Recent progress in studies on the health benefits of pyrroloquinoline quinone

Abstract

Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was identified initially as a redox cofactor for bacterial dehydrogenases. Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues because of its wide distribution in dietary sources. Importantly, nutritional studies in rodents have revealed that PQQ deficiency exhibits diverse systemic responses, including growth impairment, immune dysfunction, and abnormal reproductive performance. Although PQQ is not currently classified as a vitamin, PQQ has been implicated as an important nutrient in mammals. In recent years, PQQ has been receiving much attention owing to its physiological importance and pharmacological effects. In this article, we review the potential health benefits of PQQ with a focus on its growth-promoting activity, anti-diabetic effect, anti-oxidative action, and neuroprotective function. Additionally, we provide an update of its basic pharmacokinetics and safety information in oral ingestion.

Graphical abstract

Structure and redox reaction of pyrroloquinoline quinone (PQQ).

Key words:

• pyrroloquinoline quinone (PQQ)
• redox cofactor
• anti-diabetes
• anti-oxidant
• neuroprotection

Pyrroloquinoline quinone (PQQ, Fig. 1) is an aromatic tricyclic o-quinone that serves as a redox cofactor of a number of prokaryotic dehydrogenases, such as alcohol and sugar dehydrogenases.^1,2) More recently, the first eukaryotic PQQ-dependent sugar oxidoreductase has been discovered in a mushroom, the basidiomycete Coprinopsis cinerea.³⁾ Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues at picomolar to nanomolar levels,⁴⁾ and an especially large amount has been found in human milk⁵⁾ because of its presence in daily foods, including vegetables and meats.^6–8) PQQ is a ubiquitous molecule that influences a multitude of physiological and biochemical processes and has been established to be beneficial for growth and stress tolerance in both bacteria and higher organisms.^9,10) Most importantly, nutritional studies have revealed that PQQ deficiency in mice and rats exhibits various systemic responses, including growth impairment, compromised immune responsiveness, abnormal reproductive performance, and reduced respiratory quotient.^11–13) Moreover, in 2003, Kasahara and Kato reported that PQQ could qualify as a newcomer to the B group of vitamins.¹⁴⁾ These authors cloned a presumed mouse homolog (U26) of the yeast gene, 2-aminoadipic acid reductase (LYS2), and proposed that U26 could be involved in the metabolic degradation of dietary lysine, acting as a PQQ-dependent 2-aminoadipic 6-semialdehyde dehydrogenase, because U26 contained the putative PQQ-binding motif that is conserved among bacterial PQQ-dependent dehydrogenases.¹⁴⁾ However, claims for a mammalian vitamin have been questioned because conclusive evidence for the existence of a mammalian PQQ-dependent enzyme is lacking.^15,16) Although currently there remains controversy over whether PQQ is indeed an essential vitamin in mammals, PQQ has been discovered to have a diverse range of physiological properties that could be beneficial to human health over the last decade. The objectives of this review were to, first, present an overview of the recent insights gained on the potential health benefits of PQQ in anti-diabetic, anti-oxidative, and neuroprotective actions, and second, update its metabolism and safety information in pharmacological applications.

Fig. 1. Structure and redox reaction of pyrroloquinoline quinone (PQQ).

