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Year-long validation study and reference values for urinary amino acids using a reversed-phase HPLC method.

Analysis of the urinary amino acids is a crucial part of the diagnosis and clinical management of disturbances of amino acid metabolism, including both primary and secondary disorders such as those related to renal and hepatic drug toxicity. Likewise, analysis of urinary amino acids has been applied to studies of bone and muscle turnover as well as in nutritional assessment (1, 2).

Physiological amino acids have been determined extensively in clinical laboratories by automated ion-exchange chromatography in combination with postcolumn ninhydrin detection (3, 4). This technique provides simplicity in sample preparation and highly reproducible results (5, 6) and has been considered as the reference method (2). On the other hand, reversed-phase HPLC (RP-HPLC) [1] has progressively become an alternative in the last decade for both protein hydrolysates and biological fluids (7,8).

RP-HPLC offers higher sensitivity, versatility, and speed of analysis than classical ion-exchange separations (9,10). The more usual methods involve precolumn derivatization with phenylisothiocyanate (PITC) (11,12) or o-phthalaldehyde (OPA) (7,13). Although OPA methods show a higher sensitivity and more suitable features for automation than PITC methods, OPA derivatives are highly unstable, and direct detection of secondary amino acids (proline and hydroxyproline) and sulfur amino acids (cysteine and homocysteine) is not possible (3). OPA methods have been recommended for quantification of primary amino acids in physiological fluids, whereas PITC methods have been proposed when both primary and secondary amino acids are to be determined (9).

Every laboratory should always provide methodrelated reference values. Amino acid concentrations in biological fluids are related to different factors, such as diet, protein metabolism, circadian rhythms in metabolism, and excretion of each amino acid (1). Particularly, urinary amino acid excretion shows age-related physiological variations mainly related to the progressive maturation of the tubular reabsorption systems (1,14). This leads to an age-dependent decrease of amino acid excretion, which prompts laboratories to define reference values for several classes (15, 16).

In analysis of urinary amino acid excretion in infants, incontinent patients, or general outpatients, it often is very difficult to obtain 24-h urine collections. Therefore, random urine samples frequently are collected for amino acid analysis, and the amino acid values are expressed relative to creatinine (15,16). The excretion rates of amino acids may vary independently of creatinine over a 24-h period. Nevertheless, creatinine has been shown to be the reference compound with the lowest variance both within and between individuals (17,18). Moreover, the variance of urinary amino acid excretion has been shown to improve when the concentrations in urine are related to creatinine (17).

The range of any reference interval can be considered as the sum of both biological and methodological variability; therefore, the latter must be continuously assessed by a long-term quality-control program to guarantee the clinical utility of the established intervals (19), and validation of the long-term method performance should always be included during the production of new reference values. Appropriate urinary amino acid reference values for RP-HPLC methods have not been comprehensively reported. On the other hand, good correlation of RP-HPLC data with those derived by the reference ion-exchange chromatography method has been shown (11,12), but differences in sensitivity and sample preparation procedures may yield potential differences in the reference values. Therefore, we studied the long-term precision and recovery of a commercial PITC RP-HPLC method and derived specific age-related reference values for urine amino acid excretion in relation to creatinine in both untimed and 24-h urine collections. These reference intervals were compared with those published for ion-exchange methods and with the amino acid concentrations observed in patients diagnosed with specific pathologies.

Materials and Methods


A total of 226 urine samples of random and 24-h collections from infants, children, and adults were prospectively analyzed throughout a 5-year period and selected for the study. All samples were obtained from outpatients and inpatients being seen for a routine pediatric check-up or preoperative testing before minor elective surgery, or for psychomotor dysfunction screening studies. All patients with metabolic, hepatic, and renal diseases were eliminated from consideration. The study was performed in accordance with the Helsinki Declaration of 1975, as revised in 1996, and oral informed consent was obtained from parents and patients. All participants were in a good nutritional state and consuming their usual food intake; no special precautions were taken regarding drug therapy during the time of sample collections. Untimed and 24-h clean-voided specimens were collected without addition of any preservative. At admission, urine samples were frozen at -20[degrees]C until assayed, usually within 2 weeks.


