THE FREE AMINO ACIDS OF HUMAN BLOOD PLASMA

BY WILLIAM H. STEIN AND STANFORD MOORE

(Prom the Laboratories of The Rockefeller Institute for Medical Research, New York 91, New York)

(Received for publication, July 6, 1954)

The presence of amino acid nitrogen in the non-protein nitrogen fraction of blood plasma was first established in 1912 by Van Slyke and Meyer (1). Chemical, microbiological, and chromatographic techniques have subse- quently been employed to determine many of the individual amino acids which contribute to the total or-amino nitrogen as currently measured by the ninhydrin-CO2 method of Hamilton and Van Slyke (2). In the studies reported in this communication, chromatographic methods in which col- umns of Dowex 50-X4 are employed (3) have been applied to this problem (Fig. 1). It has been possible to identify with a high degree of probability twenty-eight ninhydrin-positive substances in protein-free plasma and to determine most of them quantitatively. The results, taken in conjunc- tion with the values for glutamine obtained by Archibald (4) and Hamilton (5), account for nearly 100 per cent of the total a-amino N of postabsorptive plasma.

EXPERIMENTAL

Unless otherwise specified, 25 or 50 ml. samples of blood were drawn from the cubital vein before breakfast from normal adult males. Each 25 ml. portion was delivered into a 50 ml. centrifuge tube containing 5 mg. of heparin previously dried as a film on the walls. The freshly drawn blood ,:as centrifuged, and the plasma was withdrawn and centrifuged again in a on-heparinized tube. The samples were deproteinized immediately or lored at -20” in plastic vials. It has been found in the present study, ‘wever, that changes in the concentrations of some of the amino acids

! cur if the whole plasma is stored. Che chromatographic analyses were carried out by the Dowex 50-X4

~,~hod (3) with columns initially equilibrated at pH 2.2 in order to obtain ; “timal separation of taurine and urea.

Removal of Proteins-Equilibrium dialysis, ultrafiltration, and precipi- .ion with picric acid were investigated as methods for the deproteiniza- ‘.i of the plasma. For each of the dialysis and precipitation experiments,

1, 10 ml. aliquots of a single sample of plasma were taken. To one ali- ,here were added about 2.5 mg. of a synthetic mixture of amino acids

!, 1.;; ml. of a 1: 1 dilution of the mixture used in testing the Dowex 50-X4

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916 AMINO ACIDS OF BLOOD PLASMA

method (3)). In the ultrafiltration experiments the procedure was sim- ilar, but the quantities were halved. After deproteinization, each pair of samples was analyzed on the ion exchange columns and the recoveries of the added amino acids were determined.

The equilibrium dialysis was carried out for 16 hours at 4” according to the procedure of Hamilton and Archibald (6). The dialysate was con- centrated to about 6 ml. on a rotary evaporator (7), and the entire sample was placed on the Dowex 50 column for analysis.

Ultrafiltration was carried out for 16 hours at 4” according to the pro- cedure of Prescott and Waelsch (S).l After measurement of the volume, the ultrafiltrate was adjusted to pH 2 for chromatography.

Deproteinization with picric acid was effected in general as described by Hamilton and Van Slyke (2). An additional step has been introduced, however, to remove the excess picric acid. For this purpose, a bed of an ion exchange resin is employed under conditions which are known to per- mit the amino acids to pass through quantitatively. The resin used for this purpose is the strongly basic anion exchanger Dowex 2-X8 in the chlo- ride form.2 The fines were first removed by settling the resin in water, and the product was thoroughly washed with N HCl. The resin, suspended in water, was poured into a 2 X 20 cm. chromatograph tube, allowed to settle for 5 minutes under gravity, and packed under a pressure of 2 to 5 cm. of mercury. Sufficient resin was used to give a bed 2 X 2 cm. A circle of soft filter paper was placed on the surface of the resin and the bed was washed with 15 ml. of N HCl, followed by water until the effluent was neutral. If not used immediately, the column was stored with a few mm. of water over the resin surface.