I. Chemical nature of PQQ

PQQ (4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid) is a redox active o-quinone that can be reversibly reduced to pyrroloquinoline quinol (4,5-dihydroxy-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid, PQQH₂) through a semiquinone intermediate (Fig. 1).¹⁷⁾ It has been demonstrated that PQQ stably acts as an efficient electron transfer catalyst from a number of organic substrates to molecular oxygen (O₂), constructing quinoprotein model reactions. In the presence of ascorbate, NAD(P)H, and thiol compounds such as glutathione, PQQ undergoes a two-electron reduction to form PQQH₂.^18–20) Subsequently, the generated PQQH₂ is oxidized back to the original quinone via the reduction of two equivalents of O₂ to super oxide anion (${\text{O}}_2^{-}$), which spontaneously or enzymatically dismutates to hydrogen peroxide (H₂O₂).²¹⁾ It is noteworthy that PQQ has the ability to catalyze continuous redox cycling so that picomole amounts of PQQ are capable of generating micromolar amounts of product.^17,22) Meanwhile, PQQ can exert pro-oxidant actions by the formation of reactive oxygen species (ROS), such as ${\text{O}}_2^{-}$ and H₂O₂, via its redox cycling under certain conditions and induce oxidative protein modifications, including the oxidation of cysteinyl thiols.²³⁾ PQQ also catalyzes the oxidation of primary amines, including the ε-amino group of lysine residues in elastin and collagen, via Schiff base formation under aerobic conditions.²⁴⁾ Elastin oxidation by PQQ in the presence of Cu²⁺ results in the formation of 2-aminoadipic semialdehyde residues and eventually its derived covalent cross-links. On the other hand, PQQ easily reacts with amino acids to form imidazole derivatives, such as imidazolopyrroloquinoline quinone, in biological samples, and these derivatives are biologically active in some cases.^5,8) The protonated form of PQQ shown in Fig. 1 dissolves only slightly in water, and the tricarboxylic acid of PQQ dissociates in neutral pH water. Therefore, PQQ disodium salt (PQQ Na₂) is generally used in various examinations because of its high solubility in waster. The atomic geometry of PQQ Na₂ (commercially available as BioPQQ™, a trademark of Mitsubishi Gas Chemical Co., Inc., (Tokyo, Japan)) was resolved using single-crystal X-ray structure analysis as PQQ Na₂ tri-hydrate.²⁵⁾ PQQ Na₂ has an advantage in the application to various experiments compared with free-form PQQ, because it can be handled easily owing to its water-soluble property. BioPQQ™, chemically well-characterized, was used in the following studies.

II. Natural source of PQQ

It is well known that PQQ is distributed ubiquitously in nature and found in numerous dietary sources, including fermented soy beans (natto), tea, green peppers, parsley, kiwi fruit, and human milk.^5,6) Various methods for instrumental analyses and bioassays for PQQ have been developed, but the PQQ content in foods varies in different reports because PQQ is chemically reactive and prone to form derivatives or condensation products with other nutrients.^5,6,8,26,27) Kumazawa et al. have developed a method based on gas chromatography/mass spectrometry (GC/MS) with isotopic dilution for free PQQ after derivatization with phenyltrimethylammonium hydroxide.^4,6,7) Using this analytical method, the levels of free PQQ in various foods, including vegetables, fruits, and teas, were determined to be in the range of 3.7–61 ng/g wet weight or ng/mL in liquid foods. Recent analyses of PQQ using a reliable liquid chromatography/electrospray-ionization tandem mass spectrometry (LC/MS/MS) method elucidated that free PQQ was present in various food samples in the range of 0.19–7.02 ng/g fresh weight or ng/mL in liquid foods.⁸⁾ Based on available food composition data,^4–7) it is estimated that humans consume 0.1–1.0 mg PQQ and its derivatives per day.²⁸⁾

The biosynthesis of PQQ in higher organisms has not been shown, and therefore, the major source of PQQ in these organisms, including plants and animals, is believed to be derived from microorganisms. The details of PQQ biosynthesis have not been resolved yet, but a putative pathway has been proposed on the basis of the functions of conserved genes in numerous bacteria.²⁹⁾ The majority of PQQ-producing bacteria contain six genes (pqqABCDEF) in an operon.^10,30) These genes have been expressed in Escherichia coli, a non-PQQ producer and lead to the production of PQQ.^31,32) Genetic knockout studies of each of these genes show that four of the six gene products (PqqA, PqqC, PqqD, and PqqE) are absolutely required for PQQ production.^32,33) In all cases, pqqA encodes a small polypeptide, typically 20–30 amino acids in length, containing a conserved glutamate and tyrosine that serve as the backbone in PQQ biogenesis.^34,35) The glutamate and tyrosine undergo post-translational modifications to form the intermediate 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ).^32,34) PqqC is the most characterized and has been shown to catalyze the eight-electron oxidation and ring cyclization of AHQQ to form PQQ.³⁵⁾ A large number of bacteria extracellularly excrete PQQ and is indicated as micrograms per mL of broth culture.³⁶⁾ On the other hand, common strains of bacteria in the human intestinal tract appear to synthesize little PQQ,^37,38) and hence, it seems that dietary intake is the major source of PQQ in the human body.

