- Email: [email protected]
Gas chromatographic determination of hydroxyproline in urine hydrolysates
305
Clinica Chimica Acta, 88 (1978) 305-310 0 Elsevier/North-Holland Biomedical Press
CCA 9593
GAS CHROMATOGRAPHIC DETERMINATION IN URINE H’YDROLYSATES
M. MAKITA *, S. YAMAMOTO Faculty
of Pharmaceutical
OF HYDROXYPROLINE
and YUKIKO TSUDAKA
Sciences,
Okayama
University,
Tsushima,
Okayama
700 (Japan)
(Received March 17th, 1978)
Summary A simple and specific method for the determination of hydroxyproline in urine hydrolysates has been described. Hydroxyproline was converted into its N-isobutyloxycarbonyl methyl ester derivative without elaborate cleanup, which was analyzed by gas chromatography. Hydroxyproline was clearly’ separated from other urinary constituents on a 0.60% FFAP on dimethyldichlorosilane-treated Gas-Chrom P column. Kainic acid was used as the most convenient internal standard available. The relative standard deviations of peak height ratios were l-15-2.51% at the lo-150 pg levels. Percent recoveries of hydroxyproline added to urine hydrolysates ranged from 98.8 to 107.3%.
Introduction Increasing interest has been directed towards the determination of hydroxyproline (Hyp) in urine, since it has been found that the urinary excretion of Hyp can be used as an important indicator of various diseases associated with collagen metabolism [l-4]. Calorimetric methods have been most commonly used for the determination of Hyp in urine [ 5-111, but they lack specificity and are generally timeconsuming. Gas chromatographic (GC) techniques have been employed to determine Hyp in urine as its trifluoroacetyl n-butyl ester [12] and its trimethylsilyl derivative [ 131. However, it may be difficult to accomplish sound quantitative works by these methods because both Hyp derivatives are unstable to moisture [141. Recently, a practical GC method for the determination of amino acids has been developed in our laboratory, based on the preparation and gas chromatog* To whom comespondence should be addressed.
306
raphy of the N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives [ 15, 161. This method is rapid, convenient and reliable, and adaptations of the method to biological samples have already been demonstrated [17,18]. In the present work, the determination of Hyp in urine by the use of this GC techniaue was investigated. Materials and method Reagents
Standard Hyp solution. The stock standard solution was made up in water at a level of 0.5 mg/ml. Working solution was made up by diluting the stock standard solution with water to provide a level of 100 pg/ml. Internal standard solution. Kainic acid (Nakarai Chemical Ltd., Kyoto, Japan) was used as the most convenient pure internal standard available. Working standard in water was prepared at a level of 100 pg/ml. The standard solutions were used for the preparation of calibration curve and for the calculation of recovery rate. The standard solutions were stored in capped glass bottles at 4°C. Isobutyl chloroformate (isoBCF) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) and used without further purification. N-Methyl-N-nitroso-p-toluenesulfonamide for the evolution of diazomethane gas was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Gas chromatography
A Shimadzu Model CC-5AP gas chromatograph equipped with a dual-column oven bath, dual differential hydrogen flame ionization detectors and on-column injection ports was used for programmed temperature gas chromatography. The support, Gas-Chrom P (100-120 mesh), glass columns (2 m X 3 mm I.D.) and quartz wool plugs placed in each end of the column used in this work were silanized with 5% dimethyldichlorosilane in toluene. The liquid phase, FFAP was purchased from Varian Associate Instrument Devision (Palo Alto, Calif., U.S.A.). The column packing, 0.60% FFAP on dimethyldichlorosilane-treated
TABLE
I
GLC OPERATING _
CONDITIONS
Column Initial temperature Isothermal Temperature program Final temperature Injection and detector Gas flow-rate Carrier gas (NZ) Hydrogen Air Chart speed
rate temperature
* Soon after elution of Hyp, the column and held for 5 min.
