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Factors in blood influencing the determination of thyrotropin releasing hormone
Clinica Chimica Ada, 46 (1973) 377-382 8 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
377
CC* 5790
FACTORS TROPIN
IN BLOOD
INFLUENCING
RELEASING
THE
DETERMINATION
OF THYRO-
HORMONE
P. MAY AND R. K. DONABEDIAN The Department of Laboratory necticut 06504 (U.S.A.) (Received
Medicine,
Yale University
School of Medicine,
New Haven, Con-
March 20, 1973)
SUMMARY
This study demonstrates that when thyrotropin releasing hormone (TRH) is incubated in normal human serum or heparinized plasma there is IOO~/~ loss of immunoreactivity. When either high (2.4 pg/ml) or low (50 rig/ml) concentrations of TRH are incubated in EDTA anticoagulated plasma for 90 min at 37O there is 7585 y0 loss of immunoreactivity. When British-anti-Lewisite (BAL) or 8-hydroxyquinoline sulfate are added to serum prior to incubation with TRH, 48 and 29% remains after 90 min, respectively. When BAL and 8-hydroxyquinoline are added to EDTA anticoagulated plasma in combination, 88% of TRH immunoreactivity is recovered after 90 min incubation at 37O. The addition of enzyme inhibitors to urine was found to be unnecessary for urinary TRH measurement since IOO~/~ of TRH was recovered from urine after z-h incubation at 37O.
INTRODUCTION
The recent introduction of a radioimmunoassay for thyrotropin releasing hormone (TRH) (ref. I) has provided a simple and sensitive way for investigation of the metabolism of TRH. However, serum inactivation2-5 and rapid renal excretion6 of TRH present problems of measurement. In addition, it is important to know the stability of TRH under various in vitro conditions. It has been demonstrated that TRH is rapidly degraded by serum at 37’, pH 6 to 8 (ref. 5) but the stability of TRH in serum containing various inhibitors of enzyme activity and the stability in urine has not been reported. The purpose of this communication is to describe conditions for the radioimmunoassay of TRH and to study the stability of TRH in serum and plasma with and without various enzyme inhibitors.
378 MATERIALS
MAY,DONABEDIAN
AND
METHODS
The eflect of serum
on standard
cw’ues separated
by double
antlbody
and dextrawcoated
charcoal
techniques In all cases radioimmunoassay of TRH was accomplished by methods previously describedl. A series of six standard doseeresponse tubes (three pairs) were prepared using identical antibody titers (I :3000). One hundred ~1 of normal human serum were added to two of the sets of tubes while IOO ,~l of 0.15 M NaCl were added to two additional sets. One hundred ,~l of serum that had been heated at 56” for I h were added to the remaining two sets of tubes. After 18-h incubation at 4’, bound and free fractions from one of the three pairs of dose-response tubes was separated by 1.0 ml of dextran-coated charcoal prepared as previously describeds, and the other member of the three pairs was separated by immunoprecipitation with goat anti-rabbit globulin. Comfiarison of stability of TRH incubated i?a serum a& urine One ml of normal human serum and 1.0 ml of urine were placed in 12 x 75-mm test tubes. One of the urine samples and one of the serum samples was then cooled to 4’ while the hundred ng was allowed in duplicate, All samples the standard to all points
other urine and serum samples were heated to 37’ in a water bath. Two of TRH were then added to each urine and serum sample and incubation to proceed. Fifty-$ aliquots of serum were obtained at IO, 30 and 60 min while 50 ,~l of urine was obtained at IO, 30, 90 and 120 min in duplicate. were assayed immediately. Fifty ,~l of serum were added to all points of curve used to measure the serum samples, and 50 ~1 of urine were added of the standard curve used to assay the urine samples.
