J Clin Endocrinol Metab 41: 801, 1975 Problems in the Measurement of Urinary TRH A.G. Vagenakis, E. Roti, J. Mannix, and L.E. Braverman Department of Medicine, University of Massachusetts Medical School, Worcester, Ma., 01605 ABSTRACT. Many studies of TRH physiology have been based on the radioimmunoassay of TRH in urine. In view of the inability of serum to deactivate urine TRH and the interference of urea with the TRH assay, measurement of urine TRH by radioimmunoassay may not be a true reflection of the actual concentration of TRH. The isolation, characterization and synthesis of thyrotropin-releasing hormone (TRH) and the development of a specific and sensitive radioimmunoassay (1) has led to a greater understanding of TRH physiology. It is now recognized that TRH is present in considerable quantities not only in the hypothalamus but also in the extrahypothalamic areas of the brain (2,3). Immunoreactive TRH has also been found in the urine of man and rat (4-6), human cerebrospinal fluid (7), and human and rat plasma (5,8). During studies in our laboratory, it became evident that urine TRH detected by radioimmunoassay might not be TRH. The present report describes these findings. MATERIALS AND METHODS A radioimmunoassay for TRH has been developed according to the method of Bassiri and Utiger (1). A final dilution of TRH antibody of 1:30,000 was employed. The assay detected as little as 1-2 pg of synthetic TRH. In close agreement with others (6,9), considerable quantities of TRH (6-10 ng/hypothalamus) were detected in methanol extracts of rat hypothalami.. In contrast, no TRH was detected in hypothalami which had remained at room temperature prior to methanol extraction, suggesting that the various deactivated products of TRH do not cross-react in the immunoassay. Since enzymes present in serum rapidly deactivate TRH (10), the
ability of serum to deactivate urine TRH was evaluated. One ml of freshly voided urine with or without the addition of synthetic TRH was incubated with various quantities of fresh human serum (0.2-1.0 ml) at 37°C for two hours. Urine pH was adjusted to 7.0 before the addition of the serum. The various samples were then extracted twice with 3 ml 90% methanol, the extracts brought to dryness at room temperature, and then reconstituted in 1 ml phosphate saline buffer prior to assay. In another series of experiments, 4 ml aliquots of urine were treated with charcoal to extract TRH by the method of Oliver et al (5). The charcoal precipitate was extracted twice with 4 ml 90% methanol, and brought to dryness at room temperature, reconstituted in 1 ml buffer and then 0.1 ml was directly assayed for TRH. A 0.5 ml aliquot of the reconstituted buffer solution was incubated with 0.2 ml serum to inactivate TRH as described above. The latter solution was extracted with 90% methanol, dried, and reconstituted in 0.5 ml buffer and 0.1 ml assayed for TRH. The recovery of TRH added to urine was 50-70% in the charcoal-treated urine. In contrast, recovery of added TRH from a methanol extract of urine without charcoal extraction was 98-100%. Since urea is present in high concentration in the urine and has been reported to interfere with the radioimmunoassay for ADH in urine (11), studies were carried out on the effect of urea on the TRH immunoassay. Urea was added to saline or phosphate buffer at concentrations of 1-50 mg/ml
Submitted July 17, 1975
801
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JCE & M • 1975 Vol 41 • No 4
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802
which encompassed the range of urea concentrations in normal human urine. The urea solutions were then assayed for TRH. A portion of the urea solution was also extracted with charcoal and assayed for TRH as described above. To determine whether urea present in normal urine would affect the immunoassay for TRH, 1 or 2 ml of freshly voided urine was incubated for one hour at 37°C with urease in order to degrade the urea (12). Urease (50 mg/ml) was dissolved in 1 ml 50% (v/v) glycerol-double distilled water. 