Cerebrospinal Fluid Leptin in Anorexia Nervosa:Correlation with Nutritional Status and Potential Rolein Resistance to Weight Gain*

CHRISTOS MANTZOROS†, JEFFREY S. FLIER, MICHAEL D. LESEM,TIMOTHY D. BREWERTON, AND DAVID C. JIMERSON

Division of Endocrinology, Department of Medicine (C.M., J.S.F.), and the Department of Psychiatry(D.C.J.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts02215; the Department of Psychiatry, University of Texas Medical School (M.D.L.), Houston, Texas77025; and the Department of Psychiatry and Behavioral Sciences, Medical University of SouthCarolina (T.D.B.), Charleston, South Carolina 29425

ABSTRACTStudies in rodents have shown that leptin acts in the central ner-

vous system to modulate food intake and energy metabolism. Toevaluate the possible role of leptin in the weight loss of anorexianervosa, this study compared cerebrospinal fluid (CSF) and plasmaleptin concentrations in anorexic patients and controls. Subjects in-cluded 11 female patients with anorexia nervosa studied at low weightand after treatment, and 15 healthy female controls. Concentrationsof leptin in blood and CSF were measured by RIA. Patients withanorexia nervosa, compared to controls, had decreased concentrationsof leptin in CSF (98 6 26 vs. 160 6 58 pg/mL; P , 0.0005) and plasma

(1.75 6 0.46 vs. 7.01 6 3.92 ng/mL; P , 0.005). The CSF to plasmaleptin ratio, however, was higher for patients (0.060 6 0.023) than forcontrols (0.025 6 0.007; P , 0.0001). At posttreatment testing, al-though patients had not yet reached normal body weight, CSF andplasma leptin concentrations had increased to normal levels. Theseresults demonstrate the dynamic changes in plasma and CSF leptinduring positive energy balance in anorexia nervosa. The results fur-ther suggest that normalization of CSF leptin levels before full weightrestoration during treatment of anorexic patients could contribute toresistance to weight gain and/or incomplete weight recovery. (J ClinEndocrinol Metab 82: 1845–1851, 1997)

THE RECENTLY discovered adipocyte hormone leptin,the obese (ob) gene product, is an important component

of the physiological system for regulation of energy balanceand body weight (1–5). In the mouse, defects in the leptincoding sequence (ob/ob) (1) or the leptin receptor/signalingsystem (db/db) (6) cause severe obesity. Leptin-deficient ob/obmice respond dramatically to treatment with recombinantleptin by decreasing food intake, increasing motor activity,and increasing energy metabolism/body temperature (2–4,7). Previous studies of human obesity have shown elevatedleptin messenger ribonucleic acid levels in adipose tissue andelevated leptin levels in plasma, suggesting that human obe-sity in most cases is not a result of defective leptin production(8–13).

Administration of small amounts of leptin into the cerebralventricles decreases food intake in rodents, indicating thatleptin acts directly in the central nervous system (CNS) (4,

14). As the long form of the leptin receptor messenger ribo-nucleic acid is expressed in the hypothalamus (15), and leptinhas direct effects on isolated hypothalamic tissue (16), it islikely that the initial step in leptin action occurs at hypotha-lamic leptin receptors.

It is not yet known whether leptin reaches its sites of actionin the CNS by transport across the blood-brain barrier, bydirect transport from blood to cerebrospinal fluid (CSF), orby diffusion to sites (e.g. in the hypothalamus) functionallyoutside the blood-brain barrier. Binding sites have been dem-onstrated for leptin in the choroid plexus as well as theleptomeninges (15, 17, 18), complementing other evidencefor a saturable transport system for leptin into the brain (19).

Measurement of the leptin concentration in the CSF and itsrelationship to the plasma leptin concentration may providean important research probe for investigation of disorders ofbody weight regulation in humans. Recent reports have dem-onstrated a significant positive correlation between CSF andplasma leptin levels in individuals ranging from normalweight to obese (20, 21). In overweight individuals, there wasevidence for a decrease in the ratio of CSF to plasma leptin,suggesting that saturation of the leptin transporter at ele-vated plasma leptin levels could contribute to leptin resis-tance in human obesity (20, 21).

The present investigation evaluated the relationship be-tween CSF and plasma leptin concentrations in a combinedgroup of healthy volunteers at normal weight and in lowweight patients with anorexia nervosa. Recent reports indi-cate that plasma leptin concentrations are decreased in an-orexia nervosa (22, 23). This study evaluated whether levels

Received December 10, 1996. Revision received February 20, 1997.Accepted March 5, 1997.

