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Removal of glutathione interference in blood amino acid analysis
BIOCHEMICAL
MEDICINE
15,
282-288 ( 1976)
Removal of Glutathione Interference Blood Amino Acid Analysis JAMES
A. LEMONS, Division University
CECILIA
of Perinatal of Colorado
TENG,
AND MICHAEL
in A. NAUGHTON
Medicine, Colorado General Hospital, Medical Center. Denver, Colorado 80220
Received January 12, 1976
Recent studies have documented the importance of the red blood cell in the interorgan transport of free amino acids (l-5). Therefore, in order to determine the net transfer of amino acids to a tissue, amino acid concentrations must be measured in whole blood rather than in plasma. However, whole blood analysis has been hampered in the past by the presence of high concentrations of glutathione in the erythrocyte. During ion exchange chromatography, this tripeptide uniformly interferes with the accurate determination of several important amino acids. Oxidized glutathione (GSSG) is eluted in most systems as a broad peak which overlaps those of alanine, glycine, and proline, and may also interfere with the recoveries of citrulline and cu-aminobutyric acid. Reduced glutathione (GSH) is eluted as a single sharp peak superimposed upon that of aspartic acid. The present technique was developed in order to remove glutathione (GSSG and GSH) from its normal position in the amino acid chromatogram by oxidation with performic acid. MATERIALS
AND METHODS
Performic acid was prepared according to the method described by Hirs (6), by the addition of 0.5 ml of 50% hydrogen peroxide (Eastman Kodak) to 9.5 ml of 99% formic acid (Fisher Scientific). The resulting solution was then allowed to stand at room temperature for 30 min. Standard amino acid samples were prepared by dissolving either individual or prepared mixtures of standard amino acids (Pierce Chemical) in 0.01 M HCl (pH 2.0) to a final concentration of 0.1 pmole/ml. Glutathione standards were prepared in the same manner, with or without the addition of standard amino acids in the concentration described. Whole blood samples taken from fetal and adult sheep were prepared by the addition of 2.0 ml of blood to 3.0 ml of demineralized water, and snap frozen in ’ Author to whom reprint requests should be addressed: Division of Perinatal Medicine, Colorado General Hospital, Container B-198, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220. 282 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
GLUTATHIONE
AND
AMINO
ACID
ANALYSIS
283
acetone/dry ice in order to induce complete lysis of all cellular components. Each sample was then thawed under warm water and deproteinized by the addition of 1 .O ml of 15% sulfosalicylic acid containing the internal standard norleucine (0.6 kmole/ml). The supernatant was stored at -75°C until the time of analysis (7). In order to determine the effects of per-formic acid on glutathione, as well as individual amino acids in the standards and whole blood samples, the following analyses were performed. (a) Aliquots of 10, 20,40, and 60 ~1 of per-formic acid were added to 1.5 ml of standard amino acid solution containing 0.75 pmole each of GSSG and GSH. (b) Twenty microliters of per-formic acid were added to 1.5 ml of 0.01 M HCl containing 0.75 and 1.5 pmoles of GSH and 0.6 and 0.9 pmole of GSSG. (c) Twenty microliters of performic acid were added to 1.5 ml of HCI containing the individual amino acids methionine, cystathionine, and cystine (0.1 pmole/ml). (d) Twenty and forty microliters of per-formic acid were added to 1.5 ml of the sheep blood sample prepared as described above. Following the addition of performic acid, the sample was gently mixed by vortexing for approximately 15 set and loaded immediately on to the JEOL-6AH amino acid analyzer. Three samples were analyzed within a 36-hr period by an automated program, and included the treated samples and an untreated control sample. The chromatography system used was adapted from Jeppson (8), and utilizes 2-column, high pressure ion exchange with JEOL resin LCR-2, three citrate buffers per column, and column temperatures of 36 and 55°C. The basic amino acids and carnosine (P-alanylhistidine) are eluted on the short column and are not interfered with by glutathione. The neutral and acidic amino acids are analyzed on the long column, and are eluted with the interfering glutathione. Performic acid was added to samples analyzed on both the short and long columns in the present investigation in order to determine the effects of oxidation on as many amino acids as possible. During chromatography, peak sizes were determined according to OD at 570 nm following reaction with ninhydrin at 95°C by the JEOL Integrator 15-SK and recorded. Concentrations of individual amino acids were calculated by comparison with the internal standard norleucine. RESULTS
