plasma amino acids: screening, quantitation, and...

methods in nutrition

876The American Journal of Clinical Nutrition 24: JULY 1971, pp. 876-890. Printed in U.S.A.

Plasma amino acids: screening, quantitation,and interpretation”2

Charles R. Scriver, Carol L. Clow and Peter Lamin

Information about the steady-state concen-tration of amino acids in human plasma hasaccumulated steadily in recent years, partlyas the result of new methodology, but alsobecause of the relevance of this information

to human health and disease. There are twoareas in particular that have both served andbenefited from the growth and application ofthis knowledge. One is the field of nutrition;

the other pertains to hereditary and acquireddiseases that affect amino acid metabolism.

A number of simple qualitative or semi-quantitative methods have been developed todetect perturbation of the normal amino acidpattern in plasma. As a result, it has beenpossible to detect, with mass screening earlyin life, the few subjects with abnormal amino

acid metabolism among the many who are

normal (1 , 2). Nonetheless, abnormal pat-terns and their exact significance can berecognized only when the normal is known.

Consequently, there have been many studies,often using the newer semiautomatic elution

chromatographic methods of quantitative

analysis, to define the normal interindividual

variation in steady-state distributions of

amino acids in human plasma. When these

parameters are known, variation more subtle

than that generated by inborn errors of me-tabolism can also be identified. Hence weknow that circadian rhythms and nutritionalfactors generate intraindividual variation in

the steady-state levels of amino acids in

plasma.It is our intention to describe the methods

that have been widely used to study plasmaamino acids in man. We will also discuss the

advantages and shortcomings of the methods

with which we are familiar. The mean, stand-

ard deviation, and range of values for theprincipal amino acids in the plasma of normal

human subjects from birth to maturity arecompiled from the literature so that this re-

port may assist the clinical investigator.

The plasma free amino acid pool;

a steady state?

Measurements of plasma amino acids areusually made at a single point in time, andit is upon such measurements that we must

often decide whether a subject manifests“normal” or “abnormal” amino acid metabo-

lism, even though the dynamics of amino acid

metabolism have not been evaluated. Christ-ensen (3) has discussed the factors that regu-late the apparent steady-state levels of amino

acid in plasma; these are indicated in Fig. 1.The equilibrium of amino acids in plasma is

a reflection of their inflow (dietary intake, in-testinal absorption, release from endogenousprotein stores, and net endogenous synthesis

of nonessential amino acids), and their out-flow (uptake into liver and nonhepatic tis-sues, endogenous protein synthesis, and ca-tabolism). The fact that plasma levelsfluctuate remarkably little, indeed usuallyless than ±50% , despite intermittent dietaryinfluxes that may be tenfold greater than the

‘From the Debelle Laboratory for BiochemicalGenetics, McGill University-Montreal Children’sHospital Research Institute, Montreal 108, Quebec,Canada.

2 Supported by the Department of NationalHealth and Welfare Grant 604-7-643, Medical Re-search Council Grant MT-1085, and the NationalGenetics Foundation, New York.

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PLASMA AMINO ACIDS 877

Intestinal lumen

I

Metabolites

ILiver __ Extracellular free Nonhepatic

Free amino ‘ amino acids � free amino �-+ Metabolitesacids acids

I __ ICellular �-�- Plasma proteins *-� Cellularproteins proteins

Fio. 1. Equilibrium system for free amino acids in extracellular space, including plasma. -II- Indicatesflux across cell membranes.

amounts of amino acid present initially in theextracellular space, indicates the importanceof tissue uptake and metabolism in the con-trol of amino acid levels in plasma.

Membrane transport. Uptake of amino

acids into cells against a tissue : plasma gradi-

ent is achieved by conjugate forces in themembrane, which utilizes ionic interactionsand energy to regulate the flux of the chem-ically unmodified substrate (4, 5). Geneticevents can alter amino acid transport in man(6, 7) and this, coupled with evidence for

specificity in the selection of the substrateby the transport process, indicates that themembrane possesses transport proteins (8).

Uptake of amino acids by human cells is evi-dently constitutive and operates independ-ently of metabolic events that may affect thesize of the intracellular pool. For example,

the renal transport of an amino acid, thecatabolism of which is blocked, is no differentin the “blocked catabolic” mutant than in the

normal person. Thus, one can conclude that

transport regulates plasma steady-state levelsto some extent by allowing amino acids to

equilibrate with the intracellular pools thatare many times larger than the extracellularpool.

