- Email: [email protected]
On steroid conjugates in plasma VIII. Extraction of 17-ketosteroid conjugates
I54
SHORT
COMMUNICATIONS
with reagent A) can be done with an automatic diluting pipette. Mixing of reagent C and deproteinised supemate can also be carried out with an automatic pipette”. Measurements can be made with the aid of an automatic cuvette. (d) Blood glucose values obtained by the method show good correlation with the values obtained with the method of Hagedorn-Jensen. Thus, the usual normal values can be maintained, when this method is introduced. The standard deviation of the method is about 3 mg%. (e) The recently published o-toluidine method5 was also investigated in our laboratories. Comparison with the phenylhydrazine method showed blood glucose values which were 30-40 rng% lower (deproteinisation with zinc acetate). Moreover, the o-toluidine method has an optimal heating time of 8 min; prolonged heating causes a reduction of the colour. A standard must be run through the same procedure in each series and readings should be made within 30 min, as the colour is not stable at room temperatur?. CONCLUSION
The phenylhydrazine method for the determination of glucose in biological fluids is attractive in routine analysis. The method is rapid, especially since the heating time, necessary for colour development, can be reduced to IO min. This investigation was carried out within the framework of the Buitengewone Normcommissie “Klinisch-Chemische methoden”, BNC 42, van het Nederlands Normalisatie-Instituut.
Clinical-Chemical
Laboratory,
J. A. P. STROES H. A. ZONDAG
St. Elisabeth’s Hos$ital,
Haarlem and Chemical Department, National Institute of Public Health, Utrecht (The Netherlands)
P. J. H. CHR. CORNEIJSSEN
1 A. H. HOLTZ, HA. J. VAN DREUMEL AND E. J. VAN KAMPEN, Clin. Chim. .4cta, 6 (1961) 467 a N. WAHBA, S. HANNA AND M. M. EL-SADR, Analyst, 81 (1956) 430. 3 H. H. KREUTZER AND M. BOSMAN, Ned. Tijdschr. Gsneesk., 104 (1960) 379. 4 -4. MATHER, Am. J. Clin. Pathol., 33 (1960) 186. 5 A. HYV~~RINEN AND E. A. XIKKILA, C&z. Chim. Acta, 7 (1962) 140.
Received
June r&h,
1962 Clin.Chim. Acta,8 (1963)152-154
On steroid conjugates in plasma VIII. Extraction of 17-ketosteroid conjugates
Recently, a reversible association between dehydroepiandrosterone (DHEA) sulfate and serum albumin has been demonstrated by in vitro techniques’. At the same time solvolysable I7-ketosteroid (17-KS) conjugates could be extracted from native
SHORT COMMUNICATIONS
plasma
following its dilution
with 300/~sodium sulfate.
155
These findings, together
with
the isolation of 17-KS sulfates from plasma213 and their estimation by various methods 4,5 suggested an anionic binding of 17-KS sulfates to serum albumin also under physiological conditions. On the other hand, the presence of non-polar 17-KS conjugatesassumedly represented by sulfatidyl-r7-KS and phosphatidyl-r7-KS-in cr-globulin containing serum fractions has been reported E- *. Since such contradictory evidence may have resulted in part from different extraction procedures, the following experiments were performed. Of a serum pool 50 ml samples were extracted in duplicate, using (A) 4 x 4 ~01s. chloroform-methanol (I : I v/v) (ref. g) ; (B) 5 ~01s. ethanol-acetone (I : I v/v) (ref. IO) ; (C) 3 ~01s. ethanol (ref. II); (D) 4 x 3 ~01s. ethyl acetate, after dilution with z ~01s. 30% sodium sulfate (ref. I) ; (E) 3 x 1.5 ~01s. chloroform, after addition of pyridinium sulfate to give a 1.5 M solution (ref. 5) ; (F) h exane, methylene chloride, and methanol, employing continuous extraction of lyophilized serum (ref. 2); (G) z ~01s. methanol and 3 ~01s. carbon tetrachloride (ref. 12). All extracts were evaporated to dryness was dissolved a column (I x water, 0.1 N water (I : 9 : 2
in a rotovac
at 35-45”.
