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Reproducibility of plasma catecholamine metabolites in normal subjects
Reproducibility of Plasma Catecholamine Metabolites in Normal Subjects Farooq Amin, Aqeel Hashmi, Adriana E. Stroe, Oladele Adebogun, Dianna Densmore, and Peter J. Knott B a c k g r o u n d : Plasma homovanillic acid (HVA) and me-
thoxy-hydroxy-phenylglycol (MHPG) are often used in repeated measurement designs to assess dopamine and norepinephrine metabolism in clinical research. However, only limited data on the reproducibility of these metabolites are currently available. Methods: Plasma HVA and MHPG were measured in normal volunteers on five nonconsecutive mornings under fasting conditions and restricted smoking and physical activity. Results: Intraclass correlation coefficients (ICC) for plasma HVA and MHPG across 5 days were determined to be 0.85 and 0.79, respectively. ICC improved to 0.90for HVA and 0.91 for MHPG when the average of the first 2 days was examined against the average of the last 2 days. Conclusions: Results suggested that under controlled conditions plasma HVA and MHPG measurements have good reproducibility. Biol Psychiatry 1998;43: 233-235 Published by the Society of Biological Psychiaoy 1998 Key W o r d s : Plasma, catecholamine, metabolite, reproducibility, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol
Introduction
p
lasma homovanillic acid (HVA) and methoxy-hydroxy-phenylglycol (MHPG) concentrations are frequently used to assess dopaminergic and noradrenergic metabolism, respectively, in clinical studies of psychiatric disorders (Amin and Friedhoff 1997; Amin et al 1992; Elsworth et al 1982). Measurements of these plasma metabolites are very convenient and, therefore, are often used in repeated measurements study designs. This implicitly assumes that these measurements are reproducible.
However, only limited data on the reproducibility of plasma HVA and MHPG (Baker et al 1988) are currently available in the published literature. This study was done to investigate the reproducibility of these plasma metabolites on several study mornings under well-controlled conditions.
Methods and Materials Nine normal volunteers were studied on 5 nonconsecutive study days. All subjects were men from the ages of 25-66 years, and were physically healthy as determined by medical history, physical examination, and routine laboratory tests. A structured clinical interview was employed to exclude major psychiatric disorders using DSM-III-R criteria. Prior to each study morning subjects were instructed to observe a tow monoamine diet for 72 hours and fast overnight for 14 hours. They were also instructed not to take any over-the-counter medication for at least 3 days, avoid strenuous physical activity and smoking after midnight, and arrive at the medical center by 8:15 A.M. on the study mornings. The 5 study days were completed over a mean +_ SD period of 47 _+ 9 days, ranging from 16-103 days. Blood samples were collected at 9:00 A.M. in heparinized vacutainer tubes and centrifuged for 15 minutes at approximately 2000 G wh}le at 10°C. Plasma was separated and immediately stored at -80°C until assayed using high performance liquid chromatography methods described elsewhere for HVA (Knott et al 1990) and MHPG (Yang et al 1988). The intraassay and interassay coefficients of variation for the laboratory were estimated to be 2.4% and 6%, respectively, for HVA and 5.2% and 13%, respectively, for MHPG. The association of metabolites between days was assessed by Pearson correlation coefficient. The intraclass correlation coefficient (ICC) was used to determine the reproducibility across days. Plasma MHPG data were not available in one subject.
Results From the Department of Psychiatry, Houston VAMC and Baylor College of Medicine, Houston, Texas (FA, AH, AES, OA, DD); and Department of Psychiatry, Bronx VAMC and Mount Sinai School of Medicine, New York, New York (PJK). Address reprint requests to Farooq Amin, M.D., Psychiatry Service (116 A), Houston VA Medical Center, 2002 Holcombe Blvd., Houston, TX 77030. Received March 26, 1997; revised July 2, 1997; accepted July 9, 1997.
