Total and reduced/oxidized forms of coenzyme Q₁₀ in fibroblasts of patients with mitochondrial disease

Abstract

Coenzyme Q₁₀ (CoQ₁₀) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ₁₀ receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ₁₀ has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ₁₀ or the ratio of reduced to total CoQ₁₀ (reduced/total CoQ₁₀) in patients with mitochondrial diseases.

We measured reduced and oxidized CoQ₁₀ in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ₁₀ deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ₁₀ ratio.

In primary CoQ₁₀ deficiency patients, total CoQ₁₀ levels were significantly decreased, however, the reduced/total CoQ₁₀ ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ₁₀ deficiency patients, total CoQ₁₀ levels did not decrease. However, the reduced/total CoQ₁₀ ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency.

Measurement of CoQ₁₀ in fibroblasts proved useful for the diagnosis of primary CoQ₁₀ deficiency. In addition, the reduced/total CoQ₁₀ ratio may reflect the metabolic status of mitochondrial disease.

Highlights

• •Total and reduced/oxidized forms of CoQ₁₀ contents were examined in fibroblasts of patients with mitochondrial disease.
• •Measurement of CoQ₁₀ in fibroblasts is useful for the diagnosis of primary CoQ₁₀ deficiency.
• •Reduced/total CoQ₁₀ does not change in primary CoQ₁₀ deficiency.
• •Reduced/total CoQ₁₀ is higher in complex IV and V deficiency than in complex I deficiency.

1. Introduction

CoenzymeQ₁₀ (CoQ₁₀), also known as ubiquinone, is a lipophilic molecule composed of a redox-active benzoquinone head group and species-specific isoprenoid side chain (10 subunits in humans) [1,2]. CoQ₁₀ takes three forms depending on the redox state of the benzoquinone ring; oxidized (CoQ₁₀, Ubiquinone), fully-reduced (CoQ₁₀H₂, Ubiquinol), and semi-reduced (CoQ₁₀⁺, Semiubiquinone) forms [2]. It presents ubiquitously in all cellular membranes and cells [3]. The amount of CoQ₁₀ and the proportion of reduced CoQ₁₀ differ between organs and cells; CoQ₁₀ is distributed in high amounts in the heart, kidneys, liver, and muscles, and the proportion of reduced CoQ₁₀ is lower in the brain and lungs [3]. In cells, it is mostly localized in the mitochondria [3].

CoQ₁₀ has multiple functions. One of the main roles of CoQ₁₀ is as a component of the mitochondrial respiratory chain. As a mobile electron carrier, CoQ₁₀ accepts electrons from complex I and II and transfers them to complex III [2]. In the mitochondrial inner membrane, CoQ₁₀ is proposed to exist as two independent pools: a CoQ_NADH pool in the super-complex (CI, CIII, CIV) involved in the oxidation of NADH; and a CoQ_FADH pool involved in the oxidation of CII and other enzymes that use CoQ as a cofactor [4]; CoQ_NADH receives electrons from NADH, and CoQ_FADH receives electrons from FADH₂ and other enzymes, such as glycerol phosphate dehydrogenase (GPDH), choline dehydrogenase (CHDH), sulphide:quinone oxidoreductase (SQOR), dihydroorote dehydrogenase (DHODH), and electron transfer flavoprotein dehydrogenase (ETFDH), and is reduced [5]. In respiratory chain complex III, electrons are transferred from reduced CoQ₁₀ to cytochrome c in a process called the Q cycle. The Q cycle results in the oxidation of two molecules of reduced CoQ₁₀, the reduction of two molecules of cytochrome c, and the formation of one additional molecule of reduced CoQ₁₀ [2]. This normal forward electron transfer results in the creation of an electrical gradient and a pH gradient (proton gradient) between the mitochondrial matrix and intermembrane space. The energy generated by this proton motive force enables complex V(ATP synthase) to synthesize ATP. On the other hand, reverse electron transfer (RET) of CoQ₁₀ is also known to occur; in RET, electrons from reduced CoQ₁₀ are returned to complex I, reducing NAD+ to NADH, and generating ROS [6] (Supplemental Fig. 1).

