3种检测吡咯喹啉醌的方法比较

目的:研究和比较3种检测吡咯喹啉醌(PQQ)的方法,确定各种方法的特点和适用范围。方法:设计和改进了3种检测PQQ的方法,分别为活性电泳法、光谱法和NBT-Gly法,探讨其检测限和线性范围、精密度、样品检测和加样回收率。结果:活性电泳法专一性好,具有很高的灵敏度,可检测到12.6 ng/mL PQQ,可靠但准确性较差;NBT-Gly法操作简便,可用于大量样品的检测,但重复性不佳;光谱法精密度较好,但样品中存在吸光物质时对检测结果影响较大。结论:活性电泳法、光谱法和NBT-Gly法均可用于PQQ的检测,活性电泳法适于培养上清等复杂样品的粗略定量,NBT-Gly法适于大量样品的检测,光谱法适于纯度较高的PQQ的定量。     

Extractions of pyrroloquinoline quinone from crude biological samples

The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaCl did not improve the solvent extraction. PQQ, which had been extracted into n-butanol, could be re-extracted into an aqueous phase by addition of either n-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of 1 micrograms PQQ, which had been added to 1 g human liver, brain and 1 ml plasma and had undergone the n-butanol and the Sep-Pak extractions, was 50, 75 and 105%, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 micrograms/g or ml, respectively, if present.     

Synthesis of pyrroloquinoline quinone in vivo and in vitro and detection of an intermediate in the biosynthetic pathway.

In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.     

Factors relevant in bacterial pyrroloquinoline quinone production

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.     

Factors relevant in bacterial pyrroloquinoline quinone production.

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.     

Factors relevant in the reaction of pyrroloquinoline quinone with amino acids. Analytical and mechanistic implications

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several alpha-amino acids, the formation of a yellow coloured compound (lambda max = 420 nm) was accompanied by oxygen uptake and disappearance of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ-containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.     

PQQ and quinoprotein research--the first decade

On the occasion of the first international symposium on pyrroloquinoline quinone (PQQ) and quinoproteins (Delft, September 1988), a review of this novel field in enzymology is presented. Quinoproteins (PQQ-containing enzymes) are widespread, from bacteria to mammalian organisms (including man), and occur in several classes of enzymes. Indications already exist that PQQ is a versatile cofactor, involved not only in oxidation but also in hydroxylation, transamination, decarboxylation and hydration reactions. The current list of quinoproteins shows that it was overlooked in several well-studied enzymes where the presence of a common cofactor had already been established. Up until now, all eukaryotic quinoproteins have covalently bound PQQ (or perhaps pro-PQQ), while free PQQ occurs exclusively in a number of (bacterial) dehydrogenases and in the culture fluid of certain Gram-negative bacteria. Biosynthesis of free PQQ in methylotrophic bacteria starts with tyrosine and glutamic acid as precursors while intermediates in the route have not been detected and the presence of free PQQ is not required for synthesis of the covalently bound form of the cofactor in glutamic acid decarboxylase from Escherichia coli. Therefore, the assembly of covalently bound cofactor might occur in situ, i.e. in the quinoproteins themselves. If the latter also applies to mammalian quinoproteins, this implies that PQQ is not a vitamin. On the other hand, positive effects have been reported upon administration of PQQ to test animals. Methods suited to detach and to detect PQQ with a derivatized o-quinone moiety may answer questions on the uptake and processing of the compound.     

Pyrroloquinoline quinone (coenzyme PQQ) and the oxidation of SH residues in proteins.

Pyrroloquinoline quinone (PQQ) catalyzes the oxidation of cysteamine at neutral pH with a second order rate constant K2 = 0.45 M-1 s-1. The reduction of PQQ was monitored by absorption and fluorescence spectroscopy, whereas the oxidation of cysteamine to cystamine was followed by titration with 5,5'-dithiobis(2-nitrobenzoic acid). PQQ also catalyzes the oxidation of thiol groups critically connected with the function of two proteins, i.e. thioredoxin and phosphoribulose kinase. The reaction of PQQ with reduced thioredoxin brings about the oxidation of two thiol groups of the oxireductase, whereas the enzyme phosphoribulose kinase is inactivated at 25 degrees C. The oxidized disulfide bond of phosphoribulose kinase is reduced by dithiothreitol and the enzyme recovers catalytic activity. The ability of PQQ to catalyze the oxidation of vicinal cysteinyl residues to generate disulfide bonds under mild experimental conditions can be exploited to define the precise role of modified thiol residues in either catalysis or stabilization of protein structure.     

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry.

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.     

A combined spectroscopic and crystallographic approach to probing drug–human serum albumin interactions

The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.     

Pyrroloquinoline Quinone is a Potent Neuroprotective Nutrient Against 6-Hydroxydopamine-Induced Neurotoxicity

Pyrroloquinoline quinone (PQQ), which is an essential nutrient, has been shown to act as an antioxidant. Reactive oxygen species (ROS) are thought to be responsible for neurotoxicity caused by the neurotoxin 6-hydroxydopamine (6-OHDA). In this study, we investigated the ability of PQQ to protect against 6-OHDA-induced neurotoxicity using human neuroblastoma SH-SY5Y. When SH-SY5Y cells were exposed to 6-OHDA in the presence of PQQ, PQQ prevented 6-OHDA-induced cell death and DNA fragmentation. Flow cytometry analysis using the ROS-sensitive fluorescence probe, dihydroethidium, revealed that PQQ reduced elevation of 6-OHDA-induced intracellular ROS. In contrast to PQQ, antioxidant vitamins, ascorbic acid and α-tocopherol, had no protective effect. Moreover, we showed that PQQ effectively scavenged superoxide, compared to the antioxidant vitamins. Therefore, our results suggest the protective effect of PQQ on 6-OHDA-induced neurotoxicity is involved, at least in part, in its function as a scavenger of ROS, especially superoxide.     

