Effects of dietary CoQ 10 and α-lipoic acid on CoQ 10 levels in plasma and tissues of eggs laying hens

In this paper we described the effect of administrated CoQ10, and alfa-lipoic acid on the concentration of total CoQ10 in plasma end body tissues of eggs laying hens. Organisms raise a complex network of enzymes, metabolites and molecules with antioxidant activities in order to prevent oxidative damage of theirs bodies. Adequate blood concentrations of small weight molecules ingested with food and food additives are important for the proper functioning of the antioxidant defense. To test this hypothesis we prepared following experiment. Forty weeks old hens were selected from two genotypes; Ross 308 broiler mothers and Lohmann breed hens. Animals were fed for a period of 84 days. Concentrations of supplemented CoQ10 and ALA were calculated from feed instruction tables so each hen received an average of approximately 5 mg of CoQ10 and 50 mg of ALA per kg of animal weight per day. During the experiment blood samples were taken and at the end of the experiment different body tissues (heart, liver, breast, legs) were collected and analyzed with originally developed HPLC-MS/MS method based selective ionization with LiCl on MRM scanning. We found a number of interesting and unexpected results. Supplemented CoQ10 increased concentrations of coenzyme CoQ10 in plasma and different hen’s tissues. Increased concentration of CoQ10 is the result of its transfer with chylomicrons from the digestive tract to various organs of the body and to the liver where exogenous and endogenous CoQ10 has been re-redistributed through lipoproteins. Supplemented ALA caused much greater concentration of CoQ10 in different tissues and plasma then CoQ10. Plausible explanation of our results is such that ALA may regenerates the antioxidants and accelerate the formation of endogenous CoQ10 which is distributed with lipoprotein carriers and increases overall concentration of CoQ10. Our experiments definitely show that Lipoic acid beside glutathione promotes also a synthesis of CoQ10 and increases the total concentration especially in liver and heart tissues.


INTRODUCTION
Living organisms have to raise a complex system of enzymes, metabolites and molecules with antioxidant activities in order to prevent oxidative damage of theirs bodies [1,2].Until recently, scientists believed that each antioxidant worked separately, independently of the others.Research performed at the Packer Lab at the University of California at Berkeley showed that there is a dynamic connection among certain key antioxidants.These special antioxidants operate together and represent a dynamic defense of an organism.Antioxidants in this network terminate oxidation processes by removing or quenching free radicals and are capable of slowing or preventing the oxidation [3].
The expression antioxidant network was first presented by Packer [4], who stated that antioxidants do not act alone but are linked together into a network.Interaction of antioxidants had been already noticed before Packer, but he was the first who outlined a concept of a network based on the five molecules; CoQ 10 , ascorbic acid (vitamin C), tocopherol (vitamin E), glutathione and lipoic acid.The diagram of the antioxidant network built from reduced and oxidized forms of: lipoic acid, glutathione, CoQ 10 , vitamin C and vitamin E is presented in Figure 1.On the top at standard redox potential of less than -0.315V is the net supplied with protons from NADH (the reduced form of Nicotinamide adenine dinucleotide NAD + a coenzyme found in all living cells), and NADPH (the reduced form of Nicotinamide adenine dinucleotide phosphate NADP + ).At -0.220 V FADH 2 (the reduced form of a redox cofactor flavin adenine dinucleotide FAD involved in several important reactions in metabolism) supports reduced form of CoQ 10 .In hydrophilic phases a considerable protection is produced from degradation product with antioxidant activity, like uric Administered food may increase concentrations of vitamins and coenzymes in the network.The antioxidants from plants; carotenoids, flavonoids and polyphenols also protect antioxidant network but only in the redox range between +0.400 V and +0.700 V. From the Figure 1, it is possible to conclude that the operation of antioxidant network is complex function [5][6][7], but also very logical.Regeneration of net strongly depends on high concentration of NADH and NADPH.
Our previous research work connected with industrial poultry farming indicates the hypothesis that chickens and hens could be very suitable candidates for scientific estimation of the intensity of oxidative stress and protecttive effect of Low Molecular Weight Antioxidants [8,9].The aim of this study was to determine the effects of a scientifically selected diet on the content of several antioxidants in blood plasma and some animal tissues.Administered food provided necessary conditions for existence of adequate blood levels of enzymes, coenzymes, which together with the large number of administered small weight molecules were responsible for correct functioning of body antioxidant defense.To test this hy-pothesis we had to develop new reliable analytical methods for assessing the amount of antioxidants in plasma and animal tissues.In present study the concentrations of total amount of CoQ 10 in different body tissues and blood plasma of laying hens are presented.Oxidized form of CoQ 10 after prolonged feeding with food fortified with CoQ 10 , and α-Lipoic acid (ALA) were measured with originally developed HPLC-MS/MS method [10].

