Method for detecting cystine and cysteine

Method for detecting cystine or cysteine which comprises adding a reducing agent and a metallic compound which liberates nickel ion or cobaltous ion and observing a color change.

This invention relates to a coloring composition for the detection of 
cystine or cysteine in a solution and a method of detection therefor. It 
is known that both cystine and cysteine are sulfur-containing amino acids 
and are easily convertible to each other by oxidation and/or reduction. 
There have been various methods to detect cystine or cysteine. They include 
polarographic, cyanide-nitro-prusside, iodine-nitride, Sullivan and 
Fleming Vassel methods. However, these known methods require a long time 
for detection. These methods also require reagents which should be 
prepared at the time of use, and which are poisonous, thus causing careful 
and troublesome handling. It is generally known that organic thiol 
compounds reveal color by forming complexes with various kinds of heavy 
metallic ions, but methods for detecting cysteine or cystine by making use 
of this reaction are not practical because the color reaction is not 
sensitive. 
We have conducted further research and finally found a coloring composition 
for detecting cystine or cysteine and a detecting method therefor. The 
principle is as follows. 
Cysteine itself or cysteine which is produced by the reduction of cystine 
forms complexes with heavy metallic ions in the presence of certain kinds 
of heavy metallic ions and reducing agents. 
In addition to this reaction, sulfide ion (S.sup.2.sup.-) is released from 
the reducing agent itself or by the reaction between the reducing agent 
and cysteine. 
The sulfide ion binds to the complex and forms a mixed ligands complex. We 
have found that by the formation of the mixed ligands complex, the 
presentation of color becomes remarkable and the coloration occurs in a 
short time. 
On the basis of these findings, we have developed a very sensitive coloring 
composition to detect cystine or cysteine easily and quickly: said 
coloring composition comprises a reducing agent which reduces cystine to 
cysteine and releases sulfide ion by itself or by the reaction with 
cysteine, and a metallic compound which liberates nickel ion or cobaltous 
ion. We have also developed a method for detecting cystine or cysteine in 
solution by using said coloring composition. 
Metallic compounds of nickel ion or cobaltous ion used in the present 
invention are organic acid salts such as acetates, and inorganic acid 
salts such as sulfates, nitrates, chlorides and hydroxides. 
Reducing agents used in the present invention include alkali dithionites 
and alkali borohydrides. They reduce cystine to cysteine and also release 
S.sup.2.sup.- by the reaction with cysteine. 
It is also possible to use an alkali sulfide or an alkali hydrosulfide as a 
reducing agent. An alkali sulfide or alkali hydrosulfide forms a mixed 
ligands complex with S.sup.2.sup.- ions which have already been present; 
however, at the same time, it reacts with cobaltous ion or nickel ion to 
form a metallic sulfide which sometimes makes color indistinct. In such 
case, however, it is possible to make it clear with the separation of the 
metallic sulfide. 
Brown color is developed when a solution containing cystine or cysteine is 
added to the coloring composition of the present invention. 
It is easy to check the absence of cystine or cysteine in solution if the 
color of the solution is different from brown. For this purpose, it is 
desirable to add a chelating agent to the coloring composition, which 
forms a water-soluble chelate compound by reaction with nickel ion or 
cobaltous ion to give such a color as blue, green or red when a solution 
contains no cystine or cysteine. Suggested chelating agents include 
ethylenediaminetetraacetates (EDTA), nitrilotriacetate, tartrate and 
citrates. 
It is desirable to maintain the solution at pH 6.0-8.5 because the color 
reaction proceeds at pH 6.0-8.5 easily. It is therefore desirable to add a 
buffer agent such as tris (hydroxymethyl) aminomethane phosphate, 
dipotassium hydrogen-phosphate-sodium dihydrogen phosphate, boric 
acid-sodium borate or a combination thereof. 
The coloring composition of this invention can be used in the form of a 
solution of each compound, but it can also be offered in the form of 
powders, granules, pellets and tablets because it is more convenient and 
practical to use. The coloring composition can be used without using 
fillers, but it is recommended to add such water-soluble fillers as 
soluble starch, glucose, sorbitol, sucrose, mannitol, etc., because it is 
quite convenient for use and efficient for weighing out the coloring 
composition. 
It is also possible to change the degree or limit of coloration by changing 
the amount of the component in the coloring composition. It is possible to 
determine cystine or cysteine content by absorptiometry. But it is also 
possible to estimate cysteine or cystine content by eye-observation. 
