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Determination of human erythrocyte zinc: Hemoglobin ratios
534
.DETERMINATION
OF HUMAN
ZINC: HEMOGLO’BIN
ERYTHROCYTE RATIOS*
A number of current publications affirm that the quantitation of trace amounts of zinc is of inferest2-?. Recently, a technic involving differential demasking was applied to the determination of serum zinc 8- IO. The process eliminated the effect of interfering metals such as copper and iron by fosming non-ionogenic cyano-complexes. Then the zinc compound formed simultaneously in the same milieu was selectively destroyed by chloral hydrate with the subsequent homogeneous generation of blue zinc-&neonate. This color was measured before the more stable iron and copper coordination compounds decomposed and interfered with the determination. The present investigation describes the application of the procedure to erythrocyte zinc, and encompasses a study of several phases in&ding the following: 1. The contamination of reagentP. 2. The effect of the release of iron on the zinc determinations. .T*The replication of the zinc :hemoglobin ratios. 4. The recovery of varying amounts of zinc from the same base value. 5. The determination of normal zillc:b~~~~globin ratios.
Zincon
(z-carboxy-z’-hydroxy-t;‘-sull’oformazylbenzene) reagent : Prepare a solution by dissolving the material in acationox and then diluting to volume with metal-free water. Borate buffer, PH 9 :53 ml of 4 _niNaOH and 31 g of AX boric acid are diluted to a liter. Standards: 0.0 to 3.0 ml of 50 mgll zinc are each diluted to IOO ml with HIfl (~5 ml), 1056 trichlomcetic acid (25 ml) and metal-free distilled water.
0.137; solution of zincon in 2 5; aqueous acationos***
~~~~~~~u~~~~~ of samfife. The blood was centrifuged and the supernatant plasma was removed by aspiration. Three washes with saline solution wese carefully carried out before an equal volume of metal-free distilled water was added to hemolyze the cells. This solution, spun free of its stroma, was used for the analysis of hemoglobin and zinc. * Supported in part by a Grant-in-Aid from the Receiving Hospital Research Corporation. ** Present address, The Learv Laboratory, Ekston, Massachusetts. **+ Arationox is the trade n&e of a metal-iree non-ionic detergent used in ftame photometry as a wetting agent. It is sold by the Scientific Products Division, American Hospital Supply Co., 40-q xGSth St., Flushing 58, New Wok.
DETERMINATION
OF
ERYTHROCYTE
ZINC
: HEMOGLOBIN
RATIOS
635
Detewnination of hemoglobin. The cyanmethemoglobin technic I2 was selected for the determination of hemoglobin using 0.02 ml of the hemolysate. Detevminatiofz of zinc. Pipette 3.0 ml of the hemolysate into a heavy-walled centrifuge tube. Add 1.5 ml of 1.0 N HCl solution. Cover the tube to prevent evaporation and heat the tube in a boiling water bath for 5 min. Cool, add 1.5 ml of 100/b trichloracetic acid, mix well and centrifuge for 5-10 min in a high speed centrifuge. To a 4.o-ml aliquot, add 0.4 ml of the neutralizing 4.0 N NaOH solution, 1.0 ml of the borate buffer, 0.1 of 3% NaCN solution and 0.6 ml of the zincon reagent. Set the instrument to zero at 630 m,u with this tube as a blank and then add 0.2 ml of 6oq/’ chloral hydrate solution. Mix quickly, and within I min read the increase in absorbance of this same solution at 630 mp. All of the samples and standards can be carried to the self-blanking stage before the chloral hydrate step is performed for each tube, one at a time. Calibratiolt of standads. 4 ml of each of the standards are treated in the same manner described for the samples. The blank absorbance difference is subtracted from the standards in graphing the calibration curve. DISCUSSION