0.60% FFAP on 100-120 (2 m X 3 mm I.D.) 170°c 1 min 4’ C /min 215OC * 27O’C
mesh silanized Gas-Chrom
P
40 ml/min 50 ml/min 800 ml/min 0.5 cm/min temperature
was raised
quickly
to 26O’C
by manual
Operation
307
Gas-Chrom P was prepared using n-BuOH/CHC13 (1 : 1, v/v) as a coating solvent according to the procedure of Horning et al. [ 191. The glass columns were filled with the stationary phase by gentle tapping under suction and pre-conditioned with a carrier gas (nitrogen) flow-rate of 20 ml/min at 270°C for 22 h. The chromatographic conditions are shown in Table I. Method Twenty-four hour urine samples were collected under toluene, and 5 ml of the urine was hydrolyzed at 100°C for 22 h in a closed tube with 6 M HCl under nitrogen pressure according to the procedure of Mee [ 121. The tube was cooled and the content was filtered through Whatman No. 30 filter paper. The filtrate was evaporated to dryness in reduced pressure and the residue was dissolved in water to make it up to a volume of 5 ml. One ml of this solution and 0.5 ml of the internal standard solution were pipetted into a lo-ml polyethylene-stoppered vial. To this solution, 0.5 ml of 10% Na2C!03 and 0.1 ml of isoBCF were added. The solution was shaken with a shaker for 10 min at room temperature. The reaction mixture was extracted twice with 3 ml of diethyl ether and the ethereal extracts were discarded. The aqueous layer was saturated with NaCl, acidified to pH l-2 with 10% H3P04 (thymol blue test paper), and then extracted 3 times with 3 ml of diethyl ether with vigorous shaking by hand for 1 min. To the combined ethereal extracts was added 1 ml of methanol, and esterification was carried out by bubbling diazomethane, generated according to the micro-scale procedure of Schlenk and Gellerman [ 201, through this solution until a yellow tinge persisted. After standing at room temperature for 5 min, the solvents were quickly evaporated to dryness at 50°C under a stream of air. The residue was dissolved in 0.2 ml of ethyl acetate, and 4 ~1 of this solution was injected into the gas chromatograph. Each peak of Hyp and the internal standard was measured and the peak height ratio (PHR = Hyp/internal standard) was calculated. Results and discussion The process of acid hydrolysis is important to the actual amount of urinary Hyp analyzed. Mee [ 121 has indicated that the classical overnight acid hydrolysis gave an incomplete hydrolysis and higher urinary Hyp values were observed at 100°C for 22 h under nitrogen pressure. Therefore, in the present study this condition was adopted. After acid hydrolysis, an aliquot corresponding to 1 ml of the 24-h urine was derivatized and analyzed. This amount was sufficient for GC analysis of Hyp in a normal urine. In the cases of some diseases in which excessive amounts of Hyp are excreted the sample volume to be analyzed may be diminished. The derivative preparation and GC analysis were readily achieved without elaborate cleanup and also no precaution to exclude moisture was necessary in the preparation, handling and storage of the derivative. The derivative preparation was established within ca. 20 min and several samples were treated simultaneously . A 2 m X 3 mm I.D. glass column packed with 0.60% FFAP on 100-120
308
(A)
L
L
, 0
, 5
10
I
0
5
10
0
5
10
MIN
Fig. 1. Gas chromatograms obtained from (A) standard, (B) urine hydrolysate, and (C) urine hydrolysate plus internal standard (IS.), kainic acid (50 /.&I. The peaks of hydroxyproline (HYP) in (B) and (0 are 29.1 pg. GLC conditions are given in Table I.
mesh dimethyldichlorosilane-treated Gas-Chrom P was used for the separation of Hyp from other amino acids or other components present in the sample mixture. The chromatograms are shown in Fig. 1. Each derivative of Hyp and the internal standard in the standard run showed a single and symmetrical peak and kainic acid was well suited as an internal standard. No extraneous peak nor interference was observed at the retention time of the internal standard in various urine samples. The peak of Hyp derived from a urine sample was carefully analyzed by gas chromatography-mass spectrometry to check for interference, and it was confirmed that there were no overlapping peaks with that of Hyp. A complete run of GC is less than 20 min for a urine sample. The
TABLE II REPRODUCIBILITY
OF PEAK HEIGHT RATIO
Amounts derivatized (jog)
10 25 50 100 150
(PHR) RSD (%) **
PHR * 1
2
3
4
5
Ave.