Another important role of CoQ₁₀ is as an antioxidant. CoQ₁₀ is the sole lipid-soluble antioxidant that is endogenously synthesized. Reduced CoQ₁₀ inhibits both the initiation and the propagation of lipid peroxidation [7]. NADH-quinone oxidoreductase 1 and cytochrome b5 reductase, have been known to be the major oxidoreductases in the plasma membrane [8]. In addition, ferroptosis suppressor protein 1(FSP1) was also found to be an important oxidoreductase. FSP1 reduces extra-mitochondrial CoQ₁₀ and acts as a lipophilic radical-trapping antioxidant to suppress lipid peroxides, resulting in the inhibition of cell death, called ferroptosis [9,10]. Moreover, reduced CoQ₁₀ also regenerates the other antioxidants—α-tocopherol and ascorbate—into an active reduced form [7]. CoQ₁₀ is also involved in the β-oxidation of fatty acids [11], de novo pyrimidine biosynthesis [12], sulfide oxidation [13], an essential cofactor for uncoupling proteins (UCPs) [14], and modulation of the mitochondrial permeability transition pore [15]. Thus, the importance of the distinct state of CoQ₁₀ is gaining growing attention [5].

Primary CoenzymeQ₁₀ (CoQ₁₀) deficiency is an autosomal recessive mitochondrial disease caused by a decrease in CoQ₁₀ due to mutations in genes involved in CoQ₁₀ biosynthesis (COQ genes) [16]. To date, defects in at least 10 COQ genes (COQ2, COQ4, COQ5, COQ6, COQ7, COQ8A, COQ8B, COQ9, PDSS1, PDSS2) have been found to cause this disease [17]. Primary CoQ₁₀ deficiency was first reported in 1989 as familial mitochondrial encephalomyopathy [18]. Currently, over 280 patients from 180 families have been reported [17]. Secondary CoQ₁₀ deficiencies occur in a wider variety of pathologies, including mitochondrial disease [19,20]. Measuring the CoQ levels is known to be useful for diagnosing CoQ₁₀ deficiency [21]. As described above, the ratio of reduction varies among organs and cells. As a result, in addition to the total amount of CoQ₁₀, evaluating the reduced/oxidized CoQ₁₀ ratio may also be useful for further elucidating various pathophysiological states in cells.

Methods to measure CoQ₁₀ in fibroblast and the reduced/oxidized state of CoQ₁₀ have been reported [22,23]. However, to our knowledge, there are no reports on the measurement of the reduced/oxidized CoQ₁₀ ratio in primary CoQ₁₀ deficiency or mitochondrial diseases. Therefore, we decided to measure the total CoQ₁₀ levels as well as the levels of reduced and oxidized CoQ₁₀ in skin fibroblasts from patients affected by various mitochondrial diseases.

2. Material, methods, and patients

2.1. Subjects

We studied fibroblasts from 24 patients with mitochondrial disease, including primary CoQ₁₀ deficiency (Table 1). The inclusion criterion for patients with primary CoQ₁₀ deficiency was childhood onset with biallelic pathogenic variants in the COQ gene encoding proteins for the biosynthesis of CoQ₁₀ [16]. Fibroblasts were obtained from patients at Kanagawa Children's Medical Center, Chiba Children's Medical Center, and Jichi Medical University under the approval of the Ethics Committee of Jichi Medical University. Written informed consent was obtained from the parents of each patient.

Table 1.

Fibroblast cell lines from patients with mitochondrial disease, including primary CoQ₁₀ deficiency.

