Orally ingested ubiquinol-10 or ubiquinone-10 reaches the intestinal tract and is absorbed by the small intestine of mice mostly in its original form

Abstract

Coenzyme Q10 (CoQ10) is an important lipid-soluble antioxidant and an essential component of the mitochondria. The oral bioavailability of the reduced form of CoQ10, ubiquinol-10, has been reported to be greater than that of the oxidized form of CoQ10, ubiquinone-10, in some studies. In contrast, it has also been highlighted that the oral bioavailability of ubiquinol-10 is not superior to that of ubiquinone-10 because ubiquinol-10 may be oxidized during digestion. In fact, it has not been shown which form of CoQ10 exists in the process from oral intake to absorption in the gastrointestinal tract. In this study, the amounts of ubiquinol-10 and ubiquinone-10 were measured in the gastrointestinal content and small intestine tissue after oral administration of ubiquinol-10 or ubiquinone-10 to C57BL/6J mice. The form of CoQ10 detected in the gastrointestinal content and small intestine tissue was almost the same as that when administered orally. The results of our study suggested that the orally administered ubiquinol-10 and ubiquinone-10 mostly reached the small intestine without oxidizing to ubiquinone-10 and reducing to ubiquinol-10, and both were absorbed by the small intestine tissue in almost their original forms.

Introduction

Coenzyme Q (CoQ) is an important lipid-soluble antioxidant against oxidative stress^(1–3) and essential for mitochondrial respiratory chain.⁽⁴⁾ CoQ is present in its reduced or oxidized form in nearly all tissues. CoQ10, which is found in humans, has a polyisoprene chain containing 10 isoprene units, whereas CoQ9 is the major CoQ in rodents.⁽⁵⁾

The level of CoQ10 in the body is thought to be maintained by endogenous synthesis and exogenous sources, i.e., dietary intake. A study on patients with total parenteral nutrition revealed that the serum level of CoQ10 in the patients decreased to nearly half of the level before total parenteral nutrition treatment.⁽⁶⁾ In fact, many foodstuffs contain CoQ10,^(7–9) and a portion of CoQ10 in foodstuffs is in the reduced form of CoQ10 (ubiquinol-10).⁽¹⁰⁾

Some studies on rats, dogs, and humans revealed that the plasma level of CoQ10 in the ubiquinol-10 treatment group was higher than that in the ubiquinone-10 treatment group when the same amount of ubiquinol-10 or ubiquinone-10 was orally administered.^(11,12) In the rat study, the area under the concentration versus time curve (AUC) of total CoQ10 in the ubiquinol-10 administration group was shown to be approximately two times higher than that in the ubiquinone-10 administration group.⁽¹¹⁾ Similarly, in the dog study, the AUC of total CoQ10 in the ubiquinol-10 administration group was shown to be approximately 1.4 times higher than that in the ubiquinone-10 administration group.⁽¹¹⁾ In addition, in the human study, the increase in the total CoQ10 level after 4 weeks of ubiquinol-10 intake was approximately two times higher than that of ubiquinone-10 intake.⁽¹²⁾

In contrast, some researchers have noted that the bioavailability of ubiquinol-10 is not superior to that of ubiquinone-10. They pointed out that one of the reasons for this is that orally ingested ubiquinol-10 may be oxidized to ubiquinone-10 before reaching the enterocytes in the small intestine due to the instability of ubiquinol-10 in gastric and small intestine digestive fluids.^(13,14) This view is not adequately supported by evidence since it is mostly based on in vitro studies and is not based on appropriate in vivo studies.

So far, there is little finding on the process from oral intake of CoQ10 to absorption in the gastrointestinal tract. However, understanding which form of CoQ10 exists in the gastrointestinal tract lumen and the small intestine tissue after the administration of ubiquinol-10 or ubiquinone-10 is considered as useful information to clarify the mechanism of ubiquinol-10 and ubiquinone-10 absorption. In this report, this was demonstrated using mouse model.

Materials and Methods

Reagents and animals

Ubiquinol-10 and ubiquinone-10 were obtained from Kaneka Corporation (Osaka, Japan). Corn oil was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ubiquinol-10 or ubiquinone-10 was dissolved in corn oil to prepare solutions of 60 mg/ml. Male C57BL/6J mice aged nine weeks and rodent diet CE-2 were purchased from CLEA Japan, Inc. (Tokyo, Japan). The mice were fed the diet and water ad libitum. All animal experimental procedures were approved by the Animal Care and Use Committee of Kaneka Corporation (approval number: 2016–12 and 2021–8, approval date: 24 March 2016, and 24 March 2021, respectively), and conducted in accordance with the guidelines for animal experiments of Kaneka Corporation following the Act on Welfare and Management of Animals (Ministry of the Environment, Japan) and the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Ministry of the Environment, Japan).