III. Growth-promoting activity

Although no enzymes in animals have been identified that exploit PQQ as a cofactor, PQQ has been shown to be essential for normal growth and development in animals. When PQQ is omitted from a chemically defined diet fed to mice and rats, various systemic responses are observed including growth impairment, immune dysfunction, decreased reproductive performance, and reduced respiratory quotient.^11–14) Oral supplementation of PQQ (above 300 ng/g diet) improves reproduction and enhances neonatal rates of growth compared with the response from diets devoid of PQQ.¹²⁾ More recently, dietary supplementation of PQQ Na₂ in broiler chicks has been shown to improve growth performance, carcass yield, immunity, and plasma status.³⁹⁾ Thus, this unique compound is characterized as an important growth factor or putative essential nutrient in animals, whereas the nutritional benefits of PQQ for human growth and development are still unknown. Although the detailed mechanism of PQQ action in animals still remains unclear, the ability to carry out continuous redox cycling suggests a role for PQQ as a cofactor, redox signaling molecule, or anti-oxidant.

In cultured human and mouse cells, PQQ also functions as a potential growth factor to promote cell proliferation when added to culture media.^40–42) PQQ enhances the incorporation of [³H]-thymidine into human skin fibroblasts cultured in medium containing PQQ at concentrations as low as 3 nM. Kumazawa et al. have observed that PQQ treatment stimulates activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) in c-Ha-ras transformed NIH/3T3 mouse fibroblasts, resulting in increased cell proliferation.⁴¹⁾ ERK, one of the mitogen-activated protein kinases, activates transcription in the ras-signaling pathway and plays a pivotal role in cell proliferation and survival.⁴³⁾ This signal transduction by sequential phosphorylation often is initiated by the binding of peptide growth factors to receptor tyrosine kinases (RTKs). Recently, we showed that PQQ also significantly enhanced proliferation of human epithelial A431 cells at concentrations above 10 nM.⁴²⁾ Moreover, we found that PQQ induces the activation (tyrosine autophosphorylation) of epidermal growth factor receptor (EGFR), a RTK of the ErbB family, and its downstream target ERK 1/2 in a ligand-independent manner. The activation of the ERK pathway accompanying EGFR phosphorylation via binding of EGF plays a prominent role in the proliferation of epithelial cells. On the other hand, EGFR signaling is negatively regulated by protein tyrosine phosphatase 1B (PTP1B), which catalyzes tyrosine dephosphorylation of activated EGFR, and the inhibition of PTP1B has been reported to evoke a ligand-independent activation of EGFR.^44,45) Recent findings also indicate that PTP1B activity is modulated by post-translational modification, such as oxidation and alkylation of an extremely reactive cysteine residue at the catalytic center.⁴⁶⁾ On the basis of these facts, we have elucidated that PQQ inhibits PTP1B through the oxidation of catalytic cysteinyl thiol by H₂O₂ produced during its redox cycling, thereby inducing the ligand-independent activation of EGFR (Fig. 2). PTP1B has a substrate-specific ability to dephosphorylate RTKs, including the insulin receptor (IR),⁴⁷⁾ insulin-like growth factor-I receptor,⁴⁷⁾ platelet-derived growth factor receptor,⁴⁸⁾ vascular endothelial growth factor receptor,⁴⁹⁾ and nerve growth factor (NGF) receptor,⁵⁰⁾ implicating the modulation of multiple growth factor-activated signaling pathways. Hence, our data suggests that inhibition of PTP1B via redox cycling by PQQ might induce a diverse range of physiological effects through potentiated RTK-mediated signaling and gene expression and exert a growth factor-like action.