(a) GSSG and GSH. When performic acid was added to samples in excessive amounts (40 and 60 pl), poor separation of several amino acids resulted (aspartate, threonine, and se&e). On the other hand, 10 ~1 of performic acid in 1.5 ml of sample failed to completely oxidize glutathione
284
LEMONS,
TENG
AND
NAUGHTON
present in the blood. In the present system, 20 ,ul ofperformic acid in 1.5 ml of sample provides optimal resolution of all amino acids and complete oxidation of 1.5 pmoles of GSH and 0.9 pmoles of GSSG. The oxidized GSH and GSSG were eluted at the extreme front of the chromatogram (Fig. 1). (b) Amino acids. Pet-formic acid was found to affect the following amino acids (Table 1): methionine, cystathionine, cystine, tyrosine, tryptophan, I-methyl histidine, 3-methyl histidine. histidine, aspartate. and TABLE RECOVERIES OF 25 AMINO FOLLOWING PERFORMIC
Amino
acid
Ornithine Lysine Histidine” l-Methyl histidine” 3-Methyl histidine” Carnosine Tryptophan Arginine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Citrulhne cY-Aminobutyrate Vahne Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine” Phenylalanine
1 ACIDS ACID
AND C’ARNOSINE OXIDATION
Untreated sample
Perfornnc treated sample
100 100 loo 100 100
f -t t t f
0.8” 1.3 0.9 1.3 I.5
YX.0 f 7.h 100.4 i 3.1
100 100 100 100 100 100 IO0 100 100 100 100 100 100 100 100 100 loo 100 100 IOU 100
i2 + -c z t _+ i t IT -+ ?I r I t f tk t i I
6.3 X.6 I.5 I.2 1.x
Y3.2 f II 100.6 .k 9.5. I .A 99.7 f
-.
I .o
6.2 2.0 11.3 2.6 I.4 ‘4.7 2.5 I.9 I.1 I.2 I.3 1.7 2.4 2.7 I.5
u P value by Student’s r test for significance samples. ’ Percentage recovery t 1 SD. c Not significant. ‘t See text and Fig. 3.
2. I 1.3 2.4
Y9.l
i
1.9
101.2 98.X 106.X 99.2 9X.6
t +I? 2 rt
5.0 I.7 3.0 2.1 I.1
100.8
t_ 2 5
loo.?
? 2 I
101,s
2 2.0
0 it II Y8.P i 97.1 t XI 7 z Y4.9 i of difference
4.3
between
1.X 3.4 x.7 I 6 treated
and
untrsatcd
GLUTATHIONE
AND
AMINO
ACID
ANALYSIS
286
LEMONS,
TENG
AND
NAUGHTON
phenylalanine. Methionine was oxidized to methionine s&one (9). which was then eluted as a sharp peak following aspartic acid ( 10). Cystathionine was converted to three small peaks, which eluted near the positions of serine and valine. The magnitude of these peaks was such that it did not interfere with the analysis of serine and valine (Table 1). Cystine was converted to cysteic acid and eluted at the front of the chromatogram (10). The tyrosine peak was reduced in size in proportion to the amount of performic acid added, but eluted in the normal position. Modified tyrosine residues (mono- and dichlorotyrosine) presumably formed as described by Thompson (1 l), but were not identified on the present chromatogram. Recoveries of phenylalanine and aspartate were slightly decreased, whereas tyrosine, histidine, l-methyl histidine, and 3-methyl histidine were all markedly reduced in the second and third samples of the 36-hr analysis (Fig. 2). The remaining 15 amino acids and carnosine were unaffected by the performic acid, and complete recoveries were obtained from both the standard mixture of free amino acids and from the sheep blood samples (Table I). In this laboratory. performic acid is not used in the analysis of the basic amino acids (short column), and therefore recoveries of histidine, l-methyl histidine. 3-methyl histidine. and tryptophan are not affected. DISCUSSION
Because of the importance of the erythrocyte in carrying and transferring free amino acids, measurement of whole blood amino acid concentrations is required to determine net transfer of an amino acid to or from
100
& ____...........I..._.)
a0
&
I-5
k-+
60
kt 8 L
.. . . .. . . . ...""---*
TlnoSIWE
I
L 1,
1w
23
.,,_._...._.....
&--."
35
p....-““......, . ... . , \ +-t HISTIDINE L 2 14 20
.."..."....,*
___._..__..___.__. &.--..-
. . . -4
P 80 E
I_
2 /'\
t00
I.n*
2
I
HISTIDINE
I4
26
+-t
lI-zL2
I4
20
FIG. 2. The effect of performic acid on the recoveries of four amino acids is depicted in relation to retention time during analysis (untreated samples, ------- 3 -------: treated samples, . -).
