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Simultaneous determination of cortisol and corticosterone in urine
379
CLINICA CHIMICA ACTA
SIMULTANEOUS DETERMINATION OF CORTISOL AND CORTICOSTERONE IN U R I N E *
MALCOLM M. M A R T I N AND A R L I N E L. A. M A R T I N with the assistance of M. E. McMANUS
Department of Pediatrics, School of Medicine, Georgetown University :ffedical Center, Washington, D.C. aooo7 (U.S.A.) (Received October 28, 1969)
SUMMARY
Urinary cortisol and corticosterone have been measured sinmltaneously without prior purification or chromatography using a fluorometric method which incorporates a correction for non-specific fluorescence. Non-corticoid fluorescence was corrected for by the introduction of a sample blank in which corticosteroid fluorescence has been altered by interaction with hydroxylamine. Daily excretion of cortisol in normal subjects from I8 to x2o pg with a mean of 5I ~ 24 pg S.D. and of corticosterone o to I6 p g with a mean of 3 o ± 3.2 pg S.D. The demonstration of a circadian rhythm, low levels in patients with adrenocortical hypofunction, elevation in the third trimester of pregnancy and the expected responses to metyrapone and corticotropin illustrate the close correlation with ,,drenocortical activity.
INTRODUCTION
A year ago we reported a relatively simple and yet specific method for the simultaneous fluorometric determination of cortisol and col~icosterone in human plasma ~. The method incorporates oxime formation with alteration in steroid structure and fluorescence to provide a ~ample blank. The procedure has now been applied to the simultaneous determination of cortisol and corticosterone in urine and amniotic fluid, and it has been found sensitive, specific and practical. MATERIALS AND METHODS
Materials and procedure were as described for plasma I except that 4 ml of a z4-h collection of urine or 4 ml of amniotic fluid were extracted with 6 volumes of methylene chloride which, prior to drying down, was washed once with 5 ml of o.75 N NaOH followed by 5 ml of o.o2 M acetic acid. Separa~on of cortisol and corticcz* This investigation was supported in part b y U S P H S Grant AM 1o910 from the Institute of Arthritis and Metabolic Diseases, National Institutes of Health.
Clin. Chim. Acta, 27 (I97 o) 379-383
380
MARTIN, MARTIN
sterone, fluorescence development and calculatioa of the results followed the original technique t. RESULTS
Adequacy of the method was evaluated by investigating the recovery of known amounts of cortisol and corticosterone added to urine (Table I). The simultaneous determination of cortisol and corticosterone in I2 a!iquots of the same urine showed a mean of 48 ~ 5.o pg per 24 h S.D. for cortisol and 6.o ~ o.8 pg per 24 h S.D. for corticosterone. Reproducibility of the method was further analyzed by estimating the standard deviation (s) for duplicate determinations according to the formula TABLE
1
SIMULTANEOUS
RECOVERY
OF CORTISOL AND CORTICOSI ERONE ADDED
TO URINE
pg Steroid added Cortisol Corticosterone
°/o Cortisol recow, red Mean -~: S.D. Range
°/o Corticosterone recovered Mean ~ S.D. I¢~ange
o.1oo o.2oo
78 -_k_ 13.6 96 A= 7.9
81 ~ 11.3 83 :k 6.3
0,025 o.o5o
63-98 88-i1o
69-Io 5 76-97
where d = difference of tile duplicate determinations and n = number of duplications (Table I I). All calculations are based on duplicate determinations using 4 ml of urine o" amniotic fluid and 2.0 ml plasma as previously described'. The coefficient of variation for cortisol determinations was 8%, that for corticosterone determinations 20% in the range o-4. 9 pg per 24 h and 15% above that level. s =: x / S d 2 / 2 n
TABLE
II
S T A N D A R D D E V I A T I O N (S) O F [ ' " P L I C A T E DETERMINATIONS FOR GIVEN RANGES OF EXCRETION URINARY CORTISOL AND CORTICOSTERONE AND THE PERCENT ERROR FOR EACH RANGE
pg/24 h
No. of pairs
s
% Error
Cortisol o-99 t°°-499 5 °°'-999 > Iooo
60 23 t3 8
't .° I9.o 52.0 t33,o
! 8 • 9 i 7 ._Jr= 9
Corticosterone 0-4.9 5-20 > 2o
59 20 IO
0.58 1.49 6.40
oF
_-J: 2o :k_ I5 -t: 15
In 60 normal subjects without endocrine, hepatic or renal disease on a free diet urinary 24-h excretion of cortisol ranged from IS.o-I2opg with a mean of 51 ~ 24pg S.D. and of corticosterone from o-16/~g per 24 h with a mean of 3.o _-4-3.2 pg per 24 h S.D. (Fig. I). In IO collections the urinary excretion of corticosterone was below the sensitivity of the method as described. Values in children were corrected for surface area to bring them into line with data obtained in adults. None of the patients were acutely ill at the time of collection and none were being exposed to a known stressful procedure. The circadian variation in urinary cortisol excretion was investigated in Clin. Chim, Acta. 27 (t97 o) 379,-383
CORTISOL AND CORTICOSTERONE IN URINE URINARY CORTISOL
:
.38i
URINARY CORTICOSTERON£
20-
!$"
10,~.
