2): 178–9. doi:10.1093/cvr/cvp307. PMID 19744948.

11 ori, Tommaso; Burstein, Jason M.; Ahmed, Sofia; Miner, Steve E.S.; Al-Hesayen, Abdul; Kelly, Susan; Parker, John D. (2001-09-04). “Folic Acid Prevents Nitroglycerin-Induced Nitric Oxide Synthase Dysfunction and Nitrate Tolerance: A Human In Vivo Study”. Circulation. 104 (10): 1119–1123. doi:10.1161/hc3501.095358. ISSN 0009-7322

12 Burton, B.; Grant, M.; Feigenbaum, A.; Singh, R.; Hendren, R.; Siriwardena, K.; Phillips, J.; Sanchez-Valle, A.; Waisbren, S.; Gillis, J.; Prasad, S.; Merilainen, M.; Lang, W.; Zhang, C.; Yu, S.; Stahl, S. (2015). “A randomized, placebo-controlled, double-blind study of sapropterin to treat ADHD symptoms and executive function impairment in children and adults with sapropterin-responsive phenylketonuria”. Molecular Genetics and Metabolism. 114 (3): 415–24. doi:10.1016/j.ymgme.2014.11.011. PMID 25533024

13 Fernell, Elisabeth; Watanabe, Yasuyoshi; Adolfsson, Ingrid; Tani, Yoshihiro; Bergström, Mats; Phd, Per Hartvig; Md, Anders Lilja; Phd., Anne-Liis von Knorring MD.; Phd., Christopher Gillberg MD.; Phd., Bengt Lángström (2008). “Possible effects of tetrahydrobiopterin treatment in six children with autism – clinical and positron emission tomography data: A pilot study”. Developmental Medicine & Child Neurology. 39 (5): 313–8. doi:10.1111/j.1469-8749.1997.tb07437.x. PMID 9236697.

14 Frye, Richard E.; Huffman, Lynne C.; Elliott, Glen R. (2010). “Tetrahydrobiopterin as a novel therapeutic intervention for autism”. Neurotherapeutics. 7 (3): 241–9. doi:10.1016/j.nurt.2010.05.004. PMC 2908599. PMID 20643376

15 Channon, Keithm. (2004). “Tetrahydrobiopterin”. Trends in Cardiovascular Medicine. 14 (8): 323–7. doi:10.1016/j.tcm.2004.10.003. PMID 15596110

16 Alp, Nicholas J.; Mussa, Shafi; Khoo, Jeffrey; Cai, Shijie; Guzik, Tomasz; Jefferson, Andrew; Goh, Nicky; Rockett, Kirk A.; Channon, Keith M. (2003). “Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression”. Journal of Clinical Investigation. 112 (5): 725–35. doi:10.1172/JCI17786. PMC 182196. PMID 12952921.

17 Khoo, J. P.; Zhao, L; Alp, N. J.; Bendall, J. K.; Nicoli, T; Rockett, K; Wilkins, M. R.; Channon, K. M. (2005). “Pivotal Role for Endothelial Tetrahydrobiopterin in Pulmonary Hypertension”. Circulation. 111 (16): 2126–33. doi:10.1161/01.CIR.0000162470.26840.89. PMID 15824200

18 Cunnington, C.; Van Assche, T.; Shirodaria, C.; Kylintireas, I.; Lindsay, A. C.; Lee, J. M.; Antoniades, C.; Margaritis, M.; Lee, R.; Cerrato, R.; Crabtree, M. J.; Francis, J. M.; Sayeed, R.; Ratnatunga, C.; Pillai, R.; Choudhury, R. P.; Neubauer, S.; Channon, K. M. (2012). “Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients with Coronary Artery Disease”. Circulation. 125 (11): 1356–66. doi:10.1161/CIRCULATIONAHA.111.038919. PMC 5238935. PMID 22315282.