ANALYTICAL BIOCHEMISTRY 70, 479-488 (1976)

A Method for Determining Amino Acid Concentrations and Specific Activities of Amino Acids and Some Other Compounds in Biological Fluids 1 NEELAKANTAN V A I D Y A N A T H , RONALD BIRKHAHN, JOHN

R. BORDER,

RAPIER M C M E N A M Y , 2 GARRET O S W A L D , GREGORY TRIETLEY, AND TRAN F . Y U A N a

Departments of Surgery and Biochemistry, the Medical School, State University of New York at Buffalo, Buffalo, New York 14214 Received June 23, 1975; accepted August 27, 1975 A method is described for obtaining plasma ultrafiltrates from which the concentrations of all amino acids, including tryptophan and ammonia, are obtained. A split-stream methodology is described for obtaining, in addition to the concentrations, the radioactivities of amino acids, glucose, and plasma water.

A number of methods for determining the free amino acid concentrations in plasma and other biological fluids have been reported. Typically, the methods have involved sulfosalicylic acid or picric acid to obtain protein-free supernatant solutions for the analysis (1-3). Extractions with these reagents generally give erroneous values for glutamine, glutamate, and tryptophan. We have further developed a technique used earlier, that of centrifugal ultrafiltration (4), to obtain ultrafiltrates from which concentrations of all amino acids, including those above mentioned as well as ammonia, can be determined directly by a resin column technology. Included is a description of the use of split stream system for simultaneously determining the concentrations of amino acids and radioactivities of amino acids and some other compounds eluted from the column.

EXPERIMENTAL Materials

Amino acids standards for physiological fluids, lithium citrate, and lithium hydroxide were obtained from Pierce Chemical Company (Rockford, Illinois). L-~-Amino-y-guanidino-butyric acid, c~-aminoisobutyric acid, 1-aminocyclopentane-l-carboxylic acid, norleucine, and /3-alanine were obtained from Calbiochem, LaJolla, California. I Supported by the National Institutes of General Medical Sciences, Grant No. GM 15768. 2 To whom reprint requests should be directed. a Recipient of NIH Fellowship No. 1-F 02 GH 55141-01. 479 Copyright © 1976by AcademicPress. Inc. All rightsof reproductionin any l)3T'mreserved.

480

VAIDYANATH

E T AL.

L-[G-3H]Threonine, L-[G-3H]valine, L-[N-4,5-3H]leucine, L-[N-3-3H] phenylalanine, L-[N-3-aH]glutamic acid, L-[U-14C]alanine, [1-14C]1aminocyclopentane-l-carboxylic acid, and D-[U-14C]glucose were obtained from New England Nuclear, Boston, Massachusetts. L-[a4C]Lactic acid was obtained from International Chemical and Nuclear Corporation, Irvine, California. a-[1-~4C]Aminoisobutyric acid was obtained from Dhom Products, North Hollywood, California. The purity of isotopes was determined by the method of Manning and Moore (4). L-Leucine-N-carboxyanhydride was obtained from the Fox Chemical Company, Los Angeles, California, lot F1524. L - [ U - 1 4 C ] Alanine initially obtained from International Chemical and Nuclear Corporation was found to contain 12% o-alanine, L-[U-~4C]Alanine from N e w England Nuclear contained approximately 1% D isomer, L-[G-3H]threonine and L-[N-3-3H]phenylalanine each contained a contaminant of 5 to 10% activity which eluted at approximately the void volume of the resin column. The other isotopic compounds showed no D isomer and little, if any, trace contaminants. All bloods were collected in a solution of 0.15M sodium ethylenediaminetetraacetate, 0.13 M NaF, pH 7.4, 50:1 v/v, placed in butyrex test tubes, size 12.5 × 100 mm (Lusteroid Container Company, Inc., Irvington, New Jersey), immediately chilled in an ice bath, and centrifuged at 10,000g for 15 rain in order to provide a platelet-poor plasma. The plasma was removed, placed in a tared vial, and the latter reweighed to obtain the weight of the plasma collected. To the plasma was added 20/xl/g plasma of a solution containing per liter 7.06 mmol of norleucine, 28.24 mmol of/~-alanine, 14.12 mmol y-guanidino-a-aminobutyric acid, and 25.5 nCi of 14C-labeled fl-alanine. These amino acids serve as internal standards to provide concentrations in a liter of plasma water of 150 /xmol of nodeucine, 600/~mol of fl-alanine, 300/~mol y-guanidino-a-aminobutyric acid, and 550 nCi fl-alanine. Plasma was taken to contain 0.92 ml of H20/g. Ultrafiltration was used to separate the plasma water from the plasma proteins (4). Visking tubing (Union Carbide Corporation, Food Products Division, Chicago, Illinois, size 8/32), was boiled 15 min in 3% Na2CO.~ solution, rinsed, then boiled 15 min × 2 in distilled water. The inside of the tubing was thoroughly flushed with water at each change. After testing for leaks 200- to 250-cm sections of the tubing were inflated with N2 and allowed to dry in an environment of low dust content. As soon as dry, the tubing was cut into 45-cm lengths and stored at 0 ° until used. All the following steps were carried out at 0-4°C: 0.05 ml of decanol and 0.05 ml of benzene were added to each gram of plasma and mixed well; the plasma was placed in a small stem volumetric pipet, a 45-cm section of the inflated, dried ultrafiltrate tubing was held approximately horizontal with the ends bent slightly upward, the pipet was inserted in one end of the tubing and the plasma allowed to slowly drain into the tubing. The ends of the tubing were

