Clinical Science (1991) 81,645-653
645
ATP production in isolated muscle mitochondria from
haemodialysis patients: effects of correction of anaemia with
erythropoietin
PETER
BARANY*, ROLF WIBOMt, ERIC HULTMANt
AND
JONAS BERGSTROM"
Departments of *Renal Medicine and tClinical Chemistry 11, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden
(Received 25 February/lO June 1991; accepted 19 June 1991)
SUMMARY
1. The ATP production rate in isolated skeletal muscle
mitochondria was measured with a bioluminescence
method, before and during erythropoietin treatment, in
2 1 anaemic haemodialysis patients. In addition, the
concentrations of ATP, phosphocreatine and total
creatine and the ratio of alkali-soluble protein to DNA
were determined in skeletal muscle. Maximal oxygen
uptake and maximal exercise capacity were determined
on a bicycle ergometer.
2. The results unexpectedly showed a 35% higher
mitochondria1 ATP production rate in the patients before
erythropoietin treatment than in sedentary control
subjects. On the other hand, mitochondrial density, as
measured by the activity of the matrix enzyme glutamate
dehydrogenase, was the same in the patients as in the
sedentary control group. After 1 year on maintenance
erythropoietin treatment, the ATP production rate per kg
of muscle decreased in five out of seven patients and
reached the same level as in the sedentary control
subjects. The ratio between ATP production rate and
glutamate dehydrogenase activity was on average 40%
higher in the patients at the start and decreased towards
the control level in six out of seven patients after 1year on
maintenance erythropoietin treatment. When related to
the mitochondrial protein content, a significant reduction
in the ATP production rate was observed.
3. The ratio of alkali-soluble protein to DNA in
skeletal muscle and the concentrations of ATP, phosphocreatine and total creatine in skeletal muscle at rest were
normal in the patients and did not change during the
study. The maximal aerobic power improved by 25%
after the correction of anaemia.
Correspondence: Dr Peter BBrBny, Department of Renal
Medicine, Huddinge University Hospital, S-, 141 86 Huddinge,
Sweden.
4. These results suggest that the enhanced mitochondrial ATP production rate in renal anaemia is a
metabolic adaptation to decreased oxygen transport and
is reversed by long-term correction of anaemia.
Key words: adenosine triphosphate; anaemia, erythropoietin, haemodialysis, mitochondria, oxidative phosphorylation, skeletal muscle, uraemia.
Abbreviations: ASP, alkali-soluble protein; GDH, glutamate dehydrogenase (EC 1.4.1.2); a-KG, a-ketoglutarate; PCM, palmitoyl-L-carnitine plus L-malate; PM,
pyruvate plus L-malate; PPKM, pyruvate plus palmitoyl-Lcarnitine plus a-ketoglutarate plus L-malate; PTH, parathyroid hormone; Succ-Rot, succinate plus rotenone.
INTRODUCTION
Skeletal muscle dysfunction is commonly found in
uraemic patients. Several effects of the illness may
contribute, such as inactivity, malnutrition, anaemia,
neuropathy and endocrine and metabolic distances. From
the results of animal experiments using a Clark electrode
for determination of oxygen consumption, a reduced
mitochondrial respiratory capacity has been suggested
[l-71 to be a cause of impaired oxidative energy metabolism in uraemia [l-91. In isolated rat liver mitochondria,
sera from uraernic rats may inhibit phosphorylation [l,21.
In uraemic rats (uraemia of 5 weeks' duration), respiration
in intestinal mucosal mitochondria decreases [3], but in
mice with acute uraemia (induced by urethral ligation for
0-48 h) no significant alterations in respiratory capacity
in isolated liver mitochondria were found [4]. Perna et al.
[6,7] have demonstrated impaired oxidative phosphorylation in isolated skeletal muscle and myocardial mitochondria from uraemic rats, and the same reduction of
mitochondrial oxygen consumption was observed after
the administration of parathyroid hormone (PTH) to
P. BBr6ny et al.
646
Table 1. Mitochondria1 ATP production rates for five dimerent substrates in 21 anaemic haemodialysis patients before
erythropoietin treatment and in the control subjects
Abbreviation: NS, not significant.
Patient no.
