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Phenylacetic acid excretion in man
ANALYTICAL BIOCHEMISTRY 99, 283-287 (1979)
Phenylacetic Acid Excretion in Man MARGARET E I L E E N M A R T I N , FAROUK K A R O U M , AND RICHARD J E D W Y A T T
Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, Intramural Research Program, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032 Received February 1, 1979 A rapid, reliable, sensitive, and highly specific mass fragmentographic method for the quantification of phenylacetic acid (PAA) in urine is described. The method was used to determine total, conjugated, and free PAA simultaneously in normal urine. The mean +_ SE excretion of total, conjugated, and free PAA in urine of 24 normal adult volunteers were, respectively, 137.4 _ 15.8, 128.8 -4- 15.0, 8.49 _+ 0.99 mg/24 h. Free PAA constitutes less than 10% of the total (free plus conjugated) urine excretion of PAA.
Phenylacetic acid (PAA) 1 and its glutamine conjugate, phenylacetylglutamine (PAG), are normal constituents of human urine (1-3). Phenylacetic acid is primarily excreted as PAG (4,5) at a rate exceeding 100 mg/24 h (2). Although PAA can be derived from phenylalanine and phenylethylamine by decarboxylation and deamination, its route of formation is not completely understood. Curtius et al. (6) have delineated a pathway from phenylalanine which may involve phenylpyruvic acid. The fact that PAA is excreted in large amounts compared to, for example, vanilmandelic and homovanillic acid (7), hints at a possible dietary origin (8). However, if it is endogenously produced, as suggested from studies on phenylketonuric subjects (6,9), then such a high rate of excretion may indicate a high rate of phenylethylamine (PEA) and/or phenylalanine turnover and metabolism. Our interest in phenylethylamine metabolism and psychiatric disorders (10) has led us to develop highly specific methods for 1 Abbreviations used: PAA, phenylacetic acid; PAG, phenylacetylglutamine, PEA, phenylethylamine; PFP, pentafluoropropionyl; PAA-dr, deuterated phenylacetic acid. 283
the assay of both phenylethylamine (11) and PAA in urine and other biological media. MATERIALS AND METHODS Deuterated PAA (PAA-dT) was purchased from Merck, Sharpe and Dohme (Quebec). All other reagents were of the highest purity obtainable. For the assay of PAA, 50/zl of urine was mixed with 50/zl of a solution of 1 mg PAA-dT/ml in 0.1 N HC1. Twenty microliters of this mixture was transferred into two 1-ml Microflex (Knotes Glass Company, Evanston, Ill.) tubes. To one tube, 5 ~g of 10% triethylamine in methanol was added and the mixture evaporated to dryness under N2. Fifty microliters of 6 N HC1 was added to the second tube. This tube was heated at 100°C for 45 to 60 min and cooled; 5/zl of 10% triethylamine was added and the mixture evaporated to dryness under N2. Triethylamine was added in order to reduce the loss of free PAA by evaporation. It forms a salt with free PAA (13), which is considerably less volalite than PAA. In each batch of analysis, a triplicate of pooled diluted urine (urine plus PAA-d0 was run. To two of these triplicates, 1 and 2/xg of free PAA or 5 and 10/zg-equiv of free P A A as phenyl0003 -2697/79/160283 -05 $02.00/0
284
MARTIN, KAROUM, AND WYATT
acetylglutamine were added, processed for free PAA or total PAA, and used for the construction of standard curves. The standard curves were used to check the concentration of PAA-d7 in the diluted urine samples. PAA in the urine samples was calculated by directly comparing its peak height with that corresponding to PAA-dT. Derivatization. The PAA in the dried residues obtained from the evaporated urine samples (with or without hydrolysis) was converted to its pentafluoropropionyl esters (12) by heating at 70°C for 30 min with 100/xl of pentafluoropropanol and 50/zl of pentafluoropropionic anhydride. Excess acylating reagents were evaporated under Nz and the residues reconstituted in 25/zl of ethylacetate (dried under Call2). Mass fragmentography. An 8-ft 1/8-in. o.d. stainless-steel column packed with 3% SE-54 on Chromosorb G was used for the separation of PAA from other urinary constituents. For single ion monitoring, we focused on the molecular ion. The mass to charge (m/e) ratio of the molecular ion of PAA and its deuterated isomer (PAA-d0 are, respectively, 268 and 275 (Fig. 1). The column was maintained at an oven temperature of 155°C. A Model 3200 Finigan gas chromatograph quadrupole mass spectrometer was used.
