Clinical trial NCT04690673
Conditions: Drug Mechanism Healthy
Keywords: electrochemical Paracetamol Lipidomics Endocannabinoid system
Sponsor: Johanna Kujala
Start date: 2021-01-15; Completion date: 2021-04-15
Enrollment: 12 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The main objective is to assess whether a novel electrochemical tool is reliable in detecting concentration of paracetamol in fingerprick- , saline-, urine-, and serum samples. We will recruit 12 healthy volunteers aged 18-45. They will get 1 g oral paracetamol. Paracetamol concentration will be detected from abovementioned samples at timely intervals for 24 hours, analyzed with the novel electrochemical method and compared to gold standard mass-spectrometry analysis.

Despite of extensive use, the mechanism of action of parasetamol is not completely understood. The central serotonergic system may play a role. Endocannabinoid system is a group of lipid mediators, that possibly is involved in mediating paracetamol effect to the serotonergic system. Serum lipidomic assessment can be used to study endocannabinoid metabolics. In this study we will try to assess changes in endocannabinoid system by looking into serum lipidomics in order to understand the mechanism of action of paracetamol.

Detailed description:

Arms: arm 1: All healthy volunteers are in this group. They receive 1g paracetamol orally. Saline-, urine-, venous blood and fingerprick samples will be collected at timely intervals.
Interventions: intervention 1: Paracetamol intervention 2: Paracetamol concentration measurements and lipidomic assessment
Intervention descriptions: intervention 1: 1 g oral paractamol intervention 2: Paracetamol concentration is measured from saline-, urine-, venous blood- and fingerprick- samples at timely intervals. Also serum lipidomic assessment is performed from venous blood samples.

Protocol documents:
Prot_SAP_000.pdf: 
This is an English translation of the original research plan in Finnish, dated September 21, 
2020. The translation was made by Johanna Kujala for ClinicalTrials.gov on request in March 
2024. 
 
FEPODPara2020-1 -research 
NCT04690673 
Detection of Paracetamol Concentration in Blood-, Saline- and Urine Samples 
With an Electrochemical Indicator in Healthy Volunteers - a Validation Study for 
a Novel Technique 
Researchers: Eija Kalso (PI), Tuomas Lilius, Janne Backman, Johanna Kujala, Terhi Lohela, Mikko 
Niemi, Elsi Mynttinen, Mikko Neuvonen, Niklas Wester 
 
Backround:  
Paracetamol is the most commonly overdosed drug, and paracetamol concentration 
measurement is a standard laboratory test. The ’gold standard’ for concentration 
determination is Mass-Spectrometry (MS), in our laboratory the High Performance Liquid 
Cromatography – Tandem Mass Spectrometry (HPLC-MS/MS).  
In practice, other laboratory methods than MS are more feasible. Our clinical laboratory 
HUSLAB uses an immunological photometric method. The analysis is carried out in the 
laboratory, which, including the transport of the sample, causes a delay in receiving the 
results. Thus, Aalto University, University of Helsinki and Helsinki and Uusimaa Health 
care district (HUS) are collaborating to develop a fast, portable drug concentration 
measurement tool for patients at the point-of-care in the FEPOD project (Fast 
Electrochemical Point-Of-Care Diagnostics for Opioids and other pain medicine). The 
project has developed a prototype sensor that uses electrochemical analysis to analyse 
opioids and other analgesics. To date, the electrode has been shown to be capable of 
quantitative and selective measurement of certain drug concentrations in vitro. The aim is 
to develop a portable, fast-to-use drug analyzer for point-of-care use. 
Paracetamol is a widely used drug used to treat pain and fever. It causes a significant 
proportion of intentional and unintentional drug poisonings. The therapeutic width of 
parcetamol is narrow, and relatively small overdoses can lead to significant liver failure 
and even death.  
Paracetamol is rapidly absorbed orally and is quickly and evenly distributed in most tissues 
and fluid spaces, where the concentration of the drug can also be measured.  
 
