Ria Leptin

Human-Leptin-RIA - Sensitive (human OB protein) Radioimmunoassay for the quantitative detection of

Human Leptin Product code: LEP-R40 (125 tubes)

0 DE/CA40/00809/11

For in-vitro use only! In the USA: For Research Use Only!

Aspenhaustr. 25 • D-72770 Reutlingen / Germany Phone: + 49 - (0) 7121 51484-0 • Fax: + 49 - (0) 7121 51484-10 E-mail: [email protected] • http://www.mediagnost.de

Table of contents FEATURES BACKGROUND INTENDED USE PRECAUTIONS General Radioactivity METHODOLOGY Assay performance Stability of Serum Leptin: MATERIALS Materials Provided for 125 tubes: Required Materials Not Provided Reagent Storage and Preparation: Sample Preparation: Suggestion for dilution protocol ASSAY PROCEDURE Standard procedure: Extended washing procedure for increased precision Procedure for increased sensitivity (non-equilibrium assay) : EVALUATION OF RESULTS Establishing the standard curve Evaluation of sample concentrations Concentration of high and low control samples EXPECTED VALUES Example Calculation of standard deviation scores (SDS; Z-scores) Example: Estimation of optimal dilution of samples REFERENCES SUMMARY-Mediagnost hLEPTIN RIA SENSITIVE LEP-R40

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FEATURES  Measures total serum leptin  Calibrated against the International Standard of the WHO/NIBSC Code 97/594  The high affinity antiserum ensures no interference from leptin binding proteins  The high sensitivity (range of standards 0 - 16 ng/ml) allows precise measurement also in lean subjects, e.g. in anorectic or cachectic patients, male adolescents, and young children  Small sample volume requirement due to high sensitivity  Allows measurement in capillary blood, e.g., in longitudinal studies  The high sensitivity allows measurement of low leptin concentrations in specimens other than serum such as urine, cerebrospinal fluid, and cell culture media BACKGROUND Leptin, the product of the ob gene (1,2), is a recently discovered singlechain proteohormone with a molecular weight of 16 kD which is thought to play a key role in the regulation of body weight. Its amino acid sequence exhibits no major homologies to other proteins (1). Leptin is almost exclusively produced by differentiated adipocytes (3-5). It acts on the central nervous system, in particular the hypothalamus, thereby suppressing food intake and stimulating energy expenditure (2,6-9). Leptin receptors - alternatively spliced forms exist that differ in length belong to the cytokine class I receptor family (10-12). They are found ubiquitously in the body (10,11,13,14) indicating a general role of leptin which is currently not fully understood. A circulating form of the leptin receptor exists, which acts as one of several leptin binding proteins (15). Besides its metabolic effects, leptin was shown to have a strong influence on a number of endocrine axes. In male mice, it blunted the starvationinduced marked decline of LH, testosterone, thyroxine, and the increase Human-Leptin RIA-Sensitiv

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of ACTH and corticosterone. In female mice, leptin prevented the starvation-induced delay in ovulation (16). Ob/ob mice, which are leptin deficient due to an ob gene mutation, are infertile. This defect could be corrected by administration of leptin, but not through weight loss due to fasting (17), suggesting that leptin is pivotal for - at least female reproductive functions. All these actions may, at least in part, be explained by the suppressive effect of leptin on neuropeptide Y (NPY) expression and secretion by neurons in the arcuate nucleus (6,18,19). NPY is a strong stimulator of appetite (20,21) and is known to be involved in the regulation of various pituitary hormones, e.g. suppression of GH through stimulation of somatostatin (22,23), suppression of gonadotropins (23) or stimulation of the pituitary-adrenal axis (21). The most important variable that determines circulating leptin levels is body fat mass (24-26). Obviously, under conditions of regular eating cycles, leptin reflects the proportion of adipose tissue (27) showing an exponential relationship. This constitutive synthesis of leptin is modulated by a number of non-hormonal and hormonal variables. Stimulators in both rodents and humans are overfeeding (28,29), insulin (3,5,30-33) and glucocorticoids (5,34-36). Suppression has been shown for fasting (27), cAMP and ß3-adrenoceptor agonists (35). From these findings it becomes clear that leptin is an integral component of various metabolic and endocrine feedback loops. For clinical purposes, it is important to note that serum leptin levels show a moderate circadian variation with a peak during the night at about 2 a.m. (37). The leptin values at this time are about 30 to 100 % higher than the levels measured in the morning or early afternoon. This variation together with the influence of food intake needs to be taken into account, when blood samples are collected. Under fairly standardized conditions, i.e. normal eating cycles and blood sampling in the morning or early afternoon, a single leptin measurement is informative. For the appropriate interpretation of measured leptin levels, reference ranges are required. Because body fat mass is the major confounding Human-Leptin RIA-Sensitiv

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variable, these ranges should be referred to measures of the percentage body fat such as body mass index (BMI) or percent body fat determined by, e.g., bioelectric impedance assessment (BIA). Leptin levels are higher in females than in males (38,39) and an age dependence was shown in children and adolescents (40). Therefore, reference ranges referring to measures of body fat should be stratified according to gender and pubertal development. Leptin levels are high in most obese patients suggesting the presence of leptin insensitivity (20,26,37,40-42). In a small percentage of patients, however, leptin levels have been found inappropriately low with respect to their fat mass. It remains for future studies to prove that these patients represent a new pathophysiologic entity: leptin deficiency. Since leptin has also been shown to be of great importance for reproductive functions, possible new pathophysiologic mechanisms may be discovered relating infertility to insufficient leptin production. The discovery of leptin has released an avalanche of research activities seeking to understand the regulation and actions of this new hormone. Most importantly, it has provided a key to better understand the physiology of body weight regulation and to unveil possible pathophysiologic mechanisms in both obesity and eating disorders. Further, it may provide new insights into certain causes of infertility. This widespread importance makes leptin an interesting parameter for physicians dealing with obesity, cachexia and metabolic disturbances, diabetologists, endocrinologists, gynecologists, andrologists, and psychiatrists treating patients with eating disorders.