Case [ref]	diagnosis	DNA mutation	variants, heteroplasmy ratea	function
1	primary CoQ10 deficiency	COQ2	c.[349G > C];[912 + 1G > del]	CoQ10 biosynthesis
2 [24]	primary CoQ10 deficiency	COQ4	c.[718C > T];[421C > T]	CoQ10 biosynthesis
3 [25]	primary CoQ10 deficiency	COQ4	c.[431C > A];[718C > T]	CoQ10 biosynthesis
4	primary CoQ10 deficiency	COQ4	c.[190C > T];[479G > A]	CoQ10 biosynthesis
5	primary CoQ10 deficiency	COQ4	c.[190C > T];[479G > A]	CoQ10 biosynthesis
6 [26]	Leigh syndrome	NDUFA1	c.[55C > T], 100% (X-linked)	Respiratory chain subunits, complex I
7 [26]	Leigh syndrome	MT-ND3	m.10158 T>C, heteroplasmic (F; 90%)	Respiratory chain subunits, complex I
8 [27]	neonatal cardiomyopathy	MT-ND5	m.13513G > A, heteroplasmic (F; 78.87%)	Respiratory chain subunits, complex I
9	infantile mitochondrial disease	MT-ND5	m.13513G > A, heteroplasmic (B; 77%)	Respiratory chain subunits, complex I
10 [28]	Leigh syndrome	MT-ND5	m.13513G > A, heteroplasmic (F; 26%)	Respiratory chain subunits, complex I
11	mitochondrial cardiomyopathy	ACAD9	c.[811 T > G];[1766-2A > G]	Respiratory chain assembly factor, complex I
12 [28]	non-lethal infantile mitochondrial disease	ACAD9	c.[1150G > A];[1817 T > A]	Respiratory chain assembly factor, complex I
13 [29]	Leigh syndrome	SURF1	c.[743C>A], homoplasmy	Respiratory chain assembly factor, complex IV
14 [25]	Leigh syndrome	SURF1	c.[367_368delAG];[572delC]	Respiratory chain assembly factor, complex IV
15 [25]	Leigh syndrome	MT-ATP6	m.8993 T > G, homoplasmy	Respiratory chain subunits, complex V
16 [30]	mtDNA depletion syndrome	DGUOK	c.[143-307_170del335];[143-307_170del335]	Deoxynucleotide triphosphate synthesis
17 [30]	mtDNA depletion syndrome	MPV17	c.[451dupC];[308_310del]	mitochondrial protein synthesis
18 [30]	mtDNA depletion syndrome	MPV17	c.[148C > T];[149G > A]	mitochondrial protein synthesis
19	Kearns-Sayre syndrome		Single mtDNA deletion (5513 bp del; m.8290–13,802)
20 [26]	MELAS	(tRNA-Leu)	m.3243 A > G, heteroplasmic (F; 21%)	Mitochondrial tRNA
21 [26]	MELAS	(tRNA-Trp)	m.5541C > T, heteroplasmic (F; 49%)	Mitochondrial tRNA
22 [31]	ECHS1 deficiency	ECHS1	c.[832G > A]	Metabolism of toxic compounds
23 [28]	cardiomyopathy	BOLA3	c.[287A > G];[287A > G]	Iron‑sulfur protein assembly
24	cardiomyopathy	BOLA3	c.[287A > G];[287A > G]	Iron‑sulfur protein assembly

^aF; fibroblasts, B; blood

We obtained fibroblasts from five patients with primary CoQ₁₀ deficiency. One patient carries biallelic COQ2 variants with the c.[349G > C];[912 + 1G > del] (Case 1). Four patients had biallelic COQ4 mutations: one with compound heterozygous biallelic variants c.[718C > T];[421C > T] (Case 2) [24], one with c.[431C > A];[718C > T] (Case 3) [25], and two with c.[190C > T];[479G > A] (Cases 4, 5).

Ten patients had disorders of mitochondrial respiratory chain subunits. Seven patients had mutations related to complex I: one with a c.[55C > T] mutation in NDUFA1 (Case 6) [26], one with an m.10158 T>C mutation in MT-ND3 (Case 7) [26], Three with an m.13513G > A mutation in MT-ND5 (Case 8) [27], (Case 9), (Case 10) [28], one with a c.[811 T > G];[1766-2A > G] mutation in ACAD9 (Case 11), and one with a c.[1150G > A];[1817 T > A] mutation in ACAD9 (Case 12) [28]. NDUFA1, MT-ND3, and MT-ND5 are subunits of complex I, and ACAD9 is the assembly factor of complex I. Two patients had mutations related to complex IV: one with a c.[743C>A] mutation in SURF1 (Case 13) [29], and one with a c.[367_368delAG]; [572delC] mutation in SURF1 (Case 14) [25]. SURF1 is the assembly factor of complex IV. One patient had an m.8993 T > G mutation in MT-ATP6 (Case 15) [25]. MT-ATP6 is a subunit of complex V.

Three patients had mitochondrial DNA (mtDNA) depletion syndrome: one with a c.[143-307_170del335];[143-307_170del335] mutation in DGUOK (Case 16) [30], one with a c.[451dupC];[308_310del] mutation (Case 17) [30] and one with a c.[148C > T];[149G > A] mutation in MPV17 (Case 18) [30]. Both DGUOK and MPV17 are involved in the maintenance of mtDNA. One patient had Kearns-Sayre syndrome with a single mtDNA deletion (5513 bp del; m.8290–13,802) (Case 19). Two patients had MELAS: one with an m.3243 A > G mutation of tRNA-Leu (Case 20) [26] and one with an m.5541C > T, mutation of tRNA-Trp (Case 21) [26]. One patient had short-chain enoyl-CoA hydratase (ECHS1) deficiency with heterozygous mutations in maternal c.[832G > A] in ECHS1 (Case 22) [31]. ECHS1 plays a role in valine and fatty acid catabolism in mitochondria. Two patients had c.[287A > G]; [287A > G] mutation in BOLA3 (Cases 23) [28],(Case 24). BOLA3 is related to iron‑sulfur cluster production and is involved in the assembly of the mitochondrial respiratory chain complex.