Fig. 2. Proposed mechanism for the ligand-independent activation of epidermal growth factor receptor (EGFR) signaling through redox cycling of pyrroloquinoline quinone (PQQ). PQQ undergoes redox cycling in the presence of reductants, such as ascorbate and glutathione, and then produces ${\text{O}}_2^{-}$ and H₂O₂. The generated H₂O₂ inactivates protein tyrosine phosphatase 1B (PTP1B) via the oxidation of catalytic cysteinyl thiol (Cys-215) to the corresponding sulfenic acid (–SOH), sulfinic acid (–SO₂H), and sulfonic acid (–SO₃H). The inhibition of PTP1B evokes the EGF-independent activation (tyrosine phosphorylation) of EGFR and subsequent activation (serine/threonine phosphorylation) of ERK 1/2.

IV. Anti-diabetic effects

Accounting for 90–95% of diabetic population, type 2 diabetes mellitus (T2DM) has increased rapidly in recent decades worldwide, and the morbidity and mortality associated with secondary complications of the disease, such as retinopathy, nephropathy, and cardiovascular disease, also have increased significantly.⁵¹⁾ T2DM is characterized by mitochondrial disorder and chronic hyperglycemia and dyslipidemia resulting from insulin resistance of the peripheral tissues and impaired insulin secretion from the pancreas.⁵²⁾ Mitochondria regulate metabolic pathways through signal transduction that is essential for metabolic homeostasis and cellular function. Recent studies show that mitochondrial dysfunction of diabetic subjects is closely related to lifestyle factors, including diet, physical activity, sleep, and stress.^53,54) Prolonged exercise and diet intervention can reverse, at least partly, the mitochondrial deficiency and improve the metabolic flexibility and insulin sensitivity in patients with T2DM.^54,55) Recently, dietary PQQ supplementation has been revealed to enhance mitochondrial function and biogenesis and improve metabolic homeostasis in mice and rats.^56–58) PQQ deficiency in young mice increases the plasma glucose level, reduces hepatic mitochondrial content by 20–30%, and suppresses mitochondrial respiration.⁵⁶⁾ Similarly, rats fed a diet deficient in PQQ exhibit elevated plasma lipid and ketone bodies owing to lower mitochondrial content and decreased energy expenditure.⁵⁷⁾ More importantly, PQQ supplementation reverses the mitochondrial alterations and metabolic impairment and significantly improves the lipid profile in diabetic UCD-T2DM rats.^56,57) Mechanistically, mitochondrial biogenesis and function are stimulated by the transcriptional coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), through activation of the nuclear respiratory factor (NRF-1 and NRF-2).⁵⁹⁾ The transcription factor cAMP-responsive element-binding protein (CREB) increases transcription of PGC-1α via a conserved CREB-binding site in the proximal promoter and is activated by exercise or fasting.⁶⁰⁾ Indeed, the exposure of mouse Hepa 1–6 hepatocytes to PQQ elevates PGC-1α promoter activity by enhancing CREB transcriptional activity and stimulating mitochondrial biogenesis (Fig. 3(A)).^57,61) PQQ exposure also increases the levels of NRF-1 and NRF-2, resulting in the upregulation of the mitochondrial transcription factor A (Tfam) and mitochondrial gene expression. However, the molecular mechanism underlying the activation of CREB-PGC-1α signaling pathway by PQQ remains unclear.