'
ATP production rate (mmol min- ' kg- of muscle)
Sex
GDH activity
(mmol of NADH
min-' kg-' of
muscle) (25°C)
PPKM
a-KG
PCM
PM
Succ-Rot
7.7
7.2
10.6
12.0
6.9
10.5
6.2
10.9
7.7
10.3
6.8
14.2
11.2
9.6
9.2
7.2
7.1
9.3
7.0
7.6
7.7
5.2
5.1
6.7
5.8
4.2
8.0
3.9
6.9
5.7
7.1
4.6
8.1
6.4
5.7
6.0
4.2
3.3
5.0
3.6
4.0
5.1
5.9
3.8
6.7
6.0
3.2
6.3
2.5
3.4
5.1
4.7
4.1
8.2
6.3
6.4
6.1
2.7
3.3
4.6
3.0
4.1
5.2
2.8
1.7
3.0
5.1
2.3
3.7
1.7
1.8
2.8
4.8
2.6
5.4
3.0
2.9
4.0
1.4
2.8
2.5
2.0
3.3
3.4
2.5
2.7
3.9
4.2
5.6
4.0
2.3
3.6
3.2
2.3
4.9
3.7
3.5
2.8
3.2
2.6
2.7
2.8
-
I .2
1.1
1.2
0.9
1.2
1.2
1.1
1.4
0.9
1 .O
0.9
1.3
1.O
1.4
1.5
0.9
I .3
1.o
1.I
1.1
Mean k SD
Range
8.9f2.1
(6.2-14.2)
5.4+ 1.4
(3.3-8.1)
4.8 rt 1.6
(2.5-8.2)
3.0+ 1.1
(1.4-5.4)
3.4 k 0.9
(2.3-5.6)
1.1 k0.2
(0.9-1.5)
Patients vs sedentary group
P
P= 0.0 13
NS
P= 0.045
P = 0.006
NS
NS
Sedentary control subjects ( n= 7)
Mean fSD
6.6k 1.3
4.6 k 0.9
3.5 k 1.0
2.2 k 0.3
1.1 *0.1
(4.7-8.9)
(3.4-6.2)
(1.7-4.9)
( 1.8-2.7)
3.2 k 0.8
(rr=2)
(2.6-3.7)
+ 1.2
5.0 f 0.6
4. I k 0.6
2.4 f 0.4
(6.2-9.5)
(4.4-6.1 )
(3.6-5.3)
(1.8-3.1)
11.0 k 2.2
8.1 f 1.8
4.3
* 1.4
3.8 k 1.5
(5.4- 10.7)
(2.6-6.8)
(2.4-7.0)
Patients ( n= 21 )
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
F
F
M
F
M
M
M
M
F
F
M
F
M
M
F
M
M
F
M
F
F
Range
Moderately active control subjects ( t z = 7)
Mean k SD
Range
Highly active control subjects ( n = 7)
Mean k SD
Range
7.6
(7.I -I 3.8)
normal rats. Treatment with verapamil reversed these
derangements in both groups of rats, which suggest that
the inhibition of mitochondrial respiration was related to
PTH-induced intracellular calcium accumulation.
Recently, Nishida et af. [S], using a 31Pn.m.r. spectroscopy
technique, investigated uraemic patients and found
reduced phosphocreatine levels during exercise. Reduced
muscle thermogenesis in haemodialysis patients has been
measured by direct microcalorimetry [9].
Exercise training has a stimulatory effect on mitochondrial respiratory capacity [lo, 111 and results in
increased activities of mitochondrial enzymes [lo]. Iron
deficiency in rats reduces respiratory capacity in isolated
mitochondria [ 121, but artifically induced iron deficiency
for 4 weeks in human subjects did not alter the activities
of oxidative enzymes [13]. In patients with reduced
2.9 k 0.4
(tz=3)
(2.7-3.4)
4.0 k 0.4
(t2=5)
(3.6-4.7)
1 .O
(0.9- 1.2)
1.4k0.2
(1.2-1.6)
1.8k0.2
(1.4-2.1)
muscle blood flow, an adaptation to ischaemia, with
increased activities of mitochondrial enzymes, has been
shown [14,15].
When the conventional method for the determination
of respiratory capacity in isolated mitochondria is used, it
is necessary to obtain 1-2 g of muscle tissue, and the
muscle biopsy must be taken by a surgical technique. This
has limited clinical studies of mitochondrial respiration.
The development of a new sensitive bioluminescence
method [ l l , 161 allowing the use of the percutaneous
muscle biopsy technique has made it possible to perform
the present study in 21 haemodialysis patients. When
human recombinant erythropoietin became available for
clinical studies [17, 181 in patients with renal anaemia, it
seemed important to investigate the effect of the correction of anaemia on skeletal muscle mitochondrial
Mitochondrial ATP production in renal anaemia
function. The ATP production rate was measured in
isolated skeletal muscle mitochondria before and after
correction of anaemia by erythropoietin treatment. In
seven patients a muscle biopsy was obtained after 1 year
on maintenance erythropoietin treatment. The concentrations of ATP, phosphocreatine and total creatine and the
ratio of alkali-soluble protein (ASP) to DNA were
measured in skeletal muscle. Exercise tolerance tests on a
bicycle ergometer were performed for measurement of
maximal exercise capacity and maximal oxygen uptake.
MATERIALS AND METHODS
Q
%
151
0
0.0
0.5
1.0
1.5
2.0
2.5
GDH activity
Patients
Twenty-one haemodialysis patients (10 females, 11
males; age 4 1 f 9 years, m e a n f s ~ )with anaemia
(haemoglobin level < 80 g/1 before transfusion) participated in the study, which was approved by the Ethics
Committee of Karolinska Institute. The patients participated voluntarily and were all fully informed about the
purpose of the study and the risks and discomfort
associated with the experiments. The primary renal
diagnoses were chronic glomerulonephritis ( 11 cases),
chronic pyelonephritis (four cases), systemic lupus
erythematosus (two cases) and unknown in two cases.
Two patients were bilaterally nephrectomized; one had
polycystic kidney disease and the other had juvenile
nephronophthisis. The mean time on regular haemodialysis treatment was 42 (range 3-280) months. All
patients were clinically stable, adequately dialysed
according to urea kinetics [19] and had no overt signs of
aluminium intoxication or malnutrition (height 172 f 10
cm, body weight 65.6 f9.9 kg, Broca’s index 0.91 k 0.1 1,
m e a n s f s ~ )PTH
.
levels, measured with an r i a . method
for detecting PTH mid-molecule, were elevated in most
patients (42.2 f32.0 ,ug/l, reference range 0.5-1.5 pgll),
but serum calcium (2.48 f0.21 mmol/l) and phosphate
(1.9 f0.4 mmol/l) levels were controlled with phosphatebinders, calcium carbonate and, in some cases, calcitriol.
Acidosis was corrected by the administration of sodium
bicarbonate (bicarbonate concentration 22.7 f 2.2 mmol/
1). Routine medication also included vitamins B and C.
Seven patients were treated with anti-hypertensive drugs.
