Isolation, Purification and Quantitative Analysis of Coenzyme Q 10 from Fermentation Broths

 Coenzyme Q10, also known as ubiquinone and decenoquinone, is a class of fat-soluble quinone compounds, with the chemical name of 2,3-dimethoxy 5-methyl 6-decyloisopentenyl benzoquinone.  Coenzyme Q10 is yellow or orange crystalline powder, odorless and tasteless, insoluble in water, soluble in chloroform, benzene, acetone or petroleum ether, melting point 48 . 0 ~ 50 . 0 °C, relative molecular mass 863.4. 4; in plants and animals, microorganisms and other cell bodies and mitochondrial inner membrane conjugation, is an important organism in the respiratory chain hydrogen delivery body.

 

As a multifunctional biochemical drug, coenzyme Q 10 has a wide range of clinical applications [1, 2]; coenzyme Q10 can be produced by animal and plant tissue extraction, chemical synthesis, and microbial fermentation; compared with other methods of preparation, microbial fermentation has the features of low raw material cost, easy control and large-scale production, which can meet the clinical needs of this drug.

 

Coenzyme Q 10 is an intracellular product of microbial fermentation; therefore, the fermentation product Coenzyme Q 10 must be isolated and purified. High performance liquid chromatography (HPLC) is a demanding, expensive and time-consuming analytical method. Therefore, a simple, accurate and relatively rapid method for the determination of coenzyme Q10 in fermentation broth is required. The separation and determination of coenzyme Q10 in the fermentation broth were carried out by organic solvent extraction, thin-layer chromatography and ultraviolet spectrophotometry, which can be used as a reference for further large-scale fermentation production control and compositional analysis.

 

1 Materials and Methods

1 . 1 Instruments

Chromatography silicon GF254-plate (10 cm × 20 cm); EZ585Q Freeze Dryer: manufactured by FT's systems Inc; High-speed Centrifuge; Ultrasonic Cell Breaker; TU-1800 Automatic Scanning Visible Ultraviolet Spectrophotometer: manufactured by Beijing Pudian General Instrument Company Limited; High Performance Liquid Chromatograph: shimadzu model LC-6AD liquid chromatograph. High Performance Liquid Chromatograph: shimadzu model LC- 6AD .

 

1 . 2 Main reagents

Anhydrous ethanol, methanol, acetone, petroleum ether, chloroform, benzene, ethyl ether, hexane, iso-octane, isopropanol, etc. are analytically pure.

 

1 . 3 Samples

Imported Coenzyme Q10 standard: product of siGma, a gift from Zhoushan Hailixiang Pharmaceutical Co.

 

1 . 4 Preparation of Coenzyme Q 10

Preparation procedure: Fermentation broth (prepared in the laboratory, strain Rhizobi- um radiobacter WsH2061) → centrifugation at 6,500 r/min for 15 min → freeze-drying → extraction with stirring of organic solvents → centrifugation to remove the bacterial body → evaporation and drying under pressure → residue extraction with a small amount of petroleum ether, concentration under reduced pressure of the collected petroleum ether layer and then washed with water → dried with anhydrous sodium sulfate → dissolution with a fixed amount of anhydrous ethanol → thin layer chromatography separation → scraping off spots with the same R Dissolve with a fixed amount of anhydrous ethanol → thin layer chromatography → scrape off the spots with the same R『 value as the standard → petroleum ether extraction → evaporation and drying → dissolve the residue with about 0.5 mL of ethanol → low pressure evaporation and drying → remove the bacteria by centrifugation. Dissolve with about 0.5 mL of ethanol → store at low temperature for use.    

 

1 . 5 Analytical Methods for Coenzyme Q 10

UV analysis: UV scanning of the sample to be measured at a wavelength of 190-300 nm, determination of its maximum UV absorption wavelength, and quantitative determination at 275 nm.

 

High performance liquid chromatography (HPLC): the column was sPherisorbC18 (10 cm×4.6 mm ID). The column was sPherisorbC18 (10 cm×4.6 mm ID), filled with octadecylsilane-bonded silica gel, methanol/anhydrous ethanol (1:1, v/v/v) as the mobile phase, with a column temperature of 35 ℃, a 10,000-checksquare-meter wavelength of 275 nm, and a sample volume of 20 μL.