By virtue of this speedy check ability, there are many fields of 
application of this composition. Medical diagnosis, for example, for 
cystinuria which shows abnormally increased cystine in urine, is one 
interesting field of application. Namely, with the coloring composition, 
the procedure required is only to add urine to the composition. If it 
contains an abnormal degree of cystine (cystinuric urine), it turns brown 
within a few minutes at room temperature, and that is one important 
feature of the present invention. Hence, this invention offers a novel 
coloring composition and novel method for not only the diagnosis of a 
cystinuria, but also the detection in an earlier stage of latent 
cystinuria by group medical examinations. 
The present invention is further illustrated by the following examples, but 
they are not to be construed as limiting the present invention.

EXAMPLE 1 
(1) 9.0 ml. of 5% borate buffer (pH 7.2) containing 100 .mu.g/ml. cystine 
was prepared. Into this buffer 1.0 ml. of 3.44 mM metallic compound (3.44 
.mu.mol) listed hereunder and 15 mg. sodium dithionite (86 .mu.mol) were 
added. Thus, the mol ratio is 1/25 (metallic compound/dithionite). After 
standing for 5 minutes, the color of this sample solution was observed 
with the eye and its absorbance was also determined at 400 m.mu. against a 
control solution. The control solution had been prepared in the same 
manner as the sample solution except for the use of 5% borate buffer 
containing no cystine. As shown hereunder, cobaltous and nickel compounds 
are useful to detect cystine among metallic compounds. 
__________________________________________________________________________ 
Color with the Eye 
Sample Control 
Metallic Compound Absorbance 
Solution 
Solution 
__________________________________________________________________________ 
Cobaltous 
acetate 
(4 H.sub.2 O) 
1.360 brown light pink 
sulfate 
(7 H.sub.2 O) 
1.365 " " 
nitrate 
(6 H.sub.2 O) 
1.364 " " 
hydroxide 0.323 " " 
chloride 
(6 H.sub.2 O) 
1.310 " " 
Nickel 
nitrate 
(6 H.sub.2 O) 
0.358 " light green 
sulfate 
(6 H.sub.2 O) 
0.286 " " 
hydroxide 
(1 H.sub.2 O) 
0.307 " " 
carbonate 
(4 H.sub.2 O) 
0.154 " " 
Cupric 
sulfate 
(5 H.sub.2 O) 
0.062 light blue 
light blue 
chloride 
(2 H.sub.2 O) 
0.060 " " 
nitrate 
(3 H.sub.2 O) 
0.060 " " 
acetate 
(1 H.sub.2 O) 
0.059 " " 
Ferric 
sulfate 
(6 H.sub.2 O) 
0.015 light yellow- 
light yellow- 
ish brown 
ish brown 
Ferrous 
sulfate 
(7 H.sub.2 O) 
0.010 " " 
Ferric 
chloride 
(3 H.sub.2 O) 
0.017 " " 
Ammonium 
ferrous 
(6 H.sub.2 O) 
0.036 " " 
sulfate 
Ammonium 
ferric 
(24 H.sub.2 O) 
0.015 " " 
sulfate 
__________________________________________________________________________ 
(2) The experiments of (1) were modified in such a way as to change the 
concentration of metallic compound in order to study the effect on the 
coloration. Nickel nitrate was used as the metallic compound. As shown 
hereunder, the degree of coloration increases with the nickel ion 
concentration. 
______________________________________ 
Concentration of 
nickel nitrate 
Nitrate/Dithionite 
Absorbance 
(mM) Molar Ratio After 1 minute 
______________________________________ 
1.13 1/76 1.503 
0.85 1/101 0.455 
0.71 1/121 0.252 
0.57 1/150 0.043 
0.43 1/200 0.003 
0.28 1/307 0.000. 
______________________________________ 
(3) 10 ml. of 5% borate buffer (pH 7.2) containing 100 .mu.g/ml. cystine 
and 2.0 ml. of 100 .mu.g/ml. nickel nitrate (0.7 .mu.mol) were mixed. 
Various kinds of reducing agents listed hereunder were added to the 
resulting solution. After 5 minutes, the absorbance and color of each 
sample solution were observed against a control solution. As shown 
hereunder, all the reducing agents gave visible brown color to the sample 
solution, although some deeply and others lightly. 