Five fibroblasts from healthy individuals were purchased: two fibroblasts from the PromoCell Company (#C-12300, GmbH, Heidelberg, Germany), two fibroblasts from Japanese Collection of Research Bioresources Cell Bank (#TIG-120, #HT-2020, Japan), and fibroblasts from Lonza Japan (#CC-2509, Tokyo, Japan). Another five fibroblasts from patients without mitochondrial disease were used as controls. Cells from passages 4–29 were used for assays.

2.2. Cell culture and growth conditions

The fibroblasts were maintained in 1.0 g/L low glucose Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C under 5% CO₂.

2.3. CoQ₁₀ measurement in fibroblasts

2.3.1. CoQ extraction

The methods for the extraction CoQ₁₀ were based on a previously reported method with slight modifications [32]. To extract CoQ₁₀ from fibroblast in 60 mm dishes, cells were washed twice with PBS, and pellets were re-suspended in 500 μL of lysis buffer (0.25 mM Sucrose, 2 mM EDTA, 10 mM Tris, and 100 UI/mL heparin, pH 7.4.), and sonicated twice for 5 s. These homogenates were also used to citrate synthase and protein quantification. To measure CoQ₁₀, nine hundred microliters of ethanol containing internal standard CoQ₁₀-d9 (IsoSciences, Ambler, PA) and 20 μM tert-butyl hydroquinone (TBHQ) (FUJIFILM Wako, Osaka, Japan) was added to 100 μL of homogenates. TBHQ was added to prevent oxidation of reduced CoQ_10. The cell suspensions were vortexed and centrifuged at 15,700 ×g for 10 min (4 °C).

2.3.2. Reduction of ubiquinone

Reduced CoQ₁₀ was required for use in the calibration curve measurement. However, since reduced CoQ₁₀ is easily oxidized, reduced CoQ₁₀ was prepared just before the analysis by reducing oxidized CoQ₁₀ following a previously reported method with slight modification [33]. Briefly, 50 μL of CoQ₁₀ was diluted in 1.95 mL hexane in a glass tube. Twenty milligrams of NaBH₄ was added and followed by the addition of 100 μL methanol, vortexed for 3 min, then placed in the dark for 5 min at room temperature. After reduction, 1 mL of water containing 100 μM EDTA was added to stop the reaction, vortexed for 1 min, and centrifuged 1500 ×g for 5 min at 4 °C. The upper layer containing reduced CoQ₁₀ was transferred to a glass tube.

2.3.3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

The method for measuring reduced and oxidized CoQ₁₀ was based on the previously reported method with slight modifications [23]. An LC-MS/MS analysis was performed on an LC-electrospray ionization-MS (LC-ESI-MS) with triple quadrupole (Nexera X2 and LCMS-8060, Shimadzu, Kyoto, Japan). A Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm, Phenomenex) and a guard column filled with the same packing material were used. The column temperature was kept at 40 °C. The mobile phase was isocratic with 2 mM ammonium formate in methanol. The flow rate was 0.8 mL/min, the injection volume was 2 μL, and the run time was 6 min. The interface temperature was 300 °C, the desolvation line temperature was 250 °C, and the heat block temperature was 400 °C. The nebulizing gas flow was 3 L/min, the heating gas flow was 10 L/min, and the drying gas flow was 10 L/min. The samples were kept at 4 °C before injection by the autosampler. The MS/MS conditions for each target were optimized using the automated multiple reaction monitoring (MRM) optimization procedures in LabSolutions (Shimadzu). The MRM used for quantification was m/z 880.5 > 197.1 for oxidized CoQ₁₀, 882.4 > 197.0 for reduced CoQ₁₀, and 890.4 > 206.2 for CoQ₁₀-d9 (internal standard). Standards and samples were quantified using the LabSolutions software program to determine the peak area for oxidized CoQ₁₀, reduced CoQ₁₀, CoQ₁₀-d9, and the standard curves were used to determine the total amount of CoQ present in the samples.