Address all correspondence and requests for reprints to: Dr. David C.Jimerson, Department of Psychiatry, Beth Israel Deaconess MedicalCenter, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail:djimerso@bidmc.harvard.edu.; or to Dr. Jeffrey S. Flier, Division of En-docrinology, Department of Medicine, Beth Israel Deaconess MedicalCenter, 330 Brookline Avenue, Boston, Massachusetts 02215. Email:jflier@bidmc.harvard.edu

* This work was supported in part by USPHS Grant DK-R37–28082from the NIDDK (to J.S.F.).

† Member of the Clinical Investigators Training Program, Beth IsraelHospital-Harvard-Massachusetts Institute of Technology, Departmentof Health Science and Technology, in collaboration with Pfizer.

0021-972X/97/$03.00/0 Vol. 82, No. 6Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.Copyright © 1997 by The Endocrine Society

1845

of leptin in the CNS, as reflected in CSF leptin concentrationsand in the CSF to plasma leptin ratio, are abnormal in pa-tients with anorexia nervosa, and whether such levels arelikely to contribute to the onset or maintenance of weight losscharacteristic of this disorder. Additionally, given the role ofCNS serotonin in postingestive satiety (24), the relationshipbetween CSF concentrations of leptin and the serotonin me-tabolite 5-hydroxyindole acetic acid (5-HIAA) was assessed.

Subjects and MethodsSubjects

The control group included 15 healthy, normal weight women (age,25 6 5 yr). All were free of current or past major psychiatric illness, asassessed by standardized interview (25). The patient group included 11women (age, 24 6 6 yr) who met criteria for anorexia nervosa based onthe Diagnostic and Statistical Manual of Mental Disorders, Third Edition(26). Patients were medically stable and had no other concurrent medicalillness. Subjects were medication free for a minimum of 3 weeks beforethe study. Before study participation, all subjects gave written informedconsent, as approved by the institutional review board.

Study procedures

After a prescreening assessment, healthy volunteers were admittedovernight to a clinical research unit at the Laboratory of Clinical Science,Intramural Research Program, NIMH. Patients were admitted to thesame clinical research unit for a combined treatment and longitudinalstudies program. After postadmission psychiatric and medical assess-ments, patients began a 3-week behavioral and nutritional stabilizationprogram. They consumed a diet of 800-1200 Cal/day, designed to main-tain their admission body weight. During this low weight phase, patientswere introduced to the behaviorally oriented treatment program. Thefirst set of plasma and CSF samples was obtained at the end of the lowweight stabilization phase.

During the supervised weight restoration phase of the treatmentprogram, patients’ dietary intake was progressively increased to achieveweight gain at a rate of approximately 1.5 kg/week. The second studyphase occurred an average of 81 days after the initial studies, whenpatients were approaching their goal weight (approximately 85% of age-and height-adjusted expected body weight). After reaching their goalweight, patients participated in the third phase of the treatment pro-gram, which focused on weight stabilization at the goal weight. The thirdstudy phase occurred an average of 32 days after the goal weight studyphase, approximately 3 weeks after patients had achieved their goalweight. As shown in Table 1, body mass index (BMI) had increasedsubstantially by the time of the weight gain study phase and showed asmall significant further increase at the goal weight phase. Body com-position was assessed by anthropometry in seven of the anorexic pa-tients during the three study phases and in six of the controls, as pre-viously described (27).

Studies were conducted in the morning after overnight bed rest andovernight fast (except water). Subjects were allowed brief bathroomprivileges 2 h before sample collection. Blood samples were obtained byvenipuncture. CSF samples were obtained by lumbar puncture withpatients in the lateral decubitus position. Heparinized plasma samplesand CSF were collected on ice and were aliquoted immediately forstorage at 270° C.

Laboratory methods

RIA for leptin was performed with components obtained from LincoResearch (Indianapolis, IN). For measurement of plasma leptin, recom-binant human leptin or 50 mL test plasma, in duplicate, were incubatedfor 24 h at 4 C with phosphate-buffered saline containing 0.1% TritonX-100, a 1:2000 dilution of antileptin antiserum, and 125I-labeled leptin(;15,000 cpm). Antibody-bound leptin was precipitated by the additionof 500 mL precipitating reagent. Tubes were centrifuged for 45 min at2,500 rpm, after which supernates were decanted, and pellets werecounted in a g-counter. For the standard plasma leptin assay, the limitof detection was 0.5 ng/mL, and the within-assay coefficient of variationwas 4.4% for low levels (2.9 ng/mL) and 5.7% for high levels (14.1ng/mL). Interassay variations were 6.9% and 9.0%, respectively. Allassays were run twice in duplicate, and results of the two assays weresimilar.