A small fraction of paracetamol is metabolised to highly reactive N-acetyl-p-benzoquinone 
imine (NAPQI), which is normally bound inactive by intracellular glutathione. In case of 
overdose, glutathione is insufficient and reactive NAPQI binds to other proteins in the liver, 
causing cell damage and destruction. As a treatment for overdose, N-acetylcysteine 
infusion is used. According to current therapeutic practice, a nomogram is used to 


determine the initiation of N-acetylcysteine infusion, in which the measured serum 
paracetamol concentration is contrasted to the time elapsed since the time of drug 
administration. In practice, in a suspected overdose situation, the drug infusion is started 
before the drug concentration analysis is completed, so there is a clear clinical need for a 
rapid analysis method.  
 
Paracetamol concentrations can be measured in plasma, saliva, urine and capillary blood 
samples by MS and UV methods (1,2,3). There appears to be a correlation between 
saliva, capillary blood and plasma concentrations, but the results are partly contradictory. 
Capillary blood and saliva samples are of interest due to their non-invasive nature, 
especially in paediatric and elderly patients. The electrochemical analysis method used in 
this study is also interesting because of the speed of the analysis. The analysis will be 
completed in a few minutes. 
 
In addition to determining the concentration of paracetamol, this study aims to investigate 
the effect of paracetamol on serum lipidomics. The mechanism of action of paracetamol, 
despite its abundant use, is unclear. There are several suggestions: cyclo-oxygenase 
blockade, serotonin, opioid, adenosine, catecholamine, nitrate and endocannabinoid-
mediated mechanisms. The site of action is likely to be in the central nervous system, 
especially activation of the descending pain regulation pathway is possible. 
 
The central serotonin-mediated mechanism of action is one of the significant possible 
mechanisms of action of paracetamol. The endocannabinoid system is considered an 
important link between paracetamol and the serotonin system: AM404, the active 
metabolite of paracetamol, may strengthen the cannabinoid system via CB1 receptors, 
which strengthens the bulbospinal serotonergic brainstem inhibitory pain pathway. 
Antinociception would occur at the spinal cord level, where activation-dependent serotonin 
receptors would block the transmission of the pain impulse. In animal studies, inhibition of 
the serotonin descending pathway reduced pain relief induced by paracetamol (4). AM404 
indirectly activated the cannabinoid receptor CB1 (5), and CB1 receptor blockade inhibited 
the analgesic effect of paracetamol in rats (6). 
 
The endocannabinoid system is a group of lipid mediators that regulate ion channel activity 
and neurotransmitter release. Physiological, pathological, or drug-induced changes affect 
the communication of the endocannabinoid system. Targeted lipidomics research can aim 
to understand the metabolism of endocannabinoids.  
 
This study has two objectives: 1) Is the electrochemical sensor developed by the FEPOD 
project reliable for measuring paracetamol concentration in capillary, urine, venous blood 
and saliva samples compared to the result analysed with a Mass Spectrometry from a 
venous blood sample? 2) Does paracetamol affect serum lipidomics?  
 
Course of the study 
 
This is a one-day trial on healthy volunteers. The study will be carried out in the research 
laboratory of the University of Helsinki and HUS Clinical Pharmacology Unit at 
Biomedicum 2 in Helsinki (Tukholmankatu 8C).  
 
