de Nemours & Co., Wilmington, DE. 2. Sthre JD, Grady HJ, ... E. I. du Pont de Nemours & Co., Inc. Wilmington, ..... Wellesley HilLs, MA 02181. Renal Handling of ...
In summary, while we agree that ion-exchange chromatography is somewhat more susceptible to matrix effects than are other methodologies, we do not agree that the studies presented by Drs. Stein and Bohner accurately reflect the magnitude of these effects in human sera or their impact on the clinical utility of the Du Pont CK-MB method. A meaningful assessment of matrix effects would require comparison of results in native samples that
are grouped according to concentrations of protein, NaCl, and CK-MM. Finally, our experience is that traditional method-comparison studies between CK-MB methods do rately reflect the relative performance of the methods. regressive statistics show
not accudiagnostic Often the a bias or
correlation coefficient such as those reported by Stein and Bohner. However, when these same results are interpreted in light of a sequential sampling protocol and when the results are compared with the final diagnosis, the Du Pont DK-MB method shows a diagnostic performance comparable with that of alternative methods. Support for this statement can be found in several published studies, both those sponsored by Du Pont (2, 5-7) and those done independently (8, 9). References 1. CKMB Test Methodology, E. I. du Pont de Nemours & Co., Wilmington, DE. 2. Sthre JD, Grady HJ, Baillie EE, et al. An Evaluation of a Creatine Kinase Isoenzym.e Method for the Du Pont aca5. Idem, 1979.
3. Storey JD. Creatine Kinase ‘MB” Determination in the Clinical Diagwsis of Acute Myocardial Infarction. Idem, 1980. 4. Technical Bulletin: Interpretation of Results from the Du Pont CKMB Method. Idem, 1983. 5. Clement GE, Havassey J, Gull J. Diagnostic performance of combined isoenzyme analysis using the Du Pont aca5 CK-MB and LDH isoenzymes. Clin Chem 28, 1618 (1982). Abstract. 6. Hustad KO, Howanitz PJ, Preston BA, Helak JW. Evaluation of a modified column method for CK-MB using a Du Pont aca5 II and aca5 HI. Clin Chem 28, 1618 (1982). Abstract. 7. WeeksRB, Malhotra HC. Clinical evaluation of an automated column method for CK-MB determination. Clin Chem 27, 1024 (1981). Abstract. 8. Forsman RW, O’Brien JF, Jones JD, Annesley TM. Evaluation of the Du Pont CKMB method in the prediction of myocardial infarction. Clin Chem 27, 1024 (1981). Abstract. 9. Maturen A, Pietras RJ, Kondos GT, et al. A protocol for use of the aca5 CKMB method in acute myocardial infarction. Clin Chem 29, 1183 (1983). Abstract.
William F. Naccarato E. I. du Pont de Nemours Wilmington, DE 19898 342
& Co., Inc.
Gas-Chromatographic Procedure, with Pyrolytic Methylation of Serum Fatty Acids, in the Diagnosis of Refsum’s Syndrome To the Editor:
Refsum’s syndrome is a rare inherited disorder of lipid metabolism characterized by deficiency of phytanic acid ahydroxylase (no EC no. assigned) and, consequently, by storage of phytanic acid (3,7,1 1,15-tetramethylhexadecanoic acid) in tissue lipids (1). The origin of this
branched-chain
fatty
acid
is
exclusively exogenous, with phytanic acid and phytol, its precursor, being present in most diets. Identification of phytanic acid among the total fatty acids in plasma confirms the diagnosis of this syndrome (2, 3). Total fatty acids in plasma generally
are measured by gas chromatography after methylation of the fatty acids liberated by saponification of a lipid extract of the sample (3). In 1974, MacGee and Allen (4) proposed a simple procedure for methylating total fatty acids obtained by direct saponification of a serum sample. Instead of the methylation reagent tnmethyl(a , a, a-tnifluoro-m -tolyl)ammonium hydroxide they proposed, the more easily available and more stable trimethyiphenylammonium hydroxide (TMPAH) was later suggested (5). To our knowledge, back-extraction into TMPAH and pyrolytic methylation of total fatty acids have not yet been applied to the analysis of specimens from patients with clinical findings suggesting
Refsum’s
syndrome.
We
have found that thissample preparation can be applied to determination of phytanic acid with results equivalent to those obtained by the longer classical methods. We used, with some modifications, the procedure of Williams and MacGee (5): shake 200 jiL of serum or plasma for 5 mm on a rotary mixer with 3.8 mL
of chloroform/methanol
(2/1,
(“MethElute”; Pierce Chemical Co., Rockford, IL 61105) and dissolving the residue in 0.5 volume of methanoll water
(1/2, by vol)I.
