ANALYTICAL
BIOCHEMISTRY
65,
298-304 (i 975)
Determination
of
L-l-a-Alanine
‘“C-labeled
Specific
Plasma
Radioactivity
P. E. B. REILLY department
of B~oc~~~~stry. Uni~~er~~ty of Liverpoot, L69 3BX, England
Received September 26. 1974: accepted November
11, 1974
A method is described which anables the specific radioactivity of W-labeled L + a-alanine in plasma to be determined. Plasma alanine concentration is determined spec~ophotometrically using alanine dehydrogenase. In a separate procedure, this enzyme is also used to convert ‘*C labeled alanine and added carrier alanine, to pyruvate. The phenylhyd~zone derivative of the pyruvate is then prepared and is assayed for radioactivity after crystallization to constant specific radioactivity. A maximum error of 1.5% for any one specific radioactivity determination was found.
During preparations for an investigation into the effects of adrenalectomy on alanine cycle activity (1) in sheep, it became apparent that no conveniently rapid, simple, and accurate method was available for determination of ‘*C-labeled plasma L + a-alanine specific radioactivity. Ion exchange elution chromatography appears to be the method most frequently used, determination of radioactivity being made on the eluent either using a continuous flow cell (2) or batch scintillation counting (3). This method has the advantage of enabling specific radioactivities of many of the amino acids to be determined in 1 aliquot of any particular plasma sample. It sufIers however from the disadvantages of lengthy elution times and extensive dilution of sample activity in the elution buffers. A further, overriding, disadvantage is that it can only be conveniently conducted using relatively elaborate, commercially available, automated systems which necessitate heavy capital expenditure and skilled personnel commitment for maintenance and preparative procedures. Paper chromatography, thin layer chromatography, and gas liquid chromatography are all methods that have been used to separate amino acids or amino acid derivatives from each other and in some cases from other ionic materials, (4-7). None of these methods is generally used to facilitate determination of amino acid specific radioactivity owing to severe limitations, imposed by the need for small sample sizes, on the accuracy of the assay for radioactivity, A method has accordingly been developed for determination of i4Clabeled plasma L + cw-alanine specific radioactivity which overcomes 298 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
L +
(Y-ALA
SPECIFIC
RADIOACTIVITY
DETERMINATION
299
these problems. It is simple, accurate, and reasonably rapid. It makes use of the reverse isotope dilution principle and involves enzymic conversion of alanine, after its extraction from plasma, to pyruvate with subsequent assay of pyruvate phenyl hydrazone for radioactivity after crystallization to constant specific activity. MATERIALS
AND
METHODS
Analytical grade reagents and deionized water were used throughout. Buffer I. pH 10.0, 200 mM aqueous sodium carbonate: bicarbonate containing 100 pmol EDTA and 20 mmol hydrazine hydrate/ 100 ml. Buffer 2. pH 10.6, 1.0 M aqueous piperazine hydrate, 1 mM EDTA adjusted with concentrated HCl. Triethylamine solution. 2.0 M in 20% aqueous acetone. Phenyl hydrazine hydrochloride solution. Leaflets freshly recrystallized from ethanol and dried at 6o”C, aqueous 10% (w/v) solution. Alanine dehydrogenase (E.C. 1.4. I. 1.) Suspension in ammonium sulphate, Boehringer Corporation, (30 units/mg protein, 5 mg protein/ml). Nicotinamide adenine dinucleotide (free acid Beohringer Corporation) 36 mg/ml aqueous solution prepared freshly for each period of use. L + cY-Alanine. Beohringer Corporation: solution 1, 174 mg/lOO ml, stock solution, diluted 1 to 20 for use; solution 2, 20.0 mg/ml. [U-14C]L + a-alanine. Radiochemical Centre, Amersham. Fifty microcuries in 25-m] aqueous stock solution is stored at - 15°C. Diluted with carrier L + a-alanine to 2.0 nCi and 10.0 mg alaninel0.5 ml for use. [ U-14C] L + -Lactate sodium salt. Radiochemical Centre Amersham. 