JOURN4L OF DIALYSIS, 1 ( 7 ) , 697-703 ( 1 9 7 7 )
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AMINO ACID LOSS DURING HEMOFILTRATION H. Mann Department of Internal Medicine I 1 Techn. University of Aachen Goethestrasse 27/29 5100 Aachen, German Federal Republic
ABSTRACT Loss of free amino acids during hemodiaf i 1 tration therapy. was investigated in 5 dogs. Hemodiafiltration was performed with a cellulose-acetate membrane with a limit of molecular separation at MW 19,000. The concentration of amino acids was investigated in ultrafiltrate and in blood at the beginning and at the end df di af i 1 tration. Loss of amino acids in the ultrafiltrate was seven times as much as free amino acid content o f plasm, whereas amino acid concentration in plasma decreased only slightly (20 X ) . Applying these results to hemodiafiltration in man, it is concluded that loss o f amino acids in hemodiafiltration i s not greater than in conventional hemodialysis therapy.
INTRODUCT f ON The mean molecular weight o f amino acids is 140 (extremes 75 and 240). Hence all amino acids permeate the membranes used
in dialysis (membranes such as phane and the more permeable membranes used in hemdiafiltration).
The molar concentration of
amino acids in the plasma o f uremic patients is very low compared
697 Copyright 0 1977 by Marcel Dekker, Inc. All Rights Raened. Neither this work nor any put may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfdming. and recording, or by any information storage and retrieval system, without permission in vrriting from the publisher.
698
MA"
Table I
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Solute urea creatinine uric acid p ho spha te glycine glutamic acid cyst ine glucose
Mw
Concentration
60
ml ml ml ml
200 m g / l O O I0 mg/lOO 168 10 mg/lOO 95 6 mg/100
113
32000 p ~ o i / i
8000 PAOl/l 5900 f l I o l / l 10000 pNol/l
75
300 pNol/l
240 180
100 pMol/l
147
50 pNol/l
70 mg/100 ml
3800 pMol/l
w i t h s o l u t e s normally excreted by t h e kidneys such as urea and c r e a t i n i n e (Table I ) .
Since i n h e m o d i a f i l t r a t i o n , mass trans-
p o r t through t h e membrane i s brought about by t h e pressure depende n t amount o f u l t r a f i l t r a t e and n o t by a c o n c e n t r a t i o n difference o f s o l u t e s on b o t h sides o f t h e membrane, as i n d i a l y s i s , i t can be expected t h a t w i t h h e m o d i a f i l t r a t i o n , amino a c i d s may be exc r e t e d t o a g r e a t e r e x t e n t than i n conventional d i a l y s i s therapy. MATERIALS AND METHODS Loss o f f r e e amino acids was i n v e s t i g a t e d i n uremic dogs
(20
- 25
kg).
H e m o d i a f i l t r a t i o n was performed w i t h a 1 M 2 c e l l u -
lose-acetate membrane.
The l i m i t of molecular separation of t h e
membrane was MW 19,000.
I n these experiments blood f l o w was 50
80 ml/min and u l t r a f i l t r a t e f l o w about 50 ml/min.
-
The d i l u t i n g
f l u i d was added t o t h e blood before e n t e r i n g t h e u l t r a f i l t e r . Fran these data t h e clearance o f a l l penneable substances can be c a l c u l a t e d as 30 ml/min.
During a 3
- 5 hr.
average u l t r a f i l t r a t e o f 13.4 l i t e r s was obtained. sponds t o 3 . 3 l i t e r s / h .
treatment, an This corre-
699
AMINO ACID LOSS DURING HEWFILTRATION
Two days prior t o hemodiafiltration the animals d i d not i n gest food. In 5 experiments concentration o f amino acids was fnvestigated i n arterial blood before starting, d u r i n g and a t the end of
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
each hemodiafiltration, and i n the u l t r a f i l t r a t e (Technicon Analyzer).
RESULTS
The results are sumnarized i n Table 11. All data are means of a l l experiments.
In the f i r s t column the normal values for
nonuremic man are shown.