The deproteinization of 10 ml. of plasma was carried out in a glass-stop- pered flask by the addition of 50 ml. of 1 per cent picric acid solution. After a few seconds of shaking, the suspension was centrifuged for 10 minutes. The supernatant liquid was poured off completely and exactly 50 ml. (cor- responding to 8.34 ml. of the original plasma) were passed through the Dowex 2 bed under a pressure of 1 to 4 cm. of mercury. The resin column removed any traces of precipitate remaining in the supernatant solution.

1 It is a pleasure to acknowledge the cooperation of Dr. H. Waelsch, who placed some of his ultrafiltration equipment at our disposal.

2 Dowex 2-X8 (200 to 400 mesh) from Technical Service and Development, The Dow Chemical Company, Midland, Michigan. Quantitative tests on columns of the chloride form of the resin have shown that 0.02 N HCl elutes all of the amino acids without retardation, with the exception of tryptophan, which is held back slightly as a result of its affinity for the aromatic groups of the resin phase. Moreover, the amino acid is not fully stable on Dowex 2, the recovery being only about 85 per cent. The fact that the amino acid is also labile on Dowex 50-X4 columns (3) precludes the determination of tryptophan by the present method. The small amount of Dowex 2 used to remove picric acid in each experiment was discarded after use.

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W. H. STEIN AND S. MOORE 917

The walls of the chromatograph tube and the resin bed were washed with five 3 ml. samples of 0.02 N HCl. The clear, colorless effluent and wash- ings were concentrated on a rotary evaporator to a volume of 5 ml. and a few mg. of Celite (analytical filter aid) were added. The suspension was filtered by gravity through a paper previously washed with N HCl and water, and the filtrate and washings were concentrated to a volume of about 1 ml. The concentrate was washed into a plastic vial, the final vol- ume being 4 to 5 ml., at which point it frequently was convenient to store the tube at -20’ overnight. The following day, the solution was brought to pH 7 to 8 (Hydrion paper) by the dropwise addition of N NaOH and allowed to stand at room temperature for 4 hours, conditions which were shown by analysis for -SH groups to convert cysteine (which cannot be determined on Dowex 50-X4) to cystine. The sample was adjusted to pH 2 by the addition of N KC1 and the solution was stored at -20” until an- alyzed.

RESULTS AND DISCUSSION

Deproteinixation-Removal of protein by precipitation with picric acid proved to be the most satisfactory of the three methods tested. Difficulty was experienced in obtaining reproducible recoveries of the individual amino acids added to plasma when either equilibrium dialysis or ultrafil- tration was employed. Both processes are relatively slow, requiring about 16 hours at 4”, and apparently during this interval secondary changes can take place. In the picric acid procedure, on the other hand, removal of proteins from the plasma can be accomplished within 30 minutes after the blood is drawn. The recoveries of added amino acids were consistently within 10 per cent of theory. Small losses of tryptophan and also of hip- puric acid are incurred, however, when Dowex 2 is used for the removal of picric acid.

Interpretation of Efluent Curves-A typical effluent curve obtained from a picric acid filtrate of normal postabsorptive plasma is shown in Fig. 1. Identification of the peaks on the effluent curve was aided by paper chroma- tography. In a duplicate experiment, alternate fractions were analyzed by the ninhydrin method to locate the peaks. Aliquots from the remain- ing tubes were desalted on small beds of ion exchange resins and chro- matographed on paper in exactly the manner already described in the work with urine (9). Aspartic acid, ethanolamine, and the isomeric methyl- histidines, because of their extremely low concentrations, were the only substances in Fig. I which could not be subjected to this additional test. For the identification of ornithine, the specific color reaction of Chinard (10) was applied in the manner already outlined (11).

Qualitatively, t~he picture in Fig. 1 is in general agreement with many of the results obtained previously by microbiological assays, chemical de-

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Glutamine tAsparaglne--” ?POline Threonine