 

2 Results and Discussion

2 . 1 Determination of Coenzyme Q10 Extraction and Preparation Methods

Since coenzyme Q10 is an intracellular substance, which mainly exists in the inner membrane of mitochondria, its extraction effect has a direct impact on the purity and yield of the product. The most commonly used extractant for the extraction of intracellular quinones is organic solvents [3, 4]. The results of the extraction of intracellular quinones from bacterial cells using 1) methanol and ether at a volume ratio of 2:1; 2) iso-octane and isopropanol at a volume ratio of 3:1; 3) methanol and chloroform at a volume ratio of 1:2; 4) acetone direct extraction; and 5) ultrasonic crushing of acetone suspension are shown in Fig. 1. The results were shown in Fig. 1. The results showed that the methanol-chloroform mixture was easy to separate multiple layers after extraction, and the subsequent treatment was difficult, so we finally decided to use acetone suspension ultrasonication method.

 

Fig. 1 Effect of different solvent treatments on the extraction rate of coenzyme Q10

 

2 . 1 . 1 Determination of Solvent Stirring Extraction Time

Freeze-dried cells were ultrasonicated with acetone suspension at 0.5, 1, 3, 5 and 8 h. The results are shown in Fig. 2 . 5, 1, 3, 5 and 8 h of continuous stirring to compare the extraction effect, the results are shown in Figure 2 . The finalized organic extraction time was 5 h .

 

Fig.2 Effects of extracting time on the extract yields of CoQ10

 

2 . 1 . 2 The Effect of Extraction Temperature on the Extraction Effect

Adopting room temperature, 40, 60 ℃ for extraction, the results are shown in Figure 3 . Considering the continuous heat preservation treatment on the volatilization of organic solvents and the purpose of the composition of the possible impact, and finally determine the organic extraction temperature for room temperature (25 ℃) combined with intermittent heating method of extraction .

 

 

Fig.3 Effects of extracting temperature on the extract effectiveness of CoQ10

 

2 . 2 Determination of Coenzyme Q10 by TLC-UV Method

2 . 2 . 1 Thin-layer chromatography for the separation of coenzyme Q10 Spreading agent selection

Thin-layer chromatographic separation of the crude extract of coenzyme Q10 was carried out using chloroform and benzene (1:1, v/v) or petroleum ether and ether (8:2, v/v) as the unfolding agent, and iodine vapor as the color rendering agent or under ultraviolet light directly [5]. 5 μL of sample was used as the sample volume, and the reference concentration of the standard sample was 100 μg/mL, and the spots displayed were compared with the R『 value of the standard. At the same time, a series of standard solutions with mass concentrations of 10 ~ 150 μg/mL were prepared, and their minimum detection mass concentrations were determined, and the results are shown in Table 1.

Table 1 Selection of unfolding agents for thin layer chromatography and minimum mass concentration for detection

Developer	Chloroform/benzene Petroleum ether/ ether Benzene/acetone (volume ratio 85 : 15 1 : 1) 8 : 2) 93 : 7)
R『   Value Minimum detectable mass concentration/ (μg/mL)		0 . 5   35	0 . 67   24	0 . 85   50	-   -

 

Benzene/acetone was colorless by iodine vapor and UV light; the size of color spots of other developers was not related to the mass concentration, but only the color shade was related to the mass concentration of the samples, so it was necessary to combine with the UV method for the determination. According to the suitable range of R『 value and detection sensitivity, chloroform and benzene were selected as the unfolding agents and analyzed by TLC.

 

2 . 2 . 2.2.2 Preparation of UV Spectrophotometric Standard

Curve 20 mg of Coenzyme Q10 standard was accurately weighed, dissolved in anhydrous ethanol and concentrated to 50 mL, and then the solution was diluted to a certain volume to obtain a series of mass concentrations of 8, 20, 40, 80, 120, 160, 200 μg/mL, etc., and the absorbance was measured at 275 nm. The results showed that the linearity of coenzyme Q10 in the range of 80~120 μg/mL was good, and the regression curve equation was y = 59 . 111x - 1 . 577 5, correlation coefficient R2 = 0 . 999 6 .