GLUTATHIONE
AND AMINO
ACID ANALYSIS
287
an organ. However, in order to perform whole blood analysis and accurately measure alanine and glycine, a method is required to remove GSSG and GSH from the normal chromatogram. This has been accomplished by the application of the method of Hits (6), utilizing the oxidant per-formic acid. Pet-formic acid may be expected to react with many free amino acids, as originally suggested by the data of Toennies (9). The results of the present investigation support this work, in that 10 of the 25 amino acids were altered by performate oxidation. However, if the described method of 2-column analysis is employed, the recoveries of only six amino acids is significantly decreased (i.e., aspartate, phenylalanine, tyrosine, cystine, methionine, and cystathionine). Furthermore, 95% of aspartate and phenylalanine are recovered in the presence of performic acid. The concentration of glutathione in sheep blood is approximately 70 mg/lOO ml of red blood cells, or about 0.7 pmole/ml of whole blood (12), similar to the concentration in human blood (13). Since 20~1 of performic acid in 1.5 ml of sample oxidizes 0.9 pmole of GSSG and 1.5 pmole of GSH, and because there is a one to three dilution of blood prior to analysis, there is a large margin of safety for complete removal of glutathione present in the whole blood. In summary, the use of performic acid to remove glutathione interference from the whole blood sample is simple and effective. The technique permits the accurate measurement of the majority of amino acids, including glycine and alanine, both of which are important gluconeogenic precursors (14,15). SUMMARY
Whole blood amino acid analysis, which is necessary to determine the net transfer of free amino acids to an organ, is hampered by the presence of high concentrations of glutathione in the erythrocyte. Reduced and oxidized glutathione interfere with the accurate measurement of alanine, glycine, and aspartate during ion exchange chromatography. Oxidation with per-formic acid provides a simple and expedient method of removing interfering glutathione peaks from the blood sample during analysis. This technique permits the precise quantitation of the majority of amino acids (including alanine and glycine) commonly measured in blood samples. ACKNOWLEDGMENTS This work was supported in part by Public Health Service Research Grants No. HD-00781, No. HD-01866 and No. HD-05111.
REFERENCES 1. Winter, C. G., and Christensen, H. N. J. Biol. Chem. 2. Christensen, H. N., Fed. Prof. 32, 19-28 (1973).
239, 872-878
(1964).
288
LEMONS.
TENG AND NAUGHTON
3. Elwyn, D. H., Fed. Proc. 25, 854-861 (1966). 4. Elwyn, D. H.. Launder, W. J., Parikh, H. C.. and Wise, E. M.. Jr.. Amrr. J. P-‘i~~sioi. 222, 1333-1342 (1972). 5. Felig, P., Wahren, J.. and Raf. L., Proc. Nut. A(,&. S(,i.. USA 70, 177% 1779 ( 1973). 6. Hirs, C. H. W., J. Bid. Chem. 219. 61 I-621 ( 1956). 7. Lemons, J. A.. Adcock. E. W., Jones, M. D.. Naughton. M. 4.. Meschia. G.. and Battaglia, F. C. (in preparation). 8. Jeppson, J. 0.. and Karlsson. I. M.. J. Chromutogr. 72. 93-103 (1972) 9. Toennies. G.. and Homiller. R. P.. J. Amer. Chem. SW. 64, 3054-3056 (19421. IO. Zacharius, R. M., and Talley. E. A.. Anal. Chem. 34. 1551-1556 (1962). Il. Thompson, E. 0. P., Biochim. Rioplzys. Acrtr 15,440-441 (1954). 12. Tucker, E. M.. it7 “The Blood of Sheep” (M. H. Blunt. Ed.). p. 146. Springer-Verlag. New York. 1975. 13. Gerald, P. S., and Scott. E. M., in “The Metabolic Basis of Inherited Disease“ (.I. B. Stanbury. J. B. Wyngaarden. and D. S. Fredrickson, Eds.). p. 1095. McGraw-Hill Book Co.. New York, 1966. 14. Felig. P., Pozefsky. T., Marliss. E.. and Cahill. G. F.. Sc,re,~c,r 167, 1003-IOU4 (1970). 15. Marliss, E. B., Aoki, T. T.. Pozefsky. T.. Most. A. S., and Cahill. G. F.. .I. C/i,?. inrest. 50, 814-817 (1971).