Studies on the hydrolysis of phenylacetylglutamine. In order to determine the time required to completely hydrolyze phenylacetylglutamine (PAG), quadruplets of three urines were hydrolyzed with 50/zl 6 N HC1 for periods of time ranging from 0 to 120 min. For this, 0.5 ml of urine was mixed with 100/zl of a 50/zg/ml solution of PAA-dT. Twenty microliters of this diluted urine was mixed into 50/zl of 6 N HCI. In the same experiment, equal volumes of 20/zg/ml PAG solution and PAA-d7 (20/zg/ml) were mixed and 20-/xl portions of the solution were hydrolyzed with 50/zl of the 6 y HC1 for different periods. After hydrolysis, the samples were
I00
............
100
25
M.+~. m/e 268
~m/e 119
r L, ,,i ................................. ,.............................................,................I
......
150
200
250
FIG. 1. Partial mass spectrum of phenylacetic acid (PAA) pentafluoropropionyl (PFP) ester. The left-hand vertical scale represents relative intensity compared to the base peak (m/e 119), and the right-hand vertical scale represents the percentage intensity compared to the total intensity produced by the various fragments derived from PAA.
processed and derivatized as described above. Urine collection. Urines were collected from 9:00 AM to 9:00 PM on 1 day and 9:00 PM to 9:00 AM the following day from 13 normal adult volunteers working at the National Institute of Mental Health. After collection, the urines were mixed with 10 ml 10% EDTA, the volume was measured, and aliquots were frozen at -40°C until analyzed. These urines were analyzed for total PAA. Twenty-four-hour urine samples were also collected from 24 normal subjects and analyzed for free, total, and conjugated PAA. The addition of 10% EDTA did not alter the urine pH. All urines were individually checked and found to have pH values between 5 and 7 with a mean of around 6. Storage of urine at -40°C did not appear to effect free PAA nor PAG concentrations. Analysis of some urines repeated about 6 months apart gave essentially the same results. RESULTS AND DISCUSSION
As shown in Fig. 2, PAG underwent complete hydrolysis after heating at 100°C for 50 min in 6 N HCI. The curves shown in Fig. 2 are representative of several similar results obtained in the course of developing the method. The linearity of the hydrolysis
285
PHENYLACETIC ACID EXCRETION IN MAN
25
w
'~
2.0
20
~
n5
1.5
n~
10
1.0 /
/
--
URINE SAMPLE (QUADRUPLICATE)
....
PHENYLACETYLGLUTAMINE
/
0.5
(20 /z.g)
I0
2o
30
40
50
60
//' 100
I I0
120
HYDROLYSIS TIME (MIN)
FIG. 2. Production of PAA from phenylacetylglutamine (PAG) in urine and standard after hydrolysis at 100°C for different periods of time in 6 N HC1. Solid line represents the production of PAA from PAG in urine and the broken line represents the production of PAA from PAG equivalent to 20/xg free PAA. All samples contain the same amount of deuterated PAA-dT. The left-hand scale represents the ratio of free PAA to PAA-d7 for peaks produced after the hydrolysis of urinary PAG for different time periods. The right-hand scale corresponds to the known 20/zg of PAG also compared to the deuterated standard. See text for details.
of PAG is illustrated in Fig. 3. To test the reliability of the assay, samples from 10 individuals were aliquoted into two sets of vials and blindly analyzed 1 week apart, The intraclass correlations for total, conjugated, and free PAA were 0.98, 0.85, and 0.95, respectively. On the other hand, duplicate analysis of several urines carded out in
the same batch showed correlation coefficients well over 0.95. The method described here is currently being used to evaluate PAA excretion in mental illnesses and is linear for all values so far examined. For example, in the above mentioned analysis, excretions of total PAA as low as 5 mg/24 h and as high as 400 mg/24 h were
(AFTER 50 MIN. HYDROLYSIS) 4.0 ,~ ,~ 3.0
u_ 2.0 0
_o
~
n.-
~.o
0
i
i
i
t
5
10
15
20
H-g OF
PHENYLACETYLGLUTAM)NE
FIG. 3. A typical standard curve o f different amounts o f PAG added to urine and assayed as described
in the text. The vertical axis represents the ratios of the peak heights of free PAA determined to those corresponding to a known amount of PAA-dr standard included in each urine sample. The points on the vertical axis corresponding to the differences in the above ratio for the same urine volumes with (as expressed by the horizontal axis) and without added PAG. All urines were hydrolyzed and processed as described in the text.
