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Interference with urine 17-oxogenicsteroid analysis
Clin. Biochem. 1, 172-175 (1967)
LABORATORY
NOTE
I N T E R F E R E N G E W I T H URINE 17-OXOGENICSTEROID ANALYSIS
MARY G. METCALF* Medical Unit, Princess Margaret Hospital, Christchurch, New Zealand (Received May 20, 1967)
INTERFERENCE WITH ANALYTICAL PROCEDURES is of tWO types. Direct interference occurs when a substance directly influences an analytical procedure. Indirect interference occurs when a drug or one of its metabolites induces a change in the production or excretion of the test substance b y the patient. In this note, examples of direct interference with the 17-oxogenicsteroid (17-OGS) analysis are reported and discussed. T h e chemical reactions involved in the m e a s u r e m e n t of 17-OGS in urine are outlined in Fig. 1. ?.2-
-
CHOH ~
O
C=O
H
CH 2 - / CHOH
~ , ~ O H
+ ~~'~"~ O"
reO duc H 4 ti°nN with a~E~~ H
d 17- oxogenicstePoids oxidation with • Na IO 4
17) 20-dials alkaline • rn- dinit, Pobenzene I
17-oxostePoids
NO 2 J
(Ema x .520nrn )
FIO. 1. Chemical principles underlying the measurement of 17-oxogenic-steroids in urine. In theory, a n y substance competing with the 17-OGS for either NaBH4, NaIO4 or m-dinitrobenzene would be expected to interfere with the analysis. I n a d e q u a t e a m o u n t s of N a B H 4 would cause unpredictable results, and complete absence would lead to the inclusion of 17-oxosteroid glucuronides as 17-oxogenicsteroids and the exclusion of 17-hydroxy-20-oxosteroids from the assay. Substances destroying NaIO4 would be expected to cause low results b y preventing the conversion of 17,20-diols to 17-oxosteroids, while those reacting with m-dinitrobenzene would cause high results if the non-steroldal colour complex *Supported by a New Zealand MlZC grant.
LABORATORY NOTE
173
absorbed light a t 520 n m and low results if it did not. In practice, these four types of interference h a v e been experienced. Glucose is a c o m m o n constituent of urine which interferes with the 17-OGS analysis b y destroying both N a B H , and NaIO4. Provided sufficient extra N a B H 4 and NaIO~ is added to c o m p e n s a t e for the glucose present (1), 17-oxogenicsteroids m a y be measured in urine containing as much as 6 g % glucose. Other substances which interfere in this m a n n e r should be amenable to similar treatment. METHODS Urine for analysis was acidified to p H 2-3 and stored a t 4 °. 17-Oxogenicsteroids 17,20-dihydroxysteroids and 17-hydroxy-20-oxosteroids) were measured as 17-oxosteroids after t r e a t m e n t with N a B H 4 and NaIO4 (2), !7-hydroxycorficosteroids as the yellow phenylhydrazones (3) and "free cortisol" (mainly cortisol, corticosterone and 21-desoxy-cortisol) b y the fluorimetric procedure described by De M o o r and co-workers (g). In the absence of glucose, 0.05 g N a B H 4 and 0.2 g NaIO4 was added to each 5 ml urine analysed for 17-OGS. INTERFERENCE DUE TO CLOXACILLIN
T h e antibiotic Cloxacillin (5-methyl 3-o-chlorophenyl-4-isoxazolyl penicillin) is the only non-steroid drug currently found to interfere with the analysis by causing the excretion in urine of a substance t h a t mimics a 17-oxogenic-steroid and forms a purple complex with alkaline m-dinitrobenzene. A p p a r e n t l y gross excretions of 17-oxogenicsteroids are observed in patients taking Cloxacillin (Table 1). T h e increase was shown to be a p p a r e n t , and not due to increased TABLE
1
THE EFFECTOF CLOXACILLINON URINE17-OXOGENIC-STEROID ANALYSIS
Patient
Day
1
1 2 3 5 6 7 8 9 1
2
3
1 2
Antibiotic (g/day) Cloxac~lin
1.5 2.0 2.0 2.0 1.0 0 0 12 capsule cases 1.0 1.0 0 Ampic~n
0 2.0
