Canadian Journal of Physiology and Pharmacology
Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of tissue microsomes or recombinant human cytochrome P-450 oxidoreductase (CPR); implications for possible roles in gasotransmission.
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2019-0376.R1
Manuscript Type: Article
Date Submitted by the 19-Oct-2019 Author:
Complete List of Authors: Vukomanovic, Dragic; Queens University Jia, Zongchao; Queens University Nakatsu, Kanji;Draft Queen's University, Pharmacology & Toxicology Smith, Graeme; Queen's University, Anatomy and Cell Biology; Kingston General Hospital, Obstetrics and Gynecology Ozolinš, Terence; Queen's University, Biomedical and Molecular Sciences
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue:
• Riboflavin, • Pyrroloquinoline quinone, • Carbon monoxide, • Keyword: Cytochrome P450 oxidoreductase, Human placenta microsomes
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1 Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of
2 tissue microsomes or recombinant human cytochrome P-450 oxidoreductase (CPR);
3 implications for possible roles in gasotransmission.
4
5 Dragic Vukomanovica,b, Zongchao Jiaa, Kanji Nakatsua, and Graeme N. Smitha,b Terence R.S.
6 Ozolinša
7 a. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario,
8 Canada, K7L 3N6
9 b. Department of Obstetrics and Gynaecology, Kingston General Hospital, Kingston, Ontario,
10 Canada, K7L 3N6
11 Draft
12 CORRESPONDING AUTHOR:
13 Dr. Terence R.S. Ozolinš, Department of Biomedical & Molecular Sciences,
14 Queen’s University, Kingston, ON, Canada
15 K7L 3N6
16 Tel 613-533-3306 Facsimile 613-533-6412
18
19
20 There are no declarations of interest for any of the authors.
21
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22 ABSTRACT
23 Carbon monoxide (CO), an endogenously produced gasotransmitter, regulates inflammation and
24 vascular tone, suggesting delivery of CO may be therapeutically useful for pathologies like
25 preeclampsia where CO insufficiency are implicated. Our strategy is to identify chemicals that
26 increase the activity of endogenous CO producing enzymes, including cytochrome P-450
27 oxidoreductase (CPR). Realizing both riboflavin and pyrroloquinoline quinone (PQQ) are
28 relatively non-toxic, even at high doses, and they share chemical properties with toxic CO
29 activators we previously identified, our goal was to determine whether riboflavin or PQQ could
30 stimulate CO production. Riboflavin and PQQ were incubated in sealed vessels with rat and
31 human tissue extracts and CO generation was measured with headspace-gas chromatography.
32 Riboflavin and PQQ increased CO productionDraft ~ 60% in rat spleen microsomes. In rat brain
33 microsomes, riboflavin and PQQ increased respective CO production ~four-fold and two-fold,
34 compared to baseline. CO production by human placenta microsomes increased four-fold with
35 riboflavin and five-fold with PQQ. In the presence of recombinant human CPR, CO production
36 was three-fold greater with PQQ than riboflavin. These observations demonstrate for the first
37 time that riboflavin and PQQ facilitate tissue-specific CO production with significant
38 contributions from CPR. We propose a novel biochemical role for these nutrients in
39 gastransmission.
40 Keywords:
41 Riboflavin
42 Pyrroloquinoline quinone
43 Carbon monoxide
44 Human placenta microsomes
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45 Cytochrome P-450 oxidoreductase
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47 Novelty Bullets:
48
49 Riboflavin or PQQ stimulated CO generation in a tissue-specific manner
50 Recombinant human cytochrome P-450 reductase (CPR) generates CO in the presence of
51 riboflavin or PQQ
52 CO production is a novel biochemical function for these nutrients
53 Riboflavin or PQQ may increase CO production in conditions of CO insufficiency such as
54 preeclampsia Draft 55
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64 INTRODUCTION
65 Carbon monoxide (CO) is an important gasotransmitter critical in certain signalling
66 pathways related to vascular tone and the production of inflammatory mediators (reviewed in
67 Motterlini & Foresti 2017). Thus, the actions of CO may have clinical applications in many
68 diseases associated with vascular dysfunction and/or inflammation, including preeclampsia
69 (Phipps et al. 2019). The development of therapeutic CO delivery is an area of active research
70 and includes exogenous CO delivery methods and the development of CO-releasing molecules
71 (CORMs (Ismailova et al. 2018; Kim & Choi 2018). Another strategy, one that we favour, is to
72 generate CO in situ by activating endogenous CO-producing enzymes.
73
74 The primary endogenous sourceDraft of CO is from heme oxygenase during heme degradation
75 (Motterlini & Foresti 2017). There are two primary forms of mammalian heme oxygenases
76 (HO). HO-2 is predominantly expressed in brain and testes, and although HO-1 is highly
77 inducible following oxidative stress, the spleen is a rich source of its constitutive expression
78 (Vukomanovic et al. 2014). Recently, we demonstrated CO is also produced by cytochrome P-
79 450 oxidoreductase (CPR) (Vukomanovic et al. 2017), indicating at least three significant
80 enzymatic sources of CO. The CO-producing enzymes HO-1, HO-2 and CPR reside in the
81 endoplasmic reticulum (Riddick et al. 2013; Motterlini and Foresti 2017), and when cells are
82 disrupted, the endoplasmic reticulum forms heterologous vesicles, which after differential
83 centrifugation yield a highly enriched source of endoplasmic reticulum enzymes, i.e.