Published by the Society of Biological Psychiatry 1998
Plasma HVA and MHPG concentrations on 5 days are presented in Figures 1 and 2, respectively. Means and standard deviations (SD) of these metabolites on all 5 days are presented in Table 1. Each metabolite measurement was generally strongly correlated across days with corre0006-3223/98/$0.00 Pll S0006-3223(97)00378-8
234
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BIOL PSYCHIATRY 1998;43:233-235
30
25 m
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Figure 1. Plasma HVA across 5 nonconsecutive days (n = 9).
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lation coefficients ranging from 0.90-0.99 for plasma HVA and from 0.62-0.94 for plasma MHPG. ICCs across 5 days were 0.85 for plasma HVA and 0.79 for plasma MHPG. This reproducibility improved to 0.90 and 0.91 for plasma HVA and MHPG, respectively, when the average measurement for the first 2 days was examined against the average of the last two days (Table 1).
6.0
Discussion The results suggested that under controlled conditions plasma HVA and MHPG measurements have good reproducibility. To our knowledge, this is a first study assessing the reproducibility of these plasma metabolites on more than 2 days. The only previous report that examined the
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Figure 2. Plasma MHPG across 5 nonconsecutive days (n = 8).
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Day-2
Day-3
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Study Days
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1998;43:233-235
Table 1. Reproducibility of Plasma HVA and MHPG at 9:00 A.M. across 5 Nonconsecutive Days Daily plasma metaboliteconcentrationsin ng/ml, mean _+ SD
HVA MHPG
Day 1
Day 2
Day 3
Day 4
Day 5
ICC~ across 5 days
10.7 _ 3.8 3.2 -+ 0.9
12.5 _+6.3 3.2 + 0.7
10.8 - 4.5 3.5 +- 1.0
10.3 _+3.6 3.2 - 0.9
10.7 -4-3.6 3.0 - 0.9
0.85 0.79
ICC, first 2 daysb against last 2 days 0.90 0.91
lntraclass correlation coefficient. b The averaged measurement of the first 2 days was examined against the averaged measurement of the last 2 days.
reproducibility of these metabolites assessed ICC across 2 days and under less well-controlled naturalistic conditions (Baker et al 1988). The reproducibility of plasma H V A in our study was observed to be higher (ICC = 0.85) than in the previous report (ICC = 0.67), although plasma MHPG results were the same. It is possible that better plasma HVA reproducibility in our study was due to more stringent study conditions (e.g., control of physical activity and smoking). Our results also support the view that by designing studies that use an average of two or more measurements more stable assessment of these plasma metabolites may be possible. This work is based on support provided to Dr. Farooq Amin by the Medical Research Service of Department of Veterans Affairs.
References Amin F, Davidson M, Davis KL (1992): Homovanillic acid measurement in clinical research: A review of methodology. Schizophr Bull 18:123-148.
Amin F, Friedhoff AJ: Plasma homovanillic acid as a tool to investigate brain dopaminergic activity. In Friedhoff AJ, Amin F (eds), Plasma Homovanillic Acid in Schizophrenia: Implications for Presynaptic Dopamine Dysfunction. Washington DC: American Psychiatric Press, 1997, pp 1-16. Baker NJ, Adler LE, Waldo M, Gerhardt G, Drebing C, Cox B, Berry S, Phillips W, Freedman R (1988): Reproducibility of the measurement of plasma noradrenergic and dopaminergic metabolites in normal subjects. Psychiatry Res 23:119-130. Elsworth JD, Redmond DE, Jr., Roth RH (1982): Plasma and cerebrospinal fluid 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) as indices of brain norepinephrine metabolism in primates. Brain Res 235:115-124. Knott PJ, Yang RK, Cheng H, Warne P, Haroutunian V, Davidson M, Davis KL (1990): An improved, sensitive method for measurement of plasma homovanillic acid by HPLC with coulometric detection. 14th International Symposium on Column Liquid Chromatography (Abstract). Yang RK, Campbell G, Cheng H, Tsuboyama GK, Davis KL (1988): Highly sensitive and simple method for determination of free 3-methoxy-4-hydroxyphenylglycolin plasma by highperformance liquid chromatography using a Sep-Pak alumina B cartridge. J Liquid Chromatogr 11:3223-3231.