N ~:,'...::~"
~
;'~;.-."2;~
~"::::.':
4O
IO
110
i
!
!
4
8
lZ
CORTICO$IERONE lpg per day}
CORTISOL tpg per day)
Fig. I. Cortisol and corticosterone excretion by normal subjects.
6 subjects. In all 6 studies a definite and highly significant diurnal rhythm could be demonstrated (Table III). The urinary excretion of cortisol and corticosterone in, esponse to ACTH stimulation and the administration of metyrapone, an x x/~-hydroxylase inhibitor, was studied in x6 subjects. Metyrapone given orally in a dose of z5 mg/kg body weight every 4 h for two days had no discernible effect on the -~4-h urinary excretion of cortisol or corticosterone. Corticotropin stimulation (40 U ACTHAR-Gel intramuscularly b.i.d.) in contrast, caused a greater than five-fold increase in urinary cortisol and corticosterone on day x an(i a greater than ten-fold ini:rease on day z. Patients with hypopituitarism and ACTH deficiency had a mean urinary cortisol excretion of x9 ~: 5 Pg/z4 h which differed significantly from the normal TAI]L1- III URINAR~ ° EXCRETION PHYSIOLOGICAL
OF CORTISOL
CONDITIONS
No. of obs. Normal subjects Circadian r h y t h m
AND CORTICOSTERONE
AND IN DIFFERENT
60 6
Time of day
6 a.m.-noon noon--6 p.m. 6 p.m.-mdn m d n - 6 a.m.
IN
NORMAL
SI'BJEI~T~
UNI)ER
VARIOUS
PATHOLOGICAL STATES
Cortisol pg/z 4 h Mean ± S.D.
Covticosterone ug/-'4 h Mean ~ S.D. (Range)
5~ ::k z4
3 .0 ~: 3 .z
z 7 ~: 6
A C T H gei: D a y i 4 ° U i/hi bM. Day z
t6
16 -,= 8 9 ~: 6 8 ± 4 02o ( I I S - 2 o z o )
1 z l o (55o-2o4 o)
55.3 (zz.o-q9.o)
Metyrapone: Day I
I6
5 z -): z3
4.4 :]: z.6
15 m g / k g q 4hp-o. Day z A C T H deficiency
57 _4= z 4
4.2 :~ 2.5
6
3 Adrenal suppression Pregnancy 3rd trimester Io Urine 9 Plasma I6 Amniotic fluid
IO. 4 (z.I-75.o)
I9 ~ 5 Io, 19, 16
78 4-_=29
3.6 ~ 3.3
z3.4 ~ 5.4 p g / I o o ml 3 0 ± 1.3 p g / I o o ml
Clin. Chim. Acta, 27 (I97 o) 379-383
382
....