84 microsomes. We use microsomes as an in vitro screening tool to understand the important
85 structure activity relationships (SAR) necessary to stimulate CO production (Vukomanovic et al.
86 2011; Vukomanovic et al. 2014). Briefly, we came to realize that the capacity of a compound to
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87 enhance CO production was not associated with a traditional pharmacophore, but instead
88 appeared related to redox activities inherent to the structure; a potent prototypic CO generator is
89 menadione, a synthetic analogue of vitamin K (K3). Using menadione, a subsequent series of
90 studies identified a novel enzymatic source of CO production. Briefly, the use of specific HO-2
91 inhibitors with rat brain microsomes, and recombinant forms of HO-2 and CPR allowed us to
92 conclude that the menadione-mediated CO production that we had previously noted in rat brain
93 extracts was primarily the result of CPR, not HO-2 (Vukomanovic et al. 2017).
94 We have identified a number of synthetic isoalloxazine derivatives as potent CO
95 producers (unpublished data). Isoalloxazine is a tricyclic heterocycle from which flavins are
96 derived, and we reasoned that a biochemical source of flavin may provide an acceptable safety
97 profile. Riboflavin is the most abundantDraft biochemical source of flavin and although the riboflavin
98 recommended daily allowance (RDA) is 1.3 milligrams daily for men and 1.1 mg for women, it
99 may be consumed orally as high as 400 mg/day to treat migraine headaches with few adverse
100 side effects (Powers 2003; Thakur et al. 2016). Thus, there is compelling rationale to test
101 whether riboflavin stimulates CO generation.
102
103 Naphthoquinones, are also potent CO generators (Vukomanovic et al. 2011), but the dose
104 of menadione required to increase CO levels in vivo was toxic (Odozor et al. 2018).
105 Recognizing the need to identify less toxic naturally occurring naphthoquinones, we investigated
106 vitamin K1 (phylloquinone) and K2 (menaquinone; MK-4), but disappointingly, neither was
107 effective at producing CO in spleen or brain microsomes (Vukomanovic et al. 2014). This
108 prompted a search for alternate compounds with a quinone or quinone-like nucleus, and evidence
109 of clinical safety. Pyrroloquinoline quinone (PQQ; 4,5-dioxo-4,5-dihydro-1H-pyrrolo[2,3-
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110 f]quinoline-2,7,9-tricarboxylic acid), an aromatic tricyclic o-quinone, is an important nutrient,
111 found in a variety of foods, although its role as a vitamin remains controversial (reviewed in
112 Misra et al. 2012; Akagawa, et al. 2016; Ames 2018). PQQ has a number of proposed health
113 benefits including reduction of pro-inflammatory markers (Harris et al. 2013), a
114 pathophysiological factor in preeclampsia. Together, these observations, and its limited toxicity
115 when taken orally (Akagawa et al., 2016), made PQQ a promising candidate for CO production.
116
117 The goal of our study was to determine whether riboflavin and PQQ promote CO
118 production in microsomes from several tissue sources, including human placenta, the organ in
119 which CO insufficiency has been implicated in the etiology of preeclampsia (Levytska et al.
120 2013; Venditti and Smith, 2014). Draft
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123 MATERIAL AND METHODS
124
125 Preparation of Riboflavin and PQQ
126 A 10.0 mM stock solution of riboflavin 5’-phosphate sodium salt hydrate (Sigma-Aldrich Ltd,
127 Toronto, Canada, catalogue #R7774) was prepared in double distilled water. A 10.0 mM stock
128 solution of PQQ (ChemScene LLC, Monmouth Junction NJ, USA, catalogue # CS-W009276)
129 was prepared in DMSO.
130
131 Preparation of rat spleen and brain microsomal fractions
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132 Male rats were obtained from Charles River Ltd (St-Constant, Canada) and the organs were
133 harvested under approved protocols and in accordance with Queen’s University Animal Care
134 Committee and the Canadian Council on Animal Care’s Guide to the Care and Use of
135 Experimental Animals. The microsomal fractions of rat spleen (Braggins et al. 1986) and rat
136 brain (Trakshel et al 1988), were prepared by differential centrifugation of tissue homogenate as
137 previously described (Appleton et al. 1999; Kinobi et al. 2006). Protein concentration of the
138 microsomal fraction was determined by a modification of the Biuret method (Cook et al. 1995).
139 The microsomal fraction was resuspended in 100 mM phosphate buffer/20% (v/v), glycerol
140 solution containing 10 mM EDTA and stored at –80 °C until used (Kinobi et al. 2006).