AND
RESULTS
The described demasking technic includes a built in process of self-blanking for the determination of the extent of zinc contamination of reagentsg. Along with the recovery of absolute quantities, self-blanking ensured that the analysis was free enough from contamination of reagents to perform the procedure safely. In the scheme of analysis, zinc was differentially demasked in the presence of other metals (Fe, Cu). The homogeneous generation of ionic zinc in buffered zincon allowed almost immediate color formation. The sample served as its own spectrophotometric blank because the cuvet in which the demasking process was carried out had been read prior to the addition of concentrated chloral hydrate. When such a procedure was used along with a reagent blank, the presence of zinc contamination beyond the limits of good control was readily apparent. In the case of contamination, the absorbance change for the blank, before and after the addition of the demasking agent, was considerably greater than the 0.01-0.03 usually encountered, and the 0.18 total absorbance per IOO ,ug of zinc standard per IOO ml of solution was enhanced. Unless the precautions in the handling of the syringes and samples described by VALLEE et a1.13 are observed, determinations will contain rather large, positive errors. Efiect
of release of i~o~t on zinc determiuations
Since the process involved in releasing zinc from its binding site was somewhat rigorous, the effect of the high iron concentrations which might be released from hemoglobin into the filtrate was studied. Two approaches were employed. 1. The effect of increasing iron concentrations on fixed amounts of zinc was determined. 2. The analysis for iron in filtrates of erythrocyte hemolysates was carried out. In the first instance the procedure was investigated while continually increasing the concentration of iron. It was found that in the physiological range for zinc, one could achieve a concentration of 20 ,ug of iron per aliquot in the presence of o-6 ,ug Clin. Chim. Acta, 7 (1962) 634-638
B. z.m et al.
636
of zinc per aliyuot without any serious deleterious effect on the reaction. At 20 ,ug of iron per aliquot, a figure well beyond the average found, a slight fading was encountered which would cause an average loss of several percent in the sample. For the second experiment, the analysis of a number of hemolysates was made where the TABLE
3’5 395 390 390 400 350 370 375 375 390
8.9 8.9 8.9 8.9 8.9 7.9 7.9 7.9 7.9 7.9
I
(35.5)* 45.0 44.0 44.0 46.0
6.2 6.2 6.2 6.2 6.2
44.5 47.0 47.5 37.5 49.0 -._-..
4.6 4.6 4-6 4.6 4.6
i ..________
290 290
300 295 295 230 2Oj 270 110
205 _.. _.-_
47.0 47.0 48.5 47.5 47.5 50.0 44.5 45.5 45.5 34.5
* Not used in calculations.
hemoglobin concentration varied from 6.4-13.5 g/r00 ml. The average iron value found was 13.2 ,ug of iron per aliquot against 5.8 ,ug of zinc per aliquot. This was less than the concentration of iron which interfered in the described procedure. Therefore, it follows that little interference from released iron would be encountered. TABLE
II
RECOVERYOF ADDITIONSOF ~~~/~~o
--.. ____ Sanzple
-4 dded
I*
0
2 3
55 5.5
Fouxd
Theoretical
190 190 190 190 190 285
12
13 r4 ‘5 16
27:
5 b
95
;
255 260 255
9 10
95 95 95 95
‘55
257 257 257 257
260
Ijg
Awrage
of triplicate anaiysis.
