色氨酸.Tryptophan

  

Introduction


Tryptophan is one of eight essential amino acids, necessary for the development of the vitamin niacin. It cannot be synthesized in the human body and thus must be obtained from food or supplements. Tryptophan is necessary for the production of several crucial substances in the body, including the neurotransmitter serotonin (5-hydroxytryptamine). Because serotonin (a melatonin precursor) plays a key role in mood and sleep patterns, tryptophan supplements have been used for some time as antidepressants, sleep aids, and weight-loss aids.

Tryptophan, however, is a substance with a colorful political history. In 1989, tryptophan supplements (specifically, the optical isomer L-tryptophan) were linked to a series of deaths and subsequently removed from the market. As the production of these supplements involved genetically engineered bacteria, L-tryptophan has become an example for those opposed to genetic engineering, as well as those condemning the involvement of government in drug development. Currently, this drug is banned by the FDA as a supplement for human use. It is, however, still available in many countries including Canada, Germany, the Netherlands and Great Britain. 

 


 

General Chemical Information


Common name:

Tryptophan

Chemical name:

[2-Amino-3-(3-indolyl)propionic acid]

Isomerism:

The Tryptophan molecule has a chiral center at the carbon connected to the carboxylic acid and the amine groups. Hence, it has 2 optical isomers: D-tryptophan or [(R)-2-Amino-3-(3-indolyl)propionic acid], and L-tryptophan [(S)-2-Amino-3-(3-indolyl)propionic acid]. L-tryptophan is the amino acid precursor of serotonin and melatonin.

Empirical formula:

C11H12N2O2

Molecular mass:

204.225 amu

Melting point:

295°C

PhysChem constants for the zwitterionic form

LogP :

-1.46 ± 1.0

Bioconcentration factor (BCF):

-1.94 ± 1.0

Adsorption coefficient (Koc):

-0.56 ± 1.0

Solubility:

0.021 mol/L in water at 25°C

 

 


 

General Chemical Information


Tryptophan is an amino acid essential to survival. Amino acids serve as building blocks for proteins, as well as serving as starting points for the synthesis of vitamins and many other crucial cellular molecules. While most plants and microorganisms can produce all the amino acids they need, tryptophan is one of eight amino acids that cannot be produced by animals.

The synthesis of tryptophan is one of the most complicated of all amino acids in the human diet. The overall pathway for the biosynthesis of tryptophan from chorismate can be viewed here.

Tryptophan is the least abundant of the essential amino acids. However, it is also one of the most crucial, as it is involved in the formulation of niacin and the neurotransmitter serotonin. Serotonin is crucial for regulation of mood and appetite; as well, since it is necessary for the production of the hormone melatonin, it is necessary for regulation of the sleep cycle.

Serotonin is produced from tryptophan in a two-step process, with the molecule 5-hydroxytryptophan as an intermediate step.

The compound 5-hydroxytryptophan can be extracted from natural sources as well as synthesized from tryptophan; as a result of the controversy surrounding tryptophan, it has been offered as an alternative to tryptophan supplements.

  

 


 

Metabolic Role


 

Dietary Supplement


Tryptophan has often been hyped as "nature's prozac" or "nature's serotonin solution." While certainly an exaggeration, these extravagant claims have some basis in fact.

Serotonin deficiency is often a major factor in depression, anxiety, sleep disturbances, and weight gain, to name a few. Simply supplementing serotonin when there is a deficiency would appear to be the easiest solution; however, since serotonin cannot pass through the blood-brain barrier, direct supplementation with serontonin is in fact ineffective.

Most antidepressant drugs available today fall into the category of Selective Serotonin Reuptake Inhibitors (SSRIs) such as Prozac, Zoloft and Paxil. These drugs work by preventing the excessive uptake of serotonin in the brain, leaving more available in the synapses between neurons. However, many of these drugs have considerable side effects, in addition to being fairly expensive.

Unlike serotonin, tryptophan (or more accurately, its breakdown product 5-hydroxytryptophan) can pass through the blood-brain barrier. Thus supplementation of tryptophan would appear to be a simple and natural alternative to SSRI drugs. Since tryptophan can be cheaply produced and is not regulated as a drug, it is a much cheaper alternative. As well, it does not have the side effects of many drugs as it is naturally occurring in the body.

Tryptophan on its own works best in depression cases where psychobiologic arousal is high, and serotonin production is inadequate to balance the effects of dopamine and norepinephrine - resulting in a anxious, agitated state. For more apathetic, vegetative depressive cases, tryptophan supplementation is more effective when combined with supplementation of tyrosine, a precursor to dopamine/norepinephrine.