286
MARTIN, K A R O U M , A N D WYATT
Urine sample
0
1.0
Same urine + 2150g PAG
2.0 0
MIN
Same
urine +
5 ~g PAG
1.0 2.0 0
1.0
2.0
MIN
MIN
FIG. 4. Typical mass fragmentograms of 0, 2.5, and 5.0/zg of PAG added to three urine aliquots. The urines were analyzed for total PAA as described in the text. The solid line represents the trace produced by focusing on PAA molecular ion (m/e 269) and the broken line to that of PAA-d~ (m/e 275).
accurately and reproducibly quantified in the same batch of analysis and from the same standard curve. Figure 4 shows typical fragmentograms of a urine sample with two added levels of PAG as normally used for the construction of a standard curve. The urine samples analyzed for the 12-h excretion of PAA were not the same as those analyzed for the 24-h excretion, hence the apparent difference between the sum of the two 12-h total PAA and the 24-h excretions shown in Table 1. However, we have compared the mean of half of all the 24-h total PAA with both of the two 12-h urine collec-
tions for total PAA excretions. The differences were not statistically significant. The urinary excretion of total PAA during two different 12-h periods, as well as the excretion of free and conjugated PAA, were summarized in Table 1. Free PAA constitutes about 7% of the total PAA excreted which is similar to reports published by other workers (4,13,14). There appears to be no difference in total PAA excretion in urines collected from 9:00 AM to 9;00 PM and from 9:00 PM to 9:00 AM. The fact that PAA excretion during the most active part of the day (9:00 AM to 9:00 PM) is not significantly different from that during the night, suggests that PAA excretion is not related to activity and that it does not come directly as free PAA from the diet. Our conclusion on the diurnal rhythm in PAA excretion and the influence of diet on this one metabolite is consistent with those reported by Seakins (8). In an extensive study on PAA excretion, Seakins observed no consistant diurnal variation in PAA excretion in normal adults. The effects of fruit juice, coffee, and bananas on PAA excretion were also studied and found to be small (8). In a study carried out by Stein et al. (2) fasting was reported to have produced no change in urinary excretion of conjugated PAA. PAA excretion is increased in two hereditary abnormalities of phenylalanine metabolism, phenylketonuria and hyperphenylalanimia (6,9). Restriction of dietary
TABLE 1 MEAN (-+SEM) URINE EXCRETION OF PAA IN NORMAL ADULT SUBJECTSa Time of collection
No. of excretions
Free PAA
Total PAA
24-h Urine collection 12-h Urine collection 9:00 AM to 9:00 PM 12-h Urine collection 9:00 PM to 9:00 AM
24
8.40 ± 0.99
137.4 -+ 15.8
13
--
80.4 ± 13.2"
--
13
--
87.8 ± 14.2"
--
The results are expressed as rag/collection period. * The two values are not statistically significantly different.
Glutamine (Free PAA/conjugated PAA) conjugated PAA x 10 128.8 _+ 15.0
0.69 _+_ 0.06
PHENYLACETIC ACID EXCRETION IN MAN
phenylalanine in these two abnormalities decreased PAA excretion. Furthermore, studies on growth requirements have shown that phenylpyruvic acid can replace phenylalanine in diet (15,16). This acid when fed by mouth gave rise to PAA (16). Phenylethylamine is quantitatively metabolized to PAA (8). Therefore, it appears that phenylalanine and probably its decarboxylated product (PEA), and phenylpyruvic are the major sources of urine PAA. According to Seakins (8), a large portion of urine PAA is derived from gut (especially the large bowel) metabolism of phenylalanine via its decarboxylation to phenylethylamine. In view of some recent observations, however, this suggestion requires confirmation. For example, the ingestion of neomycin (M. Sandier, personal communication) was not found to reduce significantly total urine PAA excretion. Although a number of assays exist for measuring PAA in the urine (2-5,9,13,14), the method described here is the first to use mass spectrometry. It is specific, reliable and as many as 50 samples can be assayed in a day. The use of deuterated PAA to correct for losses of PAA during evaporation considerably improves the consistancy of the overall procedure. Because of its volatility, free PAA is easily lost when extracted into organic solvents and evaporated in v a c u o . Goodwin et al. (13), as did we, overcame the problem of volatility by forming a salt with triethylamine. Because of the large amount of PAA normally excreted and the fact that a small volume of urine (10/zl) is analyzed, the method does not appear to be sensitive to interference by other urine constituents. We have recently completed the analysis of urines obtained from patients on a variety of antipsychotic drugs and failed to observe a single sample with peaks that emerge near
287
or close to those corresponding to PAA and PAA-dT. As shown in Fig. 4, the mass fragmentograms of urine contain essentially two peaks which correspond to PAA and P A A - d r .