Intra-assay coefficients of validation (CVs) and relative errors (REs), as measurements of precision and accuracy, respectively, were determined in five parallel analyses of the same cell. To evaluate inter-assay precision and accuracy, one cell line was independently evaluated on three different days. Precision was calculated as (standard deviation/mean concentration) × 100 (%), and accuracy was calculated as (quantitative value/theoretical value) × 100 (%). The intra-assay precision (CV) of reduced CoQ₁₀ and oxidized CoQ₁₀ was 0.58% and 1.39%, respectively. The intra-assay accuracy (RE) of reduced CoQ₁₀ and oxidized CoQ₁₀ was 4.20% and 2.50%, respectively. The inter-assay precision (CV) of reduced CoQ₁₀ and oxidized CoQ₁₀ was 1.27% and 1.84%, respectively. The intra-assay accuracy (RE) of reduced CoQ₁₀ and oxidized CoQ₁₀ was 10.54% and 1.18%, respectively (Supplemental Table 1, QC1).

2.3.4. Citrate synthase and protein quantification

Fibroblast CoQ₁₀ levels were expressed as citrate synthase (CS) activity (measured CoQ₁₀ values/CS units, nmol/CS units). CS activity was measured spectrophotometrically referring to the method described by Srere (1969), with 0.1 mM DTNB, 0.3 mM Acetyl-CoA, 0.5 mM Oxaloacetate, and 12–20 μg protein in 200 μL total incubation volume. CS units are determined as follows: CS Units (μmol/min/mL) = (ΔA₄₁₂/min x V (mL) × dil)/ 13.6 × L (cm) × V_enz (mL), V (mL); the reaction volume, dil; the dilution factor of the original sample, 13.6(mM⁻¹ cm⁻¹); the extinction coefficient of TNB at 412 nm, L (cm); pathlength for absorbance measurement (0.552 cm), V_enz(mL); the volume of the enzyme sample. Protein concentrations were quantified using Qubit™ Protein Assay Kits and a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA).

2.4. Statistical analyses

Statistical analyses were performed using the GraphPad Prism software program (version 9.01, GraphPad Software Inc., La Jolla, CA). Comparisons between samples were performed using a one-way ANOVA. The results are expressed as the mean (standard deviation). P values of <0.05 were considered to indicate statistical significance.

3. Results

3.1. Total CoQ₁₀ levels were observed to decrease in all patients with primary CoQ₁₀ deficiency

We showed the reduced, oxidized, and total (sum of reduced and oxidized) CoQ₁₀ values corrected for the CS unit (nmol/CS unit) (Table 2). In addition, we also measured the CoQ₁₀ values corrected for protein levels (nmol/g protein) (Supplemental Table 2). Six patients showed decreased the total CoQ₁₀ values (<70% of the control): five with primary CoQ₁₀ deficiency (Cases 1–5), and one with Kearns-Sayre syndrome (Case 19) (Table 2). The total CoQ₁₀ levels were significantly decreased in primary CoQ₁₀ deficiency than in controls (primary CoQ₁₀ deficiency(n = 5)1.00 ± 0.19 nmol / CS unit (mean ± SD), controls (n = 10)2.30 ± 0.24 nmol / CS unit, p < 0.0001) (Fig. 1). However, total CoQ₁₀ levels were the same in mitochondrial disease other than primary CoQ₁₀ deficiency as in controls (mitochondrial disease(n = 19)2.23 ± 0.26 nmol / CS unit, p = 0.93).

Table 2.

Reduced and oxidized CoQ₁₀ values and total CoQ deficiency in fibroblasts.

Case	reduced CoQ10		oxidized CoQ10		total CoQ10 (nmol/CS unit)		% CoQ deficiency (%)
	mean	SD	mean	SD	mean	SD
1	0.37	0.15	0.37	0.02	0.74	0.13	32
2	0.12	0.00	0.16	0.00	0.28	0.00	12
3	0.57	0.45	0.59	0.18	1.16	0.63	50
4	0.64	0.03	0.74	0.20	1.38	0.17	60
5	0.78	0.01	0.64	0.02	1.42	0.01	62
6	0.48	0.01	1.65	0.00	2.13	0.01
7	1.03	0.04	1.00	0.32	2.03	0.29
8	1.22	0.04	0.82	0.18	2.04	0.22
9	1.40	0.54	0.96	0.03	2.36	0.51
10	0.86	0.16	1.34	0.30	2.20	0.46
11	1.02	0.08	2.17	0.53	3.19	0.44
12	1.18	0.56	1.31	0.17	2.49	0.38
13	1.33	0.03	0.57	0.02	1.90	0.00
14	1.77	0.31	0.58	0.10	2.35	0.21
15	1.54	0.09	0.71	0.00	2.25	0.09
16	0.86	0.02	1.46	0.01	2.32	0.01
17	1.37	0.40	1.28	0.14	2.65	0.25
18	1.01	0.20	1.52	0.08	2.53	0.29
19	0.57	0.01	0.72	0.02	1.29	0.02	56
20	1.41	0.40	0.51	0.11	1.92	0.51
21	1.24	0.28	0.65	0.28	1.89	0.56
22	1.19	0.11	0.87	0.23	2.06	0.34
23	0.81	0.19	1.84	0.46	2.65	0.27
24	0.92	0.01	1.23	0.00	2.15	0.01
Reference (n = 10)	1.19	0.15	1.11	0.10	2.30	0.24