141
142 Collection and perfusion of human placentasDraft
143 Term human placentas (>37 weeks) from uncomplicated pregnancies were obtained following
144 elective Caesarean section at the Kingston General Hospital (Kingston, Ontario), under the
145 approval of the Health Sciences Research Ethics Board at Queen’s University (OBGY-290-16).
146 Maternal identifiers were not collected in order to maintain confidentiality and anonymity.
147 Following removal from the uterus, the umbilical cord remained clamped to maintain blood
148 pressure and the placenta was placed on ice for transport to the laboratory.
149
150 After removal of the amnion on the placental surface, a peripheral chorionic plate artery
151 was cannulated and continuously perfused using a peristaltic pump (Gilson Minipuls 3, Gilson
152 Inc. USA, Middleton WI) with ice cold Krebs’ buffer (Ahmed et al. 2005) at the rate of 20
153 mL/min. Perfusate and blood were allowed to drain from the paired vein during perfusion of the
154 cotyledon. When the cotyledon on the fetal side became pale, perfusion was initiated on the
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155 maternal side by inserting a catheter into the cotyledon until the entire cotyledon appeared
156 bloodless. The perfused cotyledon was then excised, and the rest of the placenta disposed.
157
158 Preparation of human placenta microsomal fractions
159 The microsomal fractions from perfused human placenta tissue were prepared by differential
160 centrifugation of tissue homogenate as previously described (Vukomanovic et al. 2010). Protein
161 concentration of the microsomal fraction was determined using a modification of the Biuret
162 method, as we described previously (Cook et al. 1995). The microsomal fraction was
163 resuspended in a 100 mM phosphate buffer/20% (v/v), glycerol solution containing 10 mM
164 EDTA and stored at -80 °C.
165 Draft
166 Preparation of recombinant human CPR (rh-CPR)
167 The hCPR-pET22b vector encoding full-length human CPR was a generous gift from Dr. Paul
168 Ortiz de Montellano (University of San Francisco). Expression and purification were based on
169 protocols previously described (Dierks et al. 1998). In brief, the plasmid was transformed into
170 BL21 (DE3) pLysS cells that were cultured in Terrific Broth (TB) supplemented with trace
171 elements and riboflavin in the presence of ampicillin and chloramphenicol. Induction of
172 expression was initiated with 0.4 M isopropyl-β-D-thiogalactopyranoside (IPTG). When the
173 absorbance (A) at 600 nm had reached ~0.4 the cells were allowed to grow further overnight at
174 30°C. Spheroplasts were generated from the harvested cells prior to lysis by sonication. Lysates
175 were centrifuged at 10,000 r/min (Ti45 rotor) and the supernatant further centrifuged at 35,000
176 r/min (Ti45 rotor) to pellet the membrane fraction which was subsequently stored at –80°C for
177 future purification.
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178
179 Membrane pellets were thawed, then solubilized in 20 mM potassium phosphate (pH
180 7.4), 20% (v/v) glycerol, 0.2% Triton X-100, 0.1 mM EDTA, 1 mM PMSF, 0.2% sodium cholate
181 with one Roche EDTA-free protease cocktail tablet. After clarification, the supernatant was
182 purified over a 2′5′-ADP-Sepharose column, which had been equilibrated with Buffer A (20 mM
183 potassium phosphate (pH 7.4), 20% glycerol, 0.1 mM EDTA, 0.2% sodium cholate). The column
184 was washed with Buffer A, followed by washes in the presence of 250 mM NaCl, and in the
185 presence of 2 mM adenosine. Elution of the rh-CPR protein was performed with 5 mM 2’AMP.
186 Purified protein was dialyzed against 40 mM potassium phosphate, 20% glycerol overnight at
187 4°C, concentrated, and quantified by Bradford assay using bovine serum albumin (Sigma-
188 Aldrich USA Inc.) as a standard. ProteinDraft was aliquoted, flash-frozen in liquid nitrogen and stored
189 at –80°C.
190
191 Enzymatic assay for CO generation
192 CO generation by human placenta microsomal fractions was determined with a gas
193 chromatographic method using headspace-gas analysis as described by (Vukomanovic et al.
194 2014). The incubation conditions had been previously optimized with respect to concentrations
195 of reaction components and the duration of incubation (Vukomanovic et al. 2017). Briefly, a
196 reaction mixture (150 μL) containing 100 mM phosphate buffer (pH 7.4), 50 μM
197 methemalbumin and enzyme (1 mg microsomal protein or 10 μM recombinant protein), was pre-
198 incubated with an riboflavin or PQQ concentration ranging from 0.01 to 100 μM, for 10 min at
199 37 °C in a shaking water-bath and then each vial’s head space was purged with CO-free air for
200 15s. The reaction was initiated by adding nicotinamide adenine dinucleotide phosphate
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201 (NADPH; Sigma-Aldrich Ltd, Toronto, Canada) to a final concentration of 1.0 mM, and was
202 continued for 15 min at 37 °C. The reaction was stopped by instantly freezing the reaction
203 mixture on dry ice. The generated CO in the head-space was quantified using a specially
204 designed gas-chromatograph for CO analysis (ta 3000R Process Gas Analyzer; Ametek Inc.