0.2 or 0.4 ml of the above solution was added to 1 or 2 ml urine, respectively, and incubated at 37°C for one hour. The solution was then centrifuged and the supernatant was diluted 1:4 and 0.1 ml was assayed for TRH. Urine without urease was also assayed in a 1:4 dilution. In order to determine the effect of urease per se on the immunoassay for TRH, solutions of urea in buffer (20 mg/ ml) with or without added synthetic TRH were incubated with and without urease for one hour at 37°C and then assayed for TRH. Similar experiments were also carried out by adding TRH to urine with and without urease. All samples in individual experiments were assayed in duplicate in the same assay. RESULTS Serum deactivation of urine TRH. When TRH (1-80 ng) in buffer was incubated with 0.2 ml serum, approximately 80100% of the TRH was deactivated. These findings are similar to those of Bassiri and Utiger (10). In contrast, little or no deactivation of apparent endogenous urine TRH occurred when urine was incubated with serum (Table 1). The mean concentration of urine TRH was 610 ± 292 pg/ml before and 691 * 369 pg/ml after serum deactivation. Similarly, charcoal extracted urine TRH could not be deactivated by serum (Table 2 ) . The mean concentration in five different
urine samples was 222 ± 93 pg/ml before and 294 ± 196 pg/ml after deactivation by serum. In order to exclude the possibility that urine contains an inhibitor of the serum enzyme, various concentrations of TRH were added to urine. The urine samples were then incubated with serum. The inactivation of added TRH was similar to that found when TRH was added to buffer alone, suggesting that urine had no inhibitory effect on the serum TRH deactivating enzymes. Urine was collected following the iv administration of 400 yg TRH. Aliquots of these urine samples were then incubated with serum. Deactivation of the excreted TRH was almost complete. Effect of urea on the TRH radioimmunoassay. The addition of urea to saline or phosphate-saline buffer was detected as TRH in the radioimmunoassay (Table 3 ) . Large quantities of urea (mg/ml) were interpreted as pg/ml quantities of TRH. Following charcoal extraction, the urea effect was markedly attenuated but apparent TRH was still detected. Inhibition of the urea effect by urease (Table 4). Since urea exhibited a nonspecific effect on the TRH immunoassay, urea was eliminated from the urine by incubating urine with urease. The nonspecific effect of urea on the TRH assay was abolished in the presence of urease. The recovery of TRH added to urine plus urease was approximately 90%, suggesting that urease did not alter the TRH molecule or interfere with its immunoassay. Furthermore, the recovery of TRH added to buffer containing urea and urease was also 90%. Finally, the nonspecific effect of urea in buffer on the TRH assay was abolished by the addition of urease. DISCUSSION The present studies clearly demonstrate that urine TRH as measured by radio imrauno as say is probably not TRH but is a nonspecific effect of urea on the immunoassay. These findings seem justified since the urinary immunoreactive substance which we detected was completely
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803
RAPID COMMUNICATIONS inactivated by urease. Urease itself does not affect the inununoassay or deactivate TRH. Furthermore, the inability of serum to deactivate urine TRH suggests that what is assayed in urine as TRH in whole and methanol or charcoal-extracted urine may not be TRH. The possibility that TRH was excreted in the urine in a form which is protected from serum deactivation seems unlikely since TRH in urine from a patient following the iv administration of TRH was deactivated by serum. These findings suggest that if the chemical form of TRH which is released from the brain is similar to synthetic TRH, it is not bound to a "protector" substance in the urine. If TRH is present in urine, its concentration is probably low and may not be detected by present assay techniques. Thus, reported production rates for TRH which depend upon urine TRH values in animals and man may not be valid (4,13). The present findings also may help to explain why urine TRH concentration does not change in various abnormalities of the thyroid or following exogenous T4 and T3 administration (14). Furthermore, the failure to detect significant changes in urine TRH in animals with hypothalamic lesions which are accompanied by significant decreases in TRH content in the hypothalamus and stalk median eminence may also .be due to an inability to accurately measure true urine TRH (15). Oliver et al (5) have recently reported that ethanol extracts of charcoal-treated urine displayed an elution pattern similar to synthetic TRH as judged by G-10 Sephadex filtration and paper electrophoresis. However, close inspection of their data clearly reveals that the patterns are not identical. Furthermore, it is possible that the immunoreactive substance which they reported may have been urea. This possibility is supported by our findings. First, serum had no deactivating efrect on urine TRH in methanol extracts of charcoal-treated urine.