MARTIN, MARTIN
(p < O.OI). Suppression with Prednisone resulted in urine values for cortisol similar to those found in patients with hypopituitarism. Plasma cortisol levels as well as the urinary excretion of cortisol were elevated in the third trimester of pregnancy. Despite considerable overlap, mean daily cortisol excretion differed significantly from the non-pregnant mean (p < 0.05). Cortisol concentration in amniotic fluid obtained by amniocentesis between the 32nd and 36th week was 3.0 ~ 1.3 pg/loo ml. When cortisol was administered orally to a child, increasing the dose by 2o mg every other day to Ioo mg and then to 15° mg for two days, urinary cortisol excretion rose from a mean control value of 30 pg per day in an exponential fashion to reach a maximum of 118o pg per day, comparable to the levels observed following ACTH stimulation. DISCUSSION
The method described here is a relatively simple, rapid, highly sensitive photofluorometric method for the simultaneous determination of cortisol and corticosterone, well suited for the clinical laboratory. It can be applie¢~. directly to biological fluids without prior purification or chromatography. Non-specihc, non-corticoid fluorescence is corrected for by the introduction of a sample blank in which corticosteroid fluorescence is altered, by interaction with hydroxylamine. The criteria used for the identity of the substances determined as cortisol and corticosterone are steroid oxime formation, behavior in the extraction procedure and fluorescence in lO% ethanolic 85% aqueous sulfuric acid under conditions found optimal for these compounds. Thin-layer chromatography of a sample extract confirmed absence of the characteristic fluorescence spectra of cortisol and corticosterone in the hydroxylamine-treated blank. Approximately 5% to lO% of cortisol is found in the carbon tetrachloride extract and a similar fractio;l ui corticosterone in the methylene cldoride extract. The difference in fluorescence intensity of the two steroids compensates to some extent "~|or this relative exchange and this shortcoming in the separation does not detract from the usefulness of the method for clinical application. The accuracy and specificity of the method are attested to by the low levels found in patients with known hypofunction of the adrenal .-:,rtex, the demonstration of a circadian rhythm and values in normal subjects in keeping with those in the literature (Table IV), the elevated levels in the third trimester of pregnancy and the response to metyrapone and corticotropin stimulation 2,3. The concentration of cortisol in the amniotic fluid obtained by amniocentesis as measured by this method is also in excellent agreement with the value obtained by a procedure involving acetate formation and chromatography 4. These results and the levels obtained in pregnancy urine lend further support to the specificity of the method, as does the lack of interference from other fluorogenic steroids, particularly the estrogens. The results obtained for urinary cortisol compare well with those reported for the competitive protein-binding radioassay recently published s. The latter method is applicable to micro-quantities, which offers a definite advantage for the determination of cortisol in plasma, but is of little importance as regards urine. The photofluorometric method described, furthermore, permits the determination of two urinary steroids simultaneously. An additional important aspect is its practicability, reflected Clin. Chim. Acta, 27 (I97o) 379-383
CORTISOL AND CORTICOSTERONE IN URINE
383
T A B L E IV N O R M A L U R I N A R Y C O R T I S O L V A L U E S IN R E P R E S E N T A T I V E
Urinaty cortisol (pg/z4 h) Mean Range 35 44 71 93 48 5t
13-86 o-I2o o-x8i 6o-x59 O-lO8 t8-i2o
No. o/obs, 24 xzo 38 z4 23 60
LARGE SERIES
Urinary corticosterone (pg/z4 h) 3lean Range No. o[ obs.
Corrected for recovery
Re[erence
5.8
2.2-9-.0
7
3.0
o.o-16.o
60
yes no yes no no no
Ayers et al., 1957 (5) Cope and Black, I959 (2) Rosner, x963 (6) Pal and Smith, i965 (7) Murphy, x968 (~) present study
in the time required for the analysis, the skill required and the cost involved. We find that zo samples can be completed without pressure by a single laboratory technician in one working day, with just average technical skill. If only cortisol determinations are required, the carbon tetrachloride extracts may be discarded, which permits 3o and more samples to be included in one run. The usefulness of the method rests in its high sensitivity and close correlation to adrenocortical activity. REFERENCES M. M. MARTIN A,~D A. L. MARTIN, J. Clin. Endocrinol., 28 (1968) t37. C. L. COPE AHD E. G. BLACK, Brit. Med. J., It (1959) Ix7. B. E. P. MURPHY, J. Clin. Endocrinol., 28 (t968) 343C. W. BAIRD AND I. E. BUSH. Acta Endocrinol, 34 (I96o) 97. P. J- AVERs, O. GARROD, S. A. SIMPSON AND J. F. TAIT, Biochem. J., 65 (1957) 639. J. M. ROSSER, J. J. Cos, E. G. BIGLIERI, S. HAHE A.~D P. H. FORSHAM, J. Clin. Endocrinol.. 25 (196:~) 82o. 7 S. B. PAL. A,~D V. K. SMITH, Clin. Chim..4 'a. I2 (I965) 588.