DETERMINATION OF PLASMAAMINO ACIDS

481

brought together and held tightly. The tubing was folded on itself several times and placed in a 50-ml conical plastic centrifuge tube (Amicon Corp., Lexington, Massachusetts). The latter tube was fitted with a polypropylene plug, 6 mm thick, 18 mm in diameter, conically shaped to fit the curvature of the tube. This plug upon which the ultrafiltrate tubing rests contained several slits in order that it could be removed after centrifugation by inserting an instrument such as a bent paper clip beneath it. After placement in the centrifuge tube the open ends of the Visking tubing were folded over the lip of the tube and tightly secured with a No. 5 Nalgene stopper. The assembled apparatus is shown in Fig. 1. Centrifugation was conducted in a PR 2 refrigerated centrifuge (International Equipment Co., Boston, Massachusetts) at 0°, 3500 rpm ( - 3000g) for ½ to 2 hr to obtain a minimum of 1.25 ml of ultrafiltrate (sufficient for duplicate analyses if needed). The ultrafiltrates were stored at - 8 0 ° until analyzed. Amino acid analyses, two-column methodology, were conducted with a Beckman 121 HP analyzer equipped with an Infotronics Integrator (Columbia Scientific Company, Austin, Texas), using the general procedure ofBenson et al. (1967), as described in the company's operator's manual for physiological fluids. To each milliliter of plasma ultrafiltrate sample was added 20 mg of citric acid. On the long column was placed 500 /zl and on the short column 150/~1 of the ultrafiltrates. The stream from the long column was split, approximately 2/7 diverted to the ninhydrin coil for determining amino acid concentrations, and 5/7 diverted to an isotope flow analyzer to detect zones of radioactivity. The latter instrument was a Model 3241 dual-channel analog flow monitor system equipped with a 2-ml

FIG. 1. Tube and membrane assemblyfor centrifugalultrafiltration.(A) assemble tube; (B) plug for insertion intotubes to support membrane;(C) Nalgene stopper;(D) foldedmembrane (normally with plasma inside) ready to be placed into centrifugetube.

482

VAIDYANATH E T AL.

flow cell packed with anthracene crystals and with a graphic readout (Packard Instrument Company, Downers Grove, Illinois). The eluant from the flow cell was collected in tubes of 5 ml each. Concentrations of amino acids were generally obtained from the integrator values. It was, however, necessary to give careful attention to the uniformity of the integrated peaks, and a peak-heights method was used when uncertainty was indicated in the integrator values. For plasma concentrations greater than 25/ZM, duplicate sample analyses and analyses of standards indicated a coefficient of variation of __-3%. For values less than 25/~M, accuracy was generally considerably less. To determine the radioactivity in the zones from the isotope flow analyzer, the zones were collected, volumes measured, and except for 3H20, 10-ml aliquots taken to dryness at 85°C. The residues were dissolved in 2 ml of H20, 16 ml of Aquasol (New England Nuclear, Boston, Massachusetts) added, and counting conducted in a Tri Carb Liquid Scintillation Spectrometer, Model C3320 (Packard Instrument Co., Chicago, Illinois). Aliquots of the 3H~O zones (2 ml) were taken directly and mixed with the Aquasol solution for counting. Counting for 14C, 3H, and 14C-3H double isotopes were conducted with the spectrometer set for 5000 counts or 100 min, whichever occurred first. The values were normalized with the internal standard of 14C-labeled/3-alanine. RESULTS AND DISCUSSION

