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The effect of excess L-phenylalanine on mothers and on their breast-fed infants
176
August, 1967 T h e J o u r n a l o[ P E D I A T R I C S
The effect of excess L-pbenylalanine on mothers and on their breast-fed infants Four lactating mothers--two who were heterozygous [or phenylketonuria and two nonheterozygous primiparas--were given oral doses o[ L-phenylalanine. The concentrations o[ phenylalanine and tyrosine in the mothers' and their in[ants' serum, as well as the concentrations o[ phenylalanine and its metabolites in the breast milk and in the mothers" and in[ants' urine, were measured. The heterozygous mothers exhibited higher concentrations o[ these substances than the nonheterozygous ones. Since the breast milk o[ untreated homozygous mothers would presumably contain more phenIyalanine and its metabolites than that o[ heterozygous ones, the question o[ whether or not they should nurse their in[ants deserves serious consideration.
Robert O. Fiseh, M.D., "x"Robert Jenness, Ph.D., Doris Doeden, M.S., and John A. Anderson, M.D., Ph.D. MINNEAPOLIS~
MINN.
I N r R A U r E R I N ~ growth retardation has been observed in two infants of an untreated phenylketonuric mother? One was homozygous and the other was heterozygous for phenylketonuria. The infants were breast fed for eleven and nine months, respectively. During these periods both infants exhibited significant growth retardation. This study was undertaken to determine whether or not the infants' growth retardation may have been associated with an excessive intake of phenylalanine and its metabolites from the breast milk.
SUBJECTS AND METHODS Four mothers and their breast-fed newborn infants were studied. Two of the ~Address: Box 323, Mayo Memorial Hospital, University o1 Minnesota, Minneapolis, Minn. 55455.
VoI. 71, No. 2, pp. 176-180
mothers were known heterozygotes for phenylketonuria (Nos. 1 and 3) and had borne one and two phenylketonuric children, respectively. Each of the two normal mothers (Nos. 2 and 4) was a primipara. The data pertinent to these mothers and their infants are presented in Table I. The two infants of the known heterozygous mothers were not phenylketonuric. The mothers and their infants were studied in the following manner: Mothers 1 and 2 were given a total of 800 mg. of L-phenylalanine per kilogram of body weight in five separate doses over a period of 24 hours. The L-phenylalanine was given at the following times and in the following doses: 8:00 A.Z~., 100 mg. per kilogram; 8:30 A.M., 100 mg. per kilogram; 2:00 P.~., 200 mg. per kilogram; 8:00 P.g., 200 mg. per
Volume 71 Number 2
Effect of excess L-phenylalanine
177
Table I. D a t a on the mothers and their offspring
Mothers
Age (yr.)
No. 1~ 33 No. 2 23 No. 3~ 39 No. 4 21 '~Heterozygotes.
Weight ] Baby's (Kg.) sex 85.9 67.7 68.2 70.9
Boy Girl Girl Boy
Birthdate
Weight (Gm.)