ACKNOWLEDGMENT The authors are grateful to Dr. B. L. Goodwin of Bernhard Baron Memorial Research Laboratories, London, for his gift of phenylacetylglutamine.
REFERENCES 1. Thierfelder, H., and Sherwin, C. P. (1914) Ber. Deut. Chem. Ges. 47, 2630-2634. 2. Stein, W. H., Paladini, A. C., Hirs, C. H. W., and Moore, S. (1954)J. Amer. Chem. Soc. 76, 2848-2849. 3. V611min, J. A., Bosshard, H. R., MOiler, M., Rampini, S., and Curtius, H. Ch. (1971) Z. Klin. Chem. Klin. Biochem. 9, 402-404. 4. James, M. O., Smith, R. L., Williams, R. T., and Reidenberg, M. (1972)Proc. Roy. Soc. Ser. B 182, 25-35. 5. James, M. O., and Smith, R. L. (1973) Eur. J. Clin. Pharmacol. 5, 243-246. 6. Curtius, H. Ch., V611min, J. A., and Baerlocher, K. (1972) Clin. Chim. Acta 37, 277-285. 7. Karoum, F., Anah, C. O., Ruthven, C. R. J., and Sandier, M. (1975) Clin. Chim. Acta 24, 341-348. 8. Seakins, J. W. T. (1971) Clin. Chim. Acta 35, 121-131. 9. Blau, K. (1970) Clin. Chim. Acta 27, 5-18. 10. Wyatt, R. J. (1978) in Nature of Schizophrenia (Wynne, L., Cromwell, R., and Mattysse, S., eds.), pp. 116-125, Wiley, New York. 11. Karoum, F., Nasrallah, H., Potkin, S., Chuang, L., Moyer-Schwing, J., Phillips, I., and Wyatt, R. J. (1979) J. Neurochem. 33,201-212. 12. Watson, E., Wilk, S., and Roboz, J. (1974) Anal. Biochem. 59, 441-451. 13. Goodwin, B. L., Ruthven, C. R. J., and Sandier, M. (1975) Clin. Chim. Acta 62,443-446. 14. Van der Heiden, C., Wadman, S. K., Ketting, D., and DeBree, P. K. (1970) Clin. Chim. Acta 31, 133-141. 15. Rose, W. C. (1937) Science 86, 298-300. 16. Bulb, E. C., and Butts, J. C. (1949) J. Biol. Chem. 180, 839-843.
Phenylacetic Acid Excretion in Man MARGARET E I L E E N M A R T I N , FAROUK K A R O U M , AND RICHARD J E D W Y A T T
Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, Intramural Research Program, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032 Received February 1, 1979 A rapid, reliable, sensitive, and highly specific mass fragmentographic method for the quantification of phenylacetic acid (PAA) in urine is described. The method was used to determine total, conjugated, and free PAA simultaneously in normal urine. The mean +_ SE excretion of total, conjugated, and free PAA in urine of 24 normal adult volunteers were, respectively, 137.4 _ 15.8, 128.8 -4- 15.0, 8.49 _+ 0.99 mg/24 h. Free PAA constitutes less than 10% of the total (free plus conjugated) urine excretion of PAA.
