Pharmacological Investigation of Hypoalbuminemia on the Prolonged and Potentiated Action of Midazolam in Rats

Abstract

Midazolam (MDZ) is clinically used for its sedative and anticonvulsant properties. However, its prolonged or potentiated effects are sometimes concerning. The main binding protein of MDZ is albumin, and reduced serum albumin levels could lead to MDZ accumulation, thereby potentiating or prolonging its effects. Previous investigations have not thoroughly examined these phenomena from a behavioral pharmacology standpoint. Consequently, this study aimed to evaluate both the prolonged and potentiated effects of MDZ, as well as the effects of serum albumin levels on the action of MDZ in low-albumin rats. Male Wistar rats were classified into control (20% protein diet), low-protein (5% protein), and non-protein groups (0% protein diet) and were fed the protein-controlled diets for 30 d to obtain low-albumin rats. The locomotor activity and muscle relaxant effects of MDZ were evaluated using the rotarod, grip strength, and open-field tests conducted 10, 60, and 120 min after MDZ administration. Serum albumin levels decreased significantly in the low-protein and non-protein diet groups compared with those in the control group. Compared with the control rats, low-albumin rats demonstrated a significantly shorter time to fall, decreased muscle strength, and a significant decrease in the distance traveled after MDZ administration in the rotarod, grip strength, and open-field tests, respectively. Decreased serum albumin levels potentiated and prolonged the effects of MDZ. Hence, serum albumin level is a critical parameter associated with MDZ administration, which should be monitored, and any side effects related to decreased albumin levels should be investigated.

INTRODUCTION

Midazolam (MDZ) is a benzodiazepine with muscle relaxant properties and is commonly used in intensive and emergency care because of its sedative and anticonvulsant effects.¹⁾ Although the onset time and duration of action are limited, the sedative and muscle relaxant effects are prolonged and enhanced by various factors, including continuous use, hepatic and renal dysfunction, and obesity.^2–5) In clinical practice, prolonged stay in the intensive care unit is often a concern due to the prolonged and enhanced effects of MDZ.

In addition to the factors described previously, we focused on the protein-binding rate of MDZ as a factor related to the prolonged and potentiated action of MDZ. The main binding protein of MDZ is albumin, with a high binding rate of approximately 97%.⁶⁾ At physiological pH, it becomes lipophilic and accumulates in adipose tissue, following redistribution to the central tissues.⁷⁾ Furthermore, MDZ is metabolized via CYP enzymes, yielding the metabolite 1-hydroxymidazolam, which retains roughly half the activity of the parent compound.⁸⁾ Considering these properties, a reduction in serum albumin levels may lead to prolonged and enhanced MDZ action due to an elevated free fraction of MDZ, increased distribution volume leading to adipose tissue accumulation,³⁾ and an enhanced formation of 1-hydroxymidazolam. While there are reports addressing the effects of hypoalbuminemia on MDZ action from a clinical perspective,^9,10) investigations from a behavioral pharmacology perspective remain scarce.

Therefore, this study aimed to investigate the effect of blood albumin on the action of MDZ in rats with low-albumin levels, and a pharmacological behavioral analysis was conducted to evaluate the prolongation and potentiation of MDZ action.

MATERIALS AND METHODS

Animals

Six-week-old male Wistar rats were obtained from Jackson Laboratory Japan, Inc. (Kanagawa, Japan). Two to three rats per cage were housed at 24 ± 2 °C with a 12-h light period (7:00 a.m. to 7:00 p.m.).

Dietary Composition

The dietary composition (Table 1) was based on previous reports, with few modifications.^11,12) The diets were prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan). The basic diet used was AIN93G, modified to a 20% protein diet (control diet), 5% protein diet (low-protein diet), and 0% protein diet (non-protein diet).

Table 1. Dietary Composition of the Rat Feed

	20% protein content (control diet)	5% protein content (low-protein diet)	0% protein content (non-protein diet)
	Mixing ratio (%)	Mixing ratio (%)	Mixing ratio (%)
Casein	20	5	0
L-Cystine	0.3	0.075	0.08
Corn starch	39.7486	47.7236	43.9486
Alpha corn starch	13.2	20.45	29.3
Sucrose	10	10	10
Soya oil	7	7	7
Cellulose powder	5	5	5
AIN93G mineral	3.5	3.5	3.5
N93 vitamin	1	1	1
Choline bitartrate	0.25	0.25	0.25
Butyl hydroquinone-3	0.0014	0.0014	0.0014
Total	100	100	100
Total calories (kcal)	368	368	368

Materials

MDZ used for behavioral analysis was purchased from Sandoz K.K. (Tokyo, Japan). Isoflurane^®, used for liver and kidney extractions, was purchased from Viatris, Inc. (Tokyo, Japan).