of various conditiow on the rate of degradation of TRH in seyum TRH (2.4 ,~g) was added to a series of tubes containing 1.0 ml normal human serum that had been heated at 56” for 30 min, 1.0 ml of normal human serum containing 2 ,~l of a 0.806 M solution of BAL in benzyl benzoate (Schwarz-Mann), 1.0 ml of normal human serum containing IO ,~l of a 0.170 M solution of 8-hydroxyquinoline sulfate (Analabs), 1.0 ml of plasma anticoagulated with EDTA, 1.0 ml of heparinized plasma, I.0 ml of normal human serum containing I0 ~1 of a 0.5% solution of the monoamine oxidase inhibitor pargyline and 1.0 ml of normal human serum. Incubation was allowed to proceed at 37’ in a constant temperature water bath. Five-$ aliquots were taken in duplicate after 30 min and again after 90 min of incubation. All samples were assayed immediately for TRH. Five ~1 of serum were added to all points of the standard curve. Effect
E_fect of BAL and %hydroxyquinoline in combination on the rate of degradation of TRH in plasma anticoagulated with EDTA Fifty ng of TRH were added to either 1.0 ml of EDTA plasma alone or 1.0 ml of EDTA plasma containing BAL and 8-hydroxyquinoline as described above. Incubation was allowed to proceed at 37’ and IOO-,~l aliquots were taken in duplicate at 30 min and again at 90 min. TRH was assayed immediately utilizing two separate standard curves. All points of standard curve No. I contained IOO ,~l of normal human plasma and was used to measure the TRH incubated in the EDTA plasma alone. All
RIA
FACTORS AFFECTING
OF
TRH
379
points of standard curve No. 2 contained IOO ,~l of EDTA plasma containing BAL and 8-hydroxyquinoline in the same concentrations as used in the samples, and was used to assay the samples containing
BAL
and 8-hydroxyquinoline.
RESULTS
Effect of serum 01%stnndard cwues separated by de&an-coated charcoal and by doubleantibody techniques Fig. I demonstrates the marked effect that serum has on the bound: free ratios of standard curves separated by both double-antibody and dextran-coated charcoal techniques. In both cases the addition of IOO ,d of serum at the beginning of the incubation period resulted in an upward shift of the standard curves so that the bound : free ratios of the curves containing serum were all greater than those containing 0.15 M NaCl. This apparent “enhancement” of antibody binding was most apparent with the double-antibody technique and was partially destroyed by heat inactivation. In addition, the initial slopes of those standard curves containing heated serum or saline were greater than those containing normal serum, whether separated by doubleantibody or by dextran-coated charcoal.
1.2
L
1.0
Double
‘L\ Double
Antibody-Normal
Serum
Antibody
Serum
Chorcool-
-0
I 0
1 5 TRH
-.
O----~_,,_
I
I
10
15
Normal
Chorcool -Heated Double Antibody
-A------A -.
-4
-Heoted
Serum Serum - Soline
Charcoal-Saline
20
(ng/ml)
Fig. I. Effect of serum on standard coated charcoal techniques.
curves separated
by both
double-antibody
and dextran-
Stability of TRH in serum and wine Fig. 2 demonstrates the stability of TRH in urine and serum incubated at 37’ and at 4’ for 2 h. Each point represents the average of two separate determinations. There was no demonstrable breakdown of TRH in urine, whereas only IZO/ of the TRH remained after I h incubation in serum at 37O. While the rate of breakdown of
380
MAY,
DONABEDIAN
Urine-37’C Urine-4°C
-we
50
Serum-4’C
-
0
1
1
I
15
30
45 INCUBATION
I
I
60
75 TIME
I
90
I
I
105
120
(minutes)
Fig. 2. Comparison of stability of TRH incubated in human serum and urine at 37” and 4”. Each point represents the average of duplicate determinations.
Heat
Inactivated
BAL
Serum
Serum
6-Hydrcxyquinotine EOTA
lormol
Plasma
Serum
leporinired
Plasma
barpytins 0
30 INCUBATION
60 TIME
40
(minutes)
Fig. 3. Effect of various conditions on the rate presents the average of duplicate determinations.
of serum
degradation
of TRH.
TRH incubated in serum at 4’ was substantially less than that significant breakdown did occur with 50% remaining after I h.