Compared w i t h the values o f the uremic
dogs before starting hemodiafiltration (Cbo)
there are no appreci-
able differences except for a higher Concentration of cysteine and threonine i n the dog. During hemodiafiltration there is a decrease i n the concen-
tration of most amino acids fran s t a r t (Cbo) t o the end of treat-
ment (Cbl). Mean decrease i n concentration is fran 305 mg/l t o i.e. about 20 X .
238 mg/l
Plasma alanine and tyrosine, on the
other hand, increased during hemodiafiltration. In the ultraf i l t r a t e there i s about half the concentration of amino acids as i n plasma.
T h i s corresponds t o the expected concentration after
diluting the blood entering the ultrafilter w i t h the diluting fluid.
Thus, penneation of amino acids through the membrane is
not limited. Their clearance is 28.9 ml/min and corresponds t o the theoretically expected clearance of 30 ml/min.
7 a0
MA”
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Table I1 UF
normal values m a n mg/l
‘bo mg/l
mg/l
mg/l
rng
35-4
40.7
14.3
8.1
288.6 84.8
2.5 2.9
GlY
17.4 12.4
3-5 5.8 27.1 7.3 9.8 15.4
21.5 6.3
CYS
30.7 14.3 2.2 5.3 17.7
Pro
9-2 27.1
17.1
Se 1:
11.8
12.1
Tyr
9.1 7.1 13.2
4.7 9.3 19.6 20.3 5.8 11.3 52.6 30.6
Ala kt3 ASP
Cit
Glu
8.6
His OrIl
Ileu Leu
LYS
Met Phe The Val
23.4
3.2 9.5 19.4 79.9
‘bl
16.8
4.2 I .o 10.0
14.5 41.5 178.2 15.3 69.4 55.7 13.7 143.4
9.7
10.1
735.3
18.i
5.6
9.1 8.2 2.8
2.8
6.4
6- 9 12.9 77.3 3.5 11.5 34.2
20.6
1.1
3.1 13.3 I .I
5-2
3.1 5.5
10.1
12.9 4.5 6.8
18.3
15.2
41.5 73.8
136.0
773-1 60.4
90.8 252.3 203 7
From the amino acid concentration (CUF) and the amount of the u l t r a f f l t r a t e the quantity o f amino acids eliminated per hemodiaf i l t r a t i o n can be derived (UF).
I t is about seven times the quan-
t i t y of amino acids i n one l i t e r o f plasma, i.e. seven times as
much a s the entire quantity o f plasma amino acids i n the dog. Average amino acid loss was 2.07 g during 4 hrs. o f t r e a t ment or about 0.5 g/h. DISCUSSION To evaluate the importance o f amino acid loss i n hemodiafil-
t r a t i o n i t is necessary t o compare amino acid loss i n hemodiafilt r a t i o n w i t h amino acid l o s s In conventional d i a l y s i s therapy and
ACID LOSS DURING HEMOFILTRATION
AMINO
w i t h normal amino acid loss i n the urine.
701
I t is also necessary
to apply the results found i n the dog t o the human. Normal mean daily amino acid excretion i n man i s 2.2 g (1.3 3.2 9).
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
(1,3,7)
During 6
- 12 hrs.
-
o f hemodialysfs by several authors
loss of amino acids of 4.5
- 10.0 g has been described,
average 8.5 g per dialysis run or 1.2 g/h respectively. According to Salisbury the distribution of amino acids i n plasma i n dialyzed and nondialyzed uremics is not different from normals. Rubini and Gordon found no change i n the amino acid spectrum
during dialysis, although the amino acid loss was e i g h t times
greater than the total plasma content. From these results i t can be concluded that the amino acids eliminated from plasma during dialysis are replaced instantly from the intracellular space. loss of about 1
- 2 g/h
T h i s also is the reason why amino acid
throughout one dialysis run is very con-
stant. Similarly, i n our experiments i n dogs, the concentration of free amino acids decreases only about 20 X, although seven times as
much amino acids have been excreted by hemodiafiltration as were i n plasma.
T h i s result also corresponds to the results ,found i n h e m -
dialysis therapy (7). Since the amino acid concentration i n blood i n our experiments corresponds t o the amino acid concentration i n man, our results i n the dog can be conditionally applied t o man.
