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Cystine metabolism in the dog
BIOCHEMICAL
MEDICINE
3, 384-396
Cystine J. C. CRAWHALL,
( 1970)
Metabolism
in the
P. PURKISS,
AND
E.
Dog P.
YOUNG1
Department of Medicine, McGill University ‘Clinic and Division of Clinical Biochemistry, Royal Victoria Hospital, Montreal, Canada, and the Professorial Medical Unit, St. Bartholomew Hospital, London, ECZ, England
Received October 27, 19691
Several diseases associated with hereditary metabolic disorders of the sulfur amino acids have been described. Two of these, cystinuria and cystinosis, are associated with disorders of cystine transport and metabolism, respectively, but little is known of the quantitative aspects of the metabolism of this amino acid. In preparation for future studies on the rate of metabolism of cystine in the human, some animal studies have been carried out. It was decided that a fairly large mammal would be required for these experiments in order that repeated samples of blood could be taken over a short period of time without significantly depleting the total blood volume of the animal; the dog had certain obvious advantages for this kind of study. A total of four dogs was studied, and the problem of the quantitative isolation of cystine from the plasma, free of sulfur-containing metabolites, was approached differently in each successive dog. MATERIALS
AND
METHODS
Reagents 35S-L-cystine was obtained from the Radiochemical Centre, Amersham. Performic acid was prepared by mixing formic acid (50 ml, 98%) with hydrogen peroxide (5 ml, 100 vol %). Animals Dogs were all greyhounds, and further details are shown on Table 1. They were starved overnight but given water ad Zib. The next morning they were anaesthetised with a minimal quantity of Nembutal and maintained throughout the experiment with a minimum of the same anaesthetic. The animals were placed on a warmed operating table and ’ Present address: Institute
for Child
Health, London, 384
England
CYSTINE
XXPERIXENTAL
Pkperiment 110.
Weight of dog, kg
1
20.9
2 :;
25.4
4
30 8 18 0
DETAILS
Blood vol, ml Not determined
2765 2600 1720
:355
METABOLISh
TABLE 1 OF SEPAR.~TE
Total radioactivity injected, pCi
Total cystine injected, mg
48.4
0.26
DOG
EXPER~MEXTS
Plasma cystine concenRadiot&ion, activity, mg/lOO ml &i/kg 0..5
16
Hematocrit, $& 67
1X2
1.38
0 .5s
IO
,I5’1
428 277
2.30 1 ,3') II
(I 4s 0 :zs
Ii 1.-I
7.7 6:i
two intravenous cannulae and one femoral arterial cannula were inserted. The latter was sealed with a three-way stainless steel stopcock with luer fittings. In the last experiment a catheter was inserted into the bladder. The last three experiments were carried out for 8 hours. In the fourth dog, 350 ml of dextrose saline (4% w/v dextrose and 0.18% w/v NaCl was infused into one vein, and 160 ml of mannitol (20% w/v) was infused into the other vein, both infusions being equally spread throughout the 8-hour experimental period. 35S-cystine was dissolved in 0.1 ml of 1 N HCl and diluted to 10 ml with phosphate buffer ( pH 7.4, 0.15 M). Radioactive cystine solution (2 x 0.5 ml) was taken for assay of the radioactivity, and the remainder was injected iv at time zero. Blood samples (10 ml) were collected from the arterial cannula into a heparinised syringe at intervals of time shown on the successive figures. Isotonic saline solution (10 ml) was injected into the contralateral cannulated vein as each blood sample was taken. Urine was collected in thr last dog experiment at 30-minute intervals. At the completion of the experiment, Evans blue (1 ml, 0.2% w/v) was injected iv, and blood was sampled after 10 minutes for the determination of the total blood volume ( 1). Blood was also taken for haematocrit determination at several periods during the experiment. For the measurement of radioactivity, a measured volume of plasma (generally 2.5 ml) was removed from the cells after centrifugation. An equal volume of salicylsulfonic acid (3% w/v) was added, and the precipitated proteins were removed by centrifugation. The precipitate was washed once with deionised water ( 1 ml). The original supernatant and washings were combined. A portion was removed for determination of total radioactivity. The treatment of the remainder varied with each experiment according to the procedure used for isolation of the cystine. Radioactivity was determined on 0.2-ml por-
386
CRAWHALL,
PURKISS,
AND
l-OlrN(:
tions of the deproteinised plasma or other aqueous fractions. Kadioactivity was measured by liquid scintillation counting techniques using either a gel suspension procedure or a dioxan-based phosphor ( 2). Counting was carried out in a refrigerated Nuclear-Chicago liquid scintillation counter. Automatic amino acid analysis was carried out on a Technicon amino acid analyser using gradient elution as previously described (3). In order to carry out all the determinations for the fourth experiment, a short column procedure was devised. This was a column (0.9 X 60 cm) packed with Chromobeads A resin (Technicon) at 65”. The column was eluted at 1.4 ml/min. The effluent was divided with 0.9 ml/min going to a fraction collector and 0.5 ml/min going to the calorimeters. Only th- first five chambers of the autograd were used. The first three chambers contained sodium citrate buffer (0.2 M with respect to sodium ions, pH 2.87). The fourth and fifth chambers contained buffers modified as specified for chambers 4 and 5 in a previous report (3 ) In this system, cystine was srparated from other amino acids and elutecl after 2 hours. RESULTS
The first which the of the total exact zero but in the
IO3
experiment was designed to give preliminary information on design of successive experiments was based. The rate of fall plasma radioactivity is shown for all four dogs in Fig. 1. The time of radioactivity is not known as mixing has to take place, first dog experiment the count/min/ml of plasma at 5 minutes
0. -.._ =---OS___
=--_o_
----___ -----------o 3
4
5 TIME
FIG. 1. Uncorrected teinisation after single 0, Dog 4.