Urea Aspartic lSer/ne

acid , Taumne 1

I I f

, A. d\ ml. 50 100 150 200 250

“-30” +-----50”- pH2.2 +I+ Influent 0.2 N pH 3.1

02

0.1

300 350 400 450 500 550 -,.o Gradually increasinq PH

600 650 700 750 ̂800 850

and [Na+] (OZN pH 3.1 L1.4~ pH 5.1) 75” ----+I

I FIG. 1. The ninhydrin-positive components of protein-free human blood plasma

obtained from adult males in the postabsorptive state. Chromatography of 8.34 ml. of plasma was carried out on a 150 X 0.9 cm. column of Dowex 50-X4. The terms in brackets refer to substances present in quantities too small to permit paper chro- matography to be used as an additional method of identification. The dash curve in the glycine plus citrulline peak represents citrulline. The open circles under the ammonia peak represent the amount of ninhydrin-positive material remain- ing in the fractions after removal of ammonia. The observed NH1 is predomi- nantly derived from the decomposition of glutamine during the experiment. The proline and cystine (as half cystine) peaks have been corrected for their relatively low color yields to bring the concentrations (in leucine equivalents) into approxi- mately correct molar proportions relative to those of the other amino acids. The urea peak would require multiplication by about 30 to bring it similarly into scale.

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W. H. STEIN AND S. MOORE 919

terminations, and paper chromatography. The presence in plasma of taurine and ar-amino-n-butyric acid has already been revealed by paper chromatography (12, 13). The two compounds have been found in rel- atively constant amounts in all samples examined by ion exchange chro- matography. The existence of asparagine in plasma was detected by Agren and Nilsson (14) by paper chromatography. The concentration of the compound was determined by Barry (15) employing the Dowex 50-X8 procedure and has been measured in the present study by rechro- matography of a sample from the glutamine-asparagine peak (3). The presence of asparagine is not unexpected in view of its occurrence in urine (9)s and in tissue extracts (16). The concentration of asparagine in plasma, however, is less than one-tenth that of glutamine. Thz presence of orni- thine in plasma has not been established heretofore. Agren and Nilsson (14) observed a spot corresponding to ornithine on paper chromatograms of plasma desalted by the procedure of Consden, Gordon, and Martin (17), but this observation was not conclusive proof for the existence of ornithine in plasma, since it has been shown that this amino acid can arise from argi- nine during the electrolytic desalting procedure (11).

The separat,ion of citrulline from glycine requires a separate experiment in the Dowex 50-X4 method. Archibald (18) demonstrated the presence of citrulline in blood plasma, and its occurrence has been confirmed by rechroniatography of a sample from the glycine zone (3). The quantity is indicated by the dotted curve in Fig. 1.

The ammonia peak is primarily an artifact arising as a result of the de- composition of glutamine during the deproteinization and storage of the sample prior to chromatography. The shoulder on the peak comes from ammonia generated from glutamine during the chromatographic process. Identification of the isomeric methylhistidines is not certain because of their very low concentration, but they are the only amino acids known to emerge at the given positions and both are present in relatively large amounts in human urine (9, 19).

The almost complete absence of aspartic acid in plasma and the low concentration of glutamic acid, some of which doubtless results from the decomposition of glutamine, are noteworthy. These two amino acids are absent from freshly voided urine (9), but are found in relatively high concentrations in the extracts of most tissues (16). Even after the in- gestion of 50 gm. of casein, the concentration of free aspartic acid is not significantly greater than that shown in Fig. 1. Thus, it is predominantly

3 In previous studies with normal urine (9), it was not appreciated that the serine + asparagine peak also contained some glutamine, and, therefore, the asparagine values are too high. Experiments performed in connection with the present work have indicated that the asparagine output by a normal adult male is of the order of 20 mg. per day.

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920 AMINO ACIDS OF BLOOD PLASMA

the amides of both of these amino acids that are brought to the tissues by the blood stream. These facts may have a bearing on the nausea and vomiting frequently observed to accompany the intravenous administra- tion of protein hydrolysates from which aspartic and glutamic acids have not been removed.

Hydroxyproline has not been found in chromatograms of plasma, indi- cating that its concentration must be less than 0.2 mg. per cent.

The plasma pattern in Fig. 1 is very different from the curve obtained with urine (9). In urine, taurine is a major component, while the quanti- ties of proline and valine are very low; glycine exceeds alanine in amount and histidine, I-methylhistidine, and 3-methylhistidine are present to a far greater extent than is lysine. In plasma, the situation is the converse. Moreover, there are only two small unidentified peaks in Fig. 1, one be- tween urea and aspartic acid, the other to the right of leucine, whereas a number of unknown substances render the urine picture less clear-cut.