INTENDED USE This radioimmunoassay kit is suited for measuring human leptin in serum or plasma, other biological fluids (e.g. urine or cerebrospinal fluid), and conditioned adipocyte culture media for scientific and diagnostic purposes also in very low concentrations. Human-Leptin RIA-Sensitiv

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For standard purposes (0 - 64 ng/ml leptin) we recommend our HumanLeptin RIA - Standard, product code LEP-R42. Under conditions of normal eating cycles, measurement in a single blood sample collected in the morning or early afternoon is sufficient. The comparison with BMI-related reference ranges may be useful to detect conditions of relative leptin deficiency as a possible cause of obesity. Due to its high correlation with body fat mass leptin measurements under standardized conditions may be used as a simple and inexpensive test for determination of body fat. PRECAUTIONS General All reagents are for in vitro use only! In conducting the assay, follow strictly the test protocol. The acquisition, possession and use of the kit is subject to the regulations of the national nuclear regulatory authorities. Reagents with different lot numbers should not be mixed. Reagents contain Sodium-Azide as preservative, however, highly diluted (0.02%). Sodium-Azide is very toxic, R-Phrases: 28, 32, 50/53 and SPhrases 28, 45, 60, 61 must be considered. First aid procedures: Skin contact: Wash affected area thoroughly with water at least 15 minutes. Discard contaminated cloths and shoes. See a physician. Eye contact: In case of contact with eyes, rinse immediately with plenty of water at least 15 minutes. In order to assure an effectual rinsing spread the eyelids. See a physician. Ingestion: If swallowed, wash out mouth throughly with water, provided that the person is conscious. Immediately see a physician.

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The handling of radioactive and potentially infectious material must comply with the following guidelines: The material should be stored and used in a special designated area. Do not eat, drink or smoke in these areas. Never pipette the materials with the mouth. Avoid direct contact with these materials by wearing laboratory coats and disposable gloves. Spilled material must be wiped off immediately. Clean contaminated areas and equipment with a suitable detergent. Unused radioactive material and radioactive waste should be disposed according to the recommendations of the national regulatory authorities. Radioactivity Before ordering or using radioactive materials, it is necessary to take the appropriate actions to ensure compliance with national regulations governing their use. Local rules in each establishment, which define actions and behaviour in the radioactivity working areas, should also be adhered to. The advice given here does not replace any local rules, instructions or training in the establishment, or advice from the radiation protection advisers. It is important to follow the code of good laboratory practice in addition to the specific precautions relating to the radionuclide I-125 used. Iodine-125 has a radioactive half-life T1/2 of 60 days and emits 35,5 keV gamma radiation, 27 – 32 keV x-rays and no beta radiation. Shielding is effective done by lead, first half value layer is 0.02 mm lead, reduction to 10 % is made by 0.2 mm. To reduce the radiation dose time spent handling radioactivity should be minimized (plan ahead), and distance from source of radiation should be maximized (doubling the distance from the source quarters the radiation dose).

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Formation of aerosols, e.g. by improper opening and mixing of vials or pipetting of solutions which may cause minute droplets of radioactivity become airborn, is a hazard and should be avoided. Solutions containing iodine should not be made acidic, because this might lead to the formation of volatile elemental iodine. As some iodo-compounds can penetrate rubber gloves, it is advisable to wear two pairs, or polyethylene gloves over rubber. For cleaning of contaminated areas or equipment, the Iodine-125 should be rendered chemically stable by using alkaline sodium thiosulphate solution together with paper or cellulose tissue. METHODOLOGY Assay characteristic The radioimmunoassay for human leptin utilizes a high affinity polyclonal antibody specific for this protein. It recognizes quantitatively human leptin. Standards and

125

I-tracer are prepared from recombinant leptin.

No disturbances from leptin binding proteins due to the use of high affinity antiserum. Assay performance Range of standards: 0 - 16 ng/ml Half maximal displacement: (50% B/B 0): at about 1.8 ng/ml or 0.8 ng/ml (procedure for increased sensitivity see page 16). Optimal range: - about 2 ng/ml, one overnight incubation step. - Procedure for increased sensitivity: about 1 ng/ml, with two overnight incubation steps.

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Specificity: This assay is specific for human leptin. The antibody shows only little cross-reactivity with mouse, rat, horse, sheep and chicken leptin. No cross-reactivity was found with other proteins such as insulin or IGF-I. Sensitivity: 0.04 ng/ml (0.01 ng/ml with procedure for increased sensitivity) Intra- and interassay variation: The intraassay variation has been found to be lower than 5%, the interassay variation did not exceed 7.6% (two serum samples containing different concentrations of leptin were determined 10 times each in eight separate assays). Recovery: Serum spiking experiments with recombinant human leptin at about 2 ng/ml yielded a recovery of 97% (± 2%). Calibration of the Assay The Assays is calibrated against the International Standard for human leptin. The standard preparation of the WHO with code 97/594 (y) is available from the NIBSC (z). One ampoule of the preparation, reconstituted in 1 ml Assay Buffer, will be quantified with the nominal content of 5000 ng human leptin. Parallelism with serum samples: Serial dilutions of human serum samples gave excellent parallelism with the standard curve (see figure 1). Stability of Serum Leptin: Leptin appears to be stable under common conditions of sample collection, handling and storage.The constant results of a large number of samples show that immunoreactive serum leptin appears to be stable at 20°C for several years. Even repeated freezing and thawing up to 10 cycles had no influence on leptin levels. No decrease of immunoreactive serum leptin was observed after incubation of serum samples at 4°C and Human-Leptin RIA-Sensitiv

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at room temperature up to 8 days. However, at 37°C, leptin levels began to decline after 1 day.

100 80 60 40 20 0 128

64

32

16

8

4

2

1

Verdünnungsfaktor Figure 1:

Serial dilution of leptin standard (filled circle) and different sera with varying concentrations of leptin. The leptin level of the undiluted standard was 16 ng/ml. Means of duplicate measurements are shown.