Date of L-phenylaIanine loading
Total amount of L-phenylalanine (mg./ Kg./ day)
1/ 2/65 11/23/65 1/20/66 11/25/65
5,280 3,750 4,370 3,440
1/11/65 11/27/65 1/27/66 11/29/65
800 8OO 600 6O0
kilogram; and at 12:00 P.M., 200 mg. per kilogram. Mothers 3 and 4 were given a total of 600 mg. of L-phenylalanine per kilogram of body weight in four separate doses over a period of 24 hours. The L-phenylalanine was given at the following times and in the following doses: 8:00 *.M., 100 mg. per kilogram; 8:30 A.M., 100 rag. per kilogram; 4:00 P.M., 200 rag. per kilogram; and at 12:00 p.M., 200 mg. per kilogram. The mothers fasted from 10:00 P.M. until 5 hours following the first L-phenylalanine loading, and drank only water during this period. For the rest of the study day they ate regular meals. At 12 hours prior to and at 12 and 24 hours after the first loading, 10 ml. of breast milk were collected. After the infant had been nursed, the breasts were emptied. The milk samples were collected prior to the next breast feeding. The blood samples for serum phenylalanine 2 and tyrosine 3 determinations were obtained immediately prior to loading, during fasting, and at 1, 2, 3, 4, 5, and 24 hours after the first loading. Urine was collected 24 hours before loading and for periods of 5 and 19 hours following the first loading. Blood samples were obtained from the infants for determinations of serum phenylalanine 2 and tyrosine ~ immediately before and 24 hours after the first loading. Urine specimens were collected from the infants on the preloading day and about 10 and 24 hours after the first loading. T h e infants were breast fed approximately every 3 to 4 hours through the day. T h e studies were carried out on the nonheterozygous control mothers and their in-
rants in the University of Minnesota Hospitals and on the heterozygous mothers and their infants in their homes. The phenylalanine content of the breast milk was determined according to a modified form of the fluorimetric method of McCaman and Robins. 2 One volume of milk was mixed with one volume of 0.6N trichloroacetic acid (TCA) and centrifuged to remove the protein and the layer of fat that floats to the top; then, 0.1 ml. of supernatant fluid (equivalent to 0.05 ml. of milk) was used for analysis. The analysis was done on two 0.1 ml. aliquots to which 0.1 ml. of 0.3N T C A was added. The internal control standard contained 0.1 rot. of 0.3N T C A and 0.025 /zm of phenylalanine. The tenfold quantity of reagent specified in the original McCamanand-Robins method was used. The incubation was done at 90.0 ~ C. for fifteen minutes. The tubes were read in a Coleman Model 12C Photofluorimeter with filter B-1 PC-1. The breast milk was further analyzed for the p-hydroxyphenolic acid derivatives of tyrosine (p-hydroxyphenylproprionic acid, phydroxyphenylacetic acid, and p-hydroxybenzoic acid) and for o-hydroxyphenylacetic acid by gas claromatography. 4 Assays were done on the filtrate following the precipitation of the protein from 3.5 ml. of breast milk by adding 1.0 ml. of 10 per cent zinc sulfate and 0.5 ml. of 1N N a O H . The internal standards for comparison were prepared by adding milk to known standards. T h e indole-3-acetic acid in the milk and urine was measured. ~ The urine specimens from mothers and infants were measured for the p-hydroxyphenolic acid derivatives of tyrosine (p-hydroxyphenylproprionic acid, p-hydroxyphenylacetic acid, p-hydroxybenzoic acid), for o-hydroxyphenyl-
178
F i s c h e t al.
acetic acid, and for indole-3-acetic acid. 4 The serum tyrosine response to the oral L-phenylalanine load was used as a measure of heterozygosity for phenylketonuria. G RESULTS
AND DISCUSSION
The serum phenylalanine concentration in the heterozygous mothers (Nos. 1 and 3) 2 hours after loading was greater than in the nonheterozygous control mother's (Nos. 2 and 4). At 3, 4, and 5 hours following loading, the differences between the serum phenylalanine concentration in the heterozygous and control mothers were even greater. The serum phenylalanine in one heterozygous mother (No. 1) at 24 hours following loading ~" was 66.7 my. per cent (4.04/~mj~ per 1 ml.) and in the control mother (No. 2), 3.9 rag. per cent (0.24 /~m per milliliter). The serum phenylalanine in the other heterozygous mother (No. 3) at 24 hours following loading was 13.2 rag. per cent (0.80 t*m per milliliter) and in the control mother (No. 4), 0.9 mg. per cent (0.05 ~m per milliliter). Following the increase of phenylalanine in the mothers' serum, there was an increase of phenylalanine in the breast milk of three of the mothers. There was no increase of phenylalanine in the breast milk of one mother (No. 4) whose serum phenylalanine level had increased the least (Table III). The initial concentration of phenylalanine in the breast milk of the four mothers varied from 0.15 to 0.40 micromoles per milliliter. The derivatives of phenylalanine and tyrosine were detectable in the breast milk only following loading. The greatest increase was found with respect to the concentration of p-hydroxybenzoic acid in the breast milk of the heterozygous mothers (Nos. 1 and 3). It is of interest that one mother (No. 1) had headaches and vertigo at the end of the loading day. During the loading period the urine specimens of the four mothers contained signifi~It is significant that Mothers 1 and 2 received 800 my. per kilogram of body weight and that Mothers 3 and 4 received 600 my. of L-phenylalanine per kilogram of body weight. tOne micromole .~ 0.1652 my. of phenylalanine.