Phenylacetic acid (PAA) 1 and its glutamine conjugate, phenylacetylglutamine (PAG), are normal constituents of human urine (1-3). Phenylacetic acid is primarily excreted as PAG (4,5) at a rate exceeding 100 mg/24 h (2). Although PAA can be derived from phenylalanine and phenylethylamine by decarboxylation and deamination, its route of formation is not completely understood. Curtius et al. (6) have delineated a pathway from phenylalanine which may involve phenylpyruvic acid. The fact that PAA is excreted in large amounts compared to, for example, vanilmandelic and homovanillic acid (7), hints at a possible dietary origin (8). However, if it is endogenously produced, as suggested from studies on phenylketonuric subjects (6,9), then such a high rate of excretion may indicate a high rate of phenylethylamine (PEA) and/or phenylalanine turnover and metabolism. Our interest in phenylethylamine metabolism and psychiatric disorders (10) has led us to develop highly specific methods for 1 Abbreviations used: PAA, phenylacetic acid; PAG, phenylacetylglutamine, PEA, phenylethylamine; PFP, pentafluoropropionyl; PAA-dr, deuterated phenylacetic acid. 283
the assay of both phenylethylamine (11) and PAA in urine and other biological media. MATERIALS AND METHODS Deuterated PAA (PAA-dT) was purchased from Merck, Sharpe and Dohme (Quebec). All other reagents were of the highest purity obtainable. For the assay of PAA, 50/zl of urine was mixed with 50/zl of a solution of 1 mg PAA-dT/ml in 0.1 N HC1. Twenty microliters of this mixture was transferred into two 1-ml Microflex (Knotes Glass Company, Evanston, Ill.) tubes. To one tube, 5 ~g of 10% triethylamine in methanol was added and the mixture evaporated to dryness under N2. Fifty microliters of 6 N HC1 was added to the second tube. This tube was heated at 100°C for 45 to 60 min and cooled; 5/zl of 10% triethylamine was added and the mixture evaporated to dryness under N2. Triethylamine was added in order to reduce the loss of free PAA by evaporation. It forms a salt with free PAA (13), which is considerably less volalite than PAA. In each batch of analysis, a triplicate of pooled diluted urine (urine plus PAA-d0 was run. To two of these triplicates, 1 and 2/xg of free PAA or 5 and 10/zg-equiv of free P A A as phenyl0003 -2697/79/160283 -05 $02.00/0
284
MARTIN, KAROUM, AND WYATT
acetylglutamine were added, processed for free PAA or total PAA, and used for the construction of standard curves. The standard curves were used to check the concentration of PAA-d7 in the diluted urine samples. PAA in the urine samples was calculated by directly comparing its peak height with that corresponding to PAA-dT. Derivatization. The PAA in the dried residues obtained from the evaporated urine samples (with or without hydrolysis) was converted to its pentafluoropropionyl esters (12) by heating at 70°C for 30 min with 100/xl of pentafluoropropanol and 50/zl of pentafluoropropionic anhydride. Excess acylating reagents were evaporated under Nz and the residues reconstituted in 25/zl of ethylacetate (dried under Call2). Mass fragmentography. An 8-ft 1/8-in. o.d. stainless-steel column packed with 3% SE-54 on Chromosorb G was used for the separation of PAA from other urinary constituents. For single ion monitoring, we focused on the molecular ion. The mass to charge (m/e) ratio of the molecular ion of PAA and its deuterated isomer (PAA-d0 are, respectively, 268 and 275 (Fig. 1). The column was maintained at an oven temperature of 155°C. A Model 3200 Finigan gas chromatograph quadrupole mass spectrometer was used.
Studies on the hydrolysis of phenylacetylglutamine. In order to determine the time required to completely hydrolyze phenylacetylglutamine (PAG), quadruplets of three urines were hydrolyzed with 50/zl 6 N HC1 for periods of time ranging from 0 to 120 min. For this, 0.5 ml of urine was mixed with 100/zl of a 50/zg/ml solution of PAA-dT. Twenty microliters of this diluted urine was mixed into 50/zl of 6 N HCI. In the same experiment, equal volumes of 20/zg/ml PAG solution and PAA-d7 (20/zg/ml) were mixed and 20-/xl portions of the solution were hydrolyzed with 50/zl of the 6 y HC1 for different periods. After hydrolysis, the samples were
I00
............
100
25
M.+~. m/e 268
~m/e 119
r L, ,,i ................................. ,.............................................,................I
......
