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Indole analysis


Tryptophan analysis in feed, food and pharmaceutical samples

Tryptophan (= 3-Indole-alanine) is an essential amino acid. Tryptophan can be metabolized or gradated into a variety of indoles. These indoles can have special (agriculture) functions as e.g. the auxins, which are plant growth regulators.

Tryptophan is detectable as free amino acid, but is also bound in peptides or proteins. Free tryptophan can be detected in vegetable and physiological fluids, but also in feed and food samples. The bound tryptophan in peptides and proteins has to be liberated by alkaline hydrolysis. Acid hydrolysis is not advised, because tryptophan is acid labile. Ansynth Service B.V. is specialized in the analysis of free tryptophan and in the analysis of tryptophan bound in peptides and proteins after alkaline hydrolysis. If in the sequence of a peptide or protein valine, leucine or isoleucine are directly bound to tryptophan, then even after alkaline hydrolysis the dipeptides of tryptophan Val-Trp, Leu-Trp or Ile-Trp will remain. Ansynth Service B.V. can also estimate the dipeptide bound tryptophan by quantitative analysis of these dipeptides.

Tryptophan analysis in physiological samples

Tryptophan as essential amino acid needs to be liberated in the small intestine by protein and peptide digestion. Then tryptophan has to be resorbed in the small intestine. A small part of the tryptophan is converted in the wall of the small intestine into serotonin, which is regulating the peristaltic movement of the intestine. Serotonin leaking from the intestinal wall to the blood is absorbed by blood platelets. After the intestinal resorption tryptophan is transported by the vena portae to the liver. See Figure 1. In blood tryptophan is the only amino acid partly bound to albumin. About 15% of tryptophan is free and about 85% is albumin bound. Free tryptophan is needed for protein biosynthesis. In the liver tryptophan has to be metabolized via the kynurenine pathway into NAD+ (Nicotinamide-Dinucleotide). NAD+ is important for e.g. catabolism of Carbohydrates, Lipids and Proteins by oxidative processes, and is then converted into NADH + H+. The respiratory chain regenerates NAD+ from NADH while producing ATP. In rest, a human being uses about 40 kg of ATP/24 hrs for energy required for e.g. muscle contraction, active ion transport and thermogenesis. Intestinal Trypthophan uptake scheme
After transport of tryptophan over the blood/brain barrier tryptophan is converted into serotonin in the central neural system. Serotonin is a very important neurotransmitter in the central nervous system. The break-down product of serotonin is 5-hydroxy-indoleacetic acid.

Tryptophan not resorbed in the small intestine is partly metabolized by bacteria in the colon. These intestinal metabolites are passively absorbed over the colonic wall and transported directly into the great blood circulation. These metabolites can be metabolized, e.g. in the liver. Finally tryptophan and all tryptophan metabolic and degradation products might be excreted in the urine by the kidney.

Ansynth Service B.V. can estimate free and total tryptophan in blood, and tryptophan, kynurenine pathway metabolites and many indole tryptophan metabolites and degradation products in urine.

Disorders of Tryptophan intestinal resorption, transport and metabolism can cause severe Somatic, Neurological and Behavioural Abnormalities.

  • Intestinal malabsorption of tryptophan can lead to tryptophan deficiency. An excess of tryptophan is then entering the colon, and is metabolized or gradated by colonic bacteria. One important degradation reaction is the conversion of tryptophan into free indole by splitting of the alanine side-chain. Indole absorbed by the colon, will be transported by the great blood circulation to the liver. In the liver indole is sulfonated to indican (= 3-indolesulfonic acid). Indican is excreted in the urine by the kidney. A high excretion of indican in the urine, mostly combined with a low urinary tryptophan, is a symptom of tryptophan malabsorption in the small intestine. See Figure 1.
  • Intestinal tryptophan malabsorption leads to defecation problems, because the serotonin synthesis in the intestinal wall is decreased, which causes less peristaltic movement.
  • Tryptophan deficiency in the liver decreases the production of NAD+ via the kynurenine pathway. The result will be a lower level of NAD+ catalysed oxidation processes and diminished production of ATP. This has e.g. as consequence that intracellular enzyme ATP-ase with cofactors ATP and Mg2+ needed for the Na+/K+-pump and Ca2+/Mg2+-pump is not working properly. These Ion-pumps are very important for the regulation of the osmolarity within the cell and the working of Ion-channels in relation to neurotransmission. A low production level of NAD+ and ATP are responsible for a lot of clinical problems. By measurement of tryptophan, kynurenine pathway metabolites, indican and other indole metabolites in urine, the metabolic consequences of tryptophan deficiency can be indicated.
  • Tryptophan deficiency in blood results also in a lower level of tryptophan transport over the blood/brain barrier. This will decrease the tryptophan availability as precursor for the serotonin biosynthesis in the central nervous system, leading to serotonin neurotransmission problems. The tryptophan analysis of free and bounded tryptophan in blood might indicate a lower tryptophan transport over the blood/brain barrier.

By measurement of amino acids and free + total tryptophan in blood, and amino acids + tryptophan and many metabolites in urine Ansynth Service B.V. can indicate the severity of nutritional deficiencies, and tryptophan transport and metabolism disorders.

Somatic

Anorexia
Glossitis
Constipation or diarrhea
Dermatitis
Pellagra-like skin problems

Neurological

Neurological
Epilepsy
Bulbar palsy
Tremor
Loss of position sense
Spasticity
Exaggerated tendon reflexes
Hyperesthesia
Paresthesia

Behavioural

Apathy
Anxiety
Depression
Obsession- compulsion
Hostility
Hyperirritability
Mania
Memory deficits
Delirium
Organic dementia
Emotional lability
Sleep difficulties
AD or ADHD
Autism like symptoms

N.B. Depending on age, nutritional status or severity of deficiencies, and/or metabolic abnormalities not every symptom needs to be present, or the symptoms differentiates in severity.

Treatment

Based on the amino acid analysis and many years of metabolic disorder research Ansynth can together with a medical specialist define a personalised treatment. Follow up on regular base is essential to keep monitoring the patient and to be able to adjust the therapy whenever needed. It is wishful and necessary that a Family Doctor, Paediatrician, Internist, Neurologist or (Child)-Psychiatrist is consulted before diagnostic laboratory investigations are performed, and to prescribe and control therapy.

Typical Tryptofan metabolites and Kynurenine pathway metabolites that can be analysed by Ansynth:
3-hydroxy-kynurenine5-hydroxy-indole-3-acetic acid
5-hydroxy-tryptamineAnthranillic acid
KynurenineIndole-3-lactic acid
5-hydroxytryptophanacetyl-tryptophan
3-hydroxy-anthranillic acidindole-3-acetic acid
Tryptophanindole
indican (3-indoxyl-sulfate)indole-3-propionic acid
kynurenic acidindoleacryllic acid
xanthurenic acid


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