Carbon monoxide secretion into ophthalmic venous blood of a crossbreed of wild boar and pig during light and nocturnal phase of day

Marek Koziorowski 1, Stanisława Stefańczyk-Krzymowska2, Anna Tabęcka-

Łonczyńska1, Przemysław Gilun2 and Marian Kamiński3

1Department of Animal Physiology and Reproduction, University of Rzeszow, 35-959

Rzeszów, Poland 2Department of Local Physiological Regulation, Institute of Animal Reproduction and Food

Research of the Polish Academy of Sciences, 10-747 Olsztyn, Poland 3 Chemical Faculty, Gdańsk University of Technology, 80-233 Gdańsk, Poland

Running title: Carbon monoxide secretion into ophthalmic venous blood

Key words: carbon monoxide, eye, circadian rhythm

Total number of words: 2772

Subject area: Endocrinology

Corresponding author: Marek Koziorowski, ul. Rejtana 16C, Rzeszów 35-959, Poland;

e-mail: mkozioro@univ.rzeszow.pl; Tel.: (17) 872 32 57; Fax: (17) 872 32 61.

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Introduction

In both human and other animal tissues, two isoforms of heme oxygenase (HO); inducible

(HO-1), and constitutive (HO-2) break the rings of the heme to produce carbon monoxide

(CO), ferrous iron and biliverdin/bilirubin (Tenhunen et al. 1968; Maines, 1988; Ortiz de

Montellano, 1998). Functional HO-2 was found in the neurons of the cortex, hypothalamus,

thalamus and cerebellum (Vincent et al. 1994, Maines, 2000). It was documented, that CO as

a gaseous transmitter was synthesized rapidly following neural depolarization, and HO-2 was

activated during neuronal stimulation by phosphorylation by casein kinase-2 (Boehning et al.

2009).

In the eye, bright light stimulated HO in the retina and red blood cells to produce CO

(Kutty et al. 1995). High level of HO-1 mRNA and immunocytochemically localized HO-2

was found in the photoreceptors, amacrine, and both bipolar and ganglion cells of the retina in

the turtle, as well as in the ganglion and amacrine cells of the inner retina in rats (Cao et al.

2000). Stimulation of the retina, in combination with CO and nitric oxide (NO) application,

dramatically increased cGMP synthesis in the retina in comparison to NO or CO application

alone (Cao et al. 2000). Moreover, HO-1 appeared to be involved in the regulation of

intraocular pressure via CO production (Prifitera et al. 2007). HO-1 was also upregulated in

central retinal artery occlusion (Goldenberg-Cohen et al. 2008). CO has been found to be a

very important vasodilator (Pannen & Bauer, 1998; Kozma et al. 1999; Wu & Wang, 2005;

Abraham & Kappas 2008). When CO diffuses into the vascular smooth muscle cells, it elicits

vasodilatation by increasing cGMP production, and consequently it hyperpolarizes the muscle

cells by opening potassium channels (Furchgott & Jothianandan, 1991; Christodoulides et al.

1995; Morita & Kourembana, 1995). Furthermore, bath perfusion of tissues with exogenous

CO produced an increase in potassium current and hyperpolarized resting membrane potential

(Rich et al 1994). CO also induced vasodilatation as well as NO release (Thorup et al. 1999).

The presence and distribution of both isoforms of HO has been demonstrated using

immunohistochemical methods in the epithelium of the seromucous glands, endothelium of

human nasal mucosa and in erythrocytes (Andersson et al. 2000; Lo et al. 2005).

In 1996, Dan A. Oren published an inspirational hypothesis on humoral

phototransduction. This paper assumed that the information of light intensity might be

transmitted from the retina to the brain, not only by neural pathway, as generally accepted, but

also by a humoral pathway. CO and NO could be transported with venous blood and

transferred by counter current mechanism to the arterial blood in the area of the

perihypophyseal vascular complex. If CO and NO were transferred to the arterial blood in the

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area of the perihypophyseal vascular complex then they could influence some of the neural

centres of the brain (Oren,1996). According to our knowledge, this concept has never been

studied. There is also lack of information on the relationship between the phase of the day and

CO production in the eye as well as the concentration of CO in venous blood flowing out of

the eye.