All patients were considered to have a sedentary life-style
and were not regularly performing physical training.
Twelve patients were transfusion-dependent ( > 3 units
transfused during the last 6 months) and had recently
been transfused before entry into the study. Seven
patients had not been transfused during the 6 months
before their inclusion in the study. Five patients had iron
overload (serum ferritin level a 4 0 0 pg/l) and five
patients had serum ferritin levels below SO pg/l.
The patients received intravenous recombinant human
erythropoietin treatment (50-200 i.u./kg body weight)
after haemodialysis 3 times a week. Erythropoietin was
supplied by Cilag AG ( n= 14)and Boehringer Mannheim
Gmbh ( n = 6). One patient had spontaneously increasing
haemoglobin levels before erythropoietin treatment and is
therefore not included in the continuation of the study.
647
(rnrnolof NADH min-’ kg-’ of muscle)
Fig. 1. Muscle mitochondrial ATP production rate (at
25°C) for the substrate combination PPKM in relation to
GDH activity (at 25°C) in control subjects and haemodialysis patients (before erythropoietin treatment). The
regression line for the control subjects is shown ( r = 0.88,
P<O.OOl). 0, Haemodialysis patients; A , sedentary
control subjects ( n= 7); 0 , moderately active control
subjects ( n= 7); .,
highly active control subjects ( n= 7).
Seven patients received a kidney graft during the study.
One of them was re-investigated 16 months after early
graft loss, when he had been on maintenance haemodialysis and erythropoietin treatment for more than 1 year.
Other reasons for discontinuation were: the patients’ wish
to withdraw ( n = 3), intercurrent diseases ( P I = 3) and
death ( n = 1). Three muscle specimens could not be
analysed because of problems with insufficient material.
Muscle biopsy and an exercise tolerance test were performed on the day between dialysis treatments, before
erythropoietin therapy (biopsy I, haemoglobin level
75 k 11 g/l, n = 21) and at the time (1-7 months) when the
target haemoglobin level of 100 g/1 was reached (biopsy
11, haemoglobin level 1 1 0 f 8 g/l, n=9). The patients
were then transferred to maintenance erythropoietin
treatment, and in seven patients a third biopsy (111,
haemoglobin level 106 f 13 g/l, n = 7) was obtained after
approximately 12 months on maintenance erythropoietin
therapy.
Muscle biopsies
Thirty-seven biopsy samples of the quadriceps femoris
muscle were taken under local anaesthesia, using a
Bergstrom-Stille biopsy needle 1201.
Mitochondrial ATP production rate
The mitochondrial ATP production rate was determined by a bioluminescence technique in a suspension of
mitochondria isolated from fresh muscle specimens
(weight 4 8 f 12 mg), as previously described [ l l , 161.
Five different substrates were used: pyruvate plus Lmalate (PM), palmitoyl-L-carnitine+ L-malate (PCM), a-
P. Bargny et al.
648
Table 2. Mitochondria1 ATP production rate for the different substrates in the patients
investigated before and after correction of anaemia and before and after 1 year on maintenance
erythropoietin treatment
Biopsy I, at the start (haemoglobin level 75 f 11 g/l); biopsy 11, after correction of anaemia
(haemoglobin level 110f 8 g/l, 1-7 months); biopsy 111, after 1 year of maintenance erythropoietin therapy (haemoglobin level 106 f 13 g/l, 15-18 months). Values are means SD.
+
ATP production rate
(mmol min- kg- of muscle)
Before and after correction of anaemia
PPKM ( IZ = 9)
a-KG ( I I = 9)
PCM (17 = 9)
PM(n=9)
Succ-Rot ( ) I = 8)
GDH activity ( n = 9)
(mmol of NADH min-' kg-' of muscle)
Before and after 15-18 months on erythropoietin
PPKM (11 = 7)
u-KG(II=~)
PCM(n=7)
PM ( n= 7)
Succ-Rot ( I I = 7)
GDH activity ( 1 2 = 7 )
(mmol of NADH min-' kg-' of muscle)
Biopsy I
Biopsy I1
8.7f 1.9
5.9f 1.4
4.Gf 1.5
2.7f1.0
3.5? 1.0
8.1 f 1.G
5.4f 1.3
4.3f 1.2
2.7 k 0.7
3.0 f0.8
1.2 0.2
1.2 f0.3
Biopsy 1
8.9 f 2.2
5.4 f 1.2
4.7 f 1.4
2.8 f 1.1
3.1 f 0.7
Biopsy 111
7.1 f 1.8
4.5 f 1.8
3.9f1.4
2.8 k 1.0
2.7 f0.9
1.2f0.2
1.2f0.3
*
ketoglutarate (a-KG), succinate plus rotenone (Succ-Rot)
and a combination of the substrates: pyruvate plus
palmitoyl-L-carnitine plus a-KG plus L-malate (PPKM).
The ATP production rates were determined at 25°C. The
activity of the enzyme glutamate dehydrogenase (GDH;
E C 1.4.1.2), specific to mitochondria, was determined in
whole muscle and in intact mitochondria in the suspension. The ratio between the activities was used to convert
measurements of mitochondrial ATP production rate in
the mitochondrial suspension to activity per muscle mass
[11].The mitochondrial yield in the final suspension was
calculated from the GDH ratio and was 17 f 7%. The
activity of GDH was determined by the method of
Schmidt [21]. The protein content in the mitochondrial
suspensions was measured by the method of Lowry et al.
[221.
