Biotransformation or Detoxification Pathways

This page contains educational material about Biotransformation (detoxification) pathways of the body. This information is for educational purposes only. Nothing in this text is intended to serve as medical advice. All medical decisions should be made only with the guidance of your own personal medical authority. I am doing my best to get this data up quickly and correctly. If you find errors in this data, please let me know.

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Our bodies need to have a mechanism to remove unwanted chemicals or toxins from the body. We are exposed to both exogenous (xenobiotics) substances as well as endogenous substances (products of metabolism) that need to be removed from the body. Many toxins need to be altered by specific metabolic pathways before the body can remove them. Generally, the idea is to get the more lipophilic or fat-soluble substances changed into more water-soluble compounds, which are easier to remove from the body. Water soluble substances can be more easily disposed of in the urine or the bile. However, we do use our sweat, breath, hair, skin and even nails to get rid of toxic elements.

Basic Ideas

1. Biotransformation/detoxification is a metabolic process the body uses to remove unwanted hydrophobic substances (water hating) from either exogenous (foreign) or endogenous (made in the body) sources through transforming them into more hydrophillic (water loving) substances.

2. Biotransformation/detoxification occurs throughout the whole body. However, the main areas for serious biotransformation to take place are in the liver and intestines.

3. There are three steps involved: Phase I, Phase II, Phase III - Phase 1 is about functional group modifiction and making the substance more polar. Phase two is about conjugation of the functional groups making the substance even more polar. Phase III is about using transport mechanisms to get past cell membranes and remove these substances.

4. The Liver, Intestines and Kidneys are key players in transformation/detoxification

Some Details

Cell membranes are primarily lipid or oil based. They are largely impermeable to polar (water-soluble) substances.  A water-soluble substance requires assistance to get inside the cell. This assistance is available from specialized transport proteins in the cell membrane. The cell only allows desirable water-soluble molecules into the cell and will not allow entry of water-soluble toxins. The cell excretes unwanted water-soluble substances with transport proteins also.

Since the cell membrane is made  out of lipids,  fat-soluble substances can move into and through the lipid membrane more easily and without assistance. This allows lipid-soluble toxins easier access to the inside of the cell.  It also makes it harder for the cell to remove the lipid-soluble toxins.

Ingested toxins that are fat soluble enable these substances to be absorbed by the intestines and it also makes elimination of many of the xenobiotics difficult. Fat-soluble (non-polar) substances often end up stored in fat tissue.

The body has provided an enzymatic transformational/detoxification system to change these lipid-soluble toxins into less active/toxic water-soluble substances. Generally this is done via the “Phase II enzymatic system” by the attachment (conjugation) of water-soluble molecules to the lipid-soluble toxin at specific attachment points. When the toxic substance does not have attachment points, the body can use parts of the enzymatic system called “Phase I”  to molecularly transform the toxin so it has attachment points.

Once the substance is made water-soluble it can be excreted out of the cell by means of the transport proteins which is called “Phase III” of the transformational/detoxification system. Phase III can involve transport of transformed toxins both out of or into a cell. Phase III transport proteins have the job of shuttling toxins into the bile or urine for excretion.

A main route for toxins to enter the body is through the intestines. The idea is to remove these toxins in the intestines or if they get past the intestines to remove them in the liver. These two organs are where you will find transformation/detoxification going at full speed. However biotransformation of toxins is available through-out the body. There are other routes of entry into the body. Toxins/chemicals/drugs can also enter from the lungs, the skin, the eyes, vaginally, rectally.

Technically there are three phases of Biotransformation (AKA Metabolic Detoxification). These are classified as Phase 1, Phase II and Phase III. In phase 1, enzymes act on a compound that transforms the compound into a form that can be excreted or to a form that needs additional transformation by Phase II. Often times compounds made in Phase 1 actually become more toxic and this is called a bioactivation reaction. Phase II uses conjugation to make these compounds easier to remove them from the body. Phase II also acts directly on compounds to conjugate them without the need of Phase I. It can only do this if the substance already has a functional group on it that can be conjugated. Phase III is an efflux stage where the altered compound is removed from the cell using transporter proteins. You can imagine stage III as the stage where the compound takes a taxi as it is leaving the neighborhood.

A substance that activates Phase I and Phase II is known as a bifunctional inducer. If it activates only Phase II it is known as a monofunctional inducer. For the safest detoxification a substance undergoes a slow Phase I, followed by a more rapid Phase II. This prevents accumulation of Phase I metabolites which can be more toxic than their precursors.

The body attempts to eliminate anything that is toxic or is in excess. Besides the usual things we think of as toxins, it can include everything form hormones and vitamins to inflammatory molecules and prescription drugs. Therefore the bodies biotransformational or detoxification processes protects us from endogenous as well as exogenous toxins. It also provides a mechanism of homeostasis.