The Journal o[ Pediatrics August 1967
cantly increased amounts of p-hydroxyphenolic acids, and the urine specimens of three mothers (Nos. 1, 3, and 4) contained an increased amount of o-hydroxyphenylacetic acid. Indole-3-acetic acid, which was not found in measurable amounts in the milk before or following loading, was found to be noticeably increased in the urine of all four mothers following loading. The serum phenylalanine concentration in the infants was found to be within the normal range: it did not exceed 2 mg. per cent 24 hours following the first loading even though they had been routinely nursed by their mothers during this period. It should be noted, however, that the modified McCaman-and-Robins fluorimetric method cannot be used in our laboratory to measure serum phenylalanine in quantities smaller than 1 my. We can assume that the infants' serum concentrations of phenylalanine did not increase significantly during the testing period. On the other hand, the infants' tyrosine levels were measured and found to be normal prior to and 24 hours following the first loading of their mothers. There were no consistent changes in the concentration of phenylalanine and tyrosine metabolites in the urine specimens of the infants during the loading day. However, the urine specimens of the infants represent samples and not complete 24 hour collections. The purpose of giving excessive doses of L-phenylalanine to the four mothers was to simulate the metabolic circumstances of phenylketonuric mothers during lactation. It can be assumed that the metabolic changes in a lactating phenylketonuric mother would cause her breast milk to differ even more from a "normal" mother's breast milk. This assumption is supported by the fact that there was proportionately more phenylalanine and its metabolites in the breast milk of the two heterozygous mothers than in the breast milk of the nonheterozygous control m o t h e r s during the testing period. Mother No. 1 exhibited a 29-fold increase and Mother No. 3 exhibited a 12-fold increase of the serum phenylalanine level 24 hours following the first loading with L,phenyl-
Volume 71
Effect of excess L-phenylalanine
Number 2
179
Table II. Serum phenylalanine and tyrosine values of the mothers Values
Mothers
Fasting* (rag. per cent)~,
1 hr.'~ (rag. per cent)
2 hr, (mg. per cent)
3 hr, (mg. per cent)
4 hr. (rag. per cent)
5 hr. (mg. per cent)
24 hr. (rag. per cent)
2.3 (0.14) 1.3 (0.07)
31.4 (1.90) 2.5 (0.14)
45.8 (2.77) 2.1 (0.12)
44.0 (2.66) 1.2 (0.07)
37.0 (2.24) 1.6 (0.09)
29.7 (1.80) 1.1 (0.06)
66.7 (4.04) 5.8 (0.32)
No. I Phenylalanine Tyrosine
No. 2 Phenylalanine Tyrosine
<
15.3
19.7
20.6
9.2
6.0
3.9
<(0.06)
1.0
(0.93)
(1.19)
(1.25)
(0.56)
(0.36)
(0.24)
1.3 (0.07)
2.7 (0.15)
2.4 (0.13)
3.7 (0.20)
3.7 (0.20)
2.9 (0.16)
3.7 (0.20)
1.1 (0.07) 1.5 (0.08)
16.6 (1.00) 1.7 (0.09)
26.1 (1.58) 2.2 (0.12)
25.0 (1.51) 2.3 (0.13)
20.8 (1.26) 2.2 (o.12)
17.5 (1.06) 2.2 (0.12)
13.2 (0.80) 3.3 (0.18)
No. 3 Phenylalanine Tyrosine
No. 4 Phenylalanine
< 1.0 9.8 16.0 11.9 9.5 4.5 < 1.0 <(0.06) (0.59) (0.97) (0.72) (0.57) (0.27) <(0.06) Tyrosine 1.5 3.4 4.0 7.2 5.9 5.6 3.2 (0.08) (0.19) (0 22) (0.40) (0.32) (0.31) (0.18) *Fasting values represent those obtained on the days before loading took place, i'The number of hours is computed from the time of the first loading. ++Serum phenylalanine and tyrosine values are given in milligramsper cent and (in parentheses) in micromolesper milliliter.
