287

Clinica Chimica Acta, 66 (1976) 287-293 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

CCA 6995

QUANTITATIVE GASCHROMATOGRAPHIC DETERMINATION OF SHORT CHAIN ALDEHYDES AND KETONES IN THE URINE OF INFANTS

CLAUDE BACHMANN ‘, REGULA BAUMGARTNER JEAN-PIERRE COLOMBO a

b, HUGO WICK b and

aDepartment of Clinical Chemistry, Inselspital, University of Berne, Berne and bUniversity Children’s Hospital, Base1 (Switzerland) (Received

September

19,1975)

Summary A gaschromatographic method for the quantitation of short chain aldehydes and ketones in urine is described. The method is reliable and sufficiently easy to be used for clinical investigations. Reference values for infants’ urine are given for acetaldehyde, acetone and butanone. Acetone concentrations in urine closely correspond to the normal range of the blood concentration reported in the literature.

Introduction Ketosis is often encountered in children with cyclic vomiting and particularly in the inborn errors of the metabolism of the branched chain amino acids, where ketosis can already be found in early infancy. Its pathogenesis is not understood. Menkes identified 2-butanone, 2- and 3-pentanone and 2-hexanone in the urine of such a patient with propionic acidemia [l] . Abnormal ketones were also found in connection with methylmalonic acidemia and 2-methylacetoacetic aciduria [ 21. Recently it has been shown that exposure to 2-hexanone causes axonal degeneration in rats and probably neuropathy in men [3] . We developed a simple qualitative method to determine these compounds. It led to the discovery of abnormal ketones in the urine of a patient with biotin responsive methylcrotonylglycinuria [4]. The method has now been modified to allow the quantitation of aldehydes and ketones in urine.

288

Materials and methods Materials

1. 2,4_Dinitrophenylhydrazine p.a., Merck, Darmstadt, 10 mM in 2 N HCl. 2. Benzene (Uvasol), Merck, Darmstadt. 3. Aluminium oxide neutral, Woelm, Eschwege, containing 3% aqua bidest. 4. Dichloroethane, Merck, Darmstadt. 5. Dinitrophenylhydrazones of aldehydes and ketones (standards): an excess of free aldehyde or ketone (Fluka AG, Buchs) was added to the dinitrophenylhydrazine solution. The dinitrophenylhydrazones were extracted with benzene and dried in a vacuum desiccator. 6. Gaschromatograph Perkin Elmer, model 900, with a 6 ft, 2 mm ID glass column containing 3% Dexsil 300 GC on Supelcoport 100/120 mesh (Supelco, Bellefonte, Pa.). 7. Digital integrator, Infotronics, model CRS-208. Methods Preparation

Urine collected in plastic bags was frozen immediately upon voiding and stored at -20°C without preservative. Before analysis it was thawed at 0-4°C. 2-8 ml were added to 4 ml of the dinitrophenylhydrazine solution in a glass stoppered 60-ml centrifuge tube. After standing in the dark (1 hour) 3 g of sodium chloride were added. The dinitrophenylhydrazones were extracted with three times 15 ml of benzene. The combined organic extracts were dried under nitrogen, then dissolved in 0.5 ml of benzene and applied to a short column of 1 g aluminium oxide. After addition of three washings (0.5 ml each), the dinitrophenylhydrazones of the aldehydes and ketones were eluted with 8 ml of benzene. The eluate was dried down under nitrogen and taken up in 1.0 ml of dichloroethane prior to gaschromatography. For blank runs bidistilled water was used instead of urine. The peaks were identified by their retention times relative to added formaldehyde-dinitrophenylhydrazones (-DNPH). Gaschromatography

The conditions were: injection port 26O”C, oven 215°C isothermal, flame ionization detector 28O”C, nitrogen flow 46 ml/min at an inlet pressure of 48 psi. 1 ~1 of the extract was applied with a 5 ~1 S.G.E. guided plunger syringe (11.5-cm needle). Quantitation was done with the integrator at a track rate of 100 pV/min and a peak sensor gain of 10 with the gaschromatograph at attenuation 2 in range 1. Reference

urines

The urines were collected from 6 newborns (5 days of age) and 16 infants, 8 of which were prematures (l-8 weeks of age). They were fed only with commercial cow’s milk formula (Nan@, Eledon@, Humana@). None had a metabolic disorder.