% CoQ deficiency: <70% of control CoQ₁₀ value.

Fig. 1.

Total CoQ₁₀ values.

Total (sum of reduced and oxidized) CoQ₁₀ values of mitochondrial patient fibroblasts. CoQ₁₀ deficiency (CoQ₁₀ levels <70% of control CoQ₁₀) was found in six cases. All cases of primary CoQ₁₀ deficiency showed decreased CoQ₁₀ levels. In mitochondrial disease, CoQ₁₀ values were not decreased, with the exception of Case 19 (Kearns-Sayre syndrome). Data are expressed as **** P < 0.0001, and n.s. indicates no significance.

3.2. The reduced/total CoQ₁₀ ratio was unchanged in primary CoQ₁₀ deficiency, but was higher in complex IV or V deficiency

We showed the ratio of reduced CoQ₁₀ to total CoQ₁₀ (reduced/total CoQ₁₀) of fibroblasts (Table 3). The ratio of reduced and oxidized CoQ₁₀ to total CoQ₁₀ in control fibroblasts was 52% and 48%, respectively (Table 3). In primary CoQ₁₀ deficiency, the reduced/total CoQ₁₀ ratio did not change compared to the control (reduced/total CoQ₁₀ ratio of primary CoQ₁₀ deficiency (n = 5) 49 ± 7% (mean ± SD), controls (n = 10) 52 ± 1%, p = 0.92) (Fig. 2). Regarding the cases with respiratory chain complex deficiency, there was no difference between control and complex I deficiency (reduced/total CoQ₁₀ ratio of complex I deficiency (n = 7) 44 ± 7%, p = 0.37). However, the reduced/total CoQ₁₀ ratio in complex IV or V deficiency was increased in comparison to the control (complex IV or V deficiency (n = 3) 71 ± 3%, p = 0.022). In addition, the reduced/total CoQ₁₀ ratio in complex IV or V deficiency was higher in comparison to complex I deficiency and primary CoQ₁₀ deficiency (complex IV or V deficiency vs. complex I deficiency: p = 0.0021, complex IV or V deficiency vs. primary CoQ₁₀ deficiency: p = 0.015).

Table 3.

Ratio of reduced / total CoQ₁₀.

Case	reduced/total CoQ10 (%)		Reduced/total CoQ10 in cases versus reduced/total CoQ10 in controls (%)
	mean	SD
1	50	11	96
2	43	1	83
3	49	14	95
4	47	8	90
5	55	1	106
6	23	0	44
7	51	9	97
8	60	4	115
9	59	10	114
10	39	1	75
11	32	7	61
12	47	15	91
13	70	1	135
14	75	7	145
15	68	1	131
16	37	1	71
17	52	10	100
18	40	3	77
19	44	0	85
20	74	1	142
21	66	5	127
22	58	4	111
23	30	10	59
24	43	0	83
Reference (n = 10)	52	1

The reduced/total CoQ₁₀ ratio decreased (< 80% of control value); cases 6, 10, 11, 16, 18, 23.

The reduced/total CoQ₁₀ ratio increased (120% < of control value); cases 13, 14, 15, 20, 21.

Fig. 2.

Comparison of the reduced/total CoQ₁₀ ratio.

Comparison of the reduced/total CoQ₁₀ ratio in cases with primary CoQ₁₀ deficiency, complex I deficiency, and complex IV or V deficiency. In primary CoQ₁₀ deficiency, the reduced/total CoQ₁₀ ratio was the same as that of the controls. In complex I deficiency, 3/7 cases showed a decreased (< 80% of control value) reduced/total CoQ₁₀ ratio. In complex IV or V deficiency, 3/3 cases showed an increased (120% < of control value) reduced/total CoQ₁₀ ratio. Data are expressed as *P < 0.05, **P < 0.01, and n.s. indicates no significance.

