Clinica Chimica Acta, 197 (1991) 27-34 Elsevier Science Publishers B.V. ADONIS 000989819100078B

27

CCA 04941

Carnitine in dried blood spots: a method suitable for neonatal screening Robert J. Barns ‘, Francis G. Bowling 2, Gail Brown’,+, Alan E. Clague ’ and Alison Thompson ’ ’ Division of Chemical Pathology, Royal Brisbane Hospital, Brisbane, Queensland and 2 Neonatal Screening Unit, State Health Laboratories, (Received

31 January

Key words: Sudden

infant

Brisbane (Ausiralia)

1990; revision received 23 October

death syndrome;

Camitine; Medium Neonatal screening

1990; accepted

15 November

chain acylCoA

dehydrogenase

1990)

deficiency;

Summary

A method is described which enables the quantitative determination of both free and total carnitine levels in dried blood spots. This method is suitable for neonatal screening for either primary or secondary carnitine deficiency. The 95% confidence interval for free carnitine was 26-76 pmol/l (median = 44) and for total camitine was 35-102 pmol/l (median = 60).

Introduction

Carnitine has an essential role in the translocation of long chain fatty acyl residues from the cytosol into the mitochondrial matrix where they undergo poxidation [l]. In addition, camitine also has a role in detoxification of some of the organic acids which are produced in excessive amounts in certain organic acidaemias [2-41. The acyl camitines formed are then excreted in the urine. This excessive loss of carnitine in urine can result in a secondary camitine deficiency, particularly in infants up to one year of age, as they have low levels of the final synthetic enzyme,

’ Deceased. Abbreviations: SIDS, sudden infant death syndrome; MCADD, medium chain acylcoenzyme A dehydrogenase deficiency; MADD, multiple acylcoenzyme A dehydrogenase deficiency; HEPES, N-2-hydroxyethylpiperazine-N ‘-2-ethane sulphonic acid. Correspondence: Dr. R.J. Barns, Division of Chemical Pathology, Department of Pathology, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.

28

y-butyrobetaine hydroxylase [5-71. Hence, in addition to low plasma concentrations of carnitine in these patients, there may be a relatively high proportion of acylcarnitine in plasma. Some cases of the sudden infant death syndrome (SIDS) have been shown to occur in infants with various genetic diseases, including medium chain acylcoenzyme A dehydrogenase deficiency (MCADD), McKusick 20145, multiple acylcoenzyme A dehydrogenase deficiency (MADD), McKusick 23168, and systemic camitine deficiency [8,9], McKusick 21214. It has been suggested that some lo-15% of all SIDS patients have such a genetic component, most commonly MCADD, but data are sparse and largely anecdotal. Both MCADD and MADD have been shown to cause a secondary carnitine deficiency [lo-131. These diseases are treated by a diet low in fat and high in carbohydrate and in the case of carnitine deficiency by carnitine replacement. Also, their incidence is possibly 1 : 5 000 (10% of the This group most of the requirements for but a screening has not screening for group of SIDS could worthwhile. One possible approach is the of and in the blood for other screening tests. an assay, based the method of McGarry and Foster is described. Materials and methods

L-Carnitine, acetylcoenzyme A and HEPES were purchased from Sigma Chemical Company. Acetylcoenzyme A, [acetyl-l-l4 Cl- was a product of New England Nuclear and sodium tetrathionate was purchased from Fluka Chemical Company. Camitine acetyltransferase was a product of Boehringer Mannheim. All other chemicals were the purest available. The scintillation fluid used was either Emulsifier-safe from Packard or Ready-Safe from Beckman. Dowex AG l-X8(Cl- form) (200-400 mesh) was purchased from BioRad Chemical Company. The resin was recycled by treating it with successive washes of 250 mmol/l NaOH, water, 250 mmol/l HCI and water. Dried blood spots routinely collected from newborns onto Schleicher and Schuell (No. 903) filter paper were used for the assay. Quality control dried blood spots were made by applying 75 ~1 of pooled whole blood to the neonatal screening paper and air drying. When required, two 5-mm diameter discs were punched from each newborn dried blood spot and from the quality control card. Each pair of discs was eluted overnight at room temperature in 300 ~1 154 mmol/l NaCl. Two aliquots (125 ~1 each) of the saline extract were taken for the determination of free and total car&me, respectively, using a method modified from that of McGarry and Foster [15]. Total carnitine To each of the extracts for total carnitine determination, 10 1.11 600 mmol/l NaOH were added with mixing. The mixtures were then incubated at 37OC to hydrolyse any acylcamitine to free carnitine. After 60 min, 50 ~1 250 mmol/l HEPES (Na+), pH 7.5, and 10 ~1600 mmol/l HCl were added with mixing in that order.

