2.1. General Properties

Pyrroloquinoline quinone (PQQ; 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f]quinoline-2,7,9-tricarboxylic acid) is an aromatic water-soluble quinone whose chemical properties are analogous to combining the chemical features of ascorbic acid, riboflavin and pyridoxal-5-phosphate into one molecule. For example, both the oxidized (PQQ_ox) and reduced (PQQH₂) forms of PQQ carry out redox cycling. PQQ also serves as a dehydrogenase cofactor, a free radical scavenger, and an amine oxidase catalyst [12,13]. PQQ is highly electrophilic and reacts with many substances (e.g., various carbonyl reagents, such as hydrazine, hydroxylamine and semicarbazides). Moreover, PQQ forms complexes with acetone, aminoguanidine, urea, various diamines, and divalent metals [12,13]. In addition, PQQ reacts with ammonia to produce an iminoquinone, and under acidic conditions, internal lactones are formed, in contrast to the formation of dihydrates under neutral and basic conditions [14].

PQQ does not easily self-oxidize or condense into inactive forms. Accordingly, when compared on a molar basis, PQQ can be 100 to 1000 times more efficient in redox cycling assays than other enediols, such as ascorbic acid and menadione, as well as many isoflavonoids, phytoalexins and polyphenolic compounds [12]. As a redox cofactor, PQQ is capable of catalyzing continuous redox reactions involving oxidation of thiols [12], riboflavin [12], ubiquinone, terminal cytochromes [15], α–tocopheroxyl radicals [16] and nicotinamide adenine dinucleotide cofactors [17]. As an antioxidant, PQQH₂ can act as an aroxyl radical scavenger, even more so than α-tocopherol, against peroxyl radicals [16]. Moreover, in combination with α-tocopherol, PQQH₂ can protect α-tocopherol by reducing α-tocopheroxyl radicals generated in the oxidation of polyunsaturated lipids. Figure 1 summarizes relationships seminal to PQQ’s role in redox processes and as an antioxidant.

Oxidized PQQ does not exert appreciable antioxidant activity in the presence of metals like copper; however, PQQox can catalyze the non-enzymatic oxidation of the epsilon amine function of lysyl groups to aldehydic functions in proteins, such as elastin and collagen, and the oxidation of pyridoxamine and pyridoxamine-5-phosphate to pyridoxal and pyridoxal-5-phosphate [12].

2.2. PQQ as an Enzyme Cofactor

Of the bacterial enzymes for which PQQ serves as a cofactor, most are glucose or alcohol dehydrogenases. The one exception is lupinine hydroxylase, from the genus Pseudomonas [13]. This PQQ—dependent quinoenzyme serves as an amine dehydrogenase, with the quinolizidine alkaloid from lupin plants, lupinine, as the substrate. Mechanisms involving hydride ion transfers are proposed for the oxidation of glucose and alcohol substrates. Metals ions, such as K⁺, Mg²⁺, Li⁺ or Ca²⁺ also serve catalytic roles [13].

Of fundamental importance, Akagawa and coworkers [18,19] have reported that PQQ serves a functional role in mammalian dehydrogenases such as lactate dehydrogenase (LDH). PQQ bound to LDH enhances NADH oxidation to NAD+ and pyruvate production [18,19]. Optimizing oxidative metabolism is inextricably linked with improved antioxidant defense and enhanced immune function. In this regard, NAD+ is a substrate for the deacylation reactions catalyzed by sirtuins, particularly sirtuin 1 and 3 (SIRT1 and SIRT3). The sirtuins are a family of signaling proteins essential to metabolic regulation related to oxidative processes. Some of the relationships are outlined in Figure 2.