thoxy-hydroxy-phenylglycol (MHPG) are often used in repeated measurement designs to assess dopamine and norepinephrine metabolism in clinical research. However, only limited data on the reproducibility of these metabolites are currently available. Methods: Plasma HVA and MHPG were measured in normal volunteers on five nonconsecutive mornings under fasting conditions and restricted smoking and physical activity. Results: Intraclass correlation coefficients (ICC) for plasma HVA and MHPG across 5 days were determined to be 0.85 and 0.79, respectively. ICC improved to 0.90for HVA and 0.91 for MHPG when the average of the first 2 days was examined against the average of the last 2 days. Conclusions: Results suggested that under controlled conditions plasma HVA and MHPG measurements have good reproducibility. Biol Psychiatry 1998;43: 233-235 Published by the Society of Biological Psychiaoy 1998 Key W o r d s : Plasma, catecholamine, metabolite, reproducibility, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol
Introduction
p
lasma homovanillic acid (HVA) and methoxy-hydroxy-phenylglycol (MHPG) concentrations are frequently used to assess dopaminergic and noradrenergic metabolism, respectively, in clinical studies of psychiatric disorders (Amin and Friedhoff 1997; Amin et al 1992; Elsworth et al 1982). Measurements of these plasma metabolites are very convenient and, therefore, are often used in repeated measurements study designs. This implicitly assumes that these measurements are reproducible.
However, only limited data on the reproducibility of plasma HVA and MHPG (Baker et al 1988) are currently available in the published literature. This study was done to investigate the reproducibility of these plasma metabolites on several study mornings under well-controlled conditions.
Methods and Materials Nine normal volunteers were studied on 5 nonconsecutive study days. All subjects were men from the ages of 25-66 years, and were physically healthy as determined by medical history, physical examination, and routine laboratory tests. A structured clinical interview was employed to exclude major psychiatric disorders using DSM-III-R criteria. Prior to each study morning subjects were instructed to observe a tow monoamine diet for 72 hours and fast overnight for 14 hours. They were also instructed not to take any over-the-counter medication for at least 3 days, avoid strenuous physical activity and smoking after midnight, and arrive at the medical center by 8:15 A.M. on the study mornings. The 5 study days were completed over a mean +_ SD period of 47 _+ 9 days, ranging from 16-103 days. Blood samples were collected at 9:00 A.M. in heparinized vacutainer tubes and centrifuged for 15 minutes at approximately 2000 G wh}le at 10°C. Plasma was separated and immediately stored at -80°C until assayed using high performance liquid chromatography methods described elsewhere for HVA (Knott et al 1990) and MHPG (Yang et al 1988). The intraassay and interassay coefficients of variation for the laboratory were estimated to be 2.4% and 6%, respectively, for HVA and 5.2% and 13%, respectively, for MHPG. The association of metabolites between days was assessed by Pearson correlation coefficient. The intraclass correlation coefficient (ICC) was used to determine the reproducibility across days. Plasma MHPG data were not available in one subject.
Results From the Department of Psychiatry, Houston VAMC and Baylor College of Medicine, Houston, Texas (FA, AH, AES, OA, DD); and Department of Psychiatry, Bronx VAMC and Mount Sinai School of Medicine, New York, New York (PJK). Address reprint requests to Farooq Amin, M.D., Psychiatry Service (116 A), Houston VA Medical Center, 2002 Holcombe Blvd., Houston, TX 77030. Received March 26, 1997; revised July 2, 1997; accepted July 9, 1997.
Published by the Society of Biological Psychiatry 1998
Plasma HVA and MHPG concentrations on 5 days are presented in Figures 1 and 2, respectively. Means and standard deviations (SD) of these metabolites on all 5 days are presented in Table 1. Each metabolite measurement was generally strongly correlated across days with corre0006-3223/98/$0.00 Pll S0006-3223(97)00378-8
234
Brief Reports
BIOL PSYCHIATRY 1998;43:233-235
30
25 m
20 c t< >
15
Figure 1. Plasma HVA across 5 nonconsecutive days (n = 9).
"lt~
IE ~0
10
I:L
A
~
A
I
!