Each
residue
in IO ml chloroform-methanol-water (I : 9 : 2. v/v) and transferred unto 20 cm) of DEAE cellulose, pretreated with 0.1 N hydrochloric acid, sodium hydroxide, water, 9o”/” methanol, and chloroform-methanolv/v). Elution was carried out with 15 ml of the following solvents : (I)
chloroform-methanol-water (I : 9: 2 v/v) ; (2) 90% methanol; (3) 50% methanol; (4) 25% methanol; (5) water; and (6) ~.oMacetate buffer of pH 4.75. Allchromatographic fractions containing organic solvents were evaporated and the residues redissolved in 25 ml water. After addition of 2.5 g sodium chloride and 1.0 ml cont. sulfuric acid all samples were extracted twice with 25 ml ethyl acetate, the combined extracts being incubated for 18 h at 37”, washed with sodium hydroxide and water, and evaporated to dryness. For removal of lipid material a solvent distribution of the residues between 80% methanol and n-hexane was employed. Following the evaporation of the aqueous methanol phase the free 17-KS present in the residue were separated by paper chromatography and quantitatedlO. In order to gain additional information on a second defatting technique I3 for purification of plasma extracts prior to cleavage of conjugation lo, two dry extracts prepared from two serum samples by method A were dissolved in 15 ml 70% methanol, cooled for 18 h at -20°, and centrifuged for 5 min at 4000 rev./min. The supernatant was removed and the residue washed with 2 ml icecold 70% methanol. Supernatant, as well as residue were subjected to solvolysis and analyzed for individual 17-KS. From Table I it becomes evident that the total concentrations of 17-KS conjugates obtained by methods A-G agree fairly well. However, there is a striking difference in the distribution of conjugates in chromatographic fractions 1-6. While by method A, B, and D only 18-36oj, of total 17-KS conjugates appear in fraction 6, usually containing steroid sulfates, methods E and G yield more than 8076 of total conjugates in this fraction. Purification of extracts A by the freezing technique resulted in losses of non-polar conjugates, amounting to g and 16% resp. of total 17KS conjugates, as found in the lipid residue. The fact, that in vitro a definite binding of DHEA sulfate to serum albumin takes place17 I4 which can be reversed by addition of sodium sulfate, and that solvolysable 17-KS conjugates can be extracted from plasma diluted with sodium sulfate does not
156
SHORT
COMMUNICATIONS
necessarily reflect the existence of such binding under in viva conditions; even if various 17-KS sulfates have been isolated from peripheral plasma by different methods 2$415.Extensive degradation of lipoproteins, known to contain 17-KS5 and yielding the assumed sulfatidyl-r7-KS among other lipids may equally well serve as explanation. Indeed, the chromatographic data collected during this investigation seems to favor the latter concept. The presence of varying amounts of 17-KS conjugates in
CHROMATOGRAPHY
OF DIFFERENT SERUM EXTRACTS OS DEAE
CELLULOSE
._~
Extraction @oceduve
pg IT-Ketostevoidslroo
ml in fraction
I;
2
91.7 86.2
11.4 16.1
-
11.3
18
30.0
‘3
B
79.8 84.0
14.6 ‘7.2
-
52.8 42.6
36 “9
C
60.8 54.4 68.2 67.4
9.4 7.9 12.8 1g.b
-
50.6
48
82.2
57
20.7
21
x
D
3
17.2 6.0
3.7 2.2
-
P
37.6 4'4
3.3 5."
