Press "Enter" to skip to content

Determination of melatonin

Determination of melatonin and related

compounds in plasma samples

Daniel Papp

Abstract Neurodegenerative diseases are becoming some of the most common diseases in the

world, especially in developed counties. Although the causes of these conditions are known, no

efficient treatment has been established; on the other hand, symptoms can be treated and patients’

quality of life can be kept relatively high. This review focuses on melatonin, a form of possible

medication, which is a promising compound for suppressing the symptoms of Alzheimer’s disease

(AD) and sleep disorders and its precursor compounds tryptophan and serotonin, their role in AD

and sleep disorders and their determination in human plasma samples.

Introduction

Research topics related to neurodegenerative

diseases are becoming more and more

widespread in research institutes and

universities. This increasing popularity is easy

to understand if we take a look at the numbers:

according to the World Alzheimer report 2018″,

currently about 50 million people in the world

are affected by Alzheimer’s disease (AD)

1

“,

while approximately 6 million people are

suffering from Parkinson’s disease (PD)

2

“,

making these two the most common

neurodegenerative diseases. Other typical

examples include multiple sclerosis”,

Huntington’s disease and Amyotrophic lateral

sclerosis (ALS). Moreover, prevalence of these

conditions is expected to increase as these

diseases are related to aging and oxidative

stress. Combining this with the fact that in

developed countries, average life expectancy is

becoming higher due to better medical

treatment and higher general quality of life”,

ADI prognoses over 150 million AD-related

patients worldwide

1

. Another problem that

makes these diseases highly feared is that

damage in nerve cells is irreversible, so early

diagnosis of the disease and proper medical

follow-up is crucial in the cases so that quality

of life of the patient can be kept on the same

level by treating the symptoms.

Sleep disorders and Alzheimer’s disease

As stated before, AD is currently the most

common neurodegenerative disease worldwide

with 50 million diagnosed patients. According

to the accepted theory, it is caused by the overly

high level of two proteins at the same time. In

the brain, beta-amyloid (Aβ) protein forms

plaques when it reaches a critical level. These

plaques deposit among neurons and disrupt

communication between them. The other, socalled

tau-protein was also found in high

concentration when analyzing patient samples.

If tau-protein is highly phosphorylated, it forms

aggregates called neurofibrillary tangles

(NFTs), blocking the transport system in the

neuron

1

. Apart from this, there are more

hypotheses about the causes, blaming genetics”,

oxidative stress or inflammation for

development of the disease

1

3

. Obvious

symptoms of AD can be classified in two main

groups, which are cognitive dysfunction and

behavioral problems, however, these bring

difficulties with dealing with daily problems as

well, which can be considered the third group

of symptoms

4

. Treatment of AD focuses on

suppressing these symptoms and slowing the

progression of the disease itself, however”,

promising strategies and drugs appear from

time to time, for example, Cirrito et al. found

that serotonin infusion reduces Aβ levels in

mice, furthermore, SSRI drugs showed similar

effects

5

.

Sleep disorders, commonly known as insomnia

and hypersomnia, among other causes can be

caused by several mental disorders such as AD

or PD, but can occur independently of these as

well. Since no general definitions has been

established, it is difficult to estimate the

number of people affected. In general it can be

stated that factors such as stress, age and the

already mentioned neurodegenerative diseases

can contribute to insufficient amount and/or

bad quality of sleeping

6

. Today, orally dosed

melatonin is routinely used for medication of

insomnia in elderly patients, however, other

age groups with different other disorders have

also been treated efficiently

7

.

Melatonin and precursor compounds

Melatonin (N-[2-(5-methoxy-1H-indol-3yl)ethyl]acetamide)

is

an

endogenous

hormone”,

secreted

by the pineal gland in brain. Its

biosynthesis starts in the pinealocytes using

tryptophan, which is taken up from blood.

Tryptophan is converted to serotonin in a twostep

process.