Penicillin V
5 0 6 2.0 Normalexcretion (adults20-40years)
17-Hydroxycorticosteroids (mg/day)
"Cortisol" (mg/day)
59 88 163 152 55 13 12 8 61 71 20
3 1 1 2 4 3 2
0.13 0.11 0.13 0.13 0.14 0:16
22 16
3 2
19 20 7-24
2 4 1-8
17-Oxogenicsteroids (rag/day)
2 2 3
0.09-0.34
174
METCALF
adrenal cortical activity, b y demonstrating t h a t the excretion of 17-hydroxycorticosteroids and of "free cortisol" was in the normal range in these patients. T h e brightly coloured capsule cases did not interfere with the assay, and the clinical condition of the patient appeared not to be a relevant factor. T w o other penicillins, Ampicillin (a-aminobenzyl penicillin) and Penicillin V (potassium salt of phenoxymethyl penicillin) did not interfere with the 17-OGS analysis (Table 1). T w o patients treated with 2 g / d a y Penicillin V and one patient treated with 4 X 106 u n i t s / d a y Penicillin G (Benzyl penicillin) also excreted normal a m o u n t s of 17-OGS. T h e failure of Ampicillin and Penicillin V to interfere with the 17-OGS analysis presumably means t h a t the interference is a function of the 3-o-chlorophenyl-5-methyl-4-isoxazolyl side chain of Cloxacillin and not of the penicillin nucleus. I t is not clear from inspection how this side chain could interfere with the analysis. OCCASIONAL INTERFERENCE BY METOPIRONE If interference by competition for reagents is suspected, it m a y be confirmed b y assaying the urine at different dilutions. An apparent increase in 17-oxogenicsteroids with dilution is indicative of interference. In general, Metopirone t r e a t m e n t does not interfere with the urine 17-OGS analysis, and in this laboratory a c h e c k t h a t interference is absent is made by diluting all "Metopirone urines" and analysing both the diluted and undiluted samples. However, in the occasional patient, Metopirone t r e a t m e n t is accompanied by the excretion of a substance which appears to interfere by competing for colour TABLE 2 THE RARE PHENOMENONOF METOPIRONE INTERFERENCE
'
Patient
Metopirone test
Sampledilution (urine : water)
Alkaline m-dinitrobenzene (ml)
17-oxogenicsteroids (mg/1)*
1
1
1:0 1:3
0.6 0.6
110 153
2
1
1:0
0.6
38
3
1
4
1
4
2
1:2 1:3 1:0 1:0 ~1 : 1 1:0 1:3 1:0 1:5
0.6 0.6 0.6 1.2 0.6 0.6 0.6 0.6 0.6
4
3
5
nil
49 50 51 62 62 116 152 79 206 120 165 164 30 29
1 :0
0.6
1:3 1:4 1:0 1:1
0.6 0.6 0.6 0.6
*Each figure is the mean of duplicate measurements. S.D. (0-20 mg/1 range) = 1 mg/l.
LABORATORY NOTE
175
reagent. This phenomenon, which was observed in less than 5% of all Metopirone tests, occurred on three separate occasions in the one patient. The a p p a r e n t l y low results were assumed due to inadequate colour reagent when it was found that excess NaBH4 and NaIO4 were present after reduction and oxidation respectively. As shown in Table 2, this type of interference can be avoided either b y diluting the urine samples or b y increasing the a m o u n t of alkaline m-dinitrobenzene used for colour development. ACKNOWLEDGMENT I t h a n k the laboratory staff at this hospital for undertaking m a n y of the steroid analyses. REFERENCES 1. METCALF, M.G.
Quantitative analysis of urinary 17-oxogenicsteroids using sodium metaperiodate oxidation. Clin. Chem. 12,258-262 (1966). 2. METCALF, M. G. A rapid method for measuring 17-hydroxy-corticosteroids in urine. J. Endocr. 26, 415-423 (1963). 9. REDDY, W. J. Modification of the Reddy-Jenkins-Thorn method for the estimation of 17-hydroxycorticoids in urine. Metabolism 3, 489-492 (1954). 4- DE MOOR, P., RASKIN,M. & STEENO, O. Dosage fluorimetrique des A4-3 ceto-ll-hydroxycorticoides plasmatiques et urinaires libres. Ann. Endocr. 21, 479-494 (1960).