50 &i in 25-m] aqueous stock solution stored at - 15°C. Diluted with carrier lithium L+ lactate (Sigma) to 2.0 nCi and 40 pg lactate/ml. [ 1-14C] Pyruvic acid, sodium salt. Radiochemical Centre Amersham. Stock: 50.0 &i in 25.0 ml aqueous solution, stored at - 15°C. Diluted for use to 2.0 nCi and 40.0 pg sodium pyruvatell .O ml. Mixed U-‘“C-labeled high specific radioactivity amino acids (CFB 104) Radiochemical Centre, Amersham. An aliquot was diluted immediately before use without addition of carrier to 10.0 nCi/ml. Each of the 14 amino acids was present at a specific radioactivity of > 45 mCi/matom of carbon. Scintillation fluid. Toluene: Triton-X 100, mixed in the ratio 2: 1, containing 5.0 g PPO and 0.6 g POPOP/litre. Bovine serum albumin. 62.5 mg protein/ml. Armour Pharmaceutical Company. Sheep jugular blood plasma (obtained using heparin as anticoagulant) stored at - 15°C prior to use. The description of determination of plasma 14C-labeled L + a-alanine specific radioactivity may be conveniently divided into four groups of procedures. These are (1) enzymic determination of plasma alanine con-
300
P. E. B. REILLY
centration; (2) Addition of carrier alanine to a l-ml aliquot of plasma and its separation from pyruvic and lactic acids using a small Dowex 50 W cation exchange resin column; (3) Enzymic conversion of alanine to pyruvate and purification of pyruvate phenyl hydrazone by recrystallization from aqueous solution; and, (4) Radioactivity assay of weighed amounts of this derivative after solubilization in methanol. PROCEDURES
DESCRlBED
1. Plasma alanine quantitatiun. To OS-ml untreated plasma was added 50 ~1 concentrated perchloric acid. After thoroughly mixing and standing at room temperature for 5 min the suspension was centrifuged for 5 min in a bench centrifuge. A 200-~1 aliquot of the clear supernatant was removed and mixed with 100 ~1 of 2.0 M aqueous KOH solution and after mixing and standing in ice for five minutes the suspension was centrifuged as described above. From the clear supernatant solution a 200~~1 aliquot was taken and added to a tube containing the following mixture; 2.5 ml buffer I, 10 ~1 bovine serum albumin, 200 ~1 NAD solution, 20 ~1 alanine dehydrogenase. A reagent blank prepared with water in place of plasma was carried through the above procedure at the same time and after 45 min incubation at 25°C samples were read against the blank at 340 nm in l-cm light path quartz cuvetts using a Pye Unicam SP1800 double beam recording spectrophotometer. This method had previously been found to be satisfactorily accurate for L + cr-alanine determination from the test results shown in Table 1. The deproteinization procedure had also been shown to be satisfactory as a result of finding that the % recovery of alanine (50 ~1 undiluted TABLE THE
1
YIELD OF NADH AMOUNTS
IN THE REACTION OF ALANINE INITIALLY
Alanine initially present in cuvette Ol.mol) 0.1953 0.0976 0.0488 0.0195
IS COMPARED WITH THE KNOWN PRESENT IN THE CUVETTE~.~
Mean NADH produced *SEM(n) (pm00 0.1992 0.0981 0.0487 0.0193
k 2 i2
0.0014(7) 0.~57(4) 0.00056(5) 0.00058(3)
% Recovery (NADHiAlanine) -cSEM(n) 101.98 100.47 99.78 98.65
i 2 + t
0.74(7) 0.59(4) 1.16(5) 3.01(3)
(1 20-200 ~1 aliquots of diluted alanine solution I were used, water was added where necessary to make a sample volume of 200 ~1. * The data were calculated on the basis that the molar extinction coefficient of NADH under these circumstances (l-cm light path. 340 nm), is 6220, and the sample volume is known (2.93 ml).