For most individuals, their diet is the largest source of toxin exposure. This can be from bacterial or fungal organisms on food or it can be from farming practices where sewage sludge, herbicides, or pesticides were used. Food related toxins can also be from food preservatives, food additives, heavy metals and even chemicals from the processing or packaging of the food.

The next source of toxins comes from the environment. The number one source of toxins in most peoples environment comes from the buildings they live in, work in or go to school in. There are also the buildings they frequent such as governmental buildings or stores. The buildings are often built with potentially hazardous materials that off-gas toxicants. Something as simple as installing a new carpet in a home, can make a person extremely ill depending on the carpet chemicals and the biotransformational capability of the person. Items in stores people frequent or the items you purchase and take home can be a potential source of off gassing depending on what chemicals that item was made out of.

Additionally, if a building has water-damage and is not remediated within 24-48 hours the growth or fungus and microbes within the mix of chemical laden building materials becomes a nightmare. You may have a release of mycotoxins, bacterial endotoxins, beta glucans, mannans, proteinases, hemolysins, c-type lectins, spirocyclic drimanes, and volatile organic compounds to name a few known toxins associated with water-damaged buildings. It is thought that as many as 50% of buildings in the United States have some sort of water-damaged. Often people do not know they are in a water-damaged building that was not remediated properly. There is no visible mold or bacteria, but the toxins still linger. They can be quite sickened by the environment without ever realizing what is causing it.

Then we have other environmental toxins which are probably the ones most people think of first. These are things that pollute our water, air and soil and are due to the things emitting from our vehicles, manufacturing processes, chemicals sprayed on lawns, ill-thought out agricultural practices and more things than I want to list. We have really created a chemical soup of toxins on the planet and our bodies are working hard to protect us all the time.

There are things people can do to limit their exposure to toxins. I suggest you visit the Environmental Working Group website to learn more about toxins in our environment and how you can protect yourself and your patients. Additionally, learning how the body transforms these toxins can give you ideas of which transformational processes are not working up to snuff and how to support them.

Keep in mind that gene polymorphism plays a role in how people are able to transform toxins into less toxic substances and remove them from the body.

Knowing about Biotransformation/Detoxification systems helps you decipher if a persons health issues are related to an issue in this area. Issues can stem from genetic polymorphisms, an over-worked system from too many exogenous or endogenous toxins, or a lack of nutrients available to support the biotransformational system. An understanding of the biochemical mechanisms involved in the regulation of these systems and the nutritional support necessary to keep these enzyme systems going, gives the practitioner a wonderful tool to help their patients.

Phase I and Phase II:
Biotransformation/Detoxification goes on everywhere in the body, but the powerhouse of detoxification is in the liver and in the intestinal wall. There is a two-step enzymatic process for the transformation , neutralization and removal of unwanted chemical compounds. This includes normal substances created by the body such as hormones and other signaling molecules as well as drugs, pesticides, insecticides, food additives, alcohol, mycotoxins, microbial toxins and other toxins from the gut. Anything that is toxic or would become toxic at high levels goes through this process. These biotransformational processes also metabolize signaling molecules into other signaling molecules, allowing for variation in the activities of these substances.

Phase I enzymes directly neutralize many chemicals, but some are transformed into intermediate forms that are then processed by phase II processes. These intermediate forms are often more chemically active and therefore can be more toxic. If the phase II detoxification systems are not working adequately, these intermediates can cause substantial damage, including the initiation of carcinogenic processes or genetic mutations. Additionally free radicals can be created by the process. Antioxidants can help protect us from the reactive oxygen intermediates. Some of these antioxidants are carotenes (vit A), Ascorbic acid (vit C), tocopherol (Vit E), copper, manganese, Selenium, zinc, coenzyme Q10, thiols (sulfurs in garlic, onions and mustard family plants), bioflavonoids, silymarin (Milk thistle).

Once toxins are transformed, the water soluble products are eliminated via the kidneys in the urine and many fat soluble items are excreted in the feces via the bile. For the intestinal transformation to be working adequately, the small intestines need to be healthy. If the intestines can't keep up with the fat-soluble toxins coming in or they are unhealthy, they will end up in the liver via the portal blood and the biotransformational system in the liver removes what it can. I want to stress that bile secretion by the liver is a critical process and the main mechanism for moving conjugated toxins out of the liver and into the intestines.The liver needs to be functioning up to par to create adequate bile and the gall bladder needs to also be intact to concentrate and store the bile for excretion into the digestive tract, along with the newly made water-soluble toxins the body wants to eliminate.


