Simultaneous determination of human plasma immunoreactive beta-lipotropin, gamma-lipotropin, and beta-endorphin using immuno-affinity chromatography

Abstract

Since plasma ACTH, beta-lipotropin [beta-LPH-(1-91)], gamma-lipotropin (gamma-LPH: [beta-LPD-(1-58)]), and beta-endorphin (beta-EP: [beta-LPH-(61-91)]) are all derived from a common precursor molecule, their quantification in the same plasma under basal and stimulatory conditions should help to elucidate factors involved in their secretion and regulation. A sequential immune affinity chromatographic procedure was used to separate immunoreactive beta-LPH, gamma-LPH, and beta-EP on individual patient samples. Basal morning plasma concentrations [femtomoles per ml (to convert values to picograms per ml, femtomoles per ml values are multiplied by 10 for beta-LPH, by 5.8 for beta-LPH, by 4.5 for ACTH, and by 3.4 for beta-EP; n = 19; mean +/- SEM)] were: beta-LPH, 6.1 +/- 0.8; gamma-LPH, 4.4 +/- 0.5; and ACTH, 11.1 +/- 1.3. beta-EP was undetectable (< 1.5 fmol ml-1) in 7 of the 19 basal samples. The mean +/- SEM for the 12 remaining samples was 2.3 +/- 0.2. Insulin-induced hypoglycemia and Pitressin administration were associated with nearly equivalent increments of immunoreactive ACTH and beta-LPH concentrations. The resolving power of the technique was tested by separately applying the immunoreactive beta-LPH, gamma-LPH, and beta-EP fractions obtained from plasma pools to Sephadex G-50 gel filtration for molecular weight estimation. Greater than 88% of all immunoreactive material eluted with a Kav similar to the appropriate standard peptide markers. This immune affinity chromatographic system, therefore, permits simultaneous quantification of these peptides on small plasma volumes more rapidly and with greater resolution than when molecular sieve chromatography is used as an adjunct to RIA.