The Low-Down on Fermented Tea, Part III: Glucuronic Acid

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In the previous post, The Low-Down on Fermented Tea, Part II: Metabolism & Detoxification, we discussed metabolism on a physiological scale, while circumnavigating the complexities of how this is accomplished on a cellular level.  We did however introduce the chemical compound glucuronic acid (GA); which is itself the metabolite of such complex cellular metabolic pathways. Namely, those of the symbiotic culture in kombucha, or "the mother", as well as that of the uronic acid pathway in liver cytoplasm - the later of which being our endogenous source of GA.

The metabolism of tea and sugar compounds in a kombucha fermentation is perhaps an excellent means of producing compounds with therapeutic effects for us humans, but will they be produced at adequate levels and with the same bio-compatibility as those endogenous to us (e.g. GA produced in the liver)?  If not, do we have any way of manipulating this?

Metabolism Thermodynamics - "maintaining the balance"

"The organism is not a static, isolated from the exterior, system that always contains identical components. Rather, it is an open system in a (quasi) stationary, or steady, state that retains its mass relations under permanent exchange of substances and energies building it, the state where some components persistently arrive from outside while other components are persistently leaving."

"Hence there is indeed something essential that should be received by all of us with food.  This is not however the simple energy, but rather the thermodynamic free energy contained in it.  A minimum amount of free energy has to be consumed each day in order that a biological organism survives; to overcome the effect of persistent entropy production in the steady state."

- Ludwig von Bertalannfy

Glucuronic Acid

Glucuronic acid (GA) has two primarily beneficial functions when we discuss health benefits: as building blocks for glycosaminoglycans and proteoglycan synthesis, and glucuronidation (phase II metabolism, or conjugation, of lipophilic xeno- and endo-biotics).

Proteoglycans are essentially composed of two basic molecules: a core protein and glycosaminoglycans (GAGs).  The major biological function of proteoglycans derives from the physico-chemical characteristics of the GAG component of the molecule. Namely, the high viscosity and low compressibility, or rigidity provides structural integrity to cells and passageways between cells, allowing for cell migration. This also provides the ideal hydrodynamic qualities for a lubricating fluid in the joints; providing hydration and swelling pressure to the tissue enabling it to withstand compressive forces 1.

Glucuronic acid (GA) is an organic acid which was first isolated in urine (hence the root name).

Glucuronic Acid - 2D Structure

An organic acid is simply an organic compound with acidic properties; and no, we don't mean organic as in it is grown without the use of pesticides, man-made fertilizers, feed additives, or growth regulators.  An organic molecule is one containing carbon atoms.

Uronic acids, such as GA, are sugar derivatives; formed from the oxidation of the aldoses' primary alcohol group.  GA, in particular, is formed by the specific oxidation of the C-6 carbon of glucose and is a key intermediate metabolite of the uronic acid pathway. Without getting too far into the chemistry of this compound, one noteworthy detail is that it can form two stereoisomers (D- & L- glucuronic acid) - due to asymmetry at the C-5 carbon.  Each stereoisomer has a respective α- and β-anomer, from the presence of another asymmetric carbon formed from the ring closure of the more common cyclic hemiacetals (resulting from nucleophilic attack at the carbonyl carbon of the aldehyde portion of the molecule) 2.

Uronic Acid Pathway

Endogenous GA Via UDP-α-D-Glucuronic Acid

Endogenous production of the active form of GA is carried out in vivo in the liver cytoplasm, via the uronic acid pathway.  The uronic acid pathway is fundamentally an alternative for the oxidation of glucose, or glycolytic pathway.  Unlike the glycolytic pathway, which is used as a major source of cellular energy, no ATP is produced from the uronic acid pathway (see below for a more detailed representation).

Uronic Acid Pathway & Metabolism

Interesting side note: in muscle, glucose-6-phosphate cannot be dephosphorylated, since it lacks the glucose-6-phosphatase enzyme. Thus, it is routed entirely through the glycolytic pathway to produce energy for muscle contraction3.

As you can see from the above diagram, the active UDP-α-D-glucuronic acid is utilized for several purposes, following phosphorylation and hydration, (i.e. Phase 2 metabolism conjugation reactions and GAG formation).

Interesting side note: humans are one of the few higher animals that are unable to synthesize L-ascorbic acid (vitamin C) because of their deficiency in L-gulono-gamma-lactone oxidase, the enzyme catalyzing the terminal step in L-ascorbic acid biosynthesis. Vitamin C is thus a dietary requirement for humans4.
 

Phase 2 Metabolism Involving GA - The Cellular Mechanism

So, at this point we have shown how to obtain the active form of GA endogenously, by means of UDP-α-D-glucuronic acid production via the uronic acid pathway, and exogenously via diet (e.g. kombucha).  Now we will discuss how these compounds are actually used for metabolism, at a cellular level.