L +
(r-ALA
SPECIFIC
RADIOACTIVITY
DETERMINATION
301
alanine Solution I containing 0.976 pmol) added to plasma was 97.6 + 0.26 (5). 2. Purijication of alanine. This procedure is a modification of that described by Harris et al. (8). Small (0.6 X 1.5 cm) Dowex 50 W cation exchange resin columns in the H+ form rinsed to pH 6-7 with water were prepared with a flow rate of approximately 0.15 ml/min. Through such columns were run in sequence: (a) a mixture (conveniently contained in a scintillation vial) of 1.0 ml untreated plasma, 0.5 ml alanine solution 2, 3 drops of 2 N HCl (to adjust the pH to 2.5-3.0); (b) 2.0 ml distilled water rinse of the vial; (c) 2.0 ml distilled water. (These three fractions constitute the column effluents referred to later in the section.) (d) Triethylamine solution (3.0 ml) (collected into a scintillation vial). This last fraction was shown in the following way to contain the alanine. Alanine was extracted from plasma as described above except that 0.5 ml (2.0 nCi) of the diluted U‘“C-labeled alanine solution containing 10.0 mg carrier alanine was added in place of the 0.5 ml unlabeled carrier alanine solution 2. The contents of the vials were evaporated to dryness by vacuum dessication (overnight) and dissolved in 1.0 ml water. After addition of 10 ml of scintillation fluid and dark adaptation for 2 h the samples were counted in a liquid Scintillation spectrometer (SL40 Intertechnique). The % recovery of 14C added to plasma was 99.13 * 0.3 (6). For the specific radioactivity of the pyruvate derivative produced from alanine accurately to reflect alanine specific radioactivity endogenous plasma pyruvate must not be presented in the alanine fraction. This argument applies also to endogenous plasma lactate since the enzyme used, although of very high purity, is specified as containing lactate dehydrogenase activity at less than 0.01% alanine dehydrogenase activity. Both these metabolites were shown to be quantitatively recovered in the column effluent fractions in the following way. To 1.0 ml untreated plasma were added 0.5-ml alanine solution 2, 3 drops 2 N HCl and either 1.O ml of diluted U-14C-labeled lactate or 1.O ml of diluted 1-14C-labeled pyruvate. Alanine was extracted as described above and each of the effluent fractions was assayed for radioactivity after addition of 10 ml scintillation fluid. The summed % recoveries of radioactivity in the effluent fractions were 99.79 2 0.22 (6). in the case of pyruvate, and in the case of lactate 99.88 + 0.19 (6). Glucose was found also to be quantitatively recovered in the effluent fractions. This was shown using the glucose oxidase method described by Krebs et al. (9) by testing a loo-p1 aliquot of a deproteinized filtrate of the pooled effluent fractions obtained as described above. The filtrate was prepared by centrifugation after addition of 2.0 ml of 0.6 M aqueous
302
P.
E.
B.
REILLY
perchloric acid to the pooled effluents. A suitably diluted aliquot of the plasma used was similarly deproteinized and used as the reference for % recovery, which was found to be 99.99 + 0.17 (6). This is a fortuitous finding since radioactivity in glucose has been shown not to interfere in the production of pyruvate phenylhydrazone. However, since glucose is radioactively labeled in experimental samples, the effluents may conveniently be used for glucose specific radioactivity determination. 3. Conversion phenylhydrazone.
of alanine
to pyruvate
and production
of pyruvate
The dried eluate prepared as described in the last section was dissolved in 2.0 ml buffer 2 and to this solution was added 50 ~1 hydrazine hydrate, 100 mg NAD, and 100 ,ul of the enzyme suspension. The final pH of this mixture was in the range 10.0-10.2. The vials were incubated for 2 h at 25°C after which time concentrated HCl was added dropwise (18-25 drops required) to adjust the pH to 2.8-3.0. To the acidified solution was then added 0.5 ml of the phenylhydrazine solution and the vials shaken gently at room temperature. Derivative started to appear within two minutes and shaking was continued for 20 min. Ice was then packed round the vials and shaking was continued for a further 20 min. The crystals were then harvested on a millipore filter (HAWP 02500) using suction, scraped off the disc and dispersed in 10 ml water in a boiling tube. The crystals were dissolved by vigorously boiling the suspension for a few moments whereupon the solution was allowed to cool to room temperature. After a few minutes small crystals started to appear at the air:liquid interface and the tubes were then shaken once gently. A crop of uniformly small needlelike yellow crystals formed within the next few minutes. These were harvested on filter pads as described previously and dried for 2 h at 60°C. They were then weighed accurately to the second place of decimals of milligrams using a Mettler M5 microbalance. The derivative prepared under these conditions has been exhaustively analysed and identified as pyruvate phenylhydrazone, (10). 4. Radioactivity assay. To each vial containing crystals prepared as described above was added 1.0 ml methanol. Gentle shaking effected complete solution within a few moments and after addition of 10 ml scintillation fluid and dark adaptation for 2 h they were assayed for radioactivity. Efficiency of counting lay in the range 83-87% and a quench curve was used to account for variations in quenching when dpm were calculated from cpm. The results of using this method are shown in Table 2, where close agreement between the theoretical value and the values found is shown. It is clear from these results that the plasma amino acids other than alanine which were present in the incubation mixture but which were not labeled with 14C did not affect the accuracy of the determination. This is
L
+ (Y-ALA
SPECIFIC
RADIOACTIVITY TABLE
RESULTS
OF SPECIFIC
RADIOACTIVITY
L+cI-ALANINE
Derivative counted (mg) 7.381 9.066 7.854 7.600 8.708 8.476
Corresponding weight of alanine (mg) 3.6905 4.533 3.927 3.800 4.354 4.238
DETERMINATION
2 DETERMINATION
EXTRACTED
303
FROM
Activity in vial counted (nCi) 0.740 0.896 0.770 0.756 0.858 0.833 Mean kSEM(n)
1 ml
OF U-T-LABELED OF PLASMA”
Specific radioactivity (nCi/mg alanine) 0.2005 0.1976 0.1961 0.1989 0.1971 0.1966 0.1978 10.00062 (6)
Error vi/c) +0.70 -0.75 -1.51 -0.11 -1.01 -1.26
” Endogenous alanine in plasma was 0.0436 mg to which 10.0 mg of unlabeled alanine containing 2.0 nCi U-“C-labeled L+cY-alanine had been added. The theoretical specific radioactivity was therefore 0.1991 nCi/mg alanine.