6
7
a
0
(hours)
total radioactivity (count/min/ml) injections of “S-L-cystine. 0,
Dog
of plasma following 1; W, Dog 0: A,
deprohg 3;
CYSTINE
METABOLISM
“87 *I
was 76,000 and had fallen to 7,600 in 96 minutes. This would give an approximate value of tl,2 for cystine in the plasma of 27.4 minutes if the rate of decay was considered as a single exponential. This method would probably measure the radioactivity of various metabolites and impurities as will be discussed later, so it was preferable to try to establish specific activity figures for cystine. The concentration of cystine in the plasma was measured at two different times by automatic amino acid analysis and was found to be 0.50 mg/ 100 ml at zero time and 0.28 mg/ 100 ml at 6 hours. This fall in concentration introduces a complication into the calculation, but for the purposes of this experiment it will be assumed that the concentration change was not significant in the first 2 hours of the experiment. To determine the radioactivity of the cystine itself. an aliquot of the plasma was passed through an ion exchange column (Zeocarb 225; 100-200 mesh, H+, 10 X 0.9 cm), and any acid metabolites or impurities were allowed to pass through in the neutral eluate (50 ml ) It was found (Fig. 2) that the amount of radioactivity in each of the neutral eluates did not change indicating that there was a small amount which was not metabolised in of an acidic impurity in the :‘S-cystine the dog. The columns were then eked with aqueous ammonium h\:droxide (2 N), and the radioactivity of the ammonia eluate again measured ( Fig. 2). This fraction should approximate to a true cystine fraction. The remainder of these fractions were oxidised with performic acid.
I
‘0
20
I
I
I
4
8
40
60
80
100
I20
hns)
3
TIME
4
5
(hours)
FIG. 2. Symbols ( A ) represent the radioactivity in the neutral eluate of a catiog exchange resin column (Zeocarb 225,H’) of the deproteinized plasma after injection of ““S-cystine IV. This fraction contained oxidised products of cystine. The colamn was then eluted with aqueous ammonia (2 N). This fraction ( 0 ) contained cystinr only.
388
CRAWHALL,
PURKISS,
AND
YOU h G
and the specimens again applied to a cation exchange column (Zeocarb 225). The cystine was now oxidised to cysteic acid and appeared in the neutral eluate. The radioactivity of this fraction was counted and in all cases corresponded to at least a 90% recovery of the radioactivity in the previous ammonia eluate fractions. From 5 to 15 minutes the initial fall of radioactivity corresponding to cystine had a tl,,:: of 10 minutes, and during the next period of 15 to 40 minutes the radioactivity again fell by one half giving a tl,:: of 25 minutes although the rest of this curve did not conform to a single exponential decay rate. During these periods the plasma cystine concentration could probably be considered as constant. The protocol was modified for the second dog experiment by administering more radioactive cystine and carrying out the experiment for a longer period of time. The rate of change of total radioactivity in the plasma is shown in Fig. 1. An aliquot of each deproteinised plasma sample was applied to the cation exchange resin column and &ted first with water and then with ammonia as described above. The amount of radioactivity in the ammonia eluate is shown in Fig. 3. The ammonia eluates were evaporated to dryness and oxidised with performic acid (2.5 ml) for 2 h ours at 4”. Water (20 ml) was then added, and the performic acid was removed by evaporation in a rotary evaporator. The residue was redissolved in water and applied to another similar ion exchange resin column. This was again eluted with water and ammonia (2 N). All the radioactive cystine had been oxidiscd to cysteic acid.
III. ‘0510
20
40
60
80 TIME
FIG. (Zeocarb fraction
3. Radioactivity in the ammonia 225,H’) of the deproteinised contained cystine only.
eluate plasma
100 (mind
120
140
160
180
of a cation exchange resin after injection of “S-cystine.
column This
CYSTINE
389
METABOLISM
A graph of the change of cystine radioactivity with time in this dog where radioactivity was plotted on a logarithmic scale showed that, after 45 minutes, the radioactivity decreased at an approximately linear rate and could thus be represented by a single exponential function. By subtracting intercepts on this line from the earlier experimental points (see Appendix), a second line was obtained representing an initial exponential decay. An equation was then obtained representing the sum of these two exponential terms, and some calculations of rate of turnover of radioactive cystine were carried out as described in the Appendix and summarized in Table 2. The radioactivity at zero time, calculated by putting t = o in the derived equation for this dog, was 47,500 dpm/ml. The total radioactivity injected into the dog was 4.04 x lo8 dpm so that the initial dilution space was 8500 ml. The plasma volume of the dog was only 1300 ml, so that an immediate dilution had occurred into a volume greater than the plasma volume. The total weight of the dog was 25.4 kg, and if the total extracellular space was taken as 15% of the gross weight and the plasma space as 5% of the gross weight then these combined volumes would amount to 5080 ml, and hence the dilution of radioactivity from 6 minutes onward was into a volume somewhat greater than the total vascular and extracellular space of the dog. The cystine content of the plasma was checked by direct automatic amino acid analysis in two of the early timed plasma specimens. An TABLE CALCULATED
1st Pool
2nd Pool
Initial IniCal
dilution dilution
Poor,
SIZE
.4m
Pool size, Turnover Mg/min/total mMoles/kg/hl il,2, mm Pool size, Turnover mg/min/total mmoles/kg/hl tlfr, mm
mg/kg rate
R.4TE
hodg body
mg/kg rate
space, ml space/kg body wt, ml Derived equation Derived
2
TI-RNOVER
equation
body
wt
FOR
DOGS
wt
DCK; 24
wt
:: 2-I II;‘, d 13 0 89
w-t
body
DATA
16
016 98 0 X.500 334 for dog 2. A = 38,OOOe-0 541 +9, 500e-~0071 t for dog 4. A = 99, T,OOe-.‘07t +20,000e-~023t
2
2 AND 4 DO(: 4 :; 4
396
CRAWHALL,
PURKISS,
AND
POUNC