Methods

Generating Rats with Low-Albumin Levels

Male Wistar rats were classified into the following three groups: control diet (n = 5), low-protein diet (n = 5), and non-protein diet (n = 6). The protein-controlled diets and water were provided ad libitum for 30 d. Body weight and food intake were measured daily at regular intervals, and the obtained values were averaged for each group.

Blood Sampling and Biochemical Analysis

Blood samples were collected from the caudal vein under isoflurane anesthesia before (day 0) administration and at 10, 20, and 30 d after administration of the protein-controlled diet. The blood samples were centrifuged (1500 rpm, 10 min, 15 °C), and the serum was collected and stored at −80 °C for a maximum of 4 weeks. The serum levels of albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose (GLU), triglycerides (TG), and creatinine (Cre) were determined by the Nagahama Life Science Laboratory of Oriental Yeast Co. (Nagahama, Shiga, Japan). The mean values for each group were calculated.

Behavior and Organ Weight Analysis of Rats with Low-Albumin Levels

Male Wistar rats were classified into the following two groups: control diet (n = 10) and low-protein diet (n = 11), resulting in rats with low-albumin levels. To determine the effect of serum albumin on rats’ locomotion and muscle strength, 1 mL/kg isotonic sodium chloride solution was administered intraperitoneally (i.p.) to both groups on the day of the study. The rotarod, open-field, and grip strength tests were conducted after 10, 60, and 120 min of administering isotonic sodium chloride solution. To determine the hepatic and renal effects of the protein-controlled diet, the liver and kidneys were excised and weighed under isoflurane anesthesia.

Behavioral Analysis after MDZ Administration to Rats with Low-Albumin Levels

To determine whether serum albumin levels affected the potentiation and prolongation of MDZ, both groups were administered MDZ (5 mg/kg, i.p.), and rotarod, grip strength, and open-field tests were conducted 10, 60, and 120 min after MDZ administration. MDZ was administered at 1 mL/kg in isotonic sodium chloride solution on the day of testing.

Rotarod Test

The rotarod test is generally performed to assess coordinated movement and motor skills.^13,14) It can also be used to confirm motor capacity impairment.¹⁵⁾ A rotating rod apparatus (Shinwa Denshi, Ugo Basile, Comerio, ITALY) with a spindle width of 6 cm and a speed range of 2–80 rpm was used at a rotation speed of 10 rpm. For the test, the rats were placed on the rod in the opposite direction of rotation and forced to walk. Exercise performance was determined by measuring the time to fall, and the maximum observation time was 180 s.

Grip Strength Test

Forelimb grip strength was measured using a grip strength-measuring device (GPM-100; Melquest, Toyama, Japan).^16,17) This device operates by holding the rat’s forelimbs on the horizontal bar of a gauge, pulling slowly on the tail, and measuring the tension at the point when the rat releases the forelimb from the horizontal bar.

Open-Field Test

The open-field test is commonly used to evaluate stress, anxiety disorders, and spontaneous locomotion.^18,19) Rats were placed individually in the center of a box-shaped apparatus (100 × 100 cm) separated by a polyvinyl chloride board, and spontaneous locomotion was measured by measuring the distance traveled in 180 s, using an automated video tracking system (Noldus Ethovision^®xt version: 13.0).

Statistical Analyses

Body weight, food intake, and serum albumin levels were analyzed using a one-way ANOVA, followed by multiple comparisons using Dunnett’s test. Results of the rotarod, open-field, and grip strength tests were analyzed using the Mann–Whitney U-test. The significance level was set at p < 0.05, and Stat Mate IV (Atoms, Tokyo, Japan) was used for statistical analysis.

Ethical Approval of the Study Protocol

This experiment was conducted after obtaining approval from the Ethics Committee for Animal Experiments of Ehime University in accordance with the guidelines of the Ethics Review Committee for Animal Experiments (Protocol Number: 05MO35-2).

