722 The American Journal of Clinical Nutrition 39: MAY 1984, pp 722-729. Printed in USA© 1984 American Society for Clinical Nutrition

Diurnal variations in plasma concentrations ofbasic and neutral amino acids and in red cellconcentrations of aspartate and glutamate:effects of dietary protein intake1-3Timothy JMaher,4 Bruce S Glaeser,5 and Richardf Wurtman

ABSTRACT The effects of dietary protein content on diurnal variations in plasma concen-trations of neutral and basic amino acids, and on red blood cell levels of acidic amino acids,were studied in seven normal humans. The subjects consumed, on three consecutive 3-dayperiods, diets containing 0, 75, or 150 g of egg protein per day; blood was collected at 4-hintervalson the 2nd and 3rd days of each diet. For each of the large neutral amino acids (LNAA;

isoleucine, leucine, tyrosine, phenylalanine, methionine, valine, and tryptophan) significant

correlations were observed between itsplasma levelsand the protein content of the diet; highestlevelswere noted after consumption of the l50-g protein diet, and lowest values after the 0-g

protein diet. For each LNAA, except tryptophan, “fed” values (ie, those at 3 PM and 7 PM) were

decreased relativeto “fasting” values (those at 3 AM and 7 AM) after consumption of the 0-gprotein-free diet, but increased after consumption of the 150-g protein diet. Threonine, serine,

and proline behaved like the LNAA: in contrast, glycine and alanine rose after protein-free mealsand fell with the high-protein diet. The basic amino acids, lysine, arginine, and histidine tendedto respond like the LNAA to variations in dietary protein content. Red blood cell concentrations

of glutamate tended to vary inversely with the protein content of the diet, while no relationshipwas noted between red blood cell aspartate and dietary protein content. Food-induced changes

in plasma LNAA have been found to affect brain levels of amino acids that are neurotransmitter

precursors, as well as the syntheses of the transmitters themselves. The present data raise thepossibility that dietary protein content might also influence brain levels of certain basic aminoacids and their products. Am iC/in Nuir l984;39:722-729.

KEY WORDS Amino acids, red blood cells, lysine, histidine, arginine, glutamate

Introduction

Considerable information is availableconcerning factors affecting plasma levels ofthe neutral amino acids, particularly theirchanges as a function of time of day (1-3)or after consumption of meals of varyingcomposition (3, 4). Moreover, abundant evi-dence has accumulated that these plasma

‘From the Laboratory of Neuroendocrine Regula-tion, Department of Nutrition and Food Science, Mas-sachusetts Institute of Technology, Cambridge, MA02139.

2 in part by grants from the NationalInstitutes of Health (AM-14228) and the National Aer-onautics and Space Administration (NAG 2-2 10). TJMis a Fellow of the Center for Brain Science and Metab-olism Charitable Trust. BSG stipend was generouslyprovided by the GD Searle Co.

changes can affect the brain, inasmuch as:1) the large neutral amino acids (LNAA) arewell transported into the brain (5); 2) therates at which each particular LNAA entersthe brain depend on a competition betweenit and the other LNAA (ie, on the ratio ofits plasma concentration to the sum of theothers) (5, 6); and 3) the changes in brainlevels of at least two such LNAA, tryptophan

3Address reprint requests to: Dr Richard J Wurt-man, Room E25-604, Massachusetts Institute of Tech-nology, Cambridge, MA 02139.

4Present address: Department of Pharmacology,Massachusetts College of Pharmacy and Allied HealthSciences, Boston, MA 02115. Present address: Re-search Department, Pharmaceutical Division, Ciba-Geigy Corp. Summit, NJ 07901.

Received August 2, 1983.Accepted for publication November 29, 1983.

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EFFECT OF DIETARY PROTEIN ON PLASMA AMINO ACIDS 723

and tyrosine, which can occur diurnally orpostprandially, affect the syntheses of mon-oaminergic brain neurotransmitters (7, 8).