of Ztt :Hb
fI
.217
LOO
Replication
Added
162
210 ZOO
GF
_. _-
Sample
220
55 55
-1-
217 2x7
ZINC
ml)
17 19 19 20
285 285 285 ‘85
Fomd 345 3-15 350 345 355 _)35 435 345 445 450
Theovetical 35’2 352 352 352 352 447 447 447 447 447
._
ratios
Replication is one criterion for accuracy in any procedure. To this end a pooled hemoglobin solution was prepared. Aliquots of several dilutions of these solutions were analyzed for hemoglobin and zinc. The results are shown in Table I. It can be seen that the zinc:hemoglobin ratios are maintained well within the limits of analytical accuracy. The mean Zn:Hb ratio was 46.3 ,ug/g with an a.verage mean deviation of 1.6 pg/g and a standard deviation of & 1.8 pg]g. The value for the first sample (ratio of 35.5 ;&g/g) was discarded in accordance with the findings of MELLOR~*, i.e.,
DETERMINATION
OF ERYTHROCYTE
ZINC : HEMOGLOBIN
RATIOS
637
this divergent value has a deviation from the mean of the o-titer determinations which is more than four times their mean deviation. Recovery
of additions
Table II shows the analytical accord in the results obtained on the addition of known amounts of zinc to a pooled hemoXysate. The base concentration of r,inc was determined in triplicate. Zi~tc : hemoglobin
rat{o
presumably normal people were selected and determinations of plasma zinc, hemolysate hemoglobin and hemolysate zinc wese made. The plasma zinc was used 20
32.1
3.x 5.6 * :,:t‘2
IIS 206
38.1 36.7
;::
42.2
‘44 238
38.1 46.0
2:;
36.4 41.8
8.7
256
29.4
6.6
4-1 4.5 6.2 3.7 5.6
138 238 288 118 “25
33-7 52.9 46.5 31.9 40.2
.5.0 4.6 4.” 4-x 5.4
33.1 41.2
--._.-
_.._ -_
33.9 39.5 52.2 45.2
as an internal control on cont~~atio~. The 20 results averaged 1x1 pg per IOO ml for plasma zinc, a figure which correlates well with presently established procedures”. The Zn:Rb ratio in these same samples showed a mean of 39.6 ,ug/g with a standard deviation af & 6.8 ,ug/g. Th e ratios ranged between 29.4 and 52.9 pg/g. The data for these findings are shown in Table III. 40 random samples drawn from patients showed a wider scatter and the values found ranged between 16.6 and 67.0 pug/g.The distribution for this data was unskewed with a majority of the samples falling between 41 to 50 pug/gwith a mean of 45.0 pug/g.
A procedure for the determination glttbin present has been described. The recoveri.es, interference, contamination ratios in normal persons. This technic everyday operation,
of erythrcxyte zinc and its ratio to the hemoseveral phases of study incfuded replication, and the determination of a range of Zn:Hb appears to be suitable to multiple analyses in
REFEREIVCES
638
B. ZAK et al.
5 W. B. HERRING, B. S. I,EAVELL, L. M. PAIXAO AND J. H. YOE, Agi~.J. Clirz. N&vitzn?i, 8 (1960)
855. ’ B. L. VALLEE, Ph.vsiol. &IX., 39 (1959) 443. i R. E. FREDERICKS, K. R. T~NAK.~ AXJ W. X. VALENTINE. ilxnl. Bioche~z., 2 (1961) 169. * F. FEIGL, Clzemistvr of Specific,Selectiw md Semitim Reartiom, Academic Press, Ne%vYork. 1949. 9 L. A. WILLIA~IS, J. S. COHEN AND B. ZAK, C&U. Chelyz.,in the press. lo J. A. PLATTE AND V. M. M+.RcH, .4iaaZ.Chem., 31 (1959) 1226. I1 K. E. THIERS, in D. GLICK (Ed.), Methods of Biochemical A-lncclysis, Vol. T’, Interscience, XWI I2 J. B. MULE, Laboratoql Medicine. Hewzafology. C. V. Mosby C.O., St. Louis. 1958, p. 653. I3 B. L. V.
3rd ed., 1949.
p.