LABORATORY
NOTE
I N T E R F E R E N G E W I T H URINE 17-OXOGENICSTEROID ANALYSIS
MARY G. METCALF* Medical Unit, Princess Margaret Hospital, Christchurch, New Zealand (Received May 20, 1967)
INTERFERENCE WITH ANALYTICAL PROCEDURES is of tWO types. Direct interference occurs when a substance directly influences an analytical procedure. Indirect interference occurs when a drug or one of its metabolites induces a change in the production or excretion of the test substance b y the patient. In this note, examples of direct interference with the 17-oxogenicsteroid (17-OGS) analysis are reported and discussed. T h e chemical reactions involved in the m e a s u r e m e n t of 17-OGS in urine are outlined in Fig. 1. ?.2-
-
CHOH ~
O
C=O
H
CH 2 - / CHOH
~ , ~ O H
+ ~~'~"~ O"
reO duc H 4 ti°nN with a~E~~ H
d 17- oxogenicstePoids oxidation with • Na IO 4
17) 20-dials alkaline • rn- dinit, Pobenzene I
17-oxostePoids
NO 2 J
(Ema x .520nrn )
FIO. 1. Chemical principles underlying the measurement of 17-oxogenic-steroids in urine. In theory, a n y substance competing with the 17-OGS for either NaBH4, NaIO4 or m-dinitrobenzene would be expected to interfere with the analysis. I n a d e q u a t e a m o u n t s of N a B H 4 would cause unpredictable results, and complete absence would lead to the inclusion of 17-oxosteroid glucuronides as 17-oxogenicsteroids and the exclusion of 17-hydroxy-20-oxosteroids from the assay. Substances destroying NaIO4 would be expected to cause low results b y preventing the conversion of 17,20-diols to 17-oxosteroids, while those reacting with m-dinitrobenzene would cause high results if the non-steroldal colour complex *Supported by a New Zealand MlZC grant.
LABORATORY NOTE
173
absorbed light a t 520 n m and low results if it did not. In practice, these four types of interference h a v e been experienced. Glucose is a c o m m o n constituent of urine which interferes with the 17-OGS analysis b y destroying both N a B H , and NaIO4. Provided sufficient extra N a B H 4 and NaIO~ is added to c o m p e n s a t e for the glucose present (1), 17-oxogenicsteroids m a y be measured in urine containing as much as 6 g % glucose. Other substances which interfere in this m a n n e r should be amenable to similar treatment. METHODS Urine for analysis was acidified to p H 2-3 and stored a t 4 °. 17-Oxogenicsteroids 17,20-dihydroxysteroids and 17-hydroxy-20-oxosteroids) were measured as 17-oxosteroids after t r e a t m e n t with N a B H 4 and NaIO4 (2), !7-hydroxycorficosteroids as the yellow phenylhydrazones (3) and "free cortisol" (mainly cortisol, corticosterone and 21-desoxy-cortisol) b y the fluorimetric procedure described by De M o o r and co-workers (g). In the absence of glucose, 0.05 g N a B H 4 and 0.2 g NaIO4 was added to each 5 ml urine analysed for 17-OGS. INTERFERENCE DUE TO CLOXACILLIN
T h e antibiotic Cloxacillin (5-methyl 3-o-chlorophenyl-4-isoxazolyl penicillin) is the only non-steroid drug currently found to interfere with the analysis by causing the excretion in urine of a substance t h a t mimics a 17-oxogenic-steroid and forms a purple complex with alkaline m-dinitrobenzene. A p p a r e n t l y gross excretions of 17-oxogenicsteroids are observed in patients taking Cloxacillin (Table 1). T h e increase was shown to be a p p a r e n t , and not due to increased TABLE
1
THE EFFECTOF CLOXACILLINON URINE17-OXOGENIC-STEROID ANALYSIS
Patient
Day
1
1 2 3 5 6 7 8 9 1
2
3
1 2
Antibiotic (g/day) Cloxac~lin
1.5 2.0 2.0 2.0 1.0 0 0 12 capsule cases 1.0 1.0 0 Ampic~n
0 2.0
Penicillin V
5 0 6 2.0 Normalexcretion (adults20-40years)