Experimental Design
Forty weeks old hens were selected from two genotypes; Ross 308 broiler mothers and Lohmann breed hens.Animals were housed in wire laying cage (one bird per cage) and fed on the commercial feed NS-val (Ross) and NSK (Lohmann) prepared in Perutnina Ptuj, Slovenia) for 2 weeks before the experiment started.Animals received the supplemented diet on the first day of the experiment and were fed for a period of 12 weeks.Concentrations of supplemented CoQ 10 and ALA were calculated from feed instruction tables [11,12].Each hen received an average of approximately 5 mg of CoQ 10 and (or) 50 mg of ALA per kg of animal weight per day.During the 84 days pilot raise, all animals were treated under identical environmental and growing conditions.Tests were done in optimal breeding and healthy conditions.The required amount of CoQ 10 was provided as the water soluble additive originally synthesized in our laboratory (Laboratory for Food Chemistry, National Institute of Chemistry, Ljubljana, Slovenia) by in-capsulation of CoQ 10 into corn dextrin.The applied food grade alfa-lipoic acid and raw CoQ 10 were purchased from Linyi Tianliheng Trade Co (China).
During the experiment the blood samples were taken five times, at the start (day 1) and 21, 42, 63, and 84 days after the experiment was introduced.Up to 2 ml of blood were taken from vene cutaneae ulnaris.After the end of the experiment hens were sacrificed and different body tissues (heart, liver, breast, leg) were separated and stored together with plasma end blood samples in cool storage at −80˚C until the start of analyses.
All experimental procedures were done according to the guidelines for the care and use of experimental animals at Biotechnical faculty, Department of Animal Science, University of Ljubljana, Slovenia.Experiments on animals were approved by Ethic Committee of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia.

Experimental Procedures
Samples were prepared with following procedures: 400 µL of heparined blood was denaturated with 200 µL of 10% perchloric acid in ethanol.Analites were extracted three times with 2 mL of n-hexane and the combined organic extracts were concentrated with rotary evaporator (Rotavapor R-144 Büchi, Switzerland).The residue was dissolved again in 200 µL of 2-propanol and analyzed with HPLC-ESI-MS/MS.
Part of chicken breasts, legs, wings, whole hearts and livers were mixed with H 2 O and homogenized for 3 minutes with Ultraturax at 20.000 rpm into a homogenous paste.10 g of the homogenized sample were weighed into 50 mL centrifuge tube.15 mL of warm (35˚C -40 ˚C) distilled water was added and intensively mixed for 5 minutes.Fat was extracted twice with 20 mL of solvent mixture consisting of chloroform and methanol (2:1, v/v).The combined extracts were concentrated and dried in a stream of nitrogen.The oil residue was dissolved again in 5 mL of 2-propanol.
Plasma and tissues concentrations of total CoQ 10 were quantified with Sciex API-4000 QTRAP LC/MS/MS system from Applied Biosystems /MDS (Sciex Concord, ON, Canada), equipped with TurboIonSpray TM ionization system and connected to HPLC system constructed from LDC Constametric 4100 pump, and SpectraSystem AS3000 autosampler.
The reduced and oxidized form of CoQ 10 were successfully separated by LC column-LUNA C18 (2), 3 μm, 100 × 4.6 mm (Phenomenex, Torrance, CA, USA).Both forms were eluted with an isocratic mobile phase (acetonitrile: 2-propanol, 55:45) at a flow rate of 0.5 mL/min.The injection volume was 2.0 µL.For efficient ionization a solution of 0.5 µM LiCl (0.5 mL LiCl/L mobile phase) was added directly into container of mobile phase.Sciex Analyst software was used to perform data analysis and peak integration.

Statistical Analysis
All statistics were run using Statgraphic plus Ver. 4.An analysis of variance (ANOVA) and a Student t-test were employed to evaluate differences between groups with respect to plasma levels, and the relationship between concentration levels and supplementation time.