MEDICINE
15,
282-288 ( 1976)
Removal of Glutathione Interference Blood Amino Acid Analysis JAMES
A. LEMONS, Division University
CECILIA
of Perinatal of Colorado
TENG,
AND MICHAEL
in A. NAUGHTON
Medicine, Colorado General Hospital, Medical Center. Denver, Colorado 80220
Received January 12, 1976
Recent studies have documented the importance of the red blood cell in the interorgan transport of free amino acids (l-5). Therefore, in order to determine the net transfer of amino acids to a tissue, amino acid concentrations must be measured in whole blood rather than in plasma. However, whole blood analysis has been hampered in the past by the presence of high concentrations of glutathione in the erythrocyte. During ion exchange chromatography, this tripeptide uniformly interferes with the accurate determination of several important amino acids. Oxidized glutathione (GSSG) is eluted in most systems as a broad peak which overlaps those of alanine, glycine, and proline, and may also interfere with the recoveries of citrulline and cu-aminobutyric acid. Reduced glutathione (GSH) is eluted as a single sharp peak superimposed upon that of aspartic acid. The present technique was developed in order to remove glutathione (GSSG and GSH) from its normal position in the amino acid chromatogram by oxidation with performic acid. MATERIALS
AND METHODS
Performic acid was prepared according to the method described by Hirs (6), by the addition of 0.5 ml of 50% hydrogen peroxide (Eastman Kodak) to 9.5 ml of 99% formic acid (Fisher Scientific). The resulting solution was then allowed to stand at room temperature for 30 min. Standard amino acid samples were prepared by dissolving either individual or prepared mixtures of standard amino acids (Pierce Chemical) in 0.01 M HCl (pH 2.0) to a final concentration of 0.1 pmole/ml. Glutathione standards were prepared in the same manner, with or without the addition of standard amino acids in the concentration described. Whole blood samples taken from fetal and adult sheep were prepared by the addition of 2.0 ml of blood to 3.0 ml of demineralized water, and snap frozen in ’ Author to whom reprint requests should be addressed: Division of Perinatal Medicine, Colorado General Hospital, Container B-198, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220. 282 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
GLUTATHIONE
AND
AMINO
ACID
ANALYSIS
283
acetone/dry ice in order to induce complete lysis of all cellular components. Each sample was then thawed under warm water and deproteinized by the addition of 1 .O ml of 15% sulfosalicylic acid containing the internal standard norleucine (0.6 kmole/ml). The supernatant was stored at -75°C until the time of analysis (7). In order to determine the effects of per-formic acid on glutathione, as well as individual amino acids in the standards and whole blood samples, the following analyses were performed. (a) Aliquots of 10, 20,40, and 60 ~1 of per-formic acid were added to 1.5 ml of standard amino acid solution containing 0.75 pmole each of GSSG and GSH. (b) Twenty microliters of per-formic acid were added to 1.5 ml of 0.01 M HCl containing 0.75 and 1.5 pmoles of GSH and 0.6 and 0.9 pmole of GSSG. (c) Twenty microliters of performic acid were added to 1.5 ml of HCI containing the individual amino acids methionine, cystathionine, and cystine (0.1 pmole/ml). (d) Twenty and forty microliters of per-formic acid were added to 1.5 ml of the sheep blood sample prepared as described above. Following the addition of performic acid, the sample was gently mixed by vortexing for approximately 15 set and loaded immediately on to the JEOL-6AH amino acid analyzer. Three samples were analyzed within a 36-hr period by an automated program, and included the treated samples and an untreated control sample. The chromatography system used was adapted from Jeppson (8), and utilizes 2-column, high pressure ion exchange with JEOL resin LCR-2, three citrate buffers per column, and column temperatures of 36 and 55°C. The basic amino acids and carnosine (P-alanylhistidine) are eluted on the short column and are not interfered with by glutathione. The neutral and acidic amino acids are analyzed on the long column, and are eluted with the interfering glutathione. Performic acid was added to samples analyzed on both the short and long columns in the present investigation in order to determine the effects of oxidation on as many amino acids as possible. During chromatography, peak sizes were determined according to OD at 570 nm following reaction with ninhydrin at 95°C by the JEOL Integrator 15-SK and recorded. Concentrations of individual amino acids were calculated by comparison with the internal standard norleucine. RESULTS