H. FCR41.4N, Eleme~~tnvz- ~zmtitntiz~e
D. Van Sostrand,
;lxul~‘sis,
63. Clin. Chim. Actu, , (1962) 634.638
THE
QUANTITATIVE IN
DETERMINATION
HOMOGENIZED
OF
TISSUE
WHOLE
BLOOD
PREPARATIONS
A. E. F. H. MEIJER Laborafovy for Pathology, Biochemical Sectim, Zirlivevsity of Leydeu (The Netherlands)
(Received January ~2nd. 1962)
After intraperitoneal administration of solutions of macromolecular substances to mice of the OzO strain (Amsterdam), part of the injected quantity was stored in the liver and spleen. There also occurred an enlargement of both organs which was very appreciable for the spleen13 %. In connection with our investigations it was desirable to determine whether a change in the blood content of the liver and spleen was the cause or one of the causes of the enlargement of the organ. Several authors have described methods for the determination of whole blood in tissue homogenates, all of which are based upon spectrophotometric measurements. The method of LOWRY AND HASTINGS” requires measurement of the extinctions in extremely LOWRY
turbid
absorption COHN”
solutions.
AND HASTINGS
The turbidity
have
tried
of light due to turbidity
measured
oxyhemoglobin
changes
increases
in the light
into hemiglobin
GORDON ANL) NURNBERGER’S
of the solutions
to overcome
linearly
absorption
or of hemiglobin
method5
causes a difficulty
by subtraction,
is based
with
produced
assuming
decreasing by
the
wavelength.
the conversion
into hemiglobin-cyanide. on the difference
which
that
of
Finally,
in shape between
The two latter methods presuppose that the degree of cloudiness is not altered by the reagents. It is our impression that the results, especially those for liver tissue, obtained by LOWRY XND HASTIKGS’ method are not consistently reliable because of the abthe absorption
sorptions
spectra
caused by turbidity.
ally brown
coloured,
ducts of hemoglobin. the analytical consistently, blood
of oxyhemoglobin
With
insoluble
the other two methods there developed
substances
These disintegration
results.
and carbosyhemoglobin.
which
were probably
products
another
method
pro-
are very likely to have influenced
Since the results by the various
we have developed
occasion-
disintegration
methods
for making
also did not agree
determinations
of whole
in tissues. Clix. Chim. Acfn, j’ (1962) 638-641
.DETERMINATION
OF HUMAN
ZINC: HEMOGLO’BIN
ERYTHROCYTE RATIOS*
A number of current publications affirm that the quantitation of trace amounts of zinc is of inferest2-?. Recently, a technic involving differential demasking was applied to the determination of serum zinc 8- IO. The process eliminated the effect of interfering metals such as copper and iron by fosming non-ionogenic cyano-complexes. Then the zinc compound formed simultaneously in the same milieu was selectively destroyed by chloral hydrate with the subsequent homogeneous generation of blue zinc-&neonate. This color was measured before the more stable iron and copper coordination compounds decomposed and interfered with the determination. The present investigation describes the application of the procedure to erythrocyte zinc, and encompasses a study of several phases in&ding the following: 1. The contamination of reagentP. 2. The effect of the release of iron on the zinc determinations. .T*The replication of the zinc :hemoglobin ratios. 4. The recovery of varying amounts of zinc from the same base value. 5. The determination of normal zillc:b~~~~globin ratios.
Zincon
(z-carboxy-z’-hydroxy-t;‘-sull’oformazylbenzene) reagent : Prepare a solution by dissolving the material in acationox and then diluting to volume with metal-free water. Borate buffer, PH 9 :53 ml of 4 _niNaOH and 31 g of AX boric acid are diluted to a liter. Standards: 0.0 to 3.0 ml of 50 mgll zinc are each diluted to IOO ml with HIfl (~5 ml), 1056 trichlomcetic acid (25 ml) and metal-free distilled water.