If these results
are t o be applied t o the conditions i n hemodiafiltration i n man,
702
MA”
d i f f e r e n t clearances induced by d i f f e r e n t f l o w r a t e s o f blood and d i l u t i n g f l u i d necessary f o r e l i m i n a t i o n o f h i g h e r q u a n t i t i e s o f urea and c r e a t i n i n e i n man must a l s o be considered. T h i s may be i l l u s t r a t e d by an example:
Blood f l o w o f 200
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m l / m i n and u l t r a f i l t r a t e f l o w o f 200 ml/min when s u b s t i t u t i n g the d i l u t i n g f l u i d before the u l t r a f i l t e r (or u l t r a f i l t r a t e flow o f 100 ml/min when s u b s t i t u t i n g t h e d i l u t i n g f l u i d behind t h e u l t r a f i l t e r ) induces a clearance o f urea and amino acids o f 100 ml/min.
H e m o d i a f i l t r a t i o n therapy i n t h i s way i s necessary t o
e l i m i n a t e t h e s o l u t e s normally excreted by t h e kidneys such as urea and c r e a t i n i n e (2,4).
Considering t h i s i l l u s t r a t i o n of
h e m o d i a f i l t r a t i o n i n man w i t h a constant amino a c i d c o n c e n t r a t i o n i n plasma o f about 300 mg/l throughout t h e t i m e o f treatment, i t can be d e r i v e d t h a t t h e r e i s a 1.8 g/h l o s s o f amino acids.
This
corresponds t o t h e l o s s o f amino a c i d s found i n hemodialysis (1,3,
5,6,7,9).
A l t o g e t h e r l o s s o f amino ac ds i n h e m o d i a f i l t r a t i o n
does n o t appear t o be g r e a t e r than i n hemodialysis. I f one decides t o conduct h e m o d i a f i l t r a t i o n i n man w i t h a
clearance o f 50 ml/min, as done by Q u e l l h o r s t e t al.,
t h e amount
o f amino a c i d s l o s t i n h e m o d i a f i l t r a t i o n would be s m a l l e r than i n hemodialysis. F i n a l l y , t h e question may be considered whether t h i s l o s s o f amino acids d u r i n g a r t i f i c f a l kidney therapy must be replaced i n t h e d i l u t i n g f l u i d o r not. i n hemodialysis therapy.
T h i s question can be answered j u s t as I n our experience, w i t h r e g u l a r p r o t e i n
d i e t s , most p a t i e n t s g a i n weight and g e n e r a l l y have no symptoms o f
703
AMINO ACID LOSS DURING HEMOFILTRATION
I t therefore appears unnecessary as a rule
protein deficiency.
t o add amino acids t o the diluting fluid. T h i s a d d i t i o n would certainly raise the price of the diluting fluid considerably.
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
ACKNOWLEDGEMENTS This study was supported by Deutsche Forschungsgemeinschaft,
Sonderforschungsberei ch 109.
REFERENCES
1.
Giordino, S., de Pascale, C., de Cristoforo, D., i n Nutrition in Renal Disease, edited by G.M. Berlyne, The Williams Company, Baltimore, 1968.
2.
Henderson, L.W., Livoti, L.G., Ford, C.A., Kelley, A.B., Lysaght, M.J., Trans. Am. SOC. Artif. Int. Organs E:119, 1973.
3.
Kopple, I.D., Swendsind, M.E., Shinaberger, J.H., Umetawa, C.Y., Trans. Am. SOC. Artif. Int. Organs XIJ:309, 1973.
4.
Mann, H., Heintz, R., Brass, H., Biomed. Technik, g:170, 1974. Pickford, I.C., Aber, G.M.,
McGale, E.H.F., 1972.
5.
3a:395,
6.
Quellhorst, E., Doht, B., Rieger, J., XIII. Hamburg, 1976.
7.
R u b i n i , M.E.,
8.
36: 1227, 1957.
9.
Young, G.A.,
C l i n . Chim. Acta.
Congr. EDTA,
and Gordon, S., Nephron, 5:339, 1968.
Salisbury, P.M.,
Dunn, M.S., Murphy, E.A.,
Paisons, F.M.,
Clin. Sci.