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MATERIALS Materials Provided for 125 tubes: The reagents listed below are sufficient for 125 tubes including the standard curve. A: Assay buffer (1 bottle, 125 ml; ready for use) B: 1st antibody: anti-human leptin containing rabbit IgG (1 bottle, 13 ml; lyophilized) C: Tracer: 125I-Ieptin; < 1.55 µCi respectively < 60 kBq (1 bottle, 13 ml; lyophilized, red-coloured) D: Rabbit immunoglobulin for non-specific binding (NSB) (1 vial, 1 ml; lyophilized) F-L: Standards: 0.25; 0.5; 1; 2; 4; 8; 16 ng/ml leptin (7 vials, 1 ml each; ready for use). M,N: High (20.1 ng/ml) and low (7.3 ng/ml)control (2 vials, 0.75 ml each; human serum; lyophilized) O: 2nd Antibody: anti-rabbit immunoglobulin (1 vial, 1 ml; lyophilized) P: Precipitation reagent (1 bottle, 65 ml; ready for use after adding reagent O) Required Materials Not Provided 1) Pipettes: 10 ml, 1 ml, 500 µl, 250 µl, 100 µl, 10 µl; Repetitive pipettes 100 µl, 250 µl and 1 ml are recommended. 2) Disposable polystyrene or polypropylene tubes. Conical tubes are highly recommended because of the small immunoprecipitates. 3 ) Vortex mixer. 4 ) Centrifuge appropriate for precipitation of immunocomplexes. Human-Leptin RIA-Sensitiv

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5 ) Device for aspiration of liquid supernatant (e.g. connected to a water pump). 6 ) Gamma counter 7 ) Ice-cold deionized water. Reagent Storage and Preparation: On receipt the kit should be stored at 2 - 8°C until expiry date. After reconstitution the lyophilized reagents should be stored at -20°C. Avoid repeated thawing and freezing. The shelf-life of the components after opening is not affected, if used appropriately. B+C) Reconstitute with 13 ml reagent A each. D) Reconstitute with 1 ml reagent A. M,N) Reconstitute with 750 µl reagent A each. O) Reconstitute with 1 ml reagent A. Transfer dissolved material to reagent P immediately before use. Add 1 vial of reagent O to 1 bottle of reagent P (or any volumes in the same ratio 1:66). The assay is unaffected by the possible occurrence of turbidity in the final reagent. Ensure that lyophilized materials are completely dissolved on reconstitution. It is recommended to keep reconstituted reagents at room temperature for half an hour and then to mix them vigorously with a Vortex mixer. This is important in particular for the controls M and N ! Sample Preparation: For testing of single blood samples, the specimens may be taken in the morning or early afternoon. For specific questions, the relationship with food intake should be taken into consideration. However, a significant stimulation by a rich meal or suppression during fasting should be expected not earlier than 6 to 8 hours later. Due to the stability of immunoreactive leptin, sample handling is unproblematic. Undiluted serum or plasma specimens may be stored frozen at -20°C for several years without loss of leptin RIA activity. Human-Leptin RIA-Sensitiv

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Repeated thawing and freezing should be avoided, although levels are unaffected by a few cycles. Serum or plasma samples can be used undiluted or should be diluted with assay buffer (reagent A) prior to measurement depending on the expected values. Usually, a dilution of 1:3 is appropriate in lean individuals and 1:20 in obese patients. However, because serum leptin levels vary over several orders of magnitude, it is recommended to dilute samples according to the patient’s BMI. The precision of measurement is highest at about 2 ng/ml. Dilution, therefore, should be such that the expected value is within this range. The relationships between serum leptin levels and BMI according to gender and age are given in the section Expected Values. Examples are given, how appropriate dilutions can be obtained. Suggestion for dilution protocol Mix 100 µl serum or plasma with 200 µl Assay Buffer (dilution1:3). 2x100 µl of this dilution should then be used in the assay. Dilution of the controls (M and N) after their reconstitution should be 1:3 with Assay Buffer.

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ASSAY PROCEDURE Standard procedure: All samples should be measured in duplicate. For optimal results, accurate pipetting and adherence to the protocol are recommended. Flow Chart of Assay Protocol Nr. Tube A F–N Samples 1,2 TC 3,4 NSB 100 5,6 B0 100 7-20 Standards F-L:100 21,22 High M:100 Control 23,24 Low N:100 Control 25,26 Sample 1 100 27,28 Sample 2 100 etc.

D

B

C

P

100 -

100 100 100

100 100 100 100 100

500 500 500 500

-

100

100

500

-

100 100

100 100

500 500

(All volumes are given in µl) 1 ) Labelling of the assay tubes (conical tubes are highly recommended) should be done in the following order (duplicates):

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1, 2

total counts (TC),

3, 4

non specific binding (NSB),

5, 6

zero standard (B 0),

7 to 20

standards (F to L),

21 to 24

controls (M and N),

25, 26 etc. samples. 2)

Add 100 µl of reagent A (Assay Buffer) to tubes 3, 4 and 5, 6.

3)

Add 100 µl of reagents F - L (standards) to tubes 7 - 20: standard 0.25 ng/ml (F) to tubes 7 and 8, standard 0.5 ng/ml (G) to tubes 9 and 10, etc.

4)

Add 100 µl of diluted reagent M (high control; dilution1: 3) and diluted reagent N (low control, dilution1: 3) to tubes 21, 22 and 23, 24, respectively.

5)

Add 100 µl of a diluted sample to tubes 25 and 26,etc.

6)

Add 100 µl reagent D (NSB) to tubes 3 and 4.

7)

Add 100 µl reagent B (1st antibody) beginning with tube 5.

8)

Add 100 µl reagent C (tracer) to all tubes.

9)

Remove tubes 1 and 2 (total counts) or seal with a stopper.

10) Mix tubes with a vortex mixer. 11) Incubate tubes at room temperature overnight (at least 15 h). 12)

Add 500 µl reagent P (after addition of reagent O !) beginning with tube 3. The reagent should be cold (2 - 4°C) !