attempt was made to quantitate the cysteic acid that was formed by the performic acid oxidation of the plasma cystine using the previously described column chromatographic techniques, The cysteic acid was determined by a manual ninhydrin procedure (4), but with the small quantities of cysteic acid present, rather variable results were obtained. However, the results showed no significant difference in the cysteic acid present in the specimens taken during the first 6 hours of the experiment. Any acid metabolites of cystine would have been expected to appear in the neutral eluate of the first column, but as in the previous experiment there was no change in the radioactivity of that fraction for 4 hours. After that there was an increase of radioactivity by about 25% which still represented only a fraction of 1% of the original cystine injected. The third experiment was on similar lines to the previous experiments. The rate of decrease of total radioactivity in the plasma was similar to that in the previous experiments (Fig. 1). Attempts were made to separate all the amino acids from each plasma specimen by automatic amino acid analysis. The results obtained on one plasma sample where all the fractions were counted showed that nearly all the radioactivity was located in the cystine peak. Minor peaks were seen which could correspond to fully oxidised sulfur compounds which elute very rapidly off the column. One small peak preceded cystine but was not identified. One small peak after cystine probably corresponded to cystathionine. It was unfortunately not possible to process all the specimens this way, and no data are available for the change of true cystine radioactivity with time in this experiment. The protocol for the fourth experiment was essentially the same as for the previous experiments except that more use was made of automatic amino acid analysis. A portion of the original ““S-cystine solution was fractionated on the amino acid analyser, and it was found that 5% of the radioactivity was eluted rapidly off the column. This finding was confirmed by submitting the 35S cystine to high voltage electrophoresis in 6.8% formic acid. The high voltage electrophoresis strip was cut into sections, and the radioactivity determined by liquid scintillation counting. In the area corresponding to substances with an overall negative charge, 1.5% of the total radioactivity was found. The area of the radioactivity did not correspond to taurine but rather to cysteic acid. The reason why column chromatography showed the presence of more impurity than high voltage electrophoresis is not known. A portion of each plasma sample was fractionated by automatic amino acid analysis using a short column technique as described in the Methods section. There was no consistent change of cystine concentration though there was some fluctuation between one sample and the next. The determination of radio-
CYSTINE
METABOLISh
:391
activity in the cystine peak could only be carried out for 1% hours after which the values observed became too low to measure in the eluates off the Autoanalyser column. These results are shown in Fig. 4. Calculations similar to these described for dog 2 in the Appendix were carried out for the results obtained from this experiment. In this case the first six points from 3 to 30 minutes comprise the first exponent, and there are only two points, at 45 and 60 minutes, representing the beginning of a second line. The results are again summarized in Table 2. Putting t = 0 in the derived equation gave a value for the initial activity of 119,500 dpm/ml plasma. The total radioactivity injected was 6.13 X lo” dpm. There was therefore a dilution volume of 5250 ml. The dog weighed 18 kg so if we consider the combined ECF and plasma of the dog as 20% of its weight, this gives a volume of 3.6 litres. The initial dilution volume was thus once more larger than the combined ECF and plasma volume. Pool size data are again summarized in Table 2. Urine samples were collected during the course of the experiment, and their radioactivity is expressed in Fig. 5 in terms of minute volumes in order to demonstrate the rate at which radioactivity was appearing in the urine. Attempts were made to identify the radioactive excretion products by high voltage electrophoresis, but these were technically unsuccessful probably because of the high salt and mannitol concentrations. However, the total sum of all the urine radioactivity was only 5% of the radioactivity injected into the dog and so did not represent a significant pathway for the excretion of radioactive metabolites.
cl
.I1 0
1
3
'
6
* 9
15
20
30
TIME FIG. obtained
40
50
60
70
(mins)
4. Radioactivity of plasma cystine for in the same way as these on Fig. 3.
dog
4. The
points
on this
graph
were
392
CRAWHALL,
PURKISS,
TIME
FIG.
initially measured
AiVD
YOUlG
(hours)
5. Radioactivity excreted in the urine of dog 4. Urine at 30-minute intervals and later at l-hour intervals, before counting aliquots.
collections were and the volumes
made were
DISCUSSION
The initial aim of these experiments was to determine the rate of disappearance of injected cystine from the plasma and if possible to observe an equilibrium state. The results, however, showed a very rapid disappearance of the radioactivity from the plasma such that no measurements could be made in an equilibrium state. The method of separating the cystine from other acidic sulfur compounds on a short cation exchange resin as employed in the first two experiments was the most satisfactory although less precise than the method of fully automatic amino acid analysis employed in the fourth dog. The disadvantage of the latter method was that the buffers employed in this procedure could not be readily .removed, and hence only a small fraction of the total eluate could be analysed for radioactivity and there was a big loss of sensitivity of the method in this respect. The two procedures could be regarded as complementary to each other, but the results obtained were not the same, either because of the difference of methodology or because of biological variation between the various dogs. In dogs 2 and 4 it was possible to show that there was a rapid equilibration of the radioactive cystine into a volume greater than the sum of the extravascular space plus the plasma which was similar in both dogs when corrected to milliliters per kilogram body weight (Table 2). In both dogs there was then a fairly rapid rate of disappearance of radioactivity for the next 35 minutes. This decrease had a tl,z of 13 minutes in dog 2 and 6.5 minutes in dog 4, when calculated from the first
CYSTINE
METABOLISM
393
exponential function of each of the equations (Table 2). The reason for the difference in the two rates is not known but could just represent biological variation between the animals or possibly be related to the different analytical techniques used in the experiment. No significant metabolites were observed in the plasma or urine so it would be reasonable to suggest that this represented intracellular uptake of cystine by the tissues from the plasma. In these two dogs, it is thought that the first exponential curve simply represents equilibration of the radioactive cystine into the intracellular space while the second exponent represents turnover of cystine. The rate of this disappearance will vary depending on the membrane transport processes of the tissues involved. These have been measured in in vitro experiments for some tissues of the rat (5). It was shown in those experiments that various tissues can maintain a concentration gradient for cystine although diaphragm muscle may be an exception. In several tissues the intracellular cystine concentration (probably in the reduced form as cysteine) has been shown to be about three times that of the plasma. The intracellular fluid volume is generally taken as being about two and a half times that of the combined extracellular and plasma volume. Hence equilibration of radioactivity would be taking place with an intracellular pool seven and a half times bigger that the plasma extracellular space pool. The apparent pool size for the equilibration of