Analysis of the mouse diet

Rodent diet CE-2 was ground into powder. Then, 0.95 ml of 2-propanol was added to 50 mg of the powder, and the mixture was centrifuged at 16,000 × g for 3 min at 4°C. Next, the supernatant was separated, and 50 μl of the extract was injected into a high-performance liquid chromatography (HPLC) apparatus.

Administration of CoQ10 and preparation of samples for analysis

Thirty of the 33 mice aged ten weeks were divided into two groups of 15 mice each and were orally administered ubiquinol-10 or ubiquinone-10 solution at 300 mg/kg. In both groups, the stomach and small intestine were quickly harvested from three mice at 0.5, 1, 2, 4, and 8 h after the administration, following euthanasia using isoflurane. The small intestine, which has a total length of about 30 cm, was divided into five sections, with each being 6 cm in length.⁽¹⁵⁾ As the control group, the remaining three mice were treated in the same manner as described above but without CoQ10 administration. All samples were rapidly stored at −80°C until analysis.

Analysis of the gastrointestinal content

The stomach and each section of the small intestine were rinsed in 10 ml of physiological saline to disperse the gastrointestinal content. The content dispersed in physiological saline was diluted 10- or 100-fold with 2-propanol and centrifuged at 16,000 × g for 3 min at 4°C. The supernatants were separated, and 50 μl of the extract was injected into the HPLC apparatus.

Analysis of the sections of small intestine tissue

The sections of the small intestine, after the rinse, were pulled up and washed once in physiological saline. These tissue samples were minced. Then, 3.9 ml of 2-propanol was added to each minced approximate 100 mg tissue sample, and the mixture was homogenized with a Polytron homogenizer. The homogenate was diluted 10-fold with 2-propanol and was centrifuged at 16,000 × g for 3 min at 4°C. The supernatants were separated, and 50 μl of the extract was injected into the HPLC apparatus.

Influence of the number of small intestine tissue washes on the CoQ10 level in the small intestine tissue

The small intestine sections of mice after CoQ10 administration were prepared independently of the above experiment to confirm the appropriate number of tissue washes. After rinsing the small intestine sections to disperse the intestinal content, the sections were divided vertically into two pieces. One was washed once in physiological saline and the other was washed twice in physiological saline. These tissue samples were treated as described above to measure the CoQ10 level.

HPLC analysis of CoQ9 and CoQ10

Ubiquinol-9, ubiquinone-9, ubiquinol-10, and ubiquinone-10 concentrations were determined using a high-performance liquid chromatography with an electrochemical detection (HPLC-ECD) system, as previously reported,⁽¹⁰⁾ with minor modification to determine CoQ9.

Results

Amounts of CoQ9 and CoQ10 in the mouse diet

The mice diet used in this study included CoQ9 (8.2 ± 1.8 μg/g) and CoQ10 (1.4 ± 0.3 μg/g). The values are means ± SE (n = 3).

Influence of the number of the small intestine tissue washes on the CoQ10 level in the small intestine tissue

All five small intestine sections showed approximately the same CoQ10 levels between once and twice washing with physiological saline (Fig. 1). Therefore, all small intestine sections were washed once to measure the CoQ10 levels.

Fig. 1.

Influence of the number of small intestine tissue washes on the CoQ10 level in the small intestine tissue. Gray and white bars indicate the level of CoQ10 in the small intestine sections that were washed once and twice with physiological saline, respectively. Five small intestine tissues (No. 1–5) obtained 2–4 h after the administration of CoQ10 were examined.

Amounts of CoQ9 and CoQ10 in the gastrointestinal content and levels of CoQ9 and CoQ10 in the small intestine tissue of mice in the control group

Amounts of CoQ9 and CoQ10 in the gastrointestinal content in the control group are shown in Fig. 2. The amount of CoQ10 in the content of the stomach and small intestine sections ranged from 0.3 to 2.7 μg, which was approximately one-fifth of the amount of CoQ9.

Fig. 2.

Amounts of CoQ9 (A) and CoQ10 (B) in the gastrointestinal content of mice in the control group. White bars are the amount of reduced form of CoQ (Q9H, Q10H), while gray bars are the amount of oxidized form of CoQ (Q9, Q10). The left-vertical axis is for the content of the stomach and the right-vertical axis is for the content of the small intestine. S1 is the first section of the small intestine, S2 is the second, S3 is the third, S4 is the fourth, and S5 is the fifth. Three bars in each section were from three mice.