Fig. 3. Pyrroloquinoline quinone (PQQ)-induced activation of cAMP-responsive element-binding protein (CREB)-peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and insulin signaling. (A) Proposed mechanism for PQQ-induced activation of CREB-PGC-1α signaling pathway. PQQ stimulates the phosphorylation and activation of CREB and enhances PGC-1α expression. Increased PGC-1α binds to and coactivates the transcriptional function of nuclear respiratory factor (NRF)-1/2 on the mitochondrial transcription factor A (Tfam) promoter. Tfam plays a crucial role in regulating mtDNA amplification and mitochondrial biogenesis. (B) Proposed mechanism for the ligand-independent activation of insulin signaling through redox cycling of PQQ. PQQ inhibits protein tyrosine phosphatase 1B (PTP1B) to oxidatively modify the catalytic cysteine through its redox cycling activity. The inhibition of PTP1B evokes the insulin-independent activation (tyrosine phosphorylation) of the insulin receptor (IR) and subsequent phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt. Phosphorylated Akt stimulates translocation of glucose transporter 4 (GLUT4) to the plasma membrane, resulting in increased cellular glucose uptake.

Insulin resistance, defined as a dysfunction of insulin target cells, such as hepatocytes, skeletal muscle cells, and adipocytes, to respond to the action of insulin, plays a pivotal role in the development of several metabolic abnormalities and T2DM. Mitigating insulin resistance has been considered as a primary clinical strategy to improve metabolic control in T2DM subjects. The underlying molecular pathophysiology of insulin resistance still is not well understood, but a number of lines of evidence point to a critical role of PTP1B in insulin resistance. PTP1B negatively regulates insulin signaling by catalyzing dephosphorylation of tyrosine residues in activated IRand IR substrate-1 (IRS-1).^47,62) Recently, a correlation between insulin resistance states and up-regulation of PTP1B expression in adipose and muscle tissues in humans has been reported.^63–65) Furthermore, transgenic overexpression of PTP1B in muscle attenuates the tyrosyl phosphorylation of IR and IRS-1, leading to insulin resistance.⁶⁶⁾ On the other hand, PTP1B-knockout mice exhibit an elevated sensitivity to insulin with increased tyrosyl phosphorylation of the IR in the liver and muscle.^67,68) Thus, the inhibition of PTP1B has emerged as a potential therapeutic strategy to treat T2DM.⁶⁹⁾ More recently, we found that PQQ elicits the ligand-independent activation of insulin signaling by inhibiting cellular PTP1B and enhances glucose uptake through the translocation of glucose transporter 4 in mouse C2C12 myotubes (Fig. 3(B)).⁷⁰⁾ In addition, we demonstrated that oral administration of PQQ (20 mg·kg⁻¹ day⁻¹) for two weeks improved impaired glucose tolerance in type 2 diabetic KK-A^y mice. Our findings clearly suggest that PQQ can be useful in anti-diabetic treatment for T2DM subjects.

V. Anti-oxidative action

PQQ is reduced easily to PQQH₂ by reaction with reducing agents such as NADPH, sodium borohydride, glutathione, or cysteine. A couple of in vitro studies demonstrated that the reduced form of PQQ (PQQH₂) exhibits anti-oxidative capacity.^71–75) The aroxyl radical-scavenging activity of PQQH₂ was 7.4-fold higher than that of vitamin C, which is known as the most active water-soluble anti-oxidant.⁷³⁾ The singlet oxygen-quenching activity of PQQH₂ was found to be 6.3-fold higher than that of vitamin C.⁷⁴⁾ Interestingly, PQQH₂ works as catalyst in the singlet oxygen-quenching reactions. Moreover, it has been clarified that PQQH₂ may rapidly convert two molecules of α-tocopheroxyl radicals to α-tocopherol.⁷⁵⁾ These results indicate that the pro-oxidant effect of α-tocopheroxyl radicals is suppressed by the coexistence of PQQH₂. The summary of the radical quenching reaction is shown in Fig. 4.