Each
incubated
point
rc-
at 37”,
of various conditions on the rate of sewwn degradation of TRH Fig. 3 illustrates that the rate of TRH degradation in serum is retarded by heat inactivation, BAL, 8-hydroxyquinoline and EDTA, in that order. On the other hand, TRH degradation in normal serum, heparinized plasma, or serum with pargyline is quite rapid, with approximately 95% degradation by 30 min. ITfect
FACTORS
RIA
AFFECTING
The effect of BAL
OF
TRH
and 8-hydroxyquinoline
381 on the rate of TRH
degradation
in EDTA
plasma In Fig. 4 the rate of TRH degradation in EDTA plasma at 37’ is compared with the rate of degradation in EDTA plasma containing BAL and %hydroxyquinoline. With EDTA plasma alone at 37’, only 20% of 50 ng/ml TRH remains after 90 min, whereas with the addition of BAL and &hydroxyquinoline 88% remains after go-min incubation.
I
0
I
/
I
30 INCUBATION
I
60 TIME
,
I
,
90
ImInutes
Fig. 1. The effect of BAL and H-hydroxyquinoline on the rate of degradation plasma. Each point represents the average of duplicate determinations.
of TRH
in EDT,\
UISCUSSION
The results described above suggest that under the proper conditions the radioimmunoassay for TRH can be a useful tool for the study of TRH metabolism. Emphasis is placed on the importance of establishing the same conditions for the standard curve and samples. These experiments have demonstrated that if IOO ~1 of serum with a TRH concentration of 200 ng/ml were assayed using a standard curve with no the sample would appear as though it contained no TRH. An additional problem with serum may be partial inactivation of standard TRH during incubation in the standard curve. This might account for the more shallow slopes of those standard curves containing normal serum as compared to those curves containing saline or heat inactivated serum. The demonstration of stability of TRH in urine at 37’ is important for interpretation of studies which involve assay of TRH in urine. If a patient is unable to void for as long as 2 h after TRH injection this should not affect recovery of TRH. The demonstration that certain chelators of heavy metals (BAL, EDTA, 8hydroxyquinoline) inhibit the degradation of TRH may have practical consequences for future attempts to measure TRH. These substances have been used in the assay for plasma renin activity7p8 and partially act by inhibiting the activity of converting enzyme, a peptidase that hydrolyzes a peptide bond between histidine and phenylalanine of Angiotensin I. The studies by Nair et ad4 suggest that the TRH inactivator is an amidase rather than a peptidase. However, Vale et ~1.~ demonstrated that the serum,
MAY,
382
DONABEDIAN
biological activity of a TRH analogue that contained no amide was also inactivated by plasma. An additional point that requires mention is that in this study the rate of degradation of TRH in EDTA plasma did not depend on the concentration of TRH. With an initial TRH concentration of either 2400 ng/ml or 50 ng/ml, only 20% remained after go-min incubation. This is in contrast to the results obtained with serum5 where the rate of degradation was greater with smaller concentrations of TRH. The exact nature of TRH inactivation appears to be complex and may involve more than one inactivator. ACKNOWLEDGEMENTS
We gratefully
acknowledge
Seligson during this study. This study was supported
the valuable by United
advice
Public
and support
Health
Service
of Dr.
Training
David Grant
5
TOI GM 00696. REFERENCES I R. 1L1.BASSIRI AND R. D. ~TTIGER, Endocrinology, 90 (1972) 722. 2 T. W. REDDING AKD A. V. SCHALLY, PYOC.Sot. Exptl. Biol. Med., 131 (1969) 415. 3 W. W. VALE, K. BURGUS, T. DUSK AND R. GUILL&IN, Hovmones, 2 (1971) 193. -1_K. WI. G. NAIR, T. W. REDUINC AND A. V. SCHALLY, Biochemistry, IO (1971) 3621. 5 R. BASSIRI AND R. D. ITTIGER, Endocrinology, 91 (1972) 657. 6 J, LEPPALUOTO, P. VIRKKUNEN AXD H. LYBECK, .I. Clin. Endocrinol., 35 (1972) 477. 7 J. W. RYAK, J, Ii. MCKENZIE AND M. R. LEE, Biochenz. J., 108 (1968) 679. 8 E. HABER, T. KOERNER, L. PAGE, B. KLIMAN AND A. PURKODE, J. Clix. Endocrinol., 29 (1969)
9 ~?~ERI~ERT, 1375.