Intestinal Activity
Drug/toxin-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Although the liver is considered the powerhouse of biotransformational processes, many toxins come into the body through the intestines. This makes the intestinal biotransformation/detoxification activities very important. The intestines perform Phase I, II and III enzymatic activity, but also contain intestinal gut microflora that produce substances which induce or inhibit transformational activities. Additionally, there are pathogenic bacteria in the gut that release toxins which add to the biotransformational load on the body. Gut microflora also remove some conjugation moieties from substances that are in the enterohepatic circulation. This means toxins which were conjugated by the liver and are sent into the bile to be removed in the fecal mass, can be unconjugated and the toxin can re-enter the circulation in its original toxic form. (Gheeze Louise, can't an enterocyte get a break!)

An additional protective system is the antiporter system in the intestines (and elsewhere) called p-glycoprotein (or known as a multidrug resistant pump) which pumps xenobiotics out of the intestinal cells into the intestinal lumen. When the cell can not keep up with the xenobiotics coming in, this gives the cell another opportunity to metabolize the toxin the second time it shows up rather than allowing the toxin to move further into the cell where it can cause damage.

Influencing Factors on Transformational Capabilities
Genetics, age, sex, lifestyle habits, nutrient availability, disease, and presence of specific xenobiotics (including medications) or the magnitude of xenobiotics can influence the person's ability to transform toxins.

The genetic polymorphism of metallothioneins can also effect biotransformation/detoxification. Metallothioneins (MT) are proteins composed of about 30% cysteine. They are used for storage, transfer, and detoxification of intracellular metal ions. They are well known for their role in protection against and elimination of toxic metals. They bind some toxic metals such as cadmium and mercury and transport them to the liver or kidneys where glutathione conjugation takes place, followed by excretion of the toxic metal in the urine or bile. They also transport and provide short-term storage for zinc and copper. Animals have shown a wide range of MT capability that is due to polymorphisms. There are also animal mutants that possess multiple copies of MT genes in their chromosomes and have prolific upregulation of MT production. There is also some polymorphism seen in humans for MT expression. Gene expression for MT production is inducible through zinc supplementation, fasting and exercise.

Substrates: Inducers, inhibitors and Nutrients
A considerable amount of research has found that various substances activate the transformational enzymes while others inhibit it. It is also known that certain nutrients are necessary to support these pathways.

Obviously, it is beneficial to improve availability of enzymes and activity of the pathways in order to eliminate toxins as soon as possible. This is best accomplished by providing the needed nutrients and non-toxic stimulants while avoiding those substances that are toxic. However, stimulation of phase I is contraindicated if the patient’s phase II systems are under active. If Phase II can't keep up with the reactive substances created by Phase I, those substances rendered more reactive by Phase I, will create additional damage before Phase II deals with them.

When there is a high load of toxins, the body can respond by increasing the necessary enzymes. A toxin that upregulates enzymes can selectively upregulate just one enzyme, or it may upregulate many enzymes.

Calcium D-glucarate found in many fruits and vegetables inhibits beta-glucuronidase which is an enzyme produced by gut bacteria and intestinal cells.Beta-glucuronidase removes (deconjugates) glucuronic acid from neutralized toxins. This reverses the glucuronidation catalyzed by UGTs. Deconjugation reverts the toxin to its previous dangerous form, and allows it to be reabsorbed as part of the enterohepatic recycling. Elevated beta-glucuronidase activity has been associated with increased cancer risk.

Many flavonoid compounds can induce biotransformation activity of many of the Phase II enzymes. The Cruciferous vegetables are known for induction of Phase II also. Cruciferous vegetables such as broccoli, bok choy and brussel sprouts, are especially known for their induction of glutathione s-transferase and glucuronyl transferases. The flavonoid eriodictyol has been shown to increase expression of phase II enzymes that correlates wtih increased cell survival in oxidative stress settings. Long term treatment showing more benefit than short term treatment. (Johnson, 2009)

D-limonene found in citrus oil, has ben shown to induce both phase I and phase II. Research has shown it increases CYP450, intestinal UGT activity and liver GST and UGT activity in rats.

Chlorophyllin derived from chlorophyll inhibits CYP450 activity as well as stimulatse glutathione activity in rats. Chlorophyll and chlorophyllin also both trap toxins similar to charcoal thereby preventing absorption in the gut. They have also been shown to trap aflatoxins and keep them from being absorbed in animals. Human research appears that co-consumption of these products with aflatoxin may limit the bioavailability of the ingested aflatoxin in humans, as they do in animal models.

Induction by erythroid 2-related factor 2 (Nrf2)
The present data indicate that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in regulating mRNA of many phase-II drug-metabolizing genes as well as a number of efflux transporters that are important for the hepatic disposition of xenobiotics. It has minimal effect on Phase I CYP450 genes. It is also important in the production of antioxidant enzymes. This is important since phase I oxidation reactions produce a lot of free-radical byproducts.

