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Leptin binding and binding capacity in serum.

The recent description of a new hormone produced by adipose tissue, called leptin, has led to an outpouring of work concerning a potential role in regulation of body weight and the physiology of feeding/fasting [for a review, see Friedman and Halaas (1)]. Much of this effort has involved the measurement of circulating leptin, generally using a RIA available commercially (2). Among the important discoveries obtained with measurements of plasma/serum leptin have been that the obesity common in Western societies is not associated with deficiency of the hormone or with mutant forms of the hormone, and that circulating leptin concentrations generally parallel measures of adiposity such as the percentage of body fat (3-5). Glucocorticoids and insulin influence leptin metabolism (6-9), and prolonged fasting depresses plasma leptin concentrations (10, 11). An important role of leptin in normal physiology may be to regulate the response to starvation or stress, by modulating the neuroendocrine response by the hypothalamus (12, 13).

Many hormones are present in circulation both as free hormone and bound to plasma protein. Binding proteins are thought to have a role in modulating the availability of free hormone (the generally metabolically active form) for interaction with target tissues [for a review, see Muller-Newen et al. (14)]. Several studies have demonstrated the presence of a bound fraction of leptin in serum/plasma (15-22). Although a few studies have identified a splice variant of the leptin receptor as responsible for a portion of the binding (particularly in pregnant mice) (17,18) and another study has identified binding of leptin to [[alpha].sub.2]-macroglobulin (19), the variety and identities of the leptin-binding proteins of serum are poorly understood. In addition, there is little information concerning the relative changes in free and bound fractions during physiological perturbations of leptin concentrations.

Several methodologies have been used in the measurement of free and bound fractions of leptin. The most common method involves addition of [sup.125]I-labeled recombinant leptin to serum, an incubation period to allow equilibration of the labeled leptin with bound hormone, and separation of the labeled species on the basis of size, generally by gel permeation chromatography in several formats (15-17,19-21). Chromatography is conducted in the cold to prevent dissociation of bound leptin during separation. This indirect method offers the advantage of high sensitivity, but it depends on efficient equilibration of all binding species with the added tracer, and it could be affected by dissociation of bound species during the lengthy chromatography required to separate bound and free species. In addition, the purity of the recombinant leptin used for labeling could be a concern; some commercial preparations of recombinant leptin are impure, and use of these preparations could lead to labeling of impurities and measurement of binding of the impurities rather than leptin itself.

Another method used gel permeation chromatography to separate bound and free fractions, and quantified leptin in the fractions by RIA with two different antibodies; one antibody was derived by immunization with a sequence (residues 26-39) near the N[H.sub.2] terminus of leptin, and the other by immunization with a sequence near the COOH terminus (residues 126-140) (18, 22). The N-terminal-specific antibody appeared to react exclusively with high-molecular weight species present in early fractions of the chromatographic separation, and the C-terminal-specific antibody reacted with a low-molecular weight region consistent with the free leptin elution position. The basis for the specificity of these antibodies has not been explained, and the validity of the measurements has not been thoroughly established.

Direct measurement of immunoreactive leptin in gel permeation chromatographic fractions by RIA has also been used (15,17). The latter method has the potential advantage that all forms of leptin (regardless of binding species) are likely to be detected, and the lengthy incubation for equilibration with tracer leptin is avoided. However, standard chromatography is slow, and analysis of the results of each chromatography required the assaying of leptin concentrations in dozens of fractions.

Accurate and facile measurement of bound and free leptin concentrations could be an important tool in future studies of leptin physiology. I have sought to develop a rapid method to quantify the bound and free fractions, using HPLC technology to improve both speed of analysis and reproducibility. The HPLC method directly measures leptin after chromatography and has the advantage of speed over indirect methods. Use of a standard RIA for quantification offers the additional advantage of facilitating direct measurement of total leptin-binding capacity. This method also offers high precision, a separation time of <30 min, and requires quantification of just two fractions per chromatography to assess the bound and free fractions of serum.

Materials and Methods

SUBJECTS

Venous blood specimens were obtained from adult volunteers, all men between the ages of 24 and 57 years, who gave informed consent to participate. All volunteers were healthy without chronic disease, and none were receiving medications. Specimens were drawn during daylight hours, between typical meals. Serum was obtained by centrifugation, and the specimens were stored at -20[degrees]C until use. Body mass index (BMI; weight in kilograms divided by height in meters, squared) was calculated from standard weight and height measurements. This study was conducted in accordance with a protocol approved by the Human Studies Committee of Washington University.