289

Preliminary experiments had shown that acetaldehyde decreases and butanone increases with repeated thawing and freezing. Since the urines of the newborns had been thawed once, we used only the acetone results of this group in our calculations. The distribution of the results was analyzed by the sign test [15] and by plotting the percentage cumulative probability on probability paper. Results The retention times of the DNPH’s of short chain aldehyde and ketone standards relative to the first formaldehyde peak are given in Table I. In Fig.1 the superposed gaschromatograms of a mixture of standards at two concentrations are shown. Aldehydes and ketones which have their carbonyl group in an asymmetrical position within the molecule give two peaks in the gaschromatogram (enantiomers over the C=N bond of the DNPH.) The method was tested with a mixture of the DNPH of formaldehyde, acetaldehyde, acetone, butanone, 3-pentanone and 2-hexanone. All those compounds have been found in urines of normal adults or of patients with ketotic syndromes [1,2,4,6]. The limit of detection was at 5 pmoles for acetone and at 10 pmoles for the other compounds tested. The response was linear to the amount injected between 50 pmoles and 10 nmoles. The precision of the method was tested in two ways: the precision of the method overall and the precision of extraction and chromatography at different concentrations. The overall precision was determined by ten replicate analyses of the urine of a patient with methylmalonic acidemia (during treatment), the precision of extraction and gaschromatographic procedure at different concentrations by adding the DNPH’s of standards to six aliquots of the patient’s urine for each concentration and then carrying through the whole procedure. The underivatized aldehydes and ketones were not used for these additions because high dilutions of the urines would have resulted from additions of some ketones because of their poor solubility in aqueous solutions. The results are summarized in Table II. From the addition experiments we also calculated the recoveries as shown in Table III TABLE

I

RELATIVE RETENTION AND KETONES Retention

TIMES

time of formaldehyde

Acetaldehyde Propanaldehyde Butyraldehyde Isobutyraldehyde Valeraldehyde Isovaleraldehyde Capronaldehyde Acrolein Crotonaldehyde

OF

THE

2,4-DINITROPHENYLHYDRAZONES

dtitrophenylhydrazone

Peak I

Peak II

1.67 2.14 2.83 2.60 4.00 3.38 5.66 2.17 3.55

1.75 2.41 3.33 2.13 4.86 3.91 7.00 2.31 4.28

OF ALDEHYDES

= 1. Peak I and II are enantiomers. Peak I 2.32 3.07 3.90 4.00 5.13 5.03 6.93 6.69 6.52

Acetone 2-Butanone P-Pentaoone 3-Pentanone 2-Hexanone 3-Hexanone P-Heptanone 3-Hep tanone CHeptanone -

Peak II

3.21 4.35 5.90 5.21 8.30 1.28

Fig.

1.

dehyde

Superposed (A),

were

injected.

Fig.

2.

gascbromatograms

acetone

(B),

butanone

Gaschromatogram

of

the

of (C).

urine

two

mixtures

3-pentanone

of

a normal

of

(D)

standard

and

newborn.

dinitrophenylhydrazones

2-hexanone

A,

(E).

acetaldehyde;

1 and

0.1

B. acetone:

of mnoles

C,

acetalof each

butanone.

(amount recovered X lOO/amount added). In this procedure pyruvate, 3_hydroxybutyrate, the ketoacids of the branched chain amino acids, or the sugar dinitrophenylhydrazones or osazones did not interfere as long as dinitrophenylhydrazine was present in excess. Additional dinitrophenylhydrazine should be added if a precipitate is noted upon addition of the reagent or if the yellow colour of the reaction mixture fades since ketoacids compete for the dinitrophenylhydrazine. TABLE

II

PRECISION:

COEFFICIENT

HYDES

KETONES

AND

HYDRAZONES

ADDED

OF IN

INITIALLY Urine

VARIATION

A PATIENT’S

(C.V.) URINE

TO

THE

OF

ALONE

REPLICATE AND

added nmoles in 5 ml

C.V.