Nrf2 increases mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification. (Kai Connie Wu, 2012)

The role of Nrf2 in protecting against oxidative and electrophilic stress has been well established, and the majority of genes involved in antioxidant defense have been identified as Nrf2 target genes in various models. For example, genes that are involved in direct reduction of reactive oxygen species (ROS), including superoxide dismutase, catalase, and glutathione peroxidases are induced by Nrf2. Genes involved in reduction of oxidized proteins, such as thioredoxin-1, thioredoxin reductase-1, and sulfuredoxin, are also Nrf2-target genes. Genes encoding enzymes that synthesize glutathione (GSH), the most abundant cellular thiol resource, namely γ-glutamate-cysteine ligase catalyze subunit (Gclc) and the modifier subunit (Gclm), as well as glutathione synthase (Gss), are known to be Nrf2 target genes. In addition, genes involved in generation of NADPH, the co-substrate to reduce oxidized GSH, such as glucose-6-phosphate dehydrogenase (G6pd) and malic enzyme (Me1), are induced upon Nrf2 activation.

Nrf2 has been variously described as an:

1. Activator of cellular defense mechanisms

2.Master redox switch and a guardian of health span

3. Gatekeeper of species longevity

The major products of Nrf2 genes are glutathione (GSH), Glutathione-S-transferase, Hemoxygenase-1, thioredoxin (non-enzyme), thioredoxin reductase, Quinone reductase, NAD(P)H:Quinone oxido-reductase, ferritin, metallothionein, peroxisome proliferator-activated receptor, nuclear factor erythroid 2-related factor 2, and NADPH regenerative enzymes. (For descriptions of these, see the terminology section at the bottom of the page.)

Activators of NRF2 are things like curcumin found in Curcuma longa, silymarin found in Silybum marianum, resveratrol found in Polygonum cuspidatum, as well as grape skin, red wine, and some berries, catechins found in dark chocolate, raw cacao, green tea. One of the strongest activators of NRF2 has been found to be the isothiocyanate called sulforaphane. Sulforaphane is the metabolite of glucoraphanin (GRN) which is contained in the cruciferous plants cell vacuole along with an enzyme called myrosinase (MYR) which are kept separate. When the plant cell wall ruptures and GRN and MYR come together, sulforaphane is enzymatically produced. All the Brassica/Crucifera plant family yields sulforaphane. The highest amounts are in broccoli and to be precise, broccoli sprouts. Cutting, chewing or otherwise opening the cell walls of these plants immediately causes synthesis of sulforaphane. However, it immediately begins to degrade. So, if you make yourself a broccoli drink you need to drink it sooner rather than later.

It might be of interest to you to know, the vitamin D receptor (VDR) is Nrf2 target gene inducible by sulforaphane. In turn, Vitamin D can increase Nrf2 expression.

The redox-modulating activity of the frequently prescribed statins and ACE inhibitors has been attributed to their Nrf2 inducer ability. Gold salts, once the mainstay of treatment for rheumatoid arthritis, are Nrf2 inducers.

There is an emerging idea that because Nrf2 is activated by a mild prooxidant signal, high doses of antioxidant supplements may blunt signals required to activate endogenous defenses . (Muthusamy VR, Kannan S, 2012 & Magbanua M. J. M., 2014)

Here is a list of various NRF2 activators listed by Life Extension from a variety of research articles: epigallocatechin gallate (EGCG), resveratrol, curcumin and its metabolite tetrahydrocurcumin, which has greater phase II activity, cinnamaldehyde, caffeic acid phenyethyl ester, alpha lipoic acid, alpha tocopherol, lycopene, apple polyphenols (chlorogenic acid and phloridzin), gingko biloba, chalcone, capsaicin, hydroxytyrosol from olives, allyl sulfides from garlic, chlorophyllin, and xanthohumols from hops


Activators of Quinone reductase

CD values(CD values refer to the concentration of a compound required to double the activity of the Phase II detoxification enzyme, quinone reductase ) for phytochemicals that activate quinone reductase: sulforaphane (0.2 μM), andrographolides (1.43), quercetin (2.50), β-carotene (7.2 μM), lutein (μM), resveratrol (21 μM), indole-3-carbinol from mature broccoli vegetable (50 μM), chlorophyll (250 μM), α-cryptoxanthin (1.8 mM), and zeaxanthin (2.2 mM), curcumin (2.7 μM), silymarin (3.6 μM), tamoxifen (5.9 μM), genistein (16.2 μM), epigallocatechin-3-gallate (EGCG) (>50 μM), and ascorbic acid (>50 μM). The comparative NQO1 inducer activity of these phytochemicals is sulforaphane > andrographolides > quercetin > curcumin > silymarin > tamoxifen > beta-carotene > genistein > lutein > resveratrol > I-3-C > chlorophyll > α-cryptoxanthin > zeaxanthin. (Christine A. Houghton, 2016)

Notably, the CD value of sulforaphane is 13.5-fold greater than that of curcumin, 18-fold greater than silymarin, and 105-fold greater than resveratrol.