(a) GSSG and GSH. When performic acid was added to samples in excessive amounts (40 and 60 pl), poor separation of several amino acids resulted (aspartate, threonine, and se&e). On the other hand, 10 ~1 of performic acid in 1.5 ml of sample failed to completely oxidize glutathione
284
LEMONS,
TENG
AND
NAUGHTON
present in the blood. In the present system, 20 ,ul ofperformic acid in 1.5 ml of sample provides optimal resolution of all amino acids and complete oxidation of 1.5 pmoles of GSH and 0.9 pmoles of GSSG. The oxidized GSH and GSSG were eluted at the extreme front of the chromatogram (Fig. 1). (b) Amino acids. Pet-formic acid was found to affect the following amino acids (Table 1): methionine, cystathionine, cystine, tyrosine, tryptophan, I-methyl histidine, 3-methyl histidine. histidine, aspartate. and TABLE RECOVERIES OF 25 AMINO FOLLOWING PERFORMIC
Amino
acid
Ornithine Lysine Histidine” l-Methyl histidine” 3-Methyl histidine” Carnosine Tryptophan Arginine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Citrulhne cY-Aminobutyrate Vahne Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine” Phenylalanine
1 ACIDS ACID
AND C’ARNOSINE OXIDATION
Untreated sample
Perfornnc treated sample
100 100 loo 100 100
f -t t t f
0.8” 1.3 0.9 1.3 I.5
YX.0 f 7.h 100.4 i 3.1
100 100 100 100 100 100 IO0 100 100 100 100 100 100 100 100 100 loo 100 100 IOU 100
i2 + -c z t _+ i t IT -+ ?I r I t f tk t i I
6.3 X.6 I.5 I.2 1.x
Y3.2 f II 100.6 .k 9.5. I .A 99.7 f
-.
I .o
6.2 2.0 11.3 2.6 I.4 ‘4.7 2.5 I.9 I.1 I.2 I.3 1.7 2.4 2.7 I.5
u P value by Student’s r test for significance samples. ’ Percentage recovery t 1 SD. c Not significant. ‘t See text and Fig. 3.
2. I 1.3 2.4
Y9.l
i
1.9
101.2 98.X 106.X 99.2 9X.6
t +I? 2 rt
5.0 I.7 3.0 2.1 I.1
100.8
t_ 2 5
loo.?
? 2 I
101,s
2 2.0
0 it II Y8.P i 97.1 t XI 7 z Y4.9 i of difference
4.3
between
1.X 3.4 x.7 I 6 treated
and
untrsatcd
GLUTATHIONE
AND
AMINO
ACID
ANALYSIS
286
LEMONS,
TENG
AND
NAUGHTON
phenylalanine. Methionine was oxidized to methionine s&one (9). which was then eluted as a sharp peak following aspartic acid ( 10). Cystathionine was converted to three small peaks, which eluted near the positions of serine and valine. The magnitude of these peaks was such that it did not interfere with the analysis of serine and valine (Table 1). Cystine was converted to cysteic acid and eluted at the front of the chromatogram (10). The tyrosine peak was reduced in size in proportion to the amount of performic acid added, but eluted in the normal position. Modified tyrosine residues (mono- and dichlorotyrosine) presumably formed as described by Thompson (1 l), but were not identified on the present chromatogram. Recoveries of phenylalanine and aspartate were slightly decreased, whereas tyrosine, histidine, l-methyl histidine, and 3-methyl histidine were all markedly reduced in the second and third samples of the 36-hr analysis (Fig. 2). The remaining 15 amino acids and carnosine were unaffected by the performic acid, and complete recoveries were obtained from both the standard mixture of free amino acids and from the sheep blood samples (Table I). In this laboratory. performic acid is not used in the analysis of the basic amino acids (short column), and therefore recoveries of histidine, l-methyl histidine. 3-methyl histidine. and tryptophan are not affected. DISCUSSION
Because of the importance of the erythrocyte in carrying and transferring free amino acids, measurement of whole blood amino acid concentrations is required to determine net transfer of an amino acid to or from
100
& ____...........I..._.)
a0
&
I-5
k-+
60
kt 8 L
.. . . .. . . . ...""---*
TlnoSIWE
I
L 1,
1w
23
.,,_._...._.....
&--."
35
p....-““......, . ... . , \ +-t HISTIDINE L 2 14 20
.."..."....,*
___._..__..___.__. &.--..-
. . . -4
P 80 E
I_
2 /'\
t00
I.n*
2
I
HISTIDINE
I4
26
+-t
lI-zL2
I4
20
FIG. 2. The effect of performic acid on the recoveries of four amino acids is depicted in relation to retention time during analysis (untreated samples, ------- 3 -------: treated samples, . -).