Measurements of amino acid concentrations in urine were performed by RP-HPLC using the Pico-Tag method (Waters) according to manufacturer's specifications (20). The analytical system consisted of a Baseline 810 workstation running on a Hewlett-Packard Vectra 486 computer, two Model 510 pumps, a Model 712 WISP autosampler, a temperature control module (set at 46[degrees]C), and a Model 484 absorbance detector (set at 254 nm). All chemicals and solvents were purchased from Sigma-Aldrich and Merck. HPLC-grade water was generated with a MilliQ water purification system from Millipore.

On the day of analysis, the samples were thawed at room temperature and centrifuged for 15 min at 30008. The clear supernatants were further cleaned up by solid-phase extraction. Briefly, 1 mL of each sample, diluted 1:1 with the internal standard (400 [micro]mol/L methionine sulfone in 0.1 mol/L HCl), was applied to a preconditioned (10 mL of methanol, 10 mL of water) Sep-Pack Plus [C.sub.18] cartridge on a Sep-Pack vacuum manifold (Waters). Purified amino acids were acquired by collecting the initial pass-through sample plus two sequential eluates of 1.5 mL of 0.1 mol/L HCl and 2.5 mL of 300 mL/L acetonitrile in 0.1 mol/L HCI, respectively.

Derivatization consisted of three steps of evaporation to dryness using a Waters vacuum station. The first drying step was carried out on the calibration mixture (25 [micro]L) and urine eluates (50 [micro]L). The second drying step was carried out after treatment with 10 /,L of methanol-1 mol/L sodium acetate-triethylamine (2:2:1 by volume), and the third step after treatment for 10 min with 20 [micro]L of derivatization reagent [methanol-triethylamine-water-PITC (Pierce), 7:1:1:1 by volume]. After derivatization, the phenylthiocarbamyl amino acid derivatives were separated on the [C.sub.18] Pico-Tag physiological free amino acid column [300 X 3.9 mm (i.d.)] using a stated binary gradient of Waters eluents 1 and 2 at a flow rate of 1.0 mL/min.

Urinary creatinine was measured by the kinetic Jaffe reaction in a Hitachi 911 analyzer (Roche Diagnostics).


Derivatized calibrators, controls, and patient samples were dissolved in 100 [micro]L of the sample diluent (Waters), and 10 [micro]L of these solutions was injected. A calibration mixture was prepared and derivatized every 2 months from two commercial solutions (Sigma-Aldrich) of acidneutral and basic amino acids in 0.1 mol/L HCI. A separate solution of glutamine and argininosuccinic acid (Sigma-Aldrich) in water, stored at -20[degrees]C, was also used. The concentration of each amino acid in the final mixture was 400 [micro]mol/L, except for cystine (200 [micro]mol/L). Amino acid derivatives were identified by their relative retention times based on the reference peaks for retention time (methionine sulfone and norleucine, both of which were added to the internal standard solution). The ratio of the peak area of each amino acid to the internal standard (methionine sulfone) was used to calculate the amino acid concentrations, and the final results were expressed as millimole of amino acid per mole of creatinine. Each urine sample was analyzed once.

Within- and between-run precision was continuously monitored with the Lyphochek Quantitative Urine Control 2 (Bio-Rad). Duplicate control samples were assayed bimonthly after fresh reconstitution. Within-run SDs were calculated from replicate differences (D) as paired SD = [([Sigma][D.sup.2]/2n).sup.l/2] (21), and between-run SDs were calculated as usual from the first result of each analytical run.

For the recovery study, two urine pools were prepared according to the creatinine concentration measured in the urine samples, and five samples of each pool were enriched with a concentrate of the amino acid calibration mixture (200 [micro]mol/L). All of these samples as well as the calibration mixture, at the same dilution in 0.1 mol/L HCI, were assayed, and the mean recovery percentage, corrected for the internal standard, was calculated.