Table III. Concentrations of phenylalanine, p-hydroxyphenolic acids (p-hydroxyphenylproprionic acid, p-hydroxyphenyl acetic acid, p-hydroxybenzoic acid), o-hydroxyphenylacetic acid, and indole-3-acetic acid in the breast milk of the mothers Concentrations (micromoles per milliliter) Second day
2 Mothers
First day
Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.40 --.
hours alter I first loading [ g or 10 P.M.
Third day
No. I 0.48 1.66 0.06 .
.
1.60 0.07 0.07
2.28 1.95 --
0.38 < 0.30 < 0.30 .
0.30 < 0.30 < 0.30
0.47 0.40 --
0.66 < 0.20 0.20
O.13 < 0.30 < 0.30 .
0.05 < 0.20 < 0.20
.
No. 2 Phenylalanlne p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.29 --.
0.22 < 0.30 < 0.30 .
.
No. 3 Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.15 --.
0.24 1.45 -.
.
.
No. 4 Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphe~ylacetic acid Indole-3-acetic acid
O. 15 --.
O. 19 < 0.30 < 0.30 .
.
1 80
Fisch et al.
alanine. Prior to the loading, the phenylaIanine concentration in the mothers' serum was lower than in their breast milk (Tables I I and I I I ) . After the loading the phenylalanine concentration increased in the mothers' serum severaltimes more than it did in their breast milk: the phenylalanine concentration in the breast milk of Mother No. 1 had almost a 6-fold increase and of Mother No. 3 a 4-fold increase. It would appear, therefore, that even when the phenylalanine concentration is increased in the serum, there is some barrier to the proportional increase of phenylalanine concentration in breast milk. There was an increase in the concentration of phenylalanine and tyrosine derivatives in the urine of some newborn infants following the loading of the mothers, but there were no noticeable cha~ages in their serum phenylalanine and tyrosine levels 24 hours following the first loading. Evidently the infants received excessive amounts of phenylalanine and/or its derivatives through their mothers' breast milk. Presumably, homozygous phenylketonuric mothers excrete even larger amounts of phenylalanine through their breast milk than do heterozygous ones. It is known that phenylketonuric patients have an excessive amount of serum phenylalanine and a decreased amount of serum tyrosine and that, because of this excessive amount of phenylalanine, they have increased amounts of see-
The Journal o[ Pediatrics August 1967
ondary phenylalanine metabolites. The excessive amount of phenylalanine in the breast milk of the nursing phenylketonuric mother is the source of a constant phenylalanine load on her offspring, whether he is homozygous or heterozygous. This load may have an effect on postnatal growth and mental development, as we have already reported? For this reason, the question of whether or not untreated phenylketonuric mothers should nurse their offspring deserves serious consideration. The authors wish to thank the four mothers for their patient and understanding cooperation in this experiment. REFERENCES
1. Fisch, R. O., Walker, W. A., and Anderson, J. A.: Prenatal and postnatal developmental consequences of maternal phenylketonuria, Pediatrics 37: 979, 1966. 2. McCaman, M. W., and Robins, E.: Fluorimetric method for the determination of phenylalanine in serum, J. Lab. & Clin. Med. 59: 855, 1962. 3. Udenfriend, S., and Copper, J. R.: The chemical estimation of tyrosine and tyramine, J. Biol. Chem. 196: 227, 1952. 4. Szymanski, H. A.: Biomedicai applications of gas chromatography, New York, 1964, Plenum Press, Inc., p. 225. 5. Weissbach, H., King, W., Sjoerdsma, A., and Udenfriend, S.: Formation of indole 3-acetic acid and tryptomine in animals, J. Biol. Chem. 234: 81, 1959. 6. Anderson, J. A., Gravem, H., Ertel, R., and Fisch, R.: Identification of heterozygotes with phenylketonuria on basis of blood tyrosine respouses, J. PEmAT. 61: 603, 1962.
