Toxicology and Applied Pharmacology 438 (2022) 115888

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Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

The effect of nutritional status on the pharmacokinetic profile

of acetaminophen
Vinitha D Souza a, Meghashree Shetty a, Murali Badanthadka a, *, B.S. Mamatha b,
K. Vijayanarayana c
a
Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS), Department of Nitte University Centre for Animal Research and
Experimentation (NUCARE), Paneer campus, Deralakatte, Mangalore 575 018, India.
b
NUCSER, Nittte (Deemed to be University), Paneer Campus, Deralakatte, Mangalore 575 018., India
c
Dept. Of Pharmacy Practice, Manipal college of Pharmaceutical Sciences, Manipal Academy of Higher Education. Madhav Nagar, Manipal 576104, Karnataka, India

A R T I C L E I N F O A B S T R A C T

Editor: Lawrence Lash Nutritional imbalance (low protein / high fat) is a public health problem affecting many people in developing
and developed nations. Such an imbalance will influence pathophysiological homeostasis in individuals and
Keywords: thereby considerably impact drug pharmacokinetics. It was reported that short-term fasting increases acet­
Acetaminophen aminophen exposure in healthy subjects, whereas no effect was observed after a high-fat diet. These findings
Low protein diet
suggest the necessity of considering nutritional status when assessing the risk of acetaminophen-induced hep­
High fat diet
atotoxicity. Additionally, the role of nutrition status on the pharmacokinetic profile of acetaminophen (APAP) at
Pharmacokinetics
Balb/C mice toxic doses is either scanty or not available. With this background, we aimed to compare the effects of nutrition
Nutrition status on the pharmacokinetic profile of APAP at a toxic dose in three different dietary regimens like - Normal
diet (ND), Low protein diet (LPD), and High-fat diet (HFD). Balb/C female mice were divided into three groups
after weaning, and for the next 15 weeks, they were fed with their respective diets (ND, LPD, and HFD). After
that, mice were dosed with APAP (300 mg/kg p.o), and blood sampling was done at different time intervals and
centrifuged at 3000 rpm for 5 min to collect plasma samples. Plasma samples were analyzed using the HPLC
method. Data analysis was done by Non-compartment analysis using Phoenix WinNonlin 8.3 software. LPD group
shows higher values of Cmax, tmax, t1/2, and AUC0–4, AUC0-x values than ND and HFD groups. Both Cmax and AUC
follow the pattern of drug exposure where LPD > ND > HFD. In conclusion, nutrition in the diet alters APAP
pharmacokinetic profile at a toxic dose in three different diet regimes. Further study on CYP450 concentration
and activity is essential to understand the pharmacokinetics difference between these dietary regimens.

1. Introduction hepatotoxicity, where serum APAP concentration is above 150 mg/mL

at 4 h after an acute overdose (Rumack et al., 1981). However,
Acetaminophen (APAP, Paracetamol) is a widely used analgesic and consensus about the threshold of APAP for initiating N-acetylcysteine
antipyretic drug available over the counter to treat acute and chronic treatment and the use of biomarkers to envisage hepatotoxicity is a
pain of varied pathological conditions (Brune et al., 2015). From the question of concern and debate (Koppen et al., 2014; Wong and Grau­
current Covid-19 pandemic condition, its use has been extensive dins, 2017). Further, it was suggested to lower the threshold to 100 mg/
worldwide (Faqihi and Sayed, 2021). However, APAP overdose is the mL at 4 h after an overdose in definite high-risk victims.
most common cause of acute liver injury (Jóźwiak-Bebenista and Both fasting and malnutrition are considered as a risk factors for
Nowak, 2014; Shankar and Mehendale, 2014). N-acetylcysteine is used APAP-induced hepatotoxicity (Price et al., 1987; Kondo et al., 2012;
as an antidote in clinical scenarios to prevent liver damage in such Kalsi et al., 2011). Hepatotoxicity is caused by the parent molecule and
victims(Yoon et al., 2016; Bauerlein et al., 2019). Generally, its metabolite N-acetyl-p-benzoquinone imine (NAPQI) (Bender et al.,
Rumack–Matthew nomogram is used to envisage the menace for 2004). Mainly APAP is eliminated via glucuronidation and sulfation

* Corresponding author at: Department of Nitte University Centre for Animal Research andExperimentation (NUCARE), NGSM Institute of Pharmaceutical Sci­
ences, Paneer campus, Deralakatte, Mangalore 575018, Karnataka, India.
E-mail address: murali@nitte.edu.in (M. Badanthadka).

