Keywords 5-hydroxyindoleacetic acid - cerebrospinal fluid - high-performance liquid chromatography
(HPLC) - suicidal
Introduction
The cerebrospinal fluid (CSF) is a natural route by which many transmitter metabolites
leave the brain.[1 ] CSF is a clear fluid formed as an ultrafiltrate of plasma. The spinal and intracranial
compartments both contain CSF. It circulates along CSF channels in the subarachnoid
space of the brain and spinal cord. The choroid plexus continually secretes it consistently
inside the brain's ventricles. The average adult's CSF contains approximately 140 mL
and 500 to 700 mL of CSF is secreted daily, or 0.2 to 0.7 mL per minute. The primary
function of the CSF is to reduce the buoyancy of the brain. Additionally, it provides
nutrients and helps eliminate several substances, including cells, neurotransmitters,
amino acids, and metabolic waste materials.
Several studies have suggested that CSF metabolites, in particular homovanillic acid
and 5-hydroxyindoleacetic acid (5-HIAA), reflect the turnover of their parent amines
in the brain.[2 ]
[3 ] 5-HIAA is the final metabolite of serotonin.[4 ] The CSF compartment, which is anatomically connected to the brain, is a valuable
source for tracking the number of vital biomarkers that are known to have a role in
the pathophysiology of central nervous system illnesses[5 ] and suicidal cases.[6 ] Reversed-phase HPLC, microdialysis, positron emission tomography tracers, etc.,
are widely used methods for analyzing the metabolites in CSF.[7 ] Measurement of serotonin metabolite in CSF is used for the diagnosis of various
neurological and psychiatric illnesses.[8 ] Analysis for CSF from autopsy samples requires unique methods for collection and
analysis. This study involves the development of the ultraviolet high-performance
liquid chromatography (UV-HPLC) method for the estimation of CSF 5-HIAA, along with
the collection and optimization of the sample preparation technique. For the measurement
of ventricular 5-HIAA, we have used HPLC with UV detection using Waters 2489 UV/Visible
detector.
Materials
Autopsy instruments, 10 mL syringe with needle, 5 mL Eppendorf tubes, centrifugation
machine, Acrodisc sterile syringe filter having a pore size of 0.22 µm and 25 mm diameter
(Pall Corporation, Pall Membrane Technology Center. 770 Pennsylvania Drive, Suite
100. Exton, Pennsylvania, United States), compatible freezer (-80oC), 5-hydroxyindole-3-acetic
acid (Merck/sigma), acetonitrile (Sigma-Aldrich, United States), concentrated formic
acid, concentrated acetic acid, methanol, phosphoric acid, ultrafilter (Ultra 370
series RIONS India), electronic balance machine, 1,000 mL measuring cylinder, micropipettes,
HPLC (Waters 2489 UV/Visible detector).
Collection of CSF
CSF was collected by direct visualization method during autopsy. After opening the
skull and removing the dura, under direct vision, the two cerebral hemispheres are
slightly parted using the index and middle fingers. Then a sterile needle with 10 mL
syringe attached to it was inserted into the dependent and posterior part of the brain's
lateral ventricle at around 1.5 cm depth. The needle was directed posteriorly and
downward in each ventricle, and CSF was aspirated ([Fig. 1 ]). Around 4 to 5 mL of CSF was collected from the brain's lateral ventricles and
transferred to the 5 mL Eppendorf tubes. Then the CSF was centrifuged upon collection
at 15,000 rpm for 15 minutes at 4 °C and filtered with a sterile nylon syringe filter
with a pore size 0.22µm. Then CSF sample was stored at −80°C freezer until analysis.
Fig. 1 Collection of cerebrospinal fluid during autopsy after opening the skull.