Quantitative Results-The quantitative data obtained from the analyses of the plasma of five normal adult males are presented in Table I, along with values from the literature. There do not appear to have been pre- vious quantitative determinations of aminobutyric acid, taurine, orni- thine, and the I- and 3-methylhistidines. Since glutamine and tryptophan are not recovered quantitatively from the Dowex 50 columns, the average figures for these amino acids in Table I have been taken from the work of Archibald (4) and Hier and Bergeim (20). When the tryptophan peak in Fig. 1 is corrected for losses, a value similar to the microbiological estimates is obtained. The value for citrulline is that of Archibald (18), with which two chromatographic determinations were in agreement. The glycine values have been corrected for the presence of 0.50 mg. per cent of citrulline in the glycine peak. In almost every instance, agreement can be found between the average results in Table I and one or more of the values from the literature. The variation in the individual values from the literature is frequently considerably greater than that found in the present studies.

The yield of total a-amino N which would be obtained in the ninhydrin- CO2 method from the individual compounds (taurine excepted) listed in Table I (4.02 mg. per cent) is in close agreement with the average value of 4.1 mg. per cent found by Hamilton and Van Slyke (2). This fact, coupled with the absence of major unknown peaks on the curve shown in Fig. 1, i.ndicates that substantially all of the amino acids of plasma have been ac- counted for.

The Dowex 50 chromatograms also provide a measure of the urea content of plasma. Since the color yield of urea in the ninhydrin procedure is only 3.14 per cent (35), the peak in Fig. 1 should be multiplied by about 30 to place it on a molar scale comparable to that of the amino acids. The

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TABLE I

Free Amino Acids of Human Blood Plasma

Postabsorptive plasma from normal adult males.

Amino acid

[Aspartic acid]- Asparagine Glutamic acid Glutamine Glycine Alanine Aminobutyric

acid Valine Leucine Isoleucine Serine Threonine Cysteine +

cystine Methionine

Taurine Proline Phenylalanine Tyrosine Tryptophan Histidine [l-Methyl-

histidine]? [3-Methyl-

histidinelt Ornithine Lysine Arginine Citrulline

Mg. amino acid per 100 ml. plasma

Sub- Sub- Sub- Sub- Sub- Aver- ect A jectB ject C ject D jectE age

0.03 0.01 0.03 0.02 0.070.03

Values from literature*

0.65 0.55 0.540.58 0.50 0.78 0.43 0.65 1.150.70

8.301 1.52 1.51 1.61 1.73 1.341.54 3.01 3.73 3.51 3.25 3.533.41 0.33 0.30 0.30 0.22 0.350.30

0.8(21) 1.0-I .4(15), present (14) 1.1(S), 3.5(22), 4.4(21) 5.8(22), 7.5(5), 8.8(23), 9.7(8) 1.7(24), 1.8(25), 1.1(21), 2.8(26) 3.4(24),4.0(25), 4.2(21), 3.7(27) Present (12)

2.94 2.37 2.84 2.56 3.712.88 2.9(20), 2.7(28), 3.4(21), 3.0(29) 1.53 1.42 1.65 1.53 2.301.69 2.0(20), 1.9(28), 2.4(21), 2.0(29) 0.76 0.69 0.87 0.87 1.280.89 1.7(20), 1.3(28), 1.8(21), 1.5(29) 1.05 1.20 1.08 1.25 1.011.12 1.2(29) 1.59 1.21 1.21 1.72 1.211.39 2.1(20), 1.7(28),3.1(21), 1.3(30) 1.09 1.30 1.20 1.25 1.081.18 1.5(28), 1.0(31), 2.0(29)

0.33 0.40 0.39 0.36 0.430.38

0.82 0.50 0.56 0.45 0.410.55 3.34 2.17 2.01 2.40 1.842.36 0.76 0.94 0.86 0.69 0.950.84 0.81 1.11 0.88 0.89 1.451.03

l.ll! 1.08 1.48 1.15 0.79 1.231.15 0.17 0.12 0.04 0.11

0.04 0.13 0.07 0.10 0.070.08

0.52(28), 0.9(21), 0.85(32), 0.29

(29) Present (13) 2.8(21), 2.9(30) 1.4(20), 1.0(33), 1.9(13), 1.6(21) 1.5(20), 1.0(28), 1.3(21), 1.0(30) 1.1(34), 1.3(28), 1.7(21) 1.4(20), 2.1(21), 1.4(30), 1.0(29)