Periodicity of control systems. The pres-

ence of circadian periodicity produces meas-

urable intraindividual variation (9-1 1), mdi-eating that there are subtle regulatorymechanisms that control steady-state levelsof amino acids in plasma. Analysis of theperiodicity of plasma amino acid levels mdi-cates that it is partly dependent on the exoge-nous input of the amino acids themselves, but

that it is also modulated by other nutrients

and by hormonal events that influence cellu-lar uptake of amino acids. Periodicity ofenzyme activities that control entry of amino

acids into catabolic pathways further de-termines the intracellular fate of these sub-

strates. Awareness of circadian periodicitymay be important when the investigator usesthe concentration of an amino acid in plasma

to define, for example, the heterozygousphenotype of an aminoacidopathy (12).

Nutritional state. The state of nutrition

must also be considered when interpretinginterindividual and intraindividual variationin amino acid levels in plasma. Obese sub-

jects have a particular pattern of hyperamino-acidemia indicative of the insulin ineffective-

ness characteristic of obesity (13). Althoughpre- and postprandial sampling is likely towitness only subtle changes in the plasma

pattern when the normal subject is eating anormal diet (14), prolonged fasting in obese

and nonobese subjects may modulate theconcentrations of plasma amino acids to amuch greater extent. For example, in theinitial few days there may be a two- to three-

fold increase in the plasma concentration ofbranched-chain amino acids, whereas anequivalent increase in glycine and threonine

appears only after this initial period; as fast-ing continues, a dramatic fall in a-alanineoccurs. The initial rise in amino acid levelspresumably reflects a fall in plasma insulin

activity, whereas the latter changes reflect, inturn, increased peripheral release (threonineand glycine) and increased splanchnic utiliza-tion (x-alanine) of amino acids.

These observations may seem extreme andirrelevant. However, many patients withaminoacidopathies are first seen when acutelyill, often at times of diminished food intake.Under such conditions, it will be necessary

to segregate the amino acid imbalance causedby secondary illness from that associatedwith the primary disease. Furthermore, if

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878 SCRIVER ET AL.

screening programs are extended to countrieswhere malnutrition is common, as may in-deed be the case (2), the investigator willneed to distinguish the amino acid imbalancedue to primary malnutrition (17, 18) from theprimary inherited aminoacidopathy. Finally,the investigator should remember that selec-

tive modification of protein intake may affectplasma amino acids (1 4, 19); this may be-come significant, for example, when patients

with hyperammoniogenic aminoacidopathies

are treated with low protein diets. Many

additional viewpoints (20) on the influenceof protein nutrition on the steady state of freeamino acids in plasma are available for theinterested reader.

Qualitative methods of amino acid

analysis

The adaptation of simple partition chro-matographic methods to the analysis of amino

acids has provided an opportunity to evaluatethe normal amino acid pattern in plasma or

whole blood and its variations at birth andthereafter. Consequently abnormalities rep-resenting hereditary or acquired variation in

amino acid metabolism can now be easilyrecognized in mass screening programs.

Two qualitative methods in particular (21,22) have been widely used to survey the hu-man plasma amino acid profile; these areindicated in Fig. 2. Both techniques combinedeproteinization of the sample directly on thefilter paper, with development of the chro-matogram in an n-butanol, acetic acid, watersolvent (1 2 : 3 : 5) (23); the use of various

location reagents increases the sensitivity withwhich various amino acids can be detected.The ease with which samples in large num-

bers may be processed with these filter paper

methods, makes them preferable to thin-layermethods for purposes of mass screening.

Plasma method. A sample of whole bloodis drawn from a skin puncture into heparmn-ized microcapillary tubes (75 mm x 1.4 mm,

outer diameter) (21). After sealing one endof the tube with plasticine, the plasma isseparated from the cells by centrifugation inan International microcapillary centrifuge(Model MB). A lO-�d aliquot of plasma3 is

applied directly to a line drawn 2.5 cm fromone edge of a 25.4-cm square, corner-

punched Whatman No. 3 MM ifiter paper(A pattern). Eleven samples can be appliedat about 2-cm intervals to one sheet of filter

paper. Transfer of plasma is simplified if acut segment of tube, the length of which isequivalent to 10 �d plasma, is held in thevertical position against the paper and theplasma is allowed to flow onto the paper in asingle application. The mean error in obtain-

ing a 1 � volume by this method is ± 3%

(24). Approximately 1 mm is required toprocess one sample completely. The filter

papers are then mounted on “universal”frames with spacers (25). They are then de-veloped overnight by ascending partitionchromatography in a freshly prepared n-buta-nol, acetic acid, water mixture (12 : 3 : 5). Inthe morning, the papers are removed fromthe solvent, dried for 1 hr at room tempera-ture in a stream of air, then removed from

the frame and stained in a mixture of ninhy-drin (0.25% w/v), isatin (0.01 % w/v), and