Subjects  
 


Test subjects are recruited by means of a recruitment advertisement sent to students of 
higher education institutions and/or universities and/or published on the Internet. 12 
healthy volunteers between the ages of 18 and 45 representing both sexes will be selected 
for the study. Subjects referred to in sections 7 to 10 § of the "Medical Research Act" 
(incapacitated, minor, pregnant, breastfeeding or prisoner) are excluded. Before inclusion 
for the study, the subjects undergo a medical examination, which includes a health 
questionnaire. 
The test subject's weight must be a minimum of 50 kg and the body mass index (BMI) 
must be between 18.5 and 30. Subjects should be healthy and non-smoking and should 
not take any continuous medication, including birth control pills or other hormonal 
contraceptives. During the medical examination, the test subjects are examined for blood 
counts and thrombocytes (B-PVKT), liver function tests (P-ALAT, P-AFOS and P-GT), 
creatinine (P-Krea), electrolytes P-K and P-Na, and a pregnancy test for women (P-hCG-
tot). Haemoglobin should be at least at the lower limit of the reference range. For other 
results, minor deviations from normal estimates, which are considered clinically 
insignificant in the opinion of the study physician, are acceptable. Blood pressure should 
be within normal range. The participants fill in a questionnaire on substance use,  
Participation in other drug trials and blood donation is prohibited 3 months before the start 
of the study, within its period and 3 months after its completion. Subjects should not use 
any medication or dietary supplements for a week before the trial or the day of the trial. 
The consumption of alcohol is prohibited on the day before and during the day of the trial.  
Admission criteria • signed informed consent • age 18-45 years • healthy • acceptable 
values in laboratory tests (determined in HUSLab): haemoglobin at least at the lower limit 
of the normal range (men 134 g/l, women 117 g/l), other results (B-PVKT, P-ALAT, P-
AFOS, P-GT, P-Krea, P-K, P-Na) small deviations from normal values that are clinically 
insignificant according to the investigator's assessment are accepted. In women, the 
pregnancy test (P-hCG-tot) should be negative. • normal blood pressure • no evidence of 
substance abuse in screening 
 
Exclusion criteria • major illness • alcohol consumption exceeding the risk threshold • 
smoking • birth control pills or other regular medication • pregnancy, its planning or 
breastfeeding • less than 3 months after the previous trial • less than 3 months after blood 
donation • veins that are considered difficult to cannulate • weight less than 50 kg, body 
mass index (BMI) less than 18.5 or over 30. 
 
Medication 
 
Subjects receive 1 g of paracetamol orally 
 
Meals 
 
The subjects fast from the evening before the trial day beginning at 24 o'clock. On the day 
of the trial, the subject is offered a standardized lunch after 4 hours and a standardized 
snack 8 hours after the dose of paracetamol.  
 
Samples  
 


On the day of the trial, the test subjects arrive at the examination facilities at about 7.30 in 
the morning.  A cubital vein is cannulated, and a venous blood sample is taken (sample 0), 
followed by repetitious samples at 0,5h; 1h; 1,5h; 2h; 3h; 4h; 6h; 8h; and 12h. If the 
cannula becomes blocked,  repetitive samples are collected by venipuncture. 
Simultaneously with venous sample, a capillary blood sample is collected from the 
fingertip. Saliva samples are collected at 0; 1h; 4h; 8h; and 12 h. The urine that 
accumulates during the day is collected in plastic canisters. A Salivette collection tube is 
used for saliva sampling. On the morning following the day of the examination, the 
subjects visit the laboratory to give the last blood sample from a vein.  
 
 
 
 
 
K2-EDTA tubes are taken protected from light and placed in an ice bath immediately after 
sampling. The plasma is separated from the K2-EDTA samples by centrifugation at +4 °C 
immediately after sampling, divided into four tubes and frozen immediately. The 4 ml K2-
EDTA tube is frozen as a whole blood sample to measure the whole blood content of 
drugs. The urine that accumulates on the study day is collected in plastic canisters. At the 
end of the study day, urine volume is measured and frozen into four 5 ml tubes to measure 
paracetamol concentrations. The lipidomics samples are divided into four tubes and frozen 
immediately at -80°C.  
The reference samples are analysed at the Department of Clinical Pharmacology.  
 
Capillary blood samples are analysed immediately after collection in the study facilities 
with FEPOD-sensor, and duplicate samples are analysed by Mass Spectrometry from 
fingertip Mitra dry blood samples later at the Department of Clinical Pharmacology 
laboratory. Saliva samples are immediately analyzed or frozen for later analysis with Mass 
Spectrometry and electrochemical methods with FEPOD-sensor. 
From frozen whole blood samples, the concentration of paracetamol is determined using 
the FEPOD sensor, and the results are compared with the drug concentrations determined 
in the capillary blood sample.  
Lipidomics samples are used to study changes in lipids. Lipidomics samples are analysed 
together with samples collected from other similar studies. If the samples are analysed 
together with samples from other studies, it may be possible that the statistical power is 
sufficient to demonstrate changes in lipidomics.  
 