0.76 0.62 0.73 0.80 0.700.72 3.02 2.51 2.77 2.55 2.732.72 1.93 1.64 1.34 1.22 1.431.51

0.5011

3.0(20), 2.2(28), 2.6(30), 2.8(29) 2.3(20), 1.6(28), 1.5(30), 2.5(29) 0.50(18), present(14)

Total.. . . . . . 37.07 Calculated yield of cu-NH,-N in ninhydrin-

COzmethod........................... 4.02 Average cu-NHs-N found by Hamilton and

Van Slyke (2) . . . . . . 4.1

* The values in reference (26, 27,29) were obtained with serum. t These compounds were present in too low a concentration to permit unequivocal

identification. $ Archibald (4). $ Hier and Bergeim (20). ]I Archibald (18) ; chromatographic determinations on two plasmas, 0.45 and 0.48

mg. per cent of citrulline.

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922 AMINO ACIDS OF BLOOD PLASMA

plasma urea values for the individuals referred to in Table I were 27, 42, 31, 28, and 30 mg. per cent, all within the normal range. Approximate ethanolamine values of from 0.05 to 0.20 mg. per cent were also obtained in these studies.

In one case, an opportunity was afforded to compare the values obtained with plasma stored for 7 months at -20’ with those secured when the plasma was deproteinized and analyzed immediately. With the excep- tion of aspartic acid, glutamic acid, and cystine, the values for all of the amino acids in the fresh and the stored frozen plasma agreed within the experimental error. Upon storage, there was a slight increase in the quan- tity of aspartic acid and a large increase in the amount of glutamic acid, both doubtless a result of the decomposition of the respective amides. No cystine was found in the stored plasma. If, as seems likely (Fujita and Numata (36)), cysteine rather than cystine is present in blood and tissues, the oxidative formation of intermolecular bridges involving cysteine and the --SH groups of proteins would explain the absence of cystine in the stored plasma. In freshly deproteinized plasma, on the other hand, cys- teine-protein linkages are not given an opportunity to form, and oxidation by air of the protein-free filtrates leads only to the formation of cystine. Irrespective of the mechanism, the fact that the apparent cystine or cys- teine content decreases with time emphasizes the,need for prompt depro- teinization of plasma whenever this amino acid is to be determined.

Conjugated Amino Acids-The absence of major quantities of conjugated amino acids in plasma is well established. In recent experiments, Christen- sen (37) and Dent and Schilling (38) found only a very slight rise in total a-amino N after acid hydrolysis of the deproteinized plasma of the dog. The results of studies showing in detail the effect upon the quantity of each amino acid brought about by the hydrolysis of protein-free plasma are given in Table II. For a fasting subject (E), hydrolysis produces no significant changes in the concentration of most of the amino acids. Only aspartic acid, glutamic acid, glycine, and possibly alanine, serine, and threonine are increased in amount by hydrolysis. Most of the aspartic acid arises from asparagine, and the glutamic acid formed could all come from glutamine. There is evidence for the existence of a small quantity of conjugated glycine in plasma, probably in the form of hippuric acid.

In the absence of conjugated forms of serine and threonine, the values for these two amino acids might be expected to decrease as a result of de- composition during hydrolysis. Actually there is a slight rise, indicating traces of bound forms. There is unequivocal evidence for the decomposi- tion or alteration during hydrolysis of cystine, methionine, proline, and tyrosine. Cystine seems to be converted largely to cysteic acid, and methi- onine partially to the sulfoxide. The fact that the amount of material in

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W. H. STEIN AND S. MOORE 923

TABLE II

Amino Acid Content of Protein-Free Plasma after Acid Hydrolysis

The hydrolysis was carried out at 110” for 16 hours in 10 ml. of 6 N HCI. The samples corresponded to the concentrated protein-free filtrate from 8.34 ml. of plasma. For the “post-protein” sample, blood was drawn 2 hours after the inges- tion of 50 gm. of casein as Protinal (a 2:l casein-carbohydrate mixture, The Na- tional Drug Company, Philadelphia).