Ii. S. Lnu,
C. \
25 (1965)
Amsterdam
- Printed in The Netherlands
377
CC* 5790
FACTORS TROPIN
IN BLOOD
INFLUENCING
RELEASING
THE
DETERMINATION
OF THYRO-
HORMONE
P. MAY AND R. K. DONABEDIAN The Department of Laboratory necticut 06504 (U.S.A.) (Received
Medicine,
Yale University
School of Medicine,
New Haven, Con-
March 20, 1973)
SUMMARY
This study demonstrates that when thyrotropin releasing hormone (TRH) is incubated in normal human serum or heparinized plasma there is IOO~/~ loss of immunoreactivity. When either high (2.4 pg/ml) or low (50 rig/ml) concentrations of TRH are incubated in EDTA anticoagulated plasma for 90 min at 37O there is 7585 y0 loss of immunoreactivity. When British-anti-Lewisite (BAL) or 8-hydroxyquinoline sulfate are added to serum prior to incubation with TRH, 48 and 29% remains after 90 min, respectively. When BAL and 8-hydroxyquinoline are added to EDTA anticoagulated plasma in combination, 88% of TRH immunoreactivity is recovered after 90 min incubation at 37O. The addition of enzyme inhibitors to urine was found to be unnecessary for urinary TRH measurement since IOO~/~ of TRH was recovered from urine after z-h incubation at 37O.
INTRODUCTION
The recent introduction of a radioimmunoassay for thyrotropin releasing hormone (TRH) (ref. I) has provided a simple and sensitive way for investigation of the metabolism of TRH. However, serum inactivation2-5 and rapid renal excretion6 of TRH present problems of measurement. In addition, it is important to know the stability of TRH under various in vitro conditions. It has been demonstrated that TRH is rapidly degraded by serum at 37’, pH 6 to 8 (ref. 5) but the stability of TRH in serum containing various inhibitors of enzyme activity and the stability in urine has not been reported. The purpose of this communication is to describe conditions for the radioimmunoassay of TRH and to study the stability of TRH in serum and plasma with and without various enzyme inhibitors.
378 MATERIALS
MAY,DONABEDIAN
AND
METHODS
The eflect of serum
on standard
cw’ues separated
by double
antlbody
and dextrawcoated
charcoal
techniques In all cases radioimmunoassay of TRH was accomplished by methods previously describedl. A series of six standard doseeresponse tubes (three pairs) were prepared using identical antibody titers (I :3000). One hundred ~1 of normal human serum were added to two of the sets of tubes while IOO ,~l of 0.15 M NaCl were added to two additional sets. One hundred ,~l of serum that had been heated at 56” for I h were added to the remaining two sets of tubes. After 18-h incubation at 4’, bound and free fractions from one of the three pairs of dose-response tubes was separated by 1.0 ml of dextran-coated charcoal prepared as previously describeds, and the other member of the three pairs was separated by immunoprecipitation with goat anti-rabbit globulin. Comfiarison of stability of TRH incubated i?a serum a& urine One ml of normal human serum and 1.0 ml of urine were placed in 12 x 75-mm test tubes. One of the urine samples and one of the serum samples was then cooled to 4’ while the hundred ng was allowed in duplicate, All samples the standard to all points
other urine and serum samples were heated to 37’ in a water bath. Two of TRH were then added to each urine and serum sample and incubation to proceed. Fifty-$ aliquots of serum were obtained at IO, 30 and 60 min while 50 ,~l of urine was obtained at IO, 30, 90 and 120 min in duplicate. were assayed immediately. Fifty ,~l of serum were added to all points of curve used to measure the serum samples, and 50 ~1 of urine were added of the standard curve used to assay the urine samples.