Another Brassicaseae plant derivative that stimulates Phase II and not Phase I is indole-3-carbinol. It is from the glucosinolate known as glucobrassicin and like sulforaphane is also a strong inducer of Nrf2. The enzyme myrosinase is needed for the hydrolysis of glucobrassicin into indole-3-carbinol(IC3). Myrosinase and glucobrassicin are contained in the brassica family plants but seperated from each other inside vaculoes. Once the plant cells are opened by chewing or cutting myrosinase is release along with glucobrassicin and hydrolysis changes glucobrassicin into indole-3-carbinol. Research shows myrosinase is also created by gut bacteria which would mean this would help make IC3 from glucorophanin. This means when cooking inactivates myrosinase, any glucorophanin still left from cooking can be altered to IC3 to some degree by gut bacteria.

Although, we may be interested in our biotransformation systems working up to par to keep us all healthy, we must remember we can have too much of a good thing at which point it is no longer beneficial. A case in point would be the newly tested drug Bardoxolone Methyl which was known to enhance glomerular filtration rate in patients with chronic kidney disease, a disease known to have significant oxidative stress. This drug was a ysnthetic analog of the triterpenoid called oleanolic acid, which is found in edible plants. and is known to have cytoprotective properties thought to be due to Nrf2 induction. The trial on this drug had to be stopped due to adverse events including 57 deaths out of 2185 trail participants. The inducer activity of this drug compared to sulforafance when studying a range of triterpenoids showed it to be 230 fold more potent than sulforafane as a NQO1 inducer. (Christine A. Houghton, 2016) The amount of triterpenoids produced by foods are considered to be non-toxic. The amount in Bardoxolone Methyl appears to be toxic. The researchers are assuming the issue with the oleanolic acid was simply due to the increased dose, but I also am aware that it is not a natural substance and it is given out of context of all the other lovely accompanying substances that are in the foods rich in oleanolic acid.


Both Phase I and Phase II can be inhibited.

When there is more than one compound that is transformed by a single enzyme, there is what is called competitive inhibition. In this case one compound is unable to be transformed due to the competition of the other compound.

An increase in toxins can also lead to what is called inhibition due to increased toxic load. It is simply a case of using up available enzymes faster than they can be made.


Clinical Presentation of someone who has toxicity and may have a Phase I/II issue
History of increasing sensitivity to exogenous exposures.

Abundant use of medications and or supplements

Paradoxical responses or sensitivity to medications or supplements

Significant past or present use of toxic chemicals in home, work or environment

Sensitivity to odors

Trouble going into new buildings or buildings with a lot of new products

Musculoskeletal symptoms such as fibromyalgia

Inflammatory disorders

Cognitive dysfunction

Autonomic dysfunction

Worsening of symptoms after anesthesia or pregnancy

Supporting Biotransformation/Detoxification
Remove offending toxins/foods/drugs/heavy metals

Remove environmental exposures in home or work or remove the person from the environment. May need to add air filters, water filters, hepa filters on vacuums, etc.

Remove any unwanted organisms, such as bacterial, parasitic, viral that can not be lived with.

Make sure their entire digestive system from mouth to anus is in good condition as well as their liver/gall bladder - (The liver and intestines are needed for necessary biotransformation as well as for the digestive process which brings in nutrients for support of biotransformation.)

Ingest necessary nutrients, clean water (stay suficiently hydrated), clean air, only organic food, including organic meat

Provide increased nutrients that function as cofactors or are required in enzymatic steps of the biotransformational/detox pathways

Get adequate sun, rest/sleep and use meditation/prayer daily

Clean and monitor heating and air conditioning systems in home, work and car.

Do not wear dry cleaned clothing. Beaware of toxic household cleaners, garden chemicals, personal care products, cooking pots, food storage containers, do not use dryer sheets.

Stay away from car exhaust and cigarette smoke, minimize electromagnetic radiation from radios, T.V.s, microware ovens.

Decrease ionizing radiation from medical tests such as x-rays.

Reduce heavy metal exposure from larger predatory fish, water, lead paint, mercury fillings in teeth, thimerosal-containing products.

Consider chelation, infrared saunas or steam saunas, bile sequestrants, antioxidants, to reduce toxic load

Examine genetic polymorphisms that may make them more susceptible. A single nucleotide polymorphism (SNP) can change an amino acid in the protein coding sequence and thereby alter an enzyme binding site and/or the substrate binding site, which may affect the overall function. Genetic polymorphisms in biotransformation/detox may play a significant role in the pathophysiology of certain diseases.