Acetone P-Butanone 3-Pentanone

ALDE-

with

added

amount

standard

(nmoles)

to

DNPH; 5 ml

100

200

500

5000

25

24

16

15

61

8

24

6

3

6

428

7

22

11

6

6

15

6

8

7

6

15

7

6

13

6

15

10

10

20

4

35

2-Hexanone Replicates

OF

DINITROPHENYL-

(%I

Formaldehyde Acetaldehyde

STANDARD

URINE Urine

alone

DETERMINATIONS

WITH

10

6

6

6

6

291 TABLE III

ACCURACY: MEAN RECOVERY (a) AND STANDARD DEVIATION 0~ STANDARD DINITROPHENYLHYDRAZONES ADDED TO URINE Each experiment w&scarried out six times. Amount added to 5 ml of urine (nmoles)

Formaldehyde Acetaldehyde Acetone P-Butsnone 3-Pentsnone 2-Hexsnone

x S.D. x S.D. x S.D. x S.D. X S.D. x S.D.

100

200

500

5000

56 14 88 21 84 19 87 13 93 6 101 13

71 17 99 6 89 9 93 6 91 14 99 6

83 13 98 3 99 6 96 8 96 7 98 15

74 11 94 5 94 5 95 6 95 6 94 10

We measured the peak heights of standards at 100, 200, 500 and 1000 pmoles and correlated them with the integrated peak areas. For compound8 eluting as double peaks we added the corresponding peak heights. The correlation coefficients between heights and area for 24 determinations were for

I

.

0

.

IA

I

ACETONE

I

. .own .

d.1..

lower

scale

5

Fig. 3. Acetone concentration in urine plotted on linear probability paper. Our own data of infants are represented as dots with use of the lower scale, the data of Bessette et al. [61 obtained with s different method in normal adults are shown as triangles and relate to the upper scale. Both distributions are not n0rItMl.

292 TABLE IV REFERENCE VALUES OF ACETALDEHYDE, FANTS AND NEWBORNS (NOT CORRECTED

Acetaldehyde Acetone Butanone

(N = 16) (iv = 22) (N = 16) ______

_.__~

ACETONE AND BUTANONE FOR RECOVERY)

Median (PM)

Range (PM)

360 63 1 ____

1460 15-233 o-4 ~_.__

__-_-._

IN THE URINE OF IN-

~__

formaldehyde 0.999, acetaldehyde 0.998, acetone 0.999, butanone 0.998, 3-pentanone 0.998 and 2-hexanone 0.992. In normal urines of newborns, prematures and young infants we found acetaldehyde, acetone and butanone. A typical gaschromatogram is shown in Fig. 2. No influence of the brand of formula fed could be detected. By using the sign test for examining the distribution of the normal values we could not prove an asymmetrical distribution. When we plotted the cumulative probability on linear probability paper the acetaldehyde values could not be fitted to a straight line, but showed a sigmoid shape. A log normal distribution was not found either. As can be seen from Fig. 3, the acetone plot showed a bend to the right. In Fig. 3 we have also plotted the urinary data (0 h) recalculated from Bassette et al [6] who determined acetone in blood and urine of adults with a different technique. We found an almost similar distribution in blood and urine, which obviously is not normal. For butanone 5 values were below the limit of detection. The measurable data fitted a normal distribution pattern. We did not find any significant difference between the newborns, prematures and infants. Thus we combined the three groups. Their median values and the range are shown in Table IV. Discussion The gaschromatography of the 2,4dinitrophenylhydrazones of aldehydes and ketones on DexsiI 300 GC allows the separation of enantiomers. Thus identification of peaks can often be based on two relative retention times which is of considerable help e.g. in the distinction of propanaldehyde, acetone and acrolein. For the final proof mass spectrometry is necessary. The liquid phase used here is highly suitable for coupled gaschromatography mass spectrometry because of the low bleed of DexsiI at elevated temperatures [4]. The advantage of using dinitrophenylhydrazones instead of the free compounds is that the sensitivity is increased both in GC and MS due to higher molecular weight of the derivatives and that the preparative handling can be done with non-volatile derivatives. The sensitivity can probably be even improved by using an electron capture detector [7]. The preparation of the samples is easier than the method proposed by Ronkainen and Brummer [8]. Preliminary results show that the method can also be used for whole blood or breath analysis. The precision above 50 pmoles injected is as expected for gaschromatography without internal standard. The variability of the manual injection is partly responsible for it. Duplicate determinations should be done if a high precision is needed