Amino Acid Chromatography and Separation of Isotopes A graphic presentation of amino acid ninhydrin and radioactivity peaks from analysis of a standard amino acid solution is shown in Fig. 2. Because of instability, glutamine is omitted from the standard. When present, it appears between glutamate and sarcosine, and its values are calculated using the ninhydrin values of glutamate as a reference. Although the amino acid standards contained tryptophan, the latter is clearly not stable when diluted. (There was a 30% loss in a standard solution kept at 4 ° for 4 months.) To avoid uncertainties, tryptophan values are calculated using the ninhydrin value of histidine as a reference but taking the color value of tryptophan 0.67 that of histidine. Pyruvate and o~-ketoglutarate elute at the same position as glucose. In actual analysis of the plasma ultrafiltrates, the activities of amino acids in many instances are too low to be recorded on the graphic plot. By the use of internal standards it is possible to locate the activities of compounds by overlaying the ninhydrin chart on the radioactivity chart. The appropriate tubes are then removed, pooled and counted. L-[U-3H]tryptophan was added to plasma to measure its recovery by ultrafiltration. In the absence of decanol and benzene the concentration in the ultrafiltrate was 50 to 80% of that in the plasma. Addition ofdecanol and

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570-nm absorbance of the ninhydrin reactants from the long column (Pro and 3 Hyp at 440 nm); (b) is the radioactivity zones from the long column; (c) is the 570-nm absorbance of the ninhydrin reactants from the short column. PSer, phosphoserine; Tau, taurine; PEth, phosphoethanolamine; Asp, aspartate; 3 Hyp, 3-hydroxyproline; Thr, threonine; Ser, serine; Asn, asparagine; Glu, glutamate; Sar, sarcosine; Aad, a-aminoadioate; Pro, proline; Gly, glycine; Ala, alanine; Cit, citrulline; Aib, a-aminoisobutyrate; Abu, ~-aminobutyrate; Val, vafine; Cys, cystine; Ctt, cystathionine; Met, methionine; Ile, isoleucine; Leu, leucine; Nle, norleucine; Acp, l-aminocyclopentane-l-carboxylate; Tyr, tyrosine; Phe, phenylalanine; /3-ala, B-alanine; fl-Aib, /3-aminoisobutyrate; y-Abu, 7-aminobutyrate; Orn, ornithine; 1 MeHis, l-methyl histidine; Lys, lysine; His, histidine; Trp, tryptophan, Cre, creatinine; Ans, anserine; Car, carnosine; Agb, a-amino-y-guanidino-butyrate; Arg, arginine. Long column. (69 x 0.9 cm) Spherical Chromatographic Resin HP-AN90 (Hamilton Co., Whittier, California), 37°, First buffer pH 2.83 (636 ml 1.5 M lithium citrate solution, 106.6 g of LiOH.H20, 180 ml of 25% thiodiglycol solution, 400 ml of 12N HCI, 1.8 ml of pentachlorophenol, diluted to 18 liters with distilled dionized water). At 152 min, second buffer, pH 4.15 ( 1200 ml of 1.5 M lithium citrate solution, 180 ml of 100% thiodiglycol, 200 ml of 12 N HCI, 1.8 ml of pentachlorophenol, diluted to 18 liters). At 282 rain 0.3 N LiOH solution for 30 min. Flow rate of long column 70 ml/hr; 20 ml to coil, 50 ml to flow cell for isotope detection. A makeup buffer, pH 5.15, from the short-column reservoir (see below), at 50 ml/hr was mixed with the stream sent to the coil. The flow rate of the ninhydrin solution (2 fiters 4 M sodium acetate, 6 liters of methycellosolve, 160 g of ninhydrin, 15.2 ml of titanium'tfichloride) was 35 ml/hr. Short Column. (29 × 0.9 cm), Custom Spherical Resin, type PA-35 (Beckman Instrument, Inc., Palo Alto, California), 55°C. First buffer, pH 4.0 (759.5 g of sodium citrate, 330 ml of 12 s HC1, 18 ml of caprylic acid, diluted to 18 liters). At 100 min second buffer, pH 5.15 (706.5 g of sodium citrate, 150 ml of 12 y HCI, 1.8 ml of caprylic acid, diluted to 18 liters). At 219 min a 0.4 N NaOH solution was passed through the column for 25 min. The flow rate of short column was 70 ml/hr, ninhydrin solution 35 ml/hr. 483