13) Mix tubes with a vortex mixer. Human-Leptin RIA-Sensitiv

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14) Incubate tubes in the cold (2 - 4°C) for 1 hour. 15) Add 1 ml ice-cold water. 16) Centrifuge all tubes except tube 1 and 2 at 2-4°C at 3000 x g for 20 min. 17) Aspirate the supernatant (except tubes 1 and 2 !). The remaining supernatant should not exeed 3 mm height above the faint precipitate. Take care that the precipitate remains intact. 18) Count the radioactivity of all tubes. Depending on laboratory equipment and common laboratory practice, aspiration of the supernatants in step 17 can be replaced by careful decantation. Extended washing procedure for increased precision The second incubation (step 14) is directly followed by step 16 (centrifugation) and 17 (aspiration). Proceed then with step 15 and add 1 ml of ice-cold water. This should not be done too vigorously in order to keep the precipitate intact. Do not mix again! Centrifuge the tubes at 24°C at 3000 x g for 5 min., aspirate the supernatant and count the radioactivity of all tubes in the gamma-counter (step 18). This extended procedure results in a somewhat higher precision bound up with a higher work expenditure. The higher precision may be irrelevant for most measurements and should therefore be used only in special cases. Procedure for increased sensitivity (non-equilibrium assay) : Follow the standard version protocol up to step 7. After step 7, the addition of reagent B (1st antibody), leave out step 8: do not add reagent C (tracer), but mix tubes with a vortex mixer and incubate Human-Leptin RIA-Sensitiv

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them for at least 15 hours (e.g. overnight) at room temperature. After this preincubation step, continue with step 8: add 100 µl reagent C (tracer) to all tubes. Now follow the procedure until step 18. For specific purposes, e.g., determination of leptin in other biological fluids such as cerebrospinal fluid or urine, the sensitivity may be further increased - on the expense of assay precision - by diluting the 1st antibody (reagent B) and tracer (reagent C) with a larger volume of Assay Buffer (reagent A). Also, the - undiluted - sample volume may be doubled without changing the assay procedure. For details and for support please contact Mediagnost GmbH. If necessary, we will provide you as a service with standard solutions adjusted to a deviated measurement range. EVALUATION OF RESULTS Establishing the standard curve The standards provided contain the following concentrations of leptin: Standard ng/ml

B0 0.0

F

G

H

I

J

K

L

0.25

0.5

1

2

4

8

16

1. Calculate the average counts of each pair of tubes. 2. Substract the average of NSB (tubes 3 and 4) from the mean counts of the standards, controls and samples. This corresponds to the corrected B values. 3. The corrected value of the zero standard (tubes 5 and 6) equals B 0. 4. Calculate the percent bound (% B/B0) by dividing the corrected B values by B0: %B/B 0 = B/B0 x100% Human-Leptin RIA-Sensitiv

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5. Plot % B/B0 versus the standard concentrations on a semi-logarithmic or logit-log paper respectively. For convenience, it is recommended to use computer assisted data reduction programs. 6. For quality control calculate the ‘percentage of NSB’: Average of tubes 3, 4 (NSB) divided by the average of tubes 1, 2 (total counts, TC) multiplied by 100%. It should be < 5%. 7. Calculate the ‘percentage bound of the zero standard’: Average of tubes 5, 6 (B 0) minus average of tubes 3,4 (NSB) divided by average of tubes 1,2 (total counts) multiplied by 100%. It should be > 20%. Evaluation of sample concentrations Read the concentration value (abscissa) corresponding to the % B/B 0 of the sample as in the example given below: average counts of NSB tubes:115 cpm average counts of zero standard (B0): 5494 cpm average counts of sample tube:2561 cpm %B/B0 = (sample counts - NSB) / (B0 - NSB) x 100% = (2561 - 115)/(5494 - 115) x 100% = 0.455 x 100% = 45.5% For a 45.5% value on the y-axis (ordinate) a value of 2.62 ng/ml on the xaxis (abscissa) may be obtained. Multiplication of this value determined graphically or by the aid of a computer program with the dilution factor gives the leptin concentration of the sample. Example: 2.62 x 3 = 7.86 ng/ml

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Concentration of high and low control samples The control samples M and N should fit with the following ranges: M, the high control: 17.4 - 22.8 ng/ml; average 20.1 ng/ml N, the low control: 6.2 - 8.4 ng/ml; average 7.3 ng/ml EXPECTED VALUES Serum leptin levels are mainly determined by body fat mass with low levels in lean individuals and high levels in obese subjects. In addition, there is a clear gender difference with higher levels in females at a given percentage body fat. Further, leptin levels are influenced by pubertal development. Any attempt, therefore, to give ranges of expected leptin levels must account for these relationships. Various methods for the estimation of body fat are available such as calculation of body mass index (weight (kg) divided by the square of height (m)) (BMI), bioelectric impedance assessment (BIA) or total body dual energy x-ray absorptiometry (DXA). Although the accuracy of BMI with respect to reflecting true fat mass is inferior to other more sophisticated methods such as BIA or DXA, BMI provides a number of advantages: 1) It is independent of the regression models applied. 2) It is easy to determine, only weight and height measurements are required. 3) It is retrospectively mostly available. 4) It is the most precise measure during short-term changes of fat mass, e.g. during fasting. Therefore, the following expectation ranges of serum leptin levels were referred to BMI as the major confounding independent variable and were stratified according to gender and pubertal development (see figures and tables 2 - 9). After the age of 20 years, no significant age dependence was observed. These gender and age adjusted expectation ranges may be used to compare a measured leptin level at a given BMI with normal subjects to detect pathologic deviations. Human-Leptin RIA-Sensitiv

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The best-fit regression lines for the various subgroups are exponential (b·BMI) curves of the form leptin = a·e . The 5th and 95th percentiles are given by the following equations: (b·BMI-c) (b·BMI+c) leptin = a·e and leptin = a·e respectively. In a semi-logarithmic plot (y-axis = log leptin), these curves give straight lines. The values for a, b and c are given in table 1 according to gender and pubertal stage and also for adults. Using these values, the expectation ranges of leptin levels can be easily extended to lower or higher BMI ranges, if required. Example The 50th percentile for boys at Tanner stages 3 and 4 is given by the following curve: leptin = 0.0181·e(0.2067·BMI) . The 5th percentile is given by (0.2067·BMI-1.1919) leptin = 0.0181·e and the 95th percentile is given by leptin = (0.2067·BMI+1.1919) 0.0181·e . In a semi-logarithmic plot, these lines are parallel with an equal distance to the 50th percentile. Calculation of standard deviation scores (SDS; Z -scores) A convenient method to detect any deviation of a measured leptin level from the corresponding reference range is to calculate its standard deviation score by relating the leptin level at the patient’s BMI to the average leptin value of the corresponding sex and age group and expressing its deviation by the x-fold standard deviation. This method may be considered as a normalization to the normal reference cohort. Thus, the leptin values can be adjusted for BMI, gender and pubertal stage/age (i.e., the influence of gender, age and BMI are removed) and may be pooled for further analysis. Accounting for the logarithmic distribution of leptin levels, the leptin SDS can be calculated by the following equation: leptin SDS = (ln(leptin) - ln(a) - b·BMI) / d In this equation, ln represents the natural logarithm (referring to the basis e). The constants a, b and d are given in table 1 according to gender and pubertal stage/age. Human-Leptin RIA-Sensitiv