Mg. amino acid per 100 ml. plasma

Amino acid I Total after hydrolysis

Aspartic acid*. ..... Glutamic “ ....... Glycinet .......... Alanine. ........... Aminobutyric acid. Valine. ............. Leucine ............ Isoleucine .......... Serine. ............. Threonine .......... Cysteine + cystine. Cysteic acidi ....... Methioninei. ....... Taurine ............ Proline. ............ Phenylalanine ...... Tyrosine ........... Histidine. .......... Ornithine. ......... Lysine. ............ Arginine ...........

Subject E, fasting

Subject F? Subject E, post-protern fasting

.......... 0.73 1.19 0.66 1.07

.......... 9.48 14.15 8.33 12.35

.......... 1.83 1.84 0.28 0.27

.......... 3.62 4.80 0.09 0.16

.......... 0.40 0.19 0.05 -0.05

.......... 3.72 6.50 0.01 0.12

.......... 2.23 5.10 -0.07 0.02

.......... 1.37 2.98 0.09 -0.05

.......... 1.06 1.81 0.05 0.01

.......... 1.26 2.29 0.05 0.03

.......... <0.03 <0.03 -1.08 -1.49

.......... 1.98 <0.03 1.98 <0.03

.......... 0.08 0.45 -0.35 -0.34

.......... 0.42 0.36 0.01 -0.07

.......... 1.69 5.53 -0.15 -0.20

.......... 0.97 1.50 0.02 -0.05

.......... 0.98 2.31 -0.47 -0.72

.......... 1.17 1.72 -0.06 0.12

.......... 0.71 1.34 0.01 0.12

.......... 2.75 5.82 0.02 0.32

.......... 1.43 2.42 0.00 0.12

Increase after hydrolysis

Subject r, post-protem

* The asparagine content of the plasma before hydrolysis was 0.54 mg. per cent for Subject E and 0.89 mg. per cent for Subject F.

t Not corrected for the presence of citrulline. $ The 1.08 mg. per cent of cystine present before hydrolysis in the plasma of Sub-

ject E could give rise to a maximum of 1.51 mg. per cent of cysteic acid. The plasma of Subject F stood for 4 months in the deep freeze before being deproteinized and analyzed, and hence contained no free cystine (see the text).

$ In the chromatograms of the hydrolysates, two small peaks appeared ahead of aspartic acid at the positions of the isomeric methionine sulfoxides (3). Assuming that these peaks are the sulfoxides, virtually all of the methionine present before hydrolysis can be accounted for in the hydrolysates as methionine + methionine sulfoxides.

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924 AMINO ACIDS OF BLOOD PLASMA

the aminobutyric acid peak is essentially unchanged by hydrolysis elim- inates the possibility that the peak might contain glutathione, which also emerges at this position (3). The data on Subject E in Table II confirm the view that, if conjugated amino acids, including peptides, are present in the venous blood of a fasting individual, the amount is extremely small.

A consideration of the data obtained with Subject F (Table II) shows that, even after a meal rich in protein, major quantities of conjugated amino acids do not appear in the plasma. The rise in free amino acids in the plasma of this individual after the protein meal has already been reported (cf. (39), Table VI, normal). There is some evidence for the presence of a detectable amount of conjugated lysine, and, as is to be expected, the con- centration of asparagine and glutamine rises after a meal. Otherwise, the picture is similar to that observed with the fasting subject.

The possibility still remains that certain forms of conjugated amino acids of unknown nature might exist in plasma and be removed during the deproteinization procedure, either by being bound to the protein-pi&c acid precipitate or to the Dowex 2 column. Simple peptides and most simple conjugates now known to exist in tissues or urine would be expected to survive the picric acid deproteinization procedure.

Whole Blood-In one instance, the amino acid composition of whole blood was also determined. For this purpose, 5 ml. of blood were laked with 5 ml. of water prior to deproteinization. From the hematocrit and the amino acid composition of the plasma, the amino acid composition of the formed elements of the blood was calculated. The cells were charac- terized by a high content of glutathione (about 50 mg. per 100 ml. of cells), comparable to that found in tissues. Relative to plasma, the cells con- tained more taurine, aspartic and glutamic acids, glycine, and ornithine, while the amounts of valine and cystine were less. For the remaining amino acids, the concentrations in the cells and the plasma were quite sim- ilar.