cystine was similar in the two dogs (Table 2). The tl,2 for the disappearance of radioactive cystine was greater in dog 2 than in dog 4 for both pools, and conversely the turnover rate of cystine in these hypothetical pools was lower in dog 2 than dog 4. The turnover rate of cystine in the second pool may mart’ nearly represent the true turnover rate of cystine if the first pool represents the rate of transport of cystine from the plasma-extracellular space into the intracellular space. This turnover rate of the second pool was 16 pmole/kg/hr in dog 2 and 38 ~molelkglhr in dog 4. The value for dog 4 was based on only two data points so that the true value is probably closer to that found in dog 2 which was based on five data points. Some radioactivity was excreted in the urine of the fourth dog which was infused with mannitol to maintain a diuresis. Very little urine was formed during the experiment in the previous dogs so that this was not a significant source of loss of radioactivity. Very little comparable data on amino acid turnover are available but some of the information on glycine turnover has been reviewed by Wyngaarden and Segal (6). No comparable data are available for dogs but U’atts and Crawhall (7) showed in man that the glycine pool size \vas 80 mg/kg body wt which is about ten times the cystine pool size reported
394
CRAWHALL,
PURKISS,
AND
YOUNG
for dogs in this paper. The turnover rate for glycine in man was estimated to be 44 mg/kg/hr ( .59 mmole/kg/hr ) . This value should be compared with the turnover rate found for the second pool in the dogs (because the first pool probably represented intracellular equilibration), The fractional turnover rate for glycine in man (7) (turnover rate/ hour)/pool size was found to be 55%. In these experiments for cystine in the dog the fractional turnover rate was found to be 43% in dog 2 and 146% in dog 4. These values indicate a comparable high fractional turnover rate for cystine in the dog although the pool size was much smaller. More detailed interpretation of these results was complicated by three problems. Firstly, that there was such a rapid decrease of radioactivity in the plasma during the first hour that the radioactivity was lower than the sensitivity of the isolation procedure before a true equilibrium state was reached. Secondly, the rate of decrease could not be expressed as a simple exponential, and thirdly the rate of decrease of radioactivity in the plasma was a combination of transport from the plasma into the intracellular space as well as a component of metabolism. SUMMARY
YS-L-cystine was injected intravenously into anaesthetised dogs, and the rate of disappearance of radioactive cystine was measured. Cystine was isolated from the plasma by two different techniques before determination of the radioactivity. It was shown that there was an almost instantaneous equilibration of the isotope into a volume greater than the plasma and extracellular space. The rate of decrease of radioactivity of plasma cystine was expressed for two dogs as the sum of two exponential terms. The first term may represent uptake of extracellular cystine into the intracellular space, and the second term may more closely represent the rate of turnover of radioactive cystine. The values corresponded to a turnover rate of 16 pmolel kglhr in one dog and 38 pmole/ kg/ hr in another dog. The fate of the Yi-L-cystine could not be determined, but it is believed that most of the disappearance of radioactivity could be explained by the uptake of the amino acid by the tissues and equilibration with the intracellular amino acid pool. ACKNOWLEDGMENTS We thank Dr. C. J. Thompson and Mr. Peter Berkowitz for his the regression analysis. We also thank Mr. A. Edwards
for assistance assistance with and
Mr.
P. Kiely
with the earlier part of this work the computer calculations used in who
performed
the surgical
pro-
CYSTINE
cedures. This work Grant No. 249.34.
was supported
7395
METABOLISM
in part
by the Canadian
Medical
Research
Council
APPENDIX
A plot of the logarithm of cystine radioactivity against time for dog 2 (Fig. 3) shows that the decrease in activity is approximately linear after 45 minutes and can thus be represented by a function of the form
where
A is the radioactivity (in dpm/ml) at time t B is a constant corresponding to the radioactivity at zero time, and k is the decay constant for this function. Values for B and k were deduced by regression analysis on the experimental data for the variation of radioactivity with time from 45 minutes to the end of the experiment, and a graph of the regression line so obtained was plotted and extrapolated to zero time. Intercepts on this line were drawn at the times at which determinations of radioactivity were made in the initial 45 minutes of the experiment, and the values of these intercepts on the ordinate were subtracted from the total radioactivity in the plasma at each point giving a further series of points which regression analysis showed to lie on a line represented by A’ = Bte--h-lt The sum produced exception It was different
of these two terms gave an expression which, when plotted, a curve agreeing closely with the experimental curve with the of the 2.5-minute point. assumed that each term of the form A = Be-“’ represented a cystine pool, and values for various parameters for each pool
Parameter 1. Init,ial
radioactivity
2. Initial
dilrition
Method
of derivation
A = R when space,
T’
t = 0
T’ = ;
5. Half
life,
rate,
t1j2
Radioactivity in iiiitial mixing
Total quantity p001 X
I< = ,(.I1 I
tj/4 =
=hZ
interpretation the
pool
after
Volume into which radioactivity is diluted in the pool represeiit,ed by this term
3. Pool size, J/ 4. Turnover
Physical
A
111 2 0.693 F = 7
In
of cystine
an eyliilihritnn situat,ion trunover rate represents rate of incorporatioii itito orit of the pool.
in the the the at111
Time takeii for half t,he cyst ine iii the pool to be replaced
396 were sents stant, From tivity
CRAWHALL,
PURKISS,
AND
YOUNG
calculated as shown in the following table. In this table, C reprethe concentration of cystine in the plasma, which is assumed conand X is the total radioactivity injected. the combined equations, the total volume into which the radioacwas initially diluted is given by I/tot:*1
=
s
B +
*,
A similar analysis was carried out for dog 4 where the last two points were considered as belonging to the second exponential and the earlier points as belonging to the first exponential. REFERENCES 1. PXTS, R. F., “Physiology of the Kidney and Body Fluids,” 2.nd Ed., p. 24. Year Book Medical Publishers, Chicago, 1963. 2. BRAY, G. A., Anal. Biochem. 1,279 ( 1960). 3. CRAWHALL, J. C., THOMPSON, C. J., AND BRADLEY, K. H., Anal. Biochem. 14, 405 ( 1966). 4. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211,907 (1954). 5. CRA~HALL, J. C., AXD SEGAL, S., Biochem. J. 105, 891 ( 1967). 6. WYNGAARDEN, J. B., AND SEGAL, S., in “The Metabolic Basis of Inherited Disease” J. B. Stanbury, J. B. Wyngaarden, and D. S. Frederickson, eds.), 2nd Ed., p. 341. McGraw-Hill, New York, 1966. 7. WARS, R. W. E., AND CRAWHALL, J. C., Biochem. J. 73, 277 (1959 ).