HPLC ANALYSIS

The apparatus consisted of a Waters model 510 pump and a Waters model 481 LC Lambda-Max spectrophotometer, controlled by a Waters model 680 Automated Gradient Controller (Millipore). Separation of serum proteins, based on gel permeation chromatography, was conducted with a Amersham Pharmacia Biotech Superose 12 HR 10/30 column (internal volume, ~24 mL) equilibrated and eluted with 10 mmol/L KHP[O.sub.4], 150 mmol/L NaCl, and 1 g/L Na[N.sub.3]. The flow rate was 0.50-1.00 mL/min, and all elutions were conducted at 4[degrees]C. Fractions were collected with an LKB model 2211 Superrac fraction collector.

LEPTIN ANALYSIS

Serum leptin concentrations were determined with the Sensitive Human Leptin RIA manufactured by Linco Research. This assay has a limit of detection of 0.05 [micro]/L and a linear range up to 10 [micro]g/L. Day-to-day CVs were 13% at 0.32 [micro]g/L and 5.8% at 2.14 [micro]g/L.

WESTERN BLOTTING OF LEPTIN

As described previously (2), serum and chromatography fractions were diluted fourfold and denatured in the presence of sodium dodecyl sulfate and 2-mercaptoethanol by brief heating. Proteins were separated in 8-16% gradient polyacrylamide Bio-Rad Ready gels. Proteins were transferred to nitrocellulose and incubated with an antibody made to recombinant human leptin in rabbits (Linco Research), followed by incubation with peroxidase-conjugated second antibody (Sigma Diagnostics). Leptin was visualized by chemiluminescence (ECL kit; Amersham Pharmacia Biotech) to generate autoradiograms.

STATISTICAL ANALYSIS

All results are stated as the mean [+ or -] 1 SD unless otherwise noted. Graphic correlations were analyzed by least-squares linear regression, using the Pearson test for statistically significant correlation. P <0.05 was regarded as statistically significant.

Results

CHROMATOGRAPHY OF SERUM LEPTIN SPECIES

Fractionation of serum leptin species on the basis of size revealed two peaks of leptin immunoreactivity in the resulting fractions (Fig. 1). The first elated as a broad peak consistent with a range of molecular sizes of globular proteins between 59 and 130 kDa. This peak may represent leptin bound to higher molecular mass serum proteins, as described previously (15-22). The second peak was much sharper, and eluted at a molecular mass consistent with free, monomeric leptin (~15 kDa). Recombinant human leptin analyzed under the same conditions coeluted with the latter peak. There was no obvious association of leptin with protein peaks detected by monitoring absorption at 280 run (Fig. 1).

[FIGURE 1 OMITTED]

INTRAINDIVIDUAL VARIATION IN THE BOUND AND FREE FRACTIONS OF LEPTIN

Serum specimens from 24 healthy adult male volunteers were chromatographed, and their bound and free leptin concentrations were measured. For these experiments, the collection of fractions was condensed to four (corresponding to fractions 1-14, 15-28, 29-42, and 43-56 of Fig. 1), which produced a single fraction that received all of the bound leptin peak and a similar fraction for the free leptin peak. This scheme simplified subsequent RIA analysis. Leptin quantification was corrected for dilution during chromatography. Bound leptin concentrations ranged from 0.45 to 3.94 [micro]g/L, and free leptin from 2.05 to 38.95 [micro]g/L. The highest amounts of bound leptin were found in sera with high total leptin concentrations, which was reflected as a significant correlation (r = 0.717; P <0.001) that was improved by log-transforming the total leptin concentrations (r = 0.833; P <0.001; Fig. 2). Previous studies have reported improved correlations of various parameters with transformed total leptin concentrations (4,5). Concentrations of bound leptin also correlated significantly with BMI (r = 0.648; P <0.001). The percentage of total leptin eluting in the bound fraction ranged from a high of 57% in serum with low total leptin to 8% in serum with a total leptin concentration >40 [micro]g/L. There was a strong negative correlation of the percentage of bound leptin with total serum leptin concentration, and the correlation was improved by plotting the percentage of bound leptin vs log leptin (r = 0.867; P <0.001). Thus, a larger fraction was in the bound form when total leptin was lowest. The overall recovery of leptin after chromatography averaged 64% [+ or -] 20% (n = 23).