Active Form of GA for Use in Metabolism

Recall from our previous post, The Low-Down on Fermented Tea, Part II: Metabolism & Detoxification, that the human body uses GA conjugation to make compounds such as alcohols, phenols, carboxylic acids, mercaptans, primary and secondary aliphatic amines, and carbamates more water-soluble; allowing for their subsequent excretion from the body through urine or faeces at a significantly increased rate. 

The GA carboxyl group is ionized at physiological pH, making the conjugated compound water-soluble. Compounds with larger molecular masses (i.e. > 60,000) are too large for renal excretion and will be excreted in faeces 5.
When considering the utility of an exogenous source of GA, its bio-availability must be considered.  Bio-availability refers to the degree to which an ingested nutrient undergoes intestinal absorption and metabolic function, or utilization, within the body. For a complete description of the nutritional adequacy of a food, three factors must be determined: the concentration of the compound at the time of consumption, the identity of various chemical species of the compound present, and the bio-availability of these forms of the compound as they exist in the meal consumed6.
Factors that influence the bio-availability of compound include: composition of the diet (could influence intestinal transit time, viscosity, emulsion characteristics, and pH); the form of the compound (forms may differ in rate or extent of absorption, ease of conversion to metabolically active form); and any interactions between a compound and components of the diet (e.g., proteins, starches, dietary fiber, lipids) that interfere with intestinal absorption of the compound. In living organisms it is the α-D-glucuronic acid form which is prevalent and most commonly involved in metabolic oxidation (See Oxygen is made in the heart of stars, so am I breathing sunbeams?) reactions.  For the incipient discussion we will assume both forms - that obtained endogenously from the UDP-α-D-glucuronic acid, of the uronic acid pathway, and that obtained exogenously, from kombucha - are equally bio-available. 7.

Cellular Mechanism for Phase 2 Metabolism

Recall, GA conjugation is a Phase 2 metabolism reaction in which GA acts as a conjugation molecule and binds to a substrate.  This occurs via the catalysis of glucuronosyltransferases (UGTs). First, in a series of reactions, the co-substrate UDP-α-D-glucuronic acid (UDPGA) is formed. The UGTs then catalyze the transfer of GA from UDPGA to another substrate (i.e. xeno- or endo-biotic), resulting in a glucuronidated substrate (conjugated metabolite) and leaving uridine 5'-diphosphate (UDP).

Specifically, this substitution involves the transfer of an α-D-GA from UDPGA to a functional group in the xenobiotic substrate; being linked by a β-glycosidic bond. The group may be a hydroxyl, phenol, carboxylic acid, thiol, or amine group8.

Example of GA Conjugation Reaction:

UDPGA + Xenobiotic (a Phenol) + UGTs = glucuronidated substrate (conjugated metabolite) + UDP

These reactions are essential in endogenous homeostasis for the glucuronidation of bilirubin, thyroid hormone, phenols, anthraquinones, carcinogen metabolites, and synthetic steroids9.
UGTs are a very broad and divers family of enzymes, considered as the most significant group of conjugation enzymes in xenobiotic metabolism; qualitatively because GA can be coupled to a large diversity of functional groups and quantitatively because of the divers number of substrates that are formed10.

Enzymatic kinetics studies have indicated that these enzymes follow a random sequential mechanism. UGTs are located in the endoplasmic reticulum, and their biosynthesis can be induced by a number of drugs and xenobiotics. Some forms of UGTs are coordinately induced with cytochromes P450 (CYP450) (11).

Xenobiotic-metabolizing UGTs comprise two subfamilies: UGT1 contains a single gene while UGT2 is a multi-gene family. They instantiate different ways of generating diversity.  For UGT2, diversity is generated by the conventional mechanism of having multiple individual genes, but diversity in the UGT1 family is generated by an unusual mechanism involving alternative mRNA splicing. There is only one UGT1 gene (UGT1A1, located on chromosome 2), but this encodes both phenol and bilirubin UGTs. The specificity-determining region of the gene is encoded by exon 1.  However due to the variability in exon 1 sequencing, and splicing with other exons, enzymes with different specificities can be generated from a single gene (9,12). 

Of course, when diving into genetics, especially in relation to health benefits, one must re-iterate the individuality of such an analysis (i.e. single nucleotide polymorphisms being able to alter the functionality of proteins and enzymes).

 

Exogenous vs. Endogenous

Recall how we made the assumptions that both sources of GA (exogenous and endogenous) were equally as active? This was solely for the sake of describing how the chemical is utilized for metabolism on a cellular level.  This is in fact not true. Utilizing carbon-14 labelling, studies have shown that administration of exogenous GA is in fact not very efficient; with roughly 50% lost, un-conjugated, in urine and another 30-35% lost as carbon dioxide through respiration.  That being said, a large portion of the remainder was found to be conjugated, with a small percentage (~1%) remaining in the liver (13).  These experiments did not consider timing of intake in relation to meal, or xenobiotic, intake; which would perhaps increase the necessity of exogenous GA, as endogenous sources are drained.  This would be especially important in the case of drug over-consumption.