 

2 . 2 . 3 Comparison of UV Absorption Curves of Coenzyme Q10 and Establishment of Quantitative Methods

The UV absorption curves of the coenzyme Q10 samples prepared from the above data, compared with the standards, were scanned at 190-300 nm with anhydrous ethanol as a blank, and the UV absorption curves are shown in Fig. 4.

 

Fig. 4 UV scanning absorption curves of the standard (upper panel) and sample (lower panel)

As can be seen in Figure 4, the scanned peak shapes of the crude extract and coenzyme Q10 sample obtained by organic solvent extraction and petroleum ether extraction were basically the same, with maximum absorption at (207 ± 2) nm and (275 ± 1) nm, and the peaks of the sample liquid were deviated from the peaks at 275 nm, and there were also some small peaks of impurities. This is due to the extraction of the sample by a variety of organic solvents and the dissolution of intracellular impurities, which have a large absorption near (207 ± 2) nm and (275 ± 1) nm.

 

Therefore, direct UV quantitative determination of the crude extract would bring some errors, so the content of Coenzyme Q10 was quantified by thin-layer chromatography separation, further purification and UV detection. The method is as follows: Crude extract → Spotting, unfolding on a silica gel plate → scraping off the spot with the same R『 value as the standard product → the target material is washed with a small amount of petroleum ether → drying on a rotary evaporator → the residue is dissolved in 0.3 mL of ethanol → the sample is dissolved with 0.3 mL of ethanol. Dissolve the residue with about 0.3 mL of ethanol → Sample. The sample was diluted with alcohol solvent according to the concentration of the collected sample and the absorbance value was measured at 275 nm.

 

2 . 3 Comparison of the Results of TLC-UV method and HPLC Method

The extracts obtained by organic solvent extraction, petroleum ether elution and TLC separation were dissolved in a certain amount of anhydrous ethanol and then analyzed by HPLC and compared with the results of the above experiments, as shown in Table 2 .

 

The mass concentration of Coenzyme Q10 measured by the TLC-UV method was 92% of that by the HPLC method, which may be due to the partial loss of the spots developed by TLC in the re-extraction process.

Table 2 Comparison of two assays for Coenzyme Q 10

 

Methodology	Mass concentration of coenzyme Q10/(μg/mL)
TLC-UV method	80 . 1
HPLC method	87 . 3

 

2 . 4 Coenzyme Q10 Identified by High Performance Liquid Chromatography Analysis

The crude extract was analyzed by thin plate chromatography and then by high performance liquid chromatography (HPLC) to identify the extraction effect.［6］ . The concentration of the sample was 200 μg/mL, the detection wavelength was 275 nm, and the chromatograms are shown in Figs. 5 and 6.

 Fig. 5 Chromatogram of the standard sample (200 μg/mL) analyzed by high performance liquid chromatography (detection wavelength: 275 nm).

 

As can be seen in Figures 5 and 6, the retention time of coenzyme Q10 in the chromatograms of both standard and sample was 20.6 min. 6 min. After chromatography of the sample solution separated by this coenzyme Q10 extraction method, HPLC analysis showed that there were few impurities, which did not interfere with the determination of coenzyme Q10. In addition, by changing the detection wavelength to 206 nm, the HPLC analysis of the standard sample and the test sample was carried out, and the results showed that the retention time of the standard sample and the test sample was 20.6 min, and the retention time of the standard sample and the test sample was 20.6 min. The results showed that there was one main peak at the retention time of 20.6 min for both standard and test samples, which proved that they were the same substance.

 

The sample was further reduced with 0.01 mL of sodium borohydride solution. The sample was further reduced with 0.01 mL of sodium borohydride solution and detected under the same chromatographic conditions and wavelength, and the absorption peak of the target disappeared at the original retention time, confirming that it was coenzyme Q10.

 

Fig. 6 Chromatogram of sample (crude extract) analyzed by high performance liquid chromatography (HPLC)

 

3 Summary

(1) By comparing the extraction and isolation methods of coenzyme Q10, it was determined that the method of collecting the logarithmic-phase organisms was to rotate at 6,000 r/min for 15 min; and the method of ultrasonic treatment of acetone suspension was adopted, and the extraction time was 5 h. The extraction time was 5 hours.