Much less information is available con-cerning factors affecting circulating levels ofthe basic amino acids, lysine, histidine, andarginine, or ofthe acidic amino acids, aspar-tate and glutamate. Circulating aspartateand glutamate enter the brain’s extracellularfluid very poorly (5) causing detectable brainchanges only when administered in dosesthat are large enough to produce major met-abolic changes [eg, hyperosmolarity and,probably, damage to the blood-brain barrier(9)]. Circulating basic amino acids such aslysine are able to enter the brain, their up-take mediated by a specific transport systemlocated within the capillary endothelia com-prising the blood-brain barrier (5). The ki-netics of this mechanism are compatiblewith the expectation that changes in plasmacomposition, like those that might occurpostprandially, could also affect their levelsin the brain. To our knowledge, no system-atic study has been carried out to define thenormal ranges of these basic or acidic aminoacids in the bloodstream, at all times of dayor after the ingestion of meals whose com-positions (ie, protein contents) span therange that Americans are likely to consume.

The changes in the concentrations ofplasma neutral amino acids occurringthroughout the 24-h period were previouslycharacterized for normal, healthy volunteersconsuming three meals per day, each con-taining no protein, 25 g, or 50 g (ie, 0 to 150g/day) (4). It was observed that these levelscould rise, stay constant, or fall after “break-fast,” depending on whether the initial dailymeal happened to be rich or poor in protein;the observed changes continued throughoutthe day, presumably for as long as the sub-jects were continuing to absorb that day’sfood, since the composition of all three dailymeals was always the same. For some aminoacids such as isoleucine the normal dailyvariation encompassed as much as a 5-foldrange: midday plasma isoleucine levels were5-fold higher, when subjects were consum-ing high-protein meals, than when they ateprotein-free meals. Such data made it pos-sible, for the first time, to determine whethera “spot” level of a particular plasma amino

acid (ie, the level in a sample from a subjectthat had been treated at a particular time, ina particular way) was or was not within“normal” limits.

To determine the changes that might oc-cur in plasma levels of the basic amino acidsafter ingestion of protein, we have repeatedthe previous study (4) and included meas-urements of these amino acids. Since a largeproportion of the glutamate and aspartate inthe blood is sequestered within red bloodcells (RBC) [ie, four times as much gluta-mate, and 80 to 100 times as much aspartate,as in plasma (10-12)], we have measuredthe concentrations of these acidic aminoacids in RBC. Plasma neutral amino acidlevels were determined again to provide aninternal control on the validity of the presentstudy.

Materials and methods

Subjects

Seven healthy male volunteer students at the Mas-sachusetts Institute of Technology, ranging in age from19 to 32 yr and in weight from 62 to 78 kg, wereadmitted to the Massachusetts Institute of TechnologyClinical Research Center for the study (11 days) afterhaving signed an advised consent form that stated thepurpose of the study, the nature of the experiment, andthe sampling to be done. Each subject was given apreliminary physical examination which included rou-tine blood tests and urinalysis; all values were withinnormal ranges. Urine samples collected at 12-h intervalsthroughout the study were also normal. The protocolhad received prior approval from departmental andMassachusetts Institute of Technology Committees onthe Use of Humans as Experimental Subjects.

Experimental procedure

On the 1st day in the Clinical Research Center,subjects underwent the routine tests. Thereafter theyconsumed three special diets, during three consecutive3-day periods, which provided 0, 75, and 150 g proteinper day, respectively, as three equivalent meals. Bloodsamples were drawn on the last 2 days of each of the 3-day periods, beginning at 7 AM, and thereafter at eleven4-h intervals. The samples were collected into heparin-ized tubes and immediately centrifuged; plasma andRBC were separated and frozen at -20C until assayed.

Diets

All meals were prepared in the kitchen of the ClinicalResearch Center under the supervision of the researchdietitian. Each patient’s diet was designed to suit hisindividual caloric needs: (weight in kg multiplied by45); daily caloric intakes ranged between 2776 to 3451kcal/day. Subjects consumed one-third of their dailycaloric intake at each of three identical meals, served at

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724 MAHER ET AL

8 AM, noon, and 5 PM. The staff ensured that eachsubject consumed all ofthe food provided at each meal.

The diet contained dried egg yolk or egg white asprotein, and corn oil, crisco, or butter as fat. Carbohy-drate sources included ginger ale, applesauce, junket,Danish dessert, oatmeal, and sucrose and dextromaltosein Kool-Aid and in specially formulated cookies.