The present study was designed to determine whether the secretion of CO into the

venous blood flowing out from the eye differed between the light phase and nocturnal phase

during the long day of the summertime. The concentration of CO in venous blood flowing out

from the nasal cavity, where HO is expressed but photoreceptors are not present, was utilized

as a control.

Methods

Basic statements

All the procedures were carried out in compliance with Polish legal regulations (act of 21

January 2005), which determine the terms and conditions for performing experiments on

animals and in accordance with the protocol of the Local Ethics Commission for Animal

Experiments in Lublin No 8/2007. Mature males of crossbreed wild boar and pig (12 months

of age, body mass ~ 100-120kg, n=8) were used in the study. The animals were housed in an

experimental farm (Physiology and Reproduction of Animals Department, Rzeszów

University). The experiments were performed during the long daylight season (June). During

the collection of blood samples the animals were kept in individual cages in rooms with

natural illumination and ad libitum access to water and food.

Surgical procedures and blood samples collection

Five hours before the collection of blood samples, the animals were premedicated with

atropine (0.05 mg kg-1 I.M; Biowet, Gorzów Wielkopolski, Poland) and azaperone (Stresnil 2

mg kg-1 I.M; Jansen Pharmaceutica, Beerse, Belgium). General anaesthesia was induced with

thiopental sodium (Thiopental, Sandoz GmbH, Austria). Two silastic catheters (o.d., 2.4 mm;

i.d., 1.8 mm) were inserted into the dorsal nasal vein. The first catheter was directed into the

proximal part of the nasal vein to collect nasal venous blood. The second one was inserted in

the opposite direction. This catheter passed through the angularis oculi vein ending in the

venous ophthalmic sinus. Placing a catheter in the venous ophthalmic sinus allowed for the

collection of venous blood flowing out of the eye (Fig. 1). Blood samples (10 ml) were

collected from the nasal vein and venous ophthalmic sinus for two consecutive days, every

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four hours during the day (4.00, 8.00 and 12.00 a.m. and 4.00, 8.00 and 10.00 p.m.) and every

two hours during the night (12.00 p.m., 2.00 a.m.). Simultaneously systemic arterial blood

samples were collected every eight hours during the day, whereas systemic venous blood

samples were collected every eight hours during the day and night from each animal.

Analysis of trace levels of CO in blood samples

1. Principles of the procedure for preparation of gaseous components and determination of

CO

Determination of CO concentrations in blood samples was performed at the Chemical

Faculty, Gdansk University of Technology. A standard addition method was used. Analytical

procedure was based on coupling of Head Space (HS) analytical technique (extraction of CO

from blood sample to gas phase) with the column gas chromatography technique using packed

column for separation of gaseous sample components (molecular sieve 5A, 80-100 mesh,

activated at 350oC). Trace concentrations of CO were determined as concentrations of

methane (CH4) using Flame Ionization Detector (FID) after stechiometric CO to CH4 catalytic

conversion in the packed micro-reactor (Raney Nickel) located between the outlet of the

column and the inlet of the FID detector (Col-/5A/-GC-/CO-CH4/-FID). Application of the

5A molecular sieve column ensures the separation of CO and oxygen (O2), and thus

eliminates the possible influence of O2 on determination of trace amounts of CO.

Chromatographic conditions during determination of the trace amounts of CO in blood

samples is presented in table 1. Detailed description of the procedure for CO/CO2/CH4/O2

separation as well as determination of trace amounts of CO in gas samples was presented

earlier (Kamiński et al. 2003).

2. Preparation of samples for CO determination

Five cm3 of blood was introduced into each of the two tightly sealed 16 cm3 HS vials filled

with helium, using a syringe with an injection needle. Simultaneously, the excess of helium

was removed to keep the atmospheric pressure in the vial. Then 0.5 cm3 of standard CO

mixture in helium (600 ppm [v/v]) was added to one of the vials. To equilibrate CO

concentrations between the liquid (blood) and gas phase, both vials were kept for 30 min at

37oC. Next, 5 cm3 of the gas sample was taken from each vial using a gas-tight syringe

(ensuring a constant pressure in a vial) and transferred into two tightly sealed 16 cm3 HS vials

filled with pure air. Prepared gas samples (0.5 cm3) were analyzed by gas chromatography

coupled to flame ionization detection after CO methanization (Col-/5A/-GC-/CO-CH4/-FID).

