Nephron 22: 361-365 (1978)
Serum Erythropoietin Concentration in Anephric Patients H. W. Radtke, P. M. Erbes, E. Schippers and K. M. Koch Department of Nephrology, University Hospital, Frankfurt am Main
Key Words. Bilateral nephrectomy • Anemia • Erythropoietin • Fetal mouse liver cell assay • Androgen therapy Abstract. In 13 bilateral nephrectomized patients serum erythropoietin (SEp) activity could be measured quantitatively by use of the highly sensitive fetal mouse liver cell assay. SEp con centration in the majority of the cases was below the mean of normal controls. There was a significant positive correlation between SEp levels and hematocrits, suggesting erythropoietin (Ep) deficiency to be a causative factor in the anemia of the anephric state. Androgen therapy stimulated extrarenal Ep production in all of 5 anephric patients studied.
The anephric state is nearly always accom panied by severe anemia [9,21], which is be lieved to be primarily caused by erythropoietin (Ep) deficiency [6,22], Several investigators were able to demonstrate Ep in the serum of bilateral nephrectomized patients, proving the existence of extrarenal production sites for Ep [2,5,7,13-15]. However, in the majority of the studies performed the percentage of sera with measureable Ep activity was small [5,7,14,15]. This may be a consequence of the limited sen sitivity of the polycythemic mouse assay ap plied by all investigators. In the present study we investigated by use of a highly sensitive in vitro bioassay in a group of anephric patients, whether the presence of
Ep in the serum is a regular finding, and whether detectable levels of serum erythro poietin (SEp) are correlated with the degree of anemia. Furthermore, we studied the effect of androgen therapy on extrarenal Ep pro duction.
Patients and Methods Patients 13 anephric patients (It males, 2 females) 14-49 years old were studied. The reason for nephrectomy was malignant hypertension resistant to therapy in 5, Goodpasture syndrome in 3, renal tuberculosis in 3, and renal carcinoma in 1 patient. 1 patient was nephrec tomized prior to renal transplantation. All patients were on chronic hemodialysis treatment for 2-9 years. Dialysis schedule was 6-10 h 3-4 times/wcek (Meltcc
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/31/2018 7:12:43 PM
Introduction
362
Method SEp activity was measured by use of the fetal mouse liver cell method, as described by Dunn el at. [4], and Wardle et al. [19]. Liver cells from 14-day-old mouse fetuses were suspended in a culture medium, and serial dilutions of test serum or Ep standard were added. 5 replicates were made up for every concentration step. After a 20-hour preincubation period at 37 °C, radioiron was added to the culture for another 4 h for pulse labeling. After cellular lysis in distilled water, heme was extracted and its radioactivity was measured in a liquid scintillation counter. Rate of radioiron in corporation into heme served as a measure of Ep activity. The following modifications were introduced: the maximal test serum concentration was limited to 4%, and the fetal calf serum portion was increased to 10% final dilution. Radioiron was preincubated with pure human transferrin (Behring, Marburg) before being added to the culture. In addition, as suggested by von Preyss and Goudsmit [16] test sera were inactivated at 56 C for 30 min. As laboratory standard we used the sheep plasma Ep step III (Connaught, Toronto), which was previously calibrated against the International Reference Preparation B (courteously made available through the WHO International Laboratory for Bio logical Standards, Mill Hill, London). Since the assay is sensitive down to 0.5 mU/ml culture and maximal test serum portion was 4% Ep concentrations above 12 mU/ml serum could be measured accurately. The index of precision ranged from 0.04 to 0.2. 95% con fidence limits were usually 80-120% of the mean potency. Calculations of Ep titers (expressed in inter national units) with their 95% confidence limits, and testing of significance of regression, linearity, and
parallelism were performed by analysis of variance with the aid of the Computer Center of the J. W. Goethe University, Frankfurt am Main.