2 , 6-lutidine (1 % v/v) in acetone. The chro-matograms are dried at 80 C for 1 5 mm andthen viewed with combined transmitted andreflected light upon a lighted X-ray viewingscreen. Significant elevation or diminution of

amino acid content is readily visible, and thesensitivity of the method has been evaluated(2 1). The upper three-fourths of the paper(above the glutamic acid spot) may then beoverstained with Ehrlich’s reagent (p-di-

methylaminobenzaldehyde) to identify hy-droxyproline, citrulline, and homocitrulline.If the lower portion of the chromatogram isstained with Pauly’s reagent (diazotized sulfa-

nilic acid), the histidine spot will be selec-tively identified.

Whole blood method. Whole blood istransferred from a skin puncture ontoSchleicher and Schuell (Keene, New Hamp-shire) paper No. 903 ; the paper must be

soaked through on both surfaces (22). Theair-dried sample is then dry-autoclaved at

250 F for 3 mm. A 3/16-inch punch (equiva-

lent to about 7 �l of blood) or #{188}-inch punch

of the blood spot is then withdrawn and in-

serted into holes of equivalent size in a

Aliquots of 20 �l applied in two lots can alsobe analyzed by this method without sacrificing

resolution. The sensitivity of the method can thus

be increased.

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ORIGIN (PROTEIN SPOT)

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STANDARDSTANDARD AMINO ACID SPOTS 3/16

RI ) Soetple Discs

Fio. 2. Examples of Partition chromatographic methods for screening on plasma amino acids (see text for

details of development and staining). A) Map of the spots for “plasma method” (21). B) Examples of actualsamples examined by “plasma method” showing, from left to right: 1) normal plasma; 2) hyperphenylalaninemia(in PKU); 3) hypermethioninemia; 4) transient neonatal hypertyrosinemia; 5) hypercitrullinemia (incitrulline-mia); 6) hyperhistidinemia (in histidinemia). The next channel (6’) shows Pauly overstain of sample 6 and the

last channel (5’) shows Ehrlich overstain to confirm citrulline. (Reproduced from Scriver (52).) C) Examplesof actual samples examined by “whole blood method” (22) showing from left to right: 1) “homocystinuria;2) “maple syrup urine disease;” 3) transient hypermethioninemia; 4) “phenylketonuria;” 5) “neonatal tyro-sinemia;” 6) hypertyrosinemia; 7) normal plasma; and 8) standard amino acid mixture. (Reproduced fromLevy et al. (29.) D) Map of the spots for whole blood method. (Taken from Efron et al. (22).)

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880 SCRIVER ET AL.

“carrier” sheet of filter paper (Whatman No.3 MM); the discs should be handled with

forceps to avoid contamination (26). Frictionholds the discs in place satisfactorily. The

carrier sheet, which may be charged with 25discs set 2.5 cm from one edge, is developedovernight by ascending chromatography(paper size, 63 x 30 cm, cut from 30-cmwidth rolls) or by descending chromatog-raphy (paper size, 57 x 33 cm with a wick,57 x 7-6 cm, sewn in with zigzag stitching(22)), in the n-butanol, acetic acid, watersolvent (12 : 3 : 5). Staining and location of

amino acids is performed as described above.This method is advantageous in its ease of

collection and storage of samples. However,streaking of amino acid spots on the devel-

oped chromatogram, because of hemoglobin

tailing, may be a problem. Two modifications

avoid this problem:1) The “tandem chromatography tech-

nique.” Szeinberg and colleagues (27) use25-cm square Whatman No. 3 MM filter

paper (see plasma method) as the carriersheet for the 3A6-inch Schleicher and Schuelldiscs containing the whole blood sample. Thechromatogram is developed first by ascendingchromatography for 6 hr in an isopropanol,water mixture (3 : 1). The paper is then driedfor 1 hr at room temperature in a stream ofair, after which the discs, which retain the

hemoglobin, are pushed out. The chromato-gram is then replaced in the same direction,in a second solvent mixture of n-butanol,acetic acid, water (1 2 : 3 : 5) solvent and de-veloped overnight; the developed chromato-gram is dried and stained as described above.