August, 1967 T h e J o u r n a l o[ P E D I A T R I C S
The effect of excess L-pbenylalanine on mothers and on their breast-fed infants Four lactating mothers--two who were heterozygous [or phenylketonuria and two nonheterozygous primiparas--were given oral doses o[ L-phenylalanine. The concentrations o[ phenylalanine and tyrosine in the mothers' and their in[ants' serum, as well as the concentrations o[ phenylalanine and its metabolites in the breast milk and in the mothers" and in[ants' urine, were measured. The heterozygous mothers exhibited higher concentrations o[ these substances than the nonheterozygous ones. Since the breast milk o[ untreated homozygous mothers would presumably contain more phenIyalanine and its metabolites than that o[ heterozygous ones, the question o[ whether or not they should nurse their in[ants deserves serious consideration.
Robert O. Fiseh, M.D., "x"Robert Jenness, Ph.D., Doris Doeden, M.S., and John A. Anderson, M.D., Ph.D. MINNEAPOLIS~
MINN.
I N r R A U r E R I N ~ growth retardation has been observed in two infants of an untreated phenylketonuric mother? One was homozygous and the other was heterozygous for phenylketonuria. The infants were breast fed for eleven and nine months, respectively. During these periods both infants exhibited significant growth retardation. This study was undertaken to determine whether or not the infants' growth retardation may have been associated with an excessive intake of phenylalanine and its metabolites from the breast milk.
SUBJECTS AND METHODS Four mothers and their breast-fed newborn infants were studied. Two of the ~Address: Box 323, Mayo Memorial Hospital, University o1 Minnesota, Minneapolis, Minn. 55455.
VoI. 71, No. 2, pp. 176-180
mothers were known heterozygotes for phenylketonuria (Nos. 1 and 3) and had borne one and two phenylketonuric children, respectively. Each of the two normal mothers (Nos. 2 and 4) was a primipara. The data pertinent to these mothers and their infants are presented in Table I. The two infants of the known heterozygous mothers were not phenylketonuric. The mothers and their infants were studied in the following manner: Mothers 1 and 2 were given a total of 800 mg. of L-phenylalanine per kilogram of body weight in five separate doses over a period of 24 hours. The L-phenylalanine was given at the following times and in the following doses: 8:00 A.Z~., 100 mg. per kilogram; 8:30 A.M., 100 mg. per kilogram; 2:00 P.~., 200 mg. per kilogram; 8:00 P.g., 200 mg. per
Volume 71 Number 2
Effect of excess L-phenylalanine
177
Table I. D a t a on the mothers and their offspring
Mothers
Age (yr.)
No. 1~ 33 No. 2 23 No. 3~ 39 No. 4 21 '~Heterozygotes.
Weight ] Baby's (Kg.) sex 85.9 67.7 68.2 70.9
Boy Girl Girl Boy
Birthdate
Weight (Gm.)
Date of L-phenylaIanine loading
Total amount of L-phenylalanine (mg./ Kg./ day)
1/ 2/65 11/23/65 1/20/66 11/25/65
5,280 3,750 4,370 3,440
1/11/65 11/27/65 1/27/66 11/29/65
800 8OO 600 6O0
kilogram; and at 12:00 P.M., 200 mg. per kilogram. Mothers 3 and 4 were given a total of 600 mg. of L-phenylalanine per kilogram of body weight in four separate doses over a period of 24 hours. The L-phenylalanine was given at the following times and in the following doses: 8:00 *.M., 100 mg. per kilogram; 8:30 A.M., 100 rag. per kilogram; 4:00 P.M., 200 rag. per kilogram; and at 12:00 p.M., 200 mg. per kilogram. The mothers fasted from 10:00 P.M. until 5 hours following the first L-phenylalanine loading, and drank only water during this period. For the rest of the study day they ate regular meals. At 12 hours prior to and at 12 and 24 hours after the first loading, 10 ml. of breast milk were collected. After the infant had been nursed, the breasts were emptied. The milk samples were collected prior to the next breast feeding. The blood samples for serum phenylalanine 2 and tyrosine 3 determinations were obtained immediately prior to loading, during fasting, and at 1, 2, 3, 4, 5, and 24 hours after the first loading. Urine was collected 24 hours before loading and for periods of 5 and 19 hours following the first loading. Blood samples were obtained from the infants for determinations of serum phenylalanine 2 and tyrosine ~ immediately before and 24 hours after the first loading. Urine specimens were collected from the infants on the preloading day and about 10 and 24 hours after the first loading. T h e infants were breast fed approximately every 3 to 4 hours through the day. T h e studies were carried out on the nonheterozygous control mothers and their in-