MEDICINE
3, 384-396
Cystine J. C. CRAWHALL,
( 1970)
Metabolism
in the
P. PURKISS,
AND
E.
Dog P.
YOUNG1
Department of Medicine, McGill University ‘Clinic and Division of Clinical Biochemistry, Royal Victoria Hospital, Montreal, Canada, and the Professorial Medical Unit, St. Bartholomew Hospital, London, ECZ, England
Received October 27, 19691
Several diseases associated with hereditary metabolic disorders of the sulfur amino acids have been described. Two of these, cystinuria and cystinosis, are associated with disorders of cystine transport and metabolism, respectively, but little is known of the quantitative aspects of the metabolism of this amino acid. In preparation for future studies on the rate of metabolism of cystine in the human, some animal studies have been carried out. It was decided that a fairly large mammal would be required for these experiments in order that repeated samples of blood could be taken over a short period of time without significantly depleting the total blood volume of the animal; the dog had certain obvious advantages for this kind of study. A total of four dogs was studied, and the problem of the quantitative isolation of cystine from the plasma, free of sulfur-containing metabolites, was approached differently in each successive dog. MATERIALS
AND
METHODS
Reagents 35S-L-cystine was obtained from the Radiochemical Centre, Amersham. Performic acid was prepared by mixing formic acid (50 ml, 98%) with hydrogen peroxide (5 ml, 100 vol %). Animals Dogs were all greyhounds, and further details are shown on Table 1. They were starved overnight but given water ad Zib. The next morning they were anaesthetised with a minimal quantity of Nembutal and maintained throughout the experiment with a minimum of the same anaesthetic. The animals were placed on a warmed operating table and ’ Present address: Institute
for Child
Health, London, 384
England
CYSTINE
XXPERIXENTAL
Pkperiment 110.
Weight of dog, kg
1
20.9
2 :;
25.4
4
30 8 18 0
DETAILS
Blood vol, ml Not determined
2765 2600 1720
:355
METABOLISh
TABLE 1 OF SEPAR.~TE
Total radioactivity injected, pCi
Total cystine injected, mg
48.4
0.26
DOG
EXPER~MEXTS
Plasma cystine concenRadiot&ion, activity, mg/lOO ml &i/kg 0..5
16
Hematocrit, $& 67
1X2
1.38
0 .5s
IO
,I5’1
428 277
2.30 1 ,3') II
(I 4s 0 :zs
Ii 1.-I
7.7 6:i
two intravenous cannulae and one femoral arterial cannula were inserted. The latter was sealed with a three-way stainless steel stopcock with luer fittings. In the last experiment a catheter was inserted into the bladder. The last three experiments were carried out for 8 hours. In the fourth dog, 350 ml of dextrose saline (4% w/v dextrose and 0.18% w/v NaCl was infused into one vein, and 160 ml of mannitol (20% w/v) was infused into the other vein, both infusions being equally spread throughout the 8-hour experimental period. 35S-cystine was dissolved in 0.1 ml of 1 N HCl and diluted to 10 ml with phosphate buffer ( pH 7.4, 0.15 M). Radioactive cystine solution (2 x 0.5 ml) was taken for assay of the radioactivity, and the remainder was injected iv at time zero. Blood samples (10 ml) were collected from the arterial cannula into a heparinised syringe at intervals of time shown on the successive figures. Isotonic saline solution (10 ml) was injected into the contralateral cannulated vein as each blood sample was taken. Urine was collected in thr last dog experiment at 30-minute intervals. At the completion of the experiment, Evans blue (1 ml, 0.2% w/v) was injected iv, and blood was sampled after 10 minutes for the determination of the total blood volume ( 1). Blood was also taken for haematocrit determination at several periods during the experiment. For the measurement of radioactivity, a measured volume of plasma (generally 2.5 ml) was removed from the cells after centrifugation. An equal volume of salicylsulfonic acid (3% w/v) was added, and the precipitated proteins were removed by centrifugation. The precipitate was washed once with deionised water ( 1 ml). The original supernatant and washings were combined. A portion was removed for determination of total radioactivity. The treatment of the remainder varied with each experiment according to the procedure used for isolation of the cystine. Radioactivity was determined on 0.2-ml por-
386
CRAWHALL,
PURKISS,
AND
l-OlrN(:
tions of the deproteinised plasma or other aqueous fractions. Kadioactivity was measured by liquid scintillation counting techniques using either a gel suspension procedure or a dioxan-based phosphor ( 2). Counting was carried out in a refrigerated Nuclear-Chicago liquid scintillation counter. Automatic amino acid analysis was carried out on a Technicon amino acid analyser using gradient elution as previously described (3). In order to carry out all the determinations for the fourth experiment, a short column procedure was devised. This was a column (0.9 X 60 cm) packed with Chromobeads A resin (Technicon) at 65”. The column was eluted at 1.4 ml/min. The effluent was divided with 0.9 ml/min going to a fraction collector and 0.5 ml/min going to the calorimeters. Only th- first five chambers of the autograd were used. The first three chambers contained sodium citrate buffer (0.2 M with respect to sodium ions, pH 2.87). The fourth and fifth chambers contained buffers modified as specified for chambers 4 and 5 in a previous report (3 ) In this system, cystine was srparated from other amino acids and elutecl after 2 hours. RESULTS
The first which the of the total exact zero but in the
IO3
experiment was designed to give preliminary information on design of successive experiments was based. The rate of fall plasma radioactivity is shown for all four dogs in Fig. 1. The time of radioactivity is not known as mixing has to take place, first dog experiment the count/min/ml of plasma at 5 minutes
0. -.._ =---OS___
=--_o_
----___ -----------o 3
4
5 TIME
FIG. 1. Uncorrected teinisation after single 0, Dog 4.
