Respiratory Responses to a Single Oral Dose of Caffeine in Male Sprague–Dawley Rats

Caffeine, renowned for its stimulatory effects on the central nervous system, is a prevalent ingredient in coffee, tea, and many other widely consumed beverages. ¹ The biological influence of caffeine extends beyond the central nervous system, affecting both the cardiovascular and respiratory systems and thereby inducing a variety of physiological changes. ² Upon ingestion, caffeine is swiftly absorbed in the gastrointestinal tract, where its primary molecular action includes interactions with adenosine receptors and the inhibition of phosphodiesterase enzymes. ¹ The molecular action of caffeine increases the ventilatory response of the respiratory system, potentially increasing the efficiency of oxygen and carbon dioxide exchange. ³ Because these effects lead to faster gas exchange and its broad physiological impacts, caffeine exhibits potential for alleviating respiratory symptoms associated with conditions such as chronic obstructive pulmonary disease and asthma, making it a substance of interest. ⁴ Through these biological responses, caffeine stimulates not only the central nervous system but also the respiratory system, highlighting its multifaceted potential for improving human health and performance. ⁵

Caffeine, a stimulant found in various foods and beverages, has been utilized for myriad reasons throughout history, offering a range of both beneficial and adverse effects. ⁶ In the short term, caffeine consumption is known to stimulate physiological and psychological functions, leading to improved mood, increased alertness, and improved cognitive ability. ⁷ The stimulating effect of caffeine is further evidenced by changes in electroencephalography (EEG) readings, where caffeine consumption results in a decrease in both log “theta” and log “alpha” power in brain electrical activity. ⁸ Such changes are indicative of heightened psychological function and are in stark contrast to the effects produced by substances that induce depression or have a sedative impact, ⁹ which typically increase these EEG powers. Despite these positive attributes, it is highly important to recognize the potential for negative outcomes associated with caffeine consumption. When consumed in excessive amounts, caffeine can cause a range of side effects, including but not limited to anxiety, disruptions to normal sleep patterns, and an increase in blood pressure. ¹⁰ These effects highlight the importance of moderating caffeine intake to leverage its benefits while minimizing potential harm, highlighting the complex nature of caffeine as a substance that can influence human health in markedly different ways depending on the context and quantity of consumption. ¹¹

Caffeine undergoes primary metabolism in the liver, resulting in the production of key metabolites such as theophylline, paraxanthine, and theobromine. ^{12 –14} The metabolic processes of caffeine include demethylation into dimethylxanthines and subsequently into monomethylxanthines. ¹ Various studies have shown that the vehicle used to deliver caffeine plays a critical role in its pharmacokinetics, significantly influencing the plasma concentrations of caffeine. ¹⁵ Moreover, the route of caffeine administration is a crucial factor that affects its absorption, distribution, metabolism, and excretion. Compared with oral administration, intravenous administration of caffeine is known to achieve higher systemic concentrations of caffeine more rapidly. However, oral administration facilitates relatively rapid clearance of caffeine from the bloodstream, primarily by increasing its excretion. This detailed understanding underscores the complexity of the interaction of caffeine with the human body, highlighting the importance of considering both the method and the medium of administration to fully grasp its pharmacokinetics and overall effects on metabolism.

In our previous research, which focused on understanding the significance of pharmacological responses for respiratory safety, we investigated the impact of caffeine on respiratory functions. ¹⁶ We administered varying intravenous doses of caffeine (0, 2, 6, and 20 mg/kg) to 6-week-old male Sprague–Dawley rats, and we measured their respiratory rate, tidal volume, and minute volume. ¹⁶ Our findings indicated that caffeine doses exceeding 6 mg/kg resulted in a marked increase in respiratory rate and minute volume, alongside a significant decrease in tidal volume, within 15 to 90 min postadministration. ¹⁶ However, our previous trial predominantly explored the intravenous route of administration, which might not fully represent the effects stemming from oral caffeine consumption, a more common route of exposure. To bridge this gap and explore the respiratory effects of caffeine when orally administered to Sprague–Dawley rats, we conducted a subsequent study. Caffeine was administered orally at doses identical to those used in our previous trial (0, 2, 6, and 20 mg/kg), and we monitored the same respiratory indices: respiratory rate, tidal volume, and minute volumes. Measurements of respiratory indices were taken at intervals of 0, 0.5, 1, 2, 4, and 6 h following oral administration of caffeine.