The levels of CoQ9 and CoQ10 in each section of the small intestine tissue in the control group are shown in Fig. 3. The CoQ10 levels ranged between 5 and 12 μg/g tissue and were approximately one-fourth of the CoQ9 levels. The ratio of ubiquinol-10 to total CoQ10 was approximately 25%.

Fig. 3.

Levels of CoQ9 (A) and CoQ10 (B) in each section of small intestine tissue of mice in the control group. White bars are the reduced form of CoQ (Q9H, Q10H), while gray bars are the oxidized form of CoQ (Q9, Q10). S1 is the first section of the small intestine, S2 is the second, S3 is the third, S4 is the fourth, and S5 is the fifth. The values are mean ± SE (n = 3).

Amount of CoQ10 in the gastrointestinal content after administration of ubiquinol-10 or ubiquinone-10

The amount of CoQ10 in the gastrointestinal content after ubiquinol-10 or ubiquinone-10 administration is shown in Fig. 4. The amount of CoQ10 increased mainly in the content of the stomach and upper section of the small intestine 0.5 h after CoQ10 administration. After 1–2 h of CoQ10 administration, the amount of CoQ10 increased largely in the content of all sections of the small intestine. After 4 h, the amount of CoQ10 increased mainly in the content of the lower section of the small intestine. After 8 h, the amount of CoQ10 in the content of sections of the small intestine was close to the normal condition level. In the content of the stomach and any section of the small intestine, CoQ10 was detected mainly as ubiquinol-10 when ubiquinol-10 was administrated. However, CoQ10 was detected mainly as ubiquinone-10 there when ubiquinone-10 was administrated. At any point in time, the amount of CoQ9 in the content after ubiquinol-10 or ubiquinone-10 administration was almost the same as that under normal conditions (data not shown). The mean amounts of ubiquinol-10, ubiquinone-10 and total CoQ10 (ubiquinol-10 + ubiquinone-10) measured at all points are summarized in Table 1.

Fig. 4.

Amount of CoQ10 in the gastrointestinal content after administration of CoQ10. 0.5 h after administration of ubiquinol-10 (0.5h-R), ubiquinone-10 (0.5h-O). 1 h after administration of ubiquinol-10 (1h-R), ubiquinone-10 (1h-O). 2 h after administration of ubiquinol-10 (2h-R), ubiquinone-10 (2h-O). 4 h after administration of ubiquinol-10 (4h-R), ubiquinone-10 (4h-O). 8 h after administration of ubiquinol-10 (8h-R), ubiquinone-10 (8h-O). White bars are the amount of reduced form of CoQ10 (Q10H), while gray bars are the amount of oxidized form of CoQ10 (Q10). The left-vertical axis is for the content of the stomach and the right-vertical axis is for the content of the small intestine. S1 is the first section of the small intestine, S2 is the second, S3 is the third, S4 is the fourth, and S5 is the fifth. Three bars in each section are from three mice.

Table 1.

Summary of amount of CoQ10 in the gastrointestinal content before and after CoQ10 administration