Serotonin deficiency has been implicated in cases of obesity, as it is associated with the brain's perception of hunger and satiety. Excessive intake of sugars and other carbohydrates is known to increase brain serotonin levels. By increasing serotonin levels, tryptophan can function as an appetite suppressant at low doses, and is sometimes very effective for weight loss.

As well, tryptophan has been successfully used as a sleep aid. Serotonin does play a role in sleep regulation, but more importantly, serotonin is the precursor to melatonin, a hormone crucial to the sleep cycle. On its own, melatonin supplementation has been shown to be an effective treatment for insomnia; however, combining melatonin with tryptophan may prove even more effective.

5-HTP: The New Tryptophan Alternative


The compound 5-hydroxytryptophan (5-HTP) is the intermediate product in the conversion of tryptophan to serotonin. It was first offered as an over-the-counter supplement in 1994, and is marketed as a natural antidepressant. Since tryptophan is still banned by the FDA and is available in the United States only by prescription, 5-HTP has been offered as an alternative.

Since it is a direct precursor to serotonin, 5-HTP is considerably more active than tryptophan. It is thought to have effectiveness comparable to many prescription antidepressant drugs.

Unlike tryptophan, which was and still is produced in large quantities in vats of bacteria, 5-HTP is generally extracted from the Griffonia seed. It can be and sometimes is produced synthetically from tryptophan, but it is generally cheaper to extract it naturally. This allows 5-HTP to be offered at a more reasonable price, although still considerably higher than tryptophan was when it was offered as a nutritional supplement.

At the present time little research has been done on 5-HTP, and as such its effectiveness for various conditions is still unknown. Early research has suggested that it will be most effective in combination with other drugs and nutrients. It is expected that with time 5-HTP will become a widely used and effective supplement.

 


 

History & Politics


Tryptophan was first isolated in 1901, from the milk protein casein, by Sir Frederick Gowland Hopkins. By feeding mice a diet devoid of tryptophan, he was later able to demonstrate that it is essential for animal life. As well, he demonstrated that tryptophan and several other amino acids cannot be manufactured in the body and must be obtained from the diet. He was awarded the Nobel Prize for his discovery of vitamins, produced by amino acids like tryptophan.

Tryptophan was widely available as a nutritional supplement throughout the 1980's. Like other food supplements, it has been produced for many years by fermentative processes, in which large quantities of bacteria are grown in vats, and the supplement is extracted from the bacteria and purified. In the late 1980's Showa Denko K.K. decided to use genetically engineered bacteria in the manufacture of tryptophan to speed up and increase the efficiency of this process.

Tryptophan produced by this process was placed on the market in 1988. As it was considered essentially equivalent to conventionally-produced tryptophan, it required no further testing to be deemed safe. However, in less than a year it had been linked to thousands of cases of eosinophilia myalgia syndrome (EMS) caused by toxins in the supplement. More than 1500 cases of permanent disability and 37 deaths resulted. As the genetically engineered supplement was not labelled to distinguish it from other tryptophan, it took months to establish it as the cause of the disease.

EMS results in eosinophilia (overproduction of blood cells) and myalgia (muscle pain) initially, giving it its name. Over the long term this condition causes symptoms including paralysis and neurological problems, painful swelling and cracking of the skin, heart problems, memory and cognitive deficits, headaches, extreme light sensitivity, fatigue, and heart problems. The disease was later linked to several toxins in the tryptophan supplements, most prominently EBT (1,1'-ethylidene-bis-tryptophan), a dimerization product of tryptophan. It was deduced that at the high concentrations used in production, tryptophan molecules were reacting with each other to produce this deadly toxin.

Tryptophan supplements were recalled in 1989, and then banned completely in 1990 by the FDA. It is unclear whether the disease was primarily the result of inadequate purification of the tryptophan, or the use of genetically modified bacteria. However, it is clear that only batches of tryptophan produced by one company, by one unique method, were toxic. No tests prior to or since the ban have shown toxins in any other tryptophan supplements.

Owing to this ambiguity, tryptophan is now the topic of numerous debates about food supplements. It is a prime target for those cautious of (or opposed to) the consumption of genetically engineered products, and the inadequate labelling of these products. However, the recall of this product is also subject to criticism relating to the role of politics in drug regulation. Critics point out that tryptophan supplements, aside from those produced by Showa Denko using genetically engineered bacteria, have never been shown to contain toxins.