2) The “elution technique.” Blood-soakeddiscs (12-mm diameter, Schleicher andSchuell filter paper No. 2992 containing

about 40 � whole blood) are eluted overnightin 100 �l of an ethanol and water mixture(60 :40) in capped conical microcentrifugetubes (0.4-mi capacity) (28). Forty micro-liters of the hemoglobin-free eluate is thenspotted, using several applications, ontoWhatman No. 3 MM paper (25 x 25 cm)

and developed by ascending chromatographyas described above (21). An additional spot,probably glutathione, is found between the“dibasic” amino acids and the origin. The

sensitivity of this modification is equivalent

to the plasma method (21). The size of thedisc and the volume of the alcohol mixture

could probably be reduced to diminish thenumber of transfers of eluate to the ifiter

paper.Performance. Both basic methods have

also been used satisfactorily for semiquanti-tative analysis. As a result, monitoring ofplasma amino acid concentrations in patients

who are being treated for an aminoacidopa-thy is greatly facilitated. Present experi-

ence indicates that this relaxed approach iscompatible with good clinical control of mostaminoacidopathies.

Large field trials have been performed

with both methods. Mass screening of new-born infants has been reported on about1 50,000 subjects (24, 29-3 1 ) and it is evi-dent that the methods perform well for this

purpose. A wide variety of normal transient

patterns of hyperaminoacidemia has been ob-served (24, 29-3 1). Many of the aminoacid-opathies associated with a specific hyper-

aminoacidemia have also been recognized inthe newborn period with these methods (seeTable 1). Partington (32) has evaluated the

sensitivity and specificity of the plasmamethod (21) for the specific detection ofhyperphenylalaninemia; he found it to beequivalent to the inhibition assay of Guthrieand Susi in reliability.

Ninhydrin-positive aminoacidopathies that

are presently known and could be detectedby chromatographic screening of plasma andwhole blood are listed in Table 1 ; aminoacid-

opathies still unknown might also be discov-ered by these methods in the future. The

above-mentioned methods can be used forpreventive community screening, if personnelare trained to interpret the chromatogramsand to be aware of the significance of theamino acid patterns that they observe. Theremust also be facilities to locate, retest, andfollow all subjects with abnormal initial tests(24).

Quantitative methods of amino acid

analysis

A little over a decade ago, very few in-

vestigators were able to perform more than

one complete analysis per week, i.e., of the

free amino acids in physiological fluids.

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Amino acids affected Abnormal enzyme Comment

Phenylalanine hydroxylating

system (?)

Phenylalanine

Tyrosine

Methionine

Histidine

Phenylalaninc

p-Hydroxyphenyl pyruvic acid Benign; responds to ascorbic

hydroxylase (EC 1.14.2.2) acid and reduced protein in-

take.

5-adeno- Benign; usually found with

(EC 2.5. high protein intake.

phenylke- Phenylalanine

iii ( Transient phenylke- Phenylalanine

tonuria

iv ) Benign hyperphe- Phenylalanine

nylalaninemia a

Tyrosine

Tyrosine

tyrosin- Tyrosine (and methionine)

ii) “5uper tyrosinemia”

(Oregon)”

L-Tyrosine: pyruvate amino- One case known; myasthenia

transferase (?) (EC 2.6. 1 . gravis, probably incidental

20) fInding.

ii ( Variant form Histidine

TABLE 1

PLASMA AMINO ACIDS

Aminoacidopathies that can be detected by plasma or whole blood screening

881

Condition or disease

Group I: Common perinatal

(adaptive) traits

Neonatal hyperphenylalanin-

emia#{176}

Neonatal tyrosinemia#{176}

Hvpermethioninemia’

Hyperhistidinemia a

Group II: Inherited amino-

acidopathies

1. The hyperphenylala-

ninemias

i) Classical phenylke-

tonuria

ii) Atypical

tonuria a

2. The hypertyrosinemias

i) Tyrosinosis (Medes)

iii) Hereditary

emia”

3. The hyperhistidinemias

i) Classical form#{176} Histidine (alanine in some

cases

ATP: L-methioninesyltransferase(?)

l.6

i-Histidine ammonia lyase (?)

(EC 4.3.1.3)

i-Phenylalanine tetrahydrop-

teridine: oxygen oxidore-

ductase (4-hydroxylating)

(EC 1.14.3.1)

L-Phcnylalaninetetrahydrop-



(EC 1.14.3.1)

i-Phenylalanine tetrahydrop-



(EC 1.14.3.1) (?)

i-Phenylalaninc tetrahydrop-



(EC 1.14.3.1)

Soluble (cytoplasmic) iso-zyme of tyrosine amino-

transferase

p-HHPA hydroxylase (EC 1.

