Method for predicting the risk of obesity in a subject

Abstract

A method for determining the fat processing activity and/or predicting the risk of obesity in a subject involves determining the level of pro-neurotensin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from the subject, and correlating the level of pro-neurotensin or fragments thereof with fat processing activity and/or the risk of incidence of obesity in the subject. An elevated level is indicative of enhanced fat processing activity and/or predictive for an enhanced risk of getting obesity.

Claims

1. A method for determining fat processing activity and/or for predicting risk of obesity in a subject comprising: determining by an immunoassay a level of pro-neurotensin 1-117 (SEQ ID NO: 5) or a level of peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) in a sample of bodily fluid obtained from said subject; and correlating said level of pro-neurotensin 1-117 (SEQ ID NO: 5) or said level of peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) with fat processing activity and/or a risk of incidence of obesity in said subject, wherein a level above a threshold level is indicative of an enhanced fat processing activity and/or predictive for an enhanced risk of obesity, wherein said subject is not obese when said sample of bodily fluid is taken from said subject, wherein the subject does not have metabolic syndrome, and wherein said immunoassay comprises (i) contacting said sample with one or more antibodies, at least one of which is labelled, that specifically bind to an epitope within pro-neurotensin 1-117 (SEQ ID NO: 5), to form a complex between the antibody and pro-neurotensin 1-117 (SEQ ID NO: 5) or peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5), and (ii) quantitating the level of the thus-formed complex.

2. A method according to claim 1, wherein said subject is a non-prediabetic subject.

3. A method according to claim 1, wherein a fasting level of pro-neurotensin 1-117 (SEQ ID NO: 5) or of peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) is determined.

4. A method according to claim 1, wherein the level of pro-neurotensin 1-117 (SEQ ID NO: 5) is determined.

5. A method according to claim 1, wherein the subject is non-diabetic with fasting whole blood glucose of less than 6.1 mmol/l but more than 5.4 mmol/l.

6. A method according to claim 1, wherein the subject does not have cancer.

7. A method according to claim 1, wherein the subject has no history of diagnosis of an acute cardiovascular event when said sample of bodily fluid is taken from said subject wherein said cardiovascular event is selected from myocardial infarction, stroke, and acute heart failure.

8. A method according to claim 1, wherein said subject is a subject with fasting whole blood glucose of less than 5.4 mmol/l.

9. A method according to claim 1, wherein the risk of obesity is independent of a risk for contracting diabetes mellitus and/or metabolic syndrome.

10. A method according to claim 1, wherein additionally at least one clinical parameter is determined in the subject selected from age, gender, systolic blood pressure, diastolic blood pressure, antihypertensive treatment (AHT), waist circumference, waist-hip-ratio, current smoker, diabetes heredity and previous cardiovascular disease (CVD).

11. The method according to claim 1, wherein the sample is selected from a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a saliva sample and a urine sample or an extract of any of the aforementioned samples.

12. A method according to claim 1, wherein said method is performed more than once in order to monitor the risk of incidence of obesity.

13. A method according to claim 12, wherein said monitoring is performed in order to evaluate a response of said subject to preventive and/or therapeutic measures taken.

14. A method according to claim 1, wherein said method is performed in a plurality of subjects in order to stratify said subjects into risk groups.

15. A method according to claim 1, wherein said threshold level is at least 60 pmol/L pro-neurotensin (pro-NT).

16. A method according to claim 1, wherein said threshold level is at least 90 pmol/L pro-neurotensin (pro-NT).

17. A method according to claim 1, wherein said threshold level is at least 123 pmol/L pro-neurotensin (pro-NT).

18. A method according to claim 1, wherein said threshold level is at least 180 pmol/L pro-neurotensin (pro-NT).

19. A method according to claim 1, wherein peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) are selected from peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 7.

20. A method for determining fat processing activity and/or for predicting risk of obesity in a subject comprising: determining by an immunoassay a level of pro-neurotensin 1-117 (SEQ ID NO: 5) or a level of peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) in a non-fasting or fasting sample of bodily fluid obtained from said subject, administering fat to said subject, determining by an immunoassay a level of pro-neurotensin 1-117 (SEQ ID NO: 5) or a level of peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) in a sample of bodily fluid obtained from said subject after fat-uptake, calculating a difference between said levels after and before fat-uptake, and correlating said difference between said levels after and before fat-uptake of pro-neurotensin 1-117 (SEQ ID NO: 5) or of said peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) with fat processing activity and/or a risk of incidence of obesity in said subject, wherein a higher difference is more indicative of an enhanced fat processing activity and/or more predictive of an enhanced risk of obesity than a lower difference, wherein the subject is not obese when the samples of bodily fluid are taken from said subject, and wherein said immunoassay comprises (i) contacting said sample with one or more antibodies, at least one of which is labelled, that specifically bind to an epitope within pro-neurotensin 1-117 (SEQ ID NO: 5), to form a complex between the antibody and pro-neurotensin 1-117 (SEQ ID NO: 5) or peptides that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5), and (ii) quantitating the level of the thus-formed complex.

21. The method according to claim 20, wherein the level of pro-neurotensin 1-117 (SEQ ID NO: 5) or a peptide that comprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) in the bodily fluid of said subject after fat-uptake is above at least 60 pmol/L pro-neurotensin (pro-NT).

22. The method according to claim 20, wherein the sample of bodily fluid obtained from said subject prior to administering fat to said subject is a non-fasting sample of said bodily fluid.

23. The method according to claim 20, wherein the sample of bodily fluid obtained from said subject prior to administering fat to said subject is a fasting sample of said bodily fluid.

Description

EXAMPLES

Example 1

(1) Development of Antibodies

(2) Peptides/Conjugates for Immunization:

(3) Peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal Cysteine residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfo-SMCC (Perbio-Science, Bonn, Germany) The coupling procedure was performed according to the manual of Perbio.

(4) TABLE-US-00002 Labelled antibody (LA) peptide (P-NT 1-19): H-CSDSEEEMKALEADFLTNMH-NH2 Solid phase antibody (SPA) peptide (P-NT 44-62): H-CNLNSPAEETGEVHEEELVA-NH2

(5) The antibodies were generated according to the following method:

(6) A BALB/c mouse were immunized with 100 μg Peptide-BSA-Conjugate at day 0 and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg at day 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 μg of the conjugate dissolved in 100 μl saline, given as one intraperitoneal and one intravenous injection.

(7) Splenocytes from the immunized mouse and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement). After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

(8) The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined. (Lane, R. D. “A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secreting hybridomas”, J. Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al. “Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies”, Horm. Metab. Res. 28: 11-15, (1996)).

(9) Monoclonal Antibody Production

(10) Antibodies were produced via standard antibody production methods (Marx et al, Monoclonal Antibody Production, ATLA 25, 121, 1997) and purified via Protein A-chromatography. The antibody purities were >95% based on SDS gel electrophoresis analysis.

Example 2

(11) Immunoassay for the Quantification of Human Pro-Neurotensin

(12) The technology used was a sandwich coated tube luminescence immunoassay, based on Akridinium ester labelling.

(13) Labelled compound (tracer): 100 μg (100 μl) LA (1 mg/ml in PBS, pH 7.4, was mixed with 10 μl Akridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled LA was purified by Gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified LA was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l Bovine Serum Albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

(14) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with SPA (1.5 μg SPA/0.3 ml 100 mmol/l NaCl, 50 mmol/l TRIS/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.

(15) Calibration:

(16) The assay was calibrated, using dilutions of pro-NT-containing human serum. A pool of human sera with high pro-NT-immunoreactivity (InVent Diagostika, Hennigsdorf, Germany) was diluted with horse serum (Biochrom AG, Deutschland) (assay standards).

(17) The standards were calibrated by use of the human pro-NT-calibrator (ICI-Diagnostics, Berlin, Germany). Alternatively, the assay may be calibrated by synthetic or recombinant pro-NT 1-117 or fragments thereof (see also Ernst et al., 2006).

(18) Pro-NT Immunoassay:

(19) 50 μl of sample (or calibrator) was pipetted into SPA coated tubes, after adding labelled LA (200 μl), the tubes were incubated for 16-22 h at 18-25° C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

(20) Tube-bound LA was measured by using the AutoLumat LB 953.

(21) FIG. 1 shows a typical P-NT dose/signal curve.

Example 3

(22) Population Study

(23) The Malmö Diet and Cancer (MDC) study is a population-based, prospective epidemiologic cohort of 28,449 men (born 1923-1945) and women (born 1923-1950) from the city of Malmö in southern Sweden who underwent baseline examinations between 1991 and 1996 (Minisymposium: The Malmo Diet and Cancer Study. Design, biological bank and biomarker programme J Intern Med 233, 39-79 (1993). From this cohort, 6,103 persons were randomly selected to participate in the MDC Cardiovascular Cohort (MDC-CC), which was designed to investigate the epidemiology of carotid artery disease, between 1991 and 1994 (Persson et al. 2008. Atherosclerosis 200: 191-198). Fasted plasma samples at the baseline examination were available for analysis of pro-neurotensin (pro-NT) and successfully measured in a total of 4,632 participants in the MDC-CC. Of those, complete data was available for BMI in 4,626, for waist circumference on 4,625 and for estimated degree of insulin resistance using the homeostasis model assessment of insulin resistance (HOMA-IR) (fasting blood glucose concentration×fasting plasma insulin concentration/22.5) (Matthews et al. 1985. Diabetologia 28: 412-419) in 4,468 participants. BMI was defined as body weight in kilograms divided by the square of height in meters and obesity as a BMI ≥30 kg/m.sup.2. Abdominal obesity was defined as a waist circumference of ≥94 cm in males and ≥80 cm in females, according to the International Diabetes Federation definition (Alberti et al. 2006. Diabet Med 23: 469-489). Insulin resistance was regarded present in subjects belonging to the top 25% of HOMA-IR values in the MDC-CC. ‘New-Onset Obesity’ is defined as obesity development among non-obese MDC-CC participants who were re-examined and diagnosed with obesity after an average follow-up time of 16.5±1.5 years.

(24) Pro-NT was measured in stored fasting plasma specimens that were frozen to −80° C. immediately at the MDC-CC baseline exam using a recent chemiluminometric sandwich immunoassay to detect a pro-NT precursor fragment (pro-NT 1-117) as described previously (Ernst et al. 2006. Peptides 27: 1787-1793). Analyses of blood glucose and plasma insulin were carried out at the time of baseline examination at the Department of Clinical Chemistry, Malmö University Hospital, which is attached to a national standardization and quality control system (Enhorning et al. 2010. Circulation 121: 2102-2108). Of the 4,626 subjects with baseline data on BMI and pro-NT, 2,900 subjects were re-examined with a new measurement of BMI after a mean follow-up of 16.5±1.5 years. In analyses of incident obesity, we excluded subjects who were obese already at the baseline examination, leaving a total of 2,606 non-obese subjects for analysis of pro-NT in relation to incident obesity. All participants gave written informed consent and the study was approved by the Ethical Committee at Lund University, Lund, Sweden.

(25) Statistical Analyses

(26) All subjects at the MDC-CC baseline examination were divided into ascending quartiles according to their value of fasting pro-NT. In cross sectional analyses we related baseline quartile of pro-NT to the dichotomous outcome of obesity, abdominal obesity and insulin resistance using age and sex adjusted logistic regression models. In the analyses of incident obesity, we related baseline quartile of pro-NT to the dichotomous outcome of incident obesity using logistic regression adjusted for baseline age, sex and BMI. Data are presented as odds ratios (95% confidence intervals) and subjects belonging to the lowest quartile of pro-NT were defined as the referent group (odds ratio=1). ‘P for trend’ denotes the P-value for linear trend over quartiles 1-4.

(27) Study Results

(28) We measured pro-NT levels in fasted plasma of 4,632 middle-aged subjects of the population-based Malmö Diet and Cancer Study Cardiovascular Cohort (Table 1). The age- and sex-adjusted likelihood of being obese, abdominally obese and insulin resistant significantly increased across quartiles of pro-NT (Table 2). Among non-obese subjects, the risk of developing obesity during an average follow-up time of 16.5±1.5 years increased gradually with pro-NT quartiles, independently of baseline body mass index, age and gender. Pro-NT median concentrations were 60.1 pmol/L (range 3.3-75.9 pmol/L) in quartile 1, 89.3 pmol/L (range 75.9-105 pmol/L) in quartile 2, 123 pmol/L (range 105-149 pmol/L) in quartile 3 and 190 pmol/L (range 149-1155 pmol/L) in quartile 4. Non-obese subjects in the top quartile of baseline pro-NT levels had a more than doubling (OR 2.06 (95% confidence interval of 1.38-3.06) of the risk of developing obesity as compared to subjects in the lowest quartile (Table 2).

(29) Using the same variables in the equation, we investigated different subgroups for prediction of new-onset obesity (Table 3), subjects with diabetes mellitus (DM) and impaired fasting glucose (IFG), high blood pressure/anti-hypertensive therapy (AHT), metabolic syndrome (MeSy), eGFR <60 (ml/min/1.73 m.sup.2), heredity of cancer, prevalent cancer, smokers, respectively, at baseline were excluded. Non-obese subjects in the highest pro-NT level quartile either none of the above mentioned conditions again showed a more than doubling of the risk of developing obesity compared to subjects in the lowest pro-NT quartile (Table 3). Non-obese subjects with none of these conditions (super healthy subjects) in the highest pro-NT level quartile even showed a more than 3 fold increased risk of developing obesity compared to subjects in the lowest pro-NT quartile.

Example 4

(30) PNT Concentrations Before and after an Oral Fat Up-Take Test (“Cream-Test”)

(31) A total of 54 patients, 19 healthy control subjects and 35 patients with a diagnosis of heart failure, were selected. The subjects fasted for at least 10 hours and ingested a standardized fat-enriched drinking solution (cream containing 30% of fat). Blood was taken at baseline and 1, 2 and 3 hours after cream up-take. Pro-Neurotensin was measured with the immunoassay as described above. Baseline pro-NT was defined as 100% and levels at the three different time points were related thereto. Pro-NT significantly increased 1 hour after cream intake in both, healthy controls and patients with HF and decreased after 2 and 3 hours but without reaching the baseline level (FIG. 2). Moreover, the relative concentration of pro-NT was significantly different between healthy controls and patients with HF at all three time points (p<0.05).

(32) TABLE-US-00003 TABLE 1 Clinical characteristics of the Malmö Diet and Cancer Cardiovascular Cohort (MDC-CC) Characteristic Value N Age (years) 57.7 ± 6.0  4,626 Female sex, n (%) 2661 (57.5) 4,626 Body Mass Index (kg/m.sup.2) 25.8 ± 3.9  4,626 Waist circumference (cm) 84.0 ± 12.9 4,625 Fasting blood glucose (mM) 5.2 ± 1.4 4,468 Fasting insulin concentration (mU/L) 7.0 (4.0-9.0) 4,468 HOMA-IR 1.5 (0.9-2.2) 4,468

(33) Data are given as mean±standard deviation for normally distributed variables, and as median and interquartile range for fasting insulin concentration. Categorical data are presented as numbers (percentages). “N” denotes the number with complete data; thus, included in analyses. “HOMA-IR” stands for Homeostasis Model Assessment of Insulin Resistance (fasting plasma insulin concentration×fasting blood glucose concentration/22.5)

(34) TABLE-US-00004 TABLE 2 Fasting plasma concentration of pro-neurotensin (pro-NT) in relation to prevalence of obesity and insulin resistance and in relation to incidence of new-onset obesity during long term follow-up in the Malmö Diet and Cancer Cardiovascular Cohort Odds ratio (95% confidence interval) N/N Pro-NT Pro-NT Pro-NT Pro-NT P for cases Quartile 1 Quartile 2 Quartile 3 Quartile 4 trend Prevalent 4626/604  1.0 1.00 1.13 1.34 0.01 obesity (ref) (0.78-1.29) (0.88-1.45) (1.05-1.70) Prevalent 4625/1769 1.0 1.07 1.23 1.30 0.001 abdominal (ref) (0.90-1.27) (1.04-1.46) (1.09-1.54) obesity Prevalent 4468/1140 1.0 1.30 1.43 1.70 <0.0001 insulin (ref) (1.06-1.59) (1.17-1.74) (1.39-2.06) resistance New-onset 2606/335  1.0 1.44 1.83 2.06 <0.01 obesity (ref) (0.95-2.10) (1.21-2.65) (1.38-3.06)

(35) ‘N/N cases’ denotes ‘total number of subjects in the analysis/number of cases with the disease in question.’ ‘Pro-NT’ denotes fasting plasma concentration of pro-neurotensin at the MDC-CC baseline examination. ‘Pro-NT Quartiles 1-4’ defines the MDC-CC population quartiles (lowest to highest) of pro-NT. Data are presented as odds ratios (95% confidence intervals) and subjects belonging to the lowest quartile of pro-NT were defined as the referent group (odds ratio=1). ‘P for trend’ denotes the P-value for linear trend over quartiles 1-4. Prevalent means that the subjects already had the ‘disease in question’ at baseline, whereas the subjects with prevalent obesity where excluded in the analysis for risk-prediction of new onset obesity.

(36) TABLE-US-00005 TABLE 3 Fasting plasma concentration of pro-neurotensin (pro-NT) in relation to incidence of new-onset obesity in different subgroups of patients during long term follow-up in the Malmö Diet and Cancer Cardiovascular Cohort Odds ratio (95% confidence interval) N/N Pro-NT Pro-NT Pro-NT Pro-NT P for cases Quartile 1 Quartile 2 Quartile 3 Quartile 4 trend all 2606/335 1.0 1.44 1.83 2.06 <0.01 (ref) (0.95-2.10) (1.21-2.65) (1.38-3.06) Male 1080/137 1.0 1.43 1.36 2.36 0.044 (ref) (0.79-2.59) (0.75-2.48) (1.29-4.30) Female 1526/198 1.0 1.45 2.23 1.88 0.019 (ref) (0.85-2.46) (1.32-3.75) (1.09-3.21) No hereditary 1456/197 1.0 1.65 1.68 2.37 0.014 cancer (ref) (0.99-2.77) (0.99-2.85) (1.41-3.97) No prevalent 2362/301 1.0 1.36 1.96 2.1 0.001 cancer (ref) (0.89-2.06) 1.29-2.95) (1.38-3.19) BP <140 1631/197 1.0 1.29 1.88 2.03 0.023 (ref) (0.77-2.18) (1.13-3.15) (1.21-3.39) No AHT 2269/273 1.0 1.63 2.03 2.12 0.004 (ref) (1.05-2.53) (1.31-3.13) (1.35-3.35) BP <140/no 1526/175 1.0 1.44 1.99 2.16 0.026 AHT (ref) (0.83-2.49) (1.15-3.42) (1.24-3.73) Non-smoker 2001/241 1.0 1.47 1.75 2.26 0.006 (ref) (0.93-2.32) (1.12-2.74) (1.42-3.61) No prevalent 2544/326 1.0 1.51 1.95 2.17 0.001 cardiac disease (ref) (1.00-2.26) (1.30-2.90) (1.44-3.26) eGFR >60 2319/320 1.0 1.36 1.77 2.22 0.001 (ref) (0.90-2.04) (1.18-2.64) (1.47-3.35) No IFG or DM 2152/254 1.0 1.44 2.03 2.46 <0.001 (ref) (0.91-2.28) 1.30-3.19) (1.55-3.90) No MeSy 2426/302 1.0 1.44 1.92 2.03 0.003 (ref) (0.96-2.18) (1.28-2.89) (1.34-3.07) Healthy all 1155/123 1.0 1.84 2.68 3.17 0.005 (ref) (0.95-3.57) (1.41-5.09) (1.57-6.37) Healthy Female 681/74 1.0 1.35 2.96 3.46 0.015 (ref) (0.56-3.25) (1.28-6.86) (1.37-8.73)

(37) ‘N/N cases’ denotes ‘total number of subjects in the analysis/number of cases with the disease in question.’ ‘Pro-NT’ denotes fasting plasma concentration of pro-neurotensin at the MDC-CC baseline examination. ‘Pro-NT Quartiles 1-4’ defines the MDC-CC population quartiles (lowest to highest) of pro-NT. Data are presented as odds ratios (95% confidence intervals) and subjects belonging to the lowest quartile of pro-NT were defined as the referent group (odds ratio=1). ‘P for trend’ denotes the P-value for linear trend over quartiles 1-4.

(38) Heredity of cancer means no known cancer in family history at baseline, no prevalent cancer means no diagnosis of cancer at baseline, no prevalent cardiac disease means no myocardial infarction, ischemic heart disease, stroke, heart failure (acute or chronic heart failure), atrial fibrillation and atrial flutter at baseline, BP=blood pressure, AHT=anti-hypertensive therapy, eGFR=estimated glomerular filtration rate, IFG=impaired fasting glucose, DM=diabetes mellitus, MeSy=metabolic syndrome.

(39) TABLE-US-00006 TABLE 4 Pro-Neurotensin concentration at baseline (fasting) and 1, 2 and 3 hours after cream intake in healthy control subjects and subjects with heart failure (HF) pro-NT [in %] baseline 1 h 2 h 3 h control 100 173.5 147.0 133.0 HF 100 227.5 201.8 179.3

(40) PNT values are given in % related to the baseline value, which was defined for both groups as 100%, respectively.

BRIEF DESCRIPTION OF THE FIGURES

(41) FIG. 1 shows a typical P-NT dose/signal curve

(42) FIG. 2 shows PNT level before and after cream up-take in patients with heart failure and a control group