Radioimmunoassay of bound and free melatonin in plasma

^a Author for correspondence. Fax 0171–377 7294; e-mail W.L.Di{at}mds.mw.ac.uk.

	Abstract

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We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

	Introduction

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Melatonin is secreted into the systemic circulation by the pineal gland, where it is rapidly distributed into tissue and biological fluid. In vitro experiments with [H]melatonin added to human (and rat) plasma suggested that 61–78% of melatonin was reversibly bound to albumin and that the binding capacity of albumin was greater in plasma than in an equivalent solution of 40 g/L human serum albumin (HSA)¹ (1). The authors speculated that plasma, in contrast to an HSA solution, contained low-M_r substances that enhanced the melatonin-binding sites of the albumin molecule. They concluded, however, that there was probably no biological difference in the activity of albumin-bound vs albumin-free melatonin because the addition of albumin did not affect the lightening effect of melatonin on melanophores in frog skin.

Reppert et al. (2) demonstrated that plasma melatonin did not equilibrate fully with cerebrospinal fluid (CSF) and speculated that this might be a consequence of the albumin-binding protein in plasma. Pardridge and Mietus (3) showed that albumin-bound melatonin was readily dissociable and therefore transportable through the blood–brain barrier. Hence other factors were necessary to explain the equilibration discrepancy between plasma and CSF. This was supported by Le Bars et al. (4), who demonstrated that an intravenous bolus of [C]melatonin was rapidly visualized in the brain.

There has been little follow-up to these studies, presumably because albumin binding is considered to be of such low affinity and poor specificity that it has no real influence on assay performance or on bioactivity. However, with the increasing interest in melatonin in human clinical research (5), we have undertaken a study to determine (a) whether plasma binding proteins (in particular albumin) influence the RIA of melatonin in human plasma and (b) whether specific RIA procedures distinguish between the bound and the free forms of melatonin.

	Materials and Methods

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chemicals and solvents
Melatonin and indole analogs, bovine serum albumin (BSA; A-8022), HSA (A-1653), human -globulin (G-4386), incomplete Freund's adjuvant, Iodogen (1,3,4,6-tetrachloro-3,6ß-diphenylglycouril; T-0656), Tricine (T-0377), and normal rabbit serum (R-9133) were purchased from Sigma Chemical Co. NaI (1MS 30) was obtained from Amersham. Thin-layer chromatograms (TLCs; 20 x 20 cm, DC-Alufolien, Kieselged 60 F₂₅₄) and activated charcoal (Norit PN5; formerly Norit OL) were obtained from BDH Chemicals Ltd. Precipitating antiserum (donkey anti-rabbit) was purchased from IDS (Boldon, UK).

6-Sulfatoxymelatonin was purified from human urine (6). 5-Sulfatoxy-N-acetylserotonin was synthesized as described by Leone et al. (7), and further purified with the method described by Street et al. (6). Methanol (AnalaR) and all other common reagents and solvents were of pure analytical grade and obtained from BDH.

Buffers.
Assay buffer: Tricine 0.1 mol/L, containing 9 g of NaCl and 1 g of gelatin per liter. Phosphate buffer (0.05 mol/L, pH 6.0): 44 mg of Na₂HPO₄ and 342 mg of NaH₂PO₄ · 2H₂O per 100 mL of H₂O. Saline: 8.5 g of NaCl per liter.

Antiserum.
Use of the Mannich reaction (8)(9) to conjugate 4 mg of melatonin (5-methoxy-N-acetyltryptamine) to 50 mg of BSA yielded a conjugate with a molar ratio of 12:1 for hapten:BSA, as determined by spectrophotometric analysis. Three rabbits were injected subcutaneously with 1 mg of immunogen in an equivolume solution of incomplete Freund's adjuvant/saline. At 4 weeks after the primary immunization, a booster intravenous injection of 1 mg of immunogen in 0.5 mL of saline was given and repeated every 2 weeks. The titers of antiserum were monitored after each booster injection. After the sixth booster injection, all rabbits were producing antisera. The rabbit with the highest titer was bled and the serum stored at -20 °C for assay.

The appropriate dilution of antiserum to use in the assay was determined by antibody dilution curves. The final dilutions were 1:416 000 for the extraction RIAs and 1:104 000 for the nonextraction RIA. The specificity of antiserum was assessed by comparing the potency (cross-reactivity) of 21 indoles with that of melatonin in displacing 50% of the tracer (Table 1 ). The principal cross-reacting compounds were 6-hydroxymelatonin (1.6%) and N-acetylserotonin (0.04%).

procedures
Iodination of melatonin.
Melatonin was iodinated with the method described by Vakkuri et al. (10). Briefly, 5 µg of melatonin in phosphate buffer (see above) and 200 µCi of I were combined in an Eppendorf tube containing 1 µg of dried Iodogen. After 5 min of reaction, the I-labeled melatonin was purified from the mixture by silica gel TLC with ethyl acetate as the solvent. The TLC fraction containing I-labeled melatonin was eluted with methanol and stored at 4 °C for assay.

