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Keywords:

  • melatonin;
  • microdialysis;
  • N1-acetyl-N2-formyl-5-methoxykynuramine;
  • oxidation;
  • retina;
  • serum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

Abstract: The product of melatonin oxidation, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), was synthesized and a method for its determination in biological samples was developed. High performance liquid chromatography (HPLC) with fluorescence detection provided good sensitivity and selectivity. Wavelengths of 350 and 480 nm were used for excitation and emission, respectively. Serum and retinal homogenates were extracted with chloroform prior to analysis by HPLC. Endogenous AFMK was detected in the retina of rats but the serum concentration of this melatonin metabolite was below the detection limit of the method for measurement. Retinal AFMK concentration was higher during the dark phase of the light/dark cycle, when the retinal melatonin content is maximal. Intraperitoneal administration of melatonin significantly increased serum and retinal AFMK levels. Formation of AFMK from melatonin was also confirmed by in vivo microdialysis with the probe implanted into the brain lateral ventricle. The study shows that AFMK is indeed a product of melatonin oxidation in vivo. The possible physiological significance of melatonin oxidation metabolic pathway is discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine synthesized in pineal gland and retina. One of the main roles of melatonin is its function in circadian rhythm regulation. Melatonin was also recently found to be a free radical scavenger and antioxidant [1, 2]. Melatonin has been proposed as an endogenous antioxidant, but rather little is known concerning its concentrations within cells, where free radicals are generated [3]. Much higher levels of melatonin have been found in cerebrospinal fluid [4] and bile [5] relative to those in blood. Regardless of the role of melatonin as an endogenous antioxidant, it is clear that this indolamine can be oxidized and administration of exogenous melatonin is useful for treatment of conditions associated with overproduction of free radicals. Melatonin used at pharmacological doses has been shown to be effective against free radical damage in models of Alzheimer disease [6, 7], Parkinson's disease [8–10], excitotoxicity [11, 12], porphyric neuropathy [13, 14], hyperoxia [15] as well as in newborn infants with sepsis [16] or suffering from transient asphyxia [17].

The oxidative cleavage of the pyrrole ring, resulting in kynuric type products, is well established for tryptophan [18] and melatonin [19–21]; therefore N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) is a probable endogenous melatonin oxidation product. This suggestion is supported also by previous findings indicating that AFMK can be synthesized from melatonin enzymatically [22, 23].

In this study we developed the sensitive method for determination of AFMK, suitable for work with biological samples. The main goal was to show the existence of endogenous AFMK and the formation of AFMK from melatonin in rats using an in vivo microdialysis method for investigation of melatonin oxidation in free moving animal.

Animals, chemicals and equipment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

Animal experiments were performed on male 3–4 months old Wistar rats bred in Animal Department of Sechenov Institute of Evolutionary Physiology and Biochemistry. Animals were maintained at standard light/dark schedule (12 h light/12 h darkness, light on at 23:00 hr). Melatonin was injected to rats intraperitoneally at a dose of 40 mg/kg of body weight.

Melatonin and 30% hydrogen peroxide were purchased from Sigma Chemical Co (St Louis, MO, USA). Acetonitrile, methanol and KH2PO4 of highest purity were obtained from local sources.

Two high performance liquid chromatography (HPLC) systems were used in this study. AFMK was purified from the reaction mixture using a solvent delivery pump model 303, a manometric module model 802C (Gilson, Middleton, WI, USA), a Rheodyne 7125 injection valve (Rohnert Park, CA, USA) and a UV-detector UV-308 (Budapest, Hungary). Analytical measurements were performed on Agilent Technologies 1100 series HPLC system (Foster City, CA, USA) equipped with fluorescence detector.

AFMK synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

The AFMK was synthesized by procedure described before [20] with minor modifications. Briefly, 5 mg of melatonin were dissolved in 100 μL of methanol and mixed with 500 μL of 30% H2O2. The absorbance of reaction mixture at 340 nm was increased as a function of incubation time that indicated the conversion of melatonin to AFMK (data not shown).

After incubation for 24 hr the reaction mixture was separated by HPLC with UV detection at 340 nm. Separation was performed on Separon SGX C18 (Elisico, Moscow, Russia), 5 μm, 3 × 150 mm HPLC column with the mobile phase, containing 0.1 m KH2PO4 and 20% of acetonitrile (vol/vol), pH 5.1. The mobile phase containing the main reaction product was collected, extracted with methylene chloride and dried under a nitrogen stream. The identity of synthesized substance was confirmed by mass spectrometry. Molecular ion at m/z 264 and major fragments at m/z 192, m/z 176 and m/z 150 were detected that corresponds to electron impact ionization mass spectrum for AFMK [20].

Method of AFMK determination by reversed phase HPLC with fluorescence detector

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

Fluorescence detection of AFMK provides much better sensitivity that UV-detection. Detection conditions were selected on the basis of excitation and emission spectra of AFMK. Wavelengths of 350 and 480 nm were used for excitation and emission, respectively.