not an unexpected result since the affinity of alanine dehydrogenase for alanine is very much higher than for any of the other amino acids whose deamination it will catalyse (11). Nevertheless evidence is needed to show that plasma alanine specific radioactivity values would not be erroneously high as a result of contamination of pyruvate phenyl hydrazone by those 14C-labeled phenylhydrazones of 2-0x0 acids which can crystallize and which may be produced by, even minimal, deamination of the other amino acids if these were to be present at much higher specific radioactivities than alanine. The experiment described in the following section furnishes this evidence. To 1 ml of plasma containing 10.0 mg unlabeled carrier and 2.0 nCi U-14C-labeled L + (Y alanine was added an aliquot of the diluted mixture of 14 high specific activity U-14C-labeled amino acids containing 2.0 nCi 14C. Pyruvate phenylhydrazone was prepared from this mixture and assayed for radioactivity as detailed in Sections 2 and 3, respectively. Its alanine content was determined as described in Section 1. The total alanine content was 10.0482 mg. Radioactivity in alanine was 2.20 nCi since alanine accounted for 10% of the radioactivity in the solution of mixed U-14C-labeled amino acids. The theoretical specific radioactivity of alanine in this mixture was therefore 0.2 19 nCi/mg alanine. The value found using the procedure described was 0.223 k 0.0018 (4) nCi/mg alanine. This result clearly indicates the suitability of this method of assay for plasma l*C L + a-alanine specific radioactivity. To derive the specific radioactivity of 14C-labeled alanine in a l-ml plasma sample obtained under experimental conditions to which unla-
304
P. E.
B.
REILLY
beled carrier alanine had been added the following calculation used:
should be
net cpm x 100 x 178 x total mg alanine presented for ion exchange % counting efficiency x 2220 x 89 x mg derivative X mg alanine in 1.O ml plasma giving the answer as nCi mg plasma alanine-‘. ACKNOWLEDGMENTS The author wishes to thank Miss K. J. H. Robson and Mr. J. D. Warner for their skilled technical assistance.
REFERENCES 1. Felig, P. (1973) Metabolism 22, 179. 2. Schram, E., and Lombaert. R. (1963) Organic Scintillation Detectors, Elsevier, New York. 3. Wolff, J. E., and Bergman, E. N. (1972) Amer. J. Physiol. 233, 447. 4. Pataki, G. (1968) Techniques of Thin Layer Chromatography in Amino Acid and Peptide Chemistry. Ann Arbor Science Publishers, Michigan. 5. Zweig, G., and Whitaker, J. R. (1967) Paper Chromatography and Electrophoresis, Vol. 1, Academic Press, New York. 6. Sherma, T., and Zweig, G. (1971) Paper Chromatography and Electrophoresis. Vol. 2, Academic Press, New York. 7. Cavagnol, J. C., and Betker, W. R. (1967) The Practice of Gas Chromatography. (L. S. Ettre. and A. Zlatkis, eds.), Chapter 3, Interscience Publishers, New York. 8. Harris, C. K.. Tigane. E., and Hanes, C. S. (1961) Can J. Biochem. Physiol. 39, 439. 9. Krebs, H. A., Bennet, D. A. H., de Gasquet, P.. Gascoyne, T., and Yoshida, T. (1963) Biochem. J. 86, 22. 10. Reilly, P. E. B. (1974) Anal. Biochem. (in press). 11. Yoshida, A., and Freese. E. (1970) in Methods in Enzymology (H. Tabor, and C. W. Tabor, eds.), Vol. XVIIA, Academic Press, New York.