Statistical analyses were performed according to the Expert Panel on the Theory of Reference Values from the IFCC (22). The Anderson-Darling test (23) was used to assess whether the distribution of test results was gaussian. Outliers were excluded according to the Dixon test (24). Results were expressed as mean [+ or -] 1.96 SD (0.95 reference intervals), and the estimated confidence intervals ([beta] = 0.9) were also calculated. Statistical significance of differences in mean values was calculated by the unpaired Student Mest. MS Excel 97 software (Microsoft) was used for data processing and statistical analysis.


The long-term design of this study required continuous monitoring of method performance based on precision and accuracy. Different batches of the commercial urine control were used over the 5 years of the study. Analytical data for the last year corresponding to the same lot of product are provided. During this 1-year period, three Pico-Tag columns were used. For two of the columns, the analyses included were performed during the last and the first halves of their respective useful lifetimes. The third column was used over its complete life span.

Phenylthiocarbamyl derivatives for 39 amino acids and some related compounds (such as amines and dipeptides) were identified in the calibration mixture, but only 36 derivatives were regularly identified in control samples according to their retention times. In the urine control, phosphoserine and argininosuccinic acid were undetectable, and [gamma]-aminobutyric acid and citrulline overlapped, eluting as a single, small peak with an average retention time assigned for presentation to citrulline. Within-run CVs were <0.5% for every peak, whereas the highest between-run CVs were 2.8% and 3.3% for threonine in calibration and control solutions, respectively. Retention times of peaks migrating between 8 and 20 min showed poorer precision. In general, peaks were slightly delayed in control samples, and the retention time imprecision was slightly higher for control samples.

To estimate accuracy, we performed two recovery studies during the last year using two different columns with 265 and 30 injections, respectively. In the first study, in which a dilute urine was used, mean recoveries were 87-122%, with the exceptions of argininosuccinic acid (132%) and aminobutyric acid (81%). In the second study, in which a concentrated urine was used, mean recoveries were 78-120%, with the exception of phosphoserine (50%) and glutamic acid (67%). In concentrated urine, derivatives migrating in the first 12 min seemed to have poorer recoveries than those migrating later. Tables showing the within- and between-run precision for retention times and the recovery data and within- and between-run precision for amino acid concentrations are included in an electronic supplement in the on-line version of this article (

Method precision over 1 year of study showed the well-known dependency on amino acid concentration. Within-run CVs ranged from 2.9% for 1-methylhistidine to 17% for arginine, with the exception of hydroxylysine (22%). Between-run CVs ranged from 7.1% for lysine to 46% for hydroxylysine. In general, amino acid concentrations >165 [micro]mol/L showed between-run CVs <10%, and amino acid concentrations between 30 and 165 [micro]mol/L showed CVs <20%, with the exception of ethanolamine (29%). CVs >25% were observed in some amino acids present in concentrations <15 [micro]mol/L (arginine, proline, aminobutyric acid, methionine, cystine, isoleucine, and hydroxylysine).

All amino acids detected in the urine control were also quantified in urine samples from patients to establish adequate reference intervals, regardless of the fact that some of them either were not useful for diagnosis or required special sampling for clinical management (1methylhistidine, carnosine, tryptophan, phosphoethanolamine, ethanolamine). Table 1 shows the reference intervals for amino acid excretion in selected age ranges for random urine samples and 24-h urine collections provided by separate groups of subjects. The age ranges were selected to represent physiological periods such as the newborn period, infancy, early and late childhood, puberty, and adulthood. A reference interval for anserine is not included because the peak frequently was undetectable. Positively skewed distributions were found in almost every amino acid age group; therefore, log-transformations were performed on every group of original data. As a result, log-distributions gave rise to asymmetric intervals around the mean. Taurine showed extremely skewed distributions with numerous outlying values; therefore, corresponding reference intervals are presented as the observed ranges.