__________________________________________________________________________ 
Nitrate/ Color with the Eye 
Dithionite Sample 
Control 
Reducing Agent 
Molar Ratio 
Absorbance 
Solution 
Solution 
__________________________________________________________________________ 
Sodium dithionite 
1/123 0.337 brown 
light green 
(15 mg) (86 .mu.mol) 
Potassium borohydride 
1/133 0.552 " colorless 
(5 mg) (93 .mu.mol) 
Sodium sulfide (9H.sub.2 O) 
1/30 0.955* " " 
(5 mg) (21 .mu.mol) 
Sodium hydrosulfide 
1/127 0.894* " " 
(5 mg) (89 .mu.mol) 
__________________________________________________________________________ 
*Absorbance of the supernatant after centrifugation. 
(4) The experiments of (3) were modified in such a way as to change the 
amount of the reducing agent in order to study the effect on the 
coloration. Sodium dithionite was used as the reducing agent. The results 
hereunder show that the degree of coloration increases with the amount of 
sodium dithionite. 
__________________________________________________________________________ 
Weight of Nitrate/ 
Absorbance Color with Eye 
Reducing Agent 
Dithionite 
After 
After After 
(mg) Molar Ratio 
1 minute 
5 minutes 
5 minutes 
__________________________________________________________________________ 
7.5 (43 .mu.mol) 
1/61 0.140 
0.143 brown 
15.0 (86 .mu.mol) 
1/123 0.328 
0.324 deep brown 
30.0 (172 .mu.mol) 
1/244 0.470 
0.525 " 
45.0 (258 .mu.mol) 
1/366 0.504 
0.537 " 
__________________________________________________________________________ 
(5) The effect of chelating agent on the color was studied. One ml. of 
several kinds of 0.35 M chelating agent (350 .mu.mol), 1 ml. of 0.4 M 
metallic compound (400 .mu.mol) and 10 mg. of sodium dithionite (57 
.mu.mol) were added to 10 ml. of 5% borate buffer containing 100 .mu.g.ml. 
cystine. The chelating agent/metallic compound/dithionite molar ratio was 
0.88/1/0.14. After standing for 3 minutes, the color of this sample 
solution was observed against a control solution. The control solution was 
prepared in the same manner as the sample solution except for the use of 
5% borate buffer which contains no cystine. 
As shown hereunder, all the sample solutions show brown color, while the 
control solutions show various colors more deeply than the case of no 
addition of chelating agent. It has become apparent that a chelating agent 
facilitates distinguishing between the sample solution and control 
solution. 
______________________________________ 
Metallic Compound: 
Nickel Nitrate Cobaltous Sulfate 
______________________________________ 
Sample Control Sample Control 
Chelating Agent 
Solution Solution Solution 
Solution 
______________________________________ 
no addition brown light green 
brown light pink 
disodium brown blue brown pink 
edetate (EDTA) 
nitrilotriacetate 
brown green brown pink 
sodium citrate 
brown green brown pink 
sodium tartrate 
brown green brown pink. 
______________________________________ 
(6) Relationship between the degree of coloration and pH was studied. One 
ml. of 40 mM of nickel sulfate (40 .mu.mol) and 5 mg. of sodium dithionite 
(29 .mu.mol), in a molar ratio of 1/0.73, were added to 9 ml. of several 
kinds of buffer (0.1 M) containing 100 .mu.g/ml. cystine. As shown 
hereunder, it is desirable to keep the solution at pH 6.0-8.5. 
______________________________________ 
Buffers pH Absorbance 
______________________________________ 
0.1 M borate 5.0 0.037 
" 6.0 0.185 
" 6.5 0.352 
" 7.2 0.481 
" 7.5 0.736 
" 8.5 0.148 
" 9.2 0.037 
0.1 M phosphate 7.5 0.118 
0.1 M tris(hydroxymethyl) 
7.5 0.243 
aminomethane-sodium 
dihydrogen phosphate 
______________________________________ 
EXAMPLE 2 
The color of the solution, which was prepared by mixing soluions or 
chemicals listed hereunder, was observed in a series of experiments (1) - 
(4). 
______________________________________ 
Concentration 
Chemicals or Weight Remarks 
______________________________________ 
Heavy Metallic compound: 
Nickel nitrate 40 mM 
Nickel sulfate 40 mM 
Cobaltous nitrate 
40 mM 
Cobaltous sulfate 
40 mM 
Chelating agent: EDTA 
10 mM 
Reducing agent: 
Sodium dithionite 
5 mg.* powder 
Buffer: Borate 5 % pH 7.2 
Cystine solution 
100 .mu.g/ml. 