Administration			Amount of CoQ10 (μg)
			Stomach	Small intestine
				S1	S2	S3	S4	S5
None (Control)		Q10H	0.40 ± 0.07	0.53 ± 0.10	0.81 ± 0.17	0.49 ± 0.11	0.38 ± 0.12	0.41 ± 0.14
		Q10	0.19 ± 0.09	1.02 ± 0.19	1.27 ± 0.17	0.75 ± 0.15	0.52 ± 0.19	0.48 ± 0.13
		Total CoQ10	0.59 ± 0.16	1.54 ± 0.29	2.08 ± 0.34	1.25 ± 0.26	0.90 ± 0.31	0.89 ± 0.27
Ubiquinol-10	0.5 h	Q10H	2,695 ± 666	395 ± 111	380 ± 74	344 ± 57	361 ± 137	97 ± 46
		Q10	92 ± 36	40 ± 8.9	62 ± 23	48 ± 20	85 ± 55	14 ± 5.6
		Total CoQ10	2,788 ± 702	435 ± 105	442 ± 95	393 ± 69	446 ± 192	111 ± 49
	1 h	Q10H	1,023 ± 539	519 ± 157	357 ± 77	296 ± 63	415 ± 121	327 ± 27
		Q10	41 ± 8.0	66 ± 37	69 ± 27	61 ± 23	92 ± 39	65 ± 19
		Total CoQ10	1,065 ± 547	585 ± 190	426 ± 103	357 ± 86	507 ± 158	392 ± 45
	2 h	Q10H	756 ± 256	102 ± 35	642 ± 79	698 ± 73	434 ± 45	728 ± 299
		Q10	48 ± 15	16 ± 7	132 ± 42	153 ± 32	98 ± 14	210 ± 63
		Total CoQ10	804 ± 271	118 ± 42	773 ± 121	851 ± 92	532 ± 58	938 ± 323
	4 h	Q10H	360 ± 46	25 ± 15	64 ± 25	243 ± 163	269 ± 123	577 ± 83
		Q10	39 ± 9	3.8 ± 0.8	11 ± 4	33 ± 15	57 ± 28	100 ± 7
		Total CoQ10	399 ± 48	29 ± 15	75 ± 29	276 ± 178	327 ± 151	677 ± 82
	8 h	Q10H	78 ± 12	4.2 ± 2.9	55 ± 46	20 ± 17	23 ± 12	109 ± 104
		Q10	14 ± 4	1.8 ± 0.8	10.2 ± 6.9	4.1 ± 2.5	3.2 ± 1.7	9.0 ± 7.4
		Total CoQ10	92 ± 8	5.9 ± 3.7	65 ± 53	24 ± 19	26 ± 13	118 ± 111
Ubiquinone-10	0.5 h	Q10H	2.6 ± 1.6	11 ± 3.6	17 ± 3.7	13 ± 3.5	15 ± 6.7	14 ± 4.6
		Q10	1,529 ± 442	1,038 ± 426	488 ± 44	212 ± 34	163 ± 39	169 ± 41
		Total CoQ10	1,531 ± 443	1,050 ± 427	505 ± 46	225 ± 37	178 ± 46	184 ± 45
	1 h	Q10H	1.7 ± 0.4	4.8 ± 1.8	9.8 ± 1.4	12.7 ± 1.9	9.5 ± 3.5	5.8 ± 2.4
		Q10	885 ± 271	232 ± 170	551 ± 187	648 ± 146	204 ± 69	104 ± 26
		Total CoQ10	887 ± 270	237 ± 169	561 ± 187	661 ± 146	214 ± 65	109 ± 28
	2 h	Q10H	14 ± 13	5.7 ± 1.5	7.7 ± 1.3	5.5 ± 0.3	7.7 ± 3.6	10.4 ± 5.4
		Q10	609 ± 228	85 ± 37	860 ± 499	505 ± 136	414 ± 86	430 ± 129
		Total CoQ10	623 ± 217	91 ± 38	868 ± 500	511 ± 136	421 ± 89	441 ± 133
	4 h	Q10H	2.5 ± 1.7	3.2 ± 1.3	9.1 ± 4.1	8.6 ± 2.2	5.4 ± 2.9	6.7 ± 1.6
		Q10	108 ± 2	15 ± 8	97 ± 62	65 ± 11	107 ± 72	204 ± 73
		Total CoQ10	111 ± 2	18 ± 9	106 ± 66	74 ± 11	113 ± 73	210 ± 72
	8 h	Q10H	0.24 ± 0.14	1.1 ± 0.2	2.0 ± 0.9	1.6 ± 0.4	4.5 ± 1.3	3.1 ± 0.8
		Q10	23 ± 13	1.6 ± 0.2	2.9 ± 1.7	3.5 ± 1.3	22 ± 9.1	19 ± 7.9
		Total CoQ10	24 ± 13	2.7 ± 0.4	4.9 ± 2.5	5.1 ± 1.7	27 ± 10	22 ± 8.5

Q10H, ubiquinol-10; Q10, ubiquinone-10; Total CoQ10, Q10H + Q10. S1, S2, S3, S4, and S5, first, second, third, fourth, and fifth sections of the small intestine, respectively. The values are mean ± SE (n = 3).

Levels of CoQ10 in the small intestine tissue after administration of ubiquinol-10 or ubiquinone-10

The CoQ10 levels in each section of small intestine tissue 2 h after ubiquinol-10 or ubiquinone-10 administration are shown in Fig. 5. The CoQ10 levels increased in all sections of the small intestine compared with under normal conditions. At all sections of small intestine tissue, CoQ10 was detected mainly as ubiquinol-10 when ubiquinol-10 was administrated. However, CoQ10 was detected almost as ubiquinone-10 there when ubiquinone-10 was administrated.

Fig. 5.

Levels of CoQ10 in each section of small intestine tissue 2 h after administration of CoQ10. Two hours after administration of ubiquinol-10 (A), ubiquinone-10 (B). White bars are the amount of reduced form of CoQ10 (Q10H), while gray bars are the amount of oxidized form of CoQ10 (Q10). S1 is the first section of the small intestine, S2 is the second, S3 is the third, S4 is the fourth, and S5 is the fifth. The values are mean ± SE (n = 3).