Chloroform extraction.
The extraction procedure was similar to that described by Brown et al. (11), who used 1 mL of rat serum with 5 mL of dichloromethane. We mixed 0.5 mL of plasma with 2 mL of chloroform. The contents were mixed for 30 s and centrifuged at 1500g at 4 °C for 10 min. The aqueous phase was aspirated and the organic phase was placed at -20 °C for 30 min to freeze the remaining aqueous layer, fat, and lipids. After 30 min the chloroform phase was decanted into another tube and evaporated to dryness under nitrogen. The dried residue was reconstituted in 0.5 mL of assay buffer for assay.

Methanol extraction.
Plasma (0.5 mL) was mixed with 2 mL of methanol for 30 s and centrifuged at 1500g at 4 °C for 10 min. The supernatant was transferred to another tube and dried in a rotary-vacuum evaporator (Cyrovap, Howe) under reduced pressure. The dried residue was reconstituted in 0.5 mL of assay buffer for assay.

Parallelism of extractions.
To assess parallelism of chloroform and methanol extractions, we extracted into chloroform or methanol 2-mL aliquots of a plasma sample containing added melatonin. Dilutions of the extraction residues in assay buffer were assayed against calibrators of known concentrations in assay buffer and gave parallel results.

Further, two plasma pools containing high concentrations of endogenous melatonin were extracted and assayed. The results for the endogenous melatonin extracts were parallel with that for melatonin calibrator in assay buffer (12).

Dialysis.
Dialysis was performed with custom-made dialysis equipment. The dialysis membrane separating the two cells was an 11.5-µm (pore size) cuprophan membrane. The 1 mL of solution placed in one cell (F2) was dialyzed against 1 mL of saline in the adjacent cell (F1). The two cells were placed on a shaker to ensure constant mixing (13). With use of saline solution in both cells, melatonin equilibrated between the two cells within 3 h.

RIA procedure.
Dispensed into polystyrene tubes was 200 µL of 0.0–500 ng/L melatonin calibrators in assay buffer (or in melatonin-free plasma for nonextraction RIA), or 200 µL (from 500 µL) of extracted sample (or plasma for nonextraction RIA), or 200 µL (from 500 µL) of an extracted QC sample (or plasma for nonextraction RIA). To each sample were added 200 µL of diluted antiserum, 100 µL of I-labeled melatonin (~8000 counts/min), 100 µL of 1:24 diluted donkey anti-rabbit antibody, and 50 µL of 1:50 diluted normal rabbit serum, and the contents of each tube were mixed. After overnight incubation at 4 °C, each sample was centrifuged at 1500g at 4 °C for 30 min, the supernatant aspirated, and the radioactivity of the pellet counted for 60 s with a gamma counter.

assay evaluation
Sensitivity (detection limit).
Sensitivity was determined as the minimum concentration (2 SD) that did not overlap with zero concentration (-2 SD). This value (n = 16 for minimum concentration and n = 16 for zero concentration) was 4 ng/L for the extraction RIA and 5 ng/L for the nonextraction RIA.

Precision.
Assay precision was evaluated at three points of low (~25 ng/L), middle (~90 ng/L), and high (~320 ng/L) concentration (n = 20 at each concentration for intraassay CV and n = 11 for each concentration for interassay CV). In the extraction RIAs, the mean intraassay CV was 9.91% and interassay CV was 14%. In the nonextraction RIA, the mean CVs were 7.5% (n = 9) for intraassay and 11.5% (n = 6) for interassay.

statistical analysis
All data were presented as means, SDs, and CVs. The association and agreement between different methods were analyzed by using correlation coefficients and the limits of agreement (14), defined as the mean percentage difference between two methods ± 2 SD of this difference. The mean percentage difference between methods A and B (e.g.) was calculated from the individual percentage differences, i.e., [(x_A - x_B)/x_B] x100.

specimens
Melatonin-free plasma.
Date-expired plasma was obtained from the Royal London Hospital blood transfer department and pooled. Activated charcoal was added (100 g/L) and stirred at 4 °C for 40 h. The mixture was centrifuged at 31 180g at 4 °C for 2 h, after which the supernatant was decanted and recentrifuged at 1500g at 4 °C for 15 min. The charcoal-stripped supernatant was decanted and stored at -20 °C.

The concentration of melatonin in the plasma before and after stripping, assessed with a chloroform-extraction assay, was 40 and <5 ng/L, respectively.