t z 3 4 5 6
Clin. Chim. Acta, 27 (197 o) 379-383
CLINICA CHIMICA ACTA
SIMULTANEOUS DETERMINATION OF CORTISOL AND CORTICOSTERONE IN U R I N E *
MALCOLM M. M A R T I N AND A R L I N E L. A. M A R T I N with the assistance of M. E. McMANUS
Department of Pediatrics, School of Medicine, Georgetown University :ffedical Center, Washington, D.C. aooo7 (U.S.A.) (Received October 28, 1969)
SUMMARY
Urinary cortisol and corticosterone have been measured sinmltaneously without prior purification or chromatography using a fluorometric method which incorporates a correction for non-specific fluorescence. Non-corticoid fluorescence was corrected for by the introduction of a sample blank in which corticosteroid fluorescence has been altered by interaction with hydroxylamine. Daily excretion of cortisol in normal subjects from I8 to x2o pg with a mean of 5I ~ 24 pg S.D. and of corticosterone o to I6 p g with a mean of 3 o ± 3.2 pg S.D. The demonstration of a circadian rhythm, low levels in patients with adrenocortical hypofunction, elevation in the third trimester of pregnancy and the expected responses to metyrapone and corticotropin illustrate the close correlation with ,,drenocortical activity.
INTRODUCTION
A year ago we reported a relatively simple and yet specific method for the simultaneous fluorometric determination of cortisol and col~icosterone in human plasma ~. The method incorporates oxime formation with alteration in steroid structure and fluorescence to provide a ~ample blank. The procedure has now been applied to the simultaneous determination of cortisol and corticosterone in urine and amniotic fluid, and it has been found sensitive, specific and practical. MATERIALS AND METHODS
Materials and procedure were as described for plasma I except that 4 ml of a z4-h collection of urine or 4 ml of amniotic fluid were extracted with 6 volumes of methylene chloride which, prior to drying down, was washed once with 5 ml of o.75 N NaOH followed by 5 ml of o.o2 M acetic acid. Separa~on of cortisol and corticcz* This investigation was supported in part b y U S P H S Grant AM 1o910 from the Institute of Arthritis and Metabolic Diseases, National Institutes of Health.
Clin. Chim. Acta, 27 (I97 o) 379-383
380
MARTIN, MARTIN
sterone, fluorescence development and calculatioa of the results followed the original technique t. RESULTS
Adequacy of the method was evaluated by investigating the recovery of known amounts of cortisol and corticosterone added to urine (Table I). The simultaneous determination of cortisol and corticosterone in I2 a!iquots of the same urine showed a mean of 48 ~ 5.o pg per 24 h S.D. for cortisol and 6.o ~ o.8 pg per 24 h S.D. for corticosterone. Reproducibility of the method was further analyzed by estimating the standard deviation (s) for duplicate determinations according to the formula TABLE
1
SIMULTANEOUS
RECOVERY
OF CORTISOL AND CORTICOSI ERONE ADDED
TO URINE
pg Steroid added Cortisol Corticosterone
°/o Cortisol recow, red Mean -~: S.D. Range
°/o Corticosterone recovered Mean ~ S.D. I¢~ange
o.1oo o.2oo
78 -_k_ 13.6 96 A= 7.9
81 ~ 11.3 83 :k 6.3
0,025 o.o5o
63-98 88-i1o
69-Io 5 76-97
where d = difference of tile duplicate determinations and n = number of duplications (Table I I). All calculations are based on duplicate determinations using 4 ml of urine o" amniotic fluid and 2.0 ml plasma as previously described'. The coefficient of variation for cortisol determinations was 8%, that for corticosterone determinations 20% in the range o-4. 9 pg per 24 h and 15% above that level. s =: x / S d 2 / 2 n
TABLE
II
S T A N D A R D D E V I A T I O N (S) O F [ ' " P L I C A T E DETERMINATIONS FOR GIVEN RANGES OF EXCRETION URINARY CORTISOL AND CORTICOSTERONE AND THE PERCENT ERROR FOR EACH RANGE
pg/24 h
No. of pairs
s
% Error
Cortisol o-99 t°°-499 5 °°'-999 > Iooo
60 23 t3 8
't .° I9.o 52.0 t33,o
! 8 • 9 i 7 ._Jr= 9
Corticosterone 0-4.9 5-20 > 2o
59 20 IO
0.58 1.49 6.40
oF
_-J: 2o :k_ I5 -t: 15
In 60 normal subjects without endocrine, hepatic or renal disease on a free diet urinary 24-h excretion of cortisol ranged from IS.o-I2opg with a mean of 51 ~ 24pg S.D. and of corticosterone from o-16/~g per 24 h with a mean of 3.o _-4-3.2 pg per 24 h S.D. (Fig. I). In IO collections the urinary excretion of corticosterone was below the sensitivity of the method as described. Values in children were corrected for surface area to bring them into line with data obtained in adults. None of the patients were acutely ill at the time of collection and none were being exposed to a known stressful procedure. The circadian variation in urinary cortisol excretion was investigated in Clin. Chim, Acta. 27 (t97 o) 379,-383
CORTISOL AND CORTICOSTERONE IN URINE URINARY CORTISOL
:
.38i
URINARY CORTICOSTERON£
20-
!$"
10,~.