Fig. 4. The summary of radical quenching reactions. Pyrroloquinoline quinol (PQQH₂) can be made from pyrroloquinoline quinone (PQQ) by reduction of NADPH, cysteine, and glutathione. Aroxyl radicals, singlet oxygen, and α-tocopheroxyl radicals are quenched by PQQH₂.

In experiments using cultured cells, it was reported that PQQ Na₂ prevents oxidative stress-induced neuronal death.^76,77) It has been shown that PQQ prevented 6-hydroxydopamine (6-OHDA)-induced cell death of the dopaminergic neuroblastoma cell line SH-SY5Y and primary rat neurons and that its preventive effect was stronger than that of vitamin C and E.⁷⁶⁾ 6-OHDA is a well-known neurotoxin that compromises mitochondria complex I, resulting in the production of ROS, such as ${\text{O}}_2^{-}$, hydroxyl radicals, and H₂O₂. Similar results were obtained in the experiment using H₂O₂.⁷⁷⁾

Moreover, marked decreases in ischemia damage are found in in vivo rat models, such as cardiovascular^78,79) or cerebral ischemia models.^80,81) The underlying mechanisms elucidated were that PQQ acts as an anti-oxidant by scavenging ${\text{O}}_2^{-}$ and protects mitochondria from oxidative stress-induced damage.⁵⁸⁾

In humans, following a single dose of PQQ Na₂ (0.2 mg/kg body weight), thiobarbituric acid reactive products (TBARS), which are measured by the malondialdehyde generated from lipid hydroperoxides, significantly decreased over the time course of the study.²⁸⁾ In addition, the change of TBARS values correlated significantly with the maximum plasma concentration (C_max) for PQQ Na₂. These results suggest that PQQ has a potential as an anti-oxidant.

VI. Neuroprotection and brain function

VI.I. In vitro studies

Neurons are susceptible to receive lethal damage from oxidative stress. This neuronal death is regarded as a cause of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In in vitro studies, the ability of PQQ Na₂ to protect human neuroblastoma SH-SY5Y cells from oxidative stress by 6-OHDA or H₂O₂ was tested.^76,77) The cell viabilities were recovered in a dose-dependent manner by adding PQQ Na₂. The inhibitory activity of PQQ Na₂ was much higher than that of vitamin C or vitamin E at the concentrations tested. These results suggest that the protective effect of PQQ on 6-OHDA or H₂O₂-induced neurotoxicity is involved in its function as a radical scavenger, especially ${\text{O}}_2^{-}$.

One of the interesting effects of PQQ is enhancement of NGF production. NGF, a protein composed of 118 amino acid residues, is well known as a neurotrophic factor required for the development and maintenance of peripheral sympathetic and sensory neurons. PQQ is shown to have a stimulatory effect on NGF synthesis/secretion in astroglial cells and in fibroblast cells without cytotoxicity.^82,83) The precise mechanism of enhancement of NGF by PQQ is not yet clear; however, cyclooxygenase activation is supposed to be an essential process, because the induction of NGF is inhibited by cyclooxygenase inhibitor or dexamethasone.⁸⁴⁾

VI.II. In vivo studies

The effects of PQQ on the learning and memory function of young rats were investigated using the Morris water maze test.⁸⁵⁾ The rats in this study were fed a diet supplemented 20 mg PQQ Na₂/(kg body weight/day) for nine weeks. Rats fed a PQQ Na₂-supplemented diet showed significantly better learning ability than the control rats. In addition, after receiving hyperoxia to induce oxidative stress for 48 h, rats fed PQQ Na₂-supplemented diets showed better memory function than the control rats. The combination of PQQ Na₂ (20 mg PQQ/(kg body weight/day)) with Coenzyme Q10 (300 mg/(kg body weight/day)) showed synergistic effects on memory function. The effect is independent of vitamin E, because the vitamin E-deficient rats did not show the effect with PQQ Na₂-supplemented diets. Similar effects were observed in aged rats.⁸⁶⁾ These results suggest that PQQ is potentially effective for preventing neurodegeneration caused by oxidative stress.