Phase I Biotransformation (Detoxification)
Phase I transformation of most chemical toxins involves a group of isoenzymes. Although there are several types of phase I enzymes, the most common enzymes are the cytochrome P450 (CYP450). Usually when people talk about Phase I enzymes, they are talking about the CYP450 system enzymes. The CYP450 enzymes are also essential for the production of numerous agents including cholesterol and steroids. There is so much known now about CYP450 enzymes and their activity that even a cursory explanation needs its own page. Please see the detailed explanation of CYP450 System here.


Phase II Transformation (Detoxification)
Phase II transformation typically involves conjugation in which various enzymes in the liver attach small chemicals to the toxin. This conjugation reaction either neutralizes the toxin or makes the toxin more easily excreted through the urine or bile. Phase II enzymes act on some compounds directly. Examples would be ciprofloxacin, propranolol, bilirubin, diflunisal, acetaminophen, and thyroxin. Other compounds must first be activated by the phase I enzymes to add a functional group before conjugation can take place. There are essentially six phase II transformation pathways: They are Glutathione conjugation, Amino acid conjugation, Methylation, Sulfation, Acetylation, Glucuronidation. Conjugation reactions generally serve as a detoxifying step in metabolism of drugs and other xenobiotics as well as endogenous substrates. However, these conjugations may can add to toxicity of some substances due to the metabolic formation of toxic metabolites such as reactive electrophiles.

In order to work, both Phase I and II enzyme systems need nutrients. In addition, they utilize metabolic energy to function and to synthesize some of the small conjugating molecules. Thus, mitochondrial dysfunction, a magnesium deficiency or physical inactivity, can cause phase II detoxification to slow down, allowing the build-up of toxic intermediates. Phase II is especially effected by inadequate energy in the form of ATP to carry out conjugation.

The 6 transformation pathways of Phase II

The major phase II reactions are glucuronidation, sulfation, conjugation with amino acids, conjugation with glutathione, methylation and acetylation. Below are short synopsis of each conjugation reaciton. For more details on each one go to glucuronidation, sulfation, conjugation with amino acids, conjugation with glutathione, methylation and acetylation. (Sorry the sulfation, acetylation and amino acid conjugation pages are not up yet.)

Some of the conjugation pathways seem to share transporter proteins that move the conjugated items out of the cells. The multidrug resistance protein (MRP) 1 (encoded by ABCC1) and the related MRP2 (ABCC2) are adenosine triphosphate (ATP)-binding cassette transporter proteins that can work synergistically with phase II conjugation pathways to reduce the accumulation of a broad range of glutathione- (GSH/GS−, γ-Glu-Cys-Gly), glucuronide- and sulfate-conjugated organic anions. In addition, MRP1 and MRP2 require GSH for the transport of certain unconjugated and conjugated compounds.

1. Glutathione conjugation (see more data on this important nutrient here)
A primary phase II transformation route is conjugation with glutathione (a tripeptide composed of three amino acids—L-cysteine, L-glutamate, and glycine). Through direct conjugation, it detoxifies many xenobiotics (foreign compounds) and carcinogens, both organic and inorganic. This includes heavy metals, such as mercury, lead, and arsenic. Glutathione conjugation is probably the most important detoxification pathway for industrial toxins and carcinogens and 60% of toxins excreted in the bile are excreted in this way. Glutathione conjugation also produces water-soluble mercaptates which are excreted via the kidneys. The elimination of heavy metals like mercury and lead, is dependent upon adequate levels of glutathione, which in turn is dependent upon adequate levels of methionine and cysteine. When increased levels of toxic compounds are present, more methionine is utilized for cysteine and ultimately glutathione synthesis. Methionine and cysteine have a protective effect on glutathione and prevent depletion during toxic overload. (See the methylation cycle diagram to understand how they are necessary in the creation of glutathione.) This, in turn, protects the liver from the damaging effects of toxic compounds and promotes their elimination.

Glutathione is also an important antioxidant. This combination of detoxification and free radical protection, results in glutathione being one of the most important anticarcinogens and antioxidants in our cells, which means that a deficiency is cause of serious liver dysfunction and damage. Exposure to high levels of toxins depletes glutathione faster than it can be produced or absorbed from the diet. This results in increased susceptibility to toxin-induced diseases, such as cancer, especially if phase I detoxification system is highly active and Phase II can't keep up. Disease states due to glutathione deficiency are not uncommon. Health conditions like mold susceptibility (CIRS due to water-damaged buildings), Parkinson's and Alzheimer's have been linked to glutathione deficiency. Glutathione is needed to protect us from damage due to cigarette smoke, radiation exposure, and alcohol to name a few issues. Glutathione provides the major intracellular defense against mercury-induced neurotoxicity. High levels of mercury will use up glutathione.