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Example: 2 A boy at Tanner stage 3, BMI = 25 kg/m , measured leptin concentration = 5 ng/ml. leptin SDS = ln(5) - ln (0.0181) - 0.2067·25) / 0.6850 = (1.6094 - (-4.0118) - 5.1675) / 0.6850 = 0.66 Estimation of optimal dilution of samples Because serum leptin levels vary widely over several orders of magnitude, depending mainly on the body fat mass, adequate dilution is a prerequisite for precise measurement. The best precision of this sensitive leptin RIA kit is given at leptin concentrations about 2 ng/ml. Therefore, samples should be diluted such that the leptin concentration is near this value in order to take maximum advantage of the precision of the kit. The reference ranges provide a useful tool for a good estimation of the expected leptin value according to BMI, gender and age. Example 1: Adult woman, BMI = 35 kg/m2. From the reference range for adult women, the average leptin level at a BMI of 35 kg/m2 is approximately 50 ng/ml. The optimal dilution would be 1:25.

Example 2: 2 Prepubertal boy, BMI = 24 kg/m . From the reference range for boys at Tanner stages 1&2, a leptin value of approximately 10 ng/ml would be expected. The optimal dilution would be 1:5. Example 3: 2 Girl at Tanner stage 5, BMI = 15 kg/m . From the reference range of girls at this pubertal stage, a leptin value of approximately 2 ng/ml would be expected. The sample should not be diluted. Human-Leptin RIA-Sensitiv

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Table 1: Constants a, b, c and d for calculation of leptin reference ranges and leptin SDS based on BMI. Groups of normal healthy individuals were stratified according to gender and pubertal stage/age. TS= Tanner stage, n= number of subjects, a,b,c,and d = constants as defined in the text.

(n)

a

b

c

d

TS 1&2

136

0.0146

0.2706

0.8821

0.5379

TS 3&4

50

0.0181

0.2067

1.1919

0.6850

TS 5

112

0.0316

0.1462

1.0821

0.6558

Adults

380

0.0130

0.2200

1.1053

0.6740

TS 1&2

136

0.0422

0.2499

0.7849

0.4786

TS 3&4

43

0.0543

0.2357

0.5745

0.3379

TS 5

157

0.2550

0.1508

0.7053

0.4301

Adults

587

0.3042

0.1467

0.8548

0.5212

Cohort Males:

Females:

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BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

1

Percentile (µg/L) 5 50 95

99

0.22

0.30

0.66

1.45

1.99

0.28 0.36

0.39 0.50

0.85 1.09

1.86 2.38

2.56 3.29

0.46 0.60

0.64 0.82

1.40 1.79

3.06 3.93

4.22 5.42

0.76 0.98

1.05 1.35

2.30 2.95

5.04 6.47

6.96 8.93

1.25 1.61 2.07

1.73 2.22 2.85

3.79 4.87 6.25

8.31 10.7 13.7

11.5 14.7 18.9

2.65 3.41

3.66 4.70

8.03 10.3

17.6 22.6

24.3 31.2

4.37 5.62

6.03 7.75

13.2 17.0

29.0 37.2

40.0 51.4

7.21 9.26 11.9

9.95 12.8 16.4

21.8 28.0 35.9

47.8 61.4 78.8

65.9 84.7 109.0

15.3 19.6

21.1 27.0

46.1 59.2

101.0 130.0

140.0

15.2 32.3

34.7 44.6

76.1 97.7

41.5 53.2

57.2 73.4

125.0

68.4 87.8 113

94.3 121.0

Figure 2: Reference ranges of human serum levels referring to BMI: GirlsTanner stage 1 & 2 (see text for details)

145

Table 2: Girls:Tanner stages 1 and 2.

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BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

1

Percentile (µg/L) 5 50 95

99

0.08

0.12

0.29

0.69

0.99

0.01 0.14

0.16 0.20

0.38 0.49

0.91 1.19

1.30 1.71

0.19 0.24

0.26 0.35

0.65 0.85

1.56 2.04

2.24 2.93

0.32 0.41 0.55

0.46 0.60 0.79

1.11 1.45 1.90

2.68 3.51 4.60

3.84 5.04 6.60

0.72 0.94

1.03 1.35

2.50 3.27

6.03 7.90

8.66 11.3

1.24 1.62

1.77 2.33

4.29 5.62

10.4 13.6

14.9 19.5

2.12 2.78

3.05 3.99

7.37 9.66

17.8 23.3

25.5 33.5

3.65 7.78 6.27

5.24 6.87 9.0

12.7 16.9 21.7

30.6 40.1 52.5

43.9 57.5 75.4

8.22 10.7

11.8 15.5

28.5 37.4

68.9 90.3

98.8 129.0

14.1 18.5

20.3 26.6

48.9 64.2

118.0

24.3 31.8 41.7

34.8 45.6 59.8

84.1 110.0 144.0

54.6 71.6

78.4 102.0

Figure 3: Reference ranges of human serum levels referring to BMI: Boys Tanner stage 1 & 2 (see text for details).

93.9 134.0 123.0

2.

Human-Leptin RIA-Sensitiv

24

LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

1

Percentile (µg/L) 5 50 95

99

0.32

0.41

0.73

1.29

1.63

0.41 0.52

0.52 0.66

0.92 1.16

1.63 2.07

2.06 2.61

0.65 0.83

0.83 1.05

1.47 1.87

2.61 3.31

3.31 4.19

1.05 1.33

1.33 1.68

2.36 2.99

4.19 5.30

5.30 6.71

1.68 2.13 2.69

2.13 2.69 3.41

3.78 4.79 6.06

6.71 8.5 10.7

8.49 10.8 13.6

3.41 4.32

4.31 5.46

7.67 9.71

13.61 17.2

17.2 21.8

5.46 6.91

6.91 8.75

12.3 15.6

21.8 27.6

27.6 34.9

8.75 11.1 14.0

11.1 14.0 17.7

19.7 24.9 31.6

34.9 44.2 56.0

44.2 56.0 70.9

17.8 22.5

22.5 28.4

39.9 50.5

70.9 89.7

89.7 114.0

28.4 36.0

36.0 45.6

63.9 80.9

114.0 144.0

144.0

45.6 57.7

57.7 73.0

80.2 144.0 102.0

Figure 4: Reference ranges of human serum levels referring to BMI: Girls Tanner stage 3 & 4 (see text for details).