Results In every patient investigated SEp concen tration could be measured quantitatively. The results together with the corresponding hema tocrits are presented in table I. The individual values range from 30 to 215 mU/ml, the mean is 104 ± 66 (SD) mU/ml. As shown in figure 1, there is a significant positive correlation be tween hematocrit and SEp concentration (r= 0.68; p
Serum Erythropoietin Concentration in Anephric Patients H. W. Radtke, P. M. Erbes, E. Schippers and K. M. Koch Department of Nephrology, University Hospital, Frankfurt am Main
Key Words. Bilateral nephrectomy • Anemia • Erythropoietin • Fetal mouse liver cell assay • Androgen therapy Abstract. In 13 bilateral nephrectomized patients serum erythropoietin (SEp) activity could be measured quantitatively by use of the highly sensitive fetal mouse liver cell assay. SEp con centration in the majority of the cases was below the mean of normal controls. There was a significant positive correlation between SEp levels and hematocrits, suggesting erythropoietin (Ep) deficiency to be a causative factor in the anemia of the anephric state. Androgen therapy stimulated extrarenal Ep production in all of 5 anephric patients studied.
The anephric state is nearly always accom panied by severe anemia [9,21], which is be lieved to be primarily caused by erythropoietin (Ep) deficiency [6,22], Several investigators were able to demonstrate Ep in the serum of bilateral nephrectomized patients, proving the existence of extrarenal production sites for Ep [2,5,7,13-15]. However, in the majority of the studies performed the percentage of sera with measureable Ep activity was small [5,7,14,15]. This may be a consequence of the limited sen sitivity of the polycythemic mouse assay ap plied by all investigators. In the present study we investigated by use of a highly sensitive in vitro bioassay in a group of anephric patients, whether the presence of
Ep in the serum is a regular finding, and whether detectable levels of serum erythro poietin (SEp) are correlated with the degree of anemia. Furthermore, we studied the effect of androgen therapy on extrarenal Ep pro duction.
Patients and Methods Patients 13 anephric patients (It males, 2 females) 14-49 years old were studied. The reason for nephrectomy was malignant hypertension resistant to therapy in 5, Goodpasture syndrome in 3, renal tuberculosis in 3, and renal carcinoma in 1 patient. 1 patient was nephrec tomized prior to renal transplantation. All patients were on chronic hemodialysis treatment for 2-9 years. Dialysis schedule was 6-10 h 3-4 times/wcek (Meltcc
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/31/2018 7:12:43 PM
Introduction
362
Method SEp activity was measured by use of the fetal mouse liver cell method, as described by Dunn el at. [4], and Wardle et al. [19]. Liver cells from 14-day-old mouse fetuses were suspended in a culture medium, and serial dilutions of test serum or Ep standard were added. 5 replicates were made up for every concentration step. After a 20-hour preincubation period at 37 °C, radioiron was added to the culture for another 4 h for pulse labeling. After cellular lysis in distilled water, heme was extracted and its radioactivity was measured in a liquid scintillation counter. Rate of radioiron in corporation into heme served as a measure of Ep activity. The following modifications were introduced: the maximal test serum concentration was limited to 4%, and the fetal calf serum portion was increased to 10% final dilution. Radioiron was preincubated with pure human transferrin (Behring, Marburg) before being added to the culture. In addition, as suggested by von Preyss and Goudsmit [16] test sera were inactivated at 56 C for 30 min. As laboratory standard we used the sheep plasma Ep step III (Connaught, Toronto), which was previously calibrated against the International Reference Preparation B (courteously made available through the WHO International Laboratory for Bio logical Standards, Mill Hill, London). Since the assay is sensitive down to 0.5 mU/ml culture and maximal test serum portion was 4% Ep concentrations above 12 mU/ml serum could be measured accurately. The index of precision ranged from 0.04 to 0.2. 95% con fidence limits were usually 80-120% of the mean potency. Calculations of Ep titers (expressed in inter national units) with their 95% confidence limits, and testing of significance of regression, linearity, and
parallelism were performed by analysis of variance with the aid of the Computer Center of the J. W. Goethe University, Frankfurt am Main.
Results In every patient investigated SEp concen tration could be measured quantitatively. The results together with the corresponding hema tocrits are presented in table I. The individual values range from 30 to 215 mU/ml, the mean is 104 ± 66 (SD) mU/ml. As shown in figure 1, there is a significant positive correlation be tween hematocrit and SEp concentration (r= 0.68; p