First”,

Other essay:   Grow your web site traffic with custom internet development

tryptophan

is

hydroxylated

by

tryptophan-5-hydroxylaze to 5hydroxytryptophan

(5HTP), which is

decarboxylized by 5HTP-decarboxylaze”,

resulting serotonin. Melatonin is formed from

serotonin after N-acetylation by serotonin Nacetyltransferase

(serotonin NAT) and

methylation by hydroxyindole-O-methyl

transferase (HIOMT) in a cascade reaction

2

8

. In

human, melatonin is metabolized in the liver by

cytochrome P450 enzymes CYP1A2 and

CYP1A1 to 6-hydroxymelatonin, but it is also

possible that melatonin is demethylated to Nacetylserotonin

(NAS). Hydroxylated

melatonin is subsequently conjugated to yield

6-sulfatoxymelatonin and excreted in urine.

Other metabolic pathways, namely

deacetylation, nonenzymatic hydroxylation

and the so-called kynuramine pathway were

also understood and described

9

.

Figure 1. Biosynthesis of melatonin

Tryptophan, the starting compound of the

biosynthesis is one of the twenty proteinbuilding

amino acids. Since it is only

synthetized in plants and bacteria from

chorismite in several enzyme-catalyzed steps

10

“,

human tryptophan demand must be satisfied by

consuming tryptophan-containing food, such as

egg, soy products and different seeds

11

. Apart

from its significance as protein building block”,

tryptophan serves as precursor for bioactive

compounds niacin

12

and so-called

phytohormones

13

.

The key intermedier of melatonin biosynthesis”,

serotonin or 5-hydroxy-tryptamine (5-HT) is a

neurotransmitter with numerous functions in

human body. Serotonin has both regulatory

functions (for example, via 5HT1

and 5HT2

receptor families) and cognitive functions (via

5HT6

receptors)

14

, thus it is involved in many

psychiatric and neurodegenerative diseases. It

is also worth mentioning that SSRI

antidepressants are considered to be one of the

most successful drugs in pharmaceutical

history compared to other ones against

common diseases

15

.

Use of melatonin and related

compounds in treatment of AD

As it was discussed previously, proportion of

elderly people in the population is increasing

due to different reasons, so AD as a typical agerelated

disease affects more and more people

every year. It was found that tryptophan”,

serotonin and melatonin levels decrease during

aging due to several causes. Melatonin has role

in neutralizing free-radicals and as antioxidant

in itself but also its metabolites along the

AMFK pathway have similar properties. It has

also been described that melatonin can be

useful in hindering the formation of Aβ

plaques. Masilamoni et al. showed that

introduction of melatonin in mice directly

prevents aggregation of Aβ proteins

3

16

. Also”,

synthesis and maturation of amyloid precursor

protein (APP) can be affected by suppressing

cAMP as APP promoter gene has cAMP

responsive regions. Furthermore, melatonin

activates two kinase enzymes that turn off APP

processing via phosphorylation of glycogen

synthase-kinase (GSK3)

17

. These effects help

to slower the progression of the disease, but

melatonin is also used for treatment of sleep

disorders of patients via its effect on the

circadian rhythm, as proved by phase-shifting

experiments and activating melatonin

receptors

7

. Medication usually consists of oral

dosage of melatonin so that plasma

concentration reaches the level of endogenous

melatonin

18

.

Role of tryptophan in AD is understood as

precursor of serotonin and melatonin

consequently administration of only tryptophan

is rarely part of AD protocols, however, studies

show that depletion of it leads to lower

cognitive performance

19

. Another group

studied the connection between tryptophan

depletion and aggression

20

. Regarding sleep

disorders, dosage of 0.25-1 g tryptophan is

reported to improve sleeping quality and other

symptoms of AD have also shown

improvement on tryptophan medication

21

.

Serotonin, or rather indirectly as SSRIs are

primarily used for suppressing behavioral

symptoms of AD. SSRI drugs are widely used

for treating depression since the 1970s with

great success and often used as part of

combined medication in AD patients. In

treatment of depression, reuptake of serotonin

is inhibited by drugs, so that it stays in the

synaptic gap for longer time, thus stimulating

neurotransmission between nerve cells. As a

result, signaling is enhanced and in long term”,

downregulation of post-synaptic receptors

occurs. SSRIs have effect also on cognitive

performance. Rozzini et al. conducted a study”,

which compared AD patients regarding their

reaction for SSRI medication. Tests showed

that cognitive functions of non-treated subject

had worsened compared to SSRI-treated and

non-depressed ones

22

. Other studies show

Other essay:   4 actions you need to take when someone harms your child

enhancement of cognitive performance as

well

23

, however, it was also discovered that

there is a greater risk for quick development of

dementia

24

.