205 USA, Newark, DE) which is sensitive and selective for CO by virtue of headspace sampling, size
206 exclusion gas chromatography and mercuric oxide reduction detection (Vreman and Stevenson,
207 1988). Peak heights and areas were used to correlate detector response between standards and
208 unknowns in the linear range. CO production by samples and blanks, analyzed in triplicate, was
209 calculated and HO activity is presented as the net difference between total and blank reactions
210 expressed as pmoles of CO. Controls in which methemalbumin, NADPH or enzymes were
211 omitted have been reported previously (VukomanovicDraft et al. 2011).
212
213 Western Blot Analysis
214 The rh-CPR preparation (5 μg of protein) was run on a 10% SDS-PAGE gel, and stained with
215 Coomasie Brilliant Blue. A molecular weight ladder used to confirm protein identity (Precision
216 Plus ProteinTM Standards; Biorad Ltd., Mississauga Canada, cat#161-0373).
217
218 Statistical analysis
219 Data were expressed as the mean ± SD and analyzed by one-way ANOVA, followed by a
220 Newman-Keuls multiple comparison test for a significant F statistic (P < 0.05) (Prism 7;
221 GraphPad Software, San Diego CA). A P value less than 0.05 was considered to be significant.
222
223 RESULTS
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224 All microsomal preparations showed significant baseline CO production of approximately 70
225 pmol (Figs.1-3).
226
227 Riboflavin and PQQ are activators of CO production in spleen
228 At concentrations below 25 µM, riboflavin was a more potent activator of CO production than
229 PQQ. For example, riboflavin stimulated increased CO production at concentrations as low as
230 100 nM, whereas the threshold for PQQ-mediated CO production was 25 µM (Fig 1). At 100
231 µM, the highest concentration used, riboflavin and PQQ increased CO production to 169±10 and
232 157±15 respectively, reflecting an approximately two-fold over baseline. Rat spleen microsomes
233 contain CPR and HO-1, but comparatively little HO-2, implicating the former two enzymes at
234 the catalytic source of CO. Draft
235
236 PQQ is superior to riboflavin at activating CO production in brain
237 The baseline for CO generated by rat brain microsomes was approximately 75 pmol (Fig. 2). The
238 addition of PQQ over the range of 0.01 to 1 µM did not increase CO production significantly.
239 After the addition of 25 µM of PQQ the production of CO increased to 148±12 pmol, and the
240 presence of 100 µM PQQ, increased CO to 303±70 pmol (Fig. 2). The CO generation curve for
241 riboflavin was similar to that of PQQ except at the highest concentration (100 µM) riboflavin
242 increased CO production to 175±9 pmol.
243
244 Riboflavin and PQQ stimulate CO production in human placenta
245 Having demonstrated CO production by riboflavin and PQQ in rodent tissue, we tested whether
246 human tissue contained similar biochemical machinery by preparing placental microsomes from
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247 healthy infant deliveries. The baseline for CO generated by human placenta microsomes was
248 approximately 70 pmol, and addition of 1 µM PQQ increased CO production to 112±18 pmol
249 (Fig. 3). The addition of 10 and 25 µM PQQ increased CO production to 210±35 and 299±48,
250 respectively. The CO production curve of riboflavin was shifted approximately one order of
251 magnitude to the right with 10 µM producing no detectable increase in CO and the addition of 25
252 µM riboflavin only yielding 129±24 pmol CO production. At 100 µM PQQ and riboflavin
253 increased CO generation to 370±27 and 280±62 pmol, reflecting respectively, an approximate
254 five- and four-fold change from 10 nM.
255
256 PQQ produces significantly more CO than riboflavin in the presence of rh-CPR
257 To determine the extent to which CPR mayDraft contribute to CO production in the human placenta,
258 experiments were conducted where riboflavin and PQQ were incubated in the presence of
259 recombinant human CPR (rh-CPR) and the necessary substrates and cofactors; methemalbumin
260 and NADPH, respectively. Unlike the microsomal preparations, baseline levels of CO
261 production were nearly undetectable in the reconstituted system (Fig. 4A). In the presence of 1
262 µM PQQ, 9±3pmol of CO was generated while 10 µM PQQ increased this to 75±17 pmol CO.
263 The further escalation to 100 µM increased CO production to 159 ± 17 pmol. Relative to the
264 PQQ CO generation curve, the riboflavin CO generation curve was shifted approximately two
265 orders of magnitude to the right. Here, the first significant increase in CO generation occurred at
266 100 µM riboflavin (47±15 pmol CO; Fig 4A). The rh-CPR containing preparation used in Fig. 4
267 was run on 10% SDS-PAGE (Fig. 4B). A very dense over-saturated band corresponding to rh-
268 CPR was identified at approximately 75 kDa with several fainter minor contaminants identified
269 at higher and lower molecular weights.