14.2.2) Primary or second-

ary defect?

L-HiStidine ammonia lyase

(EC 4. 3. 1 .3) (liver and

skin)

L-HiStidine ammonia lyase

(EC4.3.1.3) (liver only)

Benign; may respond to folic

acid; often occurs with tyro-

sinemia.

Benign; related to high protein

intake.

i) Plasma phenylalanine >16

mg/lOO ml; causes mentalretardation. When treated,

i-phenylalanine tolerancein diet is 250-500 mg phe

day.

ii) Plasma phenylalanine > l6

mg/lOO ml; similar to i) butdietary tolerance for L-phe-

nylalanine is >500 mg/day

iii) Plasma phenylalanine >16

mg/l0O ml; change in sta-tus to iv), or normal, sev-

eral months or years after

birth.

iv) Plasma phenylalanine <16

mg ‘100 ml; on normal

diet. Benign trait.

One case; associated mentalretardation.

Hepat.ic cirrhosis, and renal

tubular failure, eventually

fatal; responds to tyrosine

restriction.

Usually associated with mental

retardation and speech de-

feet.

As above.

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Branched-chain a-keto acid

oxidase(s) (partial activity)

Leucine, isoleucine, valine

Leucine, isoleucine, valine

Same

Same

Valine

Methionine and homocystine

Cystathionine

Milder form of trait.

ii) Type II

Glycine

Sarcosine (ethanolamine)

Proline

Proline

Hydroxyproline

Lysine (and glutamine)

Lysine, arginine, (NHs)ii) Type II

iii) Saccharopinunia�’

882 SCRIVER ET AL.

Lysine, saccharopine, citrul-

line

TABLE 1-Continued

Condition or disease Amino acids affected Abnormal enzyme Comment

4. The branched-chain hy-

peraminoacidemias

I) Classical “maplesyrup urine dis-

ease” a

ii) Intermittent form

iii) Mild form

iv) Thiamin-respoissive

v) Hypervalinemia

5. Sulfuraminoacidemias

i) Homocystinuria5

ii) Cystathioninuriab

6. The hyperglycinemias

i) Ketotic form”

ii) Non-ketotic form”

7. Sarcosinemiab

8. The hyperprolinemias

i) Type I”

9. Hydroxyprolinemia

10. The hyperlysinemias

1) Type I

Glycine and other glucogenic

amino acids

Branched-chain a-keto acid

oxidase(s)

Same

Same

Valine aminotranaferase (EC

2.6. 1.-)

i-Serine hydrolyase (deami-

nating); (“cystathionine

synthetase”) (EC4.2. I .13)

L-Homoserine hydrolyase (de-

aminating) (EC 4.2. 1.15)

Propionyl-CoA :carbondiox-ide ligase(ADP) (EC 6.4.

1.3)

Same or “glycine decarboxy-

lase” (?)

Sarcosine: oxygen oxidoreduc-

tase (demethylating) (EC 1.5.3.1)

L-Proline:NAD(P) 5-oxidore-

ductase (“proline oxidase”)(EC 1.5.1.2)

“i�-pyrroline-5-carboxylate

dehydrogenase”

“Hydroxyproline oxidase”

Lysine:a-ketoglutarate: tn-

phosphopynidine nucleo-

tide (TPNH), oxidoreduc-

tase (e-N-[L-glutaryl-2]-L-

lysine forming)

(Partial defect of above [10,

i]) or different enzyme?

“Saccharopinase” (?)

Postnatal collapse, mental re-

‘ tardation in survivors; diet

therapy can be effective.

Intermittent symptoms; de-� velopment may be otherwise

normal.

� Unremittent; milder than i)

� Mild form; Bi-responsive.

Retarded development and

vomiting; responds to diet.

� Usually associated with throm.� bo-embolic disease, mental

� retardation, and Marfan

� phenotype.

� Probably benign trait; vitamin‘ Bt corrects biochemical trait

in most patients.

Ketosis, neutropenia, mental

retardation; often fatal.

Benign trait (probably).

Benign trait, which is some-

times associated with heredi-

tary nephritis.

Possibly benign trait, some-

times associated with CNS

disease; .XPC excreted in

urine; proline concentration

greater than in Type I.

Two cases, associated with

CNS disease; others normal

Associated with mental retar-

dation and hypotonia.

Hyperammonemia symptoms,

related to protein intake.

One case; associated with

mental retardation and short

stature.