Three groups of healthy subjects (10 females, 11 males,
aged 18-45 years) with different degrees of physical
activity were used as controls for the mitochondrial
investigations. The results from the control subjects have
been published in a previous paper [ 111.
tissue-free solids were powdered and intracellular ASP,
i.e. non-collagen protein, and DNA were determined in
3-4 mg of the powder after precipitation of the protein.
The powder was washed twice with perchloric acid (0.2
mol/l) and the supernatant was discarded. The precipitate
was incubated for 1 h in KOH (0.3 mol/l) and ASP was
determined in a portion by the method of Lowry et al. [20,
221. DNA analysis was based on the Schmidt-Thunhauser method [23]. DNA was extracted by the method of
Schneider & Greco [24] and was determined by a micromodification of the diphenylamine method [25,26].
Specimens from 41 healthy control subjects (20 males,
21 females, age 36.1 13.7 years, m e a n f s ~ )were
processed and analysed by uniform procedures.
For the determination of ATP, phosphocreatine and
total creatine, 4-10 mg of the fat and connective tissuefree powder were analysed by enzymic methods as
previously described [27], with the modification of a 2
min (145 f 25 s) delay in freezing [28]. Values from nine
healthy control subjects [28] were recalculated with
respect t o fat-free tissue for comparison.
Determination of ASP/DNA and ATP, phosphocreatine
and total creatine concentrations in skeletal muscle
Exercise tolerance tests
After careful and rapid removal of visible fat and
connective tissue, the specimen was weighed and then
immediately frozen in liquid nitrogen. The frozen material
was freeze-dried and the fat was extracted with petroleum
ether. The dried muscle sample was carefully dissected
under a magnifying glass to remove remaining flakes of
connective tissue. The dried fat-free and connective
+
An exercise tolerance test was performed on an electrically braked bicycle ergometer using a stepwise increase
in workload every minute until exhaustion. The initial
load was 30 W followed by 10 W steps. Oxygen uptake
was measured by the Douglas bag technique at rest and
during the two highest workloads. Ten healthy subjects
(five females, five males, age 4 2 + 9 years, m e a n f s ~ )
were used as controls.
Mitochondria1 ATP production in renal anaemia
649
Statistics
The tests of significance for the differences between the
means of two independent samples were made by using
the Student's t-test when the variances were the same.
When the variances were different the comparisons were
made using the Behrens-Fischer test with Cochran's
modification. The test of equality of the variances was
made by using the F-test with a 5% level of significance
(two-tailed). The Student's t-test was used to assess the
statistical significance for paired data. A P value of less
than 0.05 was considered significant [29]. Values are
expressed as means fSD unless stated otherwise.
RESULTS
In Table 1 the individual values of the mitochondrial ATP
production rate before the start of erythropoietin treatment are shown. The mitochondrial ATP production rate
is expressed as mmol of ATP min-' kg-l of muscle
(25°C). There was a significantly higher mitochondrial
ATP production rate for the substrates PPKM (sedentary
control subjects 6.6 f 1.3 versus patients 8.9 f2.1 mmol
min-' kg-' of muscle, P<0.05), PCM (3.5 f 1.0 versus
4.8 f 1.6 mmol min-' kg- of muscle, P< 0.05) and PM
(2.2f0.3 versus 3.0k 1.1 mmol min-' kg-' of muscle,
P< 0.01) in the anaemic haemodialysis patients than in
the sedentary control subjects. GDH activity did not
differ significantly between the groups (1.1f0.1 versus
1.1 f 0.2 mmol of NADH min-' kg-' of muscle). In Fig.
1, the mitochondrial ATP production rates are expressed
in relation to the GDH activity in the patients before
erythropoietin treatment and in the three groups of
control subjects. The correlation was significant for the
healthy control subjects (r=0.88, P<O.OOl), but not for
the patients.
The mitochondrial ATP production rate (mmol of ATP
min-' 1- I ) for PPKM in the mitochondrial suspension was
expressed in relation to the intramitochondrial GDH
activity (mmol of NADH min-l I - ' ) in the suspension. In
the healthy control subjects this ratio was 5.6 f0.9 compared with 7.9 k 2.0 mmol of ATP/mmol of NADH in the
patients. The difference was significant ( P <0.001).
During erythropoietin treatment a second muscle
biopsy (11) was obtained from nine patients after the
correction of anaemia (1-7 months) and in seven patients
a muscle biopsy (111) was taken after 1 year on maintenance erythropoietin therapy. The mitochondrial ATP
production rate per kg of muscle (Table 2) or per GDH
activity (mean 6.9f 1.3 mmol of ATP/mmol of NADH,
P< 0.005 compared with control subjects) was still
increased after correction of the anaemia but decreased
towards the control level after 1 year on maintenance
erythropoietin treatment. The ratio between ATP production rate and GDH activity decreased in six out of seven
patients (7.8 f2.4 at the start versus 6.0 k 1.1 mmol of
ATP/mmol of NADH at biopsy 111, Fig. 2a). In one
patient, with a normal ratio at the start, an increased ratio
was observed. In these seven patients treated with
erythropoietin for more than 1 year the ATP production
rate was related to the content of protein in the mito-
'
10-1
2
\
2
" Biopsy I Biopsy II Biopsy 111
-5
u
C
8
0.50-
C
.
I
W
u
e
.