 

Use as a dietary supplement


For some time, tryptophan was available in health food stores as a dietary supplement. Since 2002, L-Tryptophan has been sold again in its original form. Many people found tryptophan to be a safe and reasonably effective sleep aid, probably due to its ability to increase brain levels of serotonin (a calming neurotransmitter when present in moderate levels) and/or melatonin (a sleep-inducing hormone secreted by the pineal gland in response to darkness or low light levels). Clinical research tended to confirm tryptophan's effectiveness as a natural sleep aid and for a growing variety of other conditions typically associated with low serotonin levels or activity in the brain. (Particularly work by Dr. Richard Wurtman at MIT). In particular, tryptophan showed considerable promise as an antidepressant alone, and as an "augmentor" of antidepressant drugs. Other promising indications included relief of chronic pain and reduction of impulsive, violent, manic, addictive, obsessive, or compulsive behaviours and disorders.

A more recent study has shown that nighttime use "modestly but significantly reduced sleepiness (P = 0.013) and improved brain-sustained attention processes (P = 0.002) the following morning".

In 1989, a large outbreak of a new, disabling, and in some cases deadly autoimmune illness called eosinophilia-myalgia syndrome (EMS) was traced to L-tryptophan. The bacterial culture used to synthesize tryptophan by a major Japanese manufacturer, Showa Denko KK, had recently been synthesized to increase tryptophan production; with the higher tryptophan concentration in the culture medium, the purification process had also been streamlined to reduce costs, and a purification step that used charcoal absorption to remove impurities had been omitted. This allowed another bacterial metabolite through the purification, resulting in the presence of an end-product contaminant responsible for the toxic effects. The FDA was unable to establish with certainty that this was the sole cause of the outbreak. Tryptophan was banned from sale in the US, and other countries followed suit.

Though it is indisputable that Showa Denko KK did produce and sell a contaminated batch of L-tryptophan, there are some concerns [2] that the FDA's handling of this accident unfairly favoured the pharmaceutical industry and the new antidepressant Prozac if only because of its curiously fortuitous timing. The March 22, 1990 ban on public sale of L-tryptophan came only four days before the media announcement of Prozac on March 26, 1990 in Newsweek magazine [3]. Both L-tryptophan and Prozac affect serotonin in the brain, though in different ways, and were promising in the treatment of depression. At the time of the ban the FDA did not know, or did not indicate, that EMS was caused by a contaminated batch [4], and yet even when the contamination was discovered and the process fixed, the FDA maintained that L-tryptophan was unsafe. In February 2001 the FDA loosened the restrictions on marketing (though not on importation), but still expressed the following concern:

In recent years, compounding pharmacies and some mail-order supplement retailers have begun selling tryptophan to the general public. ( Tryptophan has also remained on the market as a prescription drug (Tryptan) which some psychiatrists continue to prescribe, particularly as an augmenting agent for people who are unresponsive to antidepressant drugs. current FDA information Also, most health-food stores sell a cheap metabolite of tryptophan called 5-HTP to get around the resulting artificially high cost of the amino acid itself. But several high quality sources of L-Tryptophan do exist, and are sold in many of the largest health food stores nationwide. Indeed, tryptophan has continued to be used in clinical and experimental studies employing human patients and subjects. Several of these studies suggest tryptophan can effectively treat the fall/winter depression variant of seasonal affective disorder (SAD).

  


 

Tryptophan and turkey


According to popular belief, tryptophan in turkey meat causes drowsiness [6]. Turkey does contain tryptophan, which does have a documented sleep-inducing effect as it is readily converted into Serotonin by the body. However, tryptophan is effective only when taken on its own as a free amino acid. Tryptophan in turkey is found as part of a protein, and, in small enough amounts, this mechanism seems unlikely [7].

A more-likely hypothesis is that the ingestion of large quantities of food, such as at a Thanksgiving feast, means that large quantities of both carbohydrates and branched-chain amino acids are consumed. Like carbohydrates, branched-chain amino acids require insulin to be transduced through the myocyte membranes, which, after a large meal, creates a competition among the amino acids and glucose for insulin, while simultaneously creating tryptophan's reduced competition with other amino acids for the Large Neutral Amino Acid Transporter protein for transduction across the blood-brain barrier. The result is a greater availability of tryptophan, via the Large Neutral Amino Acid Transporter, for conversion into serotonin by the raphe nuclei, which is then available for conversion into melatonin by the pineal gland. Drowsiness is the result.