 

(2) The oxidative damage of coenzyme Q10 should be avoided during the separation and extraction process, especially under alkaline conditions, and antioxidants, such as pyrogallic gallic acid, should be added if necessary; the samples to be tested should be fresh or stored at low temperatures. By comparing the effects of different organic solvents on the extraction yield, the extraction method with relatively low toxicity, low cost and high extraction rate was selected; the HPLC analytical method was used to identify and confirm that the ideal fermentation method was identified method for the isolation and extraction of Coenzyme Q10 .

 

(3) Compared with HPLC quantitative analysis method, TLC-UV method is a more practical method for quantitative determination of coenzyme Q10 with simpler method, shorter operation time, lower cost, smaller relative error, and simple sample processing.

 

References:

［1］Wu Zu-Fang, Weng Pei-Fang, Chen Jian.  Progress of functional studies on coenzyme Q10［J］.  Journal of Ningbo University, 2001, 2: 85 - 88 .

［2］MORTENSEN S A, VADHANAVIKIT S, FOLKERS K .  Deficiency of coenzyme Q10 in myocardial failure [J].  Drugs Exptl Clin Res, 1984, (7): 497 - 502 .

[3] KRIVANKOVA L, DADAK V .  Methods in enzymology [M]. London: Academic Press Inc, 1980 .   [4] WANG Chunlin . Extraction, isolation and characterization of soybean coenzyme Q10 in China［J］.  Chinese Journal of Pharmaceutical Industry, 1996, 27(3):102 - 104 . [5] Zhuang Xiaolei, Yu Shuhong.  Quantitative determination of paclitaxel by TLC-ultraviolet spectrophotometry［J］.  Biotechnology, 2001, 11(1): 45 - 47 .

［6］MIKATA K, yAMADA .  The ubiquinone system in Hasegawaea japonica (yukaw et Maki) yamada et Banno: A new method for identifying ubiquinone homologs from yeast cells [J].  IFO Res C0mm, 1999, 19:41 - 46 .

Study on COQ 10 for Fatigue in Rats Simulating Acute Plateau Environment

 In recent years, with the development of the west, military operations, tourism and vacation activities, people's acute exposure to high altitude has become more and more frequent. Relevant studies have shown that labor efficiency at altitudes of 3500 and 4500 m decreases by 12.61% and 18.78%, respectively, compared to plains [1]. It takes days or even weeks for the body to adapt to the altitude environment, and serious complications are likely to occur in the early days (within a few days) of acute altitude exposure [2]. Reduced fatigue and pathophysiologic changes in the body after rapid entry to the plateau affect the normal work and life of the personnel and limit the smooth implementation of plateau activities.

 

Acute exposure to high altitude aggravates hypoxemia, which results in insufficient oxygen supply and restricted movement; at the same time, the metabolic reflexes of the respiratory muscles regulate blood redistribution, resulting in a decrease in blood supply to the muscles [3 - 4]. The decrease of oxygen partial pressure in the nervous system, the decrease of neurotransmitter conversion and synaptic transmission, the weakening of ion channels and ion pumps, and the abnormality of neuromuscular junction conduction [5-6], all these pathophysiological changes can aggravate the fatigue of operation.

 

Mitochondrial respiration produces ATP and consumes O2, which is accompanied by the production and removal of reactive oxide species (ROS). As the intensity of work increases, ROS production increases and clearance decreases, and ROS are mainly oxidized by the Na+-K+ pump, resulting in an imbalance of Na+ and K+ concentrations in and out of the skeletal muscle cell, affecting the excitability of the cell membrane, and inhibiting the release of Ca2+ from the sarcoplasmic reticulum and the sensitivity of myofibrils to Ca2+, thus leading to muscle fatigue [7]. In addition, ROS activate class IV muscle afferent nerve fibers and directly inhibit motor neurons [8].