UV-HPLC Method Development for 5-HIAA
HPLC analysis to detect the 5-HIAA in CSF samples was performed using an HLPC pump
system (Waters 515) fitted with Waters 2489 UV/visible detector and reversed-phase
SunFireTM C-18 column, 5 μm, 4.6 × 25 mm was used. The sample auto-injector used was
fitted with an in-line degasser. The screening of different mobile phases, including
(acetic acid [0.01–0.1%]: methanol in 1:9 ratio), (phosphoric acid [0.01–0.1%]: methanol
in 1:9 ratio) and (formic acid [0.05–0.1%]: acetonitrile in 1:9 ratio), was performed
at flow rate ranging from 0.5 mL/min to 1.5 mL/min. The resolution of 5-HIAA was performed
at three different wavelengths, including 220, 280, and 320 nm. The mobile phase containing
formic acid (0.05%) and acetonitrile in 1:9 at flow rate of 1.0 mL/min determined
at 280 nm was found to be optimum to resolve the peak of 5-HIAA with a running time
of 20 minutes at various dilution of 50, 100, 150, and 200 nm ([Fig. 2 ]); these parameters obtained were further used to analyze all the clinical samples.
The peak from other mobile phases (acetic acid [0.01–0.1%]: methanol in 1:9 ratio),
(phosphoric acid [0.01–0.1%]: methanol in 1:9 ratio) did not yield satisfactory results.
Fig. 2 5-hydroxyindoleacetic acid concentration graph at 100, 150, and 200 nm.
Analysis of CSF 5-Hydroxyindoleacetic Acid by HPLC
CSF samples were removed from the deep freezer (−80°C) and subjected to thawing on
ice (4°C). After thawing, the CSF samples were centrifuged again at 15,000 rpm at
4°C for 15 minutes and filtered using a syringe filter (Pall Corporation, United States);
90 µL of this filtered CSF sample was directly injected into a reversed-phase HPLC
column fitted with UV detector, using an auto-injector.
The standard solution of 100 nm 5-HIAA (Merck, India) was used to calculate the concentration
of 5-HIAA in all the clinical samples. All the runs were performed in triplicates
to check the repeatability of the results. We calculated the CSF 5-HIAA concentration
from HPLC analysis quantitatively by comparing it with the concentration of the standard
sample. A comparison of the area under the curve for the unknown sample (cases) and
that of the known sample was used to determine the amount of CSF 5-HIAA in samples.
Statistical Analysis
All the statistical analysis was performed using R Studio (version 4.2.0). Continuous
data were expressed as mean, standard deviation, or median (interquartile range) depending
on the distribution.
Results
The study was approved by our institute's Institutional Human Ethics Committee vide
LOP letter no 2020/PG/Jan/09. A total of 71 CSF samples were collected for 5-HIAA
assessment from 71 dead bodies after obtaining due consent from the next of kin.
Out of the three mobile phases used for UV-HPLC, the one containing formic acid (0.05%)
and acetonitrile in 1:9 at a flow rate of 1.0 mL/min determined at 280 nm was found
to be optimum to resolve the peak of 5-HIAA with a running time of 20 minutes at various
dilution of 50, 100, 150, and 200 nm. The peak with the standard solution was obtained
at 18.4 minutes at different dilutions mentioned. This mobile phase was then used
for the quantification of the 5-HIAA level from the CSF in all the samples.
Out of the 71 cases studied, the median of 5-HIAA level in CSF was found to be 97.46 nMol/L
with an interquartile range of 80.86 to 114.97 nMol/L. The minimum level of 5-HIAA
in CSF was 12.85 nMol/L, and the maximum level of 5-HIAA in CSF was 527.51 nMol/L.
Discussion
The present work contributes a new and straightforward method for estimating a serotonin
metabolite, that is, 5-HIAA, in CSF samples using UV-HPLC. The normal psychotic function
is disturbed by the fluctuation of neurotransmitters since serotonin plays a significant
part in sustaining ordinary psychosis.[7 ]
[8 ] A change in the concentrations of brain serotonin in disorders like depression,
schizophrenia, panic disorder, and Parkinson's disease is already established,[8 ] indicating the need for developing simple, sensitive, and reliable methods for estimating
serotonin metabolite in CSF samples. We used a sample of CSF to determine the amount
of serotonin since the level of serotonin in blood differs from that in the brain.[9 ] CSF, being in close proximity to the brain and a clear fluid with less protein content,[10 ] is the best-suited biological fluid for the estimation of 5-HIAA by HPLC. However,
the relevant literature on estimating 5- HIAA in biological samples using UV-HPLC
is limited. The CSF neurotransmitter composition is consistent under normal circumstances.