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271 DISCUSSION
272
273 There were two critical observations in this study. First, both riboflavin and PQQ stimulated
274 significant CO production in the presence of microsomes prepared from rat spleen, rat brain or
275 human placenta. The second critical observation is that riboflavin and PQQ also stimulated the
276 generation of CO in the presence of purified rh-CPR.
277 The preparation containing rh-CPR had as its major component an oversaturated band at
278 approximately 75 kDa corresponding closely to the previously reported molecular weight of
279 approximately 76.5 kDa (Uniprot.org). Although faint bands of contaminating proteins were
280 present it is unlikely they contributed toDraft CO generation. First, by comparison rh-CHR was
281 present at exponentially greater quantity than all the contaminants combined. Second, although
282 HO have been reported in a number of bacteria, only one has been reported in E. Coli, the
283 species from which the BL21 (DE3) pLysS cells, used to grow the expression vector, are
284 derived. In E. Coli, the HO is called ChuS (Maharshak et al. 2015) and is reported to run at
285 approximately 39 kDa (Uniprot.org). There is a faint shadow corresponding to this approximate
286 molecular weight (Fig 4B) but is many orders of magnitude less abundant than rh-CPR
287 suggesting it does not contribute to CO production.
288 In view of CO’s role as an important second messenger gas (reviewed in Motterlini and
289 Foresti 2017), these observations suggest that in addition to their well-described functions as
290 enzymatic co-factors (reviewed in Ames 2018; Powers 2003; Thakur et al. 2017), riboflavin and
291 PQQ may have hitherto unreported roles in the regulation of CO-dependent physiological
292 signalling. All microsomal preparations in the current study showed a significant baseline level
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293 of CO production approximating 70 pmol. In the presence of menadione, we demonstrated
294 baseline CO arises from contributions by HO, whereas the asymptotic component of the CO
295 generation curve is primarily the result of CPR (Vukomanovic et al. 2017). Therefore, we
296 ascribe the CO production observed at higher concentrations of riboflavin and PQQ to also be
297 predominantly CPR. It has long been held that CO production in mammalian systems is the
298 domain of heme-oxygenase enzymes (reviewed in Maines 1997), but these current observations
299 further underscore our recent studies showing the importance of CPR in physiological CO
300 production (Vukomanovic et al. 2017). CO generation occurred in persistent tissues such as
301 spleen and brain, as well as the placenta which exists transiently, suggesting that significant CO
302 generation may occur in any tissue containing CPR.
303 Draft
304 Our interest in identifying agents that stimulate CO generation in vivo stems from a
305 growing body of work implicating CO insufficiency in the etoiology of preeclampsia
306 (Bainbridge et al. 2005; Levytska et al. 2013; George et al. 2014; Ramma and Ahmed 2014;
307 Venditti and Smith 2014). Indeed, a mouse model of preeclampsia was used to demonstrate, that
308 direct inhalation of CO augments uterine artery blood flow and uteroplacental vascular growth
309 (Vendetti et al. 2013) and protected against associated maternal organ damage (Venditti et al.
310 2014). The present study demonstrating that riboflavin stimulates CO generation in human
311 placenta microsomes may provide a mechanistic link to the observation that riboflavin deficiency
312 is significant risk factor for preeclampsia (Wacker et al. 2000). A subsequent riboflavin
313 intervention study was unable to demonstrate a clinical benefit for the treatment of preeclampsia
314 (Neugebauer et al. 2006); however, we speculate the “low dose” 15 mg/day used in the
315 aforementioned study was insufficient. For instance, multiple migraine intervention trials have
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316 used “high dose” 400 mg/day riboflavin to effectively reduce the incidence and severity of
317 migraines (reviewed in Thakur et al. 2017). Our current studies demonstrating significant CO
318 production from brain microsomes, lead us to propose that the therapeutic efficacy of riboflavin
319 against migraines may be, in part, CO-dependent; recalling that CO is an important mediator of
320 inflammation and vascular tone (Motterlini and Foresti 2017), both of which are believed to be
321 dysregulated in migraine (Conti et al. 2019). A role for targeted riboflavin supplementation in
322 the prevention of hypertension has also been identified in patients with a specific defect in
323 methylenetetrahydrofolatereductase (MTHFR) (reviewed in McNulty et al. 2017). These
324 randomized trials showed that riboflavin has a genotype-specific role in lowering blood pressure
325 by an average of 6-13 mmHg independent of the effect of antihypertensive drugs. The
326 mechanism of action that has been proposedDraft is that riboflavin supplementation in patients with
327 MTHFR677TT genotype might lead to improved endothelial function through increased
328 bioavailability of nitric oxide via increases in both 5-methyltetrahydrofolate and
329 tetrahydrobiopterin. Our current findings suggest that CO generation may also contribute to a
330 portion of riboflavin’s antihypertensive effects. We are unaware of studies linking PQQ with
331 preeclampsia or migraines, but in a small clinical crossover study, supplementation with PQQ
332 significantly reduced circulating markers of inflammation (Harris et al. 2013), an effect also
333 ascribed to CO (reviewed in Motterlini and Foresti 2017). Taken together, our in vitro findings
334 showing riboflavin and PQQ activation of CO production are consistent with some of the in vivo
335 clinical observations ascribed to dietary supplementation with these two nutrients.