6
7
a
0
(hours)
total radioactivity (count/min/ml) injections of “S-L-cystine. 0,
Dog
of plasma following 1; W, Dog 0: A,
deprohg 3;
CYSTINE
METABOLISM
“87 *I
was 76,000 and had fallen to 7,600 in 96 minutes. This would give an approximate value of tl,2 for cystine in the plasma of 27.4 minutes if the rate of decay was considered as a single exponential. This method would probably measure the radioactivity of various metabolites and impurities as will be discussed later, so it was preferable to try to establish specific activity figures for cystine. The concentration of cystine in the plasma was measured at two different times by automatic amino acid analysis and was found to be 0.50 mg/ 100 ml at zero time and 0.28 mg/ 100 ml at 6 hours. This fall in concentration introduces a complication into the calculation, but for the purposes of this experiment it will be assumed that the concentration change was not significant in the first 2 hours of the experiment. To determine the radioactivity of the cystine itself. an aliquot of the plasma was passed through an ion exchange column (Zeocarb 225; 100-200 mesh, H+, 10 X 0.9 cm), and any acid metabolites or impurities were allowed to pass through in the neutral eluate (50 ml ) It was found (Fig. 2) that the amount of radioactivity in each of the neutral eluates did not change indicating that there was a small amount which was not metabolised in of an acidic impurity in the :‘S-cystine the dog. The columns were then eked with aqueous ammonium h\:droxide (2 N), and the radioactivity of the ammonia eluate again measured ( Fig. 2). This fraction should approximate to a true cystine fraction. The remainder of these fractions were oxidised with performic acid.
I
‘0
20
I
I
I
4
8
40
60
80
100
I20
hns)
3
TIME
4
5
(hours)
FIG. 2. Symbols ( A ) represent the radioactivity in the neutral eluate of a catiog exchange resin column (Zeocarb 225,H’) of the deproteinized plasma after injection of ““S-cystine IV. This fraction contained oxidised products of cystine. The colamn was then eluted with aqueous ammonia (2 N). This fraction ( 0 ) contained cystinr only.
388
CRAWHALL,
PURKISS,
AND
YOU h G
and the specimens again applied to a cation exchange column (Zeocarb 225). The cystine was now oxidised to cysteic acid and appeared in the neutral eluate. The radioactivity of this fraction was counted and in all cases corresponded to at least a 90% recovery of the radioactivity in the previous ammonia eluate fractions. From 5 to 15 minutes the initial fall of radioactivity corresponding to cystine had a tl,,:: of 10 minutes, and during the next period of 15 to 40 minutes the radioactivity again fell by one half giving a tl,:: of 25 minutes although the rest of this curve did not conform to a single exponential decay rate. During these periods the plasma cystine concentration could probably be considered as constant. The protocol was modified for the second dog experiment by administering more radioactive cystine and carrying out the experiment for a longer period of time. The rate of change of total radioactivity in the plasma is shown in Fig. 1. An aliquot of each deproteinised plasma sample was applied to the cation exchange resin column and &ted first with water and then with ammonia as described above. The amount of radioactivity in the ammonia eluate is shown in Fig. 3. The ammonia eluates were evaporated to dryness and oxidised with performic acid (2.5 ml) for 2 h ours at 4”. Water (20 ml) was then added, and the performic acid was removed by evaporation in a rotary evaporator. The residue was redissolved in water and applied to another similar ion exchange resin column. This was again eluted with water and ammonia (2 N). All the radioactive cystine had been oxidiscd to cysteic acid.
III. ‘0510
20
40
60
80 TIME
FIG. (Zeocarb fraction
3. Radioactivity in the ammonia 225,H’) of the deproteinised contained cystine only.
eluate plasma
100 (mind
120
140
160
180
of a cation exchange resin after injection of “S-cystine.
column This
CYSTINE
389
METABOLISM
A graph of the change of cystine radioactivity with time in this dog where radioactivity was plotted on a logarithmic scale showed that, after 45 minutes, the radioactivity decreased at an approximately linear rate and could thus be represented by a single exponential function. By subtracting intercepts on this line from the earlier experimental points (see Appendix), a second line was obtained representing an initial exponential decay. An equation was then obtained representing the sum of these two exponential terms, and some calculations of rate of turnover of radioactive cystine were carried out as described in the Appendix and summarized in Table 2. The radioactivity at zero time, calculated by putting t = o in the derived equation for this dog, was 47,500 dpm/ml. The total radioactivity injected into the dog was 4.04 x lo8 dpm so that the initial dilution space was 8500 ml. The plasma volume of the dog was only 1300 ml, so that an immediate dilution had occurred into a volume greater than the plasma volume. The total weight of the dog was 25.4 kg, and if the total extracellular space was taken as 15% of the gross weight and the plasma space as 5% of the gross weight then these combined volumes would amount to 5080 ml, and hence the dilution of radioactivity from 6 minutes onward was into a volume somewhat greater than the total vascular and extracellular space of the dog. The cystine content of the plasma was checked by direct automatic amino acid analysis in two of the early timed plasma specimens. An TABLE CALCULATED
1st Pool
2nd Pool
Initial IniCal
dilution dilution
Poor,
SIZE
.4m
Pool size, Turnover Mg/min/total mMoles/kg/hl il,2, mm Pool size, Turnover mg/min/total mmoles/kg/hl tlfr, mm
mg/kg rate
R.4TE
hodg body
mg/kg rate
space, ml space/kg body wt, ml Derived equation Derived
2
TI-RNOVER
equation
body
wt
FOR
DOGS
wt
DCK; 24
wt
:: 2-I II;‘, d 13 0 89
w-t
body
DATA
16
016 98 0 X.500 334 for dog 2. A = 38,OOOe-0 541 +9, 500e-~0071 t for dog 4. A = 99, T,OOe-.‘07t +20,000e-~023t
2
2 AND 4 DO(: 4 :; 4
396
CRAWHALL,
PURKISS,
AND
POUNC