In general, amino acid excretion decreases with age. Some exceptions include phosphoethanolamine and 3-methylhistidine, which showed a relatively constant excretion throughout childhood. 1-Methylhistidine excretion increases during the first year of life with subsequent fluctuations at higher concentrations. Taurine excretion decreases during the first year of life and afterward remains constant, with a new increase in adulthood. Statistical comparisons of age-matched intervals probed the differences between random and 24-h urine samples in unrelated amino acids ([beta]-aminoisobutyric acid, carnosine, and arginine for the 8-16 years group; and aspartic acid, [beta]-alanine, threonine, camosine, valine, and hydroxylysine for the adult group). Likewise, only isolated statistical sex differences in unrelated amino acids could be observed within groups of subjects older than 8 years (data not shown).


Chromatograms for nonpathological and pathological urine samples are shown in Figs. 1 and 2. Table 2 shows the urinary excretion of representative amino acids in random and 24-h samples from patients with cystinosis, Lowe syndrome, and phenylalanine-restricted or -unrestricted phenylketonuria as well as from a random urine sample of a diet-restricted patient with leucinosis. Table 2 also displays the cystine and dibasic amino acid excretion from phenotypically homozygous and heterozygous cystinuric patients. The lowest and highest concentrations are shown as well as ranges of the multiples of corresponding upper reference limits.


In spite of more than 10 years of routine use of PITC RP-HPLC in clinical amino acid analysis, validation data for its urine application are scant (3, 9). A linear response up to 2 mmol/L has been demonstrated as well as a 10-fold increase in sensitivity compared with ninhydrin methods (11), with even greater sensitivity and higher linear responses for non-a-amino acids such as imino acids and (3-amino acids. Likewise, >100 compounds and pharmacological metabolites have been comprehensively studied as potential interferents (11,12).


We have provided recovery and precision data obtained over the last year of our study as representative of the long-term performance of the method, but equivalent results for recovery and precision were obtained during the rest of the study. Over this 1-year period using three different columns, the peak positions for 37 amino acids were reproducible enough to ensure correct peak identifications in patients' samples. Co-elution of [gamma]-aminobutyric acid and citrulline has previously been documented (12), but it does not represent a serious problem because of the low concentrations of [gamma]-aminobutyric acid expected. The precision of retention times was similar to that seen in short-term studies in plasma with PITC HPLC methods (12), and in plasma and urine with ninhydrin automated ion-exchange chromatography methods (5).

In concentrated urine samples, based on a high creatinine concentration, peaks eluting from the column in the first 12-16 min showed poorer recoveries. These poorer recoveries may have resulted from insufficient clean-up of the sample with the solid-phase extraction procedure, which is intended mainly to clear the first part of the chromatogram. Insufficient clean-up leads to more complex chromatograms and poorer integration results. As a routine practice, dilution of urine samples with creatinine concentrations >10.0 mmol/L has been performed. The recoveries in the dilute pool were comparable to those for urine samples of the phenylhydantoin HPLC method (65-100%) (25), the 9-fluorenylmethyl chloroformate-OPA HPLC method (83-105%) (26), and the ninhydrin automated ion-exchange chromatography method (94-115% to 75-140%, depending on the de-ammoniation procedure) (6). Likewise, recoveries were equivalent to those obtained for plasma samples using the PITC HPLC method (89-117%) (11) and the OPA HPLC method (79-121%) (9).

The results of our within- and between-run precision studies over 1 year seem better than those described by Feste (12) (5.7-129% for the same method in a short-term scheme). Both studies confirm the poorer reproducibility for concentrations <15-30 [micro]mol/L occasionally found for some amino acids (such as arginine, proline, methionine, or isoleucine) with more efficient reabsorption mechanisms (14). These long-term CVs are poorer than those found with the 9-fluorenylmethyl chloroformate-OPA HPLC method (26) both within (2-24%) and between runs (4-27%) as well as with the most recently developed methods using ninhydrin automated ion-exchange chromatography [within-run CV <4-19.8% and between-run CV <10-23.1% (5)]. Although both studies report short-term CVs, the better precision could be explained by the automation of their procedures as opposed to the manual sample procedure used in the PITC HPLC method. The generalized high imprecision of the amino acid urine analyses highlights the importance of using an appropriate urine control with physiological concentrations rather than calibration mixtures or plasma controls in method validation and quality assurance.