24-h plasma samples.
Plasma samples were collected from five apparently healthy volunteers (four men and one woman, ages 20–28 years) at 2-h intervals for 24 h: samples XG-1, XH, XJ, XK-1, and XL. Two of the five volunteers had blood collected on a second occasion 1 month after the first collection: samples XG-2 and XK-2. All subjects were ambulatory during the daytime (natural daylight), and in bed (usually asleep) at night when blood was drawn. Illumination was from night lights filtering from adjacent wards. Venous blood samples (10 mL) were taken into EDTA-containing tubes, and saline solution was flushed every 30 or 60 min into the indwelling cannula throughout the 24-h study period. The plasma was separated and stored at -20 °C until assay.

These study protocols had previously been approved by the Local District Ethics Committee.

Plasma pools.
Human plasma samples were obtained from the blood bank. At least 10 samples were mixed to form a single pool. Four such pools were stored at -20 °C for assay.

	Results

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24-h plasma profile of melatonin by the different RIA procedures.
We assayed 24-h plasma samples from five volunteers (plus a repeat from two volunteers 1 month later), using all three procedures: nonextraction RIA, chloroform-extraction RIA, and methanol-extraction RIA. As Fig. 1 shows, all procedures demonstrated a diurnal rhythm of melatonin with peak values between 0100 and 0300. For most subjects, the values of melatonin by the nonextraction assay were higher than those by the methanol-extraction assay, which were higher than those by the chloroform-extraction assay. Otherwise, comparison of the three procedures showed good correlation (Table 2 ). The mean percentage difference between the procedures ranged from -1% to 39%, and the limits of agreement ranged from -111% to 109% (Table 2 ).

View larger version (25K): [in this window] [in a new window]   Figure 1. 24-h profile of endogenous melatonin in plasma after direct nonextraction assay () and after methanol () and chloroform () extraction assays in five volunteers (XG-1, XH, XJ, XK-1, and XL). Profiles for two volunteers were repeated 1 month later (XG-2 and XK-2).

Variable recovery of melatonin from plasma pools assayed by the nonextraction procedure.
Different concentrations of melatonin (0, 25, 100, and 400 ng/L) were added to four plasma pools. The concentrations were assayed with the nonextraction procedure (n = 9 for each concentration) and the values were calculated against a calibration curve prepared in melatonin-free plasma. The results were expressed as percentage recovery. As shown in Table 3 , the recovery within each pool was similar, but the recovery between pools varied greatly. For example, the samples from pool 2 gave ~80% of their expected values, whereas the samples from pool 1 gave <20% of their expected values.

Recovery of melatonin from HSA solution by the chloroform-extraction procedure.
Different amounts of melatonin were added to saline, phosphate buffer, 40 g/L HSA in saline (pH 6.0), and pooled plasma. Each solution was extracted with methanol or chloroform (n = 6 for each extraction at each concentration). The recovery of melatonin from saline, phosphate buffer, and the plasma pool lay within the ranges 83–115% (mean ± SE 99.3% ± 3.2%) for the methanol procedure, 65–110% (86.2% ± 5.8%) for the chloroform procedure (Table 4 ). The methanol procedure also provided good recovery (range 99–112%, mean ± SE 109% ± 5.1%) from the HSA solution. However, recovery from the HSA solution by the chloroform procedure was poor: 19–21% (20% ± 0.7%).

To study this difference further, we added melatonin to 40 g/L HSA solution or to a plasma pool and divided each into two aliquots. Each aliquot was further divided into two portions for either immediate extraction into either methanol (n = 2) or chloroform (n = 2) or incubation at 4 °C overnight before extraction. The results (Table 5 ) showed that the methanol extraction of plasma and HSA, and the chloroform extraction of plasma, gave similar melatonin recoveries (105–118%). However, chloroform extraction of melatonin in HSA gave low recovery (39%) when assayed after immediate extraction; this was further diminished by overnight incubation before extraction and assay (8%).

Postdialysis equilibration of melatonin from HSA solution.
To examine possible differences in postdialysis equilibration of melatonin from HSA solution that might be reflected in different recoveries after chloroform and methanol extraction, we added 0.200 pg/L melatonin to the 40 g/L HSA in saline solution, or to a plasma pool, or to a saline (control) solution. After the solutions were incubated (4 °C) overnight, 1-mL aliquots (F2) from the HSA solution or plasma or saline solutions (n = 4 for each solution) were dialyzed against 1 mL of saline (F1) for 3 h. F1 and F2 were then extracted with either methanol or chloroform and assayed for melatonin.