Recent progress in studies on the health benefits of pyrroloquinoline quinone

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.     

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.     

Structure of the pyrroloquinoline quinone radical in quinoprotein ethanol dehydrogenase

Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.     

Physiologic importance of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ, methoxatin) is a dissociable cofactor for a number of bacterial dehydrogenases. The compound is unusual because of its ability to catalyze redox cycling reactions at a high rate of efficiency and it has the potential of catalyzing various carbonyl amine reactions as well. In methylotrophic bacteria, PQQ is derived from the condensation of L-tyrosine with L-glutamic acid. Whether or not PQQ serves as a cofactor in higher plants and animals remains controversial. Nevertheless, a strong case may be made that PQQ and related quinoids have nutritional and pharmacologic importance. In highly purified, chemically defined diets, PQQ stimulates animal growth. Furthermore, PQQ deprivation appears to impair connective tissue maturation, particularly when initiated in utero and throughout perinatal development.     

Measurement of casein digestion by a fluorometric method

A qualitative and quantitative method to assay proteolytic degradation of casein with a spectrofluorometer was developed. Proteolysis produced by different pure or mixed proteinases in a pH range 2 to 7.4 quenches the fluorescence emitted at a wavelength of 350 nm by casein excited at 300 nm in less than 5 min. This method is very sensitive, fast, and requires minimal sample preparation. Proteinases that do not generate peptides appropriate for fluorescence quenching cannot be detected with this assay and proteinases with intrinsic fluorescence may require special adjustments of the spectrofluorometer. This method monitors the disappearance of intact substrate proteins continuously, omitting the separation step necessary in other methods to measure product peptides.     

Acid-promoted tautomeric lactonization and oxidation-reduction of pyrroloquinoline quinone (PQQ)

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion at M/e 330 and a base ion formed by triple decarboxylation at M/e 198. Other ions found probably arise through acid-catalyzed tautomeric lactonization of PQQ to PQQ-lactone (PQQL) with subsequent oxidation of PQQL and reduction of PQQ. We propose that a carboxyl group, presumably the 9-carboxyl, attacks a double bond in PQQ, reversibly converting the 4,5-orthoquinone into an 4,5-enediol and forming an isomeric lactone, PQQL, of 330 daltons. The masking of carbonyls may explain the low reactivity of PQQ with carbonyl reagents in acid. Acid-promoted tautomeric lactonization with carbonyl-masking is known to occur with fluoresceins, phenolphthalein and other compounds, but has not been recognized before with PQQ. Acid-treated PQQ demonstrates molecular and other ions derived from reduced PQQ (PQQ(2H] or its lactone at M/e 332 with a base ion at M/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(-2H)L), at M/e 328 with a base ion at M/e 196. We suggest that PQQ, in tautomeric equilibrium with PQQL, oxidizes PQQL to PQQ(-2H)L (328 daltons), with its concurrent reduction to PQQ(2H) (332 daltons). With acidified D2O, PQQ shows deuterated products with ions at M/e values consistent with lactonization and oxidation-reduction. An analytically useful quinoxaline adduct, formed from PQQ and 2,3-diaminonaphthalene (PQQ-DAN) of 452 daltons, also undergoes acid-tautomerization-lactonization and oxidation-reduction similar to PQQ showing molecular ions at M/e 450, 452 and 454 and decarboxylation-derived strong (base) ions at M/e 318, 320 and 322.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyrroloquinoline Quinine Protects Rat Brain Cortex Against Acute Glutamate-Induced Neurotoxicity

To investigate possible protective effects of pyrroloquinoline quinone (PQQ) on the rat cortex with glutamate injection and to understand the mechanisms linking the in vivo neuroprotection of PQQ. Adult Sprague–Dawley rats received glutamate injection into the rat cortex. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay was performed to observe influences of co-treatment with PQQ (simultaneous injection with PQQ and glutamate) on neural cell apoptosis in the rat cortex. The production of reactive oxygen species (ROS) in the rat cortex was detected by flow cytometry using 2′,7′-dichlorofluorescin diacetate labeling, and the activity of superoxide dismutase, glutathione and malondialdehyde was respectively determined. Real time quantitative RT-PCR and Western blot were applied to measure the mRNA and protein expressions of Nrf1, Nrf2, HO-1 and GCLC in the rat cortex. Western blot was used to detect the phosphorylation of Akt and GSK3β in the rat cortex. Co-treatment with PQQ protected neural cells in the rat cortex from glutamate-induced apoptosis. PQQ decreased the ROS production induced by glutamate injection. PQQ increased the mRNA and protein expressions of Nrf2, HO-1 and GCLC and the phosphorylation of Akt and GSK3β in the cortex of glutamate-injected rats. PQQ could produce neuroprotective effects on the rat cortex. The antioxidant properties of PQQ and PQQ-induced activation of Akt/GSK3β signal pathway might be responsible for the in vivo neuroprotection of PQQ.     

L-tyrosine is the precursor of PQQ biosynthesis in Hyphomicrobium X

A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of Hyphomicrobium X, growing on [13C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolyzed cellular protein, by measuring their 12C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L-tyrosine and L-phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by 1H- and 13C-NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the o-quinone and pyrrole-2-carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L-tyrosine, possible biosynthetic routes to PQQ are discussed.     

Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes

The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2′,7′-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p < 0.05). PQQ treatment dose-dependently protects H9C2 cells from OGD-induced injury by reducing apoptosis, decreasing intracellular ROS levels, and rescuing the OGD-induced decrease in mitochondrial content. The protective effect of PQQ may be related to its effects on the PI3K/Akt pathway.