The reference values obtained in random urine samples confirm previous findings of an age-related decrease in the excretion of amino acids adjusted for creatinine concentration (15,16) attributable to the phenomenon of renal adaptation during childhood (1,14). The validity of random urine samples for the diagnosis of aminoaciduria has already been established using ion-exchange chromatography methods, although it is commonly accepted that there is a higher variation of amino acid concentrations in untimed samplings (15) and in 12-h urine collections (17) compared with 24-h collections. Nevertheless, no important differences in mean amino acid concentrations of untimed, 12-h, and 24-h paired urine samples have been reported in adults (15,17). Comparisons of the current means of intervals corresponding to unpaired samplings confirm these findings, demonstrating only a few significant differences between random and 24-h urine samples in age-matched groups. Moreover, higher limits for 24-h intervals have been observed in several amino acids.

The differences observed between specimens could be explained in several ways: (a) as an energy source, amino acids are consumed in bacterial contamination (27), which could be more prominent in the 24-h urine specimens; (b) a diurnal variation has been observed in amino acid excretion (15,17), with the lowest values in early morning and the highest in late evening; and (c) random urine samples could be more affected by food intake than 24-h urine collections, with early morning, pre-breakfast specimens being the least affected (28). In our study, gross bacterial contamination was visually excluded for each sample at reception. We had little control over the time of sampling in random specimens, but based on the times of their reception in the laboratory, most of them were collected before 1200.

Some amino acids, such as phosphoethanolamine, methylhistidines, and taurine, show clearly different age-dependent patterns of excretion. Constant or increased excretion of these amino acids during childhood probably is related to food intake; for example, methylhistidine excretion can be increased by a diet rich in poultry, rabbit, or pork (1, 27). 3-Methylhistidine excretion is related to muscle protein breakdown (29), and creatinine is related to muscle mass, supporting the age-constant pattern of the ratio. The decreases in taurine excretion during the first year of life could be related to the progressive substitution of taurine-rich maternal milk for other foods (1, 30), whereas increased ingestion of meat and shellfish in adulthood could increase taurine concentrations (27). These distinct excretion patterns have been described previously by Parvy et al. (16), using a ion-exchange chromatography method.

[beta]-Aminoisobutyric acid excretion showed an extremely skewed distribution, especially in children younger than 8 years. The use of cutoff values to identify high excretors (1,16) in ion-exchange chromatography methods has been common. In our study, no clear cutoff values were found, but we tentatively used 113 and 57 mmol/mol of creatinine for children younger and older than 8 years, respectively (i.e., 1000 and 500 Amol/g of creatinine), according to the highest excretions observed. These values would provide a frequency of high excretors in children of 8%. Similar frequencies between 4% and 10% have been reported for Western countries (1).

Because of differences in age categories, direct comparison with other studies is not possible. Nevertheless, a general overview of our reference values suggests intervals similar to those reported by Tsai et al. (15) in untimed samples, particularly in children and adults. For glycine, we found lower excretion in infants and children younger than 8 years. With regard to the intervals reported by Parvy et al. (16) in pre-breakfast samples, we found higher reference intervals for almost every amino acid, particularly in newborns and infants. However, the means of the intervals for some of the most prominent amino acids, e.g., threonine, serine, glutamine, glycine, alanine, tyrosine, and phenylalanine, were similar. For 24-h urine collections in adults, the reference intervals for our HPLC method were also greater than intervals reported previously (31). All of these cited studies were performed with the ninhydrin ion-exchange chromatography method.

The observed differences in method-related reference intervals could be explained in several ways: (a) the increased sensitivity and better resolution of RP-HPLC columns (3, 9) as well as the differences in the sampling period; (b) the commercial origins of the calibration solutions, which have been considered responsible for dispersion of results in quality-control schemes (4) and therefore could partially explain the differences in reference intervals; and (c) the skewed distributions of amino acids in both plasma (32) and urine (1), which have long been known; therefore, the use of recommended statistical treatments of the data could yield reference limits different from the ranges reported in other studies.