However, the composition, quantity, and pressure can change in a variety of neurological
diseases. Since serotonin degrades quickly to 5-HIAA, this parameter is used to measure
the brain's serotonin levels.[11 ] There are advanced methods like liquid chromatography-mass spectrometry, HPLC with
fluorescence detection,[12 ] HPLC with electrochemical detection, gas chromatography-mass spectrometry, enzyme-linked
immunosorbent assay, and capillary electrophoresis, which can detect these compounds
at lower levels.[13 ] However, it requires expensive equipment, solvents, and extensive sample preparation.
Although these methods are faster and more sensitive than UV-HPLC,[14 ]
[15 ] they require more expensive instruments, preparation, and skill and are only feasible
in high-end laboratories. Although this study did not focus on assessing the sensitivity
of this method, there are studies in the literature with a detection limit of 0.2 mg/L
in 5-HIAA by HPLC in urine.[16 ] In our study, the minimum value of 12.85 nMol/L was detected in one of the samples
with a history of depression, which is comparable.
UV-HPLC is a sensitive method that detects CSF 5-HIAA levels quickly with precision
and accuracy, using inexpensive chemicals and requires significantly less sample preparation.
We used three different sample preparation methods, and the best one was ascertained
based on the accuracy of the result using a standard solution of 5-hydroxyindole-3-acetic
acid (Merck/sigma).
During the analysis of 5-HIAA with HPLC, many problems like baseline drift, poor peak
resolution, ghost peak, and system leak were faced. Contamination in the sample is
also a big problem, which can interfere with the accuracy and specificity of the measurements.
The possible sources of contamination include impurities in the solvents or reagents,
poor cleaning or sterilization of the equipment or containers, cross-contamination
from other samples or sources, and poor sample handling or storage. To avoid contamination-related
issues, high-quality solvents and reagents were used, and the equipment was cleaned
and sterilized by flushing the equipment and containers thoroughly and adequately
and handling the samples carefully. The CSF samples were centrifuged just after collection
and once more before analysis. Centrifugation removes particulate matter such as cells,
debris, and other insoluble material from the sample, which can clog the HPLC column
and affect the accuracy of the results. It also removes large molecules, such as proteins
or lipids, that may interfere with separating the analytes of interest in the HPLC
column.[17 ] This improved the sensitivity and reproducibility of the analysis. To avoid or resolve
problems in UV-HPLC, it is crucial to maintain the system and components, follow proper
operating procedures, and optimize the experimental conditions.
Numerous illnesses exhibit a pronounced decrease in 5-HIAA levels, according to research
that has been undertaken. Decreased CSF serotonin levels have also been associated
with suicidal deaths[6 ] and can be further researched to corroborate suicidal deaths. Serotonin is thus
a possible biomarker for many neurological and psychiatric illnesses, opening more
possibilities for future research into the creation of alternative biomarkers in forensics
and medicine.
Conclusion
A novel, sensitive, precise, and reliable UV-HPLC method for separating and quantifying
5-HIAA in human CSF samples has been developed. CSF is a suitable sample type for
the 5-HIAA measurement by UV-HPLC method and can be used in diagnosing and monitoring
patients with neuroendocrine tumors and various psychiatric diseases. Their use in
this context should be adopted routinely as an alternative to other methods. Commercial
kits and methods other than UV-HPLC for analyzing 5-HIAA require extensive sample
preparation. In contrast, using UV-HPLC, the serotonin metabolites, that is, 5-HIAA,
can be measured in a quick analysis. This method applies to clinical 5-HIAA UV-HPLC
analysis in CSF and provides the benefits of both fast analysis times and low-cost
sample preparation procedures.