--_
G
13.2
I.0
6.8
3.4
-
-
-
--
5
-
-
E
fraction 6 probably can be ascribed which becomes quite pronounced course of this study any formation not only depend on the nature of
1
6
IT-KS sulfczte (fraction 6)
I
-
19.2
18
109.s "7.4 86.6 73.5
84 93 68 61
I
14.8
39
109.4
92
to partial decomposition of labile sulfatidyl-I7-KS by use of methods E and G. As observed in the of 17-KS sulfates from non-polar conjugates does extracting solvents or added agents, but also on
pH and temperature. Under similar experimental conditions synthetic sulfatidyl-r7KS also give rise to measurable quantities of 17-KS sulfates. Likewise losses of 17-KS conjugates occurring through application of the freezing technique approximate those, experienced with synthetic sulfatidyl-r7-KS. They can be avoided by executing such a step only after solvolysis of conjugates. Department of Endocrinology, Institute for Hygiene and Microbiology, Saarland University, HomburglSaar (Gevmany)
GEORGW.OERTEL
1 R. PUCHE AND W. R. NES, Endocrinology, 70 (1962) 857. 2 E. E. BAULIEU, J. Clin. Endocrinol. Metab., 20 (1960) 900. 3 E. E. BAULIEU, R. EMMILIOZZIAND C. CORPECHOT, Experientia, 17 (1961) IIO. 4 S.CONRAD,V.MAHESH AND W. HERMANN, J.Clin. Invest.,40 (1961)947. 5 J. MCKENNA, Intern. Congr. Hormonal Steroids, Milano, 1962, Abstr. No. 217. 6 G. W. OERTEL. Biochem. Z., 334 (1961)431. ' G.W.OERTELUND E. KAISER, Biochem.2.. 336(1g6r) IO. 8 G.W.OERTEL. E. KAISER AND P. BRBHL, Biochem. Z., 336(1962) 154. Q W. R. NYE, C. WATERHOUSE AND G.V.MARINETTI, J.Clin. Invest., 40 (1961) 1194. lo G. mr'.OERTEL AND E. KAISER, Clin.Chim. Acta, 7 (1962) 221. Clin. (‘him. ACta, 8 (1963) 154--I j7
SHORT
COMMUNICATIONS
I.57
I1 G. W. OERTEL AND K. B. Em-NES, J.Biol.Chem., 232 (1958) 543. '2 B. HUDSON AND G. W. OERTEL, A~a~.B~~che~z., 2 (1961) 248. l3 W. R. BUTT, P. MORRIS, C. J. 0. R. MORRIS AND C. D. WILLIAMS, Biochem. 14 W.R.SLAUNWHITE AND A.A.SANDBERG, Endocrinoloffy. 62(1958)283. 15 L. E. GARDNER, Bull. Johns HoPkim Hosp., g4 (‘954) 195.
J.,
24 (1951) 434.
Received September 3rd, 1962 Clin. Chirn. Acta, 8 (1963) 154-157
Microdetermination
of blood glucose
The color instability that makes the Prussian blue reaction of ferrocyanide unsuitable for the determination of blood glucose I-$ can be very simply prevented by a proper selection of conditions for deproteinisation and color development. Reagents: (I) 13 g of 3CdS0, * SH,O dissolved in 63.5 ml of I N sulfuric acid and diluted to IOOOml with water. (2) Borate buffer (0.1 M, pH II): 6.2 g of boric acid dissolved in 150 ml of I l'vsodium hydroxide and diluted to 1000 ml with water. (3) Ferricyanide solution: 590 mg of K,Fe(CN), dissolved in IOO ml of 0.1 M sodium carbonate (10.6 g/l). Before use, this solution is diluted ten-fold with the same carbonate solution. (4) Ferric ammonium sulfate solution: 0.4317 g FeNH,(SO,), * 12H,O dissolved by gentle heating in IOO ml of I N hydrochloric acid and diluted to IOOO ml with 50% acetic acid (50 pg of Fe 3+ /ml). All the reagents are indefinitely stable. Procedure. To 4 mI of the cadmium sulfate solution, add 0.1 ml of oxalated blood in a centrifuge tube. After a few minutes, when the solution has become brown, add 5.9 ml of borate buffer; shake the mixture and after a short time centrifuge the precipitate for 5 min at about 2000 rev./min. To I ml of the water-clear supernate, add I ml of dilute ferricyanide, boil the solution for 5 min in a water bath (3 min are sufficient for a complete reduction), and then cool under running water; add 6 ml of the ferric ammonium sulfate solution. The color develops immediately, reaches a maximum after 15-20 min at room temperature (22-24”) and is stable for at least 5 h in the dark. Measure the color on a Beckman DU spectrophotometer at 725 rnp against a blank treated in the same manner or against distilled water; in the latter case, deduct a value of 0.03 from the readings. The E,$i is 2.320. Beer’s law is followed between x and 32 ,ug of total glucose, i.e. 0.12-4 pg of glucose per ml in the final solution. The accuracy is within & 27;. As the maximum absorption is represented by a large plateau, a calorimeter may be used instead of a spectrophotometer. The method may be very easily adapted for the determination of glucose on blood samples as small as IO ~1, simply by reducing the amounts of the reagents, without need of special apparatus. The values read on the standard curve, under the conditions described, indicate pg of glucose per IO ,ul of blood, thus to convert to g/l, it is sufficient to divide the value found by IO. When a spectrophotometer is used, the value in pg per IO ~1 may be obtained by dividing the absorbance found by 0.029.