Some examples of representative amino acid excretion in patients with aminoaciduria support the reliability of the reference intervals obtained. A partial overlapping with the corresponding reference intervals was found for the first sample of a 3-month-old patient with Lowe syndrome, but a subsequent sample at 17 months of age showed a characteristic generalized hyperaminoaciduria. This pattern has been described elsewhere (33). According to the recent studies of Guillen et al. (34), using the Waters Pico-Tag method, cystinuric patients can be classified as phenotypic homozygotes and heterozygotes by the use of a cutoff of 113 mmol/mol of creatinine for cystine excretion. From our 26 diagnosed cystinuric subjects, 24 patients could be correctly classified by cystine excretion. Our results confirm the conclusion of Guillen et al. regarding lysine as the better discriminator between non-type I heterozygotes and the general population, because it always shows values above the upper reference limit.

In conclusion, the advantages of the PITC HPLC method, such as high resolution, speed, and sensitivity for quantifying of imino acids, sulfur, and [beta]-amino acids, make it suitable for urine amino acid analysis, and its reliability and feasibility for clinical use has been demonstrated. However, its performance in terms of precision should be improved to achieve the precision of the reference method, automated ion-exchange chromatography using ninhydrin. The prospective selection of the reference patients in this study probably yielded wider and more practical reference intervals than those obtained using more stringent conditions. On the other hand, small deviations from reference intervals can be common and generally have no clinical significance (3). Although RPHPLC methods to analyze plasma amino acids are currently in use in some clinical laboratories, the measurement of urinary amino acids is most often performed by ion-exchange chromatography. Hopefully, these reference intervals will serve as a guide for laboratories moving to this newer technology.

We thank Juan Muniz for valuable technical assistance.

Received July 19, 2000; accepted November 28, 2000.


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[1] Nonstandard abbreviations: RP-HPLC, reversed-phase HPLC; PITC, phenylisothiocyanate; and OPA, o-phthalaldehyde.


Servicio de Analisis Clinicos, Hospital San Agustin, Aviles, and Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo, Camino de Heros 4, 33400 Aviles, Asturias, Spain. Fax 34-985-123-025; e-mail
Table 1. Age-related reference intervals (a) for urinary amino
acid excretion in mmol/mol creatinine.

 Untimed samples
acid 0-1 month 1-12 months 1-3 years

Asp 9-57 (23) (b) 10-69(26) 10-73(27)
Glu 31-96(54) 24-102(50) 22-89(44)
Aad 7-96(26) 7-110(29) 7-93(25)
Hyp (f) 11-556(100) 5-238(34) 5-23(10)
Pea 13-167(46) 12-216(51) 19-225(66)
Ser 48-509(156) 34-329 (106) 46-143 (81)
Asn 15-223(58) 18-197(59) 19-79(39)
Gly 127-2042 (510) 133-894 (345) 103-404 (204)
Gin 37-600(148) 63-446(168) 57-205(108)
[beta] Ala 2-41(8) 2-38(8) 2-27(7)
Tau (g) 12-1057 10-809 6-267
His 23-676(125) 69-392(164) 57-262(122)
Cit 1-18(5) 2-46(9) 2-29(7)
Thr 9-337(56) 12-145(41) 7-73(23)
Ala 34-358(109) 27-313(93) 12-185(47)
RAib 0-520(14) 5-258(36) 5-236(35)
Car 3-184(21) 8-160(36) 2-114(17)
Arg 0-81(10) 0-40(5) 0-15(4)
Pro 3-257(29) 2-90(14) 1-22(5)
1mh 0-78(7) 0-98(7) 2-242(24)
3mh 5-85(20) 10-95(31) 10-117(35)
Eta 33-253(91) 57-221(112) 37-144(73)
Abu 1-43(5) 1-58(7) 2-20(7)
Tyr 5-74(19) 12-64(28) 8-38(18)
Val 3-34(10) 3-43(11) 2-23(7)
Met 2-24(7) 2-18(6) 2-12(5)
Cysta 1-26(6) 1-11(4) 0-29(3)
[(Cys).sub.2] 5-109(24) 2-42(10) 1-23(5)
lie 2-37(8) 2-21(6) 1-12(4)
Leu 4-84(17) 4-72(17) 3-45(11)
Hly 3-49(11) 2-50(9) 1-14(4)
Phe 2-49(9) 7-42(17) 4-33(12)
Trp 1-46(8) 3-37(10) 2-29(8)
Orn 2-37(8) 2-19(6) 1-15(4)
Lys 6-464(54) 4-80(18) 4-37(12)
No. subjects 20 (11/9) (h) 30(16/14) 33(13/20)