Before analysis by HPLC, AFMK from biological samples was extracted into chloroform. Both rat retinas were homogenized in 200 μL of 0.066 m sodium phosphate buffer, pH 7.4. Then 0.4 mL of chloroform was added to this tissue homogenate or 200 μL of serum and vortexed for 30 s. Organic phase was dried under nitrogen stream and redissolved in 100 μL of mobile phase. The recovery of AFMK added to daytime rat serum was 83–85% at various concentrations tested. Thus, AFMK does not coelute with quenching substances that may significantly impair its fluorescence.

Separation was performed on HPLC (Agilent Technologies, Foster City, CA, USA) column Zorbax Eclipse XDB C8, 5 μm, 4,6 × 150 mm with the mobile phase, containing 0.1 m KH2PO4 and 20% of acetonitrile (vol/vol), pH 5.1. AFMK retention time differs from the retention time of other melatonin derivatives: N-acetyl-5-methoxykynuramine, 5-methoxytriptamine, 5-methoxyindolacetic acid. The activity of melatonin deacetylase is low or absent in retinas of non-amphibian vertebrates [24]. Hence, the products of oxidation of melatonin deacetylation metabolites are unlikely to exist in the retina of rats.

The detection limit of the method (baseline noise*2) is 15 pg of AFMK.

In vivo microdialysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

Microdialysis probe was constructed in our laboratory. It was made of a dialysis hollow fiber; molecular weight cutoff 6000 Da, 0.2 mm O.D., 8 mm active dialysis length (GAMBRO, Stockholm, Sweden). The dialysis fiber was twisted as shown in Fig. 1. This construction provides a large area of dialysis surface and, hence, better recovery for the measured substance.

image

Figure 1. The microdialysis probe.

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The operation was performed during the end of the light phase of the light/dark cycle (ZT10-ZT12) [zeitgeber time (ZT) 0 corresponds to the onset of light phase]. A rat was anesthetized with Nembutal (50 mg/kg body weight, i.p.) and mounted in a stereotaxic frame. Base on a brain atlas [25], the dialysis probe was implanted into the brain lateral ventricle (A, 5.6 mm from bregma; L, 1.76 mm from mid-saggital plane; V, 3.5 mm from brain surface). Two titanium hooks were placed nearby on the skull to serve as anchors. The area was cleaned and dried, and the probe was mounted on the skull with dental cement. After surgery, the rat was housed in an individual cage. The localization of microdialysis probe working surface was verified by histological examination.

After 1 day of recovery from the operation, animals were used in an experiment. The dialysis probe was perfused at a rate of 2 μL/min with Ringer's solution via PTFE tube (Gilson, Middleton, WI, USA) (0.4 O.D., 0.1 I.D.). Samples were collected every 15 or 30 min, stored in the liquid nitrogen and the dialysate was analyzed by HPLC without prior extraction.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

To confirm that melatonin is metabolized to AFMK in vivo, we measured AFMK concentrations in intact rats and after intraperitoneal injection of melatonin at a dose of 40 mg/kg of body weight. The first series of measurements was performed on serum and retinas obtained after animals were killed. The AFMK assay was not sensitive enough to measure endogenous AFMK in serum of intact animals (Fig. 2C), but 30 min after melatonin administration a peak corresponding to AFMK was easily detectable in dialysate (Fig. 2B) and serum (Fig. 2D). AFMK concentrations varied from 1 to 3.7 ng/mL of serum (Fig. 3A). In the retina both endogenous and melatonin-induced AFMK concentrations were estimated (Figs 2E, F and 3B). In the middle of the dark phase of the light/dark cycle, at ZT 18, retinal AFMK content was 1.6 times higher (P < 0.01, t-test) then during the day (ZT 6). Daytime melatonin administration resulted in a 1.7-fold increase in retinal AFMK content (P < 0.01, t-test).

image

Figure 2. Sample chromatograms of N1-acetyl-N2-formyl-5-methoxykynuramine(AFMK). (A) AFMK standard solution; (B) dialysate obtained from microdialysis probe implanted into brain lateral ventricle; the rat was injected intraperitoneally with melatonin at a dose of 40 mg/kg of body weight; (C) extract of serum from intact rats (AFMK was not detected); (D) extract of serum from rat injected intraperitoneally with melatonin at a dose of 40 mg/kg of body weight; (E) extract from retinas of intact rat dissected at ZT 18; (F) extract from retinas of intact rat dissected at ZT 6.

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image

Figure 3. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) concentrations in serum (A) and retinas (B) of rats. Samples were taken at ZT 6 (day), ZT 18 (night) and during the day 30 min after intraperitoneal melatonin administration at a dose of 40 mg/kg of body weight (day + melatonin). n.d., not detected; *, AFMK concentration is significantly higher when compared with the day group (P < 0.01, t-test). Results are the mean values S.E.M. for four determinations. Samples were obtained from four animals.

