Evaluation of intoxicating effects of liquor products on drunken mice

Abstract

Consumption of alcoholic beverages increases the risk of human health problems such as liver, heart and blood vessel diseases. In the present study, the concept of intoxicating degree (ID) is proposed as an index to demonstrate the degree of intoxicating activity for consuming liquor products. A mice model was designed for the evaluation of liquor product IDs. The intoxicating effects of liquor products were investigated by blood alcohol concentrations (BAC) and behaviour abilities of mice including righting reflex, running and forced-swim abilities. A linear regression model between comprehensive drunkenness degree (CD), calculated by integrating BAC and the behaviour abilities, and alcohol-feeding dosages (W), was established (with R² > 0.9) with a slope factor of K. The ratio of the K values of liquor products to that of purified alcohol could be used to express the ID. For ID values less than 1, the liquor product would have a lower intoxicating effect when the same amount of alcohol content was consumed and vice versa.

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1. Introduction

Alcohol acts as a strong depressant to the human central nervous system. Excessive intake of alcohol may interfere with normal brain activity in the frontal and temporal areas which will temporarily lead to cognitive and body control malfunction.^1,2 Moreover, overconsumption of alcohol can result in impairment of normal error correction or maintenance of appropriate behavior, thus causing difficulty in maintaining a standing position and decreased vigilance reaction.³ Over the past few decades, considerable attention has been focused on the intoxicating effect of liquor products, which caused human drunkenness symptoms.⁴

Liquor products are mainly composed of alcohol and water (98%, v/v, in total) with another 2% component consisting of fusel oil, esters, and organic acids.⁵ The intoxicating effect of a liquor product mostly depends on its alcohol content, which is usually expressed by alcohol by volume (ABV). The hydrophobic properties of alcohol and short-chain aliphatic alcohols can interfere with membrane fluidity by disrupting lipid bilayers, which is the main mechanism of drunkenness symptoms.^6–8 It has been thought that some trace elements generated during the processes of fermentation, aging and storage may have also contributed to the intoxicating effect of the liquor. Thus, the intoxicating effect of the liquor product should be evaluated by integrating the contribution of both alcohol and those trace elements. Traditionally, alcohol degree together with flavor and color are used as key indicators for evaluation of the overall quality of liquor or wine products.^9,10

In the present study, different behavioral assays including the open-field test and the tests of balance on rotary, swimming distance, and loss of righting reflex (LORR) were applied to investigate the intoxicating effects of liquor products on mice. The open-field test reveals the abilities of mice to function in a new environment and monitors changes in their mood.^11,12 The balance-on-rotary test is an efficient test to determine the coordination of the muscles and animal cerebellar function which reflects the coordination performance of mice.^13,14 Typically, mice have to run in the opposite direction of the roller to maintain body balance.¹⁴ As an instinctual behaviour for mice, the swimming distance is directly related to the excitement level of the central nervous system.¹⁵ In the LORR test, mice are assessed for duration of the loss of the righting reflex until righting reflex was regained for mice in V-shaped troughs.^16,17

The ABV is a worldwide standard that popularly appears on the labels of commercial alcoholic beverages. It only implies the amount of alcohol contained in a volume of an alcoholic product but cannot provide any of intoxicating information on human physiological activities. To overcome the inadequacies of using ABV as the sole indicator of the liquor, the levels of the intoxicating effect (intoxicating degree, ID) on the basis of model mice behavioral reactions and their corresponding BACs were investigated. The ID values for four distinct Chinese liquor beverages with similar ABV were determined using the models designed in this work. Our study developed a reliable quality index for the intoxicating effect by considering the integrated contribution of both alcohol and trace elements.

2. Materials and methods

2.1. Animals

SPF (KM) adult male mice, 18–22 g in weight, were housed in top-ventilated cages under standardized conditions with food (standard laboratory diet, Experimental Animal Center in Guangzhou University of Chinese Medicine) and acidified drinking water (pH = 2.5). A regular lighting regime of 9 h of light and 15 h of darkness was used. This study was approved by the Department of Science and Technology, Guangdong Province (Permission License No. SYXK-Yue, 2013-0127), and all the animal experiments were performed in accordance with Guangdong Provincial Regulations for the Administration of Affairs Concerning Experimental Animals (issued on October 1, 2010) during the light–dark cycle between 9:00 am and 6:00 pm.