Discussion

In this study, the administration amount of ubiquinol-10 or ubiquinone-10 was set as 300 mg/kg. The mouse diet also contained CoQ10 as well as foodstuffs (see results section). Furthermore, CoQ10 existed in the gastrointestinal content and small intestine tissue of mice under normal conditions (Fig. 2 and 3). Therefore, it is necessary to administer a sufficient amount of ubiquinol-10 or ubiquinone-10 to mice to be negligible of CoQ10 present under normal conditions. As the result, the administration amount of 300 mg/kg in this study was considered appropriate because the amount of CoQ10 in the gastrointestinal content after ubiquinol-10 or ubiquinone-10 administration was significantly higher than the CoQ10 amount under normal conditions.

Orally administrated ubiquinol-10 or ubiquinone-10 was considered to reach the end of the small intestinal tract lumen without converting to ubiquinone-10 and ubiquinol-10, respectively. Although there are large individual differences in the CoQ10 amount in the gastrointestinal content, CoQ10 administered orally reached the end of the small intestinal tract lumen from the stomach between 0.5 and 4 h. Until 4 h after CoQ10 administration, CoQ10 existed almost as ubiquinol-10 in the content of the stomach and sections of the small intestine when ubiquinol-10 was administrated. In contrast, CoQ10 existed almost as ubiquinone-10 there when ubiquinone-10 was administrated (Fig. 4). As mentioned above, some researchers recently noted that orally ingested ubiquinol-10 might be oxidized to ubiquinone-10 before reaching the enterocytes in the small intestine due to being unstable in gastric and small intestine digestive fluids.^(13,14) In a study using dogs, it was reported that 2 h after the oral administration of ubiquinol-10, the ratio of ubiquinol-10 to total CoQ10 in the small intestine was 8%.⁽¹⁶⁾ However, in the study, it was unclear whether the administrated ubiquinol-10 was measured because the amount of CoQ10 in the small intestine was not shown compared with its amount before ubiquinol-10 administration. Therefore, it is difficult to conclude that the ubiquinol-10 administered orally is oxidized to ubiquinone-10 during transit to the small intestine. Meanwhile, according to our data, it is reasonable that orally administrated ubiquinol-10 was considered to reach the end of the small intestinal tract lumen without oxidizing to ubiquinone-10 because the amount of CoQ10 in the gastrointestinal content increased compared with its amount before ubiquinol-10 administration, and the increase in ubiquinol-10 amount was confirmed.

Orally administered ubiquinol-10 and ubiquinone-10 that reached the intestinal tract was considered to be absorbed in all sections of the small intestine without almost converting to ubiquinone-10 and ubiquinol-10, respectively. The sections of small intestine tissue obtained 2 h after the administration were analyzed to detect the ubiquinol-10 and ubiquinone-10 absorbed there. As a result, both ubiquinol-10 and ubiquinone-10 were considered to be absorbed throughout the small intestine tissue because the levels of CoQ10 increased in all sections of the small intestine tissue compared with under normal conditions (Fig. 3 and 5). The increased CoQ10 in the sections of small intestine tissue were detected as mainly ubiquinol-10 after administration of ubiquinol-10 and almost ubiquinone-10 after administration of ubiquinone-10 (Fig. 5). Therefore, it is considered that ubiquinol-10 and ubiquinone-10 are absorbed into small intestine tissue without conversion. A study using rat have revealed that orally administered ubiquinone-10 was detected as mainly ubiquinol-10 in mesenteric lymph.⁽¹⁷⁾ Therefore, it is possible that ubiquinone-10 absorbed into the small intestine is reduced to ubiquinol-10 during transit from enterocytes in the small intestine to mesenteric lymph. In this study, no clear differences in the total CoQ10 levels in the small intestine tissue after 2 h of administration of ubiquinol-10 and ubiquinone-10 were observed (Fig. 5). An in vitro study using Caco-2 cells showed that the basolateral secretion of ubiquinol-10 was greater than that of ubiquinone-10.⁽¹⁸⁾ Differences in the transferability from enterocyte to lymph between ubiquinol-10 and ubiquinone-10 may cause the difference in bioavailability. A further study is needed to clarify this point.

Conclusively, in this study using mice, we discovered that orally administered ubiquinol-10 and ubiqunone-10 mostly reached the small intestine without oxidizing to ubiquinone-10 and reducing to ubiquinol-10, and both were absorbed to the small intestine tissue in almost their original forms.

Conflict of Interest

The first author Hiroshi Kubo is also employed by Kaneka Corporation, which manufactures the coenzyme Q10 products.