Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

<div><p>The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p = 0.029); low-dose Epoetin beta: 73.1 (17.8)% (p = 0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.</p></div

Optimization curve.

<p>For optimization of W elution buffer <i>low</i>, three samples were used: 1) UC, umbilical cord sample; 2) Plasma sample form a normal subject; and 3) rhEPO, epoetin beta (300 ng/l). All samples from the three specimen were tested with GlcNAc concentrations of 5 mM, 10 mM, 15 mM, 20 mM, and 300 mM. Values are means (SD). N = 6 for UC, N = 5 for plasma and N = 5 for rhEPO.</p

Standard curve.

<p>Concentrations of rhEPO (Epoietin beta) were 0, 10, 30, 100, 300 and 600 ng/l. All solutions were tested at 300 mM of GlcNAc. Values are means ± SD. N = 6.</p

The percent migrated isoform at 15 mM GlcNAc.

<p>PMI after 4, 11 and 25 days of either high-dose rhEPO, low-dose rhEPO, or placebo. N = 15. Values are means with 95 % confidence intervals. <b>*</b>p<0.05; ** p<0.00001 compared with placebo.</p

Haematological parameters after 4, 11 and 25 days of either placebo, low-dose rhEPO, or high-dose rhEPO.

<p>N = 16. Values are means (SD).</p>§<p>Data was log₁₀ transformed before analysis.</p><p><b>*</b>p<0.05;</p><p>**p<0.01 compared with placebo.</p><p>Haematological parameters after 4, 11 and 25 days of either placebo, low-dose rhEPO, or high-dose rhEPO.</p

Total plasma EPO concentration (mIU/ml).

<p>N = 15. Values are means (SD). Data was log₁₀ transformed before analysis.</p><p><b>*</b>p = 0.004;</p><p>**p<0.00001 compared with placebo.</p><p>Total plasma EPO concentration (mIU/ml).</p