rants in the University of Minnesota Hospitals and on the heterozygous mothers and their infants in their homes. The phenylalanine content of the breast milk was determined according to a modified form of the fluorimetric method of McCaman and Robins. 2 One volume of milk was mixed with one volume of 0.6N trichloroacetic acid (TCA) and centrifuged to remove the protein and the layer of fat that floats to the top; then, 0.1 ml. of supernatant fluid (equivalent to 0.05 ml. of milk) was used for analysis. The analysis was done on two 0.1 ml. aliquots to which 0.1 ml. of 0.3N T C A was added. The internal control standard contained 0.1 rot. of 0.3N T C A and 0.025 /zm of phenylalanine. The tenfold quantity of reagent specified in the original McCamanand-Robins method was used. The incubation was done at 90.0 ~ C. for fifteen minutes. The tubes were read in a Coleman Model 12C Photofluorimeter with filter B-1 PC-1. The breast milk was further analyzed for the p-hydroxyphenolic acid derivatives of tyrosine (p-hydroxyphenylproprionic acid, phydroxyphenylacetic acid, and p-hydroxybenzoic acid) and for o-hydroxyphenylacetic acid by gas claromatography. 4 Assays were done on the filtrate following the precipitation of the protein from 3.5 ml. of breast milk by adding 1.0 ml. of 10 per cent zinc sulfate and 0.5 ml. of 1N N a O H . The internal standards for comparison were prepared by adding milk to known standards. T h e indole-3-acetic acid in the milk and urine was measured. ~ The urine specimens from mothers and infants were measured for the p-hydroxyphenolic acid derivatives of tyrosine (p-hydroxyphenylproprionic acid, p-hydroxyphenylacetic acid, p-hydroxybenzoic acid), for o-hydroxyphenyl-
178
F i s c h e t al.
acetic acid, and for indole-3-acetic acid. 4 The serum tyrosine response to the oral L-phenylalanine load was used as a measure of heterozygosity for phenylketonuria. G RESULTS
AND DISCUSSION
The serum phenylalanine concentration in the heterozygous mothers (Nos. 1 and 3) 2 hours after loading was greater than in the nonheterozygous control mother's (Nos. 2 and 4). At 3, 4, and 5 hours following loading, the differences between the serum phenylalanine concentration in the heterozygous and control mothers were even greater. The serum phenylalanine in one heterozygous mother (No. 1) at 24 hours following loading ~" was 66.7 my. per cent (4.04/~mj~ per 1 ml.) and in the control mother (No. 2), 3.9 rag. per cent (0.24 /~m per milliliter). The serum phenylalanine in the other heterozygous mother (No. 3) at 24 hours following loading was 13.2 rag. per cent (0.80 t*m per milliliter) and in the control mother (No. 4), 0.9 mg. per cent (0.05 ~m per milliliter). Following the increase of phenylalanine in the mothers' serum, there was an increase of phenylalanine in the breast milk of three of the mothers. There was no increase of phenylalanine in the breast milk of one mother (No. 4) whose serum phenylalanine level had increased the least (Table III). The initial concentration of phenylalanine in the breast milk of the four mothers varied from 0.15 to 0.40 micromoles per milliliter. The derivatives of phenylalanine and tyrosine were detectable in the breast milk only following loading. The greatest increase was found with respect to the concentration of p-hydroxybenzoic acid in the breast milk of the heterozygous mothers (Nos. 1 and 3). It is of interest that one mother (No. 1) had headaches and vertigo at the end of the loading day. During the loading period the urine specimens of the four mothers contained signifi~It is significant that Mothers 1 and 2 received 800 my. per kilogram of body weight and that Mothers 3 and 4 received 600 my. of L-phenylalanine per kilogram of body weight. tOne micromole .~ 0.1652 my. of phenylalanine.