VI.III. Human studies

A placebo-controlled, double-blinded study using the repeatable battery for the assessment of neuropsychological status (RBANS) was conducted with the participation of 65 Japanese subjects between 50 and 70 years old who presented with self-identified forgetfulness or forgetfulness identified by a family member, colleague, or acquaintance.⁸⁷⁾ RBANS is a neuropsychological battery developed by Randolph in the United States.⁸⁸⁾ The neuropsychological battery questions allow repeated and quick evaluation of higher brain function disorders with a variety of brain disease complications. The content of the RBANS consists of five subtests of neurocognitive test paradigms [immediate memory, visuospatial/constructional, language, attention, and delayed memory]. Although the PQQ Na₂ (20 mg/day) and PQQ Na₂ (20 mg/day) + Coenzyme Q10 (300 mg/day) groups showed significantly better total score over time, a similar improvement over time was seen in the placebo group. Differences in immediate memory scores at week eight were significantly better in the PQQ Na₂ + Coenzyme Q10 group than in the placebo group. For analysis of immediate memory, subjects were stratified into two subgroups according to baseline total scores. Although no significant difference was present between groups in the high-scoring subgroup, the PQQ Na₂ + Coenzyme Q10 group in the low-scoring subgroup showed a significantly better score at week 8 and week 16 than the placebo group. This finding shows that individuals with lower RBANS scores may achieve a better degree of improvement in response to PQQ Na₂-supplementation than individuals with higher scores.

The result of another human clinical study was reported very recently.⁸⁹⁾ A randomized, placebo-controlled, double-blinded study to examine the effect of PQQ Na₂ on cognitive functions was conducted with 41 elderly healthy subjects. Subjects were administered orally 20 mg of PQQ Na₂/day or placebo for 12 weeks. For cognitive functions, selective attention by the Stroop and reverse Stroop test⁹⁰⁾ and visual-spatial cognitive function by the laptop tablet Touch M⁹¹⁾ were evaluated. In the Stroop test, the change of Stroop interference ratios for the PQQ Na₂ group was significantly smaller than for the placebo group. In the Touch M test, the stratification analyses dividing each group into two groups showed that the score significantly increased only in the lower group of the PQQ Na₂ group (initial score < 70).

Relating to cognitive functions, PQQ Na₂ shows effects on stress, fatigue, and sleep. Seventeen adult and female subjects participated in a clinical trial using an open-label trial to evaluate the effectiveness of PQQ Na₂ on stress, fatigue, quality of life, and sleep.⁹²⁾ The participants ingested 20 mg of PQQ Na₂ daily for eight weeks. The results in the Profile of Mood States–Short Form showed that all six measures of vigor, fatigue, tension-anxiety, depression, anger-hostility, and confusion significantly improved following PQQ Na₂ supplementation compared with scores for those measures before supplementation of PQQ Na₂. The results of the Oguri–Shirakawa–Azumi Sleep Inventory (Middle-aged and Aged version) showed significant improvement in drowsiness at awaking, sleep onset and maintenance, and sleep duration. For validation, the Pittsburgh Sleep Quality Index Japanese version also showed significant improvement in sleep-related behavior. Furthermore, the changes in these global scores were correlated with changes in the cortisol awakening response, i.e., the effects of PQQ Na₂ on improvement of sleep quality are supported by a biomarker.

Recently, two papers were published regarding the effect of PQQ Na₂ on health benefit in humans.^93,94) PQQ Na₂ is helpful for the improvement of skin conditions and lipid metabolism. PQQ Na₂ may be useful not only for the improvement of brain functions but also for various health benefits. The underlying mechanisms of the effects of PQQ Na₂ should be elucidated further.