[FIGURE 2 OMITTED]

DISSOCIATION OF BOUND LEPTIN

To demonstrate that the larger molecular mass leptin immunoreactivity was reversibly bound, excess recombinant murine leptin was added to serum samples. Murine and human leptin appear to be nearly equally potent physiologically (23-25), and therefore murine leptin should be effective in replacing human leptin in bound form if the immunoreactivity truly represents reversible binding. The RIA used to analyze chromatographic fractions does not detect the murine form (<2% cross-reactivity), so that the addition of murine leptin to dissociate human leptin would produce a decrease or disappearance of the bound-leptin peak. The addition of 200 [micro]g/L murine leptin to four serum specimens produced consistent reduction in the bound-leptin peak, with the extent of reduction ranging from 52% to 100%; dissociation was greatest when the endogenous total leptin was lowest, and dissociation was reduced when the endogenous leptin was higher (Table 1), presumably because high endogenous leptin concentrations diluted the mass-action potential of the added murine leptin.

LEPTIN-BINDING CAPACITY OF SERA

Recombinant human leptin was added in increasing concentrations to serum from a lean individual with an initial total leptin of 6.1 [micro]g/L, and the serum was fractionated. As exogenous leptin concentrations increased to 40 [micro]g/L, the area under the bound-leptin peak increased to 326% of the area of the untreated serum (Fig. 3). In additional experiments, recombinant human leptin (40 [micro]g/L) was added to sera from 21 adult male healthy volunteers, and untreated and treated aliquots were submitted to chromatographic analysis of the bound-leptin concentration. The amount of bound leptin determined in the treated sera was regarded as the maximum binding capacity for each serum specimen. Leptin-binding capacity was 1.80-5.33 [micro]g/L, and a modest positive trend was observed between binding capacity and total leptin concentration; binding capacity was nearly constant at total leptin concentrations between 2 and 10 [micro]g/L, and thereafter slowly increased as total leptin rose to near 60 [micro]g/L (Fig. 4). From knowledge of the maximum binding capacity and the bound-leptin concentration in untreated sera, it was possible to determine the fraction of maximal binding capacity that was saturated with endogenous leptin in the untreated specimens. The saturation of binding capacity in untreated sera was 15-104%; saturation was low when the total leptin concentration was <5 [micro]g/L but rose abruptly to a plateau near 80% at higher leptin concentrations (Fig. 5).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

RECHROMATOGRAPHY OF THE BOUND FRACTION OF LEPTIN

Fractions (4.1 mL) containing the bound leptin from chromatography of six subjects were pooled and lyophilized or ultrafiltered to concentrate the pool for rechromatography. The pooled bound fraction was kept cold until chromatography. Leptin eluted in both the bound and free forms (Fig. 6), indicating some dissociation of bound leptin during sample preparation, but most of the leptin remained in the bound fraction (71% [+ or -] 2%; n = 3). When the reconstituted leptin pool was warmed to 37[degrees]C for 15 min to promote equilibration of bound and free leptin forms, bound leptin decreased dramatically, and the immunoreactivity lost from the bound fraction appeared in the free fraction (Fig. 6).

PRECISION OF ANALYSIS

Serum from a single volunteer, split into small aliquots and frozen until use, was analyzed repeatedly (n = 20) on 19 different days. The free leptin fraction averaged 1.15 [+ or -] 0.15 [micro]g/L (CV = 13%), and the bound fraction averaged 0.31 [+ or -] 0.04 [micro]g/L (CV = 13%; not corrected for dilution).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

COMPARISON WITH WESTERN BLOT ANALYSIS

Chromatographic fractions containing bound and free leptin from several sera were denatured with sodium dodecyl sulfate, separated by polyacrylamide electrophoresis, and transferred to nitrocellulose; the leptin content of the fractions was visualized by staining with antibody to leptin. Leptin immunoreactivity migrated with a velocity similar to recombinant leptin in all fractions under these denaturing conditions. Densitometric scanning of the staining showed rough correlation of the leptin content in both bound and free fractions as well as in serum measured by RIA with that detected by Western blot (blot leptin = 1.13 RIA leptin + 5.2 [micro]g/L; r = 0.870; P = 0.002; n = 9).

