A11 (m'ui) - Clinical Chemistry

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Richmond. W. Analytical reviews in clinical biochemistry: the quantitative analysis of cholesterol. Ann Clin Biochem. 1992:29:577-97. 7. Richmond.
Clinical

Chernistiy

In a previous paper [4/, cholesterol concentrations were measured with the enzymatic and the LB reagent in split aliquots of hexane extracts from several individual sera. Statistically the enzymatic values showed a small systematic constant negative bias vs the LB values (-0.052 mmol/L, corresponding to 0.9% at 6.0 mmol/L). Assuming equivalent interference by the noncholesterol sterols in the two reactions, it is tempting to speculate that the enzymatic reaction is less sensitive to the remaining unidentified sources of interference [2], thus being in position to generate results closer to the definitive isotope dilution mass spectrometric method. Again, this appears to have a negligible practical impact. References 1.

2.

3.

4.

5.

8.

7.

8.

9.

Ellerbe P, Myers GL, Cooper GR, Hertz HS, Sniegoski LI, Welch MJ, White E. A comparison of results for cholesterol in human serum obtained by the Reference Method and by the Definitive Method of the National Reference System for cholesterol. Clin Chom 1990:36:370-5. Bemert iT, Akins JR. Cooper GR, Poulose AK, Myers GL, Sampson El. Factors influencing the accuracy of the National Reference System total cholesterol Reference Method. Clin Chem 1991:37:2053-61. Franzini C, Besozzi M. Proposed reference method for serum cholesterol assay. Giorn Ital Chin Clin 1985:10:251-6. Franzini C, Luraschi P. An extraction/enzymatic procedure for sewm cholesterol measurement: evaluation of performance characteristics. J Clin Chem Clin Biochem 1990:28:913-8. Smith AG, Brooks CJW. Cholesterol oxidases: properties and applications. i Steroid Biochem 1976:7:705-13. Richmond W. Analytical reviews in clinical biochemistry: the quantitative analysis of cholesterol. Ann Clin Biochem 1992:29:577-97. Richmond W. Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 1973:19:1350-6. AIlain CC, Poon LS, Chan CSG. Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974:20:470-5. Brooks CJW, Smith AG. Cholesterol osidase. Further specificity in relation to the analytical characterization

studies

42,

No.

3, 1996

in combination

477

with

mass spectrometry

/2,5-7/.

This

method

is

accurate and precise but is slow and requires expensive equipment. We propose a simple, fast, and reliable capillary electrophoresis method for detecting and measuring 10 organic acid markers-methylmalonate, glutarate, 3-methylglutarate, N-acetylaspartate, 2-aminoadipate, propionate, lactate, 2-oxoisovalerate, isovalerate, and homogentisate-in the screening of organic acidurias. The method comprises direct diluted urine application, electrophoretic separation at 29 kV, and ultraviolet absorption at 185 nm. Complete separation was achieved within 10 mm. This method is applicable for use in routine clinical chemistry laboratories. Upon approval by the Ethics Committee of La Paz Hospital, urine samples were collected from 65 control children ranging from 2 months to 10 years old and from two children with glutaric aciduria type I. Samples were filtered through Ultrafree Millipore (Bedford, MA) filters (Mr 10000), centrifuged 15 mm at 2000g, and then diluted with MilliQ water (Millipore) to obtain a creatinine concentration -1 mmol/L. The organic acid separation was performed on a Waters AccuSep polyimide-coated 100 cm X 75 m fused-silica capillary using a CIA#{174}Capillary Electrophoresis instrument (Waters, Milford, MA) equipped with a Millenium#{174} Chromatography Manager (Vaters). The electrolyte was a solution of 20

A11

of substrate

of steroids.

J Chro-

matogr 1975:112:499-511. Cooper GR, Duncan PH, Hazlehurst JS, Miller DI, Bayse DD. Cholesterol, enzymic method. In: Faulkner WR, Meites S. eds. Selected methods of clinical chemistry, Vol. 9. Washington, DC: AACC Press. 1982:165-74. 1.1. Duncan 1W, Mather A, Cooper GR. The procedure for the proposed cholesterol Reference Method. Atlanta, GA: Centers for Disease Control, 1982. 12. Mollies M, Glueck Ci, Sweeney C, Fallat RW, Isang RC, Ishikawa ft. Plasma and dietary phytosterols in children. Pediatrics 1976:57:60-7. 13. Bhattacha,a AK, Connor WE. 13-sitosterolemia, xanthomatosis. A newly described lipid storage disease in two sisters. J Clin Invest 1974:53:1033-43. 10.