Total protein, carbohydrate, and fat contents of eachmeal were calculated from standard food compositiontables. As protein was added to the diet, carbohydratewas removed thus allowing the fat content to be main-tamed at 36% ± 2 (mean ± SD) of total daily caloricintake. For a subject consuming 3000 kcal/day, ofmeals containing 0, 75, or 150 g ofprotein, correspond-ing carbohydrate contents would have been 480, 405,and 330 g, respectively.

Analytical methods

The heparinized blood samples were centrifuged for15 mm at 3000 x g at 4#{176}Cand the plasmas and RBCwere separated. Plasmas were prepared for amino acidanalysis by adding 50 mg of 5-sulfosalicylic acid to1-mi aliquots of plasma; the samples were then vigor-

ously shaken and centrifuged for 20 mm at 9000 x g at4#{176}C.The resulting supernatant fluids were filteredthrough a Swinex Millipore filter apparatus (filterHAWP 01300, Millipore Corp. Bedford, MA), yieldingclear solutions that were frozen until assay for aminoacids on a Beckman amino acid analyzer (BeckmanInstruments, Palo Alto, CA) using a lithium citratebuffer system as previously described (4). RBC wereprepared for amino acid analysis by the method of Levyand Barkin (10) adding 75 mg of 5-sulfosalicylic acid,3 ml distilled water, and 1 ml of the cells, and shakingand centrifuging for 30 mm at 850 x g at 4#{176}C.The clearsupernatant fluids were then collected and frozen untilanalysis. Amino acid analysis of the cells was performedas for plasma samples except that a sodium citratebuffer system was used to ensure the separation ofreduced glutathione from aspartate (13).

Data anal vsis

Data were initially subjected to a 4-way analysis ofvariance; the factors compared were: 1st versus 2nd dayof sample collection on each diet; protein content ofthe diet (0, 75, or 150 g/day); individual subjects; andtime of sample collection. This analysis revealed nosignificant differences between samples collected on the1st and 2nd days of sample collection (which corre-sponded to the 2nd and 3rd days on each diet); hencedata from the 2 days were pooled, and a second analysisof variance was performed on the remaining threefactors.

This analysis revealed highly significant variationsfor most of the amino acids (described below), relatedto time of day. Since, in general, amino acid levels at11 AM or 11 PM were intermediate between the “fast-ing” levels observed at 3 AM and 7 AM and the “fed”levels observed at 3 PM and 7 PM, we further examinedthe likelihood that fasting and fed levels (ie, 3 AM + 7AM versus 3 PM + 7 PM) differed significantly. Thisapproach facilitated presentation of data and acknowl-edged that the physiological mechanisms determiningsteady-state plasma amino acid levels in the fasting and

fed states require several hours to operate. An analysisof variance was subsequently performed with fastingversus fed as a factor.

Results

Large neutral amino acids

As previously reported (4), plasma con-centrations of isoleucine, leucine, tyrosine,phenylalanine, methionine, and valine var-ied directly with the protein content of themeal, during both the fasting and the fedperiods of the day (Table 1). Fasting-statevalues tended to be lowest in the 0-g proteingroup, highest in the 150-g protein group,and intermediate in the 75-g protein group.With tryptophan, plasma concentrationswhen people consumed protein-free mealsdiffered significantly from those after pro-tein-containing meals; however, those ob-served when subjects consumed 75 g of pro-tein daily did not differ significantly fromthose after 150 g, largely because of lowvalues in the latter group at 3 to 7 AM. Fed-state LNAA values were always decreased(compared with 3 to 7 AM levels) by con-sumption of the 0-g protein meal, and al-ways increased by consumption of the1 50-g protein meal. Ingestion of the 75-gprotein meal always increased plasma levelsof each LNAA, but to a much smaller extentthan the 150-g protein meal.