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The time course of melatonin conversion to AFMK in the cerebrospinal fluid was tested by in vivo microdialysis. Melatonin or AFMK were determined in the dialysis solution, obtained from microdialysis probe implanted into the lateral ventricle of rats injected intraperitoneally with 40 mg/kg of melatonin. Melatonin and AFMK profiles are depicted in the Fig. 4A and B, respectively. Melatonin concentrations reached their maximum during first 15 min after injection and then gradually decreased to undetectable levels. AFMK concentration in dialysis solution was maximal at 30–90 min after melatonin administration. Both melatonin and AFMK were undetectable in the dialysis solution obtained before melatonin injection even during the scotophase when endogenous melatonin concentrations are maximal.

image

Figure 4. Microdialysis of melatonin (A) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) (B) after intraperitoneal administration of melatonin at a dose of 40 mg/kg of body weight. Melatonin and its metabolite were measured in dialysate obtained from microdialysis probe implanted into brain lateral ventricle. Zero value means that melatonin or AFMK were undetectable. Hundred percent value corresponds to highest concentration detected for each profile. The absolute levels of AFMK varied from 21 to 359 pg per 50 μL of dialysate. Each curve (melatonin or AFMK) was obtained from separate animal (five animals were dialyzed).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References

We developed the method for determination of AFMK by reversed-phase HPLC. Fluorescence detection provides high sensitivity suitable for work with biological samples and good selectivity. AFMK was easily detectable in serum and retinas obtained from animals injected with melatonin but not in serum from untreated animals. A more sensitive detection method is required for serum measurements. However, we succeeded with measurements of endogenous AFMK in retinas. To our knowledge this is the first observation of endogenous AFMK in tissue.

The amount of endogenous AFMK per retina varied from 30 to 60 pg depending on the time of day. This is similar to concentration of melatonin measured in rat retina before [26, 27]. Moreover, diurnal changes in AFMK concentrations in the retina correspond to the diurnal rhythm of melatonin with the higher levels during the dark period of the light/dark cycle. The mammalian retina is able to synthesize melatonin [28]. We suggest that melatonin oxidation is one of the mechanisms regulating the concentration of this indoleamine. Light exposure during scotophase decreases retinal melatonin content suppressing the activity of arylalkylamine N-acetyltransferase (AA-NAT) [29, 30]. We hypothesize that light rapidly reduces retinal melatonin concentration by a mechanism that involves chemical reactions with free radicals formed during illumination. This assumption is supported by preliminary data that water-soluble antioxidant dimethylthiourea and a lipid-soluble free radical scavenger tocopherol prevent light-induced degradation of melatonin in the retina of chickens [31].

Intraperitoneal administration of melatonin significantly increased serum and retinal concentrations of AFMK. This fact confirms oxidation of melatonin to AFMK in vivo. Several possible mechanisms of AFMK formation have been suggested. AFMK can be synthesized from melatonin enzymatically by non-specific indoleamine 2,3-dioxygenase [22]. Activated neutrophils convert melatonin to AFMK by myeloperoxidase-catalyzed oxidation [23]. It was speculated that in the presence of the hydroxyl radical, melatonin donates an electron to neutralize the reactant and forms melatoninyl radical that may interact with superoxide anion to form AFMK [32]. The existence of a mealtoninyl radical was also supported by the findings of other research groups [33–35]. Photodegradation of melatonin from commercial formulations with the formation of a metabolite that has a structure identical to AFMK was reported [36]. Photooxidation of melatonin has been found in unicell Gonyaulax polyedra [19]. Photooxidative reactions of melatonin, with protoporphyrin IX as a catalyst, lead to the formation of AFMK as one of the main products. These data are of special interest in view of our hypothesis concerning physiological significance of melatonin oxidation in regulation of retinal melatonin by light. It was reported that melatonin can interact directly with hydrogen peroxide to form AFMK [20], however this was not confirmed in another study [37]. Which of these processes were operative in the current study remain unknown.

The time course of melatonin and AFMK was measured in cerebrospinal fluid using in vivo microdialysis. Melatonin concentration reaches its maximum very quickly after intraperitoneal injection. The rapid accumulation and clearance of melatonin is consistent with published data [38–40] and confirms that this indoleamine easily penetrates the blood–brain barrier. Melatonin is oxidized that results in an AFMK peak at 30–90 min after injection. This microdialysis evidence rule out the possibility that AFMK formation is an artifact caused by quick oxidation of melatonin by strong oxidizing agents formed in postmortem tissues during sample preparation but not in alive animals.

The current data indicate that melatonin, administered at pharmacological doses, can be used as an antioxidant. Melatonin has proven effective in a large number of experimental situations in which increase of free radical production occurs [41]. Melatonin treatment is effective in different in vitro and in vivo models of excitotoxicity or ischemia/reperfusion in multiple animal species [42], that is consistent with its antioxidative potential and its ability to pass through the blood–brain barrier. Considering melatonin as an antioxidant for treatment of human diseases, its fast removal from organism should be taking into account. The application procedure with continuous administration of melatonin might be useful. It is possible that melatonin can transiently accumulate in tissues or it can realize its effect indirectly through stimulation of antioxidative enzymes [43]. Moreover, the melatonin oxidation products, AFMK and cyclic 3-hydroxymelatonin, are also known to have antioxidant properties [44–46].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animals, chemicals and equipment
  6. AFMK synthesis
  7. Method of AFMK determination by reversed phase HPLC with fluorescence detector
  8. In vivo microdialysis
  9. Results
  10. Discussion
  11. References
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