 

 It has been reported in the literature that an increase in ROS can lead to fatigue, and that pretreatment of isolated rat tibialis anterior muscle with superoxide dismutase (SOD) injections reduced the frequency of afferent nerve excitation, which in turn significantly ameliorated fatigue. The dysfunction of mitochondrial oxidative phosphorylation increases the production of ROS, which further oxidizes and destroys the mitochondrial membrane, electron transport system and tricarboxylic enzyme, and further inhibits mitochondrial oxidative phosphorylation, thus forming a vicious cycle [9-11]. Within 72 h after acute altitude entry, the body's antioxidant capacity was significantly weakened, oxidative stress increased significantly, and lipid peroxidation level peaked at 48 h [12]. The oxidative stress at the early stage of plateau entry aggravates the fatigue of the organism and affects its ability to act.

 

Coenzyme Q10 (CoQ10) is found in a wide range of tissues and organs in the human body, with high levels in central, renal and hepatic tissues where metabolism is high, and in organelles, mainly in Golgi vesicles, mitochondrial plasma membranes and lysosomes [13]. CoQ10 is a mitochondria-targeting small molecule that promotes ATP production by participating in the electron transport chain in mitochondria. At the same time, CoQ10 is an important fat-soluble antioxidant that protects cell membranes, mitochondria and other organelles (e.g., Golgi, lysosomes, endoplasmic reticulum, and peroxisomes) from free radical-induced oxidative stress.

 

In addition to its direct role as an antioxidant, CoQ10 is also involved in the regeneration of the antioxidants vitamin C and vitamin E. CoQ10 is approved by the Food and Drug Administration (FDA) for use as a dietary supplement, and has been well tolerated, but its effects on the ability to operate in an acute plateau and on fatigue have not been studied in depth. In this study, we evaluated the anti-fatigue effect of CoQ10 in a rat model of acute plateau exposure and explored its possible mechanisms.

 

1 Materials and Methods

1.1 Animals

SPF grade male Wistar rats, body weight 200~230 g, Beijing Witong Lihua Co., Ltd, Production License No.: SCXK (Beijing) 2016-0011. The feeding environment was 40%~60% humidity, 23-27℃, 12-hour day/night cycle (light time 8:00~20:00), free intake of food and water, and the feeding process was in accordance with the guidelines of the Ethics Committee for Laboratory Animals of the Military Medical Research Institute of the Military Academy of Sciences. Ethical approval number: IACUC-DWZX-2020-778.

 

1.2 Drugs, Reagents and Major Instruments

CoQ10, sodium carboxymethyl cellulose (CMC-Na), and 4% tissue cell fixative, Beijing Solepol Technology Co. Malondialdehyde (MDA) kit and total superoxide dismutase (T-SOD) kit, Nanjing Jianjian Bioengineering Research Institute, Nanjing, China; TUNEL kit, Rexroth; DAB developer, Wuhan Xavier Biotechnology Co. 96-well enzyme labeling plate, Corning, USA; OmegaA15-3677 fully automated enzyme labeling instrument, BMGLABTECH, Germany; TDL-5M centrifuge, Sichuan, China; TDL-5M centrifuge, Sichuan, China; TDL-5M centrifuge, Sichuan, China; TDL-5M centrifuge, Sichuan, China. -Ltd., Guizhou Fenglei Aviation Ordnance Co., Ltd; Vit-ro-950 blood gas analyzer, Johnson & Johnson, USA; Vert.A1 optical microscope, Zeiss, Germany; rat swimming pool, manufactured by the laboratory.

 

1.3 Establishment and Grouping of the Rat Acute Plateau Model

Wistar rats were divided into normal pressure and normoxia (NN) group, hypobaric and hypoxia (HH) group and HH + CoQ10 group, with 12 rats in each group. The HH + CoQ10 group received CoQ10 30 mg kg-1 (prepared as a suspension with 0.5% CMC-Na) as a single daily ig-administration for 6 days, and the remaining 2 groups were given an equal volume of solvent in the same manner. After 4 d of administration, the rats in the HH and HH +CoQ10 groups were transferred to a low-pressure oxygen chamber and ascended to a simulated altitude of 6000 m at a speed of 5 m-s-1 for 2 d. The rats in the NN group were kept under NN conditions.

 

1.4 Swimming Exhaustion in Rats

One hour after the last dose, 6 rats in each group were randomly selected for the swimming exhaustion test. Before the experiment, the rats were weighed and a lead block weighing 8% of their body weight was attached to their tails. The rats were placed in a water tank with a depth of 30 cm and a water temperature of 27~30℃ for the exhaustion swimming test. The rats were judged to be exhausted when they were submerged underwater for ≥10 s. The time of exhaustion was recorded. The exhaustion time was recorded.