0.171 0.417 0.905 1.867 2.731
0.177 0.422 0.880 1.747 2.757
0.174 0.421 0.890 1.774 2.693
0.179 0.413 0.931 1.821 2.675
0.173 0.411 0.885 1.768 2.671
0.175 0.417 0.898 1.793 2.723
1.83 1.15 2.23 2.51 1.40
* Relative to internal standard, kainic acid (50 pg). Each value represents an independent analysis. ** Relative standard deviation.
309
k 0
25
I
I
I
10
50
150 J.lG
100
AMOUNTDERIVATIZED Fig. 2. Calibration curve for hydroxyproline. Peak height ratio = hydroxyproIine/I.S.. kainic acid (50 pg). Each point represents an average of the peak height ratios obtained from five independent analyses.
determination of Hyp could be easily established by measuring the PHR and then calibrating against the standard curve. An experiment was carried out to investigate the reproducibility of the PHR at different levels. Good results were obtained, as indicated in Table II, and the relative standard deviation of the PHR ranged from 1.15 to 2.51%. When the
TABLE III RECOVERY Added
OF HYDROXYPROLINE
FROM URINE HYDROLYSATES
PHR *
RSD (46) **
Wg)
Urine A +25 +50 +100 Urine B +25 +50 iI00
1
2
3
4
5
Ave.
0.420 0.856 (101.4) 1.336
0.431
0.636
0.581
1.036 (101.4) 1.517 (100.7) 2.400 (99.7)
1.048 (104.3) 1.516 (100.6) 2.462 (103.1)
0.441 0.845 (98.8) 1.347 (101.8) 2.269 (102.4) 0.623 1.039 (102.2) 1.502 (99.0) 2,424 (100.8)
0.435
(100.6) 2.225 (99.9) 0.632
0.439 0.864 (103.4) 1.360 (103.2) 2.357 (107.3) 0.591
0.433 0.855 (101.2) 1.348 (101.9) 2.284 (103.2) 0.613 1.041 (102.6) 1.512 (100.1) 2.429 (101.3)
1.92
4.07
Figures in parentheses represent the recovery rate (%). * Relative to internal standard, kainic acid (50 Mg). Each value represents an independent analysis. ** Relative standard deviation.
310 TABLE
IV
URINARY
EXCRETION
OF HYDROXYPROLINE
IN NORMAL
SUBJECTS
Sample number
Age
Sex *
PHR *+
m&
mg/24 h
1 2 3 4 5 6 1
3 6 22 22 25 34 52
M F F F F M M
2.547 2.395 0.806 0.297 0.463 0.434 0.205
14.2 18.9 4.5 1.7 2.6 2.4 1.1
66.6 93.6 40.0 15.8 28.4 20.6 11.3
* M: male, F: female. ** Relative to internal standard, kainic acid (50 fig). Each value represents an average of two independent analyses.