To elaborate further, SIRT1 is primarily found in the nucleus and lesser amounts in the cytoplasm [11,20], whereas SIRT3 is generally localized in the mitochondria and associated with mitochondrial DNA transcription [11,21]. Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-lα is one of the most critical factors controlled by sirtuin activity and is activated through deacetylation by SIRT1. PGC-lα acts as a transcriptional coactivator by enabling the assembly of other transcriptional enhancers, such as nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). Such factors are necessary elements in the cell required for maintaining energy homeostasis and engaging antioxidant response genes [22]. Moreover, Tchaparian et al. [23] have reported on the identification of other transcriptional networks responding to dietary PQQ supplementation. Among these are the 5′-adenosine monophosphate-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK), and the Janus kinase signal transducer and activator of transcription JAK-STAT) pathways, which aid in the control of cellular proliferation, mitosis, apoptosis and most aspects of the immune response [24,25,26,27,28,29,30,31,32].

3.1. PQQ Synthesis

Over one hundred prokaryotes have been identified as capable of PQQ synthesis and use PQQ as an enzymatic cofactor [33]. Most are Gram—negative and range in function from plant symbionts (e.g., root-associated rhizobacteria) to insect pathogens. The PQQ-requiring alcohol and glucose dehydrogenases in bacteria catalyze the oxidation of a wide variety of primary and secondary alcohols as well as catabolism of acyclic terpenes [13]. The glucose dehydrogenases (GDHs) exist in both membrane-bound and soluble forms [34,35]. Their DNA-derived protein sequences, however, are dissimilar, and there is little to no immunological cross-reactivity. Most attention has been given to the membrane bound GDHs localized to the periplasmic surface of cytoplasmic membranes [35], which are essential to bacterial respiration, oxidative metabolism, and growth. Moreover, their regulation is sensitive to changes in oxygen concentration and factors linked to cell growth. It is also important to note that some organisms that do not make PQQ will utilize it when available. A good example is the enteric bacterium Escherichia coli (E. coli), which has a membrane-bound PQQ-dependent GOH and an NADH-dependent GDH. The PQQ-dependent GDH is used preferentially when PQQ is available [13].

For those bacteria that make PQQ, five protein gene products, designated PqqA, PqqB, PqqC, PqqD, and PqqE, are required for PQQ production (Figure 3). Three gene products in the pqq operon, PqqB, PqqC, and PqqE, are members of large protein superfamilies. The roles for each gene product have been described by Zhu and Klinman [36]. Following synthesis, PQQ next binds to quinoenzymes, such as the bacterial glucose and alcohol dehydrogenases, at binding domains with the properties of β-propeller sequences. β-propeller sequences are characterized by 4 to 8 highly symmetrical blade-shaped beta-sheets arranged toroidally around a central axis. β-Propellers are found in all kingdoms of life. They function mainly as scaffolds for macromolecular interactions and ligand binding at catalytic sites [37].

3.2. PQQ and Fungi

The first to demonstrate a PQQ-containing enzyme in a eukaryote were Taketa, Matsumura, and coworkers [38,39,40,41], who isolated and characterized a novel PQQ-dependent sugar oxidoreductase from the basidiomycete, Coprinopsis cinerea (Cc). Basidiomycota are model organisms for studying fungal sex and mating types, mushroom development, and processes essential to multicellularity. The enzyme, an extracellular PQQ-dependent sugar dehydrogenase (CcSDH), comprises a signaling domain for extracellular secretion and domains for cellulose adsorption, PQQ binding, and cytochrome binding. Subsequently, Varnai and coworkers provided similar observations [15]. Although there are reports that yeast contains PQQ no specific PQQ-containing enzymes have yet been identified [42]. However, as Matsumura et al. [38] have emphasized, BLAST searches indicate the existence of many genes encoding homologous proteins to CcSDH in bacteria, archaea, amoebozoa, and fungi. Furthermore, phylogenetic analysis suggests those identified as quinoproteins are widely distributed in prokaryotes and the fungal family of eukaryotes.