 
Tube 
0-
sample 30 min 1h 1,5h 2h 
3h 4h 
6h 
8h 
12h 
      
24 h 
K2- EDTA-tube plasma 4 
ml 
1 
1 
2 
1 
1 
2 
1 
1 
1 
1 
 
K2- EDTA-tube 9 ml for 
lipidomics  
1 
1 
       
1 
Saliva 
X 
X 
X 
X 
X 
 
Capillary for FEPOD 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Mitra dry blood sample for 
MS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
K2-EDTA-tube 4 ml whole 
blood 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 


 
 
Pharmacokinetics 
 
The paracetamol concentrations in plasma, saliva, whole blood and capillary blood are 
determined at different timepoints with FEPOD and MS methods.   
 
Statistical methods 
 
Pharmacokinetic characteristics (e.g. Cmax, tmax, AUC, t1/2) are calculated from 
paracetamol concentrations using standard methods. The pharmacokinetics of 
paracetamol measured with the FEPOD sensor and with Mass Spectrometry are 
compared using variance analysis of repeat measurements, the Bland-Altman method and 
other appropriate statistical methods.  
 
Safety and potential harm   
 
Paracetamol is a widely used drug and a single dose  of 1 g cannot be considered to have 
adverse effects on subjects. 
Study-related venous cannulation or repeated sampling from the vein and fingertip causes 
transient discomfort and pain in subjects. Venous cannulation may be accompanied by 
local inflammation of the cannulated vein (thrombophlebitis). The risk of local or 
bacteremic infection by the cannula can be estimated to be low. Repeated punctures of a 
vein may be accompanied by nerve damage or damage to the vein as a rare side effect. 
This is a rare complication. Blood volume to be collected during the examination is less 
than 200 ml, which is less than a normal blood donation, and cannot be considered to 
pose a health risk to healthy individuals. 
 
Timetable  
 
The experimental part of the study will be carried out during autumn 2020 and spring 2021.  
 
Organization of the study 
 
The study will be conducted at the Clinical Research Unit of the University of Helsinki and 
HUS Clinical Pharmacology Unit at Biomedicum 2 Helsinki (Tukholmankatu 8 C, 00290 
Helsinki). The Clinical Pharmacology Unit has conducted similar pharmacokinetic studies 
for more than 20 years. On test days, investigators, a laboratory nurse responsible for 
sampling and processing and, if necessary, an assistant are present. The equipment 
needed for the study is ready. Researchers and the staff of the laboratory are responsible 
for processing the material and results collected in the study. The research plan is given to 
the staff in writing and its essential content is also explained orally.  
 
 
Mode of release 
 
The results of the research will be published in international scientific journals using expert 
judgement and possibly in a scientific meeting.  
 
Financing   


 
The FEPOD project is funded by Business Finland.  
Lipidomics research is funded by funding from foundations, possibly also with research 
funding from Helsinki University Hospital.  
 
Processing of research data  
 
Researchers are responsible for processing the data collected in the study. A register is 
formed from the collected data. Research data is processed in such a way that the identity 
of study subjects cannot be identified. The data is stored in a locked space for 15 years 
from the publication of the research results or the end of the study. After obtaining the 
necessary permits, a research register will be established.  
If a subject withdraws consent, the data collected about him or her will be processed as 
part of the research data as referred to in the research plan, but no new data will be 
collected. 
Before giving consent, research participants are informed that the data collected by the 
time their consent is withdrawn will be processed as part of the research data.  
Researchers  
Eija Kalso, PhD, Professor of Pain Medicine UH, Principal investigator. 
Responsibilities: study design, participation in conducting research and 
analyzing and reporting results, supervision of research.  
Tuomas Lilius PhD, Chief Physician HY/HUCH Poison Information Centre 
Responsibilities: study planning, participation in conducting the study, 
analysing and reporting the results   
Janne Backman, PhD, Professor, UH Clinical Pharmacology 
Responsibilities: study planning, participation in conducting the study, 
analysing and reporting the results  
Johanna Kujala, M.D., Doctoral Student UH Responsibilities: Participation in 
research planning, participation in conducting research and analysing and 
reporting results 
Mikko Neuvonen, chemist, UH Clinical pharmacology Responsibilities: 
Participation in study planning, participation in conducting the study, analysing 
and reporting the results  
Niklas Wester, Doctoral Student, Aalto University Responsibilities: Research 
planning, participation in research conduct and analysis and reporting of 
results  
Elsi Mynttinen, Doctoral Student, Aalto University Responsibilities: Research 
planning, participation in research conduct, analysis and reporting of results  
Mikko Niemi, Professor at UH Clinical Pharmacology Responsibilities: 
Participation in study design, participation in conducting the study, analysis of 
results and reporting  