2.2. Alcohol sources

The alcohol used for the preparation of the drinking solutions was obtained from Aladdin (Shanghai, China; chromatographically pure). Commercial liquor products were purchased from the local market (Guangzhou, China) and included products from four different brands: Shiwan Jade Ice (29%, ABV), Jiujiang Double Distilled (29.5%, ABV), Jiujiang Twelve Square (33.8%, ABV), and Shunde Red Liquor (30%, ABV).

2.3. Modeling animals

The mice were randomly divided into two groups: commercial liquor treatment or purified alcohol treatment (n = 6, for each group). Animals in the commercial liquor group were treated with liquor products at different dosages (0.356, 0.435, 0.514, 0.593, 0.672, and 0.751 g of alcohol per 100 g body weight) through an infusion method using 1 mL intragastric syringes. The dosage of 0.356 g alcohol per 100 g body weight was estimated as the point of tolerance for inducement of alcohol impairment causing slight drunkenness in mice, while 0.751 g alcohol per 100 g body weight was estimated as causing complete drunkenness in mice. Mice from the alcohol treatment group were fed with an alcohol solution to achieve an equivalent alcohol intake as the liquor group. Alcohol feeding which preceded the drunk behavioral testing was conducted only once to each mouse.

2.4. Analysis of BAC and behavioral reactions

The BAC and behavioral reactions of each mouse were examined prior to and after the feeding treatment of alcohol sources. Two different time points for BAC tests, i.e. at 0.5 h and 2 h, were selected as representing the rapidly increasing and slowly increasing intoxication states, respectively. All the measurements were performed in triplicate. The data were reported as the average value ± standard deviation.

2.5. Tests of behavioral reactions

The behaviour of each mouse was assessed using separate groups of mice that received or did not receive alcohol. The results from each of the behavioral tests, including the LORR (R_1t), rotary running (R_2t), and forced-swim (R_3t) abilities, were selected as indicators for the evaluation of the liquor intoxicating effect.^18–20

2.6. Test of LORR

For the test of LORR, mice were set initially lying on their backs. The time required for right restoration was defined as the duration of the reaction to move from a lying posture to full restoration of an upright position. The time was measured before (N_1t0) and after (N_1t) receiving alcohol. The R_1t was calculated using eqn (1):where t_m is defined as the longest righting restoration time for the mice after receiving alcohol.

2.7. Test of running abilities

For the test of running abilities, mice were first placed onto a rotating shaft (15 cm in diameter). Their running distance per min before (N_2t0) and after (N_2t) receiving alcohol was determined. R_2t was calculated using eqn (2):

For the forced-swim test, the swimming distance of the mice before (N_3t0) and after (N_3t) receiving alcohol was measured in an annular maze (550 mm × 350 mm × 200 mm) containing water. R_3t was calculated using eqn (3):

2.8. BAC assays

Blood samples (100 μL from each mouse) were collected from the tail of the mice and transferred into 1.5 mL tubes pre-coated with sodium heparin (Grade I-A, ≥180 USP units per mg, Sigma-Aldrich Corporation, St. Louis, Missouri, USA) immediately. Then, each of the blood samples was added with 500 μL of acetonitrile, 340 μL of water and 50 μL of internal standard. The mixture was centrifuged at 12 000 revolutions per minute for 5 minutes (TGL-16H, Heima Med-Equipment Co., Ltd., Zhuhai, China). For the measurement of BAC, the suspension was filtered with a 0.22 μm Nylon membrane (Sterlitech, Kent, Washington, USA) and then loaded onto a gas chromatography (GC) analysis system as described by previous studies.^21,22

Briefly, a Shimadzu GC system (2014 C, Shimadzu Corporation, Tokyo, Japan) equipped with a GC column (30 m × 0.25 mm × 0.25 μm, WondaCap WAX, Shimadzu Corporation, Tokyo, Japan) and a flame ionization detector (FID, Shimadzu Corporation, Tokyo, Japan) was employed to perform GC analysis. The temperatures of both the inlet and the detector were maintained at 250 °C. The program of the column temperature was 48 °C at the start, then it was increased to 55 °C at a rate of 3 °C min⁻¹, and then it was increased to 200 °C at a rate of 30 °C min⁻¹; this was followed by incubation at 200 °C for 2 minutes. Nitrogen was used as carrier gas with a flow rate of 1.43 mL through the column. The split ratio of the injector was 10.^23,24

2.9. Statistical analysis

The calculated behavioral indicators and BAC were reported as mean ± standard deviation (SD). The significance of the differences among alcohol treatment levels was assessed by using a 2-sided covariance (ANCOVA) method. The correlation between alcohol source levels and behavioral indicators was analyzed by linear regression. All analyses were performed using the IBM SPSS Statistics software (v19.0, IBM Analytics, New York, USA). Through the Kruskal–Wallis tests, p values less than 0.05 were considered statistically significant.