29

Free carnitine To each of the extracts for free camitine determination, 50 ~1 250 mmol/l HEPES (Na+), pH 7.5, 10 ~1 600 mmol/l HCl and 10 ~1 600 mmol/l NaOH were added with mixing in that order to make these assays equivalent with respect to ionic strength to those for total carnitine and to ensure there is no alkaline hydrolysis of the acyl camitine. Blanks and standard Blanks contained 115 ~1 154 mmol/l NaCl and 10 1.111.5 g/100 ml fat free bovine serum albumin in saline. Four camitine standards were also set up containing 0.5, 1.0, 1.5 and 2.0 nmol camitine (in 154 mmol/l NaCl) plus 10 ~1 fat-free bovine serum albumin with the volume made to 125 ~1 with saline. To each of these, 50 ~1 250 mmol/l HEPES (Na+), pH 7.5, 10 ~1 600 mmol/l HCl and 10 ~1 600 mmol/l NaOH were added with mixing in that order. Conversion of free carnitine to “C-acetylcarnitine Free carnitine was converted to radiolabelled acetylcamitine by adding 0.5 pmol sodium tetrathionate (made fresh), 4 nmol “C-acetylCoA (9 pCi/ pmol, stored frozen at pH 5.0) and 1 ~1 carnitine acetyltransferase (70 U/ml) with mixing. The mixtures were incubated at 37°C for 30 min and then centrifuged. Aliquots (200 ~1) of the supernatant were applied to 2.0 x 0.7 mm Dowex AGl x 8 columns and the eluate was collected. The columns were washed with 2 x 500 ~1 of water and all eluate was collected. The eluate was mixed thoroughly and 1000 ~1 aliquots were transferred to 10 ml scintillant for radioactivity determinations on a Packard Spectrometer with luminescence correction. With appropriate adjustments to volumes, this assay has been used for plasma (10 ~1) and muscle (0.5 mg wet weight) camitine determinations with no deproteinisation. Results Volume of whole blood in 5 mm disc Radiolabelled glucose was mixed with pooled heparinised neonatal blood and 75 ~1 aliquots were applied to the screening paper. Discs (5 mm dia) were punched from the dried papers and extracted with 150 1.11saline solution. The radioactivity was determined in 100 ~1 aliquots of the extract and compared with that in an equivalent dilution of the whole blood and the volume of blood calculated. A 5-mm diameter disc was found to contain the equivalent of 5.92 k 0.54 ~1 of whole blood. A similar result was obtained using radiolabelled camitine. Reproducibility of the assay Pooled heparinised whole blood was applied to screening cards and air dried. Two 5-mm discs were punched from replicate (n = 25) blood spots and extracted with 300 ~1 saline solution. Free and total camitine were determined for each extract as described in ‘Materials and methods’. The imprecision (CV%) of the free camitine assay was 8.5% at 41.9 f 3.5 pmol/l) and for the total camitine was 3.2% (at 56.0 + 1.8 pmol/l).