Buffer was used 
as the solvent 
Cysteine solution 
100 .mu.g/ml. 
" 
______________________________________ 
*(29 .mu.mol; nitrate/dithionite molar ratio, 1/0.73). 
(1) Effect of sodium ditionite on the cysteine coloration 
______________________________________ 
Nickel Nickel 
Nitrate Sulfate Cysteine 
Sodium Color After 
No. Solution Solution Solution 
Dithionite 
5 Minutes 
______________________________________ 
1 1 ml.** -- 10 ml. -- light reddish 
brown 
2 1 ml.** -- 10 ml. 5 mg.*** 
deep brown 
3 -- 1 ml.** 10 ml. -- light reddish 
brown 
4 -- 1 ml.** 10 ml. 5 mg.*** 
deep brown. 
______________________________________ 
**(40 .mu.mol; nitrate or sulfate/dithionite molar ratio, 1/0.73) 
***(29 .mu.mol). 
The light reddish brown solutions of Nos. 1 and 3 take their color from the 
colored complex formed by nickel ion with cysteine. This coloration is not 
sensitive to cysteine because it is faint. As can be seen in the case of 
solutions Nos. 2 and 4, addition of sodium dithionite results in deep 
brown color solutions. 
(2) Effect of sodium dithionite on the cystine coloration 
______________________________________ 
Nickel Nickel 
Nitrate Sulfate Cystine 
Sodium Color After 
No. Solution Solution Solution 
Dithionite 
5 Minutes 
______________________________________ 
5 1 ml.** -- 10 ml. 5 mg.*** 
deep brown 
6 1 ml.** -- 10 ml. -- water white 
7 -- 1 ml.** 10 ml. 5 mg.*** 
deep brown 
8 -- 1 ml.** 10 ml. -- water white. 
______________________________________ 
(** and *** as above). 
Solutions Nos. 5 and 7 show the same deep brown color as the solutions Nos. 
2 and 4, but Nos. 6 and 8 do not show any color because a reducing agent 
is absent. 
(3) Coloration in the present or absence of cystine 
__________________________________________________________________________ 
Nickel 
Nickel Color 
Nitrate 
Sulfate 
Cystine Sodium After 
No. 
Solution 
Solution 
Solution 
Buffer 
Dithionite 
EDTA.sup.a 
5 minutes 
__________________________________________________________________________ 
9 1 ml** 
-- -- 10 ml 
5 mg*** 
-- light 
green 
10 1 ml** 
-- -- 10 ml 
5 mg*** 
1 ml blue 
11 1 ml** 
-- 10 ml 
-- 5 mg*** 
1 ml deep 
brown 
12 -- 1 ml** 
-- 11 ml 
5 mg*** 
-- light 
green 
13 -- 1 ml** 
-- 10 ml 
5 mg*** 
1 ml blue 
14 -- 1 ml** 
10 ml 
-- 5 mg*** 
1 ml deep 
brown 
__________________________________________________________________________ 
(** and *** as above) 
(a = 10 .mu.mol) 
(molar ratio nitrate or sulfate/dithionite/EDTA = 1/0.73/0.25). 
Solutions Nos. 9 and 12 do not show brown color because they contain no 
cystine, but show light green color of nickel ion. Solutions Nos. 10 and 
13, which were prepared by adding EDTA to solutions Nos. 9 and 12, show 
blue color which is due to a colored complex of nickel ion with EDTA. 
Solutions Nos. 11 and 14 which contain cystine show deep brown color. 
(4) Coloration by using cobaltous ion instead of nickel ion 
__________________________________________________________________________ 
Cobaltous 
Cobaltous Color 
Sulfate 
Nitrate Cystine 
Sodium After 
No. 
Solution 
Solution 
Buffer 
Solution 
Dithionite 
EDTA 
5 minutes 
__________________________________________________________________________ 
15 1 ml. -- 10 ml. 
-- 5 mg. 1 ml. 
light 
pink 
16 1 ml. -- -- 10 ml. 
5 mg. 1 ml. 
brown 
17 -- 1 ml. 10 ml. 
-- 5 mg. 1 ml. 
pink 
18 -- 1 ml. -- 10 ml. 
5 mg. 1 ml. 
brown 
__________________________________________________________________________ 
(concentrations and molar ratios as in (3) above). 
In the case where cobaltous ion (4) was used instead of nickel ion (3), 
results were similar as to coloration reaction. 