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3 Calculation of the results

Concentration of CO (nmol/ml) in the blood samples was calculated using the following

formula:

)12(1)2(

AAwALV

AAwAwVwCxC

−⋅⋅⋅−⋅

=

Cx – concentration of CO in the blood sample

Cw – concentration of CO in the standard mixture added to HS vial containing the

sample studied

VL – volume of the liquid sample (blood) introduced into a HS vial

Vw – volume of the standard gas mixture introduced into a HS vial

A1 – CO peak area for the blood sample studied

A2 – CO peak area for the blood sample with the standard addition

Aw – CO peak area for the standard mixture

4 Statistical analysis

All the data are presented as means ± SEM. The concentration of CO in venous blood flowing

out from the eye and from the nasal area obtained during the morning, afternoon and night

(Figs 2 and 4) was compared between the vessels and between experimental periods using a

nonparametric Mann-Whitney t-test. The venous outflow of CO from the eye and nasal area

during the light phase of the long day (Fig. 3) was analysed by determining the total area

under respective curves. These data were also compared with nonparametric Mann-Whitney t-

test. All analyses were performed with the use of Prism GraphPad Software San Diego, CA.

Results

The concentration (mean ± SEM) of CO in venous blood flowing out of the eye (collected

from the ophthalmic sinus) averaged 3.32±0.71 nmol ml-1 and 3.43±0.8 nmol ml-1 in the

morning and afternoon, respectively. The data also did not differ between periods of sample

collection (Fig. 2). CO concentration in venous blood flowing out from the nasal cavity

averaged 0.75±0.06 nmol ml-1 and 1.03±0.22 nmol ml-1 in the morning and afternoon,

respectively, and these values did not differ. In both periods, CO concentration in venous

blood flowing out from the eye was significantly higher than that in nasal venous blood

during the morning (P<0.001) and the afternoon (P<0.05). The mean (± SEM) area under the

curve of CO concentration during the day in venous blood flowing out from the eye was

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significantly higher than that of CO concentration in the nasal venous blood (P<0.01) (Fig. 3).

The mean CO concentration in venous blood flowing out from the eye and nasal area during

the night is presented in Fig. 4. It averaged 0.89±0.12 nmol ml-1 and 0.86±0.2 nmol ml-1 in

venous blood flowing out from the eye and nose, respectively, and the values did not differ

between vessels. The mean concentrations of CO in systemic arterial blood during the day

averaged 1.3±0.34 nmol ml-1, with venous systemic blood averaging 1.24±0.75 nmol ml-1 and

1.26±0.69 nmol ml-1 during the day and night, respectively. The data did not differ between

the vessels examined or time periods.

The above results demonstrated that the following occurred: 1) during the long light-

day season, CO was produced in the eye and concentrations in ophthalmic venous blood was

significantly higher than CO concentration in venous blood flowing out from the nasal cavity;

2) CO concentration in venous blood flowing out from the eye during the night was threefold

lower than concentrations taken during the day, and did not differ from nasal venous blood;

and 3) CO concentration in venous outflow from the nasal cavity did not differ between the

diurnal and nocturnal phase of the day and did not differ from CO concentration in the

systemic venous and arterial blood.

Discussion

Daily measurement of CO concentration in venous blood flowing out in physiological

conditions from the eye and nose was performed for the first time in the present study. CO is

produced in many tissues, including the eye, which is based on the immunohistochemical

localization of HO-1 and HO-2 (Tenhunen et al. 1968; Maines, 1988; 2000; Vincent at al.

1994; Kutty et al. 1995; Ortiz de Montellano, 1998; Cao et al. 2000). This study was

conducted during the entire day (in the morning and afternoon) and during the short night. It

should be emphasized that blood samples were collected in physiological conditions without

making the animal uneasy. The catheters inserted earlier into the dorsal nasal vein and into the

angularis oculi vein enabled us to repeatedly collect blood samples from the sinus ophthalmic

(venous outflow from the eye) and from the nasal vein (venous outflow from nasal area)

during the diurnal and nocturnal phase of two consecutive days. The concentration of CO in

venous blood flowing out from the area of the nasal cavity, which was at the level similar to

that in systemic venous and arterial blood, was accepted as a control. The localisation of both

isoforms of HO in the human olfactory organ (Andersson et al. 2000; Lo et al. 2005)

suggested that CO may be produced in this area. However, the olfactory organ is not sensitive

to light, and our study demonstrated constant concentration of CO in venous blood flowing

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out from this area during the day and night. Contrary to the CO production in the olfactory

organ, we have revealed that CO concentration in the venous outflow from the eye differed

between the diurnal and nocturnal phase.