30 TABLE I Stability of acylcamitine in dried blood spots Year

?I

Total

Free/Total

1988 1987 1986 1985 1984 1982-1983

144 154 134 183 74 30

56.4 f 17.9 59.3 f 22.1 53.1 f 17.0 61.6k21.8 69.7k21.0 46.3f12.6

0.78 f 0.07 0.89 f 0.06 0.91 f 0.07 0.96 + 0.05 1.00~0.08 0.98 f 0.04

ratio a

’ Free and total camitine were determined as described in ‘Materials and methods’ in extracts obtained from neonatal screening cards collected in the years indicated. Results are mean + 1 SD.

Recovery of added carnitine To determine whether the assay can accurately determine camitine (both free and total), pooled whole blood (900 ~1) was mixed with carnitine in saline solution (100 ~1) to give additional carnitine of 50 and 100 pmol/l and the blood was applied to the blood spot paper. Free and total camitine were determined on the pooled blood spots and the recovery calculated. The recovery for 50 pmol/l was 98.4% for free and 102.5% for total and for 100 pmol/l was 91.5% for free and 94.2% for total (n = 6).

Stability of acylcarnitine Retrospective assay of stored blood spots (up to 6 yr) showed the free/total carnitine ratios progressively increased with age of the card to be essentially equal to unity after 4 years (Table I), suggesting that acylcarnitine is unstable in the blood spots. Cards collected in 1983 and earlier showed poor extraction of blood products as indicated by the variable haemoglobin levels in these extracts - hence the low total carnitine levels. A stability study was undertaken for a one month period to ensure that routinely collected samples would be suitable for testing. Sample cards were stored at 4°C 22°C and 37°C (Table II) for 1 month and analysed. Only in the case of storage at 37°C was there any change

TABLE

II

Effect of storage temperature on the stability of acylcamitine in dried blood spots a Storage conditions

Free (pmol/l)

Total (pmol/l)

Initial 1 month at 4°C 1 month at 23°C 1 month at 37°C

48.2k4.3 45.9+3.0 48.6 + 3.9 42.2 f 4.6

64.3 66.3 65.4 59.1

k + f f

4.3 4.3 2.2 4.7

a Pooled control blood was applied to neonatal screening cards and air dried. The cards were stored for one month either at room temperature (23’C) or 4°C or 37’C. The free and total camitine was determined on extracts from these cards as described in ‘Materials and methods’. Results are mean f 1 SD.

31

and this was accompanied by a visible denaturation failed to elute adequately.

of the blood protein which then

Reference range Dried blood spots from 1843 neonates were assessed for free and total carnitine to establish an estimate of the population distribution (Fig. 1). The 95% confidence limits were 26-76 pmol/l (median = 44) for free camitine and

300

-7

so0 B

A

250

.

.

200

..,. ,.. uI N u

N u m 150,b e r

100 . . 1

1OCI-

50l-

.........................

.....

I

....................

CI t 0

4-l 20

40

-1

60

80

Free camltlnr

loo

120

140

loo

(rmol/l)

N300 _,................... u b ;

200

100

a

20

40

w

a0

loo

120

140

loo

Total Camltlne (rmol/l)

C

m

0

.............. ,,.,..,....... +l.lI, ................

0.4

0.5

0.6

0.7

Free/Total

0.a

0.9

1

1.1

Ratlo

Fig. 1. The distribution of free (A) and total (B) camitine and the free to total carnitine ratio (C) was determined in extracts of dried blood spots as described in ‘Materials and methods’.

32

35-102 pmol/l (median = 60) for total camitine. The range for free/total ratio was 0.61-0.87 (median = 0.74). Using an equivalent assay for camitine in 10 ~1 plasma obtained from children, the 95% confidence limits were 12-60 pmol/l (median = 33) for free carnitine and 23-84 pmol/l (median = 51) for total camitine (n = 87). Discussion