0.137; solution of zincon in 2 5; aqueous acationos***
~~~~~~~u~~~~~ of samfife. The blood was centrifuged and the supernatant plasma was removed by aspiration. Three washes with saline solution wese carefully carried out before an equal volume of metal-free distilled water was added to hemolyze the cells. This solution, spun free of its stroma, was used for the analysis of hemoglobin and zinc. * Supported in part by a Grant-in-Aid from the Receiving Hospital Research Corporation. ** Present address, The Learv Laboratory, Ekston, Massachusetts. **+ Arationox is the trade n&e of a metal-iree non-ionic detergent used in ftame photometry as a wetting agent. It is sold by the Scientific Products Division, American Hospital Supply Co., 40-q xGSth St., Flushing 58, New Wok.
DETERMINATION
OF
ERYTHROCYTE
ZINC
: HEMOGLOBIN
RATIOS
635
Detewnination of hemoglobin. The cyanmethemoglobin technic I2 was selected for the determination of hemoglobin using 0.02 ml of the hemolysate. Detevminatiofz of zinc. Pipette 3.0 ml of the hemolysate into a heavy-walled centrifuge tube. Add 1.5 ml of 1.0 N HCl solution. Cover the tube to prevent evaporation and heat the tube in a boiling water bath for 5 min. Cool, add 1.5 ml of 100/b trichloracetic acid, mix well and centrifuge for 5-10 min in a high speed centrifuge. To a 4.o-ml aliquot, add 0.4 ml of the neutralizing 4.0 N NaOH solution, 1.0 ml of the borate buffer, 0.1 of 3% NaCN solution and 0.6 ml of the zincon reagent. Set the instrument to zero at 630 m,u with this tube as a blank and then add 0.2 ml of 6oq/’ chloral hydrate solution. Mix quickly, and within I min read the increase in absorbance of this same solution at 630 mp. All of the samples and standards can be carried to the self-blanking stage before the chloral hydrate step is performed for each tube, one at a time. Calibratiolt of standads. 4 ml of each of the standards are treated in the same manner described for the samples. The blank absorbance difference is subtracted from the standards in graphing the calibration curve. DISCUSSION
AND
RESULTS
The described demasking technic includes a built in process of self-blanking for the determination of the extent of zinc contamination of reagentsg. Along with the recovery of absolute quantities, self-blanking ensured that the analysis was free enough from contamination of reagents to perform the procedure safely. In the scheme of analysis, zinc was differentially demasked in the presence of other metals (Fe, Cu). The homogeneous generation of ionic zinc in buffered zincon allowed almost immediate color formation. The sample served as its own spectrophotometric blank because the cuvet in which the demasking process was carried out had been read prior to the addition of concentrated chloral hydrate. When such a procedure was used along with a reagent blank, the presence of zinc contamination beyond the limits of good control was readily apparent. In the case of contamination, the absorbance change for the blank, before and after the addition of the demasking agent, was considerably greater than the 0.01-0.03 usually encountered, and the 0.18 total absorbance per IOO ,ug of zinc standard per IOO ml of solution was enhanced. Unless the precautions in the handling of the syringes and samples described by VALLEE et a1.13 are observed, determinations will contain rather large, positive errors. Efiect
of release of i~o~t on zinc determiuations
Since the process involved in releasing zinc from its binding site was somewhat rigorous, the effect of the high iron concentrations which might be released from hemoglobin into the filtrate was studied. Two approaches were employed. 1. The effect of increasing iron concentrations on fixed amounts of zinc was determined. 2. The analysis for iron in filtrates of erythrocyte hemolysates was carried out. In the first instance the procedure was investigated while continually increasing the concentration of iron. It was found that in the physiological range for zinc, one could achieve a concentration of 20 ,ug of iron per aliquot in the presence of o-6 ,ug Clin. Chim. Acta, 7 (1962) 634-638
B. z.m et al.
636
of zinc per aliyuot without any serious deleterious effect on the reaction. At 20 ,ug of iron per aliquot, a figure well beyond the average found, a slight fading was encountered which would cause an average loss of several percent in the sample. For the second experiment, the analysis of a number of hemolysates was made where the TABLE
3’5 395 390 390 400 350 370 375 375 390
8.9 8.9 8.9 8.9 8.9 7.9 7.9 7.9 7.9 7.9
I
(35.5)* 45.0 44.0 44.0 46.0
6.2 6.2 6.2 6.2 6.2
44.5 47.0 47.5 37.5 49.0 -._-..