Terhi Lohela, MD, Specialist in HUS ATeK Responsibilities: Participation in 
research planning, participation in conducting research and analysing results, 
as well as reporting  
Assessment of the ethics of the research, consent procedure  
The purpose and course of the study are explained to the study participants both orally 
and in writing. After a sufficient period of reflection, the participants sign their consent. 
Research subjects may withdraw from the study at any time, without giving a reason.  
Research participants will be compensated (EUR 170.00 + daily allowance – taxes) for 
participating in this study (detailed grounds for compensation are presented below).  
The research plan is reviewed by a regional ethics committee by decision of TUKIJA 
and notified to the Finnish Medicines Agency FIMEA. Research subjects are covered by 
Pharmaceutical Injury Insurance and Patient Injury Insurance.  
The study will investigate the 1) new electrochemical paracetamol concentration sensor 
reliability, 2) usefulness of the saliva sample in determining paracetamol concentration, 
and 3) the effect of paracetamol administration on serum lipidomics.  
Given the benefits of the research and the fact that the harm caused to research subjects 
is likely to be minimal, we consider the research medically justified and ethically 
acceptable.  
Remuneration for participation in the study 
In accordance with the Finnish Social and Health Ministry (FSHM) Decree, research 
participants are paid EUR 170.00 as reasonable compensation for inconvenience and 
compensation corresponding to loss of earnings (from which tax is withheld according to 
the normal tax rate) for participating in this study, which includes 2 visits and includes one 
day spent entirely in laboratory facilities. The participants are first thoroughly explained the 
purpose and course of the study, after which they are asked to carefully read the written 
notice and consent document received. Only then, after a sufficient period of reflection, do 
the subjects sign a consent document and undergo a medical examination, blood samples 
are taken to confirm their state of health and the results of laboratory tests are clarified 
when they are completed. There is one actual research day. The examination day lasts 
approximately 14 hours and the subjects are not allowed to leave the examination facilities 
during the research day. The morning after the examination day, the test subjects should 
visit the laboratory for a blood test. On the actual day of the examination, the subjects 
have an intravenous cannula inserted into the bend of the elbow throughout the 
examination day, and if the cannula becomes blocked, samples are taken by puncture 
from a vein, if necessary. It should be noted that, in addition to visits to the research 
laboratory, the research limits the normal life of the research subjects. If, regardless of the 
reason, the research subjects are not completed, the research subjects will be paid a 
reasonable share of the total compensation based on the FSHM Decree. 
 
1) Rittau: 2012 Journal of Pharmacy and Pharmacology, 64, pp. 705–711  
2) Smith M et al: A comparison of serum and saliva paracetamol concentrations. Br J Clin 
Pharmacol. 1991 May;31(5):553-5  


3) Mohammed BS et al: Can finger-prick sampling replace venous sampling to determine the 
pharmacokinetic profile of oral paracetamol? BR J Clin Pharmacol 2010  
4) Pini L et al: The antinociceptive action of paracetamol is associated with changes in the 
serotonergic system in the rat brain. European Journal of Pharmacology 308 (1996) 31-40  
5) Mallet C et al: Endocannabinoid and serotonergic systems are needed for acetaminophen-
induced analgesia. Pain 139 (2008) 190-200  
6) Ottani A et al: The analgesic activity of paracetamol is prevented by the blockade of cannabinoid 
CB1 receptors. European Journal of Pharmacology 531 (2006) 280-281