Discussion

Analysis of free and bound leptin concentrations is significantly improved by use of HPLC and direct analysis with a sensitive RIA for leptin. Most earlier studies relied on the addition of radioactively labeled leptin, equilibration of the labeled leptin with endogenous bound leptin, and detection of radioactivity in the bound fraction after standard gel permeation chromatography to demonstrate binding (equilibration method) (15-17,19-21). The method advanced here avoids the time-consuming equilibration step and also increases both resolution and speed of analysis by using HPLC separation of bound and free fractions. The new method does require RIA of chromatography fractions, but because single fractions are collected for the bound and free measurements, many chromatographic runs can be conveniently analyzed in one RIA. The equilibration method also requires RIA measurement of total leptin. The new method avoids the potential pitfall of the earlier equilibration method of underestimating the bound fraction because of incomplete equilibration of tracer leptin to high-affinity binding species. A further advantage of the new method is that measurement of leptin-binding capacity is facilitated; sera are chromatographed with and without addition of a saturating amount of exogenous recombinant leptin, and the bound fraction of leptin is determined in both aliquots, similar to the measurement of iron-binding capacity.

The physiological basis of leptin binding in serum is unknown, but clearly humans possess binding capacity, this capacity is not always saturated, and both the concentration of bound leptin and the binding capacity vary physiologically. Both the concentration of bound leptin and binding capacity rose as the total leptin concentration (and BMI) increased in a cross-section of lean and obese men, reflecting the comparatively constant percentage of saturation of binding capacity: only very lean men had decreased saturation compared with men of normal and obese body composition. Both the bound leptin concentration and binding capacity rose rather slowly with increasing total leptin compared with the rise in total leptin, so that most of the change in total leptin was reflected in the free leptin concentrations. Previous studies have also observed correlations of leptin binding with total leptin/BMI and noted the disproportionate increase in free leptin concentrations as total leptin rose (15, 20-22). The protein species responsible for binding leptin have not been identified, although studies in both humans and mice have suggested that at least part of the binding capacity could be attributable to the presence in serum of a truncated form of the leptin receptor (15, 17, 18). Binding to the proteinase inhibitor [[alpha].sub.2]-macroglobulin has also been reported (19). A range of molecular sizes have been reported for the binding proteins and leptin-binding protein complexes, and there is evidence of multiple binding species. Sinha et al. (15) reported binding proteins of 80-280 kDa, Diamond et al. (20) reported a single species of 450 kDa, and Lewandowski et al. (18) reported two species of 100 and 200 kDa. The results presented here are consistent with multiple forms of binding species in serum, but the disparity in the range of reported values likely reflects the effects of methodological differences, with ultimate resolution resting on a detailed characterization of the binding species in future experiments.

The results of experiments where exogenous murine or human leptin was added to serum clearly demonstrate that the larger molecular forms of leptin detected in gel permeation chromatography are not the result of aggregation of leptin, but represent heterologous binding to serum protein components, and that these components are present in serum in excess of the amount of leptin binding. The addition of recombinant human leptin should have proportionately increased the high-molecular mass forms of leptin (no saturation of binding) if these were the result of aggregation. Instead, the amount of high-molecular mass leptin reached a plateau with the addition of increasing amounts of exogenous leptin. The addition of murine leptin, which is biologically active but undetected in the human leptin RIA, reduced or completely eliminated human leptin from the bound fraction. Elimination occurred as the result of exchange with bound human leptin and displacement from the bound fraction by mass action of the much higher murine leptin concentrations. If aggregation accounted for the observed high-molecular mass forms of leptin, addition of murine leptin should have promoted parallel increases in aggregated forms of mixed human and murine leptin, which would have led to the preservation of the high-molecular mass forms of human leptin.