of various conditiow on the rate of degradation of TRH in seyum TRH (2.4 ,~g) was added to a series of tubes containing 1.0 ml normal human serum that had been heated at 56” for 30 min, 1.0 ml of normal human serum containing 2 ,~l of a 0.806 M solution of BAL in benzyl benzoate (Schwarz-Mann), 1.0 ml of normal human serum containing IO ,~l of a 0.170 M solution of 8-hydroxyquinoline sulfate (Analabs), 1.0 ml of plasma anticoagulated with EDTA, 1.0 ml of heparinized plasma, I.0 ml of normal human serum containing I0 ~1 of a 0.5% solution of the monoamine oxidase inhibitor pargyline and 1.0 ml of normal human serum. Incubation was allowed to proceed at 37’ in a constant temperature water bath. Five-$ aliquots were taken in duplicate after 30 min and again after 90 min of incubation. All samples were assayed immediately for TRH. Five ~1 of serum were added to all points of the standard curve. Effect
E_fect of BAL and %hydroxyquinoline in combination on the rate of degradation of TRH in plasma anticoagulated with EDTA Fifty ng of TRH were added to either 1.0 ml of EDTA plasma alone or 1.0 ml of EDTA plasma containing BAL and 8-hydroxyquinoline as described above. Incubation was allowed to proceed at 37’ and IOO-,~l aliquots were taken in duplicate at 30 min and again at 90 min. TRH was assayed immediately utilizing two separate standard curves. All points of standard curve No. I contained IOO ,~l of normal human plasma and was used to measure the TRH incubated in the EDTA plasma alone. All
RIA
FACTORS AFFECTING
OF
TRH
379
points of standard curve No. 2 contained IOO ,~l of EDTA plasma containing BAL and 8-hydroxyquinoline in the same concentrations as used in the samples, and was used to assay the samples containing
BAL
and 8-hydroxyquinoline.
RESULTS
Effect of serum 01%stnndard cwues separated by de&an-coated charcoal and by doubleantibody techniques Fig. I demonstrates the marked effect that serum has on the bound: free ratios of standard curves separated by both double-antibody and dextran-coated charcoal techniques. In both cases the addition of IOO ,d of serum at the beginning of the incubation period resulted in an upward shift of the standard curves so that the bound : free ratios of the curves containing serum were all greater than those containing 0.15 M NaCl. This apparent “enhancement” of antibody binding was most apparent with the double-antibody technique and was partially destroyed by heat inactivation. In addition, the initial slopes of those standard curves containing heated serum or saline were greater than those containing normal serum, whether separated by doubleantibody or by dextran-coated charcoal.
1.2
L
1.0
Double
‘L\ Double
Antibody-Normal
Serum
Antibody
Serum
Chorcool-
-0
I 0
1 5 TRH
-.
O----~_,,_
I
I
10
15
Normal
Chorcool -Heated Double Antibody
-A------A -.
-4
-Heoted
Serum Serum - Soline
Charcoal-Saline
20
(ng/ml)
Fig. I. Effect of serum on standard coated charcoal techniques.
curves separated
by both
double-antibody
and dextran-
Stability of TRH in serum and wine Fig. 2 demonstrates the stability of TRH in urine and serum incubated at 37’ and at 4’ for 2 h. Each point represents the average of two separate determinations. There was no demonstrable breakdown of TRH in urine, whereas only IZO/ of the TRH remained after I h incubation in serum at 37O. While the rate of breakdown of
380
MAY,
DONABEDIAN
Urine-37’C Urine-4°C
-we
50
Serum-4’C
-
0
1
1
I
15
30
45 INCUBATION
I
I
60
75 TIME
I
90
I
I
105
120
(minutes)
Fig. 2. Comparison of stability of TRH incubated in human serum and urine at 37” and 4”. Each point represents the average of duplicate determinations.
Heat
Inactivated
BAL
Serum
Serum
6-Hydrcxyquinotine EOTA
lormol
Plasma
Serum
leporinired
Plasma
barpytins 0
30 INCUBATION
60 TIME
40
(minutes)
Fig. 3. Effect of various conditions on the rate presents the average of duplicate determinations.
of serum
degradation
of TRH.
TRH incubated in serum at 4’ was substantially less than that significant breakdown did occur with 50% remaining after I h.