 

1.5 Preparation of rat blood samples, blood gas analysis and assay of serum MDA content and T-SOD activity

One hour after the last dose, the remaining 6 rats in each group were anesthetized and placed in the supine position, and blood was collected from the abdominal aorta with a heparinized 1 mL syringe. Aortic blood was partially sealed, and arterial blood pH, arterial carbon dioxide pressure (PaCO2), arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SaO2), and arterial oxygen saturation (SaO2) were analyzed by a blood gas analyzer within 10 min. arterial oxygen saturation (SaO2), and alkali residual (BE).    Arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SaO2), and alkali residual (BE), arterial bicarbonate (HCO3-), serum calcium ion (Ca2+), and arterial lactate (Lac) levels were measured. A portion of aortic blood was centrifuged in a centrifuge tube at 450×g for 10 min at 4°C. The serum was extracted, and MDA and T-SOD activity were measured according to the instructions of the kit.

 

1.6 HE staining to observe the histopathological changes in heart, kidney, lung and brain tissues of rats

After blood sampling and execution, the apical tissues of heart, left kidney, left lung and left cerebral hemisphere of rats were fixed with 4% paraformaldehyde, dehydrated with ethanol gradient, transparent with xylene, embedded in paraffin and made into 5 μm paraffin sections. After HE staining, dehydrated and sealed, the pathological changes of heart, lung, kidney and brain tissues were observed under microscope.

 

1.7 TUNEL Assay for Apoptosis in Rat Heart, Liver, Kidney, Lung and Brain Tissues

Prepare heart, kidney, lung and brain tissue sections, and subject them to ethanol gradient dehydration, antigen repair and cell membrane rupture treatment, according to the instructions of the kit. After staining, sealing and microscopic observation, cells with brownish-yellow particles in the nucleus are positive cells, i.e. apoptotic cells. 

 

1.8 Statistical Analysis

The experimental data were expressed as x±s and analyzed statistically using SPSS18.0 software. The results of HH swim exhaustion time, HCO3-, BE, Lac and Ca2+ in NN did not conform to normal distribution, and the Mann-Whitney rank sum test was used for comparison between groups. The other results were normally distributed, and t-tests with independent samples were used. p<0.05 was considered statistically significant.

 

2 Results

2.1 Effect of CoQ10 on the duration of swimming exhaustion in a rat model of simulated acute altitude progression

 

After 48 h of simulated rapid advancement to 6000 m plateau, the swimming exhaustion time of rats in the HH group was significantly shorter than that of rats in the NN group (P<0.01), and the swimming exhaustion time of rats in the HH+CoQ10 group was significantly longer than that of rats in the HH group (P<0.05) (Figure 1). This suggests that CoQ10 has a certain anti-fatigue effect.

 

Fig. 1 Effect of coenzyme Q10 (CoQ10) on exhaus⁃ tive swimming time in rushing-into-plateau model rats. Wistar rats were randomly divided into three Wistar rats were randomly divided into three groups: normal pressure and normoxia [NN, vehicle (0.5% CMC-Na)] group, hypo⁃ baric and hypoxia (HH, vehicle) group, and HH +CoQ10 group (CoQ10 30 mg-kg-1). All rats except those in NN group were rushed into a high altitude of 6000 m 4 d after preventive administration . Vehicle and CoQ10 were administrated for another 2 d. x± s, n=6. **P<0 .01, compared with NN group; # P<0 .05 , compared with HH group.

 

2.2 Effect of CoQ10 on Blood Gas Indexes of Rats in the Acute Plateau Model

As shown in Table 1, after 48 h of simulated rapid advancement to a plateau of 6000 m, arterial blood pH, PaO2, SaO2 and HCO3- levels in the HH group were significantly lower (P<0.01), and PaCO2, BE, Ca2+ and Lac levels were significantly higher (P<0.01) compared with those in the NN group. Compared with rats in the HH group, arterial blood pH was significantly increased (P<0.05), and PaCO2, Ca2+ and Lac levels were significantly decreased (P<0.05) in the HH + CoQ10 group. It was suggested that CoQ10 could improve acidosis and hyperCO2emia and reduce the serum Lac level in rats exposed to the plateau environment.