flin. Cl&%A&, 8 (x963) x57-158
SHORT
COMMUNICATIONS
with reagent A) can be done with an automatic diluting pipette. Mixing of reagent C and deproteinised supemate can also be carried out with an automatic pipette”. Measurements can be made with the aid of an automatic cuvette. (d) Blood glucose values obtained by the method show good correlation with the values obtained with the method of Hagedorn-Jensen. Thus, the usual normal values can be maintained, when this method is introduced. The standard deviation of the method is about 3 mg%. (e) The recently published o-toluidine method5 was also investigated in our laboratories. Comparison with the phenylhydrazine method showed blood glucose values which were 30-40 rng% lower (deproteinisation with zinc acetate). Moreover, the o-toluidine method has an optimal heating time of 8 min; prolonged heating causes a reduction of the colour. A standard must be run through the same procedure in each series and readings should be made within 30 min, as the colour is not stable at room temperatur?. CONCLUSION
The phenylhydrazine method for the determination of glucose in biological fluids is attractive in routine analysis. The method is rapid, especially since the heating time, necessary for colour development, can be reduced to IO min. This investigation was carried out within the framework of the Buitengewone Normcommissie “Klinisch-Chemische methoden”, BNC 42, van het Nederlands Normalisatie-Instituut.
Clinical-Chemical
Laboratory,
J. A. P. STROES H. A. ZONDAG
St. Elisabeth’s Hos$ital,
Haarlem and Chemical Department, National Institute of Public Health, Utrecht (The Netherlands)
P. J. H. CHR. CORNEIJSSEN
1 A. H. HOLTZ, HA. J. VAN DREUMEL AND E. J. VAN KAMPEN, Clin. Chim. .4cta, 6 (1961) 467 a N. WAHBA, S. HANNA AND M. M. EL-SADR, Analyst, 81 (1956) 430. 3 H. H. KREUTZER AND M. BOSMAN, Ned. Tijdschr. Gsneesk., 104 (1960) 379. 4 -4. MATHER, Am. J. Clin. Pathol., 33 (1960) 186. 5 A. HYV~~RINEN AND E. A. XIKKILA, C&z. Chim. Acta, 7 (1962) 140.
Received
June r&h,
1962 Clin.Chim. Acta,8 (1963)152-154
On steroid conjugates in plasma VIII. Extraction of 17-ketosteroid conjugates
Recently, a reversible association between dehydroepiandrosterone (DHEA) sulfate and serum albumin has been demonstrated by in vitro techniques’. At the same time solvolysable I7-ketosteroid (17-KS) conjugates could be extracted from native
SHORT COMMUNICATIONS
plasma
following its dilution
with 300/~sodium sulfate.
155
These findings, together
with
the isolation of 17-KS sulfates from plasma213 and their estimation by various methods 4,5 suggested an anionic binding of 17-KS sulfates to serum albumin also under physiological conditions. On the other hand, the presence of non-polar 17-KS conjugatesassumedly represented by sulfatidyl-r7-KS and phosphatidyl-r7-KS-in cr-globulin containing serum fractions has been reported E- *. Since such contradictory evidence may have resulted in part from different extraction procedures, the following experiments were performed. Of a serum pool 50 ml samples were extracted in duplicate, using (A) 4 x 4 ~01s. chloroform-methanol (I : I v/v) (ref. g) ; (B) 5 ~01s. ethanol-acetone (I : I v/v) (ref. IO) ; (C) 3 ~01s. ethanol (ref. II); (D) 4 x 3 ~01s. ethyl acetate, after dilution with z ~01s. 30% sodium sulfate (ref. I) ; (E) 3 x 1.5 ~01s. chloroform, after addition of pyridinium sulfate to give a 1.5 M solution (ref. 5) ; (F) h exane, methylene chloride, and methanol, employing continuous extraction of lyophilized serum (ref. 2); (G) z ~01s. methanol and 3 ~01s. carbon tetrachloride (ref. 12). All extracts were evaporated to dryness was dissolved a column (I x water, 0.1 N water (I : 9 : 2
in a rotovac
at 35-45”.
