The aim of the study was to investigate blood alterations caused by altitude acclimatization which last more than few days after return and might play a role for exercise performance at sea level. Measurements were performed in 12 mountaineers before, during and either 7/8 or 11/12 days after a Himalaya expedition (26-29 days at 4900 to 7600 m altitude). [Erythropoietin] rose only temporarily at altitude (max. +11 +/- 1 [SE] mu/ml serum). After return hemoglobin mass (initially 881 +/- 44 g, CO-Hb method) was increased by 14% (p < 0.01); aspartate aminotransferase activity in erythrocytes (initially 682 +/- 25 U/l) was augmented (day 7: +964 +/- 152 U/l, day 11: +533 +/- 107 U/l) indicating reduced mean cell age. Calculated blood volume (+14%) was influenced by red cell formation at altitude but also by plasma expansion at sea level. The half saturation pressure for Hb-O2 (pH 7.4, 37 degrees C) as well as the 2.3-diphosphoglycerate concentration were already initially high (32.1 +/- 0.5 mmHg, 20.5 +/- 0.7 mumol/g Hb) and showed only a nonsignificant tendency to increase after return. Also Hill's n was consistently high in the mountaineers, whereas the Bohr coefficients were slightly increased only after descent. Probably the preparatory physical training, partly in the Alps, and the stay in the Himalaya influenced O2-affinity for a prolonged time. The adaptations might reduce the loss of physical performance capacity at altitude and be part of altitude training effects.
Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron-deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron-deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti-thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre-treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit. Results of this study argue against the model of a simple feedback regulation of EPO via hypoxic anaemia. Our data support the hypothesis that cytokines and the erythropoietic progenitor pool are involved in the regulation of EPO production. The results illustrate that serial measurements of EPO along with therapeutic interventions are necessary to identify patients who might benefit from treatment with exogenous recombinant human EPO.
Summary. Murine recombinant erythropoietin (EPO) was purified from an EPO-producing cell line and used for the production of polyclonal monospecific anti-murine EPO antibodies in rabbits. The anti-mouse EPO antibodies were purified by two affinity chromatography procedures. In order to obtain the most sensitive ELISA, different antibody combinations were tested in the ELISA sandwich assay. The best combination was achieved with an anti-human EPO antibody as coating and the biotinylated anti-murine EPO antibody as detecting antibody. With this sandwich-ELISA a sensitive standard curve in the range of 0·6-30 mU/ ml could be established. The assay provides a sensitive and reliable measure of murine EPO in serum and cell culture supernatants ranging from normal to highly elevated EPO levels.Keywords: murine erythropoietin, purification, polyclonal antibodies, sandwich-ELISA, specificity.Erythropoietin (EPO) is a glycoprotein growth factor responsible for the proliferation and differentiation of early and late erythropoietic progenitor cells. When recombinant human EPO became available, several immunological assays for measuring EPO in human serum were established (Jelkmann, 1992). In our own laboratory we developed a sensitive and reliable sandwich ELISA for human EPO (Noé et al, 1992). However, this human ELISA was not sufficiently sensitive to efficiently measure mouse EPO in serum or culture supernatants from murine-derived cells. In this report we describe a sandwich ELISA specific for mouse EPO, using polyclonal antibodies produced against murine recombinant EPO. MATERIALS AND METHODS Purification of murine EPO (m-EPO).The source for the purification of m-EPO was the cell line Ne-3, kindly provided by Dr W. Ostertag (Heinrich-Pette-Institut, Universität Hamburg, Germany). This mouse fibroblast cell line contained the entire coding sequence of the mouse EPO gene. The cells were first cultivated in CG medium (Vitromex, Vilshofen, Germany) with 10% fetal bovine serum (FBS, Boehringer Mannheim, Germany), the concentration of which was gradually reduced in order to obtain serum-free conditions. After cultivation in CG medium in the absence of FBS for 14 d, the cells were centrifuged and the supernatant was mixed with 1/20 of the volume of 20 × phosphatebuffered saline (PBS), containing 2% Tween 80, 2% sodium azide (NaN 3 ) and stored at ¹20ЊC until processed.The following three chromatographic steps were performed using a FPLC (Pharmacia, Freiburg, Germany). The first step was chromatography of the supernatant over an agarose wheat germ lectin (Pharmacia) column equilibrated with PBS, containing 0·1% NaN 3 , 0·1% Tween 80. The column was washed with the equilibration buffer and the bound protein was eluted with 0·3 M N-acetylglucosamine (Serva, Heidelberg, Germany) in equilibration buffer. The eluted fractions were measured at 280 nm. In addition, EPO present in the eluted fractions was also measured with an EPO ELISA (Noé et al, 1992) modified for recombinant m-EPO (Boehringer Mannheim, Germany). The fractions wit...
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