Second, when solutions of urea in buffer were treated with charcoal and the charcoal extracted with methanol, TRH immunoreactivity was detected. More recently, Montoya et al (16) have reported an immunoassay for urine and plasma TRH which employs sepharoseanti TRH columns to isolate TRH from the urine. The possibility that urea or some other substances also interfere with this immunoassay cannot be ruled out. Measurements of TRH in the blood by radioimmunoassay are also fraught with many difficulties. Indeed, recent studies employing the immunoassay for blood TRH have resulted in controversial findings. Montoya and coworkers (8) found a striking elevation in plasma TRH following cold exposure in rats while Emerson and Utiger (17) found no such changes. These diverse findings emphasize the fact that caution should be exercised in interpreting physiological experiments based on either urine or plasma TRH immunoassay. Table 1. Urine TRH before & after serum deactivation (1 ml serum + 1 ml urine). TRH (pg/ml) After Urine Sample Before
1 2
670
525
3
1100 310
4 5 6 7 8
300 760 660 800 280
1500 280 750 625 650 800 400
610 ± 292
691 ± 369
Mean ± SD
Table 2. Charcoal-extracted urine: before & after serum deactivation.
TRH
TRH (pg/ml) Urine Sample
1 2 3
Before
4
310 220 300 205
5
77
Mean ± SD
222 ± 93
After
340 570 310 220 30 294 ± 196
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Table 3. Effect of urea in buffer on the TRH radioinununoassay. Urea (mg/ml)
1 5 10 15 25 50
TRH (pg/ml)
REFERENCES
1. Bassiri, R.M., and R.D. Utiger, Endo-
TRH after charcoal extraction (pg/ml)
10 47 110 125 270 860
crinology 90:722, 1972.
2. Jackson, I.M.D., and S. Reichlin, Endocrinology 95:816, 1974.
0 39 45 40 105 100
3. Brownstein, M.J., M. Palkovits, 4. 5.
Table 4. Inhibition of the urea effect on the TRH assay by urease.
6.
TRH measured (ng) 1 ml urine
none
7.
2.0 ± 0.3* 0
urease
8.
1** 2 ml urine
2 ml buffer
JCE&M . 1975 Vol 41 * No 4
RAPID COMMUNICATIONS
804
none urease 5 ng TRH 5 ng TRH & urease urease 40 mg urea 40 mg urea & urease 5 ng TRH 5 ng TRH & urease 40 mg urea & 5 ng TRH & urease
43
2.0 0 7.6
9.
0 8.0 4.3
4.8
11,
10.
0 1.0
12. 13.
0 4.0 4.5 ± 0.2*
14.
4.8 15.
*Mean ± SD of four different samples **1 and 2 are different urines
16.
ACKNOWLEDGMENTS 17. Supported by Research Grant AM07917 from the NIAMDD, National Institutes af Health, Bethesda, Maryland.
J.M. Saavedra, R.M. Bassiri, and R.D. Utiger, Science 185:267, 1974. Jackson, I.M.D., R. Gagel, P.D. Papapetrou, D. Duprez, and S. Reichlin, Clin Res 23:238A, 1975. Oliver, C , J.P. Charvet, J.L. Codaccioni, and J. Vague, J Clin Endocr Metab 39:406, 1974. Jackson, I.M.D., and S. Reichlin, Life Sci 14:2259, 1974. Shambaugh, G.E., J.F. Wilber, E. Montoya, H. Ruder, and E.R. Blonsky, J Clin Endocr Metab 41:131, 1975. Montoya, E., M.J. Seibel, and J;F. Wilber, Endocrinology 96: 1413, 1975. Bassiri, R.M., and R.D. Utiger, Endocrinology 94:188, 1974. Bassiri, R.M., and R.D. Utiger, Endocrinology 91:657, 1972. Miller, M., and A.M. Moses, J Clin Endocr Metab 34:537, 1972. Koch, J., Lab Clin Med 11:776, 1926. Jackson, I.M.D., P.D. Papapetrou, and S. Reichlin, Program 50th Annual Meeting American Thyroid Association, Abstr No 1. Gagel, R.F., I.M.D. Jackson, D. Duprez, P.D. Papapetrou, and S. Reichlin, 7th International Thyroid Conference, 1975, Abstr No 22. Jackson, I.M.D., and S. Reichlin, Program 57th Annual Meeting Endocrine Society Abstr No 91. Montoya, E., M.J. Seibel, and J. Wilber, Program 55th Annual Meeting Endocrine Society Abstr No 180. Emerson, C.H., and R.D. Utiger, Program 57th Annual Meeting Endocrine Society Abstr No 200.