  

Tab. 1 Effect of CoQ10 on arterial blood gas analysis of rushing-into-plateau model rats

 

Parameter	NN	HH	HH+CoQ10
pH	7.42±0.11	6.88±0.16**	7. 10±0.15#
PaCO2 /mmHg	38±16	64±7**	40±23#
PaO2 /mmHg	107±11	43±19**	58±28
SaO2 /%	95±3	23±16**	46±28
BE/mmol-L-1	-0.7±9.8	-18.1±4.3**	-18.1±3.1
HCO3- /mmol-L-1	24.3±7.1	10. 1±1.9**	11.2±1.8
Ca2+/mmol-L-1	1.29±0.18	1.54±0.06**	1.33±0.21#
Lac/mmol-L-1	2.8±3.5	12.8±2.4**	9.5±2.0#

See Fig. 1 for the rat treatment. PaCO2 : arterial carbon dioxide pressure; PaO2 : arterial oxygen partial pressure; SaO2 : arterial oxygen saturation; BE. alkali residual; HCO : arterial bicarbonate; Ca : serum calcium ion; Lac: alkali residual; HCO3- : arterial bicarbonate; Ca2+ : serum calcium ion; Lac: arterial lactate. 1 mmHg=0.133 kPa. x±s, n=6. **P<0.01, compared with NN group;# P<0.05, compared with HH group.

 

2.3 Effects of CoQ10 on the histopathological structure of heart, lung, kidney and brain tissues in rats in the acute plateau model

HE staining results (Figure 2) showed that in the NN group, the cardiomyocytes had clear boundaries, clear nuclei, and no obvious cytoplasmic degeneration and necrosis; the renal tubules and glomeruli had normal structure; the alveolar walls were thin, the alveolar septa were clear, and the vesicles were obvious; neurons in the brain tissue were of normal morphology and size, and the nuclei were large and obvious. In HH+CoQ10 group rats, myocardial, renal, lung and brain tissues showed similar pathological changes to those in HH group rats, but to a lesser extent. It is suggested that CoQ10 can improve pulmonary edema and cerebral edema, and reduce the swelling of renal tubular epithelial cells in rats simulating acute plateau environment.

 

2.4 Effect of CoQ10 on apoptosis in heart, lung, kidney and brain tissues of rats in the acute plateau model

TUNEL staining showed that the apoptosis rates of heart, lung and brain cells in the NN, HH and HH+CoQ10 groups (data omitted) were not significantly altered (Figure 3). The apoptosis rate of renal tubular epithelial cells in the HH group was significantly increased compared with that in the NN group (P<0.01), and the apoptosis rate of renal tubular epithelial cells in the HH+CoQ10 group was significantly decreased compared with that in the HH group (P<0.01) (Figure 4). It was suggested that CoQ10 could improve the apoptosis of renal tubular epithelial cells in rats exposed to the acute plateau environment.

 

2.5 Effect of CoQ10 on serum T-SOD activity and MDA content in the acute plateau model rats

Compared with rats in the NN group, serum T-SOD activity was significantly lower (P<0.01) and MDA content was significantly higher (P<0.01) in the HH group; compared with rats in the HH group, serum T-SOD activity was significantly higher (P<0.05) and MDA content was significantly lower (P<0.05) in the HH+CoQ10 group (Figure 5). It was suggested that CoQ10 could improve the level of serum oxidative stress in rats exposed to acute plateau environment.

 

3 Discussion

The existing simulated plateau model and the altitude of human life in high altitude areas of China is more than 4000 m. According to the literature, exposure to altitude of 5000-6000 m for 24-72 h can induce tissue pathological changes in acute plateau models [15]. According to the literature, exposure to altitude of 5000-6000 m for 24-72 h can induce histopathological changes in animals with acute plateau [15]. Therefore, in the present study, we used an altitude of 6000 m for 48 h and a low-pressure oxygen chamber to simulate the plateau environment to establish a rat model of acute plateauing, which can reach the altitude quickly and effectively reduce the experimental bias caused by the habituation of the animals and other factors during transportation to the plateau.