For details on glutathione, including how to increase glutathione in the body: See more data on Glutathione here.

2. Amino Acid Conjugation
Several amino acids are needed for amino acid conjugation. Glycine, taurine, glutamine, arginine, and ornithine are used to combine with and neutralize toxins. Of these, glycine is the most commonly utilized in phase II amino acid detoxification. Patients suffering from hepatitis, alcoholic liver disorders, carcinomas, chronic arthritis, hypothyroidism, toxemia of pregnancy, and excessive chemical exposure are commonly found to have a poorly functioning amino acid conjugation system. For example, using the benzoate clearance test (a measure of the rate at which the body detoxifies benzoate by conjugating it with glycine to form hippuric acid, which is excreted by the kidneys), the rate of clearance in those with liver disease is 50% of that in healthy adults.

Even in normal adults, a wide variation exists in the activity of the glycine conjugation pathway. This is due to genetic variation, as well as the availability of glycine in the liver. Glycine, and the other amino acids used for conjugation, become deficient on a low-protein diet. Of course chronic exposure to toxins results in depletion also.

Adequate dietary protein is necessary for availability of these 5 amino acids.

3 Methylation (See details on methylation here.)
Methylation involves conjugating methyl groups to toxins. Most of the methyl groups used for detoxification come from S-adenosylmethionine (SAM). SAM is synthesized from the amino acid methionine, a process which requires the nutrients choline, vitamin B12, and active folate. SAM is able to inactivate estrogens (through methylation), supporting the use of methionine in conditions of estrogen excess. Its effects in preventing estrogen-induced cholestasis (stagnation of bile in the gall bladder) have been demonstrated in pregnant women and those on oral contraceptives. In addition to its role in promoting estrogen excretion, methionine has been shown to increase the membrane fluidity that is typically decreased by estrogens, thereby restoring several factors that promote bile flow. Methionine also promotes the flow of lipids to and from the liver in humans. Methionine is a major source of numerous sulfur-containing compounds, including the amino acids cysteine and taurine and is involved in createion of the antioxidant glutathione. Methylation has been studied extensively and it is known that some various genetic mutations can cause methylation issues. A common one is the inability to create active folate from folic acid.

Substrates that are used for methylation: choline, methionine, betaine, folate, vitamin B 6, B12, Riboflavin. methyltransferases are zinc dependent enzymes

Thorne has a nice product called Methyl-Guard Plus used to enhance methylation.

4. Sulfation
Sulfation is involved in a variety of biological processes, including detoxification, hormone regulation, molecular recognition, cell signaling, and viral entry into cells.

Sulfation has been shown to be an important pathway in the biotransformation of numerous xenobiotics such as drugs, and endogenous compounds such as hormones, bile acids, neurotransmitters, peptides, and lipids. Sulfation is the conjugation of toxins with sulfur-containing compounds. This process is catalyzed by the super-family of sulfotranferases (SULTs). The sulfation system is important for detoxifying industrial and environmental chemicals, several drugs, neurotransmitters, steroids, food additives, and toxins from intestinal bacteria. In addition, sulfation is also used to detoxify some normal body chemicals and is the main pathway for the elimination of steroid hormones such as thyroid hormones. Since sulfation is also the primary route for the elimination of neurotransmitters, dysfunction in this system may contribute to the development of some nervous system disorders. Additionally sulfation is important in the biosynthesis of proteins, petptides, gycosaminoglycans (GAGs) and intestinal mucins.

Many factors influence the activity of sulfate conjugation. For example, a diet low in methionine and cysteine has been shown to reduce sulfation.

When sulfation is inhibited a person is unable to detoxify and eliminate tylenol as well as adrenaline and dopamine from the brain.

Substrates that may be used for sulfation and sulfoxidation
: Sulfur containing foods in general, sulfate, taurine, glutathione, molybdenum and B2.

Inhibitors of Sulfation

Non-steroidal anti-inflammatory drugs (e.g. aspirin), tartrazine (yellow food dye)

5. Acetylation
Acetylation is a reaction, where an acetyl chemical group (CH3C=O) is added to another compound. This is a common reaction used in the metabolism of drugs. Conjugation of toxins with acetyl-CoA is the primary method by which the body eliminates sulfa drugs. This system appears to be especially sensitive to genetic variation, with those having a poor acetylation system being far more susceptible to sulfa drugs and other antibiotics. When salicylic acid is acetylated it becomes aspirin. While not much is known about how to directly improve the activity of this system, it is known that acetylation is dependent on thiamine, pantothenic acid, and vitamin C.

People with CIRS often have problems with sleep. We know that melatonin is necessary for good sleep and acetylation is necessary to make melatonin from serotonin. Serotonin is converted to melatonin by three steps involving a series of enzymes that add an acetyl, methyl and finally a hydroxyl group to the indole ring.