BIOCHEMISTRY
65,
298-304 (i 975)
Determination
of
L-l-a-Alanine
‘“C-labeled
Specific
Plasma
Radioactivity
P. E. B. REILLY department
of B~oc~~~~stry. Uni~~er~~ty of Liverpoot, L69 3BX, England
Received September 26. 1974: accepted November
11, 1974
A method is described which anables the specific radioactivity of W-labeled L + a-alanine in plasma to be determined. Plasma alanine concentration is determined spec~ophotometrically using alanine dehydrogenase. In a separate procedure, this enzyme is also used to convert ‘*C labeled alanine and added carrier alanine, to pyruvate. The phenylhyd~zone derivative of the pyruvate is then prepared and is assayed for radioactivity after crystallization to constant specific radioactivity. A maximum error of 1.5% for any one specific radioactivity determination was found.
During preparations for an investigation into the effects of adrenalectomy on alanine cycle activity (1) in sheep, it became apparent that no conveniently rapid, simple, and accurate method was available for determination of ‘*C-labeled plasma L + a-alanine specific radioactivity. Ion exchange elution chromatography appears to be the method most frequently used, determination of radioactivity being made on the eluent either using a continuous flow cell (2) or batch scintillation counting (3). This method has the advantage of enabling specific radioactivities of many of the amino acids to be determined in 1 aliquot of any particular plasma sample. It sufIers however from the disadvantages of lengthy elution times and extensive dilution of sample activity in the elution buffers. A further, overriding, disadvantage is that it can only be conveniently conducted using relatively elaborate, commercially available, automated systems which necessitate heavy capital expenditure and skilled personnel commitment for maintenance and preparative procedures. Paper chromatography, thin layer chromatography, and gas liquid chromatography are all methods that have been used to separate amino acids or amino acid derivatives from each other and in some cases from other ionic materials, (4-7). None of these methods is generally used to facilitate determination of amino acid specific radioactivity owing to severe limitations, imposed by the need for small sample sizes, on the accuracy of the assay for radioactivity, A method has accordingly been developed for determination of i4Clabeled plasma L + cw-alanine specific radioactivity which overcomes 298 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
L +
(Y-ALA
SPECIFIC
RADIOACTIVITY
DETERMINATION
299
these problems. It is simple, accurate, and reasonably rapid. It makes use of the reverse isotope dilution principle and involves enzymic conversion of alanine, after its extraction from plasma, to pyruvate with subsequent assay of pyruvate phenyl hydrazone for radioactivity after crystallization to constant specific activity. MATERIALS
AND
METHODS
Analytical grade reagents and deionized water were used throughout. Buffer I. pH 10.0, 200 mM aqueous sodium carbonate: bicarbonate containing 100 pmol EDTA and 20 mmol hydrazine hydrate/ 100 ml. Buffer 2. pH 10.6, 1.0 M aqueous piperazine hydrate, 1 mM EDTA adjusted with concentrated HCl. Triethylamine solution. 2.0 M in 20% aqueous acetone. Phenyl hydrazine hydrochloride solution. Leaflets freshly recrystallized from ethanol and dried at 6o”C, aqueous 10% (w/v) solution. Alanine dehydrogenase (E.C. 1.4. I. 1.) Suspension in ammonium sulphate, Boehringer Corporation, (30 units/mg protein, 5 mg protein/ml). Nicotinamide adenine dinucleotide (free acid Beohringer Corporation) 36 mg/ml aqueous solution prepared freshly for each period of use. L + cY-Alanine. Beohringer Corporation: solution 1, 174 mg/lOO ml, stock solution, diluted 1 to 20 for use; solution 2, 20.0 mg/ml. [U-14C]L + a-alanine. Radiochemical Centre, Amersham. Fifty microcuries in 25-m] aqueous stock solution is stored at - 15°C. Diluted with carrier L + a-alanine to 2.0 nCi and 10.0 mg alaninel0.5 ml for use. [ U-14C] L + -Lactate sodium salt. Radiochemical Centre Amersham. 