Organic acidurias : proceedings of the ninth symposium of the Society for the Study of Inborn Errors of Metabolism / edited by J. Stern and C. Toothill.

  • Society for the Study of Inborn Errors of Metabolism.
Date:
1972
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Organic acidurias : proceedings of the ninth symposium of the Society for the Study of Inborn Errors of Metabolism / edited by J. Stern and C. Toothill. Source: Wellcome Collection.

    117/202 (page 101)
    BENZOIC ACIDURIA 101 hydrochloric acid to pH 1-0 (pH meter), and was extracted once with a double volume of ethyl acetate, and four more times with equal volumes of ethyl acetate. The combined organic phases were dried overnight over anhydrous sodium sulphate, and the ethyl acetate was removed on a rotary evaporator. The residue was dissolved in methanol-diethyl ether (1:9) and then esterified with diazomethane (Schlenk & Gellerman, 1960). Decanoic acid, 1 -0 mg, was added as an internal standard just before the methyl esterificati on step. Methyl esters of benzoic and hippuric acid obtained from urine extracts were then determined by gas-chromatography on columns packed with 5 per cent neopentyl glycol adipate on Anakrom SD, 80 to 90 mesh (Analabs, North Haven, Connecticut) on dual column gas- chromatographs equipped with dual flame ionisation detectors. In Vancouver, a MicroTek MT-220 gas-chromatograph was used with 183 X 0-64 cm i.d. glass columns, while in Norway a Perkin-Ehner 800 gas-chromatograph was used, with stainless steel columns, 200 x 0-20 cm i.d. The columns were operated at 110°C for 10 minutes after injection of the sample, and their temperature was then raised 10°C/min to a final temperature of 210°C. The amounts of benzoic and hippuric acid present in urine specimens were calculated by measuring the peak areas of their methyl esters on chromatograms with a Technicon integrator-calculator, and comparing them with the areas of the methyl decanoate internal standards. When known amounts of benzoic or hippuric acid were added to normal urines, and the urines were then extracted and analysed as above, recovery rates were 100 per cent for benzoic acid, and 95 to 98 per cent for hippuric acid. Benzoic acid was determined in plasma (first deproteinised by the addition of four volimies of absolute ethanol), and in cerebrospinal fluid, by the same general technique. Combined gas-chromatography and mass spectrometry for identifi¬ cation of methyl benzoate was performed with a Varían CH 7 mass spectrometer coupled to a Varían 1440 gas-chromatograph, equipped with a single glass column (244 x 0-20 cm i.d.) packed with 10 per cent OV-17 on Gas-Chrom Q (Applied Science Laboratories, State College, Pennsylvania). Helium was the carrier gas. Results Screening for benzoic aciduria Seventeen of the 307 psychiatríc patients showed either a marked reduction or an apparent absence of hippuric acid in their urines when these were screened by paper chromatography. However, only foui of these seventeen patients were subsequently shown to excrete benzoic acid in their urine. The remaining thirteen patients excreted low amounts of hippuric acid (0-05 to 0-50 mg/mg of creatinine), but gas-chromatographic