 

The results of this study showed that compared with the rats in the HH group, the preventive administration of CoQ10 significantly prolonged the time of swimming exhaustion, alleviated acidosis and high CO2emia, and significantly improved the histopathological damage of the brain, lungs and kidneys as well as the serum oxidative stress, which suggests that CoQ10 can effectively alleviate fatigue at the early stage of the plateau, improve the pathological damage of the lungs, brain, kidneys and other important organs, alleviate the oxidative damage, and maintain the health of the body. It can also reduce the oxidative damage of tissues and maintain the health of the body.

 

CoQ10 has been widely used as a nutritional supplement, and 300 mg-d-1 has been generally recommended for human use, and no toxic effects have been observed [16]. In the present study, we aimed to investigate the effects of CoQ10, which is commonly used in human dosage, on simulated plateau rats. 30 mg kg-1 was administered to rats in a single daily dose according to the human-rat dosage conversion (body surface area conversion method) [17], which is one of the conventional dosages of CoQ10 in CoQ10 studies [18-19].

 

In the present study, we found that the exercise capacity of rats decreased significantly after 48 h of exposure to simulated 6000 m plateau, and the administration of CoQ10 significantly improved the physical performance of rats. At the same time, rats in the HH group showed typical pathological changes of pulmonary and cerebral edema, such as thickening of the alveolar wall, proliferation of the alveolar epithelium, edema of cerebral neuronal cells, and nuclear consolidation, which were ameliorated by the administration of CoQ10.

 

 It has been reported in the literature that CoQ10 can improve lipopolysaccharide-induced lung injury through its anti-inflammatory effect, and the administration of CoQ10 reduced serum C-reactive protein levels by 44.58%, alkaline phosphatase activity by 37.38%, and lactate dehydrogenase levels by 48.6%, resulting in a significant improvement in the pathologic damage of the lung tissue [20]. In another study on anesthesia and non-transplantation mild lung injury, it was also found that CoQ10 could play a role in lung protection by reducing the expression of tumor necrosis factor α [21]. These reports are consistent with the results of the present study.

 

Recent studies have shown that the kidney plays an important role in adaptation to the environment and plateau disease syndrome (acute high altitude sickness, high altitude cerebral edema and high altitude pulmonary edema) by regulating body fluids, electrolytes and acid-base balance, and renal injury occurs mostly in 2-4 d after acute altitude entry [12, 22-23]. In the present study, we found that the renal tubular epithelial cells in the HH group showed obvious edema and apoptosis, and the rate of apoptosis was significantly higher than that in the NN and CoQ10 groups, which indicated that CoQ10 had a certain protective effect on the early stage of renal tubular injury in the plateau-accelerated rats.

 

The results of this study showed that the serum MDA level was significantly increased and the T-SOD activity was significantly decreased in the HH group, while the administration of CoQ10 significantly decreased the serum MDA and increased the level of T-SOD. MDA is a peroxide formed by the attack of oxygen radicals on the polyunsaturated fatty acids in the biofilm, which triggers lipid peroxidation, and the level of MDA reflects the damage caused by oxidative stress in the organism [24]. T-SOD plays an important role in the redox balance of the organism, scavenging superoxide anion radicals (-O-2) and protecting cells from damage, and its activity can indirectly reflect the ability of the organism to scavenge oxygen radicals [24].

 

Goldfarb et al. [25] and Thirupathi et al. [26] found that antioxidant supplementation could reduce muscle damage, relieve fatigue, and improve work capacity. Other studies have reported that taurine supplementation can improve exercise capacity and reduce muscle damage [27-28]. However, some studies have reported that a large amount of exogenous antioxidant supplementation may interfere with the signaling pathway of the oxidative reduction response of myocytes, affecting their oxygen radical scavenging ability, and the advantages of CoQ10 as an endogenous antioxidant are more obvious [29].

 

In conclusion, CoQ10 can correct the acidosis and hyperCO2emia caused by pulmonary edema, improve the pathological damage of heart, lung, kidney and brain tissues, inhibit oxidative stress, and improve the anti-fatigue ability of the organism in the acute plateau environment.

 

References: 

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