The Journal o[ Pediatrics August 1967
cantly increased amounts of p-hydroxyphenolic acids, and the urine specimens of three mothers (Nos. 1, 3, and 4) contained an increased amount of o-hydroxyphenylacetic acid. Indole-3-acetic acid, which was not found in measurable amounts in the milk before or following loading, was found to be noticeably increased in the urine of all four mothers following loading. The serum phenylalanine concentration in the infants was found to be within the normal range: it did not exceed 2 mg. per cent 24 hours following the first loading even though they had been routinely nursed by their mothers during this period. It should be noted, however, that the modified McCaman-and-Robins fluorimetric method cannot be used in our laboratory to measure serum phenylalanine in quantities smaller than 1 my. We can assume that the infants' serum concentrations of phenylalanine did not increase significantly during the testing period. On the other hand, the infants' tyrosine levels were measured and found to be normal prior to and 24 hours following the first loading of their mothers. There were no consistent changes in the concentration of phenylalanine and tyrosine metabolites in the urine specimens of the infants during the loading day. However, the urine specimens of the infants represent samples and not complete 24 hour collections. The purpose of giving excessive doses of L-phenylalanine to the four mothers was to simulate the metabolic circumstances of phenylketonuric mothers during lactation. It can be assumed that the metabolic changes in a lactating phenylketonuric mother would cause her breast milk to differ even more from a "normal" mother's breast milk. This assumption is supported by the fact that there was proportionately more phenylalanine and its metabolites in the breast milk of the two heterozygous mothers than in the breast milk of the nonheterozygous control m o t h e r s during the testing period. Mother No. 1 exhibited a 29-fold increase and Mother No. 3 exhibited a 12-fold increase of the serum phenylalanine level 24 hours following the first loading with L,phenyl-
Volume 71
Effect of excess L-phenylalanine
Number 2
179
Table II. Serum phenylalanine and tyrosine values of the mothers Values
Mothers
Fasting* (rag. per cent)~,
1 hr.'~ (rag. per cent)
2 hr, (mg. per cent)
3 hr, (mg. per cent)
4 hr. (rag. per cent)
5 hr. (mg. per cent)
24 hr. (rag. per cent)
2.3 (0.14) 1.3 (0.07)
31.4 (1.90) 2.5 (0.14)
45.8 (2.77) 2.1 (0.12)
44.0 (2.66) 1.2 (0.07)
37.0 (2.24) 1.6 (0.09)
29.7 (1.80) 1.1 (0.06)
66.7 (4.04) 5.8 (0.32)
No. I Phenylalanine Tyrosine
No. 2 Phenylalanine Tyrosine
<
15.3
19.7
20.6
9.2
6.0
3.9
<(0.06)
1.0
(0.93)
(1.19)
(1.25)
(0.56)
(0.36)
(0.24)
1.3 (0.07)
2.7 (0.15)
2.4 (0.13)
3.7 (0.20)
3.7 (0.20)
2.9 (0.16)
3.7 (0.20)
1.1 (0.07) 1.5 (0.08)
16.6 (1.00) 1.7 (0.09)
26.1 (1.58) 2.2 (0.12)
25.0 (1.51) 2.3 (0.13)
20.8 (1.26) 2.2 (o.12)
17.5 (1.06) 2.2 (0.12)
13.2 (0.80) 3.3 (0.18)
No. 3 Phenylalanine Tyrosine
No. 4 Phenylalanine
< 1.0 9.8 16.0 11.9 9.5 4.5 < 1.0 <(0.06) (0.59) (0.97) (0.72) (0.57) (0.27) <(0.06) Tyrosine 1.5 3.4 4.0 7.2 5.9 5.6 3.2 (0.08) (0.19) (0 22) (0.40) (0.32) (0.31) (0.18) *Fasting values represent those obtained on the days before loading took place, i'The number of hours is computed from the time of the first loading. ++Serum phenylalanine and tyrosine values are given in milligramsper cent and (in parentheses) in micromolesper milliliter.