3. Result and discussion

3.1. Intoxicating effect of liquor products on mice

BAC is commonly used as a metric of alcohol intoxication for legal or medical purposes. It was reported that there was a progressive effect of BAC on human behaviour and impairments.²⁵ The BAC exhibits a good correlation with the degree of drunkenness.²⁶ In this study, BAC was used as a standard reference for reflection of the intoxicating effect.

The BAC of mice increased rapidly after receiving alcohol sources at the first 0.5 h. In the following 0.5 h to 2 h, the increase rate of BAC slowed down and finally reached the maximal value. After 2 h, BAC began to decline gradually and returned to the near normal level after the 6th hour (Fig. 1). Evaluation of the behavioral indicators of mice was conducted 0–2 h after feeding alcohol sources based on this system.

Fig. 1 Change in the BAC after feeding with 29.5% alcohol solution.

The BAC of the rapidly increasing and slowly increasing intoxication states was determined at two different time points, 0.5 h (t₁) and 2 h (t₂). The BAC of the mice group treated with liquor (C_tL) or the equivalent amount of alcohol (C_ta) was measured at each time point. The ratio of C_tL/C_ta is defined as the blood alcohol drunkenness degree (AD_t) indicated by BAC in eqn (4).

where t is the time point of testing.

The average intoxicating effect indicated by BAC (AD) is calculated using eqn (5):

where t₁ and t₂ are the time points of the rapidly increasing intoxication state and slowly increasing intoxication state, 0.5 h and 2 h after treatment, respectively.

In this study, ADs of the feeding dosages were calculated for the four liquor products. As reported in Fig. 2A–E, they are 0.9465 to 1.9424 for Shiwan Jade Ice, 0.8250 to 1.9840 for Jiujiang Double Distilled, 0.6520 to 1.4469 for Jiujiang Twelve Square, 0.5360 to 1.7867 for Shunde Red Rice Wine, and 0.6861 to 1.8557 for the equivalent alcohol solutions. These results indicated a variation of effects on blood alcohol accumulation among the commercial liquor products even with similar ABV.

Fig. 2 Behavioral indicators of mice after receiving different alcohol sources: (A) Alcohol solution (29.5%, ABV), (B) Shiwan Jade Ice (29.0%, ABV), (C) Jiujiang Double Distilled, (29.5%, ABV), (D) Jiujiang Twelve Squared, (33.8%, ABV), and (E) Shunde Red Liquor, (30.0%, ABV) with doses of (I) 0.356, (II) 0.435, (III) 0.514, (IV) 0.593, (V) 0.672, and (VI) 0.751. Raw data and results of the significance tests are available in Table S1.†

3.2. Effect of liquor products on the behaviour of mice

Three mouse behavioral indicators were tested before and after alcohol source treatment. Results of the righting reflex test suggested that mice could rapidly response to correction if their BAC value is lower than 5000 mg L⁻¹ (Fig. 3A). The correlations between BAC at various time points (C_t) and the other two behavioral indicators, i.e. rotary running and forced-swim abilities, were analyzed using linear regression models (Fig. 3B and C), respectively. The correlation between the BAC and two behavioral indicators was calculated using eqn (6):

C_t = −2682.5 × R_2t − 2681.0 × R_3t + 4656.6 (6)

Fig. 3 Regression of BAC against indicators of behavioral reactions (A: LORR; B: rotary running; C: forced-swim).

Afterwards, eqn (6) can be rearranged as eqn (7):

C_t = −706.9 + 5363.5 × [1 − (0.5 × R_2t + 0.5 × R_3t)] (7)

The running and forced-swim abilities were equally weighted by a comprehensive factor S_t, as calculated using eqn (8):

S_t = 1 − (0.5 × R_2t + 0.5 × R_3t) (0 ≤ S_t ≤ 1) (8)

Thus, the equation can be written as eqn (9):

C_t = −706.9 + 5363.5 × S_t(0 ≤ C_t ≤ 4656.6) (9)

The BAC of 4656.6 mg L⁻¹ was a critical level for determining whether the mice were totally drunk or not. The ratio of the comprehensive behavioral indicators after receiving liquor (S_tL) to the standard-dosage alcohol solution (S_ta) was defined as the behavioral drunkenness degree (BD) in eqn (10):

where t is the time of the behavioral test after feeding of the alcohol source.