Each
residue
in IO ml chloroform-methanol-water (I : 9 : 2. v/v) and transferred unto 20 cm) of DEAE cellulose, pretreated with 0.1 N hydrochloric acid, sodium hydroxide, water, 9o”/” methanol, and chloroform-methanolv/v). Elution was carried out with 15 ml of the following solvents : (I)
chloroform-methanol-water (I : 9: 2 v/v) ; (2) 90% methanol; (3) 50% methanol; (4) 25% methanol; (5) water; and (6) ~.oMacetate buffer of pH 4.75. Allchromatographic fractions containing organic solvents were evaporated and the residues redissolved in 25 ml water. After addition of 2.5 g sodium chloride and 1.0 ml cont. sulfuric acid all samples were extracted twice with 25 ml ethyl acetate, the combined extracts being incubated for 18 h at 37”, washed with sodium hydroxide and water, and evaporated to dryness. For removal of lipid material a solvent distribution of the residues between 80% methanol and n-hexane was employed. Following the evaporation of the aqueous methanol phase the free 17-KS present in the residue were separated by paper chromatography and quantitatedlO. In order to gain additional information on a second defatting technique I3 for purification of plasma extracts prior to cleavage of conjugation lo, two dry extracts prepared from two serum samples by method A were dissolved in 15 ml 70% methanol, cooled for 18 h at -20°, and centrifuged for 5 min at 4000 rev./min. The supernatant was removed and the residue washed with 2 ml icecold 70% methanol. Supernatant, as well as residue were subjected to solvolysis and analyzed for individual 17-KS. From Table I it becomes evident that the total concentrations of 17-KS conjugates obtained by methods A-G agree fairly well. However, there is a striking difference in the distribution of conjugates in chromatographic fractions 1-6. While by method A, B, and D only 18-36oj, of total 17-KS conjugates appear in fraction 6, usually containing steroid sulfates, methods E and G yield more than 8076 of total conjugates in this fraction. Purification of extracts A by the freezing technique resulted in losses of non-polar conjugates, amounting to g and 16% resp. of total 17KS conjugates, as found in the lipid residue. The fact, that in vitro a definite binding of DHEA sulfate to serum albumin takes place17 I4 which can be reversed by addition of sodium sulfate, and that solvolysable 17-KS conjugates can be extracted from plasma diluted with sodium sulfate does not
156
SHORT
COMMUNICATIONS
necessarily reflect the existence of such binding under in viva conditions; even if various 17-KS sulfates have been isolated from peripheral plasma by different methods 2$415.Extensive degradation of lipoproteins, known to contain 17-KS5 and yielding the assumed sulfatidyl-r7-KS among other lipids may equally well serve as explanation. Indeed, the chromatographic data collected during this investigation seems to favor the latter concept. The presence of varying amounts of 17-KS conjugates in
CHROMATOGRAPHY
OF DIFFERENT SERUM EXTRACTS OS DEAE
CELLULOSE
._~
Extraction @oceduve
pg IT-Ketostevoidslroo
ml in fraction
I;
2
91.7 86.2
11.4 16.1
-
11.3
18
30.0
‘3
B
79.8 84.0
14.6 ‘7.2
-
52.8 42.6
36 “9
C
60.8 54.4 68.2 67.4
9.4 7.9 12.8 1g.b
-
50.6
48
82.2
57
20.7
21
x
D
3
17.2 6.0
3.7 2.2
-
P
37.6 4'4
3.3 5."