Additional Analyses-Examinations have been made of the concentra- tions of amino acids in the plasma of fasting patients suffering from a num- ber of diseases known or suspected to be associated with aberrations in amino acid metabolism. The results of investigations of patients with Wilson’s disease have already been reported (39). In a single cystinuric patient, the concentrations of cystine (0.54 mg. per cent), arginine (0.89 mg. per cent), and alanine (2.58 mg. per cent) were slightly below normal, the concentration of glycine was elevated (2.56 mg. per cent), while the levels of all the other amino acids, including lysine, ornithine, and citrulline, were in the normal range. It would appear, therefore, that the increased excretion of cystine, lysine, arginine, and ornithine (31, 40) by this cys- tinuric was not a reflection of high blood levels of these amino acids.

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W. H. STEIN AND S. MOORE 925

In a case of phenylpyruvic oligophrenia, the concentration of phenylala- nine in the plasma was 23.5 mg. per cent and the urinary excretion was 334 mg. per day, markedly high values in agreement with previous find- ings (33, 41-43). All of the other amino acids in the blood and urine were at or near the normal range. In two patients with schizophrenia, one with cirrhosis of the liver, one with tuberculosis, and one with muscular dys- trophy, the levels of the amino acids in both the plasma and the urine were in or near the normal range.4

The authors wish to acknowledge the technical assistance of Mrs. Ger- trude C. Carey, Miss Renate Mikk, and Miss Joyce F. Scheer in the per- formance of these experiments.

SUMMARY

Deproteinized human blood plasma contains twenty-eight ninhydrin- positive compounds identifiable by chromatography on columns of Dowex 50-X4. The procedure provides quantitative determinations of all of the constituents, except glutamine. The results, taken in conjunction with the previous values for glutamine obtained by Hamilton and Archibald, account for 95 to 100 per cent of the individual amino acids which con- tribute.to the total a-amino ?S of postabsorptive plasma (4.1 mg. per cent). In agreement with previous investigations, no detectable quantities of peptides have been found in venous plasma, either in the fasting state or after a protein meal.

BIBLIOGRAPHY

1. Van Slyke, D. D., and Meyer, G. M., J. Biol. Chem., 12, 399 (1912). 2. Hamilton, P. B., and Van Slyke, D. D., J. Biol. Chem., 160, 231 (1943). 3. Moore, S., and Stein, W. H., J. Biol. Chem., 211, 893 (1954). 4. Archibald, R. M., J. Biol. Chem., 154, 643 (1944). 5. Hamilton, P. B., J. Biol. Chem., 158, 397 (1945). 6. Hamilton, P. B., and Archibald, R. M., Ind. and Eng. Chem., Anal. Ed., 16,

136 (1944). 7. Craig, L. C., Gregory, J. D., and Hausmann, W., Anal. Chem., 22, 1462 (1950). 8. Prescott, B. A., and Waelsch, II., J. BioZ. Chem., 167, 855 (1947). 9. Stein, W. H., J. BioZ. Chem., 201, 45 (1953).

10. Chinard, F. P., J. BioZ. Chem., 199, 91 (1952). 11. Stein, W. H., and Moore, S., J. BioZ. Chem., 190, 103 (1951). 12. Dent, C. E., B&hem. J., 41, 240 (1947). 13. Dent, C. F:., Biochem. J., 43, 169 (1948).

4 The studies on patients were possible through the generous cooperation of Dr. G. A. Jervis of Letchworth Village, Professor Oskar Diethelm and Dr. D. C. Greaves of Cornell Uuiversity Medical College, Dr. R. E. Weston of the Montefiore Hospital, and Dr. A. G. Beam and Dr. J. G. Hirsch of the Hospital of the Rockefeller Institute.

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ens, C. A. L., Jr., Johnson, R. B., and Kemmerer, A. R., J. Clin. Invest., 31, 375 (1952).

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William H. Stein and Stanford MooreBLOOD PLASMA

THE FREE AMINO ACIDS OF HUMAN

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