attempt was made to quantitate the cysteic acid that was formed by the performic acid oxidation of the plasma cystine using the previously described column chromatographic techniques, The cysteic acid was determined by a manual ninhydrin procedure (4), but with the small quantities of cysteic acid present, rather variable results were obtained. However, the results showed no significant difference in the cysteic acid present in the specimens taken during the first 6 hours of the experiment. Any acid metabolites of cystine would have been expected to appear in the neutral eluate of the first column, but as in the previous experiment there was no change in the radioactivity of that fraction for 4 hours. After that there was an increase of radioactivity by about 25% which still represented only a fraction of 1% of the original cystine injected. The third experiment was on similar lines to the previous experiments. The rate of decrease of total radioactivity in the plasma was similar to that in the previous experiments (Fig. 1). Attempts were made to separate all the amino acids from each plasma specimen by automatic amino acid analysis. The results obtained on one plasma sample where all the fractions were counted showed that nearly all the radioactivity was located in the cystine peak. Minor peaks were seen which could correspond to fully oxidised sulfur compounds which elute very rapidly off the column. One small peak preceded cystine but was not identified. One small peak after cystine probably corresponded to cystathionine. It was unfortunately not possible to process all the specimens this way, and no data are available for the change of true cystine radioactivity with time in this experiment. The protocol for the fourth experiment was essentially the same as for the previous experiments except that more use was made of automatic amino acid analysis. A portion of the original ““S-cystine solution was fractionated on the amino acid analyser, and it was found that 5% of the radioactivity was eluted rapidly off the column. This finding was confirmed by submitting the 35S cystine to high voltage electrophoresis in 6.8% formic acid. The high voltage electrophoresis strip was cut into sections, and the radioactivity determined by liquid scintillation counting. In the area corresponding to substances with an overall negative charge, 1.5% of the total radioactivity was found. The area of the radioactivity did not correspond to taurine but rather to cysteic acid. The reason why column chromatography showed the presence of more impurity than high voltage electrophoresis is not known. A portion of each plasma sample was fractionated by automatic amino acid analysis using a short column technique as described in the Methods section. There was no consistent change of cystine concentration though there was some fluctuation between one sample and the next. The determination of radio-
CYSTINE
METABOLISh
:391
activity in the cystine peak could only be carried out for 1% hours after which the values observed became too low to measure in the eluates off the Autoanalyser column. These results are shown in Fig. 4. Calculations similar to these described for dog 2 in the Appendix were carried out for the results obtained from this experiment. In this case the first six points from 3 to 30 minutes comprise the first exponent, and there are only two points, at 45 and 60 minutes, representing the beginning of a second line. The results are again summarized in Table 2. Putting t = 0 in the derived equation gave a value for the initial activity of 119,500 dpm/ml plasma. The total radioactivity injected was 6.13 X lo” dpm. There was therefore a dilution volume of 5250 ml. The dog weighed 18 kg so if we consider the combined ECF and plasma of the dog as 20% of its weight, this gives a volume of 3.6 litres. The initial dilution volume was thus once more larger than the combined ECF and plasma volume. Pool size data are again summarized in Table 2. Urine samples were collected during the course of the experiment, and their radioactivity is expressed in Fig. 5 in terms of minute volumes in order to demonstrate the rate at which radioactivity was appearing in the urine. Attempts were made to identify the radioactive excretion products by high voltage electrophoresis, but these were technically unsuccessful probably because of the high salt and mannitol concentrations. However, the total sum of all the urine radioactivity was only 5% of the radioactivity injected into the dog and so did not represent a significant pathway for the excretion of radioactive metabolites.
cl
.I1 0
1
3
'
6
* 9
15
20
30
TIME FIG. obtained
40
50
60
70
(mins)
4. Radioactivity of plasma cystine for in the same way as these on Fig. 3.
dog
4. The
points
on this
graph
were
392
CRAWHALL,
PURKISS,
TIME
FIG.
initially measured
AiVD
YOUlG
(hours)
5. Radioactivity excreted in the urine of dog 4. Urine at 30-minute intervals and later at l-hour intervals, before counting aliquots.
collections were and the volumes
made were
DISCUSSION
The initial aim of these experiments was to determine the rate of disappearance of injected cystine from the plasma and if possible to observe an equilibrium state. The results, however, showed a very rapid disappearance of the radioactivity from the plasma such that no measurements could be made in an equilibrium state. The method of separating the cystine from other acidic sulfur compounds on a short cation exchange resin as employed in the first two experiments was the most satisfactory although less precise than the method of fully automatic amino acid analysis employed in the fourth dog. The disadvantage of the latter method was that the buffers employed in this procedure could not be readily .removed, and hence only a small fraction of the total eluate could be analysed for radioactivity and there was a big loss of sensitivity of the method in this respect. The two procedures could be regarded as complementary to each other, but the results obtained were not the same, either because of the difference of methodology or because of biological variation between the various dogs. In dogs 2 and 4 it was possible to show that there was a rapid equilibration of the radioactive cystine into a volume greater than the sum of the extravascular space plus the plasma which was similar in both dogs when corrected to milliliters per kilogram body weight (Table 2). In both dogs there was then a fairly rapid rate of disappearance of radioactivity for the next 35 minutes. This decrease had a tl,z of 13 minutes in dog 2 and 6.5 minutes in dog 4, when calculated from the first
CYSTINE
METABOLISM
393
exponential function of each of the equations (Table 2). The reason for the difference in the two rates is not known but could just represent biological variation between the animals or possibly be related to the different analytical techniques used in the experiment. No significant metabolites were observed in the plasma or urine so it would be reasonable to suggest that this represented intracellular uptake of cystine by the tissues from the plasma. In these two dogs, it is thought that the first exponential curve simply represents equilibration of the radioactive cystine into the intracellular space while the second exponent represents turnover of cystine. The rate of this disappearance will vary depending on the membrane transport processes of the tissues involved. These have been measured in in vitro experiments for some tissues of the rat (5). It was shown in those experiments that various tissues can maintain a concentration gradient for cystine although diaphragm muscle may be an exception. In several tissues the intracellular cystine concentration (probably in the reduced form as cysteine) has been shown to be about three times that of the plasma. The intracellular fluid volume is generally taken as being about two and a half times that of the combined extracellular and plasma volume. Hence equilibration of radioactivity would be taking place with an intracellular pool seven and a half times bigger that the plasma extracellular space pool. The apparent pool size for the equilibration of