-e
0.40-
R
0.30-
C
.-u
0
c
0.20-
'CI
e
R
5
0.10-
o'ooJBiopsy I
Biopsy 111
Fig. 2. Muscle mitochondrial ATP production rate in a
suspension of isolated mitochondria for the substrate
combination PPKM in 12 haemodialysis patients before
and during erythropoietin treatment. The ATP production rate (mmol of ATP min-' I-') is expressed in relation
to ( a ) the intramitochondrial activity of GDH (mmol of
NADH min-' I-') and ( 6 ) the content of protein (g/l)
( n= 7) in the mitochondrial suspension. Biopsy I, at the
start (haemoglobin level 75 k 11 g/l); biopsy 11, after
correction of anaemia (haemoglobin level 110 2 8 g/l, 1-7
months); biopsy 111, after 1 year on maintenance erythropoietin therapy (haemoglobin level 106 k 13 g/l, 15-18
months). -Mean; ......, SD, control subjects ( n= 21);
0,patient no. 1; 0 , patient no. 2; 0, patient no. 3;
patient no. 4; A , patient no. 8; A , patient no. 11; X ,
patient no. 17. Statistical significance: *P<0.01.
.,
chondrial suspension (Fig. 2b). The ATP production
per g of protein decreased in six of the seven patients. The
difference was significant (0.389 k 0.130 versus
0.249+0.111 mmol of ATP min-' g-I of protein,
P<O.Ol).
The concentrations of ATP, phosphocreatine and total
creatine in muscle tissue, as well as the ratio of ASP to
DNA, were the same as those of the healthy subjects
(Table 3).
Maximal exercise capacity and maximal oxygen uptake
were low in the patient group initially, but increased after
650
P. BBr6ny et al.
Table 3. ASP/DNA ratio and ATP, phosphocreatine and total creatine concentrations in skeletal
muscle, maximal exercise capacity and oxygen uptake in the control subjects and haemodialysis
patients before and during erythropoietin treatment
Values are means k SD. Statistical significance: "P< 0.05 patients versus control subjects;
hP<0.005 patients versus control subjects; cP< 0.001 patients versus control subjects; " P < 0.01
I versus 11; ' P < 0.001 I versus 11. I, At the start (haemoglobin level 75 k 11 g/l); 11, after correction
of anaemia (haemoglobin level 1 1 0 k 8 g/l, 1-7 months); 111, after 1 year of maintenance
erythropoietin therapy (haemoglobin level 106 k 13 g/l, 15-18 months). Abbreviations: PCr,
phosphocreatine; TCr, total creatine.
Control
subjects
Patients
I
ASP/DNA ratio (g/kg)/(g/kg)
412f57
(n=41)
ATP concn. (mmol/kg of dry fat free muscle)26.2 f 2.1
(n=9)
PCr concn. (mmol/kg of dry fat free muscle) 90 f 4
(n=9)
TCr concn. (mmol/kg of dry fat free muscle) 141 f 5
(n=9)
PCr/TCr
0.64k0.03
(12=9)
Maximal exercise capacity ( W )
200 k 54
( n = 10)
Maximal oxygen uptake (litres/min)
2.41 f0.70
(.= 10)
the correction of anaemia (Table 3). The aerobic power
improved by 25% in the nine patients investigated after
the correction of anaemia (from 1.16k0.36 to
1.44 k 0.40 litres/min, P< 0.001), but still the values were
lower than in the healthy subjects. Blood pressure
reactions were normal during the exercise tests and no
tests were terminated because of hypertension. At the
outset of the study the ATP production rate (with PPKM
as substrate) and GDH activity were not correlated with
the maximal oxygen uptake. After the correction of
anaemia in the nine patients investigated, the maximal
oxygen uptake was significantly correlated both with the
mitochondrial ATP production rate (with PPKM as substrate) (r=0.686, P=0.041) and with the GDH activity
(r=0.811,P=0.008;Figs.3aand 3b).
DISCUSSION
In this study of anaemic haemodialysis patients treated
with human recombinant erythropoietin, we did not
observe any inhibition of the oxidative phosphorylation
related to uraemia, as suggested by previous animal
experiments [I-3,571. In contrast, and probably because
of an adaptation to the decreased oxygen transport in
anaemia, a higher mitochondrial ATP production rate was
observed before the correction of anaemia. Similar
findings with increased activity in rnitochondrial enzymes
have been noted in patients with reduced muscle blood
flow [14,15,30]and in animals with chronic hypoxia [31].
The increased mitochondrial ATP production rate, when
410k87
(n=20)
24.0 f 3.3
( n = 13)
93f7
( n = 13)
1 3 9 f 13
( n = 13)
0.67f0.06
(n=13)
llOk24'
(n=21)
1.22k0.37'
( n = 19)
I1
372 f 90
( n = 11)
23.7 k 2.4
(n=6)
98 f 10
(n=6)
145f11
(n=6)
0.67f0.05
(n=6)
1 2 8 f 29hd
(n=9)
1.44+0.4Ohc
(n=9)
I11
390 f 93
(n=7)
23.7 f 0.9
(12=5)
95f12
(12=5)
146f20
(ll=5)
0.65f0.07
(11=5)
127k39"
(n=4)
1.56f0.48;'
(n=4)
expressed in relation to GDH activity (Fig. 2a), suggests a
specific stimulation of the mitochondrial enzyme activity,
without increased synthesis of mitochondrial protein,
which is seen in healthy subjects as an effect of physical
conditioning [321.
Stimulation of mitochondrial respiratory capacity in
skeletal muscle by physical activity has been confirmed in
animals [lo] and humans [ I l l . The effect is mediated by
an increase in rnitochondrial protein, and increased
activities of the oxidative enzymes have also been demonstrated [lo, 331. There was a close correlation (r=0.88,
P < O . O O l ) between the GDH activity and the mitochondrial ATP production rate in the healthy subjects
(Fig. 1)[ll].The activity of GDH correlates significantly
with the amount of mitochondrial protein in the suspension of isolated mitochondria [ 111 and may therefore be
used as a reference for mitochondrial mass. Increasing
physical activity is followed by a similar increase in ATP
production rate and in GDH activity in the muscle tissue,
indicating that the ATP production rate per mitochondrial mass is not affected by training. In the anaemic
haemodialysis patients the described mitochondrial balance is disturbed: the ATP production rate is not correlated with the activity of GDH (Fig. 1).