https://doi.org/10.1016/j.taap.2022.115888
Received 29 July 2021; Received in revised form 19 December 2021; Accepted 14 January 2022
Available online 20 January 2022
0041-008X/© 2022 Published by Elsevier Inc.
V.D. Souza et al. Toxicology and Applied Pharmacology 438 (2022) 115888

process to non-toxic substances. At the same time, CYP2E1 and other libitum until 15th week.All the animal experiments were conducted as
CYP450 isoforms convert a small amount of APAP into the toxic NAPQI, per the guidelines of CPCSEA (Committee for the Purpose of Control and
which is detoxified by conjugation with glutathione (McGill and Supervision of Experiment on Animals) with an approval no: NGSMIPS/
Jaeschke, 2013). Lack of glutathione allows NAPQI to form protein IAEC/MAY-2020/185(for LPD experiment) and NGSMIPS/IAEC/DEC-
adducts on mitochondrial proteins causing mitochondrial oxidative 2020/2021 (for HFD experiment).
stress that leads to hepatic necrosis (Ramachandran and Jaeschke,
2019). In sharp contrast to malnutrition, a short-term high-fat diet in­
duces steatotic alterations of the liver (Hernández et al., 2017;Hydes 2.3. Study design
et al., 2021). The dietary-induced steatotic modifications of the liver
might change hepatic enzyme activity (Murray, 2006; Xu et al., 2012) Female Balb/C mice after weaning (15–18 g) were used to mimic
and, consequently, change exposure to APAP. clinical scenario under different nutritional conditions, getting exposed
The liver is the primary organ regulating the nutrition state and to APAP at reproductive age. To understand the role of nutrition, mice
energy balance (Carreiro et al., 2016). It is also the second-largest organ were divided into three groups (n = 6 in each group) and fed three
and essential for nutrients such as protein, fat, and carbohydrate for different diets as designated: normal diet (ND) (Procured from Amruth
their regulation and metabolism. Liver and nutrition (malnutrition) play feeds, Maharashtra, Pune, India), low protein diet (LPD) (Badanthadka
a prominent role in liver diseases (Owumi et al., 2015). Any consumed et al., 2021), and high-fat diet (HFD-32; CLEA-Japan) (Ganz et al., 2014)
drug primarily enters the liver before it reaches blood circulation. Since and purified drinking water ad libitum. After 15 weeks of study initia­
the liver is one of the body’s vital organs, its main job is to metabolize tion, LPD group develops a stable malnourished model as per the diet
and eliminate drugs from the body (Ramadori et al., 2008). Hence, the standardization (unpublished work). On week 15mice in different diet
liver is more susceptible to chemical-induced liver injury (Naim et al., regimen were administered with a single toxic dose of APAP (300 mg/
2021). Therefore, liver diseases have been a significant problem for the kg, dose-volume 10 mL/kg, p.o.) in saline.
national health care system worldwide. Liver diseases are complex and Pilot study for APAP dose selection: Conducted in ND, LPD, and HFD
influenced by exposure to different factors (Ozougwu, 2017).One of receiving mice. To start with, 500 mg/kg, dose was administered orally
those factors is the diet consumed by the individual (Ramadori et al., (Muhammad-Azam et al., 2019) to LPD receiving mice. Where mortality
2008; Achterbergh et al., 2019). To get a clear-cut idea on this area and (3/4 mice) was observed. Considering this dose is very high, we have
study the role of nutritional status on the pharmacokinetic profile of reduced the doseto 400 mg/kg. Here LPD group did not show any
APAP, we have compared the PK profile of APAP between ND (18% mortality (0/6 mice), but ND group did show mortality (4/6 animals),
protein) and LPD (10% protein) and HFD receiving Balb/C mice. suggesting for dose decrease. Hence, the dose was further reduced to
300 mg/kg, which did not show any mortality (0/6 mice) in ND, LPD,
2. Materials and methodology and HFD groups. Therefore, 300 mg/kg dose was selected for pharma­
cokinetic study as the highest acceptable dose for all three diet regimens.
2.1. Chemicals Nutritional composition of the diets:

Acetaminophen - Gift sample from Ce-Chem Pharmaceuticals Pvt. 2.3.1. Blood sampling
Ltd. 4th phase, #336, 9th Cross Rd, Ganapathy Nagar, Phase 3, Peenya, 0.2 mL of blood was collected through retro-orbital venous plexus
Bengaluru, Karnataka – 560,058.Methanolwas obtained from HIMEDIA using isoflurane anesthesia at selected time intervals - 0, 0.5, 1, 2 & 4 h
(Cat # AS061), and Ethyl acetate was acquired from RANKEM (Cat # post-administration, using EDTA coated tubes. Samples were centri­
LTR/RANK30200). fuged at 3000 rpm for 5 min, plasma was separated and stored at -20 ◦ C
for pharmacokinetic study
2.2. Animals

Female Balb/C mice after weaning (15-18 g) were housed in the 2.4. Pharmacokinetic study
animal house facility at NUCARE, Deralakatte, Mangalore, India. Stan­
dard laboratory conditions were maintained (12 h light/dark cycle; Plasma pharmacokinetics of APAP under the influence of three dietary
temperature 22 ± 2 ◦ C; relative humidity 60 ± 5 ◦ C) with free access to regimes were analyzed by extraction of APAP from plasma samples at 0, 0.5,
their designated diets (ND, LPD & HFD) and purified drinking water ad 1, 2 & 4 h post-administration.

S. No. Ingredients % S. No. Ingredients % S. No. Ingredients %

(ND) (LPD, 10% protein) (HFD)

1 Wheat flour 56.2 1 Normal diet 44.44 1 Milk casein 24.5

2 DCP 1.8 2 Corn oil 2.4152 2 Egg white 5.0
(rock base)
3 Calcite powder 1.0 3 Sucrose 6.038 3 L-cystine 0.430
4 LAF mix 1.0 4 Wheat bran 3.019 4 Powdered beef tallow 15.880
5 Linseed 5.0 5 Vitamin mix 0.6038 5 Safflower oil 20.000
(high oleic acid)
6 Maize gluten 5.0 6 Mineral mix 2.1133 6 Crystalline cellulose 5.500
7 Roasted gram flour 25.0 7 Maize starch 41.3603 7 Maltodextrin 8.250
8 Skimmed milk powder 5.0 8 Lactose 6.928
9 Sucrose 6.750
10 AIN93 vitamin mix 1.400
11 AIN93G mineral mix 5.000
12 Choline bitartrate 0.360
13 Tertiary butylhydroquinone 0.002

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V.D. Souza et al. Toxicology and Applied Pharmacology 438 (2022) 115888

2.4.1. APAP extraction from plasma samples 2.5. Statistics

The liquid-liquid extraction method extracted APAP from plasma
samples (Pingili et al., 2015; Liao et al., 2008). The stored plasma sample Data was presented in the form Mean ± SEM. Statistical difference
was thawed before use. To 50 μl plasma, 1.5 mL ethyl acetate was added, between groups was analyzed using Student’s t-test by Graphpad prism
vortex mixed using Remi rotor for 5 min & centrifuged at 3000 rpm for 8.0.1 software. Statistical significance level was set at p < 0.05.
5 min. The supernatant was separated, and vacuum dried. To the dried
residue, 200 μL of mobile phase was added and vortex mixed. Further, 3. Results
10 μL of residue mobile phase mixture was injected into the HPLC sys­
tem for analysis. Pharmacokinetic study was performed to examine the absorption,
distribution, metabolism and elimination of APAP in plasma samples
2.4.2. Sample analysis using HPLC method from mice receiving ND, LPD and HFD (See Fig. 1). On 15th week of the
Sample analysis was performed as per the literature (Pingili et al., study, all the mice (ND, LPD and HFD) were challenged with APAP
2015) using Waters RP-HPLC system (Model-1525 separation module (300 mg/kg, p.o.).Blood sampling was done at different time intervals
and model 2998, photodiode array detector) and quantified using a C18 under isoflurane anesthesia. Plasma was separated as specified in the
column (Waters SPHERISORB 5 μm, ODS 1, 4.6 *150 mm). Methanol: methodology, and HPLC method was used for sample analysis. Mean
water (60:40, v/v) system was used as mobile phase and filtered through pharmacokinetic parameters are presented in Table 1 and Fig. 2 for easy
a 0.45 μm nylon syringe membrane filter. The injection volume was comparison. The plasma concentration of APAP was higher in LPD fed
10 μL, and the effluent was monitored at 254 nm with a UV detector at a mice as compared to ND-fed control mice (Fig. 2). The LPD group show a
flow rate of 1 mL/min. The retention time of standard APAP was ob­ significant (P < 0.001 & P < 0.05), increase in APAP concentration at 1,
tained at 3.407 min. A similar retention time of APAP was observed in 2, and 4 h respectively. Maximum plasma concentration (Cmax) and
ND, LPD & HFD(3.409, 3.371, and 3.409 min, respectively), peak area respected time (tmax) was found to be higher in LPD group
was recorded. The amount of APAP was quantified based on the UV area (98.17 ± 28.29) & (0.67 ± 0.26), respectively, than in the ND fed control
of the standard curve. group (74.38 ± 18.63) & (0.58 ± 0.20) (Table 1).The mean plasma
concentration-time profile graph shows that LPD has higher drug con­
2.4.3. Calculation of pharmacokinetic parameters centration and a significant decrease in drug distribution and drug
The plasma concentrations versus time data obtained from each clearance compared to ND, which has lower Cmax and higher VZ/F, CL/F.
mouse were analyzed by Non-compartment analysis using Phoenix HFD receiving groups have decreased plasma concentration
WinNonlin 8.3 software. The maximum plasma concentrations (Cmax) (37.141 ± 22.98) compare to ND and HFD groups. Whereas, APAP
and times to achieve maximum plasma concentrations (tmax) were ob­ clearance and volume of distribution is 4 fold higher than LPD and 2 fold
tained directly from the individual plasma concentration-time curves. higher than ND receiving mice. Subsequently, the systemic exposure
Areas under the plasma concentration AUC0–4, AUC0-∞were analyzed. parameters (Cmax & AUC) are less in HFD comparison to ND and LPD
Other parameters such as elimination half-life period of the drug t1/2, the groups.
apparent total body clearance or oral clearance CL/F & volume of dis­ The median values for tmax of APAP in all three diet regimens ranged
tribution VZ/F were further calculated, and data were interpreted. from 0.5 h (HFD & ND) to 0.67 h (LPD), but these differences were not
statistically different (p > 0.05). The plasma concentration of APAP was