Preliminary studies comparing the assay of liquid whole blood and plasma samples had shown that whole blood carnitine levels are up to 50% higher than plasma levels [16]. Also, the observation of McGarry and Foster [15] was confirmed that deproteinisation prior to the assay was not recommended. Deproteinisation of whole blood samples resulted in a loss of up to 30% of both free and total carnitine if the protein were precipitated with trichloracetic acid and the supernatant neutralised with NaOH prior to the assay (unpubl. obs.). Deproteinisation has been used in most cases where whole blood carnitine levels have been reported [16,17]. The recovery results show that this assay gives a reliable assessment of whole blood carnitine concentration particularly at levels less than 100 pmol/l. The reproducibility is satisfactory for a dried blood spot assay (CV% = 3.2% for total carnitine, 8.5% for free camitine). The higher variability of the free carnitine determination may result from the lability of the acylcamitines. The described assay presents a method for determining free and total carnitine levels in fresh dried blood on filter paper samples collected from neonates. Acknowledgement

This work was funded by the Queensland Research Foundation.

Sudden

Infant

Death

Syndrome

References 1 Engel AG: Angelini C. Carnitine deficiency of human skeletal muscle with associated lipid storage myopathy: a new syndrome. Science 1973;179:899-902. 2 Chalmers RA, Roe CR, Stacey TE, Hoppel CL. Urinary excretion of L-camitine and acylcamitines by patients with disorders of organic acid metabolism: evidence of secondary insufficiency of L-camitine. Pediatr Res 1984;18:1325-1328. 3 Engel AG, Rebouche CJ. Camitine metabolism and inborn errors. J Inher Metab Dis 1984;7(Suppl 1):38-43. 4 Bieber LL, Emaus R, Valkner K, Farrell S. Possible functions of short chain and medium chain camitine acyltransferase. Fed Proc 1982;41:2858-62. 5 Rebouche CJ, Engel AG. In vitro analysis of hepatic camitine biosynthesis in human systemic camitine deficiency. Clin Chim Acta 1980;106:295-300. 6 Shenai JP, Borum PR. Tissue camitine reserves of newborn infants. Pediatr Res 1984;18:679-681. 7 Hahn P, Novak M. How important are carnitine and ketones for the newborn infant. Fed Proc 1985;44:2369-2373. 8 Anon. Sudden infant death and inherited disorders of fat oxidation. Lancet 1986;ii:1073-1075.

33 9 Roe CR, Millington DS, Maltby DA, Kinnebrew P. Recognition of medium chain acylCoA dehydrogenase deficiency in asymptomatic siblings of children dying of sudden infant death or Reye-like syndrome. J Pediatr 1986;108:13-18. 10 Coates PM, Hale DE, Stanley CA, Corkey BE; Cortner JA. Genetic deficiency of medium chain acyl CoA dehydrogenase: studies in cultured fibroblasts and peripheral mononuclear leukocytes. Pediatr Res 1985;19:671-676. 11 Stanley CA, Hale DE, Coates PM, Hall CL, Corkey BE, et al. Medium chain acyl CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low camitine levels. Pediatr Res 1983;17:877-884. 12 Roe CR, Millington DS, Maltby DA, Bohan CR, Kahler SG, Chalmers RA. Diagnostic and therapeutic implications of medium chain acy@rnitines in the medium chain acyl CoA dehydrogenase deficiency. Pediatr. Res 1985;19:459-466. 13 Tumbull DM, Bartlett K, Stevens DL et al. Short chain acyl CoA dehydrogenase deficiency associated with a lipid storage myopathy and secondary camitine deficiency. N Engl J Med 1984;311:1232-1236. 14 Wilson J, Junger G. Principles and practice of screening for disease. Public Health Papers, World Health Organisation 1968;34. 15 McGarry J, Foster D. An improved and simplified radioisotopic assay for the determination of free and esterified camitine. J Lipid Res 1976;17:277-281. 16 Borum PR, York CM, Bennett SG. Camitine concentration of red blood cells. Am J Clin Nutr 1985;41:653-656. 17 Mares-Perlman JA, Farrell, PM, Gutcher GR. Changes in erythrocyte and plasma camitine concentrations in preterm neonates. Am J Clin Nutr 1986;43:77-84.