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ability of serum to deactivate urine TRH was evaluated. One ml of freshly voided urine with or without the addition of synthetic TRH was incubated with various quantities of fresh human serum (0.2-1.0 ml) at 37°C for two hours. Urine pH was adjusted to 7.0 before the addition of the serum. The various samples were then extracted twice with 3 ml 90% methanol, the extracts brought to dryness at room temperature, and then reconstituted in 1 ml phosphate saline buffer prior to assay. In another series of experiments, 4 ml aliquots of urine were treated with charcoal to extract TRH by the method of Oliver et al (5). The charcoal precipitate was extracted twice with 4 ml 90% methanol, and brought to dryness at room temperature, reconstituted in 1 ml buffer and then 0.1 ml was directly assayed for TRH. A 0.5 ml aliquot of the reconstituted buffer solution was incubated with 0.2 ml serum to inactivate TRH as described above. The latter solution was extracted with 90% methanol, dried, and reconstituted in 0.5 ml buffer and 0.1 ml assayed for TRH. The recovery of TRH added to urine was 50-70% in the charcoal-treated urine. In contrast, recovery of added TRH from a methanol extract of urine without charcoal extraction was 98-100%. Since urea is present in high concentration in the urine and has been reported to interfere with the radioimmunoassay for ADH in urine (11), studies were carried out on the effect of urea on the TRH immunoassay. Urea was added to saline or phosphate buffer at concentrations of 1-50 mg/ml
Submitted July 17, 1975
801
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 04:33 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1975 Vol 41 • No 4
RAPID COMMUNICATIONS
802
which encompassed the range of urea concentrations in normal human urine. The urea solutions were then assayed for TRH. A portion of the urea solution was also extracted with charcoal and assayed for TRH as described above. To determine whether urea present in normal urine would affect the immunoassay for TRH, 1 or 2 ml of freshly voided urine was incubated for one hour at 37°C with urease in order to degrade the urea (12). Urease (50 mg/ml) was dissolved in 1 ml 50% (v/v) glycerol-double distilled water. 0.2 or 0.4 ml of the above solution was added to 1 or 2 ml urine, respectively, and incubated at 37°C for one hour. The solution was then centrifuged and the supernatant was diluted 1:4 and 0.1 ml was assayed for TRH. Urine without urease was also assayed in a 1:4 dilution. In order to determine the effect of urease per se on the immunoassay for TRH, solutions of urea in buffer (20 mg/ ml) with or without added synthetic TRH were incubated with and without urease for one hour at 37°C and then assayed for TRH. Similar experiments were also carried out by adding TRH to urine with and without urease. All samples in individual experiments were assayed in duplicate in the same assay. RESULTS Serum deactivation of urine TRH. When TRH (1-80 ng) in buffer was incubated with 0.2 ml serum, approximately 80100% of the TRH was deactivated. These findings are similar to those of Bassiri and Utiger (10). In contrast, little or no deactivation of apparent endogenous urine TRH occurred when urine was incubated with serum (Table 1). The mean concentration of urine TRH was 610 ± 292 pg/ml before and 691 * 369 pg/ml after serum deactivation. Similarly, charcoal extracted urine TRH could not be deactivated by serum (Table 2 ) . The mean concentration in five different
urine samples was 222 ± 93 pg/ml before and 294 ± 196 pg/ml after deactivation by serum. In order to exclude the possibility that urine contains an inhibitor of the serum enzyme, various concentrations of TRH were added to urine. The urine samples were then incubated with serum. The inactivation of added TRH was similar to that found when TRH was added to buffer alone, suggesting that urine had no inhibitory effect on the serum TRH deactivating enzymes. Urine was collected following the iv administration of 400 yg TRH. Aliquots of these urine samples were then incubated with serum. Deactivation of the excreted TRH was almost complete. Effect of urea on the TRH radioimmunoassay. The addition of urea to saline or phosphate-saline buffer was detected as TRH in the radioimmunoassay (Table 3 ) . Large quantities of urea (mg/ml) were interpreted as pg/ml quantities of TRH. Following charcoal extraction, the urea effect was markedly attenuated but apparent TRH was still detected. Inhibition of the urea effect by urease (Table 4). Since urea exhibited a nonspecific effect on the TRH immunoassay, urea was eliminated from the urine by incubating urine with urease. The nonspecific effect of urea on the TRH assay was abolished in the presence of urease. The recovery of TRH added to urine plus urease was approximately 90%, suggesting that urease did not alter the TRH molecule or interfere with its immunoassay. Furthermore, the recovery of TRH added to buffer containing urea and urease was also 90%. Finally, the nonspecific effect of urea in buffer on the TRH assay was abolished by the addition of urease. DISCUSSION The present studies clearly demonstrate that urine TRH as measured by radio imrauno as say is probably not TRH but is a nonspecific effect of urea on the immunoassay. These findings seem justified since the urinary immunoreactive substance which we detected was completely
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 04:33 For personal use only. No other uses without permission. . All rights reserved.
803
RAPID COMMUNICATIONS inactivated by urease. Urease itself does not affect the inununoassay or deactivate TRH. Furthermore, the inability of serum to deactivate urine TRH suggests that what is assayed in urine as TRH in whole and methanol or charcoal-extracted urine may not be TRH. The possibility that TRH was excreted in the urine in a form which is protected from serum deactivation seems unlikely since TRH in urine from a patient following the iv administration of TRH was deactivated by serum. These findings suggest that if the chemical form of TRH which is released from the brain is similar to synthetic TRH, it is not bound to a "protector" substance in the urine. If TRH is present in urine, its concentration is probably low and may not be detected by present assay techniques. Thus, reported production rates for TRH which depend upon urine TRH values in animals and man may not be valid (4,13). The present findings also may help to explain why urine TRH concentration does not change in various abnormalities of the thyroid or following exogenous T4 and T3 administration (14). Furthermore, the failure to detect significant changes in urine TRH in animals with hypothalamic lesions which are accompanied by significant decreases in TRH content in the hypothalamus and stalk median eminence may also .be due to an inability to accurately measure true urine TRH (15). Oliver et al (5) have recently reported that ethanol extracts of charcoal-treated urine displayed an elution pattern similar to synthetic TRH as judged by G-10 Sephadex filtration and paper electrophoresis. However, close inspection of their data clearly reveals that the patterns are not identical. Furthermore, it is possible that the immunoreactive substance which they reported may have been urea. This possibility is supported by our findings. First, serum had no deactivating efrect on urine TRH in methanol extracts of charcoal-treated urine.
Second, when solutions of urea in buffer were treated with charcoal and the charcoal extracted with methanol, TRH immunoreactivity was detected. More recently, Montoya et al (16) have reported an immunoassay for urine and plasma TRH which employs sepharoseanti TRH columns to isolate TRH from the urine. The possibility that urea or some other substances also interfere with this immunoassay cannot be ruled out. Measurements of TRH in the blood by radioimmunoassay are also fraught with many difficulties. Indeed, recent studies employing the immunoassay for blood TRH have resulted in controversial findings. Montoya and coworkers (8) found a striking elevation in plasma TRH following cold exposure in rats while Emerson and Utiger (17) found no such changes. These diverse findings emphasize the fact that caution should be exercised in interpreting physiological experiments based on either urine or plasma TRH immunoassay. Table 1. Urine TRH before & after serum deactivation (1 ml serum + 1 ml urine). TRH (pg/ml) After Urine Sample Before
1 2
670
525
3
1100 310
4 5 6 7 8
300 760 660 800 280
1500 280 750 625 650 800 400
610 ± 292
691 ± 369
Mean ± SD
Table 2. Charcoal-extracted urine: before & after serum deactivation.