The average BD at t₁ (30 min) and t₂ (120 min) was calculated using eqn (11) (when C_t1 ≤ 4656.6 and C_t2 ≤ 4656.6):

when the BAC was higher than 4656.6 mg L⁻¹, the behaviour abilities were completely lost. BD_t was calculated using eqn (12) or eqn (13):

BD = BD_t1(when C_t1 ≤ 4656.6 but C_t2 ≥ 4656.6) (12)

BD = BD_t2(when C_t2 ≤ 4656.6 but C_t1 ≥ 4656.6) (13)

The BD values for the four liquor products are shown in Fig. 2A–E. Although equal liquor alcohol dosages were administered, these products caused varied BD. In contrast to the BD of the pure alcohol solution, which ranged from 0.7316 to 1.4149 for the dosage of 0.356 to 0.672 g per 100 g body weight, the BD for Shiwan Jade Ice (29%, ABV) and Shunde Red Liquor (30%, ABV) ranged between 0.7243 and 1.5538 and between 0.4036 and 1.4286 after treatment with the same liquor alcohol dosage (0.356 g to 0.593 g per 100 g mice body weight), respectively. For the samples of Jiujiang Double Distilled (29.5% ABV) and Jiujiang Twelve Square (33.8% ABV), the BD ranged from 0.1385 to 1.2681 and 0.3522 to 1.0262 with the dosage of 0.356 to 0.751 g per 100 g mice body weight, respectively.

From the viewpoint of the BD study, variations of the liquor qualities were demonstrated. As AD described above was used to estimate the intoxicating effect of a certain liquor product on blood alcohol accumulation by the BAC indicator, BD assessed a certain amount of liquor consumption with respect to the resulting consequent impacts on the intensive physiological reactivity by mouse behaviour indicators. The two indicators assessed intoxicating intensity but with different constructs. As shown in Fig. 4, a relatively high AD value is not always in correspondence with a high BD value for a certain liquor product.

Fig. 4 Variation of the indicators of mice after receiving different alcohol sources with a dose of 0.593 mg per 100 g body weight. AD: blood alcohol drunkenness degree, BD: behavioural drunkenness degree, and CD: comprehensive drunkenness degree.

3.3. Prediction of the intoxicating effect by linear regression

For a comprehensive estimation of the intoxicating effect by considering the contributions from both AD and BD, a comprehensive drunkenness degree (CD) was proposed and calculated as the arithmetic mean value of AD and BD in eqn (14) (when C_t1 ≤ 4656.6 or C_t2 ≤ 4656.6):

As mentioned above, the BD cannot be used as an indicator of behavioral drunkenness degree when C_t1 ≥ 4656.6 and C_t2 ≥ 4656.6. However, there was a significant linear regression relationship between C_t and R_t. For this condition, the CD should be calculated as follows:

CD = AD (when C_t1 ≥ 4656.6 but C_t2 ≥ 4656.6).

The linear regressions of feeding alcohol dosages (W) and CD for the four commercial liquors and pure alcohol solutions are shown in Fig. 5. ID is defined as the ratio of K_L to K_a and is calculated using eqn (15):

where K_L and K_a are the correlation coefficients reflecting the intoxicating effect on mice with the same alcohol dosage of liquor and alcohol solution, respectively.

Fig. 5 Linear regression of CD against feeding dosage of alcohol sources.

The higher the K value is, the more easily the drunkenness occurs. The K_a for pure alcohol solutions was calculated as 2.2237, the K_L values of the four tested liquor products were 2.7063, 1.9165, 1.5883, and 2.2427 (Fig. 5), and the calculated IDs of the four liquor products were 1.2170, 0.8619, 0.7143 and 1.0085, respectively, for Shiwan Jade Ice (29%, ABV), Jiujiang Double Distilled (29.5%, ABV), Jiujiang Twelve Square (33.8%, ABV), and Shunde Red Liquor (30%, ABV).