average values of the PHR at different levels were plotted against the microgram of Hyp, the linearity of calibration curve for Hyp was observed as illustrated in Fig. 2. The urine hydrolysates were fortified with Hyp (2.5-10.0 mg%) and percent recoveries were calculated (Table III), ranging from 98.8 to 107.3%. The method was successfully applied to the determination of Hyp in various healthy human urine samples. The actual levels of Hyp found in urine from children and adults are presented in Table IV. The range of Hyp level was comparable to that obtained by other method previously reported [ 1,2,4]. The present method is rapid and convenient, and it is anticipated that this method will find extensive applications for the determination of Hyp in routine and research work in clinical chemistry. References 1 Prockop. D.J. and Kivirikko, K.I. (1967) AM. Intern. Med. 66, 1243-1266 2 LeRoy, E.C. (1967) in Advances in Clinical Chemistry (Bodansky, 0. and Stewart, C.P., eds.), Vol. 10, pp. 213-263, Academic Press, New York and London 3 Guzzo, C.E., Pachas. W.N.. PinaIs, R.S. and Krant. M.J. (1969) Cancer 24. 382-387 4 Nusgens. B. and Lapiere, Ch.M. (1973) CIin. Chim. Acta 48. 203-211 5 Prockop. D.J. and Udenfriend. S. (1960) Anal. Biochem. 1, 228-239 6 Kivirikko. K.I.. Laitinen. 0. and Prockop, D.J. (1967) Anal. Biochem. 19, 249-255 7 Koevoet. A.L. and Baars, J.D. (1969) CIin. Chim. Acta 25. 3943 8 Bergman, I. and Loxley, R. (1970) CIin. Chim. Acta 27.347-349 9 Goverde, B.C. and Veenkamp. F.J.N. (1972) Clin. Chim. Acta 41, 29-40 10 Bondjers, G. and BjBrkerud, S. (1973) Anal. Biochem. 52,496-504 11 Cleary, J. and Saunders, R.A. (1974) CIin. Chim. Acta 57, 217-223 12 Mee, J.M.L. (1973) J. Chromatogr. 87, 165-161 13 Gibbs, B.F.. Itiaba. K. and Crawha& J.C. (1974) CIin. Chim. Acta 54, 396-398 14 Blau. K. (1968) in Biomedical Applications of Gas Chromatography (Szymanski, H.A., ed.). Vol. 2, pp. 22. Plenum Press, New York 16 Makita, M., Yamamoto, S. and Kono. M. (1976) J. Chromatogr. 120, 129-140 16 Makita, M., Yamamoto, S., Sakai, K. and Shiraishi, M. (1976) J. Chromatogr. 124.92-96 17 Makita, M. and Yamamoto. S. (1976) Yakugaku Zasshi 96.777-782 18 Makita. M. and Yamamoto, S. (1976) Yakugaku Zasshi 96,813-816 19 Horning, E.C.. VandenHeuvel, W.J.A. and Greeck, B.G. (1963) in Methods of Biochemical Analysis (GIick, D., ed.). Vol. 11, pp. 80-85. Wiley-Interscience. New York 20 SchIenk, H. and GeIIerman, J.L. (1960) Anal. Chem. 32,1412-1414
Clinica Chimica Acta, 88 (1978) 305-310 0 Elsevier/North-Holland Biomedical Press
CCA 9593
GAS CHROMATOGRAPHIC DETERMINATION IN URINE H’YDROLYSATES
M. MAKITA *, S. YAMAMOTO Faculty
of Pharmaceutical
OF HYDROXYPROLINE
and YUKIKO TSUDAKA
Sciences,
Okayama
University,
Tsushima,
Okayama
700 (Japan)
(Received March 17th, 1978)
Summary A simple and specific method for the determination of hydroxyproline in urine hydrolysates has been described. Hydroxyproline was converted into its N-isobutyloxycarbonyl methyl ester derivative without elaborate cleanup, which was analyzed by gas chromatography. Hydroxyproline was clearly’ separated from other urinary constituents on a 0.60% FFAP on dimethyldichlorosilane-treated Gas-Chrom P column. Kainic acid was used as the most convenient internal standard available. The relative standard deviations of peak height ratios were l-15-2.51% at the lo-150 pg levels. Percent recoveries of hydroxyproline added to urine hydrolysates ranged from 98.8 to 107.3%.
Introduction Increasing interest has been directed towards the determination of hydroxyproline (Hyp) in urine, since it has been found that the urinary excretion of Hyp can be used as an important indicator of various diseases associated with collagen metabolism [l-4]. Calorimetric methods have been most commonly used for the determination of Hyp in urine [ 5-111, but they lack specificity and are generally timeconsuming. Gas chromatographic (GC) techniques have been employed to determine Hyp in urine as its trifluoroacetyl n-butyl ester [12] and its trimethylsilyl derivative [ 131. However, it may be difficult to accomplish sound quantitative works by these methods because both Hyp derivatives are unstable to moisture [141. Recently, a practical GC method for the determination of amino acids has been developed in our laboratory, based on the preparation and gas chromatog* To whom comespondence should be addressed.