TRH
TRH (pg/ml) Urine Sample
1 2 3
Before
4
310 220 300 205
5
77
Mean ± SD
222 ± 93
After
340 570 310 220 30 294 ± 196
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Table 3. Effect of urea in buffer on the TRH radioinununoassay. Urea (mg/ml)
1 5 10 15 25 50
TRH (pg/ml)
REFERENCES
1. Bassiri, R.M., and R.D. Utiger, Endo-
TRH after charcoal extraction (pg/ml)
10 47 110 125 270 860
crinology 90:722, 1972.
2. Jackson, I.M.D., and S. Reichlin, Endocrinology 95:816, 1974.
0 39 45 40 105 100
3. Brownstein, M.J., M. Palkovits, 4. 5.
Table 4. Inhibition of the urea effect on the TRH assay by urease.
6.
TRH measured (ng) 1 ml urine
none
7.
2.0 ± 0.3* 0
urease
8.
1** 2 ml urine
2 ml buffer
JCE&M . 1975 Vol 41 * No 4
RAPID COMMUNICATIONS
804
none urease 5 ng TRH 5 ng TRH & urease urease 40 mg urea 40 mg urea & urease 5 ng TRH 5 ng TRH & urease 40 mg urea & 5 ng TRH & urease
43
2.0 0 7.6
9.
0 8.0 4.3
4.8
11,
10.
0 1.0
12. 13.
0 4.0 4.5 ± 0.2*
14.
4.8 15.
*Mean ± SD of four different samples **1 and 2 are different urines
16.
ACKNOWLEDGMENTS 17. Supported by Research Grant AM07917 from the NIAMDD, National Institutes af Health, Bethesda, Maryland.
J.M. Saavedra, R.M. Bassiri, and R.D. Utiger, Science 185:267, 1974. Jackson, I.M.D., R. Gagel, P.D. Papapetrou, D. Duprez, and S. Reichlin, Clin Res 23:238A, 1975. Oliver, C , J.P. Charvet, J.L. Codaccioni, and J. Vague, J Clin Endocr Metab 39:406, 1974. Jackson, I.M.D., and S. Reichlin, Life Sci 14:2259, 1974. Shambaugh, G.E., J.F. Wilber, E. Montoya, H. Ruder, and E.R. Blonsky, J Clin Endocr Metab 41:131, 1975. Montoya, E., M.J. Seibel, and J;F. Wilber, Endocrinology 96: 1413, 1975. Bassiri, R.M., and R.D. Utiger, Endocrinology 94:188, 1974. Bassiri, R.M., and R.D. Utiger, Endocrinology 91:657, 1972. Miller, M., and A.M. Moses, J Clin Endocr Metab 34:537, 1972. Koch, J., Lab Clin Med 11:776, 1926. Jackson, I.M.D., P.D. Papapetrou, and S. Reichlin, Program 50th Annual Meeting American Thyroid Association, Abstr No 1. Gagel, R.F., I.M.D. Jackson, D. Duprez, P.D. Papapetrou, and S. Reichlin, 7th International Thyroid Conference, 1975, Abstr No 22. Jackson, I.M.D., and S. Reichlin, Program 57th Annual Meeting Endocrine Society Abstr No 91. Montoya, E., M.J. Seibel, and J. Wilber, Program 55th Annual Meeting Endocrine Society Abstr No 180. Emerson, C.H., and R.D. Utiger, Program 57th Annual Meeting Endocrine Society Abstr No 200.
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