6. Glucuronidation - See details on glucuronidation here.
Glucuronidation, the combining of glucuronic acid with toxins, requires the enzyme UDP-glucuronyl transferase (UDPGT). Glucuronidation processes potentially carcinogenic environmental toxins such as polycyclic aromatic hydrocarbons and nitrosamines, as well as excess steroid hormones. During glucuronidation, the enzyme glucuronosyl transferase catalyzes the conjugation of free carcinogens and steroid hormones to glucuronic acid. The glucuronide-bound toxins and hormones are then safely excreted in the bile and the urine. Many of the commonly prescribed drugs are detoxified through this pathway. It also helps to detoxify aspirin, menthol, vanillin (synthetic vanilla), food additives such as benzoates, and some hormones. Glucuronidation appears to work well, except for those with Gilbert’s Syndrome. Gilbert's Syndrome is now known to affect as much as 5% of the general population.


Evaluating transformation/detoxification abilities of the Various Enzymes

Genetic testing can be helpful to a degree. Challenge tests are usually the most helpful. A probe substance is ingested and their metabolites are measure in the urine, blood or saliva.

CYP1A2: The probe used here is caffeine. Elevation of depression of the rate of caffeine clearance suggests upregulation or downregulation of this enzyme.

CYP2C9: The probe is Tolbutamide

CYP2C19 The probes are mephenytoin or proguanil

CYP2D6: The probes are sparteine, dextromethorphan, or debrisoquine

CYP2E1: The probe is chlorzoxazone

CYP3A4: The probes are erythromycin(breath test), midazolam, 6Beta-hydroxycortisol

N-acetyl transferase: The probes are sulphadimidine, Isoniazid, or caffeine

Glucuronyl transferase: The probes are oxazepam, acetaminophen

Sulfation: The probe is acetaminophen

Glycination: The probes are benzoic acid or salicylate

Testing of common toxic elements such as mercury, arsenic, lead, aluminum, cadmium and nickel is important as they can impair these transformational pathways, besides causing metabolic toxicity on their own.



Each of the separate pages covering the various methods of biotransformation will have a section at the bottom of the page on mycotoxins.



Endogenous: Produced originating inside of the organism.

Exogenous: Produced or originating outside of the organism.

Ferritin: Binding of free iron to prevent its reaction with superoxide to produce hydroxyl radical.

Glutathione: A predominant intracellular sulfur-containing direct antioxidant. Essential in function of Glutathione peroxidase and reduced glutathione for redox balance and detoxification.

Glutathione-S-transferase: A Phase II detoxifying enzyme with broad spectrum of activity, depending on subclass. Best known for catalyzing conjugation of the reduced form of glutathione to xenobiotics for removal from the body.

Hemoxygenase-1: Redox-regulating, broad protection against oxidative stress.  Metabolises haem, also producing bilirubin which scavenges peroxyl radicals.  Anti-inflammatory and immune-modulating properties.

Metallothionein: Helps removal of heavy metals such as mercury and cadmium.

NADPH regenerative enzymes: Restores reducing equivalents and reduces oxidized GSH to its reduced form.

Nuclear factor erythroid 2-related factor 2: Nrf2 induces its own synthesis. It can be inuced by many substances.

Peroxisome proliferator-activated receptor: Regulator of adipogenesis and central integrator of glucose metabolism, energy homeostasis and skeletal metabolism.

Quinone oxidoreductase - NAD(P)H:Quinone oxido-reductase has a protective function for cells against the toxicity of electrophiles and reactive forms of oxygen. In addition, its induction protects cells against carcinogenesis. Therefore, quinone reductase is acknowledged as belonging to the group of enzymes classified as phase 2 detoxification enzymes.

Quinone oxidoreductase catalyzes the beneficial two-electron reduction of quinones to hydroquinones, preventing the one-electron reduction of quinones by other quinone reductases that would result in the production of radical species.

A multifunctional redox-regulating and detoxifying enzyme, including protection against oestrogen quinone metabolites. Directly scavenges superoxide but less efficiently than SOD.  Stabilises the p53 tumor suppressor protein, especially under exposure from γ-irradiation or other oxidative stress. Protective against dopamine cytotoxicity where SOD and Catalase were not. Upregulation of its activity by Nrf2 induction is described as an avenue for maintaining cellular defenses with advancing age.

Thioredoxin (non-enzyme): Ubiquitous intracellular sulfur-rich protein. Singlet oxygen quencher and hydroxyl radical scavenger.

Thioredoxin reductase: An oxido-reductase which regenerates thioredoxin and GSH.

Xenobiotic: In relattion to the human or animal body, a xenobiotic is a foregin compound that originates externally to the body in sources such as environmental toxins or drugs.

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