306
raphy of the N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives [ 15, 161. This method is rapid, convenient and reliable, and adaptations of the method to biological samples have already been demonstrated [17,18]. In the present work, the determination of Hyp in urine by the use of this GC techniaue was investigated. Materials and method Reagents
Standard Hyp solution. The stock standard solution was made up in water at a level of 0.5 mg/ml. Working solution was made up by diluting the stock standard solution with water to provide a level of 100 pg/ml. Internal standard solution. Kainic acid (Nakarai Chemical Ltd., Kyoto, Japan) was used as the most convenient pure internal standard available. Working standard in water was prepared at a level of 100 pg/ml. The standard solutions were used for the preparation of calibration curve and for the calculation of recovery rate. The standard solutions were stored in capped glass bottles at 4°C. Isobutyl chloroformate (isoBCF) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) and used without further purification. N-Methyl-N-nitroso-p-toluenesulfonamide for the evolution of diazomethane gas was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Gas chromatography
A Shimadzu Model CC-5AP gas chromatograph equipped with a dual-column oven bath, dual differential hydrogen flame ionization detectors and on-column injection ports was used for programmed temperature gas chromatography. The support, Gas-Chrom P (100-120 mesh), glass columns (2 m X 3 mm I.D.) and quartz wool plugs placed in each end of the column used in this work were silanized with 5% dimethyldichlorosilane in toluene. The liquid phase, FFAP was purchased from Varian Associate Instrument Devision (Palo Alto, Calif., U.S.A.). The column packing, 0.60% FFAP on dimethyldichlorosilane-treated
TABLE
I
GLC OPERATING _
CONDITIONS
Column Initial temperature Isothermal Temperature program Final temperature Injection and detector Gas flow-rate Carrier gas (NZ) Hydrogen Air Chart speed
rate temperature
* Soon after elution of Hyp, the column and held for 5 min.
0.60% FFAP on 100-120 (2 m X 3 mm I.D.) 170°c 1 min 4’ C /min 215OC * 27O’C
mesh silanized Gas-Chrom
P
40 ml/min 50 ml/min 800 ml/min 0.5 cm/min temperature
was raised
quickly
to 26O’C
by manual
Operation
307
Gas-Chrom P was prepared using n-BuOH/CHC13 (1 : 1, v/v) as a coating solvent according to the procedure of Horning et al. [ 191. The glass columns were filled with the stationary phase by gentle tapping under suction and pre-conditioned with a carrier gas (nitrogen) flow-rate of 20 ml/min at 270°C for 22 h. The chromatographic conditions are shown in Table I. Method Twenty-four hour urine samples were collected under toluene, and 5 ml of the urine was hydrolyzed at 100°C for 22 h in a closed tube with 6 M HCl under nitrogen pressure according to the procedure of Mee [ 121. The tube was cooled and the content was filtered through Whatman No. 30 filter paper. The filtrate was evaporated to dryness in reduced pressure and the residue was dissolved in water to make it up to a volume of 5 ml. One ml of this solution and 0.5 ml of the internal standard solution were pipetted into a lo-ml polyethylene-stoppered vial. To this solution, 0.5 ml of 10% Na2C!03 and 0.1 ml of isoBCF were added. The solution was shaken with a shaker for 10 min at room temperature. The reaction mixture was extracted twice with 3 ml of diethyl ether and the ethereal extracts were discarded. The aqueous layer was saturated with NaCl, acidified to pH l-2 with 10% H3P04 (thymol blue test paper), and then extracted 3 times with 3 ml of diethyl ether with vigorous shaking by hand for 1 min. To the combined ethereal extracts was added 1 ml of methanol, and esterification was carried out by bubbling diazomethane, generated according to the micro-scale procedure of Schlenk and Gellerman [ 201, through this solution until a yellow tinge persisted. After standing at room temperature for 5 min, the solvents were quickly evaporated to dryness at 50°C under a stream of air. The residue was dissolved in 0.2 ml of ethyl