484

VAIDYANATH E T AL.

benzene raised this to 99% with a coefficient of variation of 6%. As reported in binding studies elsewhere, one of the anomalous factors in the ultrafiltration of indole compounds is that the ultrafiltrate concentration is always less than that of the ultrafiltrasand, and one is thus required to increase the value of tryptophan in the ultrafiltrates by a factor of 1.1 (4). The technique using decanol and benzene provides the total tryptophan concentration, i.e., both the free and noncovalently bound concentration. Figure 3 shows the tryptophan assays from a study of the plasma arterial and hepatic venous concentration of a seriously injured septic patient. The good agreement between the arterial and hepatic venous values indicate, as is already known, that the concentration of tryptophan is little affected by passage of plasma through the gut and liver. In this study, some very large tryptophan concentration changes occurred with the time progress of this subject. These have been readily followed with the resin column assay system. The importance of following plasma tryptophan values in a study of metabolism in disease is to be emphasized. In animal studies, certain states of sepsis showed marked elevation in plasma tryptophan (6), with relative changes in concentrations larger than found with any other amino acid. Also in the above trauma-septic subject, there was a strong correlation between the plasma-free fatty-acid concentrations and total plasma tryptophan concentration. There are clearly interacting effects between these two plasma components which if appropriately studied may provide clarification of regulatory factors in their metabolism. 50

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485

D E T E R M I N A T I O N O F PLASMA A M I N O A C I D S TABLE 1 COMPARISON OF M E T H O D S OF A M M O N I A DETERMINATION a

Manual A~efial plasma 123 86 83 86 85 90 95 99

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238 186 234 131 133 170

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16 0 10 7 8 ± 12b

" Values were for different sampling times, or different dogs. Manual assays were conducted immediately after blood collections, resin column values were obtained from plasma which stood 4 to 8 hr at 0 ° before ultrafiltration was completed. b Standard deviation.

Several plasmas were analyzed for ammonia by the manual technique of Seligson et al. (7) and the values compared with resin column values (Table 1). The values from the resin columns are clearly not as reliable as those obtained manually, averaging 40, 44, and 8 tzmol/liter higher by the resin column method. However, resin column ammonia values do identify significant trends. Figure 4 reports ammonia values obtained for the above trauma-septic patient. These values were from the same resin column analyses that the tryptophan concentrations were determined. The arterial and hepatic venous results clearly support each other, although there were several hepatic venous analyses higher than the arterial values, which we believe is not correct. The small differences between the venous and

486

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arterial samples indicate that the liver was not functioning optimally in ammonia removal. In contrast, for example, in studies with normal or less severely ill dogs, the liver, independent of the concentration entering, demonstrated a propensity for returning the ammonia values to approximately their basal state (80-130 /zM). The liver of this patient did not demonstrate this capability. In the paired analyses by different techniques reported above, the portal venous ammonia concentrations obtained from the resin column were 30-100/ZM higher than the arterial concentrations. Similar comparisons with the manual method for determining ammonia gave differences from 40-80 /xM. The ammonia analysis by the ultrafiltration and resin column technique, albeit not as sensitive as when conducted manually, do thus provide useful complementary information in physiological amino acid analysis. The increase in ammonia in arterial and portal venous samples upon standing, such as occurs in plasma processed for the resin column assay system, is probably attributable to the presence of the enzyme glutaminase in the plasma. The fact that this did not occur in the hepatic venous samples (or occurred to a much lower extent) indicates that glutaminase is removed by the liver. The destruction of glutamine in plasma ultrafiltrates was evaluated by analyzing plasma ultrafiltrates immediately after collection and again after standing 2 weeks at 4 and -80°C. At 4° there was a 10 to 20% reduction in glutamine concentration and a 50 to 200% rise in glutamate. At -80°C there was no loss in glutamine or gain in glutamate values. The findings of stability at -80°C are consistent with studies earlier reported by Armstrong and Stave (1973).

