SIMULTANEOUS RIA OF HUMAN beta-ENDORPHIN e beta-LIPOTROPIN IN PLASMA EXTRACTS

The identification of f)-endorphin (f)-EP) as part of the larger precurs~_ f)-lipotropin (f)-LPH) in pituitary cells and the importance of f)-E P in behaviour and endogenous analgesia dictate the need for a method of measur- ing the plasma or li.quour concentrations of them in physiological and path- ological conditions. The present study concerns the deve:.opment of RIA methods to measure f)-EP ~:Id f)-LPH after extraction and pu r i f i c a t i.o n . Synt:" etic f)-EP (Peninsular Inc., San Francisco) and h f)-LPH (Dr.C.H.Li, San Fra~ cisco) and h f)-EP antiserum (Dr.C.Pert NIH, Bethesda) were used. 8nth peptides were labelled with Il~5 using the chloramine T method, an then purified by gel filtration in a Sephadex G-25 culumn. The specific activity of f)-EP-I125 was lSOuC/ug and that of f)-LPH-I125 was 242uC/ug. The specificity of the antiserum using both labelled peptides was chec~ with the following substances: leu-enkephalin, Ret-enkephalin, substance P GIF, 1-39 ACTH, f)-MSH, 61-77 f)-LPH, 79-91 f)-LPH, f)-E P and f)-LPH. No cross reaction was found with leu and met-enkephalin, substance P, GIF and 1-39 ACTH, while in the f)-EP and f)-LPH RIAs, f)-LPH and f)-EP showe d a cross r e ac t-: ion of 54.7% and 196.2% respectively. Using f)-LPH as tracer, 7S-9l f)-LP H atd 61-77 f)-LPH cross reacted very s l i gh tI y (0.29% a nd 0.01% r e s p e c t i ve Ly) ; when f)-EP-I125 was used the cross reactions were 0.91% and 0.26%; and whe an equimolecular pool of both peptides was tested the cross reaction in- creased to 5.2%. After the addition of 4000 dpm labelled f)-EP and f)-LPH, 2.Sml plasma was extracted for l hr with 150me glass powder in il rotary mixer at room temp- erature. The glass powders were washed with distilled water'and then 2N HC: and the peptides removed in a l hr rotation with 2ml acetone-HCl 2N 4:1. After nitrogen drying the residue was dissolved in 0.4ml acetic acid O.lN, 0.1% BSA, applied to a Sephadex G-7S column and eluted with the same sol- ution. Two distinct peaks of elution were collected, lyophilised and redissolveè in phosphate buffer pH 7.4, O.OSM for assay; an aliquot was utilised to evaluate the recovery which was 61.1~ + 4.5 and SS.2% + 5.7 (M + SE) for f)-EP and f)-LPH respectively. Using different dilutions of the antiserum and of the labelled compounds, two different RIA systems were perfected; the first for f)-EP having a sensitivity of lSpg and the second for f)-LPH having a sensitivity of 4Spg. The RIAs were characterised by an lShr pre- incubation with the antiserum followed by the addition of tracer for an inc- ubation time of 4Shr (both at 4°C); the complcx was then precipitated with goat antirabbit y globulin (24hr). This study was supported by the CNR project "Biology of Reproduction"