The overall goal of this RAPID method is to improve the yield of endogenous peptide hormones to be measured in human blood. The RAPID method was recently established for the use in rats, and is also suited for the use in human blood. The RAPID method improves the yield, and allows detection of the correct molecular form of endogenous peptide.
The RAPID method is easy to use. However, the RAPID method is more time consuming and more expensive compared to standard blood sampling. So it might be more applicable in the research setting.
Visual demonstration of this method is critical, as the timely dilution of the samples as well as the elution of great importance to ensure a proper peptide level. Collect venous blood at a standardized time after an overnight fast from a forearm vein. And process according to standard procedures, or the RAPID method.
Instruct the subjects not to exercise or smoke before blood withdrawal. For standard processing, collect blood in chilled EDTA containing tubes and centrifuge within 10 minutes at 3000 Gs for 10 minutes at four degrees Celsius. Collect the supernatant, and keep it minus 80 degrees Celsius until further processing by radioimmunoassay.
For RAPID processing, immediately dilute blood one to 10, in ice cold RAPID buffer. PH three point six, containing zero point one molar amoniumasitate. Zero point five molar sodium chloride, and enzyme inhibitors.
Within 10 minutes, centrifuge the RAPID sample at 3000 Gs for 10 minutes at four degrees Celsius, and collect the supernatant, using a pipette. Next, charge chromatography cartridges with 100%actitonitrile, at 10 milliliters per minute. Following equilibration with 0.1%trifluoroacetic, or TFA.
Load with the RAPID sample supernatant, at a constant rate of one milliliter per minute, using a syringe pump. After washing the cartridges with three milliliters of 0.1%TFA, at 10 milliliters per minute, slowly elute the RAPID sample with two milliliters of 70%acetonitrile containing 0.1%TFA, at two milliliters per minute. Dry the eluted samples using vacuum centrifugation, and store at minus 80 degrees Celsius, until further processing by radioimmunoassay.
Obtain iodine 125 radio labeled human peptides. Keep the peptides in powder form until the experiment. Then freshly dilute in 0.1%acetic acid.
For standard processing, directly after blood withdrawal, into chilled EDTA containing tubes, transfer one milliliter of blood in a tube with 50 microliters of radiolabel, containing 3000 to 6000 CPM. For RAPID processing, transfer one milliliter of EDTA containing blood into a tube holding nine milliliters of RAPID buffer, and 500 microliters of radiolabel contain 30, 000 to 60, 000 CPM. Due to the one to 10 dilution, use a 10 times higher volume of radiolabel for RAPID processing.
Directly after applying the different steps of the RAPID method, assess the recovery of radioactively labeled peptides by using a gamma counter. Do not dry the samples by vacuum centrifugation, and do note store at minus 80 degrees Celsius. For measurement, transfer the supernatant to tubes fitting into the gamma counter.
And assess the counts per minute. Measure the whole supernatant in standard samples. In RAPID samples, analyze one tenth of the total volume to achieve a comparable amount of radiolabel used.
As a 100%standard, use two samples with 50 microliters of iodine 125 radiolabeled peptide that do not undergo processing. Measure the radioactivity of all of the samples at the same time. Withdraw blood, in chilled EDTA containing tubes, and transfer one milliliter to tubes holding 200 microliters of radiolabeled acyl ghrelin, containing 15, 000 to 20, 000 CPM, for standard processing.
For RAPID processing, transfer one milliliter of blood into a tube holding nine milliliters of rapid buffer, and 200 microliters of radiolabeled acyl ghrelin containing 15, 000 to 20, 000 CPM. Afterwards, process samples as before. For further analysis by reversed phase HPLC, directly load the samples onto a stable bond C18 column equilibrated in 17%acetonitrile in water supplemented with 0.1%TFA.
After five minutes of equilibration, use a gradient from 17 to 40%acetonitrile, to elute the sample in 40 minutes, at one milliliter per minute. Collect fractions of one milliliter every minute, and analyze radioactivity using a gamma counter. In a separate experiment, load 200 microliters of radiolabeled acyl ghrelin containing 15, 000 to 20, 000 CPM onto the column directly and perform HPLC as before.
For radioimmunoassay, thaw frozen supernatants and vacuum dried powder at room temperature. Immediately before radioimmunoassay, re suspend dry RAPID samples in double distilled water, according to the original volume of plasma. Assess kisspeptin and total ghrelin as well as acyl ghrelin, using commercial radioimunoassays according to the manufacturer's protocols.
Use borosilicate tubes that allow stable palette formation. On day one, incubate the samples with assay buffer, and primary atibody, in the dilution provided by the manufacturer for a period of 24 hours. On day two, add the iodine 125 tracer, vortex, and incubate for a period of 24 hours.
On day three, add the percipitating reagent, vortex, and incubate as recommended by the manufacturer. Then, centrifuge the tubes at 3, 000 Gs for 20 minutes at four degrees Celsius. Remove the supernatants and count the radioactivity in the palettes, using a gamma counter.
Calculate the desacyl ghrelin, as the difference of the total ghrelin minus acyl ghrelin. Asses the acyl, desacyl ghrelin ratio, by diving acyl by desacyl ghrelin for each individual sample. If possible, process all samples in one batch, to avoid inter assay variability.
RAPID blood processing increases the yield of iodine 125 radiolabeled peptides in human blood, as compared to standard blood processing. After standard blood processing, the recovery of radiolabeled peptides ranged from 48%to 68%in nine peptides. RAPID processing improved the yield in all iodine 125 labeled peptides, with a recovery ranging from 71%to 98%Shown here is the elution profile of iodine 125 labeled acyl ghrelin in human blood following standard or RAPID blood processing.
After RAPID processing, iodine 125 labeled ghrelin eluded at the expected position. While after the standard procedure, an earlier peak was observed. Likely corresponding to desacyl ghrelin.
This represented a 62%degradation of the peptide. RAPID blood processing improves the acyl, desacyl ghrelin ratio as compared to standard processing. After RAPID processing, the acyl, desacyl ghrelin ratio in blood of normal weight subjects was one to three.
As compared to one to 23 following standard blood processing. Similar results were observed under anorexic and obese conditions. RAPID blood processing results in increased endogenous kisspeptin blood levels as compared to standard processing.
In both anorexic and normal weight subjects, circulating endogenous kisspeptin levels were significantly higher following RAPID as compared to standard processing. The difference did not reach significance under conditions of obesity. Once mastered, and performed properly, this technique can be done in less than one hour.
This method is especially important when the expected concentration of the peptide is low, or the differences between the groups are small. A general recommendation for peptides cannot be given. The potential benefit for each peptide should be tested once at the beginning.
This technique paves the way for researchers in different fields to explore the yield and more importantly, the correct molecular form of endogenous peptide hormones. After watching this video, you should have a good understanding of how to apply RAPID method for blood processing in humans.