In individual cases, the reduced/total CoQ₁₀ ratio decreased (<80% of the reduced/total CoQ₁₀ ratio in the control) in six cases; three cases with complex I deficiency (Cases 6, 10, 11), two cases with mtDNA depletion syndrome (Cases 16, 18), one case with BOLA3 mutation (Case 23)(Table 3). On the other hand, the reduced/total CoQ₁₀ ratio increased (>120% of the reduced/total CoQ₁₀ ratio in the control) in five cases; two cases with complex IV deficiency (Cases 13, 14), two cases with MELAS(Cases 20, 21), one case with complex V deficiency (Case 15) (Table 3).

4. Discussion

We showed here that the total CoQ₁₀ values of fibroblasts were significantly lower in patients with primary CoQ₁₀ deficiency. In this syndrome, early recognition and therapy can stop progression and improve the prognosis; however, established severe symptoms cannot be reversed [16,34]. Biochemical measurements of low CoQ₁₀ levels in muscle biopsy have been utilized for the diagnosis of this syndrome [16]. Moreover, the identification of biallelic pathogenic variants in the COQ genes, which encode proteins involved in coenzyme Q biosynthesis, enables a definitive diagnosis [16]. However, the invasiveness of muscle biopsy hampers this procedure and can delay the diagnosis. CoQ₁₀ levels in fibroblasts were examined and implicated in their usefulness [21]. Our data with LC-MS/MS for the measurement of CoQ₁₀ from fibroblasts also supported the usefulness for detecting CoQ₁₀ deficiency [32,35]. The fibroblasts from Case 2 showed the lowest CoQ₁₀ concentration and showed a very severe phenotype. A correlation between CoQ₁₀ levels and phenotype has been suggested [24,36,37]. Therefore, the CoQ₁₀ value from skin fibroblast may reflect the clinical severity.

In addition to primary CoQ₁₀ deficiency, various mitochondrial diseases have also been reported to decrease CoQ₁₀ in fibroblasts and muscle, particularly in mtDNA depletion syndrome [20,38]. However, in our analysis, three patients with mtDNA depletion, including DGUOK and MPV17 mutations, showed no decrease in CoQ₁₀ (Cases 16–18). Only one patient with large deletions of mtDNA showed markedly decreased CoQ₁₀ (Case 19). Therefore, decreased CoQ₁₀ was only a constant feature in primary CoQ₁₀ deficiency syndrome in our analysis. Our results support the widely accepted idea that early CoQ₁₀ therapy should is therefore indicated if decreased levels of CoQ₁₀ are found in the fibroblasts of patients with suspected mitochondrial disease.

To our knowledge, this is the first report to describe the reduced/total CoQ₁₀ ratios in patients with mitochondrial diseases, including primary CoQ₁₀ deficiency. In primary CoQ₁₀ deficiency, the reduced/total CoQ₁₀ ratio did not change. On the other hand, the reduced/total CoQ₁₀ ratio was decreased in 3/7 of cases of complex I deficiency and was increased in 3/3 of cases of complex IV or V deficiency. Primary CoQ₁₀ deficiency is caused by impaired CoQ₁₀ biosynthesis, which results in a decrease in the absolute value of CoQ₁₀; however, as expected, our data suggest that it does not affect the redox reaction in mitochondria. Since CoQ₁₀ changes from the oxidized form to the reduced form by accepting electrons from complex I, complex II and other dehydrogenases, the reduced/total CoQ₁₀ ratio is expected to decrease in complex I deficiency. However, only 3 out of 7 complex I patients in our cohort showed disturbed Q reduced/total ratios. In contrast, CoQ₁₀ changes from the reduced form to the oxidized form in complex III, and the reduced/total CoQ₁₀ ratio is expected to increase in complex III and later complex deficiencies (Supplemental Fig. 1, left panel). In fact, the reduced CoQ₁₀ ratio was significantly decreased with complex I inhibitor, whereas the reduced CoQ₁₀ ratio was increased with complex IV inhibitor [23]. Some (but not all) of our results support these observations. Among complex I deficiency patients, patients with isolated complex I deficiency tended to have a decreased reduced/total CoQ₁₀ ratio (3/4 cases; Cases 6, 10, 11) (Supplemental Table 3). On the other hand, the reduced/total CoQ₁₀ ratio was unchanged in a patient who also had a decreased complex III and IV enzyme activity (Case 8), in a patient without a decreased CI enzyme activity in fibroblasts (Case 12), and in a patient with a decreased CI enzyme activity in muscle (Case 7).