RESULTS

Generation of Rats with Low-Albumin Levels

Body weight, weight gain, and food intake are shown in Fig. 1. Body weight and weight gain were significantly lower in rats administered the low-protein and non-protein diets than in rats fed the control diet. Food intake was not significantly different between rats fed the low-protein and control diets but was significantly lower in the non-protein diet group than in the control group.

Fig. 1. Effects of Control, Low-Protein, and Non-protein Diets on the Rat Body Weight and Food Intake

(A) Changes in body weight before and after feeding. (B) Weight gain 30 d after feeding. (C) Changes in food intake after feeding. ** p < 0.01 compared with control using one-way ANOVA, and multiple comparison tests were performed using Dunnett’s test. (n = 5–6/group). The values obtained are the mean ± standard error (S.E.).

Figure 2 shows the changes in serum albumin levels and various biochemical parameters on day 30. Serum albumin levels decreased significantly in rats fed the low-protein and non-protein diets compared to those in rats fed the control diet from day 10 and continued to decrease with time.

Fig. 2. Effects of Control, Low-Protein, and Non-protein Diets on Rat Albumin Levels and Biochemical Parameters

(A) Changes in serum albumin levels before and after feeding. (B) Biochemical parameters 30 d after feeding. ** p < 0.01, * p < 0.05 compared with the control using one-way ANOVA; multiple comparison tests were performed using Dunnett’s test. (n = 5–6/group). The obtained values are shown as mean ± standard error (S.E.).

Cre, AST, and ALT levels were not significantly different among the groups. However, TG levels were significantly lower in the low-protein and non-protein diet-fed rats than in the control rats. GLU levels were similar in rats fed the low-protein and control diets but were significantly lower in those fed the non-protein diets.

Behavior and Organ Weight Analysis in Rats with Low-Albumin Levels

A pharmacological behavioral analysis was conducted to confirm the effects of serum albumin levels on spontaneous locomotion and muscle relaxation in rats. The rotarod, grip strength, and open-field test results are shown in Fig. 3. None of the tests showed any significant differences between the rats fed low-protein and control diets. Furthermore, at the end of the experiment, involving 10-week-old rats, those subjected to a low-protein diet exhibited a mean body weight of 71.4% relative to the control group. Furthermore, the comparative weights of the liver and kidneys were observed to be 81.8 and 62.9%, respectively. It is noteworthy that there were no significant differences in the relative enlargement or reduction of liver and kidney sizes due to the low-protein diet. Specifically, the liver-to-body weight ratio was calculated at 3.85 and 4.41%, respectively. Similarly, the kidney-to-body weight ratio was determined to be 0.81 and 0.71%, respectively (Table 2).

Fig. 3. Evaluating the Locomotor Activity and Muscle Strength in Rats with Low-Albumin Levels

Normal saline (NS) was administered intraperitoneally (i.p.) to the control and low-protein diet groups. Rotarod test (A), grip strength test (B), and open-field test (C) were performed at 10, 60, and 120 min. ** p < 0.01, * p < 0.05, Mann–Whitney U-test was used for analysis. (n = 10–11/group). The obtained values are shown as mean ± standard error (S.E.).

Table 2. Percentage of Body, Liver, and Kidney Weights

A
	Control (g)	Low-protein (g)	Low-protein/control (%)
Body weight	402.6 ± 24.2	287.3 ± 15.5	71.4
Liver	15.48 ± 2.1	12.66 ± 1.4	81.8
Kidney	3.24 ± 0.2	2.04 ± 0.2	62.9
B
	Liver/body weight (%)	Kidney/body weight (%)
Control	3.85	0.81
Low-protein	4.41	0.71

Percentage of body, liver, and kidney weights of control rats and those with low-albumin levels (A) and comparison of liver and kidney weight percentages (B). All organs were collected at 10 weeks of age.

Behavioral Analysis after MDZ Administration in Rats with Low-Albumin Levels

To confirm the effects of MDZ on spontaneous locomotion and muscle relaxation in rats with low-albumin levels, a pharmacological behavioral analysis was conducted using the methods above. The results of the rotarod, grip strength, and open-field tests are shown in Fig. 4. In the rotarod test, the fall time was significantly sooner in rats with low-albumin levels at 10 and 60 min after MDZ administration than in the control rats; the fall rate was also significantly higher in rats with low-albumin levels. In the grip strength test, compared to the control rats, those with low-albumin levels demonstrated a decrease in muscle strength at all time points (10, 60, and 120 min) after MDZ administration. Furthermore, the decrease in muscle strength did not improve after 120 min. In the open-field test, compared to the distance traveled by control rats, that traveled by rats with low-albumin levels significantly decreased at 10 and 60 min after MDZ administration and increased at 120 min.

