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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: A new method to quantify the free and bound forms was developed, based on HPLC separation and RIA quantification in chromatography fractions. Reanalysis of specimens after addition of exogenous leptin allowed direct determination of leptin-binding capacity and the degree of saturation of leptin-binding capacity.
Results: HPLC chromatography fractionated serum leptin into both the free form and as a broad peak of 59–130 kDa. Several experiments were conducted to validate the new method. The concentrations of bound leptin in serum were 0.45–3.94 µg/L, and they increased as total leptin (reflecting adiposity) increased in 24 lean and obese volunteers. Leptin was readily dissociated from the bound fraction by competition from exogenous leptin. Rechromatography of the bound fraction led to dissociation of leptin, which was promoted by warming the sera before chromatography. Leptin-binding capacity was 1.8–5.3 µg/L; binding capacity was nearly constant over a range of total leptin concentrations of 2–10 µg/L, and slowly increased at higher total leptin concentrations. Saturation of binding capacity was low (15%) at very low total leptin concentrations (<5 µg/L), but rose quickly to a plateau near 80% at higher total leptin concentrations.
Conclusions: The new method facilitates measurement of free and bound fractions of serum leptin, and is the first method measuring leptin-binding capacity. These experiments demonstrate that the concentration of bound leptin and leptin-binding capacity vary physiologically, with binding/binding capacity increasing with adiposity. Except in very lean individuals, binding capacity is nearly completely saturated.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Müller-Newen et al. (14)]. Several
studies have demonstrated the presence of a bound fraction of leptin in
serum/plasma (15)(16)(17)(18)(19)(20)(21)(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 
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 125I-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)(16)(17)(19)(20)(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 NH2 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
KHPO4, 150 mmol/L NaCl, and 1 g/L
NaN3. The flow rate was 0.50–1.00 mL/min, and
all elutions were conducted at 4 °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 µg/L and a linear range up to 10 µg/L.
Day-to-day CVs were 13% at 0.32 µg/L and 5.8% at 2.14
µg/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 ± 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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)(16)(17)(18)(19)(20)(21)(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 nm (Fig. 1
).
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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 µg/L, and free leptin
from 2.05 to 38.95 µg/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 µg/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% ± 20%
(n = 23).
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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)(24)(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 µg/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.
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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
µg/L, and the serum was fractionated. As exogenous leptin
concentrations increased to 40 µg/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
µg/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 µg/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 µg/L,
and thereafter slowly increased as total leptin rose to near 60 µg/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 µg/L but rose abruptly to
a plateau near 80% at higher leptin concentrations (Fig. 5
).
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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% ± 2%; n = 3). When the reconstituted leptin pool was
warmed to 37 °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
).
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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 ± 0.15 µg/L
(CV = 13%), and the bound fraction averaged 0.31 ± 0.04
µg/L (CV = 13%; not corrected for dilution).
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 µg/L; r =
0.870; P = 0.002; n = 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)(21)(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 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.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin which are recognized by the 2-microglobulin receptor/low density lipoprotein receptor-related protein. Eur J Endocrinol 1998;139:224-230.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 20 µg/L
(
), or 40 µg/L (
) exogenous recombinant human leptin.