Shake
the
tube
vigorously by hand for 30 s and centrifuge (1500 x g, 2 mm). Aspirate 1 to 1.5 L of the TMPAH extract into a 5tL syringe in such a way as to sandwich it between two 1-L layers of methyl acetate AR, and inject this into the chromatograph. The methyl acetate neutralizes the excess TMPAH (4, 5). The TMPAH extract is stableforat least a week at 4#{176}C, with no need to separate it from the hexane layer. We used a gas chromatograph equipped with a flame-ionization detector. The 1.8 m X 2mm glass column was packed with a 57/43 (by vol) mixture of 8% BDS on Chrom Wash DMCS 80-100 mesh and 15% CP-TM-Sil.84 on Chrom
WHP
100-120
mesh
(all
from Chrompack, Middelburg, The Netherlands). Under the chromatographic conditions-injector temperature, 260 #{176}C; oven programmed from 175 to 195 #{176}C at 1 #{176}C/min; detector, 235 #{176}C; carrier gas (nitrogen) flow rate, 40 mL/min; attenuation 10b0 x 32 A/ mV-phytanic acid is resolved from other fatty acids and has a retention time of 6.5 mm (Figure 1). We investigated other liquid phases such as OV-225, FFAP, SP-1000, BDS, and CP-TM-Sil.84 but found that only SP-1000 and the BDS/DP-TM-Sil.84 mixture satisfactorily resolved phytanic acid from the fatty acids normally present in serum. Other extraction solvents (toluene, diethyl ether, diisopropyl ether) and other acidification reagents (HC1, HC1IH3PO4 mixtures) yielded lower recovery of fatty acids than did the hexane/H3PO4 system. With some systems, extraction of fatty acids was not detected. We compared this method with a longer classical procedure [6, modified from Eldjarn et al. (3)1 that involves Folch’s extraction, saponification, hexane extraction, evaporation, and diazo-
by
vol) in a 10-mL stoppered centrifuge tube. Add 0.8 mL of 0.1 molIL KC1 and shake the tube again for 3 mm. After centriftigation at 1500 x g for 5 mm, transfer the lower layer to another 10mL stoppered tube, and evaporate it in a rotary evaporator at 45 #{176}C. Add 1.0 mL of a 150 g/L methanolic KOH solution and 200 L of water to the residue, and place the tube in a water-bath at 65 #{176}C for 30 mm. Cool, then add 1.5 mL of 1 mol/L H3P04, followed by 5 mL of n-hexane AR; shake the tube vigorously by hand for 1 mm and centrifuge (1500 x g, 5 mm). Transfer 4 mL of the hexane layer to a 10-mL stoppered
conical centrifuge tube, and add 20 L of a 0.4 moLIL TMPAH solution [prepared by evaporating one volume of a
0.2 molIL methanolic TMPAH solution
CLINICAL CHEMISTRY, Vol. 30, No. 2, 1984
0
5
10
15
20mm
Fig. 1. Gas chromatogram of total fatty acids in serum from a patient with Refsum’s syndrome Methylestersof (1) myristic acid, (2) palmitic acid, (3) palmitoleic acid,(4)phytanicacid,(5)stearic acid, (5) oleicacid,(?) linoleic acid, (8) eicosatrienoicacid, and ( arachidonicacid
methane methylation of a 1-mL sample for four normal sera and six sera from patients with Refsum’s syndrome. Results by the two methods were similar. For example, the regression of our method (y) on the classical method (x) for the peak ratios for phytanic acid/ total fatty acids from the six patients wasy = 1.037x + 0.0329 (r = 0.9935). When the serum is directly saponified with the KOH solution, as in the Williams and MacGee procedure (5), the peak ratios for phytanic acid/total fatty acids were lower than by our method or by the comparison classical method. For samples containing phytanic acid,therefore, one must saponify a lipid extract rather than the serum itself and, consequently, precede the Williams and MacGee method with a step for lipid extraction. References 1. Steinberg D. Phytanic acid storage (Refsum’s syndrome). In The Metabolic Basis of Inherited Disease, 5th ed., JB Stanbury, JB Wyngaarden, DS Fredrickson, JL Goldstein, MS Brown, Eds., McGraw-Hill Book Co., New York, NY, 1983, pp 731-747. 2. Kahlke
W.
Refsum-Syndrom.
Lipoidchemische Untersuchungen bei 9 Fallen. Kim Wochenschr 42, 1011-1016 (1964). 3. Eldjarn L, Try K, Stokke 0. The ability of patients with heredopathia atactica p0lyneuritiformis to w-oxidize and degrade several
isoprenoid
branched-chain
fatty
structures. Scand J Clin Lab Invest 18, 141150 (1966). 4. MacGee J, Allen
KG. Preparation
of
methyl esters from the saponifiable fatty acids in small biological specimensfor gasliquid chromatographic analysis. J Chromatogr 100, 35-42 (1974). 5. Williams MG, MacGee J. Quantitative recovery
of
polyunsaturated
fatty acids on
pyrolytic methylation of their trimethylphenylainmomum salts. J Chromatogr 234, 468-471 (1982). 6. Mardens Y. La recherche par chromatographie en phase gazeuse de l’acide phytanique dam le serum de malades neurologiques permet de confirmer le diagnostic de maladie de Refsum. Annex Thesis, Umversite Libre de Bruxelles, Brussels, Belgium, 1972.
A. Kumps Y. Mardens Lab. of Med. Biochem. Pharmaceut. Instit. Univ. Libre de Bruxelles Campus Plaine 205/3 B -1 050 Brussels, Belgium
A Questionof RubellaImmunity To the Editor: A major advantage of the RIA for rubella antibody, as modified by Musto et al. (1), is that low concentrations of
the circulating antibody can be detected.
The significance of having a titre of antibody lower than 1:8 is somewhat controversial because a 1:8 titre, as determined by the hemagglutination inhibition method, is still considered the lowest titre sufficient to provide defense against rubella infection. However, detecting any of this antibody suggests past exposure to the virus because there is, as yet, no evidence for natural immunity against the rubella virus. In this laboratory,
a substantial
pro-
portion of patients whose serum was examined by the RIA method showed titres of