4.6 4.6 4-6 4.6 4.6
i ..________
290 290
300 295 295 230 2Oj 270 110
205 _.. _.-_
47.0 47.0 48.5 47.5 47.5 50.0 44.5 45.5 45.5 34.5
* Not used in calculations.
hemoglobin concentration varied from 6.4-13.5 g/r00 ml. The average iron value found was 13.2 ,ug of iron per aliquot against 5.8 ,ug of zinc per aliquot. This was less than the concentration of iron which interfered in the described procedure. Therefore, it follows that little interference from released iron would be encountered. TABLE
II
RECOVERYOF ADDITIONSOF ~~~/~~o
--.. ____ Sanzple
-4 dded
I*
0
2 3
55 5.5
Fouxd
Theoretical
190 190 190 190 190 285
12
13 r4 ‘5 16
27:
5 b
95
;
255 260 255
9 10
95 95 95 95
‘55
257 257 257 257
260
Ijg
Awrage
of triplicate anaiysis.
of Ztt :Hb
fI
.217
LOO
Replication
Added
162
210 ZOO
GF
_. _-
Sample
220
55 55
-1-
217 2x7
ZINC
ml)
17 19 19 20
285 285 285 ‘85
Fomd 345 3-15 350 345 355 _)35 435 345 445 450
Theovetical 35’2 352 352 352 352 447 447 447 447 447
._
ratios
Replication is one criterion for accuracy in any procedure. To this end a pooled hemoglobin solution was prepared. Aliquots of several dilutions of these solutions were analyzed for hemoglobin and zinc. The results are shown in Table I. It can be seen that the zinc:hemoglobin ratios are maintained well within the limits of analytical accuracy. The mean Zn:Hb ratio was 46.3 ,ug/g with an a.verage mean deviation of 1.6 pg/g and a standard deviation of & 1.8 pg]g. The value for the first sample (ratio of 35.5 ;&g/g) was discarded in accordance with the findings of MELLOR~*, i.e.,
DETERMINATION
OF ERYTHROCYTE
ZINC : HEMOGLOBIN
RATIOS
637
this divergent value has a deviation from the mean of the o-titer determinations which is more than four times their mean deviation. Recovery
of additions
Table II shows the analytical accord in the results obtained on the addition of known amounts of zinc to a pooled hemoXysate. The base concentration of r,inc was determined in triplicate. Zi~tc : hemoglobin
rat{o
presumably normal people were selected and determinations of plasma zinc, hemolysate hemoglobin and hemolysate zinc wese made. The plasma zinc was used 20
32.1
3.x 5.6 * :,:t‘2
IIS 206
38.1 36.7
;::
42.2
‘44 238
38.1 46.0
2:;
36.4 41.8
8.7
256
29.4
6.6
4-1 4.5 6.2 3.7 5.6
138 238 288 118 “25
33-7 52.9 46.5 31.9 40.2
.5.0 4.6 4.” 4-x 5.4
33.1 41.2
--._.-
_.._ -_
33.9 39.5 52.2 45.2
as an internal control on cont~~atio~. The 20 results averaged 1x1 pg per IOO ml for plasma zinc, a figure which correlates well with presently established procedures”. The Zn:Rb ratio in these same samples showed a mean of 39.6 ,ug/g with a standard deviation af & 6.8 ,ug/g. Th e ratios ranged between 29.4 and 52.9 pg/g. The data for these findings are shown in Table III. 40 random samples drawn from patients showed a wider scatter and the values found ranged between 16.6 and 67.0 pug/g.The distribution for this data was unskewed with a majority of the samples falling between 41 to 50 pug/gwith a mean of 45.0 pug/g.