The distinct KL and ID values for the liquor products reveal that consuming products with similar ABV may still lead to different physiological reaction levels. Jiujiang Double Distilled (29.5%, ABV) and Jiujiang Twelve Square (33.8%, ABV) showed lower ID values and Shiwan Jade Ice (29%, ABV) and Shunde Red Liquor (30%, ABV) showed higher IDs. The variation of the ID values evidenced remarkable differences in the qualities of commercial liquor products with similar ABV. It would be possible to have the ID indicator introduced to the market for purposes of quality control or even guidance on the trend of liquor consumption.

3.4. Reflection of the quality and intoxicating effect of the liquor product by intoxicating degree

The increasing effects of alcohol intake on the health or behaviors of mice had been extensively reported by previous studies.^27–29 Based on this, the intoxicating effects of alcohol sources were compared with those of the model mice and the supposed ID as a metric of the intoxicating effect of the liquor product. An experimental method for determining the ID of liquor products based on the BAC and behaviour reactions of mice was developed in this study. The relationship between BAC and mouse behavioral reactions was investigated using an indicator of CD as the comprehensive drunkenness degree. A linear regression between CD and feeding alcohol dosage (W) was obtained. This regression enabled the calculation of ID values. Six dosages starting from slight drunkenness dosage to deep drunkenness dosage were used to investigate the general responses of mice after different alcohol dosage treatments. Comparing with single dosage, six-level points better reflects the relationship between CD and W with reduced experimental errors. Additionally, each experimental animal was used once to avoid the complications of alcohol tolerance.^30,31

Heavy drinking is a significant health hazard.³² Liquor can increase the risk for liver, brain and cardiovascular diseases leading to functional damage.^33,34 The intoxicating effect of a liquor product does depend on its ABV level and the composition of trace elements.³⁵ The trace elements, such as esters and organic acids, may interact with other components in the liquor to affect the intoxicating degree and liquor quality. Different distillation liquor products were applied to examine the influence of these trace elements on the ID of the liquor. Liquor blending with different supplements could affect the product quality and the ID indicator. For ID values less than 1, the liquor product would have a lower intoxicating effect when the same ABV was consumed and vice versa. Among the four liquor products tested in the present study, Jiujiang Double Distilled (29.5% ABV) and Jiujiang Twelve Square (33.8% ABV) exhibited lower ID values. These results suggested that the trace elements played an important role in reducing the intoxicating effects of the liquor products. It is reported that some trace elements could activate alcohol dehydrogenase (ADH) in liver and accelerate the metabolism of alcohol, which resulted in the reduction of the drunkenness degree in mice.^36,37 The mechanism of intoxicating effects of trace elements still remains unclear.

The different intoxicating effects were compared by the ID metric which served as a guide for distilleries to improve their production process and the quality of their liquor products. Additionally, the liquor product is popularly labelled by ABV. However, the use of ABV as a standard for the determination of the intoxicating impact induced by ethanol is inadequate as even if consuming the same amount of the liquor with similar ABV degree may lead to distinct symptom intensities of drunkenness. There was no such marking index that can better guide people to avoid drunkenness than by controlling the liquor intake within low or moderate doses. The index of ID supposed in this work could also be used as part of the required labelling indicating the intoxicating degrees of distinct liquor products, which might help counter drunkenness problems caused by excessive alcohol intake. Liquors with a lower ID (equivalent amounts of alcohol) may cause a lower-level drunkenness reaction.

4. Conclusion

This study provided insights and new data for the evaluation of the effects of alcohol consumption on proper behaviour and organ function and increased the awareness of the health risks associated with moderate and heavy drinking. A linear regression model between comprehensive drunkenness degree, calculated by integrating BAC and the behaviour abilities, and alcohol-feeding dosages was established (with R² > 0.9) with a slope factor of K. The ratio of the K values of liquor products to that of purified alcohol could be used to express the intoxicating degree (ID). The ID was applied in the investigation of four commercial liquor products from the market. All the products showed a distinct intoxicating effect even with similar ABV. It is prospective that the ID could be used to clearly identify the quality and intoxicating effect of liquor products in the market in the near future.

Acknowledgements

The authors acknowledge financial support from the key project on the Collaborative Innovation of Industry, Education and Research of Guangzhou, China (201508010012). The authors also give special thanks to Dr. Wenzhen Liao, University of Saskatchewan, for his kind comments that greatly improved the manuscript.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6md00491a

‡ The authors declare no competing interests.

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