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Simultaneous Determination of Human Plasma Immunoreactive β-Lipotropin, -Lipotropin, and β-Endorphin Using Immune-Affinity Chromatography^*

Department of Medicine, Division of Endocrinology, Mount Sinai School of Medicine New York, New York 10029

Address requests for reprints to: Dr. Dorothy T. Krieger, Division of Endocrinology, Department of Medicine, Mount Sinai School of Medicine, Mount Sinai Hospital, One Gustave L. Levy Place, New York, New York 10029.

Since plasma ACTH, β-lipotropin [β-LPH-(l–91)], -lipotropin (-LPH: [β-LPH-(l–58)]), and β-endorphin (β-EP: [β-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 β-LPH, -LPH, and β-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 β-LPH, by 5.8 for jβLPH, by 4.5 for ACTH, and by 3.4 for β-EP; n = 19; mean ± SEM)] were: β- LPH, 6.1 ± 0.8; -LPH, 4.4 ± 0.5; and ACTH, 11.1 ± 1.3. β-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 β-LPH concentrations.

The resolving power of the technique was tested by separately applying the immunoreactive β-LPH, -LPH, and β-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 K_av 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.

^* This work was supported in part by USPHS Grant NB-02893 and the Lita Annenberg Hazen Charitable Trust.

Received April 21, 1980.

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