336 In the current study, in vitro CO production from human and rat microsomes was
337 detectable when riboflavin and PQQ approached the μM range, whereas the reported in vivo
338 tissue concentrations of riboflavin and PQQ are nM (respectively, Bosch et al. 2011; Kumazawa
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339 et al. 1992). Despite this concentration discrepancy our prior studies suggest riboflavin and PQQ
340 will promote CO production in vivo. For instance, in rat brain microsomes menadione increased
341 CO production at μM and not nM concentrations (Vukomanovic et al. 2011), yet when added to
342 the drinking water of mice, menadione significantly increased maternal CO concentrations
343 (Odozur et al. 2018). Although plasma menadione concentrations were not measured in the
344 aforementioned study, it is unlikely that plasma menadione concentrations exceeded the nM
345 range. First, taste aversion reduces water consumption when menadione is added to the drinking
346 water (Odozur et al. 2018), and low μM concentrations have only been reported when
347 menadione was administered i.v. at toxic doses as an adjunct to cancer chemotherapy (Lim et al.
348 2005).
349 The present study showed that 100Draft µM riboflavin or PQQ increased CO production in
350 human placenta microsomes approximately three- or four- fold, over baseline respectively. This
351 compares favourably with the six-fold increase in CO production in the presence of 100 µM
352 menadione (unpublished data). The manner in which riboflavin and PQQ enhance CO
353 production remains to be elucidated; however, we speculate their mechanism of action may be
354 similar to that of menadione, because these nutrients share a number of electrochemical and
355 structural similarities with the vitamin K analogue. Previously, we demonstrated that the ability
356 of menadione to promote CO production from rh-CPR was reliant upon the generation of
357 reactive oxygen species in the form of superoxide radicals and hydrogen peroxide; addition of
358 superoxide dismutase and catalase significantly reduced CO production (Vukomanovic et al.
359 2017). The ability of riboflavin and PQQ to act as cofactors free radical formation has been
360 previously reported (respectively, Juárez et al. 2008; Akagawa et al. 2016).
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361 There were tissue-specific differences in CO production noted with both nutrients. The
362 elucidation of the cause of the tissue-specific effects would require further experimentation, but
363 we expect the differences to be, in part, the result of differences in the relative ratios of CPR,
364 HO-1 and HO-2 enzyme activities. For example, we previously demonstrated that menadione
365 stimulates robust CO production in rat brain microsomes, a constitutively rich source of HO-2;
366 however, in spleen an abundant source of HO-1, menadione fails to stimulate CO production
367 (Vukomanovic et al. 2014). In the present study, the addition of riboflavin and PQQ
368 significantly increased CO production in rat spleen microsomes, but the response was greater in
369 brain microsomes suggesting HO-2 and CPR activities are higher in this tissue. Although subtle,
370 riboflavin was the more effective promoter of CO production in the spleen whereas in rat brain
371 and human placenta PQQ was dominant.Draft What is more, in placenta microsomes and with
372 preparations only containing rh-CPR, there was a markedly similar rightward shift of the
373 riboflavin CO production curve relative to that of PQQ. We interpret the shared rightward shift
374 to mean, that with respect to riboflavin and PQQ, CPR rather than HO is the dominant source of
375 CO production in the human placenta. The importance of CPR may have profound clinical
376 implications. For instance, placental HO-1 expression and exhaled CO levels are both lower in
377 women with preeclampsia, consistent with a role for HO-1 as an important source of CO during
378 pregnancy (Levytska et al. 2013). Lower HO activity in cases of preeclampsia presents a
379 profound challenge, recalling that our therapeutic strategy is reliant using a compromised
380 endogenous biochemical process (HO-1) to produce CO. The observation, that CPR is likely the
381 dominant CO generation activity in the human placenta leads us to suggest that riboflavin or
382 PQQ may be used to activate CPR-based CO production to compensate for the HO insufficiency.
383 Additionally, the extraordinary increase of CO production by RB or PQQ becomes even more
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384 significant in the light of Zorn and Wells (2010) pointing out that even a modest enzymatic
385 activity enhancement of 10% is therapeutically useful. Thus, the several-fold increase in
386 placental CO production in the presence of RB or PQQ may have therapeutic potential even if
387 the endogenous process used to produce CO is compromised.
388
389 In conclusion, we have described previously that CO production occurs in chorionic villi
390 of human placenta (Ahmed et al. 2005), and the current studies demonstrate for the first time that
391 riboflavin and PQQ increase CO production in human placenta microsomes. Thus, we propose
392 that both nutrients may have novel biochemical roles in gasotransmission. A critical role for CPR
393 in the production of CO was also demonstrated, and the further study of this novel pathway
394 would be warranted because its up- or down-regulationDraft by nutrients may have therapeutic
395 applications.