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Condition or disease

11. Pipecolicacidemia1’

Amino acids affected Abnormal enzyme

“Pipecolate oxidase” (?)Pipecolicacd

Glycine, glutamine

Glutamine

Ornithine

Citrulline

ASA

ii) Type II

iii) Hyperornithinemia

iv) Citrullinentia”

v) Argininosuccinicaci-

duniat

Group Ill. Nutritional andother diseaseswhich may affect

amino acids inplasma

Protein-calorie malnutrition

Prolonged fasting

Obesity

Hepatitis

See text.

I;

a These diseases have been detected in newborn patients by the screening tests described in this paper.

b These traits may not be detectable by screening tests because plasma elevation is too small; since the

renal clearance of the relevant substances is high, urine testing would be preferable. Other traits in thisclass are phosphoethanolaminuria (in the hypophosphatasias) , hyper-�-alaninemia, and fl-aminoiso-butyricacidemia.


TABLE 1-Continued

12. The hyperammonemias

i) Type I ATP: carbamate phospho-

transferase (EC 2.7.2.2)

Carbamoylphosphate: L-or-

nithine carbamoyltransfer-

ase (EC2.l.3.3)

Ornithine - ketoacid amino-

transferase

L-Citrulline: 1-aspartase ligase

(AMP) (EC6.3.4.5)

1-Argininosuccinate arginine-

lyase (EC4.3.2.l)

Comment

Hepatomegaly and mental re-

tardation.

A group of diseases that shows

ammonia intoxication, pro-

tein intolerance, hepato-

megaly, vomiting, et cetera.

ASAuria also has trichor-

rhexis nodosa.

vi) (See 10, Type II)

Tryptophan,’leucine/isoleu-

cine/valine �; Tyrosine/

glycine/proline I

Alanine � ; threonine;

glycine I

Leucine,. isoleucine/valine,

phenylalanine/tyrosine

Glycine I

Methionine/tyrosine I

Severity of change related to

severity of malnutrition.

Reflects insulin insensitivity.

Reflects severity of liver dis-

ease.

Moreover, a large sample was often requiredto obtain reliable results. It is now possible,by virtue of better resins and instrumentation,

to perform (by elution chromatography onion exchange resins) several analyses dailyof very small samples of plasma or serum. We

and others (33-35) have found that attentionpaid to details in handling the samples isimportant (Table 2), and when certain arti-facts are avoided the investigator can obtainaccurate analysis of the free amino acids inplasma samples.

1) The effect of venipuncture. The con-centration of taurine and glutamic acid inplasma falls about 30 mm after venipuncture

and returns to normal in about 1 hr (36).This artifact should be distinguished from

physiological changes in studies requiring

multiple sequential venipunctures.2) Plasma versus serum, and choice of

anticoagulant. In general, plasma is easier to

prepare for application to the resin column.Plasma may be deproteinized immediately

after the blood cells are separated by centrif-ugation, whereas serum may experiencechanges in its amino acid composition uponstanding at room temperature during clotretraction.

The choice of anticoagulant can be im-portant. Most investigators use heparin; how-

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884 SCRIVER ET AL.

TABLE 2Artifacts in plasma amino acid composition related to technique in handling samples

Procedure

Effect on particular amino acids

-

Concentration in sample decreases Concentration in sample increases

1) Repeated venipuncture Taurine, glutamic acid

2) Clotting of serum standingroom temperature

at Glutamine, asparagine Taurine, aspartic acid, glu-tamic acid

3) Delay in deproteinization Cystine, homocystine, and mixeddisulfides

Picric acid method” Citrulline, homocitrulline, trypto-phan

4) ContaminationPlatelets and WBC Taurine, aspartic acid, glu-

tamic acid

RBC (hemolysis) Arginine, cystine Glutathione, ornithine

“Fingerprints” (sweat) Many amino acids

5) StorageTemperature above -68 C Glutamine, asparagine, tryptophan Glutamic acid, aspartic

acid

Handling and elution at tem-perature >40 C

Glutamine, asparagine Glutamic acid, asparticacid

“Losses occur during step when picric acid is removed on Dowex 1 or 2 resin.

ever, an excess of this anticoagulant maycause hemolysis leading to artifacts relatedto constituents of the red blood cells. Im-purities in some batches of ethylenediamine-tetraacetic acid (EDTA), which is sometimesused as an anticoagulant, can produce nm-

hydrin-positive peaks in the elution chro-

matogram (33).3) Deproteinization. Many investigators

prefer to deproteinize plasma with 3 % sulfo-salicylic acid (35) (1 vol plasma: 5 vol 3%sulfosalicylic acid), or with direct addition of30 mg sulfosalicylic acid powder/mi plasma.After high speed centrifugation (21 ,000 x g)for 10 mm, the supernatant may be stored orapplied directly to the ion exchange column.