Fig. 4. Effects of Midazolam (MDZ) Administration on Locomotion and Muscle Strength in Rats with Low-Albumin Levels

We administered MDZ (5 mg/kg, i.p.) to control rats and those with low-albumin levels. (A) Rotarod test. The percentage of rats that did not fall in the (B) rotarod test, (C) grip strength test, and (D) open-field test performed at 10, 60, and 120 min. ** p < 0.01, * p < 0.05, Mann–Whitney U-test was used for analysis. (n = 10–11/group). The obtained values are shown as mean ± standard error (S.E.).

DISCUSSION

This study aimed to investigate the effect of serum albumin levels on the activity of MDZ. During the generation of rats with low-albumin levels, body weight, weight gain, and serum albumin levels decreased over time, with a decrease in the dietary protein content. Compared to the control group, the low-protein diet group showed no significant differences in food intake, and only the non-protein diet group showed a decrease. In particular, the non-protein diet decreased body weight compared to that at experiment initiation, confirming that an excessive reduction in protein content causes significant weight loss, which is associated with a decrease in food intake, resulting in excessive undernutrition. However, the low-protein diet did not change the amount of food intake, although there was a significant reduction in body weight in the low-protein group compared with that in the control group. These results confirmed the possibility of generating rat models with low-albumin levels by feeding them diets with adjusted protein content.

The low-albumin and control rats showed no significant differences in Cre, AST, and ALT levels and no significant differences in the liver and kidney weight ratios at the end of the experiment. Extreme malnutrition decreases liver weight and the levels of associated liver tissue proteins.²⁰⁾ In this experiment, the liver and kidney weights of the rats with low-albumin levels decreased at a rate similar to that of body weight loss, suggesting that the effect of a decrease in liver tissue protein content was minimal. No significant changes were observed in Cre, AST, or ALT levels, suggesting that feeding a diet adjusted for protein content had little effect on the liver and kidney function. However, the TG levels were significantly lower in the low-protein and non-protein diet groups than in the control group. This can be attributed to the fact that a diet with reduced protein content lowers very low-density lipoprotein content and reduces TG supply from the liver.^21,22) GLU levels significantly decreased only in the non-protein diet group. Excessive protein restriction decreases insulin production while increasing insulin sensitivity in organs.^12,23) Similar results were obtained in the present study.

The results of feeding diets with varying protein content revealed that feeding a low-protein diet reduced serum albumin levels; however, it was presumed that the total protein levels also reduced along with a decrease in serum albumin, and there were concerns about the accompanying decrease in muscle strength and physical activity. Therefore, we conducted a pharmacological behavioral analysis study on rats with low-albumin levels and confirmed the effects of serum albumin levels on locomotion and muscle strength by conducting rotarod, grip strength, and open-field tests. The findings of these tests were similar between the control and low-albumin diet-fed rats. Thus, a low-protein diet did not affect locomotion or muscle strength, although serum albumin levels and body weight loss were observed.

In the pharmacological behavioral test, MDZ (5 mg/kg, i.p.) was administered in rats with low-albumin levels and the control to determine the effect of serum albumin on the action of MDZ. In the rotarod and open-field tests, compared to the control rats, those with low-albumin levels fell from the rotating bar, and a decrease in locomotion was observed at 10 and 60 min after MDZ administration; this impairment was ameliorated in most of the rats at 120 min after MDZ administration. Moreover, in the grip strength test, which was used to confirm muscle relaxation effects, a significant decrease in muscle strength was observed at all time points (10, 60, and 120 min) after MDZ administration in rats with low-albumin levels compared to those in the control rats; this finding differed from the results of the rotarod and open-field tests. The results demonstrated that the MDZ-induced decrease in locomotor activity and muscle relaxant effects were enhanced and prolonged by a decrease in serum albumin levels. Particularly, the muscle relaxation effect was prolonged, suggesting that it may take time to recover from this effect.

In summary, we confirmed that a decrease in serum albumin levels enhanced and prolonged the effects of MDZ. Hence, serum albumin levels should be monitored during the administration of MDZ, and if albumin levels decrease, the consequent side effects should be noted. In addition, further human clinical studies are needed to corroborate the study findings and establish their clinical applicability.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of Interest

The authors declare no conflict of interest.

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