EXAMPLE 3 
(1) ml. of 41 mM nickel nitrate (41 .mu.mol), 15 mg. sodium dithionite (86 
.mu.mol) and 1.0 ml. of 3 mM EDTA (3 .mu.mol) were added to 2 ml. of 5% 
borate buffer containing cystine in various concentrations. The molar 
ratio nitrate/dithionite/EDTA was 1/2.1/0.07. After standing for 5 
minutes, their absorbances were measured. As described hereunder, a 
calibration line which followed Beer's law was obtained from the results. 
The results showed that cystine can be assayed by this method. 
______________________________________ 
Cystine Concentration (.mu.g/ml) 
Absorbance 
______________________________________ 
0 0.009 
50 0.198 
100 0.407 
150 0.611 
200 0.805. 
______________________________________ 
(2) In the same manner as (1) except for the use of cobaltous sulfate 
instead of nickel nitrate, the relationship between cystine concentration 
and absorbance was examined. In this case, a calibration line was also 
obtained. 
______________________________________ 
Cystine Concentration (.mu.g/ml) 
Absorbance at 400 m.mu. 
______________________________________ 
0 0.000 
50 0.102 
100 0.260 
150 0.442 
200 0.608. 
______________________________________ 
EXAMPLE 4 
60 mg. .gamma.-globulin, which was dissolved in 1.0 ml. of water and 4.0 
ml. of 1 N HCl, was hydrolyzed by heating in a boiling water bath. After 
one hour, the solution was then neutralized by adding 1 N NaOH and diluted 
to 8.0 ml. with water. To 1.0 ml. (contains 7.5 mg. .gamma.-globulin) of 
this solution were added reagents which were employed in Example 3, above. 
The absorbance at 400 m.mu. showed 0.587 (140.mu.g) after 5 minutes. This 
value, when calculated by the calibration line in Example 3, corresponded 
to 20.0 .mu.g. cystine per 1 mg. .gamma.-globulin. This roughly equaled 
20.8 .mu.g. cystine/mg. .gamma.-globulin which is the known amount in the 
literature. Cystine content of bovine serum albumin was also measured in a 
similar manner and the resulting value, 22.0 .mu.g/mg., also roughly 
equaled the known amount 22.6 .mu.g/mg in the literature. 
EXAMPLE 5 
Reagents A, B, C and D listed hereunder were prepared. 
__________________________________________________________________________ 
Nitrate or 
Sulfate/ 
Dithionite/ 
Nickel 
Cobaltous 
Sodium EDTA, 
Reagent 
Nitrate 
Sulfate 
Dithionite.sup.x 
EDTA.sup.y 
Molar Ratio 
Buffer* 
__________________________________________________________________________ 
A 11.0 -- 2.2 8.8 1/0.89/0.62 
38.0 
(37.8 
.mu.mol) 
B 15.0 -- 2.2 8.8 1/0.45/0.46 
34.0 
(51.6 
.mu.mol) 
C 20.0 -- 2.2 8.8 1/0.28/0.34 
29.0 
(68.8 
.mu.mol) 
D -- 20.0 2.2 8.8 1/0.18/0.33 
29.0 
(71.1 .mu.mol) 
__________________________________________________________________________ 
x = 12.6 .mu.mol; y = 23.6 .mu.mol; (unit = mg.) 
*Powder consisting of 5.2 g. sodium hydrogen carbonate, and 0.4 g. sodium 
dihydrogen phosphate. 
2 ml. of urine containing cystine in various concentrations was added to 
the reagent. After standing for 1 minute, the color of the urine was 
observed with the eye. Four panelists were engaged in the examination and 
each panelist took the reagent and examined five times about one urine 
sample at random. The results of the examinations were presented by the 
marks according to the following method. If a panelist decides the urine 
to be positive, one mark is given to the judgment, 0.5 mark to Pseudo 
positive and 0 mark to negative. The results were as follows: 
______________________________________ 
Cystine 
Content 
(.mu.g/ml.) 
Reagent A Reagent B Reagent C 
Reagent D 
______________________________________ 
5 0 0 16 0 
10 0 4.5 20 0 
20 0 10.5 20 0 
30 4.5 18 20 0 
40 9.5 20 20 3 
50 16 20 20 9.5 
60 20 20 20 16 
85 20 20 20 20 
100 20 20 20 20 
200 20 20 20 20 
______________________________________ 
Note: 
If all panelists judge one urine sample as positive in 5 examinations, th 
sample gains 20 marks, i.e. 1 mark .times. 5 times .times. 4 panelists = 
20. 