I. Clin. Invest.
z:l, 1969.
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
AMINO ACID LOSS DURING HEMOFILTRATION H. Mann Department of Internal Medicine I 1 Techn. University of Aachen Goethestrasse 27/29 5100 Aachen, German Federal Republic
ABSTRACT Loss of free amino acids during hemodiaf i 1 tration therapy. was investigated in 5 dogs. Hemodiafiltration was performed with a cellulose-acetate membrane with a limit of molecular separation at MW 19,000. The concentration of amino acids was investigated in ultrafiltrate and in blood at the beginning and at the end df di af i 1 tration. Loss of amino acids in the ultrafiltrate was seven times as much as free amino acid content o f plasm, whereas amino acid concentration in plasma decreased only slightly (20 X ) . Applying these results to hemodiafiltration in man, it is concluded that loss o f amino acids in hemodiafiltration i s not greater than in conventional hemodialysis therapy.
INTRODUCT f ON The mean molecular weight o f amino acids is 140 (extremes 75 and 240). Hence all amino acids permeate the membranes used
in dialysis (membranes such as phane and the more permeable membranes used in hemdiafiltration).
The molar concentration of
amino acids in the plasma o f uremic patients is very low compared
697 Copyright 0 1977 by Marcel Dekker, Inc. All Rights Raened. Neither this work nor any put may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfdming. and recording, or by any information storage and retrieval system, without permission in vrriting from the publisher.
698
MA"
Table I
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
Solute urea creatinine uric acid p ho spha te glycine glutamic acid cyst ine glucose
Mw
Concentration
60
ml ml ml ml
200 m g / l O O I0 mg/lOO 168 10 mg/lOO 95 6 mg/100
113
32000 p ~ o i / i
8000 PAOl/l 5900 f l I o l / l 10000 pNol/l
75
300 pNol/l
240 180
100 pMol/l
147
50 pNol/l
70 mg/100 ml
3800 pMol/l
w i t h s o l u t e s normally excreted by t h e kidneys such as urea and c r e a t i n i n e (Table I ) .
Since i n h e m o d i a f i l t r a t i o n , mass trans-
p o r t through t h e membrane i s brought about by t h e pressure depende n t amount o f u l t r a f i l t r a t e and n o t by a c o n c e n t r a t i o n difference o f s o l u t e s on b o t h sides o f t h e membrane, as i n d i a l y s i s , i t can be expected t h a t w i t h h e m o d i a f i l t r a t i o n , amino a c i d s may be exc r e t e d t o a g r e a t e r e x t e n t than i n conventional d i a l y s i s therapy. MATERIALS AND METHODS Loss o f f r e e amino acids was i n v e s t i g a t e d i n uremic dogs
(20
- 25
kg).
H e m o d i a f i l t r a t i o n was performed w i t h a 1 M 2 c e l l u -
lose-acetate membrane.
The l i m i t of molecular separation of t h e
membrane was MW 19,000.
I n these experiments blood f l o w was 50
80 ml/min and u l t r a f i l t r a t e f l o w about 50 ml/min.
-
The d i l u t i n g
f l u i d was added t o t h e blood before e n t e r i n g t h e u l t r a f i l t e r . Fran these data t h e clearance o f a l l penneable substances can be c a l c u l a t e d as 30 ml/min.
During a 3
- 5 hr.
average u l t r a f i l t r a t e o f 13.4 l i t e r s was obtained. sponds t o 3 . 3 l i t e r s / h .
treatment, an This corre-
699
AMINO ACID LOSS DURING HEWFILTRATION
Two days prior t o hemodiafiltration the animals d i d not i n gest food. In 5 experiments concentration o f amino acids was fnvestigated i n arterial blood before starting, d u r i n g and a t the end of
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
each hemodiafiltration, and i n the u l t r a f i l t r a t e (Technicon Analyzer).
RESULTS
The results are sumnarized i n Table 11. All data are means of a l l experiments.
In the f i r s t column the normal values for
nonuremic man are shown.