The present results demonstrate that the concentration of CO in venous blood flowing

out from the eye averaged 3.3 nmol ml-1 (Fig. 2), whereas the mean concentration of CO in

blood flowing in opposite direction, through the small arterial vessels, was 0.95 nmol ml-1. In

the periophthalmic vascular complex, the arterial and venous blood stream are closely located

to each other, making it possible for counter current exchange of particles with the tendency

to balance concentrations. Under these conditions, CO (size of 28.01 Da) is able to penetrate

the arterial blood, supplying the eye, from the venous blood easily. Because previous studies

have demonstrated that inhaled carbon monoxide increases the CO concentration in the blood,

resulting in enhanced retinal and choroidal blood flow in healthy humans (Resch et al. 2005),

it seems obvious that the increase in CO concentration in arterial blood, due to counter current

transfer, may influence the arterial vessel wall tension and modulate blood supply to the eye.

Moreover, as stated in our earlier studies, many neurotransmitters that are much larger than

CO still permeate the venous blood flowing to the arterial blood in the perihypophyseal

vascular complex (Fig. 1), which is morphologically similar to the periophthalmic vascular

complex (Khamas et al. 1984; Ninomiya & Masui, 1999). Some neurotransmitters including

GnRH, (Krzymowski et al. 1992, Grzegorzewski et al. 1997), oxytocin (Grzegorzewski et al.

1995), beta-endorphin (Krzymowski et al. 1992; Skipor et al. 1997), dopamine (Skipor et al.

2001) and the steroid pheromone androstenol (Krzymowski et al. 1999; 2001, Stefańczyk-

Krzymowska et al. 2000) were transferred, by counter current mechanism, from the venous

cavernous sinus into arterial blood of the carotid rete mirabile that supply the brain. It could

be presumed, but it have to be experimentally documented, that particles of CO and NO,

which are much smaller than neurotransmitters, might freely penetrate into the area of the

ophthalmic vascular complex from the venous blood into the arterial blood supplying the eye,

according to their concentration gradient.

Acknowledgements

The study was supported by the Polish State Committee for Scientific Research (N N 311

1001 33) and Ministry of Science and Higher Education in 2007.

Authors dedicate this paper to Professor Tadeusz Krzymowski who inspired us to the

collection of this research.

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Table 1. Chromatographic conditions during determination of trace amounts of CO in blood

samples

Gas chromatograph Agilent 6890

Column

2m x 2.1 mm inner diameter GC

column packed with molecular sieve

5A (80-100 mesh)

Detector FID (280 oC) with Raney Ni -

methanizer

Carrier helium; 10 cm3 min-1

Oven temperature 130oC

Injection port split-less; 100oC

Sample volume 0.5 cm3

Limit of detection (LOD) 0.5 ppm (v/v) CO

12

Figures and figure legends

Figure 1. Simplified scheme of the experiment with ophthalmic and nasal venous blood

collection and arterial blood supply of the eye and venous blood effluent. 1/ maxillary artery;

2-3/ branches of the epidural rete mirabile; 4/ external ophthalmic artery; 5/ epidural rete

mirabile in the cavernous sinus; 6/ intercavernous sinus; 7/ carotis interna artery; 8/

anastomotic branch with epidural rete mirabile; 9/ emissary vein of the foramen

orbitorotundum; 10/ ophthalmic venous sinus and rete mirabile external ophthalmic artery;

11/ ophthalmic vein; 12/ ophthalmic externa artery; 13/ ciliares vein; 14/ circle of Willis; 15/

to jugular vein.

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Figure 2. Mean (± SEM) concentration of carbon monoxide in venous blood outflow from the

eye and nasal area during light phase of the long day. A – morning, B – afternoon. * P<0.05;

*** P<0.001.

Figure 3. Mean (± SEM) area under curve (AUC) of carbon monoxide concentration in

venous outflow from the eye and nasal area during entire light phase of the long day. **

P<0.01.

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Figure 4. Mean (± SEM) concentration of carbon monoxide in venous blood outflow from the

eye and nasal area during nocturnal phase of the long day.