The results (Table 6 ) showed that the melatonin of the control solution equilibrated evenly between F1 and F2 (1:1.2 for the methanol procedure and 1:1.0 for the chloroform procedure). In the HSA solution extracted with chloroform, melatonin distributed itself evenly between F1 and F2 (1:1.0); in that extracted with methanol, however, the melatonin distribution between F1 and F2 was uneven (1:3.0). In plasma, neither chloroform nor methanol extraction showed an even equilibration of melatonin between F1 and F2 (1:5.6 and 1:5.0, respectively).

	Discussion

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The results of the 24-h plasma melatonin profiles (Fig. 1 ) seem to indicate that there is little to chose between the three RIA procedures. The nonextraction, the chloroform-extraction, and the methanol-extraction procedures all show a similar pattern of melatonin secretion. The between-individual variations are more marked than the between-procedure differences within the same individual (e.g., XG-1 and XK-1). Given these results and the close correlation between the three procedures, one could argue that all three were equally valid.

More extensive investigations, however, reveal that these results probably conceal some substantial problems associated with binding proteins in the nonextraction procedure and the chloroform procedure.

The nonextraction procedure demonstrated wide variability of recovery, depending on the plasma source (Table 3 ). The cause of this variability could be a consequence of the specific characteristics of the antiserum. However, the fact that recovery was closely similar within each plasma pool but widely different between pools suggests the existence of a nonspecific plasma effect that varies between pools and affects the assay. A possible explanation for this variable plasma effect could be different concentrations of nonspecific binding proteins in the different plasma pools. In our 24-h plasma profiles, it is probable that such nonspecific plasma binding proteins were more or less constant within each series of samples, and therefore did not affect the diurnal pattern. Given the demonstration of Poeggeler and Hardeland (15) that melatonin can be oxidized in samples containing transition metals, another possible explanation for this variable plasma effect could be a variation in the amounts of such transition metals, or in the content of fat and other biomolecules that bind or chelate melatonin. As yet, we have no data that can give the exact cause of this phenomenon.

The chloroform procedure demonstrated poor recovery of melatonin from HSA solutions. This was apparent in the results from Table 4 , which showed a mean recovery of 20%, and was even more apparent after overnight incubation, which had a mean recovery of 8% (Table 5 ). The reason for the poor recovery of melatonin from an HSA solution extracted with the chloroform procedure has to be the consequence of albumin binding. This is made clear by our dialysis experiments (Table 6 ). The methanol-extraction procedure demonstrated that ~67% of melatonin is bound to albumin in the F2 cell of the HSA solution, whereas the chloroform-extraction procedure suggested there is zero binding in that cell. This can be explained only if the chloroform procedure is unable to extract albumin-bound melatonin from an HSA solution.

Our dialysis experiments after methanol extraction confirm the findings of Cardinali et al. [1], that albumin binding in plasma (80%) was greater than albumin binding in an HSA solution (67%). The surprising result, however, is that the chloroform-extraction procedure is capable of extracting the enhanced albumin-bound melatonin in plasma but is incapable of extracting the albumin-bound melatonin in HSA. From this, albumin binding in HSA solution appears to have greater avidity, but lower affinity, than in plasma.

In conclusion, we have confirmed by dialysis experiments that binding of melatonin to plasma proteins, in particular albumin, is considerable, and that these binding proteins can influence the RIA of plasma melatonin. The nonextraction procedure gives variable and uncontrollable recovery because of nonspecific binding proteins, which vary between plasma sources; the chloroform-extraction procedure will not extract albumin-bound melatonin from an albumin solution but will extract it from a plasma sample; and the methanol-extraction procedure extracts free and bound melatonin from all sources. The significance of these findings is twofold: (a) Despite the good correlation of the three assay procedures seen in Fig. 1 , there are clearly problems with using the nonextraction RIA and the chloroform-extraction RIA. Given that such procedures are in common use (16)(17)(18)(19)(20), it seems prudent to introduce alternative methods to confirm clinical findings that are based on these procedures. (b) The biological significance of albumin binding has been discounted in the past because it does not affect melatonin-related skin lightening (1). However, the bioactivity of melatonin in mammalian physiology is essentially mediated by its effect on melatonin receptors in the central nervous system (21)(22)(23). Given our findings, the receptor binding of bound and free melatonin on central nervous system receptors should be further investigated before one excludes biological significance.

	Acknowledgments

 
We thank A. Johnston in the Department of Pharmacology and R. Rountree in Biological Service Department for their advice and help. We also thank the Henry Lester Trust and The Great Britain–China Educational Trust for their generous help in the personal support for W.-L.D.

	Footnotes

 
Academic Unit of Obstetrics, Gynaecology, and Reproductive Physiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London E1 1BB, UK.

¹ Nonstandard abbreviations: HSA, human serum albumin; BSA, bovine serum albumin; TLC, thin-layer chromatogram(-graphy).

	References

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