N ~:,'...::~"
~
;'~;.-."2;~
~"::::.':
4O
IO
110
i
!
!
4
8
lZ
CORTICO$IERONE lpg per day}
CORTISOL tpg per day)
Fig. I. Cortisol and corticosterone excretion by normal subjects.
6 subjects. In all 6 studies a definite and highly significant diurnal rhythm could be demonstrated (Table III). The urinary excretion of cortisol and corticosterone in, esponse to ACTH stimulation and the administration of metyrapone, an x x/~-hydroxylase inhibitor, was studied in x6 subjects. Metyrapone given orally in a dose of z5 mg/kg body weight every 4 h for two days had no discernible effect on the -~4-h urinary excretion of cortisol or corticosterone. Corticotropin stimulation (40 U ACTHAR-Gel intramuscularly b.i.d.) in contrast, caused a greater than five-fold increase in urinary cortisol and corticosterone on day x an(i a greater than ten-fold ini:rease on day z. Patients with hypopituitarism and ACTH deficiency had a mean urinary cortisol excretion of x9 ~: 5 Pg/z4 h which differed significantly from the normal TAI]L1- III URINAR~ ° EXCRETION PHYSIOLOGICAL
OF CORTISOL
CONDITIONS
No. of obs. Normal subjects Circadian r h y t h m
AND CORTICOSTERONE
AND IN DIFFERENT
60 6
Time of day
6 a.m.-noon noon--6 p.m. 6 p.m.-mdn m d n - 6 a.m.
IN
NORMAL
SI'BJEI~T~
UNI)ER
VARIOUS
PATHOLOGICAL STATES
Cortisol pg/z 4 h Mean ± S.D.
Covticosterone ug/-'4 h Mean ~ S.D. (Range)
5~ ::k z4
3 .0 ~: 3 .z
z 7 ~: 6
A C T H gei: D a y i 4 ° U i/hi bM. Day z
t6
16 -,= 8 9 ~: 6 8 ± 4 02o ( I I S - 2 o z o )
1 z l o (55o-2o4 o)
55.3 (zz.o-q9.o)
Metyrapone: Day I
I6
5 z -): z3
4.4 :]: z.6
15 m g / k g q 4hp-o. Day z A C T H deficiency
57 _4= z 4
4.2 :~ 2.5
6
3 Adrenal suppression Pregnancy 3rd trimester Io Urine 9 Plasma I6 Amniotic fluid
IO. 4 (z.I-75.o)
I9 ~ 5 Io, 19, 16
78 4-_=29
3.6 ~ 3.3
z3.4 ~ 5.4 p g / I o o ml 3 0 ± 1.3 p g / I o o ml
Clin. Chim. Acta, 27 (I97 o) 379-383
382
....