--_
G
13.2
I.0
6.8
3.4
-
-
-
--
5
-
-
E
fraction 6 probably can be ascribed which becomes quite pronounced course of this study any formation not only depend on the nature of
1
6
IT-KS sulfczte (fraction 6)
I
-
19.2
18
109.s "7.4 86.6 73.5
84 93 68 61
I
14.8
39
109.4
92
to partial decomposition of labile sulfatidyl-I7-KS by use of methods E and G. As observed in the of 17-KS sulfates from non-polar conjugates does extracting solvents or added agents, but also on
pH and temperature. Under similar experimental conditions synthetic sulfatidyl-r7KS also give rise to measurable quantities of 17-KS sulfates. Likewise losses of 17-KS conjugates occurring through application of the freezing technique approximate those, experienced with synthetic sulfatidyl-r7-KS. They can be avoided by executing such a step only after solvolysis of conjugates. Department of Endocrinology, Institute for Hygiene and Microbiology, Saarland University, HomburglSaar (Gevmany)
GEORGW.OERTEL
1 R. PUCHE AND W. R. NES, Endocrinology, 70 (1962) 857. 2 E. E. BAULIEU, J. Clin. Endocrinol. Metab., 20 (1960) 900. 3 E. E. BAULIEU, R. EMMILIOZZIAND C. CORPECHOT, Experientia, 17 (1961) IIO. 4 S.CONRAD,V.MAHESH AND W. HERMANN, J.Clin. Invest.,40 (1961)947. 5 J. MCKENNA, Intern. Congr. Hormonal Steroids, Milano, 1962, Abstr. No. 217. 6 G. W. OERTEL. Biochem. Z., 334 (1961)431. ' G.W.OERTELUND E. KAISER, Biochem.2.. 336(1g6r) IO. 8 G.W.OERTEL. E. KAISER AND P. BRBHL, Biochem. Z., 336(1962) 154. Q W. R. NYE, C. WATERHOUSE AND G.V.MARINETTI, J.Clin. Invest., 40 (1961) 1194. lo G. mr'.OERTEL AND E. KAISER, Clin.Chim. Acta, 7 (1962) 221. Clin. (‘him. ACta, 8 (1963) 154--I j7
SHORT
COMMUNICATIONS
I.57
I1 G. W. OERTEL AND K. B. Em-NES, J.Biol.Chem., 232 (1958) 543. '2 B. HUDSON AND G. W. OERTEL, A~a~.B~~che~z., 2 (1961) 248. l3 W. R. BUTT, P. MORRIS, C. J. 0. R. MORRIS AND C. D. WILLIAMS, Biochem. 14 W.R.SLAUNWHITE AND A.A.SANDBERG, Endocrinoloffy. 62(1958)283. 15 L. E. GARDNER, Bull. Johns HoPkim Hosp., g4 (‘954) 195.
J.,
24 (1951) 434.
Received September 3rd, 1962 Clin. Chirn. Acta, 8 (1963) 154-157
Microdetermination
of blood glucose
The color instability that makes the Prussian blue reaction of ferrocyanide unsuitable for the determination of blood glucose I-$ can be very simply prevented by a proper selection of conditions for deproteinisation and color development. Reagents: (I) 13 g of 3CdS0, * SH,O dissolved in 63.5 ml of I N sulfuric acid and diluted to IOOOml with water. (2) Borate buffer (0.1 M, pH II): 6.2 g of boric acid dissolved in 150 ml of I l'vsodium hydroxide and diluted to 1000 ml with water. (3) Ferricyanide solution: 590 mg of K,Fe(CN), dissolved in IOO ml of 0.1 M sodium carbonate (10.6 g/l). Before use, this solution is diluted ten-fold with the same carbonate solution. (4) Ferric ammonium sulfate solution: 0.4317 g FeNH,(SO,), * 12H,O dissolved by gentle heating in IOO ml of I N hydrochloric acid and diluted to IOOO ml with 50% acetic acid (50 pg of Fe 3+ /ml). All the reagents are indefinitely stable. Procedure. To 4 mI of the cadmium sulfate solution, add 0.1 ml of oxalated blood in a centrifuge tube. After a few minutes, when the solution has become brown, add 5.9 ml of borate buffer; shake the mixture and after a short time centrifuge the precipitate for 5 min at about 2000 rev./min. To I ml of the water-clear supernate, add I ml of dilute ferricyanide, boil the solution for 5 min in a water bath (3 min are sufficient for a complete reduction), and then cool under running water; add 6 ml of the ferric ammonium sulfate solution. The color develops immediately, reaches a maximum after 15-20 min at room temperature (22-24”) and is stable for at least 5 h in the dark. Measure the color on a Beckman DU spectrophotometer at 725 rnp against a blank treated in the same manner or against distilled water; in the latter case, deduct a value of 0.03 from the readings. The E,$i is 2.320. Beer’s law is followed between x and 32 ,ug of total glucose, i.e. 0.12-4 pg of glucose per ml in the final solution. The accuracy is within & 27;. As the maximum absorption is represented by a large plateau, a calorimeter may be used instead of a spectrophotometer. The method may be very easily adapted for the determination of glucose on blood samples as small as IO ~1, simply by reducing the amounts of the reagents, without need of special apparatus. The values read on the standard curve, under the conditions described, indicate pg of glucose per IO ,ul of blood, thus to convert to g/l, it is sufficient to divide the value found by IO. When a spectrophotometer is used, the value in pg per IO ~1 may be obtained by dividing the absorbance found by 0.029.
flin. Cl&%A&, 8 (x963) x57-158