A procedure for the determination glttbin present has been described. The recoveri.es, interference, contamination ratios in normal persons. This technic everyday operation,
of erythrcxyte zinc and its ratio to the hemoseveral phases of study incfuded replication, and the determination of a range of Zn:Hb appears to be suitable to multiple analyses in
REFEREIVCES
638
B. ZAK et al.
5 W. B. HERRING, B. S. I,EAVELL, L. M. PAIXAO AND J. H. YOE, Agi~.J. Clirz. N&vitzn?i, 8 (1960)
855. ’ B. L. VALLEE, Ph.vsiol. &IX., 39 (1959) 443. i R. E. FREDERICKS, K. R. T~NAK.~ AXJ W. X. VALENTINE. ilxnl. Bioche~z., 2 (1961) 169. * F. FEIGL, Clzemistvr of Specific,Selectiw md Semitim Reartiom, Academic Press, Ne%vYork. 1949. 9 L. A. WILLIA~IS, J. S. COHEN AND B. ZAK, C&U. Chelyz.,in the press. lo J. A. PLATTE AND V. M. M+.RcH, .4iaaZ.Chem., 31 (1959) 1226. I1 K. E. THIERS, in D. GLICK (Ed.), Methods of Biochemical A-lncclysis, Vol. T’, Interscience, XWI I2 J. B. MULE, Laboratoql Medicine. Hewzafology. C. V. Mosby C.O., St. Louis. 1958, p. 653. I3 B. L. V.
3rd ed., 1949.
p.
H. FCR41.4N, Eleme~~tnvz- ~zmtitntiz~e
D. Van Sostrand,
;lxul~‘sis,
63. Clin. Chim. Actu, , (1962) 634.638
THE
QUANTITATIVE IN
DETERMINATION
HOMOGENIZED
OF
TISSUE
WHOLE
BLOOD
PREPARATIONS
A. E. F. H. MEIJER Laborafovy for Pathology, Biochemical Sectim, Zirlivevsity of Leydeu (The Netherlands)
(Received January ~2nd. 1962)
After intraperitoneal administration of solutions of macromolecular substances to mice of the OzO strain (Amsterdam), part of the injected quantity was stored in the liver and spleen. There also occurred an enlargement of both organs which was very appreciable for the spleen13 %. In connection with our investigations it was desirable to determine whether a change in the blood content of the liver and spleen was the cause or one of the causes of the enlargement of the organ. Several authors have described methods for the determination of whole blood in tissue homogenates, all of which are based upon spectrophotometric measurements. The method of LOWRY AND HASTINGS” requires measurement of the extinctions in extremely LOWRY
turbid
absorption COHN”
solutions.
AND HASTINGS
The turbidity
have
tried
of light due to turbidity
measured
oxyhemoglobin
changes
increases
in the light
into hemiglobin
GORDON ANL) NURNBERGER’S
of the solutions
to overcome
linearly
absorption
or of hemiglobin
method5
causes a difficulty
by subtraction,
is based
with
produced
assuming
decreasing by
the
wavelength.
the conversion
into hemiglobin-cyanide. on the difference
which
that
of
Finally,
in shape between
The two latter methods presuppose that the degree of cloudiness is not altered by the reagents. It is our impression that the results, especially those for liver tissue, obtained by LOWRY XND HASTIKGS’ method are not consistently reliable because of the abthe absorption
sorptions
spectra
caused by turbidity.
ally brown
coloured,
ducts of hemoglobin. the analytical consistently, blood
of oxyhemoglobin
With
insoluble
the other two methods there developed
substances
These disintegration
results.
and carbosyhemoglobin.
which
were probably
products
another
method
pro-
are very likely to have influenced
Since the results by the various
we have developed
occasion-
disintegration
methods
for making
also did not agree
determinations
of whole
in tissues. Clix. Chim. Acfn, j’ (1962) 638-641