17-Hydroxycorticosteroids (mg/day)
"Cortisol" (mg/day)
59 88 163 152 55 13 12 8 61 71 20
3 1 1 2 4 3 2
0.13 0.11 0.13 0.13 0.14 0:16
22 16
3 2
19 20 7-24
2 4 1-8
17-Oxogenicsteroids (rag/day)
2 2 3
0.09-0.34
174
METCALF
adrenal cortical activity, b y demonstrating t h a t the excretion of 17-hydroxycorticosteroids and of "free cortisol" was in the normal range in these patients. T h e brightly coloured capsule cases did not interfere with the assay, and the clinical condition of the patient appeared not to be a relevant factor. T w o other penicillins, Ampicillin (a-aminobenzyl penicillin) and Penicillin V (potassium salt of phenoxymethyl penicillin) did not interfere with the 17-OGS analysis (Table 1). T w o patients treated with 2 g / d a y Penicillin V and one patient treated with 4 X 106 u n i t s / d a y Penicillin G (Benzyl penicillin) also excreted normal a m o u n t s of 17-OGS. T h e failure of Ampicillin and Penicillin V to interfere with the 17-OGS analysis presumably means t h a t the interference is a function of the 3-o-chlorophenyl-5-methyl-4-isoxazolyl side chain of Cloxacillin and not of the penicillin nucleus. I t is not clear from inspection how this side chain could interfere with the analysis. OCCASIONAL INTERFERENCE BY METOPIRONE If interference by competition for reagents is suspected, it m a y be confirmed b y assaying the urine at different dilutions. An apparent increase in 17-oxogenicsteroids with dilution is indicative of interference. In general, Metopirone t r e a t m e n t does not interfere with the urine 17-OGS analysis, and in this laboratory a c h e c k t h a t interference is absent is made by diluting all "Metopirone urines" and analysing both the diluted and undiluted samples. However, in the occasional patient, Metopirone t r e a t m e n t is accompanied by the excretion of a substance which appears to interfere by competing for colour TABLE 2 THE RARE PHENOMENONOF METOPIRONE INTERFERENCE
'
Patient
Metopirone test
Sampledilution (urine : water)
Alkaline m-dinitrobenzene (ml)
17-oxogenicsteroids (mg/1)*
1
1
1:0 1:3
0.6 0.6
110 153
2
1
1:0
0.6
38
3
1
4
1
4
2
1:2 1:3 1:0 1:0 ~1 : 1 1:0 1:3 1:0 1:5
0.6 0.6 0.6 1.2 0.6 0.6 0.6 0.6 0.6
4
3
5
nil
49 50 51 62 62 116 152 79 206 120 165 164 30 29
1 :0
0.6
1:3 1:4 1:0 1:1
0.6 0.6 0.6 0.6
*Each figure is the mean of duplicate measurements. S.D. (0-20 mg/1 range) = 1 mg/l.
LABORATORY NOTE
175
reagent. This phenomenon, which was observed in less than 5% of all Metopirone tests, occurred on three separate occasions in the one patient. The a p p a r e n t l y low results were assumed due to inadequate colour reagent when it was found that excess NaBH4 and NaIO4 were present after reduction and oxidation respectively. As shown in Table 2, this type of interference can be avoided either b y diluting the urine samples or b y increasing the a m o u n t of alkaline m-dinitrobenzene used for colour development. ACKNOWLEDGMENT I t h a n k the laboratory staff at this hospital for undertaking m a n y of the steroid analyses. REFERENCES 1. METCALF, M.G.
Quantitative analysis of urinary 17-oxogenicsteroids using sodium metaperiodate oxidation. Clin. Chem. 12,258-262 (1966). 2. METCALF, M. G. A rapid method for measuring 17-hydroxy-corticosteroids in urine. J. Endocr. 26, 415-423 (1963). 9. REDDY, W. J. Modification of the Reddy-Jenkins-Thorn method for the estimation of 17-hydroxycorticoids in urine. Metabolism 3, 489-492 (1954). 4- DE MOOR, P., RASKIN,M. & STEENO, O. Dosage fluorimetrique des A4-3 ceto-ll-hydroxycorticoides plasmatiques et urinaires libres. Ann. Endocr. 21, 479-494 (1960).