5.1. Drosophila Melanogaster

Drosophila melanogaster and nematodes are often used as models in studies focused on gut microbiology and longevity [52] and surrogates to assess disease vectors at the cell signaling and gene levels [52]. For example, in D. melanogaster models of Parkinson’s disease, genetic or pharmacological activation of the D. melanogaster homolog of mammalian PGC-1α. rescues the disease phenotype by stimulating D. melanogaster PGC-1α. [53]. In turn, silencing D. melanogaster PGC-1α results in the expression of Parkinsonian phenotypes [54]. Drosophila also depend on commensal bacteria to maintain normal microbiome function. Interestingly, Shin et al. [55] reported that the PQQ-dependent alcohol dehydrogenase (PQQ-ADH) activity of Acetobacter pomorum, a commensal acetic acid-producing Drosophila bacterium, modulates insulin/insulin-like growth factor (I/IL-GF) signaling. In germ-free D. melanogaster, I/IL-GF signaling-related defects are reversed by enhancing host I/IL-GF signaling or supplementing the diet with acetic acid. I/IL-GF signaling influences D. melanogaster development, body size, energy metabolism, and intestinal stem cell activity.

5.2. Nematodes

In nematodes (Caenorhabditis elegans), PQQ exposure enhances nematode antioxidant capacity and extends lifespan by increasing the transcriptional activities of DAF-16/FOXO, SKN-1/NRF2 and SOD-3, factors involved in life extension [56]. DAF-16 is the ortholog of the FOXO family of transcription factors in C. elegans and is responsible for activating genes involved in survival (e.g., those associated with telomere extension and oxidative stress). The SKN-1 gene encodes a transcription factor that resembles mammalian NRF-2, which is associated with protection against oxidative damage and inflammation. Finally, Sasakura et al. [57] reported that PQQ activates DUOX protein BLI-3. BLI-3 controls the levels of reactive oxidant species, ensuring the levels are appropriate for optimal growth yet below those that promote inflammation.

6.1. Murine and Rodents

Diet is the apparent source of PQQ in animals given that little, if any, PQQ appears to be synthesized by the intestinal microflora (cf., 3. PQQ’s role in Prokaryotes and Fungi; [58,59]). In nutritional studies, delayed growth and neonatal development are features in mice and rats fed diets devoid of PQQ (Figure 4; [60,61]). The neonatal growth rates for mice [60,61] and rats [62] respond to PQQ in a dose-dependent manner. Optimal neonatal growth is achieved at approximately one μmol of PQQ/kg added to basal diets void of PQQ. Moreover, when PQQ exposure is initiated prior to pregnancy, the mice most deprived of PQQ have fewer litters [61].

Importantly, PQQ derivatives are readily absorbed from diets. For example, Smidt et al. [58] administered a dose of C¹⁴—PQQ (0.42 μCi/μmol) by gavage to Swiss-Webster mice. Sixty percent of the dose was absorbed, and over 80 percent of the absorbed dose was excreted renally. In addition, most of the C¹⁴—PQQ in the blood (>95%) was associated with the blood cell fraction rather than plasma.

6.2. Pigs

Regarding other animal species, Yin et al. [63] measured growth, intestinal morphology, redox status, and the levels of selected cytokines using weanling pigs. Without added PQQ the average daily weight gain was 336 g/day for the 28-day observational period. At 4.5 mg PQQ/kg diet, the rate of weight gain was −380 g/day. SOD and glutathione peroxidase (GPX) levels were also increased in the duodenum, jejunum, and ileum. In addition, PQQ exposure decreased inflammatory cytokines such as interleukin (IL)-1β, IL-2 and interferon-Y. Likewise, Wang et al. [64] examined the offspring of sows fed diets with or without PQQ supplementation. The diets were fed throughout gestation and lactation. The activity and mRNA expression levels of heme oxygenase 1 (HMOXl), SOD 1 and 2, catalase (CAT), GPX 1 and 4, and glutamate-cysteine ligase (GCL), the first-rate limiting enzyme of glutathione synthesis, were all increased in the intestine of piglets exposed to PQQ. Additional observations reported by Zhang et al. [65] are also complementary to those of Wang et al. [64] and Yin et al. [63].