Other medication approved by FDA includes

cholinesterase inhibitors (ChEI) and

memantine

25

, but these are not topic of this

review.

Analysis of melatonin and its

related compounds

Before discussing the different techniques for

analysis of the target compounds, we make

some comments regarding endogenous levels

in normal people and patients. Two out of the

three compounds have more or less constant

concentration in plasma: for tryptophan levels

are reported to be around 74 µM

26

27

, while

serotonin was found to be present in

concentration of 0.35 µM

27

. Compared to these”,

since melatonin release is regulated by light”,

significant difference between day and night

melatonin levels can be found as its

concentration is high during sleep peaking

around 2 a.m. and remains low during the

day

28

. It was also found that plasma melatonin

shows dependence of age, as it decreases

rapidly to the age of 10 and stays relatively

constant during adulthood at around 100 pg/ml

and gets lower in elderly people

4

29

. All three

compounds have decreased concentrations in

AD patients

30

31

, however, for tryptophan and

serotonin the reason might be the normal aging

process instead of the disease.

Tryptophan in itself can be analyzed with

spectrometric methods due to the light

absorption of the aromatic structure, on the

other hand, it is virtually impossible to detect it

without interference in biological matrices. In

most cases, reverse-phase liquid

chromatography (RP-HPLC) methods are used

for separation with fluorescent detector for

higher selectivity. Such methods were

described in the literature from the 1980s.

Anderson et al. determined tryptophan and

serotonin in both whole blood and plasma on

C18 column

32

, mentioning that there are

methods with amperometric detection

existing

33

with lower performance. Kema et al.

described an RP-HPLC-FL method combined

with solid-phase extraction to measure

tryptophan and related indoles in plasma

34

.

However, many research groups use ionexchange

chromatography for separation”,

usually referred to as “Beckman amino acid

analyzer”

35

36

. In this system, amino acids are

separated on cation-exchanger column and are

lead to a reaction coil to react with ninhydrin to

form light-absorbing compounds

37

. Modern

analysis methods include RP-HPLC-MS/MS

with LoQ in ng/ml level

38

39

and UPLCMS/MS

40

.

Plasma serotonin levels can be measured by

several types of immunoassays, however, both

radioimmunoassays

41

42

, and enzyme

immunoassays

43

44

were used mainly in the past

as today they are mostly replaced by

chromatographic methods. Also, fluorometric

detection is possible as reported by Evers et

al.

45

. Fluorescent detection is utilized as HPLC

detection technique today. De Benedetto et al.

developed a RP-HPLC method on C18 column

with LoQ as low as 62 ng/ml for serotonin

46

.

Mefford reported an ion-pairing

chromatography method with amperometric

detection and mentioned more amperometric

methods from the 1970s in his paper

47

.

Electrochemical detection was used by

Wallingford et al., but that method used

capillary zone electrophoresis (CZE) for

separation

48

. Lastly, LC-MS/MS methods

provide the lowest detection limits 5 nM

49

and

UPLC tandem MS methods are in the focus of

method developments. It is worth mentioning

the review of Brand et al., who assessed 101

methods to determine serotonin in platelet-poor

plasma regarding reported mean values and

sample preparation techniques

50

.

Table 1. Summary of methods in the review of Brand et al.

50

Method

Number

of

studies

Mean

(nM)

Std.dev.

HPLC

(electrochemical)

41 33.3 38.3

HPLC

(fluorometric)

27 31.5 44.3

Radioenzymatic

assay

11 8.8 11.7

Immunoassay 9 19.9 17.7

Spectrometric 4 95.1 21.4

GC-MS 1 0.8 –

LC-MS/MS 1 4.6 –

Other 7 37.8 36.1

Techniques used to determine melatonin are

basically similar to those for serotonin.

Immunoassay methods reached fmol/well

detection limits

51

and competition based

ELISA kits with sensitivity of up to 0.162

ng/ml are commercially available

52

53

.