73.0 92.4 130.0 92.4 117.0 117.0 148.0 148.0

Table 4: Girls Tanner stage 3 & 4.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1

Percentile (µg/L) 5 50 95

99

0.03 0.04

0.05 0.07

0.18 0.22

0.58 0.71

0.94 1.16

0.49 0.06

0.08 0.10

0.27 0.33

0.88 1.08

1.43 1.75

0.07 0.09 0.11

0.12 0.15 0.18

0.40 0.49 0.61

1.32 1.63 2.00

2.16 2.65 3.26

0.14 0.17

0.23 0.28

0.75 0.92

2.46 3.03

4.01 4.93

0.21 0.26

0.34 0.42

1.13 1.39

3.72 4.58

6.06 7.46

0.32 0.39 0.48

0.52 0.64 0.78

1.71 2.10 2.58

5.63 6.92 8.51

9.17 11.3 13.9

0.59 0.73

0.96 1.19

3.18 3.91

10.5 12.9

17.0 21.0

0.89 1.10

1.46 1.79

4.80 5.90

15.8 19.4

25.8 31.7

1.35 1.66

2.20 2.71

7.26 8.93

23.9 29.4

39.0 48.0

2.05 2.51 3.09

3.33 4.09 5.04

11.0 13.5 16.6

36.2 44.5 54.7

58.9 72.4 89.1

3.80 4.68

6.20 7.62

20.4 25.1

67.2 82.6

109.0 134.0

5.75 7.07

9.37 11.5

30.9 37.9

101.0 124.0

8.7 10.7

14.2 17.4

46.7 57.4

13.1

21.4

70.5

Figure 5: Reference ranges of human serum levels referring to BMI: Boys Tanner stage 3 & 4 (see text for details ).

Table 5 : Boys Tanner stage 3 & 4.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1

Percentile (µg/L) 5 50 95

99

0.50

0.66

1.34

2.71

3.62

0.58 0.67

0.77 0.89

1.56 1.81

3.15 3.67

4.21 4.89

0.78 0.91 1.05

1.04 1.21 1.41

2.11 2.45 2.85

4.26 4.96 5.76

5.69 6.62 7.70

1.22 1.42

1.64 1.90

3.31 3.85

6.70 7.79

8.95 10.4

1.66 1.93

2.21 2.57

4.48 5.20

9.06 10.5

12.1 14.1

2.24 2.60 3.03

2.99 3.48 4.04

6.05 7.03 8.18

12.3 14.2 16.6

16.4 19.0 22.1

3.52 4.09

4.70 5.46

9.51 11.0

19.3 22.4

25.7 29.9

4.76 5.53

6.35 7.39

12.9 15.0

26.0 30.3

34.8 40.4

6.43 7.48

8.59 9.99

17.39 35.2 20.2 40.9

47.0 54.7

8.70 10.1 11.8

11.6 13.5 15.7

23.5 27.3 31.8

47.6 55.3 64.4

63.5 73.9 85.9

13.7 15.9

18.3 21.2

37.0 43.0

74.9 87.0

99.9 116.0

18.5 21.5

24.7 28.7

50.0 58.1

101.0 118.0

135.0

25.0 29.1

33.4 38.8

67.6 78.6

137.0

33.8 39.4

45.1 52.5

91.4 106.0

Figure 6: Reference ranges of human serum levels referring to BMI: Girls Tanner stage 5 (see text for details).

Table 6 : Girls Tanner stage 5.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1

Percentile (µg/L) 5 50 95

99

0.03

0.05

0.16

0.47

0.73

0.04 0.05

0.06 0.07

0.18 0.21

0.54 0.62

0.84 0.97

0.05 0.06

0.08 0.10

0.24 0.28

0.72 0.84

1.12 1.30

0.07 0.08

0.11 0.13

0.33 0.38

0.97 1.12

1.51 1.74

0.1 0.11 0.13

0.15 0.17 0.2

0.44 0.51 0.59

1.3 1.50 1.74

2.02 2.34 2.7

0.15 0.17

0.23 0.27

0.68 0.79

2.01 2.33

3.13 3.62

0.20 0.23

0.31 0.36

0.91 1.05

2.69 3.12

4.19 4.85

0.27 0.31 0.36

0.41 0.48 0.55

1.22 1.41 1.63

3.61 4.17 4.83

5.62 6.5 7.52

0.41 0.48

0.64 0.74

1.89 2.19

5.59 6.47

8.71 10.1

0.55 0.64

0.86 1.00

2.54 2.94

7.49 8.67

11.7 13.5

0.74 0.86

1.15 1.33

3.4 3.94

10.0 11.6

15.6 18.1

0.99 1.15 1.33

1.54 1.79 2.07

4.55 5.27 6.10

13.4 15.6 18.0

20.9 24.2 28.1

1.54 1.78

2.39 2.77

7.06 8.17

20.8 24.1

32.5 37.6

2.06 2.38

3.21 3.71

9.46 10.9

27.9 32.3

43.5 50.3

F Figure

7: Reference ranges of human serum levels referring to BMI: Boys Tanner stage 5 (see text for details).