6.3. Chickens

Similarly, PQQ elicits responses in avian species consistent with improvements in redox and immune status. Using a basal diet containing approximately 20 μg PQQ/kg of diet, Samuel et al. [66] reported that the incremental addition of PQQ to 800μg/Kg improved antioxidant defenses, decreased inflammation, and had positive effects on growth in young 220 chickens. Moreover, Zheng et al. [67] reported that PQQ at 10 mg/kg of diet countered the inflammatory effects elicited by lipopolysaccharide (LPS). On a molar basis, PQQ was 10 to 100 times more effective than anti-inflammatory drugs such as acetylsalicylic acid and meclofenamate [68], or biofactors such as melatonin [69], in attenuating the effects of LPS.

6.4. Humans

In college—age human subjects, Harris et al. [70] reported that PQQ supplementation (5–10 mg per day) reduced C-reactive protein, interleukin-6 levels, and plasma malonaldehyde levels in plasma. In addition, the ratio of blood lactate to pyruvate and the profile of urinary metabolites (estimated by 1H-nuclear magnetic resonance) were consistent with enhanced mitochondrial oxidation. Likewise, Hwang et al. [71] show daily supplementation with 20 mg PQQ optimizes mitochondrial biogenesis in human subjects. Notably, cognitive function and memory also are improved in human subjects, following PQQ supplementation (10–20 mg per day) [72,73,74].

8.1. Kidney and Liver

Chronic kidney disease (CKD) affects almost 15% of the global population [91]. Complications include cardiovascular diseases (CVDs), diabetes, hypertension, anemia, renal disease progression, acute kidney injury, mineral and bone disorders [92], and cognitive decline [93,94,95,96]. The kidney controls body fluid composition by filtering and reabsorbing materials. Reabsorption requires ATP, most of which is generated by mitochondria [97]. The high oxidative activity of mitochondria in the kidney can elevate oxidative stress, resulting in renal dysfunction and CKD progression [98,99]. In both human and animal models, depletion of antioxidant defense activity and increased production of ROS induces inflammatory damage, predominantly targeting renal tubular epithelial cells. The nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2)—antioxidant responsive element (ARE) prevents the progression of acute kidney injury (AKI) to CKD [100,101] by regulating cellular antioxidant defenses [102]. In multiple studies, a mechanism underlying NFE2L2’s protective role is its action on its target genes, including SOD2 and heme oxygenase (HMOX1), thereby decreasing ROS in the intracellular environment. In experimental CKD models, natural bioactive compounds with kidney protective potential are associated with NFE2L2 activation [103,104]. In this regard, recent work in vivo [105] and in vitro [106] suggests that PQQ appears to target the NFE2L2 pathway. In a mouse model of nephrotoxicity induced with the chemotherapeutic cyclophosphamide (CTX, an immunosuppressive agent), Lin et al. [105] showed PQQ supplementation of CTX-treated mice ameliorates nephrotoxicity by rescuing CTX-mediated 2inhibition of NFE2L2 signaling, characterized by altered expression of NFE2L2 target genes [105].

When treated with high glucose, the HK-1 human proximal tubular epithelial cell line models the progression of diabetes in the kidney. High glucose treatment of HK-1 cells results in elevated ROS and expression of pro-inflammatory genes, with concomitant inhibition of NFE2L2 and its targets. Wang et al. [106] found that PQQ supplementation of HK-1 cells treated with high glucose activates NFE2L2 signaling by increasing NFE2L2 translocation to the nucleus. Furthermore, PQQ mitigates pro-inflammatory signaling both in vivo and in vitro, which Lin et al. showed is regulated through the NLRP3 inflammasome [105].