Fig. 1. Chromatograms of APAP (300 mg/kg; oral) in ND, LPD and HFD receiving Balb/C mice at different time points (0.5, 1, 2 and 4 h).

A. Plasma sample obtained from normal mice treated with 300 mg/kg of APAP monitored at 254 nm.
B. Plasma sample obtained from malnutrition mice treated with 300 mg/kg of APAP monitored at 254 nm.
C. Plasma sample obtained from high fat diet mice treated with 300 mg/kg of APAP monitored at 254 nm
Control – Normal diet received mice before challenging with APAP 300 mg/kg dose
Standard – APAP 4 μg/mL

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V.D. Souza et al. Toxicology and Applied Pharmacology 438 (2022) 115888

Table 1
Plasma pharmacokinetic parameters of APAP (300 mg/kg; oral) in ND, LPD and HFD receiving Balb/C mice (n = 6).
Sl. # Parametersa Unit ND LPD HFD

1. Cmax μg/mL 74.38 ± 18.63 98.17 ± 28.29 37.141 ± 22.988*

2. AUC0–4 h*μg/mL 77.37 ± 17.26 128.51 ± 38.76* 37.558 ± 28.890*
3. AUC0-∞ h*μg/mL 79.41 ± 16.71 131.59 ± 39.64* 39.328 ± 28.521*
4. tmax H 0.58 ± 0.20 0.67 ± 0.26 0.500 ± 0.000
5. t1/2 H 0.65 ± 0.18 0.69 ± 0.09 0.737 ± 0.687
6. CL/F mL/h 111.90 ± 25.58 62.31 ± 27.48** 238.788 ± 142.825
7. VZ/F L 102.25 ± 23.30 62.44 ± 32.17* 295.662 ± 378.759

* p < 0.05, p < 0.001** when compared to the normal diet grup.
AUC0-∞, area under the drug concentration-time curve from time zero to infinity; AUC0–4, area under the drug concentration-time curve from time zero to the time of
the last measurable concentration; Cmax, maximum plasma concentration; tmax, times to achieve maximum plasma concentrations; t1/2, elimination half-life period of
the drug; CL/F, the apparent total body clearance or oral clearance & VZ/F volume of distribution.
a
Parameters values are expressed as Mean ± SEM.