I
I
I
Day-1
Day-2
Day-3
Day-4
Day-5
Study Days
lation coefficients ranging from 0.90-0.99 for plasma HVA and from 0.62-0.94 for plasma MHPG. ICCs across 5 days were 0.85 for plasma HVA and 0.79 for plasma MHPG. This reproducibility improved to 0.90 and 0.91 for plasma HVA and MHPG, respectively, when the average measurement for the first 2 days was examined against the average of the last two days (Table 1).
6.0
Discussion The results suggested that under controlled conditions plasma HVA and MHPG measurements have good reproducibility. To our knowledge, this is a first study assessing the reproducibility of these plasma metabolites on more than 2 days. The only previous report that examined the
"~
5.5 ~ 5.0 ~ 4.5 ~
E 1-¢-.
(.9 0.. "l-
t~
E
¢ao
13_
3.5 ~ 3.0-
Figure 2. Plasma MHPG across 5 nonconsecutive days (n = 8).
2.5 2.01.51.0 0.5 0.0
I
I
I
I
f
Day- 1
Day-2
Day-3
Day-4
Day-5
Study Days
Brief Reports
BIOL PSYCHIATRY
235
1998;43:233-235
Table 1. Reproducibility of Plasma HVA and MHPG at 9:00 A.M. across 5 Nonconsecutive Days Daily plasma metaboliteconcentrationsin ng/ml, mean _+ SD
HVA MHPG
Day 1
Day 2
Day 3
Day 4
Day 5
ICC~ across 5 days
10.7 _ 3.8 3.2 -+ 0.9
12.5 _+6.3 3.2 + 0.7
10.8 - 4.5 3.5 +- 1.0
10.3 _+3.6 3.2 - 0.9
10.7 -4-3.6 3.0 - 0.9
0.85 0.79
ICC, first 2 daysb against last 2 days 0.90 0.91
lntraclass correlation coefficient. b The averaged measurement of the first 2 days was examined against the averaged measurement of the last 2 days.
reproducibility of these metabolites assessed ICC across 2 days and under less well-controlled naturalistic conditions (Baker et al 1988). The reproducibility of plasma H V A in our study was observed to be higher (ICC = 0.85) than in the previous report (ICC = 0.67), although plasma MHPG results were the same. It is possible that better plasma HVA reproducibility in our study was due to more stringent study conditions (e.g., control of physical activity and smoking). Our results also support the view that by designing studies that use an average of two or more measurements more stable assessment of these plasma metabolites may be possible. This work is based on support provided to Dr. Farooq Amin by the Medical Research Service of Department of Veterans Affairs.
References Amin F, Davidson M, Davis KL (1992): Homovanillic acid measurement in clinical research: A review of methodology. Schizophr Bull 18:123-148.
Amin F, Friedhoff AJ: Plasma homovanillic acid as a tool to investigate brain dopaminergic activity. In Friedhoff AJ, Amin F (eds), Plasma Homovanillic Acid in Schizophrenia: Implications for Presynaptic Dopamine Dysfunction. Washington DC: American Psychiatric Press, 1997, pp 1-16. Baker NJ, Adler LE, Waldo M, Gerhardt G, Drebing C, Cox B, Berry S, Phillips W, Freedman R (1988): Reproducibility of the measurement of plasma noradrenergic and dopaminergic metabolites in normal subjects. Psychiatry Res 23:119-130. Elsworth JD, Redmond DE, Jr., Roth RH (1982): Plasma and cerebrospinal fluid 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) as indices of brain norepinephrine metabolism in primates. Brain Res 235:115-124. Knott PJ, Yang RK, Cheng H, Warne P, Haroutunian V, Davidson M, Davis KL (1990): An improved, sensitive method for measurement of plasma homovanillic acid by HPLC with coulometric detection. 14th International Symposium on Column Liquid Chromatography (Abstract). Yang RK, Campbell G, Cheng H, Tsuboyama GK, Davis KL (1988): Highly sensitive and simple method for determination of free 3-methoxy-4-hydroxyphenylglycolin plasma by highperformance liquid chromatography using a Sep-Pak alumina B cartridge. J Liquid Chromatogr 11:3223-3231.