150
200
250
FIG. 1. Partial mass spectrum of phenylacetic acid (PAA) pentafluoropropionyl (PFP) ester. The left-hand vertical scale represents relative intensity compared to the base peak (m/e 119), and the right-hand vertical scale represents the percentage intensity compared to the total intensity produced by the various fragments derived from PAA.
processed and derivatized as described above. Urine collection. Urines were collected from 9:00 AM to 9:00 PM on 1 day and 9:00 PM to 9:00 AM the following day from 13 normal adult volunteers working at the National Institute of Mental Health. After collection, the urines were mixed with 10 ml 10% EDTA, the volume was measured, and aliquots were frozen at -40°C until analyzed. These urines were analyzed for total PAA. Twenty-four-hour urine samples were also collected from 24 normal subjects and analyzed for free, total, and conjugated PAA. The addition of 10% EDTA did not alter the urine pH. All urines were individually checked and found to have pH values between 5 and 7 with a mean of around 6. Storage of urine at -40°C did not appear to effect free PAA nor PAG concentrations. Analysis of some urines repeated about 6 months apart gave essentially the same results. RESULTS AND DISCUSSION
As shown in Fig. 2, PAG underwent complete hydrolysis after heating at 100°C for 50 min in 6 N HCI. The curves shown in Fig. 2 are representative of several similar results obtained in the course of developing the method. The linearity of the hydrolysis
285
PHENYLACETIC ACID EXCRETION IN MAN
25
w
'~
2.0
20
~
n5
1.5
n~
10
1.0 /
/
--
URINE SAMPLE (QUADRUPLICATE)
....
PHENYLACETYLGLUTAMINE
/
0.5
(20 /z.g)
I0
2o
30
40
50
60
//' 100
I I0
120
HYDROLYSIS TIME (MIN)
FIG. 2. Production of PAA from phenylacetylglutamine (PAG) in urine and standard after hydrolysis at 100°C for different periods of time in 6 N HC1. Solid line represents the production of PAA from PAG in urine and the broken line represents the production of PAA from PAG equivalent to 20/xg free PAA. All samples contain the same amount of deuterated PAA-dT. The left-hand scale represents the ratio of free PAA to PAA-d7 for peaks produced after the hydrolysis of urinary PAG for different time periods. The right-hand scale corresponds to the known 20/zg of PAG also compared to the deuterated standard. See text for details.
of PAG is illustrated in Fig. 3. To test the reliability of the assay, samples from 10 individuals were aliquoted into two sets of vials and blindly analyzed 1 week apart, The intraclass correlations for total, conjugated, and free PAA were 0.98, 0.85, and 0.95, respectively. On the other hand, duplicate analysis of several urines carded out in
the same batch showed correlation coefficients well over 0.95. The method described here is currently being used to evaluate PAA excretion in mental illnesses and is linear for all values so far examined. For example, in the above mentioned analysis, excretions of total PAA as low as 5 mg/24 h and as high as 400 mg/24 h were
(AFTER 50 MIN. HYDROLYSIS) 4.0 ,~ ,~ 3.0
u_ 2.0 0
_o
~
n.-
~.o
0
i
i
i
t
5
10
15
20
H-g OF
PHENYLACETYLGLUTAM)NE
FIG. 3. A typical standard curve o f different amounts o f PAG added to urine and assayed as described
in the text. The vertical axis represents the ratios of the peak heights of free PAA determined to those corresponding to a known amount of PAA-dr standard included in each urine sample. The points on the vertical axis corresponding to the differences in the above ratio for the same urine volumes with (as expressed by the horizontal axis) and without added PAG. All urines were hydrolyzed and processed as described in the text.
286
MARTIN, K A R O U M , A N D WYATT
Urine sample
0
1.0
Same urine + 2150g PAG
2.0 0
MIN
Same
urine +
5 ~g PAG
1.0 2.0 0
1.0
2.0
MIN
MIN
FIG. 4. Typical mass fragmentograms of 0, 2.5, and 5.0/zg of PAG added to three urine aliquots. The urines were analyzed for total PAA as described in the text. The solid line represents the trace produced by focusing on PAA molecular ion (m/e 269) and the broken line to that of PAA-d~ (m/e 275).