Factors critical to the programming of neonatal development (e.g., maternal obesity or early exposure to high fat diets) also promote hepatic inflammation. Such processes can lead to nonalcoholic fatty liver disease progression to steatohepatitis in murine models [cf., POO, intestinal barrier functions and the microbiome]. Like the kidney, the expression of NFE2L2 and its target HMOX1, along with NLRP3, is diminished in liver from the offspring of mice fed a Western-style diet and treated with PQQ [107]. In addition, supplementation of PQQ attenuates hepatic inflammation in obese rats [108]. In rats, the amelioration of cadmium- and mercury-induced liver and kidney damage has also been reported following PQQ exposure [109].

8.2. Intestinal Barrier Functions and the Microbiome

An intact intestinal barrier is now recognized as an integral regulator of health [110,111,112]. Bacteria and their products are vital contributors to impairment and permeability of the gut barrier, resulting in an increased influx of bacteria, endotoxin, bacterial DNA, and metabolites into the host circulation. Recent reviews show that microbial dysbiosis and impaired barrier function are associated with gastrointestinal disease [113,114,115], neurodegenerative disease [116,117,118,119], autoimmune disease [120,121,122,123,124,125], and an impaired metabolic status in the host manifested by obesity, insulin resistance and cardiovascular complications [126,127,128,129,130,131,132,133,134,135]. In several animal models [63,64,107,136,137], exposure to PQQ increases mRNA expression levels of tight junction proteins and improves jejunal barrier function, suggesting PQQ may act through the gut to affect tissues in the periphery. In this regard, recent evidence has emerged suggesting a short-chain fatty acid, butyrate, is a crucial modulator of barrier function and colonic homeostasis [138,139]. Interestingly, exposure to PQQ leads to increased butyrate levels [137,140], most likely because it is used preferentially as a cofactor for membrane-bound hexose and alcohol dehydrogenases in E. coli [141]. As noted previously, membrane-bound hexose and alcohol dehydrogenases are involved in the synthesis of gluconic acid, which is fermented by lactic acid bacteria into lactate and acetate products used by acid-utilizing bacteria to form butyrate [142]. Butyrate exerts a broad spectrum of positive effects, both in the intestine and systemically, including promoting energy expenditure, stimulating peroxisome proliferator-activated receptor coactivator activity, improving insulin resistance, and enhancing mitochondrial function [138,143]. Furthermore, butyrate acts as an inhibitor of histone deacetylase activity [144]; therefore, PQQ-mediated alterations in butyrate levels might result in epigenetic change. Indeed, studies in pigs [137] and rodents [107,136] demonstrated improved barrier function in offspring following supplementation with PQQ. Although studies directly investigating the effects of PQQ on microbiota composition or function are sparse, a current supposition is that some gut bacteria may require PQQ for optimal function.

Using a mouse model of developmental programming of nonalcoholic fatty liver disease, Jonscher and colleagues [107,145] were the first to detect differences in gut bacteria composition in response to PQQ exposure. PQQ supplementation to the dams significantly reversed the diet-induced decrease in Bacteroidetes and increases in Firmicutes observed in offspring bacterial composition (Figure 7). Genera within both phyla, such as Bacteroides and Allobaculum, increased in abundance while Akkermansia, from the phylum Verrucomicrobiae, decreased. Notably, PQQ attenuates the WD-induced bloom of Clostridiales spp. In aged rats fed a lard-based high fat diet, Clostridiales was the order most affected by diet, and its decrease appeared responsible for gut microbial dysfunction [146]. Whether age or type of fat affects Clostridiales abundance remains to be determined, but it is suggestive that this order may be a dynamic, longitudinal biomarker for obesity or lipotoxicity.