Table III. Concentrations of phenylalanine, p-hydroxyphenolic acids (p-hydroxyphenylproprionic acid, p-hydroxyphenyl acetic acid, p-hydroxybenzoic acid), o-hydroxyphenylacetic acid, and indole-3-acetic acid in the breast milk of the mothers Concentrations (micromoles per milliliter) Second day
2 Mothers
First day
Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.40 --.
hours alter I first loading [ g or 10 P.M.
Third day
No. I 0.48 1.66 0.06 .
.
1.60 0.07 0.07
2.28 1.95 --
0.38 < 0.30 < 0.30 .
0.30 < 0.30 < 0.30
0.47 0.40 --
0.66 < 0.20 0.20
O.13 < 0.30 < 0.30 .
0.05 < 0.20 < 0.20
.
No. 2 Phenylalanlne p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.29 --.
0.22 < 0.30 < 0.30 .
.
No. 3 Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphenylacetic acid Indole-3-acetic acid
0.15 --.
0.24 1.45 -.
.
.
No. 4 Phenylalanine p-Hydroxyphenolic acids o-Hydroxyphe~ylacetic acid Indole-3-acetic acid
O. 15 --.
O. 19 < 0.30 < 0.30 .
.
1 80
Fisch et al.
alanine. Prior to the loading, the phenylaIanine concentration in the mothers' serum was lower than in their breast milk (Tables I I and I I I ) . After the loading the phenylalanine concentration increased in the mothers' serum severaltimes more than it did in their breast milk: the phenylalanine concentration in the breast milk of Mother No. 1 had almost a 6-fold increase and of Mother No. 3 a 4-fold increase. It would appear, therefore, that even when the phenylalanine concentration is increased in the serum, there is some barrier to the proportional increase of phenylalanine concentration in breast milk. There was an increase in the concentration of phenylalanine and tyrosine derivatives in the urine of some newborn infants following the loading of the mothers, but there were no noticeable cha~ages in their serum phenylalanine and tyrosine levels 24 hours following the first loading. Evidently the infants received excessive amounts of phenylalanine and/or its derivatives through their mothers' breast milk. Presumably, homozygous phenylketonuric mothers excrete even larger amounts of phenylalanine through their breast milk than do heterozygous ones. It is known that phenylketonuric patients have an excessive amount of serum phenylalanine and a decreased amount of serum tyrosine and that, because of this excessive amount of phenylalanine, they have increased amounts of see-
The Journal o[ Pediatrics August 1967
ondary phenylalanine metabolites. The excessive amount of phenylalanine in the breast milk of the nursing phenylketonuric mother is the source of a constant phenylalanine load on her offspring, whether he is homozygous or heterozygous. This load may have an effect on postnatal growth and mental development, as we have already reported? For this reason, the question of whether or not untreated phenylketonuric mothers should nurse their offspring deserves serious consideration. The authors wish to thank the four mothers for their patient and understanding cooperation in this experiment. REFERENCES
1. Fisch, R. O., Walker, W. A., and Anderson, J. A.: Prenatal and postnatal developmental consequences of maternal phenylketonuria, Pediatrics 37: 979, 1966. 2. McCaman, M. W., and Robins, E.: Fluorimetric method for the determination of phenylalanine in serum, J. Lab. & Clin. Med. 59: 855, 1962. 3. Udenfriend, S., and Copper, J. R.: The chemical estimation of tyrosine and tyramine, J. Biol. Chem. 196: 227, 1952. 4. Szymanski, H. A.: Biomedicai applications of gas chromatography, New York, 1964, Plenum Press, Inc., p. 225. 5. Weissbach, H., King, W., Sjoerdsma, A., and Udenfriend, S.: Formation of indole 3-acetic acid and tryptomine in animals, J. Biol. Chem. 234: 81, 1959. 6. Anderson, J. A., Gravem, H., Ertel, R., and Fisch, R.: Identification of heterozygotes with phenylketonuria on basis of blood tyrosine respouses, J. PEmAT. 61: 603, 1962.