50 &i in 25-m] aqueous stock solution stored at - 15°C. Diluted with carrier lithium L+ lactate (Sigma) to 2.0 nCi and 40 pg lactate/ml. [ 1-14C] Pyruvic acid, sodium salt. Radiochemical Centre Amersham. Stock: 50.0 &i in 25.0 ml aqueous solution, stored at - 15°C. Diluted for use to 2.0 nCi and 40.0 pg sodium pyruvatell .O ml. Mixed U-‘“C-labeled high specific radioactivity amino acids (CFB 104) Radiochemical Centre, Amersham. An aliquot was diluted immediately before use without addition of carrier to 10.0 nCi/ml. Each of the 14 amino acids was present at a specific radioactivity of > 45 mCi/matom of carbon. Scintillation fluid. Toluene: Triton-X 100, mixed in the ratio 2: 1, containing 5.0 g PPO and 0.6 g POPOP/litre. Bovine serum albumin. 62.5 mg protein/ml. Armour Pharmaceutical Company. Sheep jugular blood plasma (obtained using heparin as anticoagulant) stored at - 15°C prior to use. The description of determination of plasma 14C-labeled L + a-alanine specific radioactivity may be conveniently divided into four groups of procedures. These are (1) enzymic determination of plasma alanine con-
300
P. E. B. REILLY
centration; (2) Addition of carrier alanine to a l-ml aliquot of plasma and its separation from pyruvic and lactic acids using a small Dowex 50 W cation exchange resin column; (3) Enzymic conversion of alanine to pyruvate and purification of pyruvate phenyl hydrazone by recrystallization from aqueous solution; and, (4) Radioactivity assay of weighed amounts of this derivative after solubilization in methanol. PROCEDURES
DESCRlBED
1. Plasma alanine quantitatiun. To OS-ml untreated plasma was added 50 ~1 concentrated perchloric acid. After thoroughly mixing and standing at room temperature for 5 min the suspension was centrifuged for 5 min in a bench centrifuge. A 200-~1 aliquot of the clear supernatant was removed and mixed with 100 ~1 of 2.0 M aqueous KOH solution and after mixing and standing in ice for five minutes the suspension was centrifuged as described above. From the clear supernatant solution a 200~~1 aliquot was taken and added to a tube containing the following mixture; 2.5 ml buffer I, 10 ~1 bovine serum albumin, 200 ~1 NAD solution, 20 ~1 alanine dehydrogenase. A reagent blank prepared with water in place of plasma was carried through the above procedure at the same time and after 45 min incubation at 25°C samples were read against the blank at 340 nm in l-cm light path quartz cuvetts using a Pye Unicam SP1800 double beam recording spectrophotometer. This method had previously been found to be satisfactorily accurate for L + cr-alanine determination from the test results shown in Table 1. The deproteinization procedure had also been shown to be satisfactory as a result of finding that the % recovery of alanine (50 ~1 undiluted TABLE THE
1
YIELD OF NADH AMOUNTS
IN THE REACTION OF ALANINE INITIALLY
Alanine initially present in cuvette Ol.mol) 0.1953 0.0976 0.0488 0.0195
IS COMPARED WITH THE KNOWN PRESENT IN THE CUVETTE~.~
Mean NADH produced *SEM(n) (pm00 0.1992 0.0981 0.0487 0.0193
k 2 i2
0.0014(7) 0.~57(4) 0.00056(5) 0.00058(3)
% Recovery (NADHiAlanine) -cSEM(n) 101.98 100.47 99.78 98.65
i 2 + t
0.74(7) 0.59(4) 1.16(5) 3.01(3)
(1 20-200 ~1 aliquots of diluted alanine solution I were used, water was added where necessary to make a sample volume of 200 ~1. * The data were calculated on the basis that the molar extinction coefficient of NADH under these circumstances (l-cm light path. 340 nm), is 6220, and the sample volume is known (2.93 ml).