396
397
398 ACKNOWLEDGEMENTS
399 We thank the nurses and surgeons at Kingston General Hospital for access to placentas. The
400 assistance of Laura van Staalduinen in the conduct of the Western blot analysis is gratefully
401 acknowledged.
402
403 FUNDING SOURCES
404 This research was funded by the Canadian Institutes of Health Research Grant CIP-150737
405 ABBREVIATIONS
406 CO: carbon monoxide
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407 CPR: cytochrome p450 reductase
408 HO: heme oxygenase
409 NADPH: nicotinamide adenine dinucleotide phosphate
410 PQQ: pyrroloqinoline-quinone
411 RB: riboflavin
412
413 REFERENCES
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537 Riddick, D.S., Ding, X., Wolf, C.R., Porter, T.D., Pandey, A.V., Zhang, Q.Y., Gu, J., Finn, R.D., 538 Ronseaux, S., McLaughlin, L.A., Henderson, C.J., Zou, L., Flück, C.E. 2013. NADPH- 539 cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology. Drug 540 Metab. Dispos. 41:12-23. http://doi:10.1124/dmd.112.048991. PMID:23086197. 541 542 Suits, M. D., Pal, G. P., Nakatsu, K., Matte, A., Cygler, M., and Jia, Z. (2005) Proc. Natl. Acad. 543 Sci. U. S. A.102, 16955-16960 544 545 Thakur, K., Tomar, S.K., Singh, A.K., Manda, S., Arora, S. 2017. Riboflavin and health: A 546 review of recent human research. Crit. Rev. Food Sci. Nutr. 57:17:3650-3660. 547 http://doi:10.1080/10408398.2016.1145104. PMID:27029320. 548 549 Trakshel, G.M., Maines, M.D. 1988. Detection of two heme oxygenase isoforms in the human 550 testis. Biochem. Biophys. Res. Commun. 154:285-91. http://doi:10.1016/0006-291x(88)90682-1. 551 PMID:3395332. 552 553 Uniprot.org; https://www.uniprot.org/uniref/UniRef100_A0A0K3Z2M4. Accessed 2019-10-15. 554 555 Venditti C.C., Casselman, R., Young, I., Karumanchi, S.A., Smith, G.N. 2014. Carbon monoxide 556 prevents hypertension and proteinuria in an adenovirus sFlt-1 preeclampsia-like mouse model. 557 PLOS One 9:1–7. http://doi:10.1371/journal.pone.0106502.Draft PMID:25202912. 558 559 Venditti, C.C., Casselman, R., Murphy, M.S.Q., Adamson, S.L., Sled, J.G., Smith, G.N. 2013. 560 Chronic carbon monoxide inhalation during pregnancy augments uterine artery blood flow and 561 uteroplacental vascular growth in mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 305:R939- 562 R948. http://doi:10.1152/ajpregu.00204.2013. PMID:23986360. 563 564 Venditti, C.C., Smith, G.N. 2014. Involvement of the heme oxygenase system in the 565 development of preeclampsia and as a possible therapeutic target. Women’s Health (Lond). 566 10:623-43. http://doi:10.2217/whe.14.54. PMID:25482489. 567 568 Vreman, H.J., Stevenson, D.K. 1988. Heme oxygenase activity as measured by carbon monoxide 569 production. Anal. Biochem. 168:31–38. 570 571 Vukomanovic, D., McLaughlin, B.E., Rahman, M.N., Szarek, W.A., Brien, J.F., Jia, Z., Nakatsu, 572 K. 2011. Selective activation of heme oxygenase-2 by menadione. Can. J Physiol. Pharmacol. 573 89:861–864. http://doi:10.1139/Y11-091. PMID:22039914. 574 575 Vukomanovic, D., McLaughlin, B., Rahman, M.N., Vlahakis, J.Z., Roman, G., Dercho, R.A., 576 Kinobe, R.T., Hum, M., Brien, J.F., Jia, Z., Szarek, W.A., Nakatsu, K. 2010. Recombinant 577 truncated and microsomal heme oxygenase-1 and -2: differential sensitivity to inhibitors. Can. J. 578 Physiol. Pharmacol. 88:480-6. http://doi:10.1139/y10-004. PMID:20555417. 579 580 Vukomanovic, D., Rahaman, M.N., Bilokin, Y., Golub, A.G., Brien, J.F., Szarek, W.A., Jia, Z., 581 Nakatsu, K. 2014. In Vitro Activation of heme oxygenase-2 by menadione and its analogs. Med. 582 Gas. Res. 4:1-11. http://doi:10.1186/2045-9912-4-4. PMID:24533775.