DETERMINATION

487

OF PLASMA AMINO ACIDS TABLE 2

CONCENTRATION AND SPECIFIC ACTIVITIES OF FREE AMINO ACIDS AND SOME OTHER COMPOUNDS IN THE PLASMAS OF THREE 4-DAY FASTING DOGSa Specific activities (cpm//~mol)

C o n c e n t r a t i o n s (/~M) plasma water Number PSer Tau PEth Urea Asp Thr Ser Asn Glu Gln Pro Gly Ala Abu Val Met Ile Leu Tyr Phe Orn NHa 1MeHis Lys His Trp Car Arg Glucose b Lactate ~ Wateff

Dog 414

Dog 427

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10 89

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2900 3 194 131 68 23 355 92 129 275 73 228 51 78 158 42 87 18 88 -154 79 18 49 79 7440 430 --

5572 3 136 64 55 12 68 57 94 122 53 204 29 80 162 23 55 12 139 15 88 55 22 14 40 5222 431 --

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Dog 427

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4994

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2488

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5230

4315

3571

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3906

3028

8391

5577

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413 (103) 777 8700

362 (61) -6800

400 (56) 4700

Catheters were placed in the dogs 3 days prior to the experiment. Isotopes were infused at c o n s t a n t rate to obtain p s e u d o s t e a d y state conditions. Alanine was infused with ~4C-label and the other a m i n o acids with 3H-label. See legend Fig. 1 for abbreviations. b Glucose and lactate concentrations were determined by nonresin c o l u m n techniques. c Water, cpm/ml.

To illustrate the technique, analyses of arterial samples of three normal 4-day fasted dogs are reported in Table 2. In both animals samples were drawn after continuously infusing isotopically labeled amino acids for 60 min (a time previously determined to be sufficient to obtain a

488

VA IDYANATH E T AL.

pseudo-steady state condition). The amino acid concentrations are in fair agreement with other reports (8,9). The 4-day fast preceding this sampling results in lower values than would normally be seen in animals after an overnight fast. Dog 856 has a number of substances at considerably lower concentration than in the other two animals. This probably reflects a more protein conservative state of starvation in this animal. Glutamate values are considerably lower than where acid-precipitation methods are used for deproteinization (1). This latter is believed due to the milder method of ultrafiltration used. While such calculations were not made, if one is supplied the concentration, specific activities, and infusion rates, the turnover and fractional plasma clearance rates can be obtained. Although pyruvate and a-ketoglutarate both cochromatograph with glucose, separation of these three compounds by paper chromatography in several studies indicates that after 60-min infusion of alanine and the other amino acids, only 5% of the activity in the glucose zone was due to pyruvate and a-ketoglutarate activity was negligible. In view of the low activities in these substances, the activites in the "glucose zone" were generally treated as if they were solely due to glucose. Clearly, however, one must be cautious in this assumption. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Stein, W. H., and Moore, S. (1954)J. Biol. Chem. 211, 915-926. Hamilton, P. B. (1962)Ann. N. Y. Acad. Sci. 102, 55-75. Armstrong, M. D., and Stave, U. (1973) Metabolism 22, 549-560. McMenamy, R. H., Lund, C. C., Van Marcke, J., and Oncley, J. L. (1961)Arch. Biochem. Biophys. 939 135-139. Benson, J. V., Gordon, M. J., and Patterson, J. A. (1967)Anal. Biochem. 18, 228-240. Vaidynaith, N., Birkhahn, R., Trietley, G., Yuan, T. F., Moritz, E., Weissenhoffer, W., McMenamy, R., and Border, J. (1975)J. Trauma. (In press). Seligson, D., and Hirahara, K. (1957) J. Lab. Clin. Med. 49, 962-974. McMenamy, R. H., Vang, J., and Drapanas, T. (1965)Amer. J. Physiol. 209, 1046-1052. McMenamy, R. H., Shoemaker, W. C., Richmond, J. E., and Elwyn, D. (1962)Amer. J. Physiol. 202, 407-414.