L +
(r-ALA
SPECIFIC
RADIOACTIVITY
DETERMINATION
301
alanine Solution I containing 0.976 pmol) added to plasma was 97.6 + 0.26 (5). 2. Purijication of alanine. This procedure is a modification of that described by Harris et al. (8). Small (0.6 X 1.5 cm) Dowex 50 W cation exchange resin columns in the H+ form rinsed to pH 6-7 with water were prepared with a flow rate of approximately 0.15 ml/min. Through such columns were run in sequence: (a) a mixture (conveniently contained in a scintillation vial) of 1.0 ml untreated plasma, 0.5 ml alanine solution 2, 3 drops of 2 N HCl (to adjust the pH to 2.5-3.0); (b) 2.0 ml distilled water rinse of the vial; (c) 2.0 ml distilled water. (These three fractions constitute the column effluents referred to later in the section.) (d) Triethylamine solution (3.0 ml) (collected into a scintillation vial). This last fraction was shown in the following way to contain the alanine. Alanine was extracted from plasma as described above except that 0.5 ml (2.0 nCi) of the diluted U‘“C-labeled alanine solution containing 10.0 mg carrier alanine was added in place of the 0.5 ml unlabeled carrier alanine solution 2. The contents of the vials were evaporated to dryness by vacuum dessication (overnight) and dissolved in 1.0 ml water. After addition of 10 ml of scintillation fluid and dark adaptation for 2 h the samples were counted in a liquid Scintillation spectrometer (SL40 Intertechnique). The % recovery of 14C added to plasma was 99.13 * 0.3 (6). For the specific radioactivity of the pyruvate derivative produced from alanine accurately to reflect alanine specific radioactivity endogenous plasma pyruvate must not be presented in the alanine fraction. This argument applies also to endogenous plasma lactate since the enzyme used, although of very high purity, is specified as containing lactate dehydrogenase activity at less than 0.01% alanine dehydrogenase activity. Both these metabolites were shown to be quantitatively recovered in the column effluent fractions in the following way. To 1.0 ml untreated plasma were added 0.5-ml alanine solution 2, 3 drops 2 N HCl and either 1.O ml of diluted U-14C-labeled lactate or 1.O ml of diluted 1-14C-labeled pyruvate. Alanine was extracted as described above and each of the effluent fractions was assayed for radioactivity after addition of 10 ml scintillation fluid. The summed % recoveries of radioactivity in the effluent fractions were 99.79 2 0.22 (6). in the case of pyruvate, and in the case of lactate 99.88 + 0.19 (6). Glucose was found also to be quantitatively recovered in the effluent fractions. This was shown using the glucose oxidase method described by Krebs et al. (9) by testing a loo-p1 aliquot of a deproteinized filtrate of the pooled effluent fractions obtained as described above. The filtrate was prepared by centrifugation after addition of 2.0 ml of 0.6 M aqueous
302
P.
E.
B.
REILLY
perchloric acid to the pooled effluents. A suitably diluted aliquot of the plasma used was similarly deproteinized and used as the reference for % recovery, which was found to be 99.99 + 0.17 (6). This is a fortuitous finding since radioactivity in glucose has been shown not to interfere in the production of pyruvate phenylhydrazone. However, since glucose is radioactively labeled in experimental samples, the effluents may conveniently be used for glucose specific radioactivity determination. 3. Conversion phenylhydrazone.
of alanine
to pyruvate
and production
of pyruvate
The dried eluate prepared as described in the last section was dissolved in 2.0 ml buffer 2 and to this solution was added 50 ~1 hydrazine hydrate, 100 mg NAD, and 100 ,ul of the enzyme suspension. The final pH of this mixture was in the range 10.0-10.2. The vials were incubated for 2 h at 25°C after which time concentrated HCl was added dropwise (18-25 drops required) to adjust the pH to 2.8-3.0. To the acidified solution was then added 0.5 ml of the phenylhydrazine solution and the vials shaken gently at room temperature. Derivative started to appear within two minutes and shaking was continued for 20 min. Ice was then packed round the vials and shaking was continued for a further 20 min. The crystals were then harvested on a millipore filter (HAWP 02500) using suction, scraped off the disc and dispersed in 10 ml water in a boiling tube. The crystals were dissolved by vigorously boiling the suspension for a few moments whereupon the solution was allowed to cool to room temperature. After a few minutes small crystals started to appear at the air:liquid interface and the tubes were then shaken once gently. A crop of uniformly small needlelike yellow crystals formed within the next few minutes. These were harvested on filter pads as described previously and dried for 2 h at 60°C. They were then weighed accurately to the second place of decimals of milligrams using a Mettler M5 microbalance. The derivative prepared under these conditions has been exhaustively analysed and identified as pyruvate phenylhydrazone, (10). 4. Radioactivity assay. To each vial containing crystals prepared as described above was added 1.0 ml methanol. Gentle shaking effected complete solution within a few moments and after addition of 10 ml scintillation fluid and dark adaptation for 2 h they were assayed for radioactivity. Efficiency of counting lay in the range 83-87% and a quench curve was used to account for variations in quenching when dpm were calculated from cpm. The results of using this method are shown in Table 2, where close agreement between the theoretical value and the values found is shown. It is clear from these results that the plasma amino acids other than alanine which were present in the incubation mixture but which were not labeled with 14C did not affect the accuracy of the determination. This is
L
+ (Y-ALA
SPECIFIC
RADIOACTIVITY TABLE
RESULTS
OF SPECIFIC
RADIOACTIVITY
L+cI-ALANINE
Derivative counted (mg) 7.381 9.066 7.854 7.600 8.708 8.476
Corresponding weight of alanine (mg) 3.6905 4.533 3.927 3.800 4.354 4.238
DETERMINATION
2 DETERMINATION
EXTRACTED
303
FROM
Activity in vial counted (nCi) 0.740 0.896 0.770 0.756 0.858 0.833 Mean kSEM(n)
1 ml
OF U-T-LABELED OF PLASMA”
Specific radioactivity (nCi/mg alanine) 0.2005 0.1976 0.1961 0.1989 0.1971 0.1966 0.1978 10.00062 (6)
Error vi/c) +0.70 -0.75 -1.51 -0.11 -1.01 -1.26
” Endogenous alanine in plasma was 0.0436 mg to which 10.0 mg of unlabeled alanine containing 2.0 nCi U-“C-labeled L+cY-alanine had been added. The theoretical specific radioactivity was therefore 0.1991 nCi/mg alanine.