 Untimed samples
acid 3-8 years 8-16 years >16 years

Asp 9-59(23) 4-60(15) 4-29(10)
Glu 18-53(31) 12-44(23) 9-47(20)
Aad 10-74(27) 6-45(16) 3-36(10)
Hyp (f) 0-40(5) 0-17(3) 0-10(1)
Pea 15-195(55) 22-177(63) 8-325(57)
Ser 40-94(61) 28-94(51) 15-85(36)
Asn 13-73(31) 10-86(29) 11-56(25)
Gly 100-280 (167) 83-254(145) 37-371 (132)
Gin 45-165(86) 33-125(64) 20-92(43)
[beta] Ala 2-18(6) 2-9(4) 1-11(3)
Tau (g) 7-264 28-268 13-534
His 61-259 (126) 43-211(96) 21-164(59)
Cit 1-23(5) 1-17(4) 1-15(4)
Thr 10-67(26) 10-48(22) 4-48(14)
Ala 15-97(39) 12-67(29) 10-57(23)
RAib 2-144(18) 4-48(13) 4-35(12)
Car 2-77(13) 0-49(5) 0-16(2)
Arg 0-11(3) 0-9(2) 0-5(2)
Pro 1-17(3) 1-19(3) 0-5(l)
1mh 4-359(36) 3-362(33) 2-290(22)
3mh 17-162(52) 12-86(31) 15-52(28)
Eta 26-111(54) 26-111(54) 11-88(32)
Abu 2-29(9) 1-26(6) 1-11(3)
Tyr 8-32(16) 5-30(12) 4-21(9)
Val 2-12(5) 1-16(4) 2-7(3)
Met 2-8(4) 1-10(3) 1-7(3)
Cysta 0-21(3) 0-13(2) 0-4(2)
[(Cys).sub.2] 1-20(4) 1-17(4) 0-21(3)
lie 1-7(3) 1-8(2) 1-7(3)
Leu 3-22(8) 3-17(7) 3-10(5)
Hly 1-7(3) 0-15(2) 0-4(l)
Phe 5-25(11) 2-26(8) 2-13(6)
Trp 2-27(7) 2-27(7) 2-12(5)
Orn 1-8(2) 1-5(2) 1-4(2)
Lys 5-51(17) 1-65(9) 2-63(10)
No. subjects 25(13/12) 26(13/13) 34(16/18)

 24-h collections
acid 8-16 years >16 years

Asp 8-40(17) 5-50(15) (c)
Glu 12-55(25) 11-42(22)
Aad 4-45(13) 4-32(11)
Hyp (f) 0-15(2) 0-15(2)
Pea 11-169(50) 22-187(64)
Ser 20-88(42) 18-89(40)
Asn 7-79(23) 10-65(25)
Gly 58-325 (137) 49-413(142)
Gin 14-196(51) 17-146(50)
[beta] Ala 1-23(5) 2-30 (7) (d)
Tau (g) 24-255 13-587
His 48-195(97) 30-186(75)
Cit 1-21(4) 1-22(5)
Thr 6-70(20) 6-74 (20) (e)
Ala 11-86(31) 9-89(28)
RAib 2-32 (9) (e) 3-50(12)
Car 2-40 (9) (e) 0-16(3)
Arg 0-10 (1) (d) 0-8(l)
Pro 1-9(2) 0-9(2)
1mh 4-304(34) 5-376(44)
3mh 15-83(35) 14-71(31)
Eta 14-149(46) 12-72(29)
Abu 2-15(5) 1-10(2)
Tyr 3-50(12) 3-36(9)
Val 1-12(3) 2-12 (4) (e)
Met 1-9(2) 1-5(2)
Cysta 0-9(2) 0-7(2)
[(Cys).sub.2] 1-19(5) 0-21(3)
lie 1-9(3) 1-9(2)
Leu 2-22(7) 2-16(5)
Hly 0-12(3) 0-7(2)
Phe 3-22(9) 3-13(7)
Trp 2-22(7) 3-14(6)
Orn 1-5(2) 1-5(2)
Lys 3-44(11) 2-53(11)
No. subjects 25(19/6) 33(22/11)