GLUTATHIONE
AND AMINO
ACID ANALYSIS
287
an organ. However, in order to perform whole blood analysis and accurately measure alanine and glycine, a method is required to remove GSSG and GSH from the normal chromatogram. This has been accomplished by the application of the method of Hits (6), utilizing the oxidant per-formic acid. Pet-formic acid may be expected to react with many free amino acids, as originally suggested by the data of Toennies (9). The results of the present investigation support this work, in that 10 of the 25 amino acids were altered by performate oxidation. However, if the described method of 2-column analysis is employed, the recoveries of only six amino acids is significantly decreased (i.e., aspartate, phenylalanine, tyrosine, cystine, methionine, and cystathionine). Furthermore, 95% of aspartate and phenylalanine are recovered in the presence of performic acid. The concentration of glutathione in sheep blood is approximately 70 mg/lOO ml of red blood cells, or about 0.7 pmole/ml of whole blood (12), similar to the concentration in human blood (13). Since 20~1 of performic acid in 1.5 ml of sample oxidizes 0.9 pmole of GSSG and 1.5 pmole of GSH, and because there is a one to three dilution of blood prior to analysis, there is a large margin of safety for complete removal of glutathione present in the whole blood. In summary, the use of performic acid to remove glutathione interference from the whole blood sample is simple and effective. The technique permits the accurate measurement of the majority of amino acids, including glycine and alanine, both of which are important gluconeogenic precursors (14,15). SUMMARY
Whole blood amino acid analysis, which is necessary to determine the net transfer of free amino acids to an organ, is hampered by the presence of high concentrations of glutathione in the erythrocyte. Reduced and oxidized glutathione interfere with the accurate measurement of alanine, glycine, and aspartate during ion exchange chromatography. Oxidation with per-formic acid provides a simple and expedient method of removing interfering glutathione peaks from the blood sample during analysis. This technique permits the precise quantitation of the majority of amino acids (including alanine and glycine) commonly measured in blood samples. ACKNOWLEDGMENTS This work was supported in part by Public Health Service Research Grants No. HD-00781, No. HD-01866 and No. HD-05111.
REFERENCES 1. Winter, C. G., and Christensen, H. N. J. Biol. Chem. 2. Christensen, H. N., Fed. Prof. 32, 19-28 (1973).
239, 872-878
(1964).
288
LEMONS.
TENG AND NAUGHTON
3. Elwyn, D. H., Fed. Proc. 25, 854-861 (1966). 4. Elwyn, D. H.. Launder, W. J., Parikh, H. C.. and Wise, E. M.. Jr.. Amrr. J. P-‘i~~sioi. 222, 1333-1342 (1972). 5. Felig, P., Wahren, J.. and Raf. L., Proc. Nut. A(,&. S(,i.. USA 70, 177% 1779 ( 1973). 6. Hirs, C. H. W., J. Bid. Chem. 219. 61 I-621 ( 1956). 7. Lemons, J. A.. Adcock. E. W., Jones, M. D.. Naughton. M. 4.. Meschia. G.. and Battaglia, F. C. (in preparation). 8. Jeppson, J. 0.. and Karlsson. I. M.. J. Chromutogr. 72. 93-103 (1972) 9. Toennies. G.. and Homiller. R. P.. J. Amer. Chem. SW. 64, 3054-3056 (19421. IO. Zacharius, R. M., and Talley. E. A.. Anal. Chem. 34. 1551-1556 (1962). Il. Thompson, E. 0. P., Biochim. Rioplzys. Acrtr 15,440-441 (1954). 12. Tucker, E. M.. it7 “The Blood of Sheep” (M. H. Blunt. Ed.). p. 146. Springer-Verlag. New York. 1975. 13. Gerald, P. S., and Scott. E. M., in “The Metabolic Basis of Inherited Disease“ (.I. B. Stanbury. J. B. Wyngaarden. and D. S. Fredrickson, Eds.). p. 1095. McGraw-Hill Book Co.. New York, 1966. 14. Felig. P., Pozefsky. T., Marliss. E.. and Cahill. G. F.. Sc,re,~c,r 167, 1003-IOU4 (1970). 15. Marliss, E. B., Aoki, T. T.. Pozefsky. T.. Most. A. S., and Cahill. G. F.. .I. C/i,?. inrest. 50, 814-817 (1971).