The experimental procedures were conducted in accordance with the “ICH Harmonized Tripartite Guideline S7A Safety Pharmacology Studies for Human Pharmaceuticals” (November 2000) ¹⁷ and the “Standards for Pharmacological Tests of Pharmaceuticals, Notification No. 2015–83” issued by the Ministry of Food and Drug Safety (November 11, 2015). The Institutional Animal Care and Use Committee of CentralBio Co., Ltd. approved the study (IACUC Approval No. CBIACUC_22-0320SPR1). Five-week-old specific pathogen-free Sprague–Dawley rats were obtained from Samtako Bio Korea (Seongnam, Korea). Upon arrival, the animals underwent a quarantine period and were then acclimated to and maintained in a controlled animal facility. The animal facility consisted of a controlled environment with a regular light cycle (12-h light/12-h dark), air exchange (15 ± 3 exchanges per h), an illumination intensity of 150∼300 lux, a temperature of 22.0 ± 3°C, and a humidity of 50 ± 20%. The experimental rats were fed a radiation-sterilized animal diet (1314 IRR; Altromin Spezialfutter GmbH & Co., Lage, Germany) and had ad libitum access to water that was filtered and sterilized by an ultraviolet sterilizer. ^18,19

The impact of the test substance, caffeine (Sigma–Aldrich, St. Louis, MO, USA), on the respiratory system was assessed. Three solutions of caffeine at varying concentrations—0.2, 0.6, and 2 mg/mL—were prepared in distilled water (Dai Han Pharm, Seoul, Korea) with the same method as previously described. ^16,20 The administration volume was determined to be 10 mL/kg, considering the solubility of caffeine in water and the commonly accepted oral administration volume for rodents. Prior to caffeine administration, the experimental animals were fasted (no food allowed; however, rats had ad libitum access to water) for 12 h to standardize the conditions and reduce the influence of external variables. ^{21 –24} Following caffeine administration, access to food and water was restricted for an additional 6 h to minimize any impacts of external factors on the measurement of respiratory indices. ^16,20 Various respiratory measurements were conducted following a 1-h acclimatization period over three days prior to assessment in a small animal whole-body plethysmography chamber (Buxco Electronics Inc., Wilmington, NC, USA). The average values for respiratory rate, tidal volume, and minute volume over a 10-min period were calculated with a whole-body plethysmography system (Buxco Electronics Inc., Wilmington, NC, USA). ^16,20 On the final day of respiratory measurements, all the animals used in the experiment were euthanized with CO₂ gas.

The data on respiratory parameters were analyzed via SPSS statistical software (IBM, Version 25, Chicago, IL, USA) to assess the homogeneity of variance. One-way ANOVA was conducted under the assumption of normality (P > .05), followed by Levene’s test for equality of variances. Depending on the outcome of Levene’s test, either the Dunnett test (for equal variance, P > .05) or the Dunnett T3 test (for unequal variance, P ≤ .05) was performed as a post hoc analysis to determine statistical significance. In the post hoc analysis, a P value less than .05 was considered statistically significant. ²⁵

The respiratory rate, defined as the frequency of breaths per minute, is an important vital sign indicator. In the groups that received caffeine orally at concentrations of 6 and 20 mg/kg, the respiratory rate at 0.25–1.5 h after administration was significantly higher than that in the vehicle control group (P < .05) or showed a noticeable upward trend when compared to the vehicle control group (Table 1). The respiratory rate significantly increased (P < .05) in both the 6 and 20 mg/kg dose groups at 0.5 h, with the highest dose of 20 mg/kg remaining elevated from 0.5 to 2 h when compared with that in the control group. Regardless of the administered caffeine concentration, the caffeine concentration reached C_max at 0.5 h in all groups that were administered oral caffeine. The respiratory rate then decreased at the subsequent time points.