Large neutral amino acid ratios

Significant differences were noted for theratios of tryptophan, tyrosine, phenylala-nine, and isoleucine to the other LNAAwhen correlated with dietary protein contentbut not with time of day (Table 2). Theratios decreased with increasing dietary pro-tein content for tryptophan, tyrosine, andphenylalanine, and increased (with increas-ing dietary protein) for isoleucine. For themethionine and leucine ratios, significantdifferences were associated with the time ofsample collection but not with dietary pro-tein intake. No significant time- or diet-dependent differences were noted for theratios of valine to the other LNAA. We alsocalculated these ratios with only five of theLNAA in the denominator, omitting methi-onine [which has a relatively poor affinityfor the blood-brain barrier LNAA carrier

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g prou’in/dar

0g proiesn/Ja� Tryptophan 0.084 0.106Isoleucine 0 53.1 28.1 ±0.022 ±0.022

± 12.5 ±11.7 �LNAA 75 0.078 0.06675 73.8 94.5 ±0.013 ±0.009

±23.9 ±18.9 150 0.066 0.064150 84.3 153.4 ±0.016 ±0.015

± 17.8 ±57.5Tyrosine 0 0.109 0.113

Leucine 0

75

150

107.4±24.2124.4

±33.0155.5

±36.5

57.8±2 1.5143.3

±3 1.0237.8±66.5

�LNAA 75

150

±0.0280.093

±0.0130.087

±0.016

±0.0160.093

±0.0080.093

±0.011

Tyrosine 0

75

150

50.3±14.6

54.5±14.8

60.6±11.2

30.6±9.265.2

±12.1100.6

±27.0

Phenylalanine

�LNAA

0

75

150

0.103±0.024

0.086±0.009

0.079±0.015

0.110±0.017

0.087±0.089

0.075±0.012

Phenylalanine 0

75

150

47.7±11.5

50.8±16.5

55.5±12.5

29.6±8.861.0

±9.286.7

±31.3

Isoleucine

�LNAA

0

75

150

0.117±0.027

0.130±0.014

0.122±0.012

0.101±0.021

0.141±0.011

0.140±0.018

Methionine 0

75

150

26.0±6.430.6

±14.031.2

±8.5

14.8±5.641.0

±10.964.4

±24.0

Methionine

�LNAA

0

75

150

0.054±0.0130.049

±0.0120.043

±0.011

0.052±0.0130.057

±0.0130.057

±0.011

Valine 0

75

150

196.6±108.6

263.3±91.8346.7

±119.1

114.7±30.7310.5

±41.7515.8

±198.0

Leucine

�LNAA

0

75

150

0.270±0.061

0.242±0.017

0.255±0.058

0.23 1±0.026

0.229±0.020

0.253±0.069

Tryptophan 0

75

150

39.8±11.8

46.0±12.2

45.4±7.4

28.6±7.647.6

±10.270.6

±24.3

Valine

ELNAA

0

75

150

0.817± 1.185

0.691±0.088

0.790±0.218

0.609±0.094

0.694±0.081

0.709±0.159

725

TABLE IEffect of time of day and dietary proteincontent on plasma concentrations(nmol/ml) of LNAA

TimesAmino acid Diet

3-7 AM 3-7 PM

* Each point represents the mean value (± SD) forseven subjects, sampled at two times (“fasting”: 3 to 7AM; “fed”: 3 to 7 PM), on the 2nd and 3rd days ofconsuming the diet indicated. The diets were providedas three equal meals containing, per day, the totalamounts of protein indicated. Subjects ate the 0-g dietfor 3 days, then the 75-g diet for 3 days, then the 150-gdiet. For each amino acid, except tryptophan, signifi-cant differences (p < 0.05, analysis of variance andDuncan’s multiple range test) were associated with di-etary protein content and with hours of sample collec-tion. For tryptophan, significant differences were seenwith variations in dietary protein content but not withtime of day (p = 0.06).

TABLE 2Effect of time of day and protein contenton plasma ratios of LNAA*

TimesRatio Diet

3-7 AM 3-7 PM

* Samples were collected and data analyzed as de-

scribed in the legend to Table 1 . Ratios were calculatedby dividing the concentration of the amino acid by thesum of the concentrations of the other competingLNAA (tryptophan, tyrosine, phenylalanine, leucine,isoleucine, valine, and methionine). For tryptophan,tyrosine, phenylalanine, and isoleucine, significant dif-ferences (p < 0.05, analysis of variance and Duncan’smultiple range test) were associated with dietary proteincontent but not with time of day. For methionine andleucine, significant differences were associated withhours of sample collection but not dietary protein con-tent. No significant differences were seen with valine.

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g protein/day

0 145.7±38.6

75 170.1±58.0

150 186.9±39.1

0 128.5±40.5

75 141.9±53.0

150 148.4±34.6

Threonine

Serine

Proline

Glycine

Alanine

113.2±29.5184.2

±33.7254.0

±52.0

98.5±35.2156.3

±38.1189.2

±47.6

140.7±42.7195.1

±36.3264.2

±82.5

251.5±84.5239.5

±44.3215.0

±7 1.0

515.3±222.5

548.7±104.9

518.8±78.6

0 576.8±305.2

75 356.8± 141.4

150 311.2±80.3

726 MAHER ET AL

(5)]; in all situations, significant differencesseen when methionine was included in theratio also were seen when this amino acidwas omitted from the ratio calculation.

Small neutral amino acids

Threonine, serine, and proline concentra-tions changed in a manner similar to thoseof the LNAA, although the magnitudes ofchanges tended to be somewhat smaller (Ta-

TABLE 3Effect of time of day and dietary proteincontent on plasma concentrations(nmol/ml) of neutralamino acids

TimesAmino acid Diet -________________

3-lAM 3-7PM

0 160.0±40.1

75 164.6±37.7

150 198.1±55.9

0 305.6±93.3

75 236.4±64.3

150 196.0±41.1

Samples were collected and data analyzed as de-scribed in Table 1. For each amino acid except glycine,significant differences (p < 0.05, analysis of varianceand Duncan’s multiple range test) were associated withdietary protein content and with hours of sample col-lection. For glycine, significant differences were seenwith variations in dietary protein content but not withtime of day (p = 0.12).

ble 3). With threonine, when the fasted-state(3 to 7 AM) and fed-state (3 to 7 PM) valueswere compared, amino acid concentrationsafter consumption of the l50-g protein dietwere greater than those after the 75-g proteindiet, which in turn, were greater than thoseafter the 0-g protein diet. For serine, plasmaconcentrations after consumption of the150- and 75-g protein diets did not differsignificantly, but both were greater than afterconsumption of the 0-g protein diet; forproline, concentrations after 150 g of proteinwere greater than after either of the othertwo diets.

Glycine and alanine levels were inverselyrelated to protein content, as reported pre-viously (4) (Table 3). Lowest fasted-state (3to 7 AM) levels were seen in subjects con-suming the 150-g protein diet group and thehighest levels were seen with the 0-g proteindiet. As with the LNAA, fed-state levelstended to be decreased by ingestion of the0-g protein diet and increased by the 150-gprotein diet. With both amino acids, con-centrations after 0 g of protein were greaterat any time of day than after 150 g of protein;those after 75 g were intermediate.

Basic amino acids

The effects of dietary protein and time ofday on plasma concentrations of lysine, ar-ginine, and histidine were similar (Table 4)and resembled those described for theLNAA. In all instances consumption of the150-g protein diet increased fed-state (3 to 7PM) levels while exclusion of protein fromthe diet decreased these levels; levels afterconsumption of the 75-g protein diet wereintermediate.

RBC glutamate and aspartate

RBC glutamate levels in both the fastingand fed states tended to vary inversely withthe amount of protein in the diet; no suchrelationship existed for RBC aspartate (Ta-ble 5).

Miscellaneous ninhydrin-positivecompounds

Plasma concentrations of citrulline and a-

amino butyric acid in the fasted state werelowest in the 0-g protein group and highestin the 1 50-g protein group (Table 6). In the

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EFFECT OF DIETARY PROTEIN ON PLASMA AMINO ACIDS 727

TABLE 4Effect of time of day and dietary proteincontent on plasma concentrations(nmol/ml) of lysine, arginine,and histidine*

TimesAminoacid Diet --------------

3-7 AM 3-7 PM

g protein/day

Lysine 0

75

150

176.3±40.9

161.8±40.4

193.9±42.5

120.7±3 1.0

180.5±34.8267.9

±76.5

Arginine 0

75

150

78.6±21.7

87.4±28.0

87.6±29.4

51.8±20.6

98.1±41.1125.8

±32.1

Histidine 0

75

150

70.9±16.2

75.0±22.4

81.8±43.2

52.8±14.0

75.9±36.5

99.4±3 1.0

* Samples were collected and data analyzed as de-

scribed in Table 1. For lysine, significant differences (p< 0.05, analysis of variance and Duncan’s multiplerange test) were associated with dietary protein contentand with hours of sample collection. For arginine andhistidine, significant differences were seen with varia-tions in dietary protein content, but not with time ofday (p = 0.24 and 0.90, respectively).

fed state (3 to 7 PM) levels of these com-pounds tended to decrease after consump-tion of the 0-g protein meal and increaseafter the 1 50-g protein meal. No significanteffects of diet composition or time of daywere observed in taurine concentrations (Ta-ble 6). Ornithine levels were significantlyincreased in proportion to the protein con-tent of the meal.

Discussion

As previously reported (4) the levels ofplasma LNAA were markedly influenced bythe protein content of the diet. Ingestion ofthe 0-g protein diet depressed these levels bythe afternoon or early evening (3 to 7 PM)while ingestion of the 1 50-g protein mealincreased them at this time (Table 1); con-sumption of a 75-g protein diet had inter-mediate effects. The ratios of each LNAA to

TABLE 5Effect of time of day and dietary proteincontent on red cell concentrations(nmol/ml packed RBC) ofglutamate and aspartate*

TimesAmino acid Diet

3-7 AM 3-7 PM

g protein/day

Glutamate 0 418.3 524.5±231.0 ±281.8

75 266.3 359.2±198.9 ±212.7

150 247.9 299.2±122.9 ±186.5

Aspartate 0 228.1 269.4±127.2 ±195.0

75 255.3 232.8±126.0 ±117.2

150 254.4 238.2±109.4 ±82.3

were collected and data analyzed as de-scribed in Table 1. For glutamate, a significant differ-ence (p < 0.05, analysis of variance and Duncan’smultiple range test) was associated with hours of samplecollection but not with overall dietary protein content;however, levels observed when subjects consumed theprotein-free meal were significantly greater than whenthey ate the lSO-g protein per day. No significant dif-ferences were seen with aspartate.

the sum of the others also tended to varywith diet composition and time of day: thoseof the aromatic amino acids, tryptophan,tyrosine, and phenylalanine tending to behigher when subjects consumed the protein-free meals. The physiological significance ofthese relationships derives in part from thefact that the “plasma tryptophan ratio” and“plasma tyrosine ratio” determine brain lev-els of these amino acids, which can influencethe rates at which they are converted to themonoamine neurotransmitters, serotonin,and the catecholamines (6, 14-18).

We now show that plasma levels of thebasic amino acids, lysine, arginine, and his-tidine, exhibit a similar relationship to die-tary protein content, attaining highest levelsafter consumption of meals containing thelargest amounts of protein (Table 4). In stud-ies on rats, we observe that treatments thatincrease plasma lysine can also increasebrain lysine (19). It is not yet known whetherother circulating basic amino acids influencethis relationship (eg, by competing with ly-sine for common blood-brain barrier trans-port sites), or whether brain lysine levels also

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TABLE 6Effect oftime ofday and dietary proteincontent on plasma concentrations(nmol/ml) of miscellaneousninhydrin-positive

when they ate the protein-free diet (Table

4). Administration of pure histidine to rats1� known to increase brain levels both of the

amino acid and of its product, histaminecompounds* � � - � � � � �------

Times

(25), which may be a neurotransmitter (26).Conceivably, diet-induced changes in

Compound Diet �3-lAM 3-7 PM

- �-- -�

plasma histidine levels might affect hista-mine-mediated neurotransmission.

Our observation that the concentration ofglutamate in RBCs is much lower in subjects

g protein/day

Citrulline 0 30.0 13.3±12. 1 ±6. 1

75 40.7 27.3±2 1.0 ±13.4

150 43.9 38.3±19.0 ±25.6

consuming protein-rich than protein-poormeals (Table 5) suggests that plasma levels,when examined, might also be found toexhibit diet dependence. However, the rela-

a-Amino butyrate 0 1 1.4 7.0 tionship between the glutamate concentra-±4.6 ±1.7

75 3 1.8 27.6±13.4 ±6.8

150 44.4 45.4±12.7 ± 16.8

lion of RBC and plasma is uncertain (27).RBC aspartate levels failed to display anydiet dependence.

Levels of many nitrogen-containing

Taurine 0 69.3 59.6±39.9 ±30.7

75 67.9 61.8±29.9 ±26. 1

plasma constituents failed to be affected bydiet composition (Table 6). The likelihoodthat constituents whose levels did changealso show parallel alterations in the rates at

150 67.6 67.1 which they are utilized, within brain or,±23.9 ±21.2

Ornithine 0 48.8 37.7

possibly, other tissues, warrants continuedexploration.

±1 1.8 ±16.075 62.3 69.5

±30.3 ±26.9

The authors thank Dr John D Fernstrom for assayingsome ofthe red cell samples for aspartate.

150 60.6 85.9±25.5 ±49.0 References

1. Wurtman Ri, Chou C, Rose CM. Daily rhythmS Samples were collected and data analyzed as de-

scribed in Table 1 . For citrulline, significant differences in tyrosine concentration in human plasma: per-(p < 0.05, analysis of variance, and Duncan’s multiple sistence on low-protein diets. Science 1967;158:range test) were associated with dietary protein content 660-2.and with hours ofsample collection. For ornithine and 2. Wurtman RJ, Rose CM, Chou C, Lam FF. Dailya-amino butyrate, significant differences were associ- rhythms in the concentrations of various aminoated with dietary protein content but not with hours of acids in human plasma. N Engl J Medsample collection. No significant differences were seen l968;279:171-5.with taurine. 3. Wurtman Ri. Diurnal rhythms in mammalian pro-

tein metabolism. In: Munro HN, ed. Mammalianaffect the rate at which lysine is convertedto biologically active brain products like pi-pecolic acid (20) or carnitine (21). Someevidence suggests that pipecolic acid mayaffect neurotransmission: it is released from

protein metabolism. Vol 4. New York, NY: Aca-demic Press, 1970:445-79.

4. Fernstrom JD, Wurtman Ri, Hammarstrom-Wik-lund B, Rand WM, Munro HN, Davidson CS.Diurnal variations in plasma concentrations oftryptophan, tyrosine, and other neutral amino

brain slices by high K� concentrations in thepresence of Ca� (22) and taken up intosynaptosomes by a Na�-dependent mecha-nism (23); its brain levels have also beenfound to be elevated in some patients withmental retardation (24).

Plasma histidine levels in the fed-state (3to 7 PM) were considerably higher whensubjects consumed high-protein meals than

acids: effect of dietary protein intake. Am J ClinNutr l979;32:1912-22.

5. Pardridge W. In: Wurtman Ri, Wurtman Ji, eds.Nutrition and the brain. Vol 1. Regulation ofamino acid availability to the brain. New York,NY: Raven Press, 1977:141-204.

6. Fernstrom JD, Wurtman Ri. Brain serotonin con-tent: physiological regulation by plasma neutralamino acids. Science l972;l78:4l4-l6.

�. Fernstrom JD, Wurtman Ri. Nutrition and thebrain. Sci Am l974;230:84-9l.

728 MAHER ET AL

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