accurately and reproducibly quantified in the same batch of analysis and from the same standard curve. Figure 4 shows typical fragmentograms of a urine sample with two added levels of PAG as normally used for the construction of a standard curve. The urine samples analyzed for the 12-h excretion of PAA were not the same as those analyzed for the 24-h excretion, hence the apparent difference between the sum of the two 12-h total PAA and the 24-h excretions shown in Table 1. However, we have compared the mean of half of all the 24-h total PAA with both of the two 12-h urine collec-
tions for total PAA excretions. The differences were not statistically significant. The urinary excretion of total PAA during two different 12-h periods, as well as the excretion of free and conjugated PAA, were summarized in Table 1. Free PAA constitutes about 7% of the total PAA excreted which is similar to reports published by other workers (4,13,14). There appears to be no difference in total PAA excretion in urines collected from 9:00 AM to 9;00 PM and from 9:00 PM to 9:00 AM. The fact that PAA excretion during the most active part of the day (9:00 AM to 9:00 PM) is not significantly different from that during the night, suggests that PAA excretion is not related to activity and that it does not come directly as free PAA from the diet. Our conclusion on the diurnal rhythm in PAA excretion and the influence of diet on this one metabolite is consistent with those reported by Seakins (8). In an extensive study on PAA excretion, Seakins observed no consistant diurnal variation in PAA excretion in normal adults. The effects of fruit juice, coffee, and bananas on PAA excretion were also studied and found to be small (8). In a study carried out by Stein et al. (2) fasting was reported to have produced no change in urinary excretion of conjugated PAA. PAA excretion is increased in two hereditary abnormalities of phenylalanine metabolism, phenylketonuria and hyperphenylalanimia (6,9). Restriction of dietary
TABLE 1 MEAN (-+SEM) URINE EXCRETION OF PAA IN NORMAL ADULT SUBJECTSa Time of collection
No. of excretions
Free PAA
Total PAA
24-h Urine collection 12-h Urine collection 9:00 AM to 9:00 PM 12-h Urine collection 9:00 PM to 9:00 AM
24
8.40 ± 0.99
137.4 -+ 15.8
13
--
80.4 ± 13.2"
--
13
--
87.8 ± 14.2"
--
The results are expressed as rag/collection period. * The two values are not statistically significantly different.
Glutamine (Free PAA/conjugated PAA) conjugated PAA x 10 128.8 _+ 15.0
0.69 _+_ 0.06
PHENYLACETIC ACID EXCRETION IN MAN
phenylalanine in these two abnormalities decreased PAA excretion. Furthermore, studies on growth requirements have shown that phenylpyruvic acid can replace phenylalanine in diet (15,16). This acid when fed by mouth gave rise to PAA (16). Phenylethylamine is quantitatively metabolized to PAA (8). Therefore, it appears that phenylalanine and probably its decarboxylated product (PEA), and phenylpyruvic are the major sources of urine PAA. According to Seakins (8), a large portion of urine PAA is derived from gut (especially the large bowel) metabolism of phenylalanine via its decarboxylation to phenylethylamine. In view of some recent observations, however, this suggestion requires confirmation. For example, the ingestion of neomycin (M. Sandier, personal communication) was not found to reduce significantly total urine PAA excretion. Although a number of assays exist for measuring PAA in the urine (2-5,9,13,14), the method described here is the first to use mass spectrometry. It is specific, reliable and as many as 50 samples can be assayed in a day. The use of deuterated PAA to correct for losses of PAA during evaporation considerably improves the consistancy of the overall procedure. Because of its volatility, free PAA is easily lost when extracted into organic solvents and evaporated in v a c u o . Goodwin et al. (13), as did we, overcame the problem of volatility by forming a salt with triethylamine. Because of the large amount of PAA normally excreted and the fact that a small volume of urine (10/zl) is analyzed, the method does not appear to be sensitive to interference by other urine constituents. We have recently completed the analysis of urines obtained from patients on a variety of antipsychotic drugs and failed to observe a single sample with peaks that emerge near
287
or close to those corresponding to PAA and PAA-dT. As shown in Fig. 4, the mass fragmentograms of urine contain essentially two peaks which correspond to PAA and P A A - d r .
ACKNOWLEDGMENT The authors are grateful to Dr. B. L. Goodwin of Bernhard Baron Memorial Research Laboratories, London, for his gift of phenylacetylglutamine.
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