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 YY_3-36 (+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 obesity^1,2, 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 stab....

Protocol

The protocol was approved by the local ethics committee for human research (protocol number EA1/114/10).

1. Blood Processing

1. Collect venous blood between 07:00 and 08:00 am after an overnight fast from a forearm vein and process according to standard procedure or the RAPID method. Instruct the subjects to not exercise or smoke before blood withdrawal.
2. For standard processing, collect blood in chilled EDTA-containing tubes and centrifuge within 10 min at 3,000 x g for 10 min at 4 °C. Collect the supernatant and keep at -80 °C until further processing by radioimmunoassay.
3. For RAPID processing, immediately dilute blood (wi....

Results

RAPID blood processing increases the yield of ¹²⁵I-radiolabeled peptides in human blood compared to standard blood processing.
After standard blood processing (EDTA blood on ice), the recovery of radiolabeled peptides was ~60% in 9/9 peptides (ranging from 48-68%, Figure 1A-K). RAPID processing improved the yield in all ¹²⁵I-labeled peptides, namely in somatostatin-28 (+39%, Figure 1A), glucagon-like peptide-1 (+.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 mm 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