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 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 studies^7-12 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 intake^15,16 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

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 (within 1 min after blood withdrawal) 1:10 in ice-cold buffer (pH 3.6) containing 0.1 M ammonium acetate, 0.5 M NaCl and enzyme inhibitors (diprotin A, E-64-d, antipain, leupeptin, chymostatin, 1 µg/ml). Then, centrifuge within 10 min at 3,000 x g for 10 min at 4 °C and collect supernatant using a pipette in polypropylene tubes as detailed before in rats⁶.
   1. Charge chromatography cartridges (360 mg, 55-105 µm) with 100% acetonitrile (rate 10 ml/min), equilibrate with 0.1% trifluoroacetate (TFA, rate 10 ml/min) and load with the supernatant at a constant rate of 1 ml/min using a syringe pump.
   2. Thereafter, wash cartridges with 3 ml 0.1% TFA (rate 10 ml/min) and slowly elute with 2 ml 70% acetonitrile containing 0.1% TFA (2 ml/min).
   3. Dry eluted samples using vacuum centrifugation and store at -80 °C until further processing by radioimmunoassay.
      NOTE: Conduct all steps in polypropylene (RAPID processing) and borosilicate (radioimmunoassay) tubes that exhibit significantly lower surface binding properties and thus minimize peptide loss for most of the peptides studied before²⁶.

2. Measurements

NOTE: Steps in this section should be performed in a laboratory certified for work with radioactive material. Standard precautions for the work with ¹²⁵I should be taken.

1. Recovery of Radiolabeled Peptides
   1. Obtain ¹²⁵I-radiolabeled human peptides (e.g. acyl-ghrelin, GLP-1, glucagon, insulin, kisspeptin, leptin, nesfatin-1, PYY_3-36 and somatostatin-28).
   2. Keep peptides in powder form until the experiment, then freshly dilute in 0.1% acetic acid (~100,000 cpm per ml).
   3. For standard blood processing, directly after blood withdrawal into chilled EDTA containing tubes, transfer 1 ml of blood into a tube with 50 µl of radiolabel containing 3,000-6,000 cpm (counted directly before the experiment starts).
   4. For RAPID processing, transfer 1 ml blood of EDTA containing blood into a tube containing 9 ml RAPID buffer (for composition see 1.3) and 500 µl radiolabel containing 30,000-60,000 cpm. Due to the 1:10 dilution use 10-times higher volume of radiolabel for RAPID processing.
   5. Afterwards, process samples as described in steps 1.2 to 1.3.2.
   6. For the recovery experiments, do not dry samples by vacuum centrifugation and do not store at -80 °C. Instead, assess recovery of radioactively labeled peptides directly afterwards using a gamma counter.
   7. Measure the whole supernatant in standard samples, while in RAPID samples analyze 1/10 of the total volume to obtain comparability of the amount of radiolabel used. For measurement, transfer the supernatant in tubes fitting into the gamma counter and assess the counts per minute
   8. As a 100% standard, use two samples with 50 µl of ¹²⁵I-radiolabeled peptide that do not undergo processing. Measure at the same time with other samples as described in 2.1.7.
   9. Perform the experiment five to six times for each peptide.
2. High Performance Liquid Chromatography of Radiolabeled Ghrelin
   1. Withdraw blood in chilled EDTA containing tubes and transfer 1 ml to tubes containing 200 µl radiolabeled-acyl ghrelin containing 15,000-20,000 cpm (counted directly before the experiment starts).
   2. For RAPID processing, transfer 1 ml blood into a tube containing 9 ml RAPID buffer (for composition see 1.3) and 200 µl radiolabeled acyl ghrelin containing 15,000-20,000 cpm.
   3. Afterwards, process samples as described in steps 1.2 to 1.3.2.
   4. For further analysis by reverse phase HPLC, directly load samples onto a stable bond C18 column (2.1 mm x 50 mm, 1.8 µm) equilibrated in 17% acetonitrile in water (both supplemented with 0.1% TFA).
   5. After 5 min equilibration, use a gradient from 17-40% acetonitrile to elute the sample in 40 min (flow rate of 1 ml/min).
   6. Collect fractions of 1 ml every minute and analyze radioactivity using a gamma counter.
   7. In a separate experiment, load 200 µl radiolabeled acyl ghrelin containing 15,000-20,000 cpm onto the column directly and perform HPLC as described in steps 2.2.4 to 2.2.6.
3. Radioimmunoassay
   1. For radioimmunoassay, thaw frozen supernatants (standard processing) and vacuum dried powder (RAPID method) at room temperature.
   2. Immediately before radioimmunoassay, re-suspend dry RAPID samples in double distilled H₂O according to the original volume of plasma (500 µl).
   3. Assess kisspeptin and total (including both desacyl and acyl ghrelin) as well as acyl ghrelin as described before^12,27 using commercial radioimmunoassays according to the manufacturers' protocols. Use borosilicate tubes that allow stable pellet formation.
      1. On day one, incubate the samples with assay buffer and primary antibody (e.g. anti-ghrelin) in the dilution provided by the manufacturer for a period of 24 hr.
      2. On day two, add the ¹²⁵I tracer (e.g. ¹²⁵I-ghrelin), vortex and incubate for a period of 24 hr.
      3. On day three, add the precipitating reagent, vortex and incubate as recommended by the manufacturer. Then, centrifuge tubes at 3,000 x g for 20 min at 4 °C. Remove the supernatants and count radioactivity in the pellets using a gamma counter
   4. Calculate desacyl ghrelin as the difference of total minus acyl ghrelin. Assess the acyl/desacyl ghrelin ratio by dividing acyl by desacyl ghrelin for each individual sample.
   5. Process all samples – if possible – in one batch to avoid inter-assay variability. The intra-assay variability in the present experiment was <8% for kisspeptin, <7% for total and <9% for acyl ghrelin.

3. Statistical Analysis

1. Determine the distribution of the data using the Kolmogorov-Smirnov test. Express data as mean ± standard error of mean (SEM).
2. Assess differences between two groups by t-test. Assess differences between multiple groups by all pair-wise multiple comparison procedures (Tukey post hoc test) or two-way ANOVA followed by Holm-Sidak method.
3. Consider p <0.05 significant and perform analyses using a statistics program.

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

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