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583 584 Vukomanovic, D., Rahman, M., Jia, Z., Nakatsu, K. 2017. Drug-enhanced carbon monoxide 585 production from heme by cytochrome P450 reductase. Med. Gas. Res. 7: 37-4. 586 http://doi:10.4103/2045-9912.202908. PMID: 28480030. 587 588 Wacker, J., Frühauf, J., Schulz, M., Chiwora, F.M., Volz, J., Becker, K. 200. Riboflavin 589 deficiency and preeclampsia. Obstet. Gynecol. 96:38-44. PMID:1086283. 590 591 Zorn, J.A., Wells, J.A. 2010. Turning enzymes ON with small molecules. Nat. Chem. Biol. 592 6:179-188. http://doi:10.1038/nchembio.318. PMID:20154666. 593 594
595 FIGURE LEGENDS
596
597 Figure 1. Enhanced CO production by rat spleen microsomes in the presence of riboflavin 598 (RB) and pyrolloquinolone-quinone (PQQ).Draft Heme degradation in microsomal fractions (1 mg 599 of protein) prepared from four different male rat spleens (triplicates for each) was quantified by
600 measurement of CO using a headspace gas chromatography method, after 15 minutes of
601 incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at
602 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The
603 asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or
604 PQQ value at 10 nM.
605
606 Figure 2. Enhanced CO production by rat brain microsomes in the presence of riboflavin
607 (RB) and pyrolloquinoline-quinone (PQQ). Heme degradation in microsomal fractions (1 mg
608 of protein) from four different male rat brains (triplicates for each) was quantified by
609 measurement of CO using a headspace gas chromatography method, after 15 minutes of
610 incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at
611 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The
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612 asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or
613 PQQ value at 10 nM.
614 Figure 3. Enhanced CO production by human placenta microsomes in the presence of
615 riboflavin (RB) and pyrolloquinolone-quinone (PQQ). Heme degradation in microsomal
616 fractions (1 mg of protein) from four different placentas (triplicates for each) was quantified by
617 measurement of CO using a headspace gas chromatography method, after 15 minutes of
618 incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at
619 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The
620 asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or
621 PQQ value at 10 nM.
622 Draft
623 Figure 4. Pyrolloquinoline-quinone (PQQ) produces significantly more CO than riboflavin
624 (RB) in the presence of recombinant human CPR (rh-CPR). Panel A); the heme degradation
625 in the presence of 10 μM rh-CPR (four replicates from the same preparation) was quantified by
626 measurement of CO using a headspace gas chromatography method, after 15 minutes of
627 incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at
628 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The
629 asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or
630 PQQ value at 10 nM. Panel B); The SDS PAGE (10%) of the rh-CPR preparation used to
631 generate CO in panel A) was stained with Coomasie Brilliant Blue. Lane 1 contains a molecular
632 weight ladder and Lane 2 the rh-CPR-containing preparation. The arrowhead denotes the
633 location of rh-CPR.
634
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Draft
Figure 1. Enhanced CO production by rat spleen microsomes in the presence of riboflavin (RB) and pyrolloquinolone-quinone (PQQ). Heme degradation in microsomal fractions (1 mg of protein) prepared from four different male rat spleens (triplicates for each) was quantified by measurement of CO using a headspace gas chromatography method, after 15 minutes of incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or PQQ value at 10 nM.
254x190mm (72 x 72 DPI)
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Draft
Figure 2. Enhanced CO production by rat brain microsomes in the presence of riboflavin (RB) and pyrolloquinoline-quinone (PQQ). Heme degradation in microsomal fractions (1 mg of protein) from four different male rat brains (triplicates for each) was quantified by measurement of CO using a headspace gas chromatography method, after 15 minutes of incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or PQQ value at 10 nM.
254x190mm (72 x 72 DPI)
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Draft
Figure 3. Enhanced CO production by human placenta microsomes in the presence of riboflavin (RB) and pyrolloquinolone-quinone (PQQ). Heme degradation in microsomal fractions (1 mg of protein) from four different placentas (triplicates for each) was quantified by measurement of CO using a headspace gas chromatography method, after 15 minutes of incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or PQQ value at 10 nM.
254x190mm (72 x 72 DPI)
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Draft
Figure 4. Pyrolloquinoline-quinone (PQQ) produces significantly more CO than riboflavin (RB) in the presence of recombinant human CPR (rh-CPR). Panel A); the heme degradation in the presence of 10 μM rh-CPR (four replicates from the same preparation) was quantified by measurement of CO using a headspace gas chromatography method, after 15 minutes of incubation with RB or PQQ and 1.0 mM NADPH in 100 mM phosphate buffer (pH = 7.4) at 37°C. Data are presented as mean ± standard deviation, picomoles of CO (pmol CO). The asterisk denotes a statistically significant difference of at least p< 0.05 from the respective RB or PQQ value at 10 nM. Panel B); The SDS PAGE (10%) of the rh-CPR preparation used to generate CO in panel A) was stained with Coomasie Brilliant Blue. Lane 1 contains a molecular weight ladder and Lane 2 the rh-CPR-containing preparation. The arrowhead denotes the location of rh-CPR.
254x190mm (72 x 72 DPI)
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