5HIAA

In contrast, much is known about the catabolism of catecholamines. Adrenaline (epinephrine) released into the plasma to act as a classical hormone and noradrenaline (norepinephrine) from the parasympathetic nerves are substrates for two important enzymes monoamine oxidase (MAO) found in the mitochondria of sympathetic neurones and the more widely distributed catechol-O-methyl transferase (COMT). Noradrenaline (norepinephrine) undergoes re-uptake from the synaptic cleft by high-affrnity transporters and once within the neurone may be stored within vesicles for reuse or subjected to oxidative decarboxylation by MAO. Dopamine and serotonin are also substrates for MAO and are therefore catabolized in a similar fashion to adrenaline (epinephrine) and noradrenaline (norepinephrine), the final products being homo-vanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) respectively. 

As a result of a high index of clinical suspicion and, on occasion, supporting biochemical data from other investigations, one of the first specialist investigations to ascertain whether a patient has an inborn error of biogenic amine metabolism is, as mentioned above, analysis of the CSF concentrations of HVA and 5HIAA. This is often performed in conjunction with the measurement of 3-methyldopa (3-MD), also known as 3-methoxytyrosine. 3-MD is formed from L-dopa via COMT activity and accumulates in conditions where aromatic amino acid decarboxylase activity is impaired. The chemical structures of HVA, 5HIAA and 3-MD are shown in Fig. 6.2.1. 

The chemical properties of HVA, 5HIAA and 3-MD make them amenable to reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Furthermore, the composition of CSF means that little, if any, sample preparation is required prior to analysis. However, the susceptibility of these metabolites to oxidation means that careful sample collection and storage is required in order to minimise analyte degradation. 

Fig. 6.2.1 The chemical structures of homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), and 3-methyldopa (3-MD)...

The stock standards of HVA and 5HIAA and 3-MD are made to a final concentration... 

HVA and 5HIAA using a 100 ml volumetric, dissolve 9.11 mg of HVA and 9.56 mg of 5HIAA in 80 ml of ultra pure H20. Make up to 100 ml using Milli-Q H20 and sonicate for 1 min. Mix thoroughly and aliquot immediately the solid has dissolved. Aliquots of 0.5 ml should then be transferred into labelled Eppen-dorf tubes, frozen rapidly in liquid nitrogen, and stored at -70°C. 

The data in Table 6.2.1 report reference ranges for HVA and 5HIAA when the first... 

Fig. 6.2.2a-bc High-performance liquid chromatography (HPLC) with electrochemical (EC) detection of neurotransmitter metabolites, a standard mixture b cerebrospinal fluid (CSF) sample - control c CSF sample - aromatic amino acid decarboxylase (AADC) deficiency. Peak identification 1 = 5HIAA (7.7 min), 2 = 3-MD (9.6 min), 3 = HVA (11.7 min)... 

Table 6.2.1 Age-related reference ranges for homovanillic acid (HVA) and 5-hydroxindoleacetic acid (5HIAA). Values are expressed as nmol/l. These reference ranges were established in the author s laboratory using the first 0.5 ml of lumbar cerebrospinal fluid (CSF)...

Table 6.2.2 Typical CSF profiles of HVA, 5HIAA and 3-methyldopa (3-MD) for the inborn errors of metabolism associated with a disruption of biogenic amine metabolism. A downward-pointing arrow indicates that a particular metabolite is below the established reference range. An upward pointing arrow is indicative that a metabolite is above the established reference range. WR indicates that the concentration of the metabolite is likely to be within the reference range. AADC Aromatic amino acid decarboxylase, PNPO pyridox(am)ine-5 -phosphate oxidase...

BH4 is an obligatory cofactor for both tyrosine and tryptophan hydroxylase. Consequently, the inborn errors of BH4 metabolism are associated with impaired dopamine and serotonin turnover, which is reflected by decreased concentrations of HVA and 5HIAA in the CSF. Whilst such a pattern is particularly true for the autosomal recessive disorders of BH4 metabolism, an autosomal dominant disorder of BH4 metabolism, (autosomal dominant GTP cyclohydrolase deficiency) is not always associated with marked decreases in the CSF concentration of HVA and 5HIAA [1]. 

To date, a fully documented case of tryptophan hydroxylase deficiency has not been described. However, such a deficiency would be predicted to have a characteristic CSF profile (i.e. an isolated decrease in CSF 5HIAA concentration). 

In addition to the aforementioned inborn errors of metabolism, deficiencies in the catabolism of dopamine and serotonin would also be expected to result in decreased concentrations of HVA and 5HIAA (e.g. MAO deficiency and COMT deficiency). 

Animal group Dopamine ng/gm DOPAC ng/gm 5-HT ng/gm 5HIAA ng/gm DOPAC/DA 5HIAA/HT... 

Figure 6.2. Schematic drawing of the microdialysis probe used to sample the concentration of dopamine, DOPAC, 5HIAA and HVA in the extracellular space in rat brains. The fluid is delivered by a pump and passes via the vertical inlet and the inner cannula down to a 3.0 0.5 mm part of the probe, where a semipermeable membrane surrounds the inner cannula (stippled lines). The dialysis process occurs when the fluid passes between the inner cannula and the dialysis membrane. The perfusion medium flows inside the outer cannula to the horizontal outlet and is collected in 20 min (40 pi)...

Asberg, M., Traskman, L. and Thoren, P. 5HIAA in the CSF a biological chemical suicide predictor. Arch. Gen. Psychiat. 33 (1976) 1193-1197. 

A boy (3.4 kg) was bom by cesarean section, because of fetal distress, to a mother who had taken paroxetine (40 mg/day) and olanzapine (10 mg/day) until 48 hours before delivery (84). After delivery, the baby had increased tone and was hypoglycemic. The next day he was increasingly jittery, with profuse salivation. At 44 hours there was no detectable paroxetine or olanzapine in the neonatal plasma and CSF concentrations of the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) were normal. The baby s condition improved... 

HTOL. Increases in ratios of 5-HTOL to 5HIAA in urine therefore provide a useful marker of alcohol abuse. 

Serotonin (5-hydroxytryptamine, 5HT) is an autacoid synthesized and stored in GI cells and neurons and in platelets. Metabolized by MAO type A, its metabolite 5-hydroxyinolacetic acid (5HIAA) is a marker for carcinoid. 

Serotonin (5HT) is an autocoid synthesized and stored in Gl cells, neurons, and platelets. Monoamine j oxidase (MAO) type A degrades it, forming 5-hydroxyindoleacetic acid (5HIAA), a carcinoid marker. 

Evidence against this serotonin theory includes the fact that low levels of 5HIAA (5-hydroxyindoleacetic acid, a metabolite of serotonin) are seen even in patients who have recovered and in some patients with mania. 

Indoleamine stock solutions 0.1 mg/mL in antioxidant mixture see Section 2 1, Item 8). Prepare separate stock solutions of 5HT, 5HTP, DOPAC, and 5HIAA by dissolving 5 mg of each in 50 mL of filtered antioxidant mixture Stored at 4" C in brown bottles or bottles covered with aluminum foil, the solutions are stable for 2 mo... 

HIAA standards 100,250, and 500 pg/10 pL m Ringer s solution see Note 8) Prepare as for the other indoleamine standard solutions, but pipet 1 mL from the stock solution to make 10 mL of between-stock solution. 

Using the calibration curves, calculate the amount of 5HT and 5HIAA m the dialysis samples (see Note 15). 

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