RESULTS
Reliable quantitative determination of CoQ 10 in biological samples presented in Tables 1 and 2 was enabled with HPLC-MS/MS analytical method based on improved selective ionization of reduced and oxidized form of CoQ 10 with added LiCl, and scanning in MRM scan mode.A quasi-molecular ion was formed with the added lithium ion in positive ESI-MS ionization mode.The parent ion for CoQ 10 was 869.7 m/z (M + Li) + and selected fragment ion was 241.1 m/z.The linearity rang, was from 0.02 to 5.0 mg/L (ppm), LOD was lower than 0.02 mg/kg and LOQ was 0.04 mg/kg.Obtained sensitivity is nearly 50 times higher than the sensitivity of our previously used analytical methods, mostly single step HPLC-MS.
Nevertheless the new analytical method enables simultaneous determination of reduced and oxidized form, we selected sample preparation with an oxidation step and measured the total CoQ 10 .In this way effects of uncontrolled oxidation were eliminated [13].
The plasma levels of total CoQ 10 in chicken's and hen's plasma are shown in Table 1 and Figure 2. Some results are taken from one of our previous study with chickens and demonstrate a constant increase of CoQ 10 concentration in chicken plasma [9] during the first weeks of chicken's live.In the control group the starting concentration in day 16 was 0.46 mg/L and the final concentration in day 40 was 0.92 mg/L.At the same time concentrations increased from 0.47 mg/L to 1.78 mg/L in the group which administered CoQ 10 , and in the group fed with ALA supplement, from 0.46 mg/L to 1.35 mg/L.A similar trend was observed in the experiment with hens.
In the control group the level of CoQ 10 was practically constant, and the average plasma concentration was around 2.0 mg/L.In the test group administering CoQ 10 the level slightly increased, and the average concentration was about 2.32 mg/L, at the same time in the ALA administering group the average concentration was even higher, 2.75 mg/L.The increased plasma level in animals after administering of CoQ 10 was seen in many experiments and was expected [14].Meanwhile the high increase of CoQ 10 concentration in plasma after ALA supplementation was something new that we did not expect, because so far in the literature was not possible to find such information.
After many repeated experiments we have come to believe that the results obtained are credible and logical effect of ALA antioxidant protection.The research work of Packer 1995, Han 1997 and Sen 1997 and some others [15][16][17] showed that Lipoic acid could serve as a proglutathione agent and could enhance the cellular level of glutathione (GSH).
Our experiments show that Lipoic acid increases concentrations of CoQ 10 .From obtained results it was not possible to conclude if the increased concentrations were the result of boosted production of endogen CoQ 10 or improved protection of exogenous CoQ 10 .New updated experiments will be needed if we want to clarify the obtained results.Now our opinion is that both options may be involved, increased production in liver tissue and reduction of oxidative stress which may additionally save the endogen CoQ 10 .Our experiments have also shown that the increase in CoQ 10 plasma concentrations in young chickens is greater than in adult hens during the supplementation with CoQ 10 and ALA.This result may be explained with stronger oxidative stress to which laying hens are exposed.
In Table 2 are presented concentrations of CoQ 10 in different tissues of laying hens.In our experiment two genotypes Ross and Lohmann were used.Hans were divided into three groups, control, CoQ 10 , and ALA group.In each group there were 12 animals of each genotype.
Concentrations of supplemented CoQ 10 and ALA were calculated and each hen received an average amount of approximately 5 mg of CoQ 10 or 50 mg of ALA per kg of animal weight, per day.In one group 7 animals of each genotype were selected and followed during the experiment.Plasma, meat and organ samples were taken from the same, at the start selected birds.
Measured values were evaluated in the two different ways.In the first step each genotype was processed separately.In the next step the average values taken from the both genotypes were prepared.These values are shown in Table 3.
We selected such solution, nevertheless some significant differences were observed between two genotypes, because we wanted to get enough reliable information related to the difference between supplementation with CoQ 10 and ALA, regardless of genotype.
Measured values were evaluated in the two different increase of nearly 10% was recorded.The highest increase was seen in plasma, nearly 15%.In the test group which administered ALA the increase of CoQ 10 was much higher than in coenzyme group.In heart tissue the final level of CoQ 10 was more than 5% and in liver more than 10% higher than in the control group.Concentrations in meat tissues were very high, more than 15% and in plasma nearly 40% higher than in control group.The same trend is seen in both genotypes groups.Lipoic acid produces much higher concentration of CoQ 10 then supplemented CoQ 10 alone.It is interesting that Lohmann hens have much higher response with both supplements.
It is also unexpected that concentrations in heart and liver are not increased, in reality in some cases they are even reduced.We explain these results with the influence of oxidative stress which is obviously higher in the Ross group then in the Lohmann group.Our results also show that higher concentration of CoQ 10 in plasma does not automatically mean high concentrations of CoQ 10 in tissues.ways.In the first step each genotype was processed separately.In the next step the average values taken from the both genotypes were prepared.These values are shown in Figure 3.We selected such solution, nevertheless some significant differences were observed between two genotypes, because we wanted to get enough reliable information related to the difference between supplementation with CoQ 10 and ALA, regardless of genotype.Calculated values represent the amount of CoQ 10 in control group and two experimental groups.They were obtained from measured concentrations (mg/kg) multiplied with estimated weights from instruction tables (kg) of processed organs and meat tissues.
We tried to clarify the link between distribution, accumulation, and elimination of exogenous and endogenous CoQ 10 in animal tissues with a help of a model.We wanted to determine if eaten lipoic acid busted a production of new CoQ 10 or only eliminate oxidation of it.Concentrations of processed tissues were taken from our experiments.The exogenous CoQ 10 was transported from column to liver with chylomicrons where it was prepacked to Apoproteins and redistributed through the body.In both transport paths, lipoic acid may prevented the decomposition of coenzyme.It also restored certain liver functions [18,19] and in this way boosted the synthesis, which increased the overall concentration of CoQ 10 .In In our experiments supplemented CoQ 10 was not accumulated in liver and heart, but in legs and breasts.An  Nevertheless the correlations between measured and calculated values were good, we were not able to conclude which previously described option was prevalent, and further experiments are necessary.