cystine was similar in the two dogs (Table 2). The tl,2 for the disappearance of radioactive cystine was greater in dog 2 than in dog 4 for both pools, and conversely the turnover rate of cystine in these hypothetical pools was lower in dog 2 than dog 4. The turnover rate of cystine in the second pool may mart’ nearly represent the true turnover rate of cystine if the first pool represents the rate of transport of cystine from the plasma-extracellular space into the intracellular space. This turnover rate of the second pool was 16 pmole/kg/hr in dog 2 and 38 ~molelkglhr in dog 4. The value for dog 4 was based on only two data points so that the true value is probably closer to that found in dog 2 which was based on five data points. Some radioactivity was excreted in the urine of the fourth dog which was infused with mannitol to maintain a diuresis. Very little urine was formed during the experiment in the previous dogs so that this was not a significant source of loss of radioactivity. Very little comparable data on amino acid turnover are available but some of the information on glycine turnover has been reviewed by Wyngaarden and Segal (6). No comparable data are available for dogs but U’atts and Crawhall (7) showed in man that the glycine pool size \vas 80 mg/kg body wt which is about ten times the cystine pool size reported
394
CRAWHALL,
PURKISS,
AND
YOUNG
for dogs in this paper. The turnover rate for glycine in man was estimated to be 44 mg/kg/hr ( .59 mmole/kg/hr ) . This value should be compared with the turnover rate found for the second pool in the dogs (because the first pool probably represented intracellular equilibration), The fractional turnover rate for glycine in man (7) (turnover rate/ hour)/pool size was found to be 55%. In these experiments for cystine in the dog the fractional turnover rate was found to be 43% in dog 2 and 146% in dog 4. These values indicate a comparable high fractional turnover rate for cystine in the dog although the pool size was much smaller. More detailed interpretation of these results was complicated by three problems. Firstly, that there was such a rapid decrease of radioactivity in the plasma during the first hour that the radioactivity was lower than the sensitivity of the isolation procedure before a true equilibrium state was reached. Secondly, the rate of decrease could not be expressed as a simple exponential, and thirdly the rate of decrease of radioactivity in the plasma was a combination of transport from the plasma into the intracellular space as well as a component of metabolism. SUMMARY
YS-L-cystine was injected intravenously into anaesthetised dogs, and the rate of disappearance of radioactive cystine was measured. Cystine was isolated from the plasma by two different techniques before determination of the radioactivity. It was shown that there was an almost instantaneous equilibration of the isotope into a volume greater than the plasma and extracellular space. The rate of decrease of radioactivity of plasma cystine was expressed for two dogs as the sum of two exponential terms. The first term may represent uptake of extracellular cystine into the intracellular space, and the second term may more closely represent the rate of turnover of radioactive cystine. The values corresponded to a turnover rate of 16 pmolel kglhr in one dog and 38 pmole/ kg/ hr in another dog. The fate of the Yi-L-cystine could not be determined, but it is believed that most of the disappearance of radioactivity could be explained by the uptake of the amino acid by the tissues and equilibration with the intracellular amino acid pool. ACKNOWLEDGMENTS We thank Dr. C. J. Thompson and Mr. Peter Berkowitz for his the regression analysis. We also thank Mr. A. Edwards
for assistance assistance with and
Mr.
P. Kiely
with the earlier part of this work the computer calculations used in who
performed
the surgical
pro-
CYSTINE
cedures. This work Grant No. 249.34.
was supported
7395
METABOLISM
in part
by the Canadian
Medical
Research
Council
APPENDIX
A plot of the logarithm of cystine radioactivity against time for dog 2 (Fig. 3) shows that the decrease in activity is approximately linear after 45 minutes and can thus be represented by a function of the form
where
A is the radioactivity (in dpm/ml) at time t B is a constant corresponding to the radioactivity at zero time, and k is the decay constant for this function. Values for B and k were deduced by regression analysis on the experimental data for the variation of radioactivity with time from 45 minutes to the end of the experiment, and a graph of the regression line so obtained was plotted and extrapolated to zero time. Intercepts on this line were drawn at the times at which determinations of radioactivity were made in the initial 45 minutes of the experiment, and the values of these intercepts on the ordinate were subtracted from the total radioactivity in the plasma at each point giving a further series of points which regression analysis showed to lie on a line represented by A’ = Bte--h-lt The sum produced exception It was different
of these two terms gave an expression which, when plotted, a curve agreeing closely with the experimental curve with the of the 2.5-minute point. assumed that each term of the form A = Be-“’ represented a cystine pool, and values for various parameters for each pool
Parameter 1. Init,ial
radioactivity
2. Initial
dilrition
Method
of derivation
A = R when space,
T’
t = 0
T’ = ;
5. Half
life,
rate,
t1j2
Radioactivity in iiiitial mixing
Total quantity p001 X
I< = ,(.I1 I
tj/4 =
=hZ
interpretation the
pool
after
Volume into which radioactivity is diluted in the pool represeiit,ed by this term
3. Pool size, J/ 4. Turnover
Physical
A
111 2 0.693 F = 7
In
of cystine
an eyliilihritnn situat,ion trunover rate represents rate of incorporatioii itito orit of the pool.
in the the the at111
Time takeii for half t,he cyst ine iii the pool to be replaced
396 were sents stant, From tivity
CRAWHALL,
PURKISS,
AND
YOUNG
calculated as shown in the following table. In this table, C reprethe concentration of cystine in the plasma, which is assumed conand X is the total radioactivity injected. the combined equations, the total volume into which the radioacwas initially diluted is given by I/tot:*1
=
s
B +
*,
A similar analysis was carried out for dog 4 where the last two points were considered as belonging to the second exponential and the earlier points as belonging to the first exponential. REFERENCES 1. PXTS, R. F., “Physiology of the Kidney and Body Fluids,” 2.nd Ed., p. 24. Year Book Medical Publishers, Chicago, 1963. 2. BRAY, G. A., Anal. Biochem. 1,279 ( 1960). 3. CRAWHALL, J. C., THOMPSON, C. J., AND BRADLEY, K. H., Anal. Biochem. 14, 405 ( 1966). 4. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211,907 (1954). 5. CRA~HALL, J. C., AXD SEGAL, S., Biochem. J. 105, 891 ( 1967). 6. WYNGAARDEN, J. B., AND SEGAL, S., in “The Metabolic Basis of Inherited Disease” J. B. Stanbury, J. B. Wyngaarden, and D. S. Frederickson, eds.), 2nd Ed., p. 341. McGraw-Hill, New York, 1966. 7. WARS, R. W. E., AND CRAWHALL, J. C., Biochem. J. 73, 277 (1959 ).