Peripheral tissue oxygenation, as measured by transcutaneous partial pressure of oxygen, increases significantly in erythropoietin-treated patients after the
correction of anaemia, despite reduced cardiac output
and calf blood flow [34].In the present study the high ATP
production rate was still present after the correction of
Mitochondria1 ATP production in renal anaemia
anaemia with erythropoietin. This may have been due
either to the shortness of the observation period, since
biopsy I1 was taken immediately after the target haemoglobin level had been reached (15 k 10 weeks) or to the
fact that the anaemia was only partially corrected. A
tendency towards a lower mitochondrial ATP production
rate in the five patients with the highest pretreatment
values was observed. Changes in the mitochondrial ATP
production rate in humans may be demonstrated after a
few weeks of training-detraining (R. Wibom & E. Hultman, unpublished work). After 1 year on maintenance
erythropoietin treatment the ratio between mitochondrial
ATP production rate and GDH activity decreased in six
out of seven patients and reached the same level as in the
sedentary subjects (Fig. 2a). When the ATP production
rate was related to the mitochondrial protein content the
same pattern, with a significant decrease in the ATP
production rate, was observed (Fig. 26).
That the mitochondrial respiratory capacity declines in
elderly patients has been shown by Trounce et af. [35] in
an investigation of orthopaedic patients (16-92 years). In
the present study the patients were younger (24-57 years)
and we observed no correlation between age or sex and
the mitochondrial ATP production rate.
Iron-deficient rats have a decreased mitochondrial
activity of iron-containing enzymes, but an increased
activity of mitochondrial matrix enzymes [13, 36, 371. In
our group of patients five had iron overload (serum
ferritin level > 400 ,ug/l) and five had low serum ferritin
levels ( < 5 0 ,ug/l), but no correlation with the mitochondrial ATP production rate was observed.
Massry and co-workers [5-7, 38, 391 have demonstrated that chronic renal failure and PTH treatment in
rats are associated with impaired oxidation of palmitoylCoA and a-KG by isolated skeletal muscle and myocardial mitochondria. The effect on /?-oxidation of
palmitoyl-CoA was due to reduced activity of carnitine
palmitoyltransferase in the mitochondrial membrane. It
was proposed that these alterations were mediated by
PTH-induced intracellular calcium accumulation and that
verapamil reversed the derangements. Despite the wide
range (4.3-133.4 pg/l) of PTH levels, no correlation with
mitochondrial ATP production rate (before or after
correction of anaemia) was observed in our study. The
different results of these previous animal experiments
[5-7, 38, 391 and our study may also be related to
different observation periods. The animals are probably
more affected by uraemic toxicity during a short period
(21 days of chronic renal failure) and the process of
metabolic adaptation then does not occur. The haemodialysis patients were selected for the study because they
had had severe anaemia for at least 3 months and were
adapted to the low capacity for oxygen transport to the
tissues.
The normal ASP/DNA ratio and ATP, phosphocreatine and total creatine concentrations in the present study
are in contrast to previous findings in uraemic patients,
which reported a reduced ASP/DNA ratio [40] and low
muscle concentrations of ATP and phosphocreatine [41,
421. The selection criteria for participation in this study
65 1
0
0
4
2
6
8
10
Mitochondria1ATP
production for PPKM
(mmol min- kg- of muscle)
2.51
0.0
0.5
1.0
1.5
2.0
GDH activity
(mmolof NADH min-l kg-l of muscle)
Fig. 3. Correlations between the maximal oxygen uptake
after the correction of anaemia and ( a )the mitochondrial
ATP production rate for the substrate combination
PPKM and ( 6 ) GDH activity. ( a ) r=0.686, P=0.041; ( b )
r=0.811, P=O.O08.
may have had some influence, since patients with obvious
signs of malnutrition were not included. However, some
patients in the study were underweight compared with the
healthy control subjects. The protein status in muscle, as
measured by the ASP/DNA ratio, was not correlated with
the mitochondrial ATP production rate.
Improved oxygen transport capacity after correction of
the anaemia results in increased capacity for exercise
[43] and indicates that the peripheral oxygen availability is
one limiting factor for maximal performance in uraemic
patients. In addition, low oxygen availability during
exercise is probably one important factor that induces an
increase in mitochondrial respiratory capacity. The mitochondrial ATP production rate was not correlated with
the maximal oxygen uptake when the patients were
severely anaemic. After correction of the anaemia, the
maximal oxygen uptake correlated significantly both with
652
P. B5rBny et al.
GDH activity and with the ATP production rate (Fig. 3).
Possibly, improved oxygen transport to the tissues results
in better utilization of the oxidative capacity in muscle
during exercise. Despite the observed improvement, the
maximal aerobic power was still low after the correction
of anaemia, and most of the exercise tolerance tests were
terminated because of leg muscle fatigue before the
expected maximal heart rate was reached, indicating that
factors other than mitochondrial respiratory capacity are
important for maximal performance in these patients.
Furthermore, peak blood lactate levels after exercise have
been found to be low in uraemic patients [44, 451, also
suggesting that the rate of oxidation phosphorylation is
not limiting for the exercise capacity in these patients.
Other factors, such as excitation-induced calcium release
and re-uptake from and to the sarcoplasmic reticulum,
may have significant impact on force generation during
exercise. High intracellular calcium levels in muscle have
been measured in uraemic patients [46] and a disturbed
calcium transport may impair the excitation-contraction
coupling in skeletal muscle.
ACKNOWLEDGMENTS
This work was supported by grants from the Swedish
Medical Research Council (Project nos. 01002 and
02647), Boehringer-Mannheim Gmbh, CILAG AG, the
Swedish Association of Nephrology and the National
Association of Kidney Patients in Sweden. We thank the
staff of the Departments of Renal Medicine and Clinical
Chemistry 11, Huddinge University Hospital, Karolinska
Institute, Stockholm, Sweden.
REFERENCES
1. Glaze, R.P., Morgan, J.M. & Morgan, R.E. Uncoupling of
oxidative phosphorylation by ultrafiltrates of uremic serum.
Proc. SOC.Exp. Biol. Med. 1967; 125, 172-4.
2. Yamada, T., Yoshida, A. & Koshikawa, S. Alteration of
oxidative phosphorylation in uremia. Jpn. Circ. J. 1969; 33,
59-62.
3. Russell, J.E. & Avioli, L.V. The effect of chronic uremia on
intestinal mitochondrial activity. J. Lab. Clin. Med. 1974;
81,317-26.
4. Martinelli, R., Rodrigues, L.E.A., Machado, A.E.C. &
Rocha, H. The effect of acute uremia on liver mitochondrial
activity. Nephron 1976; 17, 155-60.
5. Smogorzevski, M., Piskorska, G., Borum, P.R. & Massry,
S.G. Chronic renal failure, parathyroid hormone and fatty
acid oxidation in skeletal muscle. Kidney Int. 1988; 33,
555-60.
6. Perna, A.F., Smogorzevski, M. & Massry, S.G. Verapamil
reverses PTH- or CRF-induced abnormal fatty acids
oxidation in muscle. Kidney Int. 1988; 34,774-8.
7. Perna, A.F., Smogorzevski, M. & Massry, S.G. Effects of
verapamil on the abnormalities in fatty oxidation of
myocardium. Kidney Int. 1989; 36,453-7.
8. Nishida, A,, Umeda, M., Kubo, A., Ando, A., Nihei, H. &
Sugino, N. Impaired muscle energy metabolism in uremic
patients, measured by 31P-NMR spectroscopy [Abstract].
1 lth lnt. Cong. Nephrol. 1990, 129A.
9. Fagher, B., Monti, M., Nilsson-Ehle, P. & Thysell, H.
Reduced thermogenesis in muscle and disturbed lipoprotein
metabolism in relation to thyroid function in hemodialysis
patients. Scand. J. Lab. Invest. 1987; 47,91-7.
10. Holloszy, J.O. & Booth, E W Biochemical adaptations to
endurance exercise in muscle. Annu. Rev. Physiol. 1976;
38,273-91.
11. Wibom, R. & Hultman, E. ATP-production rate in mitochondria isolated from microsamples of human muscle. Am.
J. Physiol. 1990; 259, E204-9.
12. MacDonald, V.W., Charache, S. & Hathaway, PJ. Iron
deficiency anemia: mitochondrial a-glycerophosphate
dehydrogenase in guinea pig skeletal muscle. J. Lab. Clin.
Med. 1985; 105,ll-18.
3. Celsing, F., Blomstrand, E., Werner, B., Pihlstedt, P. &
Ekblom, B. Effects of iron deficiency on endurance and
muscle enzyme activity in man. Med. Sci. Sports Exercise
1986; 18,156-61.
4. Bylund, A.C., Hammarsten, J., Holm, J. & Schersttn, T.
Enzyme activities in skeletal muscles from patients with
peripheral arterial insufficiency. Eur. J. Clin. Invest. 1976; 6,
425-9.
5. Lundgren, F., Dahllof, A.G., Schersttn, T. & BylundFellenius, A.C. Muscle enzyme adaptation in patients with
peripheral arterial insufficiency: spontaneous adaptation,
effect of different treatments and consequences on walking
performance. Clin. Sci. 1989; 77,485-93.
6. Wibom, R., Lundin, A. & Hultman, E. A sensitive method
for measuring ATP-formation in rat muscle mitochondria.
Scand. J. Clin. Lab. Invest. 1990; 50, 143-52.
17. Eschbach, J.W., Egrie, J.C., Downing, M.R., Browne, J.K. &
Adamson, J.W. Correction of the anemia of end-stage renal
disease with recombinant human erythropoietin, results of a
combined phase I and I1 clinical trial. N. Engl. J. Med. 1987;
316,73-8.
18. Winearls, C.G., Oliver, D.O., Pippard, M.J., Reid, C.,
Downing, M.R. & Cotes, P.M. Effect of human erythropoietin derived from recombinant DNA on the anaemia of
patients maintained by chronic haemodialysis. Lancet 1986;
ii, 1175-7.
19. Farrell, P.C. & Gotch, F.A. Dialysis therapy guided by
kinetic modelling: applications of a variable-volume singlepool model of urea kinetics. Proc. 2nd Aust. Conf. Heat
Mass Transfer 1977,29-37.
20. Bergstrom, J. Muscle electrolytes in man. Determination by
neutron activation analysis on needle biopsy specimens. A
study on normal subjects, kidney patients and patients with
chronic diarrhoea. Scand. J. Clin. Lab. Invest. 1962; 1 4
( S ~ p p l68),
. 1-110.
2 1. Schmidt, E. Glutamate dehydrogenase. In: Bergmeyer, H.U.,
ed. Methods of enzymatic analysis. 2nd ed. New York
Academic Press and Weinheim: Verlag Chemie, 1974:
650-6.
22. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J.
Protein measurement with the Fohn-phenol reagent. J. Biol.
Chem. 1951; 193,265-75.
23. Munro, H.N. & Fleck, A. Recent development in the
measurement of nucleic acids in biological materials.
Analyst. 1966; 91,78-87.
24. Schneider, W.C. & Greco, A.E. Incorporation of pyrimidine
deoxyribonucleosides into liver lipids and other components. Biochim. Biophys. Acta 1971; 228,610-26.
25. Giles, K.W. & Myers, A. An improved diphenylamine
method for the estimation of deoxyribonucleic acid. Nature
(London) 1965; 206,93.
26. Burton, K. A study of the conditions and mechanism of the
diphenylamine method for the calorimetric estimation of
deoxyribonucleic acid. Biochem. J. 1956; 6 2 , 3 15-23.
27. Harris, R.C., Hultman, E. & Nordesjo, L.O. Glycogen,
glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of
man at rest. Methods and variance of values. Scand. J. Clin.
Lab. Invest. 1974; 33,109-20.
28. Soderlund, K. & Hultman, E. Effects of delayed freezing on
content of phosphagens in human skeletal muscle. J. Appl.
Physiol. 1986; 61,832-35.
Mitochondria1 ATP production in renal anaemia
29. Snedecor, G.W. & Cochran, W.G. Statistical methods. 6th
ed. Ames, I A . Iowa State University Press, 1976.
30. Elander, A., Sjostrom, M., Lundgren, F., ScherstCn, T. &
Bylund-Fellenius, A.C. Biochemical and morphometric
properties of mitochondrial populations in human skeletal
muscle fibres. Clin. Sci. 1985; 69,153-64.
31. Holm, J., Bjorntorp, P. & ScherstCn, T. Metabolic activity in
rat skeletal muscle, effect of intermittent hypoxia. Eur. J.
Clin. Invest. 1973; 3,279-83.
32. Hoppeler, H., Luthi, P., Claassen, H., Weibel, E.R. &
Howald, H. The ultrastructure of the normal human skeletal
muscle. A morphometric analysis on untrained men, women
and well-trained orienteers. Pflugers Arch. 1973; 344,
217-32.
33. Blomstrand, E., Ekblom, B. & Newsholme, E.A. Maximum
activities of key glycolytic and oxidative enzymes in human
muscle from differently trained individuals. J. Physiol.
(London) 1986;381,111-18.
34. Nonnast-Daniel, B., Deschodt, G., Brunkhorst, R. et al.
Long-term effects of treatment with recombinant human
erythropoietin on haemodynamics and tissue oxygenation in
patients with renal anaemia. Nephrol. Dial. Transplant.
1990; 5,444-8.
35. Trounce, I., Byrne, E. & Marzuki, S. Decline in skeletal
muscle mitochondrial respiratory chain function: possible
factor in ageing. Lancet 1989; i, 637-9.
36. Ohira, Y., Cartier, L.J., Chen, M. & Holloszy, J.O. Induction
of an increase in mitochondrial matrix enzymes in muscle of
iron deficient rats. Am. J. Physiol. 1987; 253 (Cell Physiol.
22), C639-44.
37. Cartier, L.J., Ohira, Y., Chen, M., Cuddihee, R.W. &
Holloszy, J.O. Perturbation.of mitochondrial composition in
muscle by iron deficiency. J. Biol. Chem. 1986; 261,
13827-32.
653
38. Bogin, E., Levi, J., Harary, I. & Massry, S.G. Effects of
parathyroid hormone on oxidative phosphorylation of heart
mitochondria. Miner. Electrolyte Metab. 1982; 7, 151-6.
39. Smogorzevski, M., Perna, A.F., Borum, P.R. & Massry, S.G.
Fatty acid oxidation in the myocardium: effects of parathyroid hormone and chronic renal failure. Kidney Int.
1988; 34,797-803.
40. Guarnieri, G., Toigo, G., Situlin, R. et al. Muscle biopsy
studies in chronically uremic patients: evidence for malnutrition. Kidney Int. 1983; 24, S187-93.
41. Del Canale, S., Elaccadore, E., Ronda, N., Soderlund, K.,
Antonucci, C . & Ciuariglia,A. Muscle energy metabolism in
uremia. Metab. Clin. Exp. 1986; 35,981-3.
42. Bergstrom, J., Lindholm, B., Alvestrand, A. & Hultman, E.
Muscle composition in CAPD patients. In: La Greca, G.,
Chiaramonte, S., Fabris, A., Feriani, M. & Ronco, C., eds.
Peritoneal dialysis - proceedings of the 2nd international
course on peritoneal dialysis. Milan: Wichtig Editore, 1986:
149-54.
43. Mayer, G., Thum, J., Cada, E.M., Stummvoll, H.K. & Graf,
H. Working capacity is increased following recombinant
human erythropoietin treatment. Kidney Int. 1988; 34,
525-8.
44. Lundin, A.P., Stein, R.A., Brown, C.D. et al. Fatigue,
acid-base and electrolyte changes with exhaustive treadmill
exercise in hemodialysis patients. Nephron 1987; 46,
57-62.
45. BBrBny, P., Freyschuss, U., Pettersson, E. & Bergstrom, J.
Physiologic effects of erythropoietin (EPO) treatment in
anemic hemodialysis (HD) patients. [Abstract]. Kidney Int.
1990; 37,1383.
46. Bergstrom, J., Bgriny, P., Hultman, E., Lindholm, B. &
Wibom, R. Bioenergetic studies of muscle in patients with
renal failure. Lecture 6th Capri Conf. Uremia 1989 (In
press).