Compared w i t h the values o f the uremic
dogs before starting hemodiafiltration (Cbo)
there are no appreci-
able differences except for a higher Concentration of cysteine and threonine i n the dog. During hemodiafiltration there is a decrease i n the concen-
tration of most amino acids fran s t a r t (Cbo) t o the end of treat-
ment (Cbl). Mean decrease i n concentration is fran 305 mg/l t o i.e. about 20 X .
238 mg/l
Plasma alanine and tyrosine, on the
other hand, increased during hemodiafiltration. In the ultraf i l t r a t e there i s about half the concentration of amino acids as i n plasma.
T h i s corresponds t o the expected concentration after
diluting the blood entering the ultrafilter w i t h the diluting fluid.
Thus, penneation of amino acids through the membrane is
not limited. Their clearance is 28.9 ml/min and corresponds t o the theoretically expected clearance of 30 ml/min.
7 a0
MA”
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
Table I1 UF
normal values m a n mg/l
‘bo mg/l
mg/l
mg/l
rng
35-4
40.7
14.3
8.1
288.6 84.8
2.5 2.9
GlY
17.4 12.4
3-5 5.8 27.1 7.3 9.8 15.4
21.5 6.3
CYS
30.7 14.3 2.2 5.3 17.7
Pro
9-2 27.1
17.1
Se 1:
11.8
12.1
Tyr
9.1 7.1 13.2
4.7 9.3 19.6 20.3 5.8 11.3 52.6 30.6
Ala kt3 ASP
Cit
Glu
8.6
His OrIl
Ileu Leu
LYS
Met Phe The Val
23.4
3.2 9.5 19.4 79.9
‘bl
16.8
4.2 I .o 10.0
14.5 41.5 178.2 15.3 69.4 55.7 13.7 143.4
9.7
10.1
735.3
18.i
5.6
9.1 8.2 2.8
2.8
6.4
6- 9 12.9 77.3 3.5 11.5 34.2
20.6
1.1
3.1 13.3 I .I
5-2
3.1 5.5
10.1
12.9 4.5 6.8
18.3
15.2
41.5 73.8
136.0
773-1 60.4
90.8 252.3 203 7
From the amino acid concentration (CUF) and the amount of the u l t r a f f l t r a t e the quantity o f amino acids eliminated per hemodiaf i l t r a t i o n can be derived (UF).
I t is about seven times the quan-
t i t y of amino acids i n one l i t e r o f plasma, i.e. seven times as
much a s the entire quantity o f plasma amino acids i n the dog. Average amino acid loss was 2.07 g during 4 hrs. o f t r e a t ment or about 0.5 g/h. DISCUSSION To evaluate the importance o f amino acid loss i n hemodiafil-
t r a t i o n i t is necessary t o compare amino acid loss i n hemodiafilt r a t i o n w i t h amino acid l o s s In conventional d i a l y s i s therapy and
ACID LOSS DURING HEMOFILTRATION
AMINO
w i t h normal amino acid loss i n the urine.
701
I t is also necessary
to apply the results found i n the dog t o the human. Normal mean daily amino acid excretion i n man i s 2.2 g (1.3 3.2 9).
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
(1,3,7)
During 6
- 12 hrs.
-
o f hemodialysfs by several authors
loss of amino acids of 4.5
- 10.0 g has been described,
average 8.5 g per dialysis run or 1.2 g/h respectively. According to Salisbury the distribution of amino acids i n plasma i n dialyzed and nondialyzed uremics is not different from normals. Rubini and Gordon found no change i n the amino acid spectrum
during dialysis, although the amino acid loss was e i g h t times
greater than the total plasma content. From these results i t can be concluded that the amino acids eliminated from plasma during dialysis are replaced instantly from the intracellular space. loss of about 1
- 2 g/h
T h i s also is the reason why amino acid
throughout one dialysis run is very con-
stant. Similarly, i n our experiments i n dogs, the concentration of free amino acids decreases only about 20 X, although seven times as
much amino acids have been excreted by hemodiafiltration as were i n plasma.
T h i s result also corresponds to the results ,found i n h e m -
dialysis therapy (7). Since the amino acid concentration i n blood i n our experiments corresponds t o the amino acid concentration i n man, our results i n the dog can be conditionally applied t o man.