Tidal volume, which measures the amount of air inhaled or exhaled during a single breath under stable conditions, is determined by converting detected pressure fluctuations during an animal’s respiration into volume changes. From 0.5 to 2 h, the tidal volume was significantly lower in the 20 mg/kg group than in the control group (P < .05; Table 2). Although there was a tendency for the tidal volume to be lower in both the low- and mid-dose groups (2 and 6 mg/kg doses) within the same time frame than in the control group, these differences did not reach statistically significant levels.

Minute volume, which represents the total volume of gas exchanged in the lungs per minute, is calculated by multiplying the respiratory rate by the tidal volume. The minute volume was significantly higher (P < .05) in both the 6 mg/kg and 20 mg/kg groups from 0.5 to 1 and from 0.5 to 2 h, respectively (Table 3), than that in the control group. This was observed regardless of the administered caffeine concentration, with the caffeine concentration reaching C_max in all caffeine-administered groups at 0.5 h. Therefore, the changes in minute volume followed a similar pattern as respiratory rate.

In the field of respiratory pharmacology, the establishment of ICH guideline S7A¹⁷ has led to the division of testing into two main categories: a pumping apparatus test and a gas exchange unit or lung function test. The pumping apparatus test evaluates the amount of gas exchange between the external environment and the airway and is a part of the core battery of pharmacological safety tests, which are generally conducted in accordance with Good Laboratory Practices. The gas exchange unit test, aimed at assessing pulmonary function, confirms efficient gas exchange between the airway and the blood and is classified as a “Follow-up and Supplemental Safety Pharmacology Study.”

In this comparative study, which is an extension of a previous study, we observed significant increases in respiratory rate and minute volume, alongside a notable decrease in tidal volume, following the oral administration of caffeine at doses of 6 (estimated equivalent to ∼400 mg [∼5 cups of coffee] for a 70 kg human) and 20 mg/kg (estimated equivalent to ∼1400 mg [∼18 cups of coffee] for a 70 kg human), within a timeframe of 0.5 to 2 h. Our findings corroborate those from earlier studies, demonstrating that intravenous doses of caffeine exceeding 6 mg/kg result in significant increases in both respiratory rate and minute volume, as well as a significant reduction in tidal volume, within a period ranging from 0.25 to 1.5 h. A distinct difference was observed between the effects of orally and intravenously administered caffeine: the changes in respiratory rate and minute volume induced by orally administered caffeine were sustained for comparatively longer durations. The results from two separate experiments suggest that the oral route of caffeine administration is associated with a slower rate of caffeine excretion than the intravenous route. Our consecutive findings provide objective insights into the mammalian response to caffeine exposure via different administration routes.

Recently, the beverage industry has seen an increase in product lines that include caffeine as a functional ingredient, targeting caffeine inclusion as a key marketing point. In addition to caffeine, taurine has also received significant attention in the beverage market to cater to consumer preferences. While numerous products are available, there has been a lack of in-depth scientific investigations into how exposure to caffeine and/or taurine affects the respiratory system. In our previous study ¹⁶ and current study, we demonstrated that caffeine exposure significantly increased the respiratory rate and minute volume but decreased the tidal volume, both with intravenous and oral administration, within the first h following administration. Taurine has been shown to increase the minute volume at a relatively delayed time point, approximately 6 h. ²⁰ However, experimental trials exploring the effects of co-administering caffeine and taurine on respiratory responses are limited. Therefore, in the future, we aim to explore how the coadministration of caffeine and taurine alters respiratory indices in mammals, with the goal of applying these findings to clinical settings by focusing on safety pharmacology and toxicity.