Table 7 : Boys Tanner stage 5.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1

Percentile (µg/L) 5 50 95

99

0.46 0.53

0.65 0.75

1.53 1.77

3.59 4.16

5.10 5.90

0.61 0.71 7.82

0.87 1.01 1.17

2.05 2.37 2.75

4.82 5.58 6.46

6.83 7.91 9.17

0.95 1.10

1.35 1.57

3.18 3.68

7.48 8.66

10.61 12.3

1.28 1.48

1.81 2.10

4.27 4.94

10.0 11.6

14.2 16.5

1.71 1.99 2.30

2.43 2.82 3.26

5.72 6.62 7.67

13.4 15.6 18.0

19.1 22.1 25.6

2.66 3.08

3.78 4.38

8.88 10.3

20.9 24.2

29.3 34.3

3.57 4.13

5.07 5.87

11.9 13.8

28.0 32.4

39.7 46.0

4.79 5.54

6.79 7.87

16.0 18.5

37.5 43.5

53.3 61.7

6.42 7.43 8.61

9.11 10.6 12.2

21.4 24.8 28.7

50.4 58.3 67.5

71.5 82.8 95.8

9.97 11.5

14.1 16.4

33.3 38.5

78.2 90.5

111.0 129.0

13.4 15.5

19.0 22.0

44.6 51.6

105.0 121.0

149.0

17.9 20.8

25.4 29.5

59.8 69.3

141.0

24.0 27.8 32.2

34.1 39.5 45.7

80.2 92.9 108.0

Figure 8: Reference ranges of human serum levels referring to BMI: Adult women (see text for details).

Table 8: Adult women.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

BMI (kg/m²) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1

Percentile (µg/L) 5 50 95

99

0.03 0.04

0.05 0.06

0.15 0.18

0.44 0.55

0.69 0.87

0.05 0.06

0.08 0.09

0.23 0.28

0.69 0.85

1.08 1.34

0.07 0.09

0.12 0.15

0.35 0.44

1.06 1.33

1.67 2.09

0.12 0.14 0.18

0.18 0.23 0.28

0.55 0.68 0.85

1.65 2.06 2.57

2.60 3.24 4.04

0.22 0.23

0.35 0.44

1.06 1.32

3.20 3.98

5.03 6.27

0.35 0.43

0.54 0.78

1.64 2.05

4.97 6.19

7.81 9.73

0.54 0.67

0.85 1.05

2.55 3.18

7.71 9.61

12.1 15.1

0.83 1.04 1.30

1.31 1.64 2.04

3.96 4.94 6.15

12.0 14.9 18.6

18.8 23.5 29.2

1.61 2.01

2.54 3.16

7.67 9.56

23.2 28.9

36.4 45.4

2.51 3.12

3.94 4.91

11.9 14.8

36.0 44.9

56.6 70.5

3.89 4.85 6.04

6.12 7.63 9.51

18.5 23.0 28.7

55.8 69.6 86.7

87.8 109.0 136.0

7.53 9.38

11.8 14.8

35.8 44.6

108.0 135.0

11.7 14.6

18.4 22.9

55.5 69.2

18.2

28.6

86.2

Figure 9: Reference ranges of human serum levels referring to BMI: Adult men (see text for details).

Table 9: Adult men.

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REFERENCES 1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. 1994 Positional cloning of the mouse obese gene and its human homologue. Nature. 372:425-432. 2. Halaas JL, Gajiwala KS, Maffei M, et al. 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 269:543-546. 3. MacDougald OA, Hwang CS, Fan H, Lane MD. 1995 Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 92:9034-9037. 4. Rentsch J, Chiesi M. 1996 Regulation of ob gene mRNA levels in cultured adipocytes. FEBS Lett. 379:55-59. 5. Wabitsch M, Jensen PB, Blum WF, et al. 1996 Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes. 45:1435-1438. 6. Stephens TW, Basinski M, Bristow PK, et al. 1995 The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 377:530-532. 7. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. 1995 Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 269:546-549. 8. Pelleymounter MA, Cullen MJ, Baker MB, et al. 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 269:540-543. 9. Levin N, Nelson C, Gurney A, Vandlen R, de-Sauvage F. 1996 Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc Natl Acad Sci U S A. 93:1726-1730. 10. Tartaglia LA, Dembski M, Weng X, et al. 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell. 83:1263-1271. 11. Chen H, Charlat O, Tartaglia LA, et al. 1996 Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 84:491-495. 12. Lee GH, Proenca R, Montez JM, et al. 1996 Abnormal splicing of the leptin receptor in diabetic mice. Nature. 379:632-635. 13. Lynn RB, Cao GY, Considine RV, Hyde TM, Caro JF. 1996 Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun. 219:884-889. Human-Leptin RIA-Sensitiv

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14. Considine RV, Considine EL, Williams CJ, Hyde TM, Caro JF. 1996 The hypothalamic leptin receptor in humans: identification of incidental sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations. Diabetes. 45:992-994. 15. Sinha MK, Opentanova I, Ohannesian JP, et al. 1996 Evidence of free and bound leptin in human circulation. J Clin Invest. 98:1277-1282. 16. Ahima RS, Prabakaran D, Mantzoros C, et al. 1996 Role of leptin in the neuroendocrine response to fasting. Nature. 382:250-252. 17. Chehab FF, Lim ME, Lu R. 1996 Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet. 12:318-320. 18. Schwartz MW, Baskin DG, Bukowski TR, et al. 1996 Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes. 45:531-535. 19.Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. 1996 Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 98:1101-1106. 20. Campfield LA, Smith FJ, Burn P. 1996 The OB protein (leptin) pathway - a link between adipose tissue mass and central neural networks. Horm Metab Res. 28:619-632. 21. Rohner-Jeanrenaud F, Cusin I, Sainsbury A, Zakrzewska KE, Jeanrenaud B. 1996 The loop system between neuropeptide Y and leptin in normal and obese rodents. Horm Metab Res. 28:642-648. 22. Chan YY, Steiner RA, Clifton DK. 1996 Regulation of hypothalamic neuropeptide-Y neurons by growth hormone in the rat. Endocrinol. 137:1319-1325. 23. Pierroz DD, Catzeflis C, Aebi AC, Rivier JE, Aubert ML. 1996 Chronic administration of neuropeptide Y into the lateral ventricle inhibits both the pituitary-testicular axis and growth hormone and insulin-like growth factor I secretion in intact adult male rats. Endocrinol. 137:3-12. 24. Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. 1995 Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med. 1:1311-1314.