Chromatographic methods are based on LC”,

although GC-ECD method after derivatization

was reported by Greiner

54

. HPLC and UHPLC

Other essay:   Jimbo James

coupled to UV or fluorescent detector is used in

most methods. Talebianpoor et al. compared

several halogen-containing solvents for

dispersive liquid-liquid extraction and used UV

detection at 220 nm. In their optimized method

they use 140 µl carbon-tetrachloride in

acetonitrile, yielding 82% recovery from

spiked plasma sample

55

. 2 ml chloroform was

used for microextraction by Munoz et al. in

2009, however, their system had a fluorometric

detector, resulting in lower necessary sample

volume and lower LLoQ

56

. On the other hand”,

solid-phase extraction was used by

Kulczykowska et al. in 1998

5

57

and Yang et

al.

58

. Several other methods were collected by

de Almeida et al. in a review in 2011

59

.

Since melatonin is in the focus of this review”,

we want to put more emphasis on its analysis

by LC-MS/MS. Zhao et al. determined

melatonin in plasma after liquid-liquid

extraction with internal standard on C18

column in only three minutes. LLoQ of the

method was found to be 0.02 mg/ml and they

achieved a fair, 79-83% recovery

60

. Yang et al.

used a Waters Oasis HLB column in front of

the ESI-MS/MS for sample clean-up

58

.

NanoLC-MS/MS method for quantification of

melatonin and NAS in human plasma was

developed by Carter et al. Their method

included protein precipitation and

centrifugation before separation on RPcolumn.

Although acquisition time was quite

long (38 minutes), they reached 11.65 pg/ml

LoQ for melatonin

61

. In all three studies”,

233→174 m/z transition was used to quantify

the analyte and melatonin-d4 internal standard

was used.

Figure 2. MRM transition of melatonin

In the last part of the review, we want to

summarize those methods that determine all

three compounds together as well as other

methods related to AD biomarkers discovery.

Rao et al. measured tryptophan, serotonin and

melatonin among other analytes in serum

matrix to determine circadian rhythm of those

in schizophrenia. However, in this study

analytes were determined by different

techniques: amino acids including tryptophan

were quantified by ion-exchange

chromatography, serotonin by RP-HPLC and

melatonin by commercially available

radioimmunoassay. Their finding was that all

three compounds have different concentration

at different times of the day in patients

62

. Chin

determined the target analytes along with three

others in rat pineal with 28 minutes acquisition

time. He used RP-IP-HPLC with sodium

octylsulfonate IP-reagent, detection was

carried out by both fluorometric and

electrochemical detectors and found good

agreement with earlier works

63

. In numerous

other communications, two of the three

compounds are determined simultaneously but

at the time of writing this review, no more

papers were found that discusses concurrent

determination of all.

Other biomarkers of AD were found in blood

and cerebrospinal fluid (CSF) as well

64

65

. Li et

al. carried out metabolic profiling of AD

patients from plasma samples with UPLC-

MS/MS and found several differences between

patient and control samples that might lead to

discovery of new biomarkers

6

66

. Sato et al. used

HPLC-APCI-MS to determine biomarkers in

plasma samples. It was found that in AD

patients, desmosterol/cholesterol ratio is

seriously decreased

67

, so it might be a possible

diagnostic target to analyze for. Also proteomic

studies were conducted to find novel

biomarkers, as compared and assessed by

Portelius et al. in 2017. In that review, 28

papers from 2012-2017 period were evaluated”,

including both MS and multi-panel

immunoassay proteomic results

68

.

Conclusion

In this review, the significance of melatonin”,

serotonin and tryptophan was discussed from

the aspect of AD as well as a compilation of

analytical methods was done. However, due to

the fact that AD-related studies are in focus of

many research groups, new communications

with new conclusions are constantly published

and results might overwrite some earlier

findings. Focusing on only these three

compounds, future studies might aim to

determine all the analytes in one short run with

new, rapid methods such as UPLC or SFC”,

which would provide a highly sensitive and

quick way for research and routine sample

analysis.

Be First to Comment

Leave a Reply

Your email address will not be published. Required fields are marked *

0 Shares
Share via
Copy link

Spelling error report

The following text will be sent to our editors: