A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood
Summary
The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.
Abstract
Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p <0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p = 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p = 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.
Introduction
In light of the worldwide increasing prevalence of obesity1,2, research in the field of food intake regulation is gaining importance. While so far only one peptide is known that is peripherally produced and centrally acting to stimulate food intake, namely ghrelin3, within the past decades, a broad range of peptides has been identified that reduce food intake, e.g. leptin, peptide YY (PYY) and also glucagon-like peptide-1 (GLP-1) and insulin4, Therefore, in studies investigating the regulatory mechanisms of hunger and satiety peptide levels are often assessed and at the same time, it is assumed that the peptide studied is stable and recovered at high yields during plasma formation. However, very often this is not the case due to rapid endogenous breakdown as shown before for e.g. ghrelin which is degraded from acyl to desacyl ghrelin5. Therefore, we recently described the RAPID method for blood processing in rats employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution6. This method improved the recovery for 11 of 12 peptides tested and allowed for the determination of the correct circulating molecular form compared to standard blood processing (EDTA blood on ice)6. This method has been used in several subsequent studies7-12 for the detection of circulating ghrelin as well as corticotropin-releasing factor13. Therefore, the method has proven useful for peptide research in rodents. However, since rodent studies are not always translatable to another species, the method should be established for the use in human blood as well.
The aim of the present study was to test the RAPID method for blood processing in humans compared to standard blood processing, EDTA blood on ice, which is widely recommended14 and frequently used in the clinical as well as research setting. We tested the recovery of a selection of 125I-labeled peptides involved in the regulation of food intake including established peptides as well as new candidates recently suggested to play a role in feeding regulation (effects on food intake are shown in Table 1) following processing with both methods. Hormones were also chosen to represent peptides of different length and charge (Table 2). Moreover, for ghrelin we investigated the molecular form(s) following the standard and RAPID method. Lastly, we assessed endogenous ghrelin (acyl and desacyl ghrelin) as well as kisspeptin levels, a peptide also recently suggested to play a role in the regulation of food intake15,16 following RAPID or standard processing. In addition, we also investigated these peptide levels in a population of subjects with a wide range of body mass index (ranging from 10.2-67.6 kg/m2) to study possible differences related to chronically altered body weight.
Diagnosis, Assessment, and Plan:
Study participants
All study participants were newly hospitalized patients (inclusion was within two days of admission to the hospital) of the Division of Psychosomatic Medicine at Charité-Universitätsmedizin Berlin and gave written informed consent. To avoid any impact of gender only female patients were included. A total of 42 subjects participated in this study and were divided into three groups: normal weight (BMI 18.5-25 kg/m2, n = 12), anorexia nervosa (BMI <17.5 kg/m2, n = 15) and obesity (BMI >30 kg/m2, n = 15). Anorexic and obese patients were diagnosed according to the International Classification of Diseases-10 and hospitalized for weight gain (anorexia nervosa) or weight reduction (obesity), respectively. All normal weight patients were hospitalized exclusively due to somatoform symptoms without relevant somatic disorders. Patients with gastrointestinal somatoform symptoms or a history of gastrointestinal surgery were excluded. Exclusion criteria also encompassed an age <18 years, current pregnancy and untreated psychotic diseases. Blood collection was performed on day 2 or 3 after hospital admission before receiving dietary treatment in order to increase or reduce body weight, respectively. Anthropometric parameters were assessed on the same day.
Protocol
Representative Results
Discussion
We reported before that the RAPID method for blood processing improved the recovery for 11/12 peptides compared to standard blood processing in rats6. In the present study we have shown that this method is also suited for the use in humans. Following RAPID processing, the recovery for 9 of 9 125I-labeled peptides tested was improved compared to standard blood processing (EDTA blood on ice). The observed improvement ranged from 19-39% which is likely to be relevant, especially under conditions when o…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by German Research Foundation STE 1765/3-1 (A.S.) and German Ministry for Education and Research 03IPT614A (C.G.). We thank Reinhard Lommel and Petra Buße for their excellent technical support as well as Karin Johansson and Christina Hentzschel for help with organization and execution of anthropometric measurements
Materials
| diprotin A | Peptides International, Louisville, KY, USA | IDP-4132 | |
| E-64-d | Peptides International, Louisville, KY, USA | IED-4321-v | |
| antipain | Peptides International, Louisville, KY, USA | IAP-4062 | |
| leupeptin | Peptides International, Louisville, KY, USA | ILP-4041 | |
| chymostatin | Peptides International, Louisville, KY, USA | ICY-4063 | |
| Sep-Pak C18 cartridges | Waters Corporation, Milford, MA, USA | WAT051910 | 360 mg, 55-105 µm |
| acyl-ghrelin | Millipore, Billerica, MA, USA | 9088-HK | Radioactive |
| GLP-1 | Millipore, Billerica, MA, USA | 9035-HK | Radioactive |
| glucagon | Millipore, Billerica, MA, USA | 9030 | Radioactive |
| insulin | Millipore, Billerica, MA, USA | 9011S | Radioactive |
| leptin | Millipore, Billerica, MA, USA | 9081-HK | Radioactive |
| kisspeptin-10 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-048-56 | Radioactive |
| nesfatin-1 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-003-26 | Radioactive |
| PYY3-36 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-059-02 | Radioactive |
| somatostatin-28 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-060-16 | Radioactive |
| ZORBAX Rapid Resolution HT SB-C18 column | Agilent Technologies, Santa Clara, CA, USA | 822700-902 | 2.1 x 50 mm, 1.8 µm |
| Agilent 1200 LC | Agilent Technologies, Santa Clara, CA, USA | HPLC, several components, therefore no single catalog number | |
| Kisspeptin RIA | Phoenix Pharmaceuticals, Burlingame, CA, USA | # RK-048-56 | Radioactive |
| Total ghrelin RIA | Millipore, Billerica, MA, USA | # GHRT-89HK | Radioactive |
| Active ghrelin RIA | Millipore, Billerica, MA, USA | # GHRA-88HK | Radioactive |
| SigmaStat 3.1 | Systat Software, San Jose, CA, USA | online download |
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