The RIA for CSF samples was performed as described above, exceptthat CSF samples (200 mL) were concentrated to a final volume of 50 mLusing a Savant lyophilizer (Farmingdale, NY), and the CSF concentrateand antileptin antiserum were incubated at 4 C for 24 h before theaddition of tracer leptin. The sensitivity of this modified RIA was 38pg/mL. CSF concentrations of 5-HIAA were measured by high pressureliquid chromatography (28).

Statistical methods

Statistical analyses were performed using SPSS software (29). Two-sided significance levels are reported, with statistical significance set ata less than 0.05. Group values for outcome measures are reported as themean 6 sd. Variables not normally distributed were log transformedand retested for normality before analysis by parametric methods. Dataon body weight and leptin concentrations in CSF and plasma obtainedat the three study phases for the patient group were compared to controlvalues by t test, with significance adjusted for multiple comparisonsusing Dunnett’s t test. For the anorexic patients, change in outcomemeasures from the low weight baseline assessment to subsequent studyphases was assessed by paired t test with Bonferroni correction formultiple comparisons. Relationships among CSF leptin, plasma leptin,and body weight were assessed by Pearson correlation coefficient or,where indicated, by Spearman rank order correlation.

ResultsCSF and plasma leptin in healthy controls

Comparative data for body weight measurements in the 15healthy controls and 11 patients with anorexia nervosa aresummarized in Table 1. In the controls, the mean CSF leptinconcentration was 160 6 58 pg/mL. There was a robustpositive correlation between the CSF leptin concentrationand BMI (r 5 0.66; df 5 13; P , 0.01; Fig. 1A). In contrast,across the relatively narrow weight range represented inthese subjects, there was only a weak positive associationbetween plasma leptin levels and BMI (r 5 0.39; df 5 13; P 50.15).

There was a significant correlation between CSF leptin andplasma leptin concentrations in the control group (r 5 0.80;df 5 13; P , 0.001; Fig. 1B). The linear relationship shown inFig. 1B had a relatively shallow slope, indicating that changesin CSF leptin levels were likely to be attenuated compared toalterations in plasma values.

Leptin in anorexia nervosa

Compared to controls, low weight patients with anorexianervosa had significantly diminished concentrations of lep-tin in CSF (t 5 3.97; df 5 24; P , 0.0005, by Dunnett’s t test;Fig. 2A). Compared to the measurements obtained at lowweight, CSF leptin values for these patients studied longi-

TABLE 1. Summary of results for body weight measurements

Anorexia nervosa (n 5 11) Healthy controls(n 5 15)Low wt Wt gain Goal wt

BMI (kg/m2) 13.5 6 1.2a 18.4 6 0.7a 19.1 6 0.5a 21.6 6 1.8Body fat (%)b 10.5 6 2.4a 19.4 6 2.9 21.8 6 3.1 19.7 6 4.0

a Significance of difference from control values, P , 0.0001.b Data for body composition include seven patients and six controls.

1846 MANTZOROS ET AL. JCE & M • 1997Vol 82 • No 6

tudinally were significantly higher during the active weightgain phase (t 5 5.70; df 5 10; P , 0.001) and during the goalweight maintenance phase (t 5 5.07; df 5 10; P , 0.005, bypaired t tests with Bonferroni adjustment; Fig. 2A). For thecombined patient and control groups, the CSF leptin con-centration was significantly correlated with BMI (r 5 0.73;df 5 24; P , 0.0001), although this relationship did not reachsignificance for the anorexic patients evaluated alone at thethree study phases.

Plasma leptin levels were significantly reduced in lowweight anorexic patients compared to control values (F 5

4.40; df 5 24; P , 0.005, by Dunnett’s t test; Fig. 2B). Therewere significant increases in plasma leptin, compared to lowweight values, when the patients were restudied duringweight gain (t 5 5.08; df 5 10; P , 0.005) and at goal weight(t 5 5.34; df 5 10; P , 0.001, by paired t tests with Bonferroniadjustment). Plasma leptin values showed a significant cor-relation with BMI in the combined subject group (r 5 0.83;df 5 24; P , 0.0001), but not in the anorexic patients assessedseparately.

In anorexic patients studied longitudinally, CSF leptinconcentrations reached normal values before full restoration

FIG. 2. Compared to controls, patients with anorexia nervosa studied at low weight had significantly decreased concentrations of leptin in CSF(***, P , 0.0005; A) and plasma (**, P , 0.005; B). When patients were restudied during weight gain and goal weight phases, leptin levels werenot significantly different from control values.

FIG. 1. Results for 15 healthy female volunteers demonstrated significant correlations between CSF leptin and BMI (r 5 0.66; df 5 13; P ,0.01; A) and between CSF leptin and plasma leptin (r 5 0.80; df 5 13; P , 0.001; B).

LEPTIN IN ANOREXIA NERVOSA 1847

of body weight, as reflected in the BMI (Fig. 3A). Thus, to theextent that CSF leptin acts as a CNS signal regarding bodyweight, this signal appeared to be prematurely normalizedas patients gained weight, as assessed by BMI. It was ofinterest that CSF leptin values for the weight gain and goalweight phases appeared to be appropriately calibrated whenplotted against percent body fat for the seven patients inwhom longitudinal anthropometric measurements wereavailable (Fig. 3B). This finding reflects the fact that duringweight restoration, the anorexic patients experienced a rel-atively rapid increase in body fat content relative to leanbody mass (27).

Relationship of CSF to plasma leptin levels

Across the wide range of values for the combined samplesfrom controls and longitudinally studied anorexic patients,there was a significant positive correlation between CSF andplasma leptin levels (r 5 0.81; df 5 46; P , 0.0001). Thisrelationship appeared to be linear, and the curve fit was notsignificantly improved by the use of logarithmic or quadraticcurves.

When the ratio of CSF to plasma leptin was plotted againstthe plasma leptin concentration, a curvilinear relationshipwas apparent (Fig. 4A). Thus, low weight anorexic patientshad higher values for the ratio of CSF to plasma leptin con-centrations than the healthy controls (t 5 5.43; df 5 24; P ,0.0001; Fig. 4B). The ratio of CSF to plasma leptin concen-tration was not significantly different from control values foranorexic patients studied during the weight restoration andgoal weight phases.

Relationship of leptin to 5-HIAA

Given the role of hypothalamic serotonin pathways inpostprandial satiety (24), it was of interest to examine the

relationship between CSF concentrations of leptin and5-HIAA in the patient and control groups. Low weight an-orexic patients had significantly reduced levels of 5-HIAA(82 6 32 ng/mL; n 5 10) compared to controls (106 6 18ng/mL; n 5 12; t 5 2.64; df 5 20; P , 0.02). CSF 5-HIAAvalues for the anorexic patients increased progressively dur-ing the treatment program, as reflected in measurementsduring weight gain (85 6 32 ng/mL) and at goal weight(100 6 43 ng/mL). CSF 5-HIAA levels were significantlycorrelated with CSF leptin levels for the combined group oflow weight anorexic patients and healthy controls (r 5 0.57;df 5 20; P , 0.01; Fig. 5), although this correlation did notreach significance for controls or patients taken separately.

Discussion

Leptin is thought to exert its primary actions in the brain,and there is, therefore, considerable interest in the levels ofleptin in CSF in health and disease, and in response to nu-tritional perturbation. The current study provides substan-tial new information in this regard. First, we have demon-strated a significant positive correlation between leptinconcentrations in CSF and plasma in healthy, normal weightsubjects. The correlation was substantially more robust thanthat between BMI and plasma leptin, suggesting that plasmalevels are influenced by transient state-related factors,whereas CSF concentrations are a more stable index of en-ergy stores. These results extend recent reports showing sig-nificant positive correlations between leptin levels in CSFand plasma in a mixed group of healthy volunteers andindividuals with Alzheimer’s disease, some of whom wereoverweight (20), and in a mixed group of lean patients (21).For the healthy, normal weight female volunteers in thecurrent study, the relationship between plasma and CSFleptin levels was linear. This contrasts with previous reports

FIG. 3. A, As 11 anorexic patients achieved increased BMI, the CSF leptin concentration increased markedly from the low weight phase (å)to the weight gain phase (f) and goal weight phase (F). Patients reached an average leptin level similar to that in 15 controls (E), even thoughtheir body weight (BMI) remained significantly lower than that in the controls. B, For 7 anorexic patients (compared to 6 controls) for whomanthropometries were available, CSF leptin values increased proportionately to the increase in percent body fat. Values are the mean 6 SE.

1848 MANTZOROS ET AL. JCE & M • 1997Vol 82 • No 6

describing a plateau at higher leptin concentrations in theslope of the regression line between CSF and plasma leptin(20, 21). Thus, the current study suggests that presumedsubstrate saturation effects on the transporter may be ap-parent only at leptin concentrations above the normal range.

We have also provided the first information on CSF leptinlevels in anorexia nervosa, an idiopathic state of self-inducedstarvation. Women with anorexia nervosa showed a marked

decrease in plasma and CSF leptin concentrations comparedto healthy, age-matched female controls, demonstrating thatanorexia nervosa does not result from a simple excess of CNSleptin levels, as might occur secondary to enhanced periph-eral levels or increased uptake. After weight gain in anorexicpatients, leptin levels increased significantly in both plasmaand CSF to levels not significantly different from normal.This is the first report of dynamic changes in CSF leptin withchanges in nutrition or body weight. These results also sub-stantially extend the findings of two other reports. In one, 10of 15 patients with anorexia nervosa had plasma leptin levelsless than the assay normal range, whereas levels in the re-maining 5 were in the normal range (22). In this same report,5 patients studied longitudinally after weight gain averaging2.1 kg/m2 had no significant change in plasma leptin levels.The significant increase in leptin levels after weight gain inthe current study is likely to reflect the larger weight gain andthe larger number of individuals studied. In a second, cross-sectional study (23), serum leptin levels were significantlycorrelated with body weight and percent body fat in 22women with anorexia nervosa and were significantly lowerthan values for healthy female controls.

In the current study, the percent decrease in plasma leptinin low weight anorexics exceeded the percent decrease intheir CSF leptin. Thus, the CSF to plasma leptin ratio wasmarkedly elevated in low weight anorexic patients comparedto controls. As we were unable to study CSF leptin in sim-ilarly low weight individuals with simple starvation, it isunclear whether this effect is a consequence of low bodyweight associated with malnutrition per se or is a specificfeature of anorexic patients. However, a similar increase inthe plasma to CSF leptin ratio was observed at low leptinlevels in patients without anorexia (20). A significant fractionof leptin in blood is bound to serum proteins (30). Additional

FIG. 4. A, The CSF to plasma leptin ratio for anorexic patients at low weight (å), during weight gain (f), and at goal weight (F) compared tothe control value (E). The CSF to plasma leptin ratio was markedly increased in subjects with low plasma leptin levels. B, Low weight anorexicpatients had significantly elevated CSF to plasma leptin ratios compared to controls (***, P , 0.0001).

FIG. 5. For the combined group of low weight anorexic patients (å)and controls (E), there was a significant correlation between CSFleptin and the serotonin metabolite 5-HIAA (r 5 0.57; P , 0.01;log-transformed variables).

LEPTIN IN ANOREXIA NERVOSA 1849

studies will be needed to evaluate the relationship betweenconcentrations of free leptin in plasma and CSF in healthyindividuals and patients with abnormal regulation of bodyweight.

It is important to consider the possible relationship be-tween diminished CSF leptin concentrations and the hypo-thalamic amenorrhea of anorexia nervosa. Leptin has beenlinked to control of reproduction in three ways. First, femaleob/ob mice with leptin deficiency have hypothalamic amen-orrhea and infertility, and chronic treatment with recombi-nant leptin can restore fertility (31). Second, the reduction inleptin levels during starvation of normal mice contributes toabnormalities in the hypothalamic-pituitary-adrenal and go-nadal axes, as leptin administration prevented the delay inreproductive cycling in this model (32). Third, administra-tion of leptin can accelerate the onset of puberty in normalmice (33, 34). If the physiology of leptin is similar in humans,treatment with leptin might restore fertility in low weightwomen with anorexia nervosa. Even if amenorrhea in lowweight patients could be treated in this manner, however, thepossibility of other adverse consequences of leptin admin-istration (e.g. delayed weight gain) would need to be care-fully evaluated.

An important observation in the current study was that asthe anorexic patients gained weight, CSF and plasma leptinconcentrations reached levels equivalent to normal values,whereas the patients were still at a significantly lower weightthan the controls. This result can be understood in terms ofour previous observation that anorexic patients participatingin a behaviorally oriented weight restoration program withprogressive augmentation of caloric intake experience a rel-atively rapid increase in percent body fat (27). Taken to-gether, these findings suggest that rapid normalization ofleptin levels during weight restoration is probably due todisproportionate accumulation of fat during the refeedingperiod and may contribute to the difficulty anorexic patientsexperience in achieving and maintaining a normal weight.

The finding of decreased CSF levels of the serotonin me-tabolite 5-HIAA in the low weight anorexia nervosa patientgroup is consistent with previously reported findings (35,36). It is of interest that the present results demonstrated asignificant positive correlation between indexes of two neu-rochemical systems thought to limit food intake. Additionalinvestigations are needed to study the possible interactionsbetween leptin and serotonin, neuropeptide Y, galanin, andother neurochemicals involved in the regulation of eatingbehavior.

In summary, this study has provided several new insightsinto the regulation of CSF leptin. First we have shown thatin normal subjects, CSF leptin correlates better with BMI thandoes plasma leptin, consistent with the ability of leptin in CSFto integrate plasma levels that may be subject to short termfluctuations. Second, we confirm a prior report that the ratioof CSF/plasma leptin is markedly increased at low plasmaleptin levels, consistent with increased efficiency of CSF up-take under these conditions. We also provide the first dem-onstration that this increased ratio decreases during weightgain. Finally, we demonstrate that patients with anorexia at

low weight have reduced CSF leptin compared to controlsand that they normalize their CSF (and plasma) leptin levelsduring weight gain at a point before achieving normal bodyweight, as assessed by BMI. This could result from the ten-dency of these patients to regain weight disproportionatelyas fat and could contribute to their resistance to regainingnormal body weight and composition.

Acknowledgments

Assistance with anthropometric measurements was provided by EvaObarzanek, Ph.D. William Z. Potter, M.D., Ph.D. assisted with laboratoryassays for cerebrospinal fluid 5-hydroxyindole acetic acid; Silke Naab,Dr. Med., provided technical assistance.

References

1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. 1994Positional cloning of the mouse obese gene and its human homologue. Nature.372:425–432.

2. Pelleymounter MA, Cullen MJ, Baker MB, et al. 1995 Effects of the obese geneproduct on body weight regulation in ob/ob mice. Science. 269:540–543.

3. Halaas JL, Gjiwala KS, Maffei M, et al. 1995 Weight-reducing effects of theplasma protein encoded by the obese gene. Science. 269:543–546.

4. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. 1995 Recombinantmouse OB protein: evidence for a peripheral signal linking adiposity andcentral neural networks. Science. 269:546–549.

5. Frederich RC, Hamann A, Anderson S, Lollman B, Lowell BB, Flier JS. 1995Leptin levels reflect body lipid content in mice: evidence for diet-inducedresistance to leptin action. Nat Med. 1:1311–1314.

6. Chen H, Charlat O, Tartaglia LA, et al. 1996 Evidence that the diabetes geneencodes the leptin receptor: identification of a mutation in the leptin receptorgene in db/db mice. Cell. 84:491–495.

7. Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS. 1996 Roleof leptin in fat regulation. Nature. 380:677.

8. Maffei M, Halaas J, Ravussin E, et al. 1995 Leptin levels in human and rodent:measurement of plasma leptin and ob RNA in obese and weight-reducedsubjects. Nat Med. 1:1155–1161.

9. Lonnqvist F, Arner P, Nordfors L, Shalling M. 1995 Overexpression of theobese (ob) gene in adipose tissue of human obese subjects. Nat Med. 1:950–953.

10. Hamilton BS, Paglia D, Kwan ATM, Deitel M. 1995 Increased obese mRNAexpression in omental fat cells from massively obese humans. Nat Med.1:953–956.

11. Considine RV, Sinha MK, Heiman ML, et al. 1996 Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med.334:292–295.

12. McGregor GP, Desaga JF, Ehlenz K, et al. 1996 Radioimmunological mea-surement of leptin in plasma of obese and diabetic human subjects. Endocri-nology. 137:1501–1504.

13. Rosenbaum M, Nicolson M, Hirsch J, et al. 1996 Effects of gender, bodycomposition, and menopause on plasma concentration of leptin. J Clin Endo-crinol Metab. 81:3424–3427.

14. Cusin I, Rohner-Jeanrenaud F, Stricker-Krongrad A, Jeanrenaud B. 1996 Theweight-reducing effect of an intracerebro-ventricular bolus injection of leptinin genetically obese fa/fa rats. Diabetes. 45:1446–1450.

15. Tartaglia LA, Dembski M, Weng X, et al. 1995 Identification and expressioncloning of a leptin receptor, OB-R. Cell. 83:1263–1271.

16. Stephens TW, Bashinski M, Bristow PK, et al. 1995 The role of neuropeptideY in the antiobesity action of the obese gene product. Nature. 377:530–534.

17. Malik KF, Young WS. 1996 Localization of binding sites in the central nervoussystem for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db)C57BL/6J mice. Endocrinology. 137:1497–1500.

18. Lynn RB, Cao GY, Considine RV, Hyde TM, Caro JF. 1996 Autoradiographiclocalization of leptin binding in the choroid plexus of ob/ob and db/db mice.Biochem Biophys Res Commun. 219:884–889.

19. Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM. 1996 Leptin entersthe brain by a saturable system independent of insulin. Peptides. 17:305–311.

20. Schwartz MW, Peskind E, Raskind M, Boyko EJ, Porte Jr D. 1996 Cerebro-spinal fluid leptin levels: relationship to plasma levels and to adiposity inhumans. Nat Med. 12:589–593.

21. Caro JF, Kolaczynski JW, Nyce MR, et al. 1996 Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance.Lancet. 348:159–161.

22. Hebebrand J, van der Heyden J, Devos R, et al. 1995 Plasma concentrationsof obese protein in anorexia nervosa [Letter]. Lancet. 346:1624–1625.

23. Grinspoon S, Gulick T, Askari H, et al. 1996 Serum leptin levels in womenwith anorexia nervosa. J Clin Endocrinol Metab. 81:3861–3863.

24. Leibowitz SF, Weiss GF, Suh JS. 1990 Medial hypothalamic nuclei mediate

1850 MANTZOROS ET AL. JCE & M • 1997Vol 82 • No 6

serotonin’s inhibitory effect on feeding behavior. Pharmacol Biochem Behav.37:735–742.

25. Endicott J, Spitzer RL. 1978 A diagnostic interview: The Schedule for AffectiveDisorders and Schizophrenia. Arch Gen Psychiatry. 35:837–844.

26. American Psychiatric Association. 1980 Diagnostic and statistical manual ofmental disorders, 3rd ed. Washington DC: American Psychiatric Press.

27. Obarzanek E, Lesem MD, Jimerson DC. 1994 Resting metabolic rate of an-orexia nervosa patients during weight gain. Am J Clin Nutr. 60:666–675.

28. Scheinin M, Chang W-H, Kirk KL, Linnoila M. 1983 Simultaneous de-termination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleaceticacid, and homovanillic acid in cerebrospinal fluid with high-performanceliquid chromatography using electrochemical detection. Anal Biochem.131:246 –253.

29. Norusis MJ. 1993 SPSS for Windows: base system user’s guide, release 6.0.Chicago: SPSS.

30. Houseknecht KL, Mantzoros CS, Kuliawat R, Hadro E, Flier JS, Kahn BB.

1996 Evidence for leptin binding to proteins in serum of rodents and humans:modulation with obesity. Diabetes. 45:1638–1643.

31. Chehab FF, Lim ME, Lu R. 1996 Correction of the sterility defect in homozy-gous obese female mice by treatment with the human recombinant leptin. NatGenet. 12:318–320.

32. Ahima RS, Prabakaran D, Mantzoros C, et al. 1996 Role of leptin in theneuroendocrine response to fasting. Nature. 382:250–252.

33. Chehab FF, Mounzih K, Lu R, Lim ME. 1997 Early onset of reproductivefunction in normal female mice treated with leptin. Science. 275:88–90.

34. Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS. 1997 Leptin acceleratesthe onset of puberty in normal female mice. J Clin Invest. 99:391–395.

35. Kaye WH, Ebert MH, Raleigh M, Lake CR. 1984 Abnormalities in CNSmonoamine metabolism in anorexia nervosa. Arch Gen Psychiatry.41:350–355.

36. Jimerson DC, Lesem MD, Hegg AP, Brewerton TD. 1990 Serotonin in humaneating disorders. Ann NY Acad Sci. 600:532–544.

LEPTIN IN ANOREXIA NERVOSA 1851