acetate, and 4 ~1 of this solution was injected into the gas chromatograph. Each peak of Hyp and the internal standard was measured and the peak height ratio (PHR = Hyp/internal standard) was calculated. Results and discussion The process of acid hydrolysis is important to the actual amount of urinary Hyp analyzed. Mee [ 121 has indicated that the classical overnight acid hydrolysis gave an incomplete hydrolysis and higher urinary Hyp values were observed at 100°C for 22 h under nitrogen pressure. Therefore, in the present study this condition was adopted. After acid hydrolysis, an aliquot corresponding to 1 ml of the 24-h urine was derivatized and analyzed. This amount was sufficient for GC analysis of Hyp in a normal urine. In the cases of some diseases in which excessive amounts of Hyp are excreted the sample volume to be analyzed may be diminished. The derivative preparation and GC analysis were readily achieved without elaborate cleanup and also no precaution to exclude moisture was necessary in the preparation, handling and storage of the derivative. The derivative preparation was established within ca. 20 min and several samples were treated simultaneously . A 2 m X 3 mm I.D. glass column packed with 0.60% FFAP on 100-120
308
(A)
L
L
, 0
, 5
10
I
0
5
10
0
5
10
MIN
Fig. 1. Gas chromatograms obtained from (A) standard, (B) urine hydrolysate, and (C) urine hydrolysate plus internal standard (IS.), kainic acid (50 /.&I. The peaks of hydroxyproline (HYP) in (B) and (0 are 29.1 pg. GLC conditions are given in Table I.
mesh dimethyldichlorosilane-treated Gas-Chrom P was used for the separation of Hyp from other amino acids or other components present in the sample mixture. The chromatograms are shown in Fig. 1. Each derivative of Hyp and the internal standard in the standard run showed a single and symmetrical peak and kainic acid was well suited as an internal standard. No extraneous peak nor interference was observed at the retention time of the internal standard in various urine samples. The peak of Hyp derived from a urine sample was carefully analyzed by gas chromatography-mass spectrometry to check for interference, and it was confirmed that there were no overlapping peaks with that of Hyp. A complete run of GC is less than 20 min for a urine sample. The
TABLE II REPRODUCIBILITY
OF PEAK HEIGHT RATIO
Amounts derivatized (jog)
10 25 50 100 150
(PHR) RSD (%) **
PHR * 1
2
3
4
5
Ave.
0.171 0.417 0.905 1.867 2.731
0.177 0.422 0.880 1.747 2.757
0.174 0.421 0.890 1.774 2.693
0.179 0.413 0.931 1.821 2.675
0.173 0.411 0.885 1.768 2.671
0.175 0.417 0.898 1.793 2.723
1.83 1.15 2.23 2.51 1.40
* Relative to internal standard, kainic acid (50 pg). Each value represents an independent analysis. ** Relative standard deviation.
309
k 0
25
I
I
I
10
50
150 J.lG
100
AMOUNTDERIVATIZED Fig. 2. Calibration curve for hydroxyproline. Peak height ratio = hydroxyproIine/I.S.. kainic acid (50 pg). Each point represents an average of the peak height ratios obtained from five independent analyses.
determination of Hyp could be easily established by measuring the PHR and then calibrating against the standard curve. An experiment was carried out to investigate the reproducibility of the PHR at different levels. Good results were obtained, as indicated in Table II, and the relative standard deviation of the PHR ranged from 1.15 to 2.51%. When the
TABLE III RECOVERY Added
OF HYDROXYPROLINE
FROM URINE HYDROLYSATES
PHR *
RSD (46) **
Wg)
Urine A +25 +50 +100 Urine B +25 +50 iI00
1
2
3
4
5
Ave.
0.420 0.856 (101.4) 1.336
0.431
0.636
0.581
1.036 (101.4) 1.517 (100.7) 2.400 (99.7)
1.048 (104.3) 1.516 (100.6) 2.462 (103.1)
0.441 0.845 (98.8) 1.347 (101.8) 2.269 (102.4) 0.623 1.039 (102.2) 1.502 (99.0) 2,424 (100.8)
0.435
(100.6) 2.225 (99.9) 0.632
0.439 0.864 (103.4) 1.360 (103.2) 2.357 (107.3) 0.591
0.433 0.855 (101.2) 1.348 (101.9) 2.284 (103.2) 0.613 1.041 (102.6) 1.512 (100.1) 2.429 (101.3)
1.92
4.07
Figures in parentheses represent the recovery rate (%). * Relative to internal standard, kainic acid (50 Mg). Each value represents an independent analysis. ** Relative standard deviation.