VII. Safety

Since 2009, dietary supplements containing PQQ Na₂ have been commercialized in the United States after the official acceptance of notification by the Food and Drug Administration, and no adverse effects have been reported. As for oral toxicity studies, a 14-day preliminary study and a 28-day repeated dose study, as acute studies, and a 13-week subchronic study were performed in rats.⁹⁵⁾ The median lethal dose was 1000–2000 mg PQQ Na₂/kg body weight in male and 500–1000 mg PQQ Na₂/kg body weight in female rats. In the 14-day study, high doses of PQQ Na₂ resulted in increases in relative kidney weights with associated histopathology in female rats only, while a follow-up 28-day study in female animals resulted in increases in urinary protein and crystals. These findings were reversible and resolved during the recovery period. In the 13-week study, a number of clinical chemistry findings and histopathological changes were noted, which were deemed to be of no toxicological significance, as the levels were within the historical control range, were not dose-dependent, occurred at a similar frequency in control groups, or occurred only in the control group. Based on these findings, a no-observed-adverse effect level (NOAEL) of 100 mg PQQ Na₂/kg body weight was determined in rats, the highest dose tested in the 13-week study. A recent study reported that the NOAEL of PQQ Na₂ in rats is considered to be 400 mg PQQ Na₂/kg body weight for both sex, the highest dose tested.⁹⁶⁾

Additionally, the genotoxic potential of PQQ Na₂ was evaluated in a core battery of genotoxicity tests.⁹⁷⁾ The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in two separate in vitro chromosomal aberration tests at the highest dosage in Chinese hamster lung fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity was noted. In the in vivo micronucleus assay in mice, PQQ Na₂ at doses up to 2000 mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. From these results, PQQ was concluded to have no genotoxic activity in vivo.

A placebo-controlled, double-blinded safety studies in humans have been reported.⁹⁸⁾ PQQ Na₂ at 20 or 60 mg/day or placebo was administered for four weeks to healthy volunteers. No adverse effects were observed in standard clinical blood tests at both dosages of PQQ Na₂. In the 60 mg PQQ Na₂/day dosage test, the urinary concentration of N-acetyl-β-d-glucosaminidase (NAG), which is a sensitive biomarker for renal tubular damage, did not change after the administration of PQQ Na₂.

[¹⁴C]PQQ was administered orally to mice to estimate absorption. PQQ was readily absorbed (62%) in the lower intestine and was excreted by the kidney (81%) within 24 h.⁹⁹⁾ Following a single dose of PQQ (0.2 mg PQQ Na₂/kg body weight), levels of PQQ peaked in the serum after 3 h of administration at a concentration around 10 nM. The rise and clearance of PQQ Na₂ in serum paralleled the change in urine.²⁸⁾ From these studies, the pharmacokinetic behavior of PQQ Na₂ seems similar to other water-soluble vitamin B group compounds. This suggests that PQQ does not accumulate considerably in the body to produce severe damage. In rats, when PQQ Na₂ was injected intraperitoneally daily for 4 days at a dose of 11.5 mg/kg body weight, functional, and morphologic changes of the kidney were observed.¹⁰⁰⁾ The oral administration does not give as high a blood concentration of PQQ as intraperitoneal injection, but it is necessary to monitor for excess dosage.

VIII. Conclusions

PQQ has been shown to be a ubiquitous molecule that influences a multitude physiological and biochemical processes. In this review, we have presented recent studies supporting the role of PQQ in maintaining and improving human health. There are potential benefits from PQQ supplementation related to lipidemic and glycemic control, prevention of cardiovascular and neurodegenerative diseases, and improvement of brain functions. Recent evidence suggests that PQQ can be useful for various health benefits through different mechanisms including redox activity, radical-scavenging activity, and modulation of cell signaling pathways. According to recent observations, PQQ shows no toxicity and genotoxicity in oral administration, and thus, oral supplementation of PQQ would be a promising approach to improving health status. On the other hand, the precise molecular mechanism underlying the action of PQQ is not understood fully. The mechanistic studies that aid in defining the function of PQQ could provide further benefits for human health.

Disclosure statement

No potential conflict of interest was reported by the authors.