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Comparison of precision of the Ness method for serum total cholesterol with a ferric chloride method
SHORT
COMMUNICATIONS
6%
Comparison of precision of the Ness method for serum total cholesterol with a ferric chloride method A new quick method for serum total cholesterol has recently been presented’, but few data were given. Moreover, whilst the authors recommended that o.I-ml samples be used their data were determined using o.z-ml samples, a procedure which they advised against in their paper. Hence a small trial was carried out to estimate the precision of the method. Thirty routine laboratory sera were analysed in duplicate by the new method, whilst the ferric chloride method of Zlatkis et aL2 as modified by Chiamori 3, was used as a comparison method of similar complexity. The same cholesterol standards in glacial acetic acid were used for both methods. RESULTS
Typical standard curves for the two methods are shown in Fig. I, readings being on a Hilger Uvispec spectrophotometer. Fig. z shows absorbance wavelength curves for the doe-mg cholesterol standard. Table I gives statistical data derived from the results of serum analyses on thirty sera.
0
V
Fig. I. Standard
curves for the Ness method
Fig. 2. Absorbance-wavelength
TABLE
L
0.1 0.2 a3 0.4 Cholesterol, g per 100 ml
460
500 550 600 Wavelength in rnp
650
and the ferric chloride method.
curves for the 4oo-mg cholesterol
standard solution.
I
RESULTS OFDUPLICATE ANALYSES ON THIRTY RANDOM SERAFOR THE NESS et al.’ METHOD AND A FERRIC CHLORIDE METHODS
Cholesterol mgjroo Mean (5) Standard deviation Coefficient of variation
SERUM
TOTAL
CHOLESTEROL
BY
ml
Ness method
FeCl, method
258 r7.r 6.6%
240 9.70 4.0%
F test: F = 3.10, P < 0.01 f test: t = 6.28, P < 0.001
CZin. Chim. Acta. 13 (1966) 669-670
SHORT
670
COMMUNICATIONS
DISCUSSION
Ness et al. read absorbance of their reaction mixture at 540 m,u, this being “the best compromise for the isosbestic point of pure cholesterol (535 mp) and of sera 