(a) Confidence intervals for these reference limits are included in an
electronic data supplement available at

(b) Range, as mean [+ or -] 1.96 SD (mean), mmol/mol of creatinine.

(c-e) Statistical significance for differences in age-matched groups
(random vs 24h urine samples: (c) P <0.01; (d) P <0.001; (e) P <0.05.

(f) Hyp, hydroxyproline; Pea, phosphoethanolamine; Tau, taurine;
[beta]Aib, [beta]-aminoisobutyric acid; Car, camosine; 1mh,
1-methylhistidine; 3mh, 3-methylhistidine; Eta, ethanolamine;
Abu, aminobutyric acid; Cysta, cystathionine; [(Cys).sub.2], cystine;
Hly, hydroxylysine.

(g) Observed ranges.

(h) M/F.

Table 2. Urinary amino acid excretion in untimed and 24-h samples
of aminoaciduria patients.

 Cystinosis Lowe syndrome Leucinosis
Age range 17 months-3 years 3 m-17 m 13 years
No. patients 1 2 1
No. samples 4 3 1
Amino acid

Ser 582-1530 (a) 281-755
 (4.6-8.1) (b) (1.6-2.3)
Asn 541-1022 198-474
 (5.2-13.5) (2.4-2.5)
Gly 1320-2303 990-3273
 (2.6-7.9) (1.7-3.7)
Gin 1572-4092 474-765
 (9.2-19.6) (1.7-2.3)
His 455-1126 364-762
 (1.8-2.9) (1.0-1.9)
Thr 473-1355 158-683
 (7.1-12.1) (2.1-4.7)
Ala 735-2000 297-542
 (6.4-18.5) (1.1-1.7)
Arg 66-177 14-59
 (3.8-18.5) (1.1-1.7)
Pro 565-1375 71-555
 (15.2-81.9) (3.3-6.1)
Tyr 214-431 37-263
 (6.8-12.6) (0.8-4.1)
Val 284-896 15-63 12
 (21.1-57.2) (0.7-1.5) (0.8)
Met 25.9-66.4 7-44
 (3.3-6.7) (0.4-2.5)
[(Cys).sub.2] (c) 41-267 21-119
 (2.0-11.7) (0.8-2.8)
Iie 74-220 8-28 9
 (6.4-31.4) (0.4-1.4) (1.1)
Leu 153-403 22-63 59
 (5.0-18.7) (0.3-0.9) (3.6)
Phe 103-337 23-62
 (4.2-12.1) (0.6-1.5)
Trp 74-135 21-36
 (2.8-4.0) (0.7-1.0)
Orn 90-270 17-106
 (11.7-24.9) (1.1-5.7)
Lys 457-1154 180-810
 (8.9-15.1) (2.3-10.1)

 Cystinuria Cystinuria
 Phenylketonuria (heterozygous) (homozygous)
Age range 9-34 years 2-31 years 2-65 years
No. patients 2 13 11
No. samples 4 23 28
Amino acid








Arg 4-321 7-527
 (0.8-69.8) (0.8-114.5)

Tyr 4-6


[(Cys).sub.2] (c) 16-113 114-324
 (0.9-5.4) (5.4-15.3)


Phe 55-337

Orn 6-189 22-284
 (0.7-47.2) (3.1-71.1)
Lys 109-858 344-1130
 (1.7-13.7) (5.3-18.0)

(a) Observed ranges, mmol/mol of creatinine.

(b) Range of multiples of the upper reference limit.

(c) Cystine.
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Title Annotation:General Clinical Chemistry
Author:Venta, Rafael
Publication:Clinical Chemistry
Date:Mar 1, 2001
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