As is obvious from the results listed above, urine containing more than 60 
.mu.g/ml., 40 .mu.g/ml. and 10 .mu.g/ml. cystine would be detected with an 
accuracy of 100% by using the reagents A, B and C, respectively. Urine 
containing more than 50 .mu.g/ml., 30 .mu.g/ml. and 5 .mu.g/ml. cystine 
could be detected with accuracy of 80 - 90% also by using the reagents A, 
B and C, respectively. By using reagent D, which contained 20 mg. of 
cobaltous sulfate instead of nickel nitrate, urine containing 60 .mu.g/ml. 
cystine can be detected with an accuracy of 80% and 85 .mu.g/ml. cystine 
with an accuracy of 100%. 
EXAMPLE 6 
Pellets were prepared which consist of 16.5 mg. nickel sulfate, 4.0 mg. 
sodium dithionite, 50.0 mg. sodium hydrogen carbonate, 4 mg. sodium 
dihydrogen phosphate and sucrose. The sulfate/dithionite molar ratio was 
1/0.37. A pellet is 400 mg. in weight by addition of an adequate amount of 
sucrose. 
Urine samples containing 30 .mu.g/ml., 50 .mu.g/ml., 100 .mu.g/ml., 150 
.mu.g/ml. and 200 .mu.g/ml. cystine were also prepared by dissolving 
cystine in normal urine (cystine content = 10 .mu.g/ml.). By the use of 
the pellets, ten panelists (A-J) examined twice the urine samples in a 
similar manner as in Example 5 except for the addition of 4 ml. of the 
sample urine. As shown in the following Table, all the panelists showed 
negative for normal urine, pseudo positive for the urine containing 30 
.mu.g/ml. of cystine, and positive for the urine containing more than 50 
.mu.g/ml. cystine. The cyanide-nitroprusside method, that is famous for 
detecting cystine, was compared with the method of the present invention. 
These results showed that the method of the present invention is superior 
to the cyanide-nitroprusside method in practical use because the latter 
method always requires fresh solutions of sodium cyanide and sodium 
nitroprusside, and it requires more than 10 minutes for the detection of 
cystine. On the other hand, the method offered by the present invention 
requires only 2 or 3 minutes for the detection of cystine and can be used 
in safety whenever one wishes to make an examination with a pellet. 
Sensitivity test of pelletized reagent for detection of cystine 
__________________________________________________________________________ 
Cystine content 
(.mu.g/ml) 10 30 50 100 150 200 
__________________________________________________________________________ 
Times of 
test 
Panelist 1st 
2nd 
1st 
2nd 
1st 
2nd 
1st 
2nd 
1st 
2nd 
1st 
2nd 
__________________________________________________________________________ 
A - - .+-. 
.+-. 
+ + + + + + + + 
B - - .+-. 
.+-. 
+ + + + + + + + 
C - - .+-. 
.+-. 
+ + + + + + + + 
D - - .+-. 
.+-. 
+ + + + + + + + 
E - - .+-. 
.+-. 
+ + + + + + + + 
F - - .+-. 
.+-. 
+ + + + + + + + 
G - - .+-. 
.+-. 
+ + + + + + + + 
H - - .+-. 
.+-. 
+ + + + + + + + 
I - - .+-. 
.+-. 
+ + + + + + + + 
J - - .+-. 
.+-. 
+ + + + + + + + 
__________________________________________________________________________ 
Judgement -: negative, .+-.: pseudo positive, +: positive 
The molar ratio of reducing agent to the metallic compound (containing Ni 
and/or Co) can vary widely as shown by experimental results given above. 
The ratios shown are from about 0.15 to about 370. Preferably, the ratio 
ranges from about 0.7 to about 250. 
When a chelating agent is used together with the reducing agent and 
metallic compound, the reducing agent/metallic compound molar ratio can 
also be varied widely, e.g., from about 0.15 to about 3 and preferably 
from about 0.15 to about 2. The molar ratio of reducing agent to chelating 
agent is not critical; preferably, it is from about 0.15 to about 30. 
Complexes of a reducing agent, a metallic compound and cystine or cysteine 
contain varying quantities of each component. Generally, the molar 
relationships are from about 7 to about 65 moles of reducing agent, from 
about 0.15 to about 100 moles of metallic compound, per mole of cystine or 
cysteine.