 

Concluding Remarks on GA

The bulk of the discussion on kombucha thus far has led to this technical crescendo surrounding the GA.  Our hopes with this discussion has been: (a) to introduce the necessary scientific principles to be able to discuss health benefits of kombucha; (b) to introduce the most promising potential health benefit of kombucha; and (c) to underscore the complex nature of such a topic and the need of us as consumers, and vendors, to be more inquisitive and approach product claims with more scrutiny.

This isn't to say that kombucha consumption does not present the possibility for health benefits, in fact I believe the opposite.  I do believe however that the manner of its preparation, as well as its consumption, is deterministic in its beneficial nature.  Most importantly, with the inefficiency of incorporating exogenous GA into the metabolic pathways, the levels of GA obtained from kombucha fermentation must be maximized.  Adequate levels must be determined, and monitored analytically, before a company can draw conclusions on any associated health benefits.  Like most things, it's not black or white, good or bad, it falls somewhere in between. 

Summary Points

  1. GA has two primarily beneficial functions when we discuss health benefits: building blocks for GAGs and proteoglycan synthesis and phase II metabolism of lipophilic xeno- and endo-biotics.

  2. GA is obtained in our liver cells via UDP-α-D-GA, by means of the uronic acid pathway.

  3. GA can be obtained by outside means via dietary supplementation, such as kombucha consumption.

  4. Endogenous GA production involves the use of enzymes and its efficiency is therefore dependent on individual factors such as epigenetics, genetic polymorphisms and diet.

  5. GA obtained from outside sources is not likely incorporated into metabolic function with high efficiency and supplementation may only be required at certain times (i.e. endogenous depletion of GA).

  6. Timing of exogenous GA consumption is important to consider - perhaps wait to drink kombucha until after a large meal or following expected exposure to toxins/toxicans requiring expelling.

 

References

  1. Yanagishita M (1993) Function of Proteoglycans in the extracellular matrix. Acta Pathol Jpn 43(6): 283-93

  2. Chhabra N (2012) Uronic acid pathway. Biochemistry For Medics. Accessed January 4, 2018http://www.namrata.co/uronic-acid-pathway-subjective-questions-solved/

  3. Pelley J (2007) Ch. 8 Gluconeogenesis and Glyogen Metabolism. In Elsevier's Integrated Biochemistry pp 65-71. Elsevier Publishing

  4. Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K (1994) Cloning and the chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J Biol Chem 269: 13685-88

  5. Peterson A, Evrin E, Berggard I (1969) Differentiation of glomerular, tubular, and normal proteinuria: determinations of urinary excretion of β2-microglobulin, albumin, and total protein. J Clin Invest 48(7): 1189-98

  6. Gregory J (1996) Ch.8 Vitamins. In Food Chemistry 3rd ed. pp 532-34. Marcel Dekker Inc., New York

  7. Fondeur-Gelinotte M, Lattard V, Oriol R, Mollicone R, Jacquinet JC, Mulliert G, Gulberti S, Netter P, Magdalou J, Ouzzine M, Fournel-Gigleux S (2006) Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of β1,3-glucuronosyltransferase I (GlcAT-I). J Prot Sci 15(7): 1667-78

  8. Strassburg C, Kneip S, Topp J, Obermeyer-Straub P, Barut A, Tukey R, Manns M (2000) Polymorphic Gene Regulation and Interindividual Variation of UDP-glucuronosyltransferase Activity in Human Small Intestine. J Biol Chem275: 36164-171

  9. Stanley LA (2017) Ch. 27 Drug Metabolism. In Pharmacognosy pp 527-45. Academic Press, Elsevier Publishing

  10. Larison L, Henrissat B, Davies GJ, Withers SG (2008)Glycosyltransferases: Structures, Functions, and Mechanisms. Annu Rev Biochem77(25): 25-35

  11. Kedderis GL (2018) Toxicokinetics: Biotransformation of Toxicants. In Comprehensive Toxicology 3rd Ed. Vol. 1 pp 128-42. Academic Press, Elsevier Publishing

  12. Rowland A, Miners J, MacKenzie P (2013) The UDP-glucuronosyltransferases: Their role in drug metabolism and detoxification. Intern J Biochem Cell Biol 45(6): 1121-32

  13. Douglas JF, King CG (1952) The Metabolism of Uniformly Labelled D-Glucuronic Acid in the Guinea Pig. J Biol Chem 198: 187-94

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