In recent years, it has been reported that the reduced/oxidized CoQ₁₀ ratio may be a marker of mitochondrial metabolic status [39]. In situations where CoQ_FADH can be excessively reduced, RET is induced, and the production of reactive oxygen species (ROS) from complex I is stimulated, which causes complex I destruction [4,6]. On the other hand, oxidizing the CoQ pool by alternative oxidase (AOX) of Ciona intestinalis xenotopically expressed in mouse mitochondria induces forward electron transport (FET) from RET [40]. Our system of measuring the reduced/total CoQ₁₀ ratio of skin fibroblasts provide the amount and redox states of CoQ₁₀.

As a limitation of our study, CoQ₁₀ is mostly localized in the mitochondria in subcellular fractions but it has also been shown to localize in Golgi, lysosomes, and other organelles [3]. In this study, we measured the whole cell CoQ₁₀ level without separating the mitochondrial and non-mitochondrial fractions. Moreover, we examined an only limited number of patients affected by only some mitochondrial diseases, which can be caused by >400 gene mutations [41].

In conclusion, we measured the reduced/total CoQ₁₀ ratio in fibroblasts from a cohort of patients with mitochondrial disease for the first time. The reduced/total CoQ₁₀ ratio tended to show no change in many of the cells that we measured. However, the reduced/total CoQ₁₀ ratio was increased in complex IV or V deficiency, while the reduced/total CoQ₁₀ ratio tended to decrease in some cases of complex I deficiency.

The following are the supplementary data related to this article.

Supplemental Fig. 1

Electron transfer in mitochondria. (A) Forward electron transfer. CoQ_NADH receives electrons from NADH and CoQ_FADH receives electrons from FADH₂ and other enzymes, and goes from the oxidized form to the reduced form. In complex III, electrons are transferred from reduced CoQ₁₀ to cytochrome c and CoQ₁₀ becomes the oxidized form. (B) Reverse electron transfer: electrons from excess reduced CoQ₁₀ are returned to complex I and reduced NAD+ to NADH. As a result, reactive oxygen species occur. GPDH, glycerol-3-phosphate dehydrogenase; CHDH, choline dehydrogenase; SQOR, sulphide:quinone oxidoreductase; DHODH, dihydroorote dehydrogenase; ETFDH, electron transfer flavoprotein dehydrogenase.

Supplemental Table 1

Precision and accuracy of reduced and oxidized CoQ in fibroblasts and neat diluent.

Supplemental Table 2

Reduced and oxidized CoQ₁₀ values corrected for protein levels and total CoQ deficiency in fibroblasts.

Supplemental Table 3

Results of enzyme assay for mitochondrial respiratory chain complexes in fibroblasts or muscle biopsy with ComplexIdeficiency.

Determination of enzyme activities

Author contributions

CW, HO contributed to the conceptualization and performance of the statistical analysis of the data and wrote the manuscript; MW, AM, EFJ, TT, HU, OT, ET, and KA performed sampling and data acquisition; YK, YO performed genetic testing; KM, AO recruited patients, provided clinical information, collected samples; TY conducted supervision of the project. All authors read and approved the final manuscript.

Ethics

All procedures followed were by the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of the World Medical Organization. This study was approved by the Ethics Committee of Jichi Medical University and written informed consent was obtained from all participants.

Funding

This work was supported in part by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, AMED to H·O (JP21im0210625, JP21ek0109511), K.M (JP21ek0109468, JP19ek0109273) and Y·O (JP21kk0305015). Health and Labor Sciences Research Grant to H·O (JP21FC1015). The Acceleration Program for Intractable Diseases Research utilizing Disease-specific iPS cells to H·U (JP22bm0804018). JSPS KAKENHI to H. O. (JP20H03648).

CRediT authorship contribution statement

Chika Watanabe: Conceptualization, Writing – original draft. Hitoshi Osaka: Conceptualization, Writing – review & editing, Funding acquisition. Miyuki Watanabe: Investigation. Akihiko Miyauchi: Investigation. Eriko F. Jimbo: Investigation. Takeshi Tokuyama: Resources. Hideki Uosaki: Resources. Yoshihito Kishita: Resources. Yasushi Okazaki: Resources. Takanori Onuki: Resources. Tomohiro Ebihara: Resources. Kenichi Aizawa: Resources. Kei Murayama: Resources. Akira Ohtake: Resources. Takanori Yamagata: Supervision.

Declaration of Competing Interest

None.

Data availability

No data was used for the research described in the article.