For most hormones and cytokines that circulate in bound and free forms, the free form is thought to be the biologically active form of the hormone, although exceptions exist (14). It is commonly assumed that soluble binding proteins act as antagonists by binding the hormone and preventing interaction with physiological receptors. Binding proteins likely also serve as a depot for hormones, allowing for longer half-life of the total hormone in circulation and continuous equilibration of the free concentration by dissociation from the bound depot. There is little evidence to suggest whether it is the free or bound forms of leptin that are biologically active. However, when free and bound leptin concentrations were measured before and after a 24-h fast (which dramatically reduces total leptin concentrations), the free but not the bound fraction was significantly decreased (15). Current theory calls for transport of leptin from the blood to the cerebrospinal fluid as the means to gain access to the hypothalamus, where leptin appears to exert effects on food intake and neuroendocrine function (1). It will be informative to examine the free/bound forms of leptin in cerebrospinal fluid because a finding of exclusively free leptin in that fluid would offer a further indication that the free form of leptin is biologically active.

This work was supported by NIH Grants DK 20579 and RR-00036. Expert technical assistance was provided by Michael Morris. Invaluable assistance with manuscript preparation was provided by Barbara J. Hartman.

Received November 2, 1999; accepted December 29, 1999.

References

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(9.) Utriainen T, Malmstr6m R, Makimattila S, Yki-Jarvinen H. Supraphysiological hyperinsulinemia increases plasma leptin concentrations after 4 h in normal subjects. Diabetes 1996;45:1364-6.

(10.) Boden G, Chen X, Mozzoli M, Ryan I. Effect of fasting on serum leptin in normal human subjects. J Clin Endocrinol Metab 1996;81:3419-23.

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(12.) Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250-2.

(13.) Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS. Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology 1997;138:3859-63.

(14.) Muller-Newen G, Kbhne C, Heinrich PC. Soluble receptors for cytokines and growth factors. Int Arch Allergy Immunol 1996;111:99-106.

(15.) Sinha MK, Opentanova I, Ohannesian JP, Kolaczynski JW, Heiman ML, Hale J, et al. Evidence of free and bound leptin in human circulation. J Clin Investig 1996;98:1277-82.

(16.) Houseknecht KL, Mantzoros CS, Kuliawat R, Hadro E, Flier JS, Kahn BB. Evidence for leptin binding to proteins in serum of rodents and humans: modulation with obesity. Diabetes 1996;45:1638-43.

(17.) Gavrilova O, BarrV, Marcus-Samuels B, Reitman M. Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor. J Biol Chem 1997;272:30546-51.

(18.) Lewandowski K, Horn R, O'Callaghan CJ, Dunlop D, Medley GF, O'Hare P, Brabant G. Free leptin, bound leptin, and soluble leptin receptor in normal and diabetic pregnancies. J Clin Endocrinol Metab 1999;84: 300-6.

(19.) Birkenmeier G, Kampfer I, Kratzsch J, Schellenberger W. Human leptin forms complexes with %macroglobulin which are recognized by the a2 microglobulin receptor/low density lipoprotein receptor-related protein. Eur J Endocrinol 1998;139:224-30.

(20.) Diamond FB Jr, Eichler DC, Duckett G, Jorgensen EV, Schulman D, Root AW. Demonstration of a leptin binding factor in human serum. Biochem Biophys Res Commun 1997;233:818-22.

(21.) Eichler DC, Root AW, Duckett G, Moore KL, Diamond FB. A spun-column assay for determination of leptin binding in serum. Anal Biochem 1999;267:100-3.

(22.) Leonhardt W, Horn R, Brabant G, Breidert M, Temelkova-Kurktschiev T, Flicker K, Hanefeld M. Relation of free and specifically bound leptin to insulin secretion in patients with impaired glucose tolerance (IGT). Exp Clin Endocrinol Diabetes 1999;107:46-52.

(23.) Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269:543-6.

(24.) Van Dijk G, Donahey JCK, Thiele TE, Scheurink AJW, Steffens AB, Wilkinson CW, et al. Central leptin stimulates corticosterone secretion at the onset of the dark phase. Diabetes 1997;46:1911-4.

(25.) Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Investig 1996;98:1101-6.

The Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, One Children's Place, St. Louis, MO 63110. Fax 314-454-2274; e-mail landt@kids.wustl.edu.
Table 1. Dissociation of bound leptin.

 Total serum Bound
 leptin, BMI, leptin, Dissociation,
Subject [micro]g/L kg/[m.sup.2] % of total %

 1 14.7 29.4 21 100
 2 19.1 34.4 21 91
 3 39.6 39.6 8 83
 4 58.1 47.6 9 52
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Title Annotation:Endocrinology and Metabolism
Author:Landt, Michael
Publication:Clinical Chemistry
Date:Mar 1, 2000
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