Fig. 2. Mean plasma concentration-time profiles of APAP (300 mg/kg;

oral) single dose administration in ND, LPD and HFD receiving Balb/C
mice.
Mean plasma concentration–time profiles of APAP following an oral
administration of APAP to mice (n = 6). ( )Normal Diet Control
(APAP 300 mg/kg); ( ) Low protein Diet (APAP 300 mg/kg). ( )
High fat Diet (APAP 300 mg/kg). All values are Mean ± SEM. Bars
represent the standard error. p < 0.05*, p < 0.001** when compared to
the normal diet group.

observed in the proportion of 1:2:3 (HFD: ND: LPD) respectively. This hydrocarbons, and methylxanthines are examples (Anderson, 1988).
data confirms the effect of nutritional status on the PK profile of APAP in Only a few drugs for these enzyme systems have had the effects of such
Balb/C mice. nutritional components are validated (Uchida et al., 2017; Tang et al.,
2018; Li et al., 2018). The effect of food on drug bioavailability, on the
4. Discussion and conclusions other hand, has received more attention and varies according to the drug
type (Toothaker and Welling, 1980).
Diet contents (low protein or high fat) are associated with the vari­ Furthermore, nutrition can have varying effects on drug bioavail­
ations in ADME profile of a molecule. Consequently, drug toxicity may ability, receptor binding, metabolism, and clearance (Maret, 2010;
increase, and the response to treatment may alter. (Murry et al., 1998). Raiten, 2011). Recognizing the role of individual nutrient deficiency on
However, few studies have evaluated the influence of malnutrition, low drug disposition and predictors of altered PK profiles for various drugs in
caloric diet, diet restriction or high fat on the pharmacokinetics of drugs clinics is the question of concern. It is more likely that the in­
using patients (Wessels et al., 1992; Allegaert et al., 2014; Achterbergh terrelationships between nutrition and marketed drugs will remain
et al., 2019). Carrying out such studies in patient populations is very established, necessitating further research. Nutritional effects on drug-
difficult because of the cost, time, availability, etc. Therefore, the pre­ metabolizing enzymes have implications for endogenous substances
sent study aimed to compare the PK profile of APAP in a mice model such as hormones and exogenous chemicals such as pharmaceuticals
under different nutrition conditions (ND, LPD, and HFD) in a separate that are metabolized by the same or related enzymes (Basu and Dick­
set of mice. erson, 1974; Rodríguez-Fragoso et al., 2011).
The human diet is a complex and unpredictable mix of nutrients that Our study demonstrated the PK profile of APAP in three different diet
may alter drug metabolism (Giacaman, 2018). The nutritional effects on patterns - ND, LPD, and HFD. APAP at overdose is known to cause liver
hepatic drug metabolism in humans were emphasized in publications damage. A small amount of APAP is metabolized by the CYP450 system
(Havel, 2005), because these reports are on the influence of starvation to N-acetyl-p-benzoquinoneimine, which can be toxic during this pro­
and dietary contents. However, data obtained from preclinical studies cess. The mechanism of APAP-induced liver damage is well documented
can be difficult to extrapolate to humans at times. It is clear from healthy (McGill and Jaeschke, 2013; El-Bakry et al., 2017). However, research
clinical subjects that specific nutrition can influence drug metabolism on the effect of diet on drug metabolism is limited. Comparison of the
via the mixed-function oxidase system and other conjugating enzymes pharmacokinetic profiles of mice fed with ND, LPD, and HFD after an
(Anderson, 1988; Yang et al., 1992). Healthy protein, cruciferous veg­ APAP challenge shows that initial absorption is rapid in all conditions,
etables, charcoal-grilled beef containing polycyclic aromatic but the extent of absorption is most remarkable in LPD groups compared

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V.D. Souza et al. Toxicology and Applied Pharmacology 438 (2022) 115888

to ND and HFD groups. APAP absorption in the HFD group is the lowest Declaration of Competing Interest
compared to the other two groups. Our findings show that Cmax and AUC
follow drug exposure patterns where LPD > ND > HFD. Previous The authors declare that they have no known competing financial
research indicates that protein-calorie malnutrition reduces CYP450 interests or personal relationships that could have appeared to influence
enzyme activity (Bidlack et al., 1986; Kim et al., 2003) and thereby lacks the work reported in this paper.
enzyme availability for converting parent molecules into active metab­
olites. This low availability of CYP450 enzyme is the likely reason for the Acknowledgements
highest concentration of parent molecule (APAP) in the LPD receiving
group in our study. Conversely, protein malnutrition affects enzyme Authors are thankful to Nitte (Deemed to be University) for the
activity (Mishra et al., 1998) and thereby reduces drug metabolism, financial support.
attributed to reduced or decreased substrate binding to CYP450 enzyme,
thereby drug concentration of APAP found to be higher in LPD receiving
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