310 TABLE
IV
URINARY
EXCRETION
OF HYDROXYPROLINE
IN NORMAL
SUBJECTS
Sample number
Age
Sex *
PHR *+
m&
mg/24 h
1 2 3 4 5 6 1
3 6 22 22 25 34 52
M F F F F M M
2.547 2.395 0.806 0.297 0.463 0.434 0.205
14.2 18.9 4.5 1.7 2.6 2.4 1.1
66.6 93.6 40.0 15.8 28.4 20.6 11.3
* M: male, F: female. ** Relative to internal standard, kainic acid (50 fig). Each value represents an average of two independent analyses.
average values of the PHR at different levels were plotted against the microgram of Hyp, the linearity of calibration curve for Hyp was observed as illustrated in Fig. 2. The urine hydrolysates were fortified with Hyp (2.5-10.0 mg%) and percent recoveries were calculated (Table III), ranging from 98.8 to 107.3%. The method was successfully applied to the determination of Hyp in various healthy human urine samples. The actual levels of Hyp found in urine from children and adults are presented in Table IV. The range of Hyp level was comparable to that obtained by other method previously reported [ 1,2,4]. The present method is rapid and convenient, and it is anticipated that this method will find extensive applications for the determination of Hyp in routine and research work in clinical chemistry. References 1 Prockop. D.J. and Kivirikko, K.I. (1967) AM. Intern. Med. 66, 1243-1266 2 LeRoy, E.C. (1967) in Advances in Clinical Chemistry (Bodansky, 0. and Stewart, C.P., eds.), Vol. 10, pp. 213-263, Academic Press, New York and London 3 Guzzo, C.E., Pachas. W.N.. PinaIs, R.S. and Krant. M.J. (1969) Cancer 24. 382-387 4 Nusgens. B. and Lapiere, Ch.M. (1973) CIin. Chim. Acta 48. 203-211 5 Prockop. D.J. and Udenfriend. S. (1960) Anal. Biochem. 1, 228-239 6 Kivirikko. K.I.. Laitinen. 0. and Prockop, D.J. (1967) Anal. Biochem. 19, 249-255 7 Koevoet. A.L. and Baars, J.D. (1969) CIin. Chim. Acta 25. 3943 8 Bergman, I. and Loxley, R. (1970) CIin. Chim. Acta 27.347-349 9 Goverde, B.C. and Veenkamp. F.J.N. (1972) Clin. Chim. Acta 41, 29-40 10 Bondjers, G. and BjBrkerud, S. (1973) Anal. Biochem. 52,496-504 11 Cleary, J. and Saunders, R.A. (1974) CIin. Chim. Acta 57, 217-223 12 Mee, J.M.L. (1973) J. Chromatogr. 87, 165-161 13 Gibbs, B.F.. Itiaba. K. and Crawha& J.C. (1974) CIin. Chim. Acta 54, 396-398 14 Blau. K. (1968) in Biomedical Applications of Gas Chromatography (Szymanski, H.A., ed.). Vol. 2, pp. 22. Plenum Press, New York 16 Makita, M., Yamamoto, S. and Kono. M. (1976) J. Chromatogr. 120, 129-140 16 Makita, M., Yamamoto, S., Sakai, K. and Shiraishi, M. (1976) J. Chromatogr. 124.92-96 17 Makita, M. and Yamamoto. S. (1976) Yakugaku Zasshi 96.777-782 18 Makita. M. and Yamamoto, S. (1976) Yakugaku Zasshi 96,813-816 19 Horning, E.C.. VandenHeuvel, W.J.A. and Greeck, B.G. (1963) in Methods of Biochemical Analysis (GIick, D., ed.). Vol. 11, pp. 80-85. Wiley-Interscience. New York 20 SchIenk, H. and GeIIerman, J.L. (1960) Anal. Chem. 32,1412-1414