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25. 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-reduced subjects. Nat Med. 1:1155-1161. 26. 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. 27. Kolaczynski JW, Considine RV, Ohannesian J, et al. 1996 Responses of leptin to short-term fasting and refeeding in humans: A link with keto-genesis but not ketones themselves. Diabetes. 45:1511-1515. 28. Harris RB, Ramsay TG, Smith SR, Bruch RC. 1996 Early and late stimulation of ob mRNA expression in meal-fed and overfed rats. J Clin Invest. 97:2020-2026. 29. Kolaczynski JW, Ohannesian J, Considine RV, Marco C, Caro JF. 1996 Response of leptin to short-term and prolonged overfeeding in humans. J Clin Endocrinol Metab. 91:4162-4165. 30. Saladin R, De-Vos P, Guerre-Millo M, et al. 1995 Transient increase in obese gene expression after food intake or insulin administration. Nature. 377:527-529. 31. Cusin I, Sainsbury A, Doyle P, Rohner-Jeanrenaud F, Jeanrenaud B. 1995 The ob gene and insulin. A relationship leading to clues to the under-standing of obesity. Diabetes. 44:1467-1470. 32. Kolaczynski JW, Nyce MR, Considine RV, et al. 1996 Acute and chronic effects of insulin on leptin production in humans: studies in vivo and in vitro. Diabetes. 45:699-701. 33. Malström R, Taskinen M-R, Karonen S-L, Yki-Järvinen H. 1996 Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM. Diabetologia. 39:993-996. 34. De-Vos P, Saladin R, Auwerx J, Staels B. 1995 Induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake. J Biol Chem. 270:15958-15961. 35. Slieker LJ, Sloop KW, Surface PL, et al. 1996 Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. J Biol Chem. 271:5301-5304.

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36. Miell JP, Englaro P, Blum WF. 1996 Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects. Horm Metab Res. 28:704-707. 37. Sinha MK, Ohannesian JP, Heiman ML, et al. 1996 Nocturnal rise of leptin in lean, obese, and non-insulin- dependent diabetes mellitus subjects. J Clin Invest. 97:1344-1347. 38. Havel PJ, Kasim-Karakas S, Dubuc GR, Mueller W, Phinney SD. 1996 Gender difference in plasma leptin concentrations. Nature Med. 2:949-950. 39. Rosenbaum M, Nicolson M, Hirsch J, et al. 1996 Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab. 81:3424-3427. 40. Hassink SG, Sheslow DV, de Lancey E, Opentanova I, Considine RV, Caro JF 1996 Serum leptin in children with obesity: relationship to gender and development. Pediatrics. 98:201-203. 41. Scholz GH, Englaro P, Thiele I, et al. 1996 Dissociation of serum leptin concentration and body fat content during long term dietary intervention in obese individuals. Horm Metab Res. 28:718-723. 42. 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. 43. Robinson CJ, Gaines-Das R,Woollacott D, et al. 2001 The first international standard for human leptin and the first international standard for mouse leptin: comparison of candidate preparations by in vitro bioassays and immunoassays. J Molecular Endocrinol. 27: 69-76. 44. Address NIBSC: Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain. 45. Blum WF, Juul A; Reference ranges of serum leptin,s.318-326. In: Leptin -the voice of adipose tissue, Blum WF et al, eds., Johann Ambrosius Verlag, Heidelberg, 1997.

46. Holtkamp K, Blum WF, et al., 2006, Elevated Physical Activity and Low Leptin Levels Co-Occur in Patients with Anorexia Nervosa, The Journal of Clinical Endocrinology Metabolism 88(11) 5169-5174 47. Guebra-Egziabher, Bernhard J., et al. 2005, Adiponectin in chronic kidney disease is related more to metabolic distrubances than to decline in renal function, Nephrol Dial Transplant 20: 129-134 Human-Leptin RIA-Sensitiv

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Mediagnost Leptin, Immunoassays: LEP-R42 LEP-R44 E07 E077 E06

Human-Leptin-Standard-RIA Kit radioimmunoassay for 125 tubes Human- Leptin- RIA- CT Kit radioimmunoassay including 125 coated tubes Human- Leptin- Standard- EIA Kit enzyme immunoassay, 96 tests Human- Leptin- Sensitive- EIA Kit enzyme immunoassay, 96 tests M ouse/Rat-Leptin EIA Kit enzyme immunoassay, 96 tests

Additional Endocrinolgical Immunoassays for Energy Regulation: Ghrelin, Immunoassay R90 Human-GHRELIN RIA Kit radioimmunoassay for 100 tubes Resistin, Immunoassay E50 Human-RESISTIN EIA Kit enzyme immunoassay, 96 tests Adiponectin, Immunoassay E09 Human- ADIPONECTIN EIA Kit enzyme immunoassay, 96 tests

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

SUMMARY-Mediagnost hLEPTIN RIA SENSITIVE LEP-R40 Reagent preparation: 1. Antibody (B) Tracer (C) NSB (D) Controls (M)+(N) 2. Antibody (O)

Reconstitution Dilution in 13 ml Assay Buffer (A) in 13 ml Assay Buffer (A) in 1 ml Assay Buffer (A) in 0.75 ml Assay Buffer (A) each 1:3 with Assay Buffer (A) in 1 ml Assay Buffer (A) Mix directly before use with 65 ml Reagent (P). 100 µl Serum- or Plasma Samples are diluted with 200 µl Assay Buffer A (Factor 1:3). Proposed Assay Procedure for Double Determination Nr.

1,2 3,4 5,6 7-20 21,22 23,24 25,26 27,28 etc.

Contents of Tubes

Addition of Reagents [µl] D: A F – L: NSB (AssayStandards Buffer) M - N:Control Samples 100 100 -

TC NSB B0 Standards High Control Low Control Sample 1 Sample 2

F-L:100 M:100 N:100 100 100

B

(1.Antibody)

100 -

100 100 100 100 100 100

C

(Tracer)

100 100 100 100 100 100 100 100

Nr.:1,2 remove until counting the activity. Mix other tubes with a Vortex-Mixer. Incubation at RT, over night, at least 15 h Add 500 µl P (after addition of reagent O) in all Tubes The reagent-mix should be cold (2-8°C). Mix tubes with a Vortex-Mixer. Incubation at 2-4°C, 1 h Add 1 ml ice cold A.dest. carefully in all tubes. Centrifuge at 3000 x g, 20 min at 2-4°C Aspirate the supernatant without destroying the precipitate. (As a precaution e.g. approx. leave 2 mm as a remaining supernatant above the precipitate). Count the activity of all tubes with a Gamma counter.

Human-Leptin RIA-Sensitiv

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LEP-R40 e 04.07.05

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