at low concentrations. The high coefficient of variation of the formaldehyde determinations is irrelevant for clinical work because the complications expected for the sample collection of free formaldehyde (boiling point: -2l”C!) are such, that its quantitation does not seem feasible. In reference urines of infants we found acetaldehyde, acetone and butanone and rarely traces of 2-pentanone. The distribution pattern of the reference values is certainly not normal for acetaldehyde and acetone. We thus give only the median values and range. The analysis of our data of acetone and of those recalculated from Bassette et al. [6] for adults suggests that there are at least two populations involved. Our results on acetone concentration in the urine are in the range reported for acetone in whole blood for this age group [9] . The data of Bassette et al. [6] also show a good correlation (r 0.91) between blood and urine acetone, except for very high concentrations in diabetics. It is not surprising that the urinary concentration of the highly lipid and water soluble acetone reflects the blood concentration, because free diffusion across tubular and bladder cell walls occurs [lo]. The same may be expected for butanone. Further investigations will be necessary to show whether this is also true for acetaldehyde. Thus for these compounds the only values to be considered in urine are those of concentration. There is no purpose in relating them to the 24-h volume or to the creatinine concentration. The determination of total ketone bodies in 24-h urines [ll] must be interpreted with care since acetoacetate and 3-hydroxybutyrate excretion is regulated by the tubules, whereas the acetone is freely diffusible. The presence of butanone and even 2-penfanone in the urine of infants which had no inborn error of metabolism indicates that their qualitative detection per se in urine is not an indicator of a genetic disorder of the branched chain amino acid metabolism. Acknowledgements We thank Mr. S. Sansano for his enthusiastic

technical

assistance.

References 1 Menkes. J.H. (1966) J. Pediatr. 69.413421 2 Daum. R.S.. Striver. C.R.. Mamer, O.A., Deluin. E., Lamm. P. and Goldman, H. (1973) Pediatr. Res. 7.149-156 3 Spencer. P.S., Scbaumburg, H.H., Raleigh, R.L. and Terhasr, C.J. (1975) Arch. Neural. 32. 219-222 4 Bachmann. C.. Nyhan. W.L. and Sweetman. L. (1974) in Application of Gaschromatography-Mass spectrometry to the Investigation of Human Disease (Mamer, O.A.. Mitchell, W.J. and Striver, C.R.. eds.). PP. 165-178, McGiB University, Montreal 5 Document+ J.R. Geigy SA, Basel. Wissenschaftliche Tabellen (1968). p. 162 6 Bassette, R., Kundiger, M. and Hanson, O.L. (1970) Microchem. J. 15.42-52 7 Kallio. H., Linko, R.R. and Kaitaranta, J. (1972) J. Chromatogr. 65,355-360 8 Ronkatnen. P. and Brummer. S. (1967) J. Chromatogr. 28,253-268 9 Akerblom. H.. Ahola. T. and Somersalo. 0. (1965) Ann. Paediatr. Fenn. 11.108-113 10 Sulway, M.J. and Mahns, J.M. (1970) Lancet ii, 736-740 11 G5schke. H. (1970) Clfn. Chim. Acta 28.359-364

Quantitative gaschromatographic determination of short chain aldehydes and ketones in the urine of infants.

287 Clinica Chimica Acta, 66 (1976) 287-293 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands CCA 6995 QUANTITATIV...
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