not an unexpected result since the affinity of alanine dehydrogenase for alanine is very much higher than for any of the other amino acids whose deamination it will catalyse (11). Nevertheless evidence is needed to show that plasma alanine specific radioactivity values would not be erroneously high as a result of contamination of pyruvate phenyl hydrazone by those 14C-labeled phenylhydrazones of 2-0x0 acids which can crystallize and which may be produced by, even minimal, deamination of the other amino acids if these were to be present at much higher specific radioactivities than alanine. The experiment described in the following section furnishes this evidence. To 1 ml of plasma containing 10.0 mg unlabeled carrier and 2.0 nCi U-14C-labeled L + (Y alanine was added an aliquot of the diluted mixture of 14 high specific activity U-14C-labeled amino acids containing 2.0 nCi 14C. Pyruvate phenylhydrazone was prepared from this mixture and assayed for radioactivity as detailed in Sections 2 and 3, respectively. Its alanine content was determined as described in Section 1. The total alanine content was 10.0482 mg. Radioactivity in alanine was 2.20 nCi since alanine accounted for 10% of the radioactivity in the solution of mixed U-14C-labeled amino acids. The theoretical specific radioactivity of alanine in this mixture was therefore 0.2 19 nCi/mg alanine. The value found using the procedure described was 0.223 k 0.0018 (4) nCi/mg alanine. This result clearly indicates the suitability of this method of assay for plasma l*C L + a-alanine specific radioactivity. To derive the specific radioactivity of 14C-labeled alanine in a l-ml plasma sample obtained under experimental conditions to which unla-
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REILLY
beled carrier alanine had been added the following calculation used:
should be
net cpm x 100 x 178 x total mg alanine presented for ion exchange % counting efficiency x 2220 x 89 x mg derivative X mg alanine in 1.O ml plasma giving the answer as nCi mg plasma alanine-‘. ACKNOWLEDGMENTS The author wishes to thank Miss K. J. H. Robson and Mr. J. D. Warner for their skilled technical assistance.
REFERENCES 1. Felig, P. (1973) Metabolism 22, 179. 2. Schram, E., and Lombaert. R. (1963) Organic Scintillation Detectors, Elsevier, New York. 3. Wolff, J. E., and Bergman, E. N. (1972) Amer. J. Physiol. 233, 447. 4. Pataki, G. (1968) Techniques of Thin Layer Chromatography in Amino Acid and Peptide Chemistry. Ann Arbor Science Publishers, Michigan. 5. Zweig, G., and Whitaker, J. R. (1967) Paper Chromatography and Electrophoresis, Vol. 1, Academic Press, New York. 6. Sherma, T., and Zweig, G. (1971) Paper Chromatography and Electrophoresis. Vol. 2, Academic Press, New York. 7. Cavagnol, J. C., and Betker, W. R. (1967) The Practice of Gas Chromatography. (L. S. Ettre. and A. Zlatkis, eds.), Chapter 3, Interscience Publishers, New York. 8. Harris, C. K.. Tigane. E., and Hanes, C. S. (1961) Can J. Biochem. Physiol. 39, 439. 9. Krebs, H. A., Bennet, D. A. H., de Gasquet, P.. Gascoyne, T., and Yoshida, T. (1963) Biochem. J. 86, 22. 10. Reilly, P. E. B. (1974) Anal. Biochem. (in press). 11. Yoshida, A., and Freese. E. (1970) in Methods in Enzymology (H. Tabor, and C. W. Tabor, eds.), Vol. XVIIA, Academic Press, New York.