CONCLUSION
Lipoic acid is the most potent member of antioxidant protection in a body.With electric potential of (−320 mV) it may regenerate all other antioxidants.Results undoubtedly confirm the existence of an antioxidant network and synergistic effect of administered low weight substances.Our work demonstrates that ALA is able to influence not only on the regeneration of glutathione but according to our results also on regeneration of CoQ 10 .

Figure 1 .
Figure 1.The diagram of the antioxidant network built from reduced and oxidized forms of: lipoic acid, glutation, CoQ 10 , vitamin C and vitamin E is presented.The net is embedded between the endogenous cellular reduction system and exogenous antioxidants from a diet.acid at +0.590 V.Administered food may increase concentrations of vitamins and coenzymes in the network.The antioxidants from plants; carotenoids, flavonoids and polyphenols also protect antioxidant network but only in the redox range between +0.400 V and +0.700 V. From the Figure1, it is possible to conclude that the operation of antioxidant network is complex function[5][6][7], but also very logical.Regeneration of net strongly depends on high concentration of NADH and NADPH.Our previous research work connected with industrial poultry farming indicates the hypothesis that chickens and hens could be very suitable candidates for scientific estimation of the intensity of oxidative stress and protecttive effect of Low Molecular Weight Antioxidants[8,9].The aim of this study was to determine the effects of a scientifically selected diet on the content of several antioxidants in blood plasma and some animal tissues.Administered food provided necessary conditions for existence of adequate blood levels of enzymes, coenzymes, which together with the large number of administered small weight molecules were responsible for correct functioning of body antioxidant defense.To test this hy-

Figure 3 .
Figure 3. Average CoQ 10 levels in tissues and plasma of laying hens in control group and after oral administration of CoQ 10 and ALA are shown.Increased concentrations of CoQ 10 are expressed in percent.

Table 1 .
CoQ 10 content (mg/L) in the hens and chickens plasma samples.(a) Hens (genotype Ross) started to administer 5 mg CoQ 10 or 50 mg lipoic acid in 37 th week on day 266 and experiment was stopped after 50 weeks on day 350; (b) Laying hens (genotype Lohmann) started with fortified feed, (5 mg CoQ 10 or 50 mg lipoic acid) on 35 th week and experiment was stopped in 47 th week; (c) CoQ 10 content (mg/L) in the chicken plasma (genotype Ross) after daily intake of CoQ 10 (5 mg) and lipoic acid (50 mg) on kg of body weight.Chickens started with fortified feed on day 16 and experiment was stopped on day 41.

Table 2 .
Concentration of CoQ 10 in different hens tissues after 84 days of supplementation with CoQ 10 and ALA.

Table 3 .
Calculated values (mg/unit) of CoQ 10 in different organs and body parts of hens of Ross and Lohmann genotype are shown.Measured concentrations of CoQ 10 (mg/kg) were multiplied with estimated weight of selected body parts.cellsLipoic acid took care of antioxidant network and protected lipid membranes by elimination of uncontrolled oxidation which resulted in higher levels of CoQ 10 . *