8.3. Cardiac and Skeletal Muscle Protection

Skeletal and cardiac muscles require a high rate of ATP turnover for efficient contraction. In this regard, rats deficient in PQQ are markedly compromised when subjected to cardiac ischemia/reperfusion injury protocols. For example, Bauerly et al. [147] reported that 20% of rats fed a PQQ deficient diet did not survive ischemia reperfusion, whereas all PQQ supplemented rats survived. These studies were an extension of previous work [148,149,150] in which the cardioprotective effectiveness of PQQ was compared with metoprolol, a beta(l)—selective adrenoceptor antagonist. Rats underwent 30 min of left anterior descending coronary artery occlusion, followed by 2 h of reperfusion. Although both PQQ and metoprolol improved indices of cardiac function, PQQ was superior to metoprolol in protecting mitochondria from ischemia/reperfusion oxidative damage. Whether PQQ supplementation influences cardiac and skeletal muscle function in healthy individuals and animal models remains unclear. For example, Hwang et al. [71] investigated the effects of PQQ supplementation on aerobic exercise performance and indices of mitochondrial biogenesis in men following a six-week endurance training/supplementation program. No differences in aerobic performance were observed; however, there were improvements in peak oxygen consumption and total exercise test duration and recovery. In addition, the PQQ group had a significant increase in PGC—lα protein levels from baseline to post endurance training compared to non-supplemented subjects. In mice, Lui et al. [151] demonstrated that PQQ protects against exercise-induced fatigue and reduced oxidative damage, presumably by improving mitochondrial function.

From a marketing perspective, there are numerous claims made regarding PQQ and exercise performance. The claims are often made without considering that oxygen is the most limiting nutrient influencing exercise performance. Although exposure to compounds with biologic properties like those of PQQ increases mitochondrial content, regular exercise may have similar (or even greater) effects. Body heat regulation is also not often considered. The surface temperature of well-functioning mitochondria is estimated to be 50 °C [152]. Cardiac cell mitochondria appear to decrease ATP production and increase inner-mitochondrial membrane permeability when heat dispersal is compromised, or temperatures are sustained at greater than 40 °C [153]. Thus, it is too soon to conclude that PQQ supplementation can independently improve exercise performance. Indeed, Hwang et al. [71] have recently reported that PQQ supplementation in men undergoing endurance exercise training has little effect on performance apart from an elevation of PGC-lα.

8.4. PQQ and Neuroprotection

PQQ is protective in cases and models of brain aging and neurodegeneration, including Parkinson’s disease, stroke, and traumatic brain injury. Perhaps the best of current examples are PQQ’s ability to protect against neuronal agents, such as rotenone [154,155,156,157,158,159,160]. Moreover, the ability of PQQ to protect against neurodegeneration may go beyond mitochondriogenesis, given that PQQ can reduce α—synuclein fibril formation [161]. PQQ also confers resistance to neurocognitive loss in rodent models of stroke and brain injury. Jensen and associates [162] were among the first to use carotid ligation to assess neuroprotective properties following intra peritoneal injection of PQQ. The interruption of blood flow resulted in most of the cortex displaying signs of infarction. Rats without PQQ supplementation had infarctions across −95 percent of cortices compared to −70 percent in rats pretreated with PQQ. In a similar study, Zhang et al. [163] used reversible middle cerebral artery occlusion to simulate interruption of blood circulation to adult rat brains. When PQQ was injected into the jugular vein, either at the same time as occlusion or 3 h after the start of ischemia, markedly improved neurobehavioral scores were observed, along with a reduction in infarct size. Regarding traumatic brain injury, Zhang and associates [155,163,164] assessed spatial memory in rats using the Morris water maze test and demonstrated that PQQ administered intraperitoneally three days before injury improves spatial memory.

In human clinical trials, PQQ was reported to promote cognitive function and improved regional blood flow in older adults [74]. In a randomized placebo-controlled, double-blinded clinical trial, PQQ administered orally (20 mg PQQ/day) to elderly adults resulted in improved cognitive measures. The PQQ dosage was based on a previous animal study showing that PQQ improves spatial memory in rats, as measured by Morris water maze [164].