0.02 0.01 0.0

Determination sis in Screening Angel Hernanz

of Organic Acids by Capillary Electrophoreof Organic Acidurias, Car/os M. Jariego (Servicio de Bioquimica, Hospital La

Castellana 261, 28046 dence: fax 34-1-3582733) Pathologic alterations acids, carbohydrates,

Madrid,

of normal cholesterol,

Spain;

*author

catabolism biogenic

for

and Paz,

correspon-

of amino acids, fatty amines, and steroids

frequently result in abnormal excretion of urinary organic acids. These metabolic disorders manifest themselves either by excretion of abnormal organic acids in the urine that are usually not detected in healthy individuals or by excretion of massive amounts of certain acids that are normally present in the urine in small ited and severe

amounts acquired neurologic

Organic

acids

[1,2/. Neonatal screening of urine for inherorganic acidurias is important for preventing disease,

mental

are quantified

and death [3,4/. by gas chromatography

retardation,

in urine

0.02

0.01 0.0

JLItI Thie

(m’ui)

Fig. 1. Electropherograms of (A) a mixture of 250 tmoI/L of each of the following organic acids: methylmalonate (1), glutarate (2), 3-methylglutarate (3), N-acetylaspartate (4), 2-aminoadipate (5), propionate (6), lactate (7), 2-oxoisovalerate (8), isovalerate (9), and homogentisate (10): (B) a normal urine sample: and (C) urine from a patient with glutaric aciduria type-I, showing the high amount of glutarate in the sample.

478

Technical

mmol/L sodium sulfate, 0.35 mmol/L calcium chloride, and 25 mL/L CIA-Pak OFM anion-OH (Waters), prepared daily, filtered, and degassed. Stock solutions were prepared by dissolving the sodium salts of methylmalonic, glutaric, 3-methylglutaric, N-acety!aspartic, 2-aminoadipic, propionic, lactic, 2-oxoisovaleric, isovaleric, and homogentisic acids (ICN Biomedicals, Costa Mesa, CA). The working calibrator solutions were prepared by diluting the stock solutions to 1000, 750, 500, 250, 125, 50, and 25 mol/L to obtain a calibration curve. The injection mode used was hydrostatic for 30 s, and the electrophoretic separation was carried out at 29 kV and thermostated at 25 #{176}C. Ultraviolet direct absorption at 185 nm was used as detection Figure

mode. lA shows

the

electropherogram

obtained

from

a

standard mixture of 250 j.tmol/L of each of the 10 organic acids. Electrolyte composition, working temperature, voltage applied, and wavelength detection were optimized to obtain baseline resolution of all organic acids. Some critical organic acids were difficult to separate. Thus, to obtain baseline separation of glutarate and 3-methylglutarate we found that the optimal concentration of sodium sulfate was 20 mmol/L. Separation of lactate, propionate, and oxoisovalerate was accomplished by optimizing the amount of calcium chloride added to the electrolyte. Concentrations between 25 and 1000 j.mo1/L for all organic acids gave a linear calibration curve (r >0.980). Withinrun CVs were 0.9-1.3% (n = 20) and between-run were 3:1. Figure lB shows the electropherogram of a normal urine sample, and Fig. 1C shows the electropherogram of a patient with glutaric aciduria type I, showing the high amount of glutarate in the urine sample. In conclusion, capillary electrophoresis is a new, simple, reliable, fast, and low-cost method for detecting and measuring was

organic

acids

in the screening

of organic

acidurias.

References 1. Shih VE. Detection of hereditary metabolic disorders involving amino acids and organic acids. Clin Biochem 1991:24:301-9. 2. Iuchman M, McCann MI, Johnson PE, Lemieux B. Screening newborns for multiple organic acidurias in dried urine samples: method development. Pediatr Rex 1991:30:315-21. 3. Ozand PT, Gascon GG. organic acidurias: a review. Part 1. i Child Neurol 1991:6:196-219. 4.

Mardens

procedures spectrometry. 5.

Marsden Curr

Opin

V. Kumps

A, Planchon for urinary organic J Chromatogr

C, Wurth C. Comparison of two extraction acids prior to gas chromatography-mass

1992:577:341-6.

DL, Nyhan WL. Neurological Neurol

Neurosurg

diseases

in disorders

of organic

acids.

1992:5:349-54.

6. Guneral F. Bachmann C. Age-related reference values for urinary organic acids in a healthy Turkish pediatric population. Clin Chem 1994:40:862-8. 7. Thompson J, Miles B, Fennessey P. Urinary organic acids quantitated by age groups in a healthy pediatric population. Clin Chem 1977:23:1734-8.