 

 

Tryptophan Hydroxylase (phospho S260) antibody images


 

 

All lanes : Tryptophan Hydroxylase (phospho S260) antibody (ab30574) at 1/1000 dilution
Lane 1 : rat brain stem lysate (control)
Lane 2 : rat brain stem lysate, incubated with S260 phosphopeptide (negative control)
Lane 3 : rat brain stem lysate, incubated with corresponding non phosphopeptide.
Lysates/proteins at 15 µg per lane.
Predicted band size : 51 kDa
Observed band size : 55 kDa
Additional bands at : 48 kDa,59 kDa,<43 kDa. We are unsure as to the identity of these extra bands.
In lane 2, the labeling is specifically blocked by the S260 phosphopeptide used as antigen.
In lane 3, the corresponding nonphosphopeptide did not block the

 

 

Tryptophan Metabolites



Tryptophan metabolism is of significant biological importance, since altered levels have been seen in patients suffering from a range of disorders, including depression, schizophrenia, Downs Syndrome and alcoholism*. The major and minor routes of tryptophan catabolism lead to a wide range of related compounds which may be acidic, basic or zwitterionic. The use of polymeric reversed phase columns, PLRP-S 100Å, offers a difference in selectivity compared to conventional alkyl-bonded silica columns. The absence of residual silanols eliminates potential band broadening of basic components. Using a PLRP-S column in conjunction with PL's evaporative light scattering detectors, all of the compounds may be analysed without the need for derivitization and with similar response factors - leading to easier, reliable quantification.

 

Column: PLRP-S 100Å 5µm, 150x4.6mm
Eluent: 93% 40mM Ammonium acetate, pH 4.5 : 7% ACN
Flow Rate: 1.0ml/min
Detector: PL-ELS 1000 (neb=85°C, evap=90°C, gas=1.0 SLM)

KEY
1. Quinolinic acid
2. 3-Hydroxykynurenine
3. Kynurenine
4. Kynurenic acid
5. Trytophan

 

 

 

Tryptophan aids the body to synthesize the B-vitamin, niacin. With the new niacin created, the body produces Serotonin. "Serotonin is a chemical that acts as a calming agent in the brain and plays a roll in sleep."(?) A person that lacks the gene which produces an enzyme allowing Tryptophan to synthesize niacin to in turn produce serotonin, will experience an 80 percent loss of the neurotransmitter, serotonin. Patients clinically diagnosed with depression are 10 times more likely to carry this mutant gene than non-depressed patients. If one were diagnosed with low serotonin levels resulting in depression, a common treatment is prescribing a patient with SSRI drugs. Serotonin selective reuptake inhibitors are drugs that allow cells to uptake serotonin in the brain. This helps patients deal with many forms of depression. A mutant gene is responsible for the body’s inability to take up tryptophan. Genetic mutation results from a single base being other than what it should be. A study was conducted at Duke University which stated, "the mutant gene was carried by 87 depressed mice, 3 of 219 healthy controls and none of 60 bipolar disorder mice. This was reported by Drs. Marc Caron, Xiadong Zhang, and colleagues in January 2005." (3)(5)(7) The depressed mice showed 50-70 less serotonins. Genetic testing for tryptophan resistance can be a possible indicator of sensitivity to depression in an individual. "Once testing confirms sensitivity to tryptophan-serotonin production, serotonin-selective antidepressants can be prescribed for the sensitive individual." (6) Tryptophan is also the subject of an urban legend. Suggested myths state that it is concentrated in turkey and that eating large amounts of turkey, i.e., Thanksgiving is what causes post-feast stupor. Other suggestions may say that this stupor is caused from alcohol, a full stomach, and the metabolism working twice as hard to process the unusually large quantity of food that is ingested. Since tryptophan is an essential amino acid that resides in many other foods besides turkey, it is something that we consume on a daily basis. (5) Also, nutritionists do not believe that increasing the concentration of tryptophan within the body will increase the level of serotonin within the brain. It is not a good idea to take an excess amount of synthesized tryptophan to try and enhance your mood. It won’t work and it can be a possible threat to your health. Perhaps someday with further research, we could predict a patient’s genetic vulnerability to depression. This may be possible if we consider how tryptophan acts in the body. Because we know tryptophan has the ability to synthesize niacin which in turn produces serotonin in every individual, maybe we can learn more and help many patients.

 

Absorption

 

This is a graph of the molar extinction coefficient of Tryptophan dissolved in water, 0.1 M phosphate buffer, pH 7. Tryptophan has a molar extinction coefficient of 5,579 M-1cm-1 at 278 nm [G. D. Fasman, Handbook of Biochemistry and Molecular Biology, Proteins, I, 183-203, CRC Press, 3 ed., 1976].


 

 

Fluorescence

 

This is the fluorescence emission spectrum of Tryptophan dissolved in water, 0.1 M phosphate buffer, pH 7. The spectrum was taken by R. A. Fuh on summer, 95 using an excitation wavelength of 270 nm