MARTIN, MARTIN
(p < O.OI). Suppression with Prednisone resulted in urine values for cortisol similar to those found in patients with hypopituitarism. Plasma cortisol levels as well as the urinary excretion of cortisol were elevated in the third trimester of pregnancy. Despite considerable overlap, mean daily cortisol excretion differed significantly from the non-pregnant mean (p < 0.05). Cortisol concentration in amniotic fluid obtained by amniocentesis between the 32nd and 36th week was 3.0 ~ 1.3 pg/loo ml. When cortisol was administered orally to a child, increasing the dose by 2o mg every other day to Ioo mg and then to 15° mg for two days, urinary cortisol excretion rose from a mean control value of 30 pg per day in an exponential fashion to reach a maximum of 118o pg per day, comparable to the levels observed following ACTH stimulation. DISCUSSION
The method described here is a relatively simple, rapid, highly sensitive photofluorometric method for the simultaneous determination of cortisol and corticosterone, well suited for the clinical laboratory. It can be applie¢~. directly to biological fluids without prior purification or chromatography. Non-specihc, non-corticoid fluorescence is corrected for by the introduction of a sample blank in which corticosteroid fluorescence is altered, by interaction with hydroxylamine. The criteria used for the identity of the substances determined as cortisol and corticosterone are steroid oxime formation, behavior in the extraction procedure and fluorescence in lO% ethanolic 85% aqueous sulfuric acid under conditions found optimal for these compounds. Thin-layer chromatography of a sample extract confirmed absence of the characteristic fluorescence spectra of cortisol and corticosterone in the hydroxylamine-treated blank. Approximately 5% to lO% of cortisol is found in the carbon tetrachloride extract and a similar fractio;l ui corticosterone in the methylene cldoride extract. The difference in fluorescence intensity of the two steroids compensates to some extent "~|or this relative exchange and this shortcoming in the separation does not detract from the usefulness of the method for clinical application. The accuracy and specificity of the method are attested to by the low levels found in patients with known hypofunction of the adrenal .-:,rtex, the demonstration of a circadian rhythm and values in normal subjects in keeping with those in the literature (Table IV), the elevated levels in the third trimester of pregnancy and the response to metyrapone and corticotropin stimulation 2,3. The concentration of cortisol in the amniotic fluid obtained by amniocentesis as measured by this method is also in excellent agreement with the value obtained by a procedure involving acetate formation and chromatography 4. These results and the levels obtained in pregnancy urine lend further support to the specificity of the method, as does the lack of interference from other fluorogenic steroids, particularly the estrogens. The results obtained for urinary cortisol compare well with those reported for the competitive protein-binding radioassay recently published s. The latter method is applicable to micro-quantities, which offers a definite advantage for the determination of cortisol in plasma, but is of little importance as regards urine. The photofluorometric method described, furthermore, permits the determination of two urinary steroids simultaneously. An additional important aspect is its practicability, reflected Clin. Chim. Acta, 27 (I97o) 379-383
CORTISOL AND CORTICOSTERONE IN URINE
383
T A B L E IV N O R M A L U R I N A R Y C O R T I S O L V A L U E S IN R E P R E S E N T A T I V E
Urinaty cortisol (pg/z4 h) Mean Range 35 44 71 93 48 5t
13-86 o-I2o o-x8i 6o-x59 O-lO8 t8-i2o
No. o/obs, 24 xzo 38 z4 23 60
LARGE SERIES
Urinary corticosterone (pg/z4 h) 3lean Range No. o[ obs.
Corrected for recovery
Re[erence
5.8
2.2-9-.0
7
3.0
o.o-16.o
60
yes no yes no no no
Ayers et al., 1957 (5) Cope and Black, I959 (2) Rosner, x963 (6) Pal and Smith, i965 (7) Murphy, x968 (~) present study
in the time required for the analysis, the skill required and the cost involved. We find that zo samples can be completed without pressure by a single laboratory technician in one working day, with just average technical skill. If only cortisol determinations are required, the carbon tetrachloride extracts may be discarded, which permits 3o and more samples to be included in one run. The usefulness of the method rests in its high sensitivity and close correlation to adrenocortical activity. REFERENCES M. M. MARTIN A,~D A. L. MARTIN, J. Clin. Endocrinol., 28 (1968) t37. C. L. COPE AHD E. G. BLACK, Brit. Med. J., It (1959) Ix7. B. E. P. MURPHY, J. Clin. Endocrinol., 28 (t968) 343C. W. BAIRD AND I. E. BUSH. Acta Endocrinol, 34 (I96o) 97. P. J- AVERs, O. GARROD, S. A. SIMPSON AND J. F. TAIT, Biochem. J., 65 (1957) 639. J. M. ROSSER, J. J. Cos, E. G. BIGLIERI, S. HAHE A.~D P. H. FORSHAM, J. Clin. Endocrinol.. 25 (196:~) 82o. 7 S. B. PAL. A,~D V. K. SMITH, Clin. Chim..4 'a. I2 (I965) 588.
t z 3 4 5 6
Clin. Chim. Acta, 27 (197 o) 379-383