Each
incubated
point
rc-
at 37”,
of various conditions on the rate of sewwn degradation of TRH Fig. 3 illustrates that the rate of TRH degradation in serum is retarded by heat inactivation, BAL, 8-hydroxyquinoline and EDTA, in that order. On the other hand, TRH degradation in normal serum, heparinized plasma, or serum with pargyline is quite rapid, with approximately 95% degradation by 30 min. ITfect
FACTORS
RIA
AFFECTING
The effect of BAL
OF
TRH
and 8-hydroxyquinoline
381 on the rate of TRH
degradation
in EDTA
plasma In Fig. 4 the rate of TRH degradation in EDTA plasma at 37’ is compared with the rate of degradation in EDTA plasma containing BAL and %hydroxyquinoline. With EDTA plasma alone at 37’, only 20% of 50 ng/ml TRH remains after 90 min, whereas with the addition of BAL and &hydroxyquinoline 88% remains after go-min incubation.
I
0
I
/
I
30 INCUBATION
I
60 TIME
,
I
,
90
ImInutes
Fig. 1. The effect of BAL and H-hydroxyquinoline on the rate of degradation plasma. Each point represents the average of duplicate determinations.
of TRH
in EDT,\
UISCUSSION
The results described above suggest that under the proper conditions the radioimmunoassay for TRH can be a useful tool for the study of TRH metabolism. Emphasis is placed on the importance of establishing the same conditions for the standard curve and samples. These experiments have demonstrated that if IOO ~1 of serum with a TRH concentration of 200 ng/ml were assayed using a standard curve with no the sample would appear as though it contained no TRH. An additional problem with serum may be partial inactivation of standard TRH during incubation in the standard curve. This might account for the more shallow slopes of those standard curves containing normal serum as compared to those curves containing saline or heat inactivated serum. The demonstration of stability of TRH in urine at 37’ is important for interpretation of studies which involve assay of TRH in urine. If a patient is unable to void for as long as 2 h after TRH injection this should not affect recovery of TRH. The demonstration that certain chelators of heavy metals (BAL, EDTA, 8hydroxyquinoline) inhibit the degradation of TRH may have practical consequences for future attempts to measure TRH. These substances have been used in the assay for plasma renin activity7p8 and partially act by inhibiting the activity of converting enzyme, a peptidase that hydrolyzes a peptide bond between histidine and phenylalanine of Angiotensin I. The studies by Nair et ad4 suggest that the TRH inactivator is an amidase rather than a peptidase. However, Vale et ~1.~ demonstrated that the serum,
MAY,
382
DONABEDIAN
biological activity of a TRH analogue that contained no amide was also inactivated by plasma. An additional point that requires mention is that in this study the rate of degradation of TRH in EDTA plasma did not depend on the concentration of TRH. With an initial TRH concentration of either 2400 ng/ml or 50 ng/ml, only 20% remained after go-min incubation. This is in contrast to the results obtained with serum5 where the rate of degradation was greater with smaller concentrations of TRH. The exact nature of TRH inactivation appears to be complex and may involve more than one inactivator. ACKNOWLEDGEMENTS
We gratefully
acknowledge
Seligson during this study. This study was supported
the valuable by United
advice
Public
and support
Health
Service
of Dr.
Training
David Grant
5
TOI GM 00696. REFERENCES I R. 1L1.BASSIRI AND R. D. ~TTIGER, Endocrinology, 90 (1972) 722. 2 T. W. REDDING AKD A. V. SCHALLY, PYOC.Sot. Exptl. Biol. Med., 131 (1969) 415. 3 W. W. VALE, K. BURGUS, T. DUSK AND R. GUILL&IN, Hovmones, 2 (1971) 193. -1_K. WI. G. NAIR, T. W. REDUINC AND A. V. SCHALLY, Biochemistry, IO (1971) 3621. 5 R. BASSIRI AND R. D. ITTIGER, Endocrinology, 91 (1972) 657. 6 J, LEPPALUOTO, P. VIRKKUNEN AXD H. LYBECK, .I. Clin. Endocrinol., 35 (1972) 477. 7 J. W. RYAK, J, Ii. MCKENZIE AND M. R. LEE, Biochenz. J., 108 (1968) 679. 8 E. HABER, T. KOERNER, L. PAGE, B. KLIMAN AND A. PURKODE, J. Clix. Endocrinol., 29 (1969)
9 ~?~ERI~ERT, 1375.
Ii. S. Lnu,
C. \
25 (1965)