The other popular method of deproteiniza-tion (37) uses picric acid, which because ofits yellow color, must be removed on Dowex

1 or 2 resin before the deproteinized samplecan be analyzed for taurine and urea. Thesulfosalicylic acid method of deproteinizationalso prevents substantial losses of tryptophan,

citrulline, and homocitrulline that occur whenpicric acid is removed from deproteinized

samples (35). The arguments are numerous,both for and against sulfosalicylic acid versus

picric acid as the agent of choice for de-

proteinization in various situations, and the

numerous discussions on this matter (33, 35,

38, 39) should be consulted by the reader.Immediate removal of protein from the

sample is important so as to avoid significant

losses of disulfide amino acids that will bind

to plasma proteins on standing at room

temperature or in the refrigerator (33, 35,

37, 40). Rapid deproteinization is necessaryin field studies, or in those situations in which

shipment of samples is required,4 or when the

treatment of patients with disorders of sulfur

amino acid metabolism is being monitored.

4 Homocystine was not found in plasma samplesobtained from the original cases of homocystinuria(41). A delay in deproteinization accounted forthis artifact.

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4) Contamination. Hamilton (26) has

mentioned that contamination of glasswarewith sweat from a fingertip can jeopardize the

reliability of analyses obtained by some highsensitivity methods.

Platelets and leukocytes contain largeamounts of taurine and dicarboxylic aminoacids (33, 36, 42). Therefore, plasma orserum samples can be contaminated withthese substances released from the cellularconstituents in the buffy coat. Plasma should

be drawn off from the cell-plasma interfacewith care to avoid disturbing the buffy coat.

Hemolysis of red blood cells may also dis-tort the apparent plasma pattern. Glutathi-one (reduced and oxidized) is present in red

cells (36); release of arginase from erythro-cytes may diminish arginine and increaseornithine in plasma; and plasma cystine maybe lowered, either by dilution with an intra-cellular pool that is low in cystine, or bybinding to protein in the hemolysate. Other

amino acids are not significantly altered, astheir concentration in mature red cells is

similar to that of plasma (42, 43).

5) Storage. The concentrations of glutamicacid and aspartic acid rise slowly, and gluta-mine and asparagine fall equivalently insamples stored for long periods at -20 C.These changes are minimized by storage at

-68 C and enhanced at -4 C, or at roomtemperature (35, 38). Evaporating and dry-ing of samples or elution of amino acidsfrom ion exchange columns at temperaturesabove 40 C will reduce the glutamine andasparagine content of plasma samples.

Methods of quantitative analysis. Thereare now many reports in the literature thatdescribe technical modifications in the basicmethods for semiautomated elution chroma-

tography of amino acids on ion exchangeresin columns. These modifications, in gen-eral, provide improved speed, resolution, and

automation of analysis; a later article in thisJournal will discuss these developments. We

will mention only two reports, which provide

the investigator with a portfolio of simple

operating programs, and which will allow him

to investigate specific aminoacidopathies

without sacrificing column time.

Shih and colleagues (44) described a series

of protocols for operation of the single col-

umn (45, 46) type of analyzer. They were

able to obtain within 1 to 2 hr quantitativeanalysis of particular groups of amino acidfrom short resin columns. The protocols arerelevant for the investigation of many of theaminoacidopathies listed in Table 1.

Our group (47) described methods forrapid operation of the two-column analyzer

system (48) as well as the simultaneous use oftwo columns. Recoveries and reproducibilityof the methods compare well with the parent

techniques (48).Experience with these modified techniques

has demonstrated their value in monitoring

patients under treatment for hereditaryaminoacidopathies and in performing investi-.gations that require analysis of many samples

for a particular amino acid.Normal values. Values for amino acid

concentration in plasma or serum of healthyhuman subjects are presented in Table 3.These data represent many studies performed

by investigators using reasonable precautionsfor the handling and analysis of the samples

of plasma or serum.5 Several different tech-niques for amino acid analysis by elutionchromatography on ion exchange resin wereused to obtain the results presented in Table

3 and yet the data are reasonably homogene-ous. One observes that essential amino acids

in plasma are lower during the period ofrapid somatic growth (49, 50) despite thehigher protein intakes on the basis of bodyweight at such times. Dickinson et al. (51)showed that amino acid concentrations inplasma fall after the first day of life, with par-

ticular reference to nonessential amino acids.Nonetheless, despite undoubted variation indiet of the subjects of the various reports, the

interindividual variation in human subjectsis relatively modest, reflecting presumably the

remarkable control exerted upon the steady-state free amino acid pattern in plasma.