If these results
are t o be applied t o the conditions i n hemodiafiltration i n man,
702
MA”
d i f f e r e n t clearances induced by d i f f e r e n t f l o w r a t e s o f blood and d i l u t i n g f l u i d necessary f o r e l i m i n a t i o n o f h i g h e r q u a n t i t i e s o f urea and c r e a t i n i n e i n man must a l s o be considered. T h i s may be i l l u s t r a t e d by an example:
Blood f l o w o f 200
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
m l / m i n and u l t r a f i l t r a t e f l o w o f 200 ml/min when s u b s t i t u t i n g the d i l u t i n g f l u i d before the u l t r a f i l t e r (or u l t r a f i l t r a t e flow o f 100 ml/min when s u b s t i t u t i n g t h e d i l u t i n g f l u i d behind t h e u l t r a f i l t e r ) induces a clearance o f urea and amino acids o f 100 ml/min.
H e m o d i a f i l t r a t i o n therapy i n t h i s way i s necessary t o
e l i m i n a t e t h e s o l u t e s normally excreted by t h e kidneys such as urea and c r e a t i n i n e (2,4).
Considering t h i s i l l u s t r a t i o n of
h e m o d i a f i l t r a t i o n i n man w i t h a constant amino a c i d c o n c e n t r a t i o n i n plasma o f about 300 mg/l throughout t h e t i m e o f treatment, i t can be d e r i v e d t h a t t h e r e i s a 1.8 g/h l o s s o f amino acids.
This
corresponds t o t h e l o s s o f amino a c i d s found i n hemodialysis (1,3,
5,6,7,9).
A l t o g e t h e r l o s s o f amino ac ds i n h e m o d i a f i l t r a t i o n
does n o t appear t o be g r e a t e r than i n hemodialysis. I f one decides t o conduct h e m o d i a f i l t r a t i o n i n man w i t h a
clearance o f 50 ml/min, as done by Q u e l l h o r s t e t al.,
t h e amount
o f amino a c i d s l o s t i n h e m o d i a f i l t r a t i o n would be s m a l l e r than i n hemodialysis. F i n a l l y , t h e question may be considered whether t h i s l o s s o f amino acids d u r i n g a r t i f i c f a l kidney therapy must be replaced i n t h e d i l u t i n g f l u i d o r not. i n hemodialysis therapy.
T h i s question can be answered j u s t as I n our experience, w i t h r e g u l a r p r o t e i n
d i e t s , most p a t i e n t s g a i n weight and g e n e r a l l y have no symptoms o f
703
AMINO ACID LOSS DURING HEMOFILTRATION
I t therefore appears unnecessary as a rule
protein deficiency.
t o add amino acids t o the diluting fluid. T h i s a d d i t i o n would certainly raise the price of the diluting fluid considerably.
Ren Fail Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.
ACKNOWLEDGEMENTS This study was supported by Deutsche Forschungsgemeinschaft,
Sonderforschungsberei ch 109.
REFERENCES
1.
Giordino, S., de Pascale, C., de Cristoforo, D., i n Nutrition in Renal Disease, edited by G.M. Berlyne, The Williams Company, Baltimore, 1968.
2.
Henderson, L.W., Livoti, L.G., Ford, C.A., Kelley, A.B., Lysaght, M.J., Trans. Am. SOC. Artif. Int. Organs E:119, 1973.
3.
Kopple, I.D., Swendsind, M.E., Shinaberger, J.H., Umetawa, C.Y., Trans. Am. SOC. Artif. Int. Organs XIJ:309, 1973.
4.
Mann, H., Heintz, R., Brass, H., Biomed. Technik, g:170, 1974. Pickford, I.C., Aber, G.M.,
McGale, E.H.F., 1972.
5.
3a:395,
6.
Quellhorst, E., Doht, B., Rieger, J., XIII. Hamburg, 1976.
7.
R u b i n i , M.E.,
8.
36: 1227, 1957.
9.
Young, G.A.,
C l i n . Chim. Acta.
Congr. EDTA,
and Gordon, S., Nephron, 5:339, 1968.
Salisbury, P.M.,
Dunn, M.S., Murphy, E.A.,
Paisons, F.M.,
Clin. Sci.
I. Clin. Invest.
z:l, 1969.