Comment

It has been several years since the merits

of mass screening for hereditary metabolicdiseases (52) became a popular topic for dis-

cussion. Although the rate of discovery of

“new” aminoacidopathies has continued to

Artifacts, with regard to glutamine and gluta-mate, are noticeable in some of the older studies.

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burgeon (53), a clearer interphase has de-

veloped between the use of mass screeningmethods and the recognition and the under-standing of many acquired and inheriteddiseases of amino acid metabolism. There islittle doubt that the proper use of screeningmethods, such as those discussed in this arti-cle, benefits the student of health and diseaseand his patient. Yet, as more use is madeof mass screening programs, there must beequally wide awareness of the limitationsinherent in these methods and in the interpre-

tation of their findings. Today’s advances indiagnosis will further burden those facilitiesthat must provide medical care to patientswith disordered amino acid metabolism; it isclear that the methods described here canalso be used to facilitate the investigationand treatment of patients with such problems.It must be a matter of some gratification forthe pioneers of partition and elution chroma-tography to see their little bits of filter paper

and their batches of resin performing yeomanservice in the mainstream of human biologytoday.

Addendum

An unfortunate delay between receipt ofthis solicited manuscript by the editors andits publication by the Journal can be offset tosome extent with the following selected ma-

terial:

Transport and metabolism

Brodehi and colleagues (54) studied tubu-lar reabsorption of phenylalanine in classical

phenylketonuric patients. Transport is nor-mal even though endogenous oxidation of thesubstrate is completely blocked. It is nowknown that phenylalanine is oxidized to a

significant extent in mammalian kidney (55,

56). Similar observations on the independ-ence of transport of a substrate from its intra-cellular fate were obtained by Glorieux et al.(57), studying sarcosine transport in normal

and sarcosinemic subjects.

Circadian variation

G#{252}ttler, Olesen and Wamberg (58) ob-

served a reversal of the normal circadian

rhythm for plasma phenylalanine in phenyl-

ketonuric patients. This finding further em-phasizes the need to standardize conditionsfor discrimination of normal subjects and

heterozygotes for phenylketonuria. Burghen

et al. (59) discovered that chloramphenicoltherapy altered normal periodicity and theinitial steady-state concentration of amino

acids in plasma.

Nutrition and plasma amino acids

Relationships between feeding and plasma

amino acids continue to be of interest. Adibiand colleagues (60) showed that starvationleading to changes in plasma insulin couldnot solely explain changes in branched chain

amino acid levels. Alanine was clearly sen-sitive to endogenous carbohydrate metabo-

lism.Synderman’s group continues to publish

data on the effect of protein intake on theplasma amino acids (61). Restricted protein

intake (2 g/kg per day) in the premature in-

fant depresses plasma ‘lysine and elevatesglycine. High protein intakes (9 g/kg per day)

increase proline, phenylalanine, tyrosine,methionine, valine, leucine, and isoleucine inplasma. The RBC : plasma distribution ratiofor most free amino acids is not significantly

altered by widely differing protein intakes.Leucine feeding depresses isoleucine andvaline in plasma (20, 62), presumably by in-fluencing catabolism of the affected amino

acid specifically. Whitehead’s group inUganda (63) reported that fasting serumamino acids provide a useful monitor of theprogress of kwashiorkor and its treatment.

Stegink and Baker (64) reported relationshipsbetween malnutrition and plasma amino acidlevels. Eskimo and Caucasian children hadsimilar postprandial amino acid levels in se-

rum despite different diets.The so-called “alanine cycle” in man has

received considerable attention (65). Alanineflux from muscle to liver serves gluconeo-

genesis in the latter organ, assuming net al-anine synthesis from pyruvate in muscle. Thecycle might also serve urea synthesis in the

transfer and removal of ammonia from pe-ripheral tissues. Formation and release of

alanine in muscle may be the rate limiting

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888 SCRIVER ET AL.

step in gluconeogenesis in human starvation.Hypoalaninemia may play a pivotal role inthe pathogenesis of ketotic hypoglycemia ofchildhood (66). Cahill’s group (67) hasshown that glutamate is taken up by muscle,whereas glutamine is released; the latter is

taken up across the splanchnic bed in man.

It is possible that this other “shunt” may be

an important source of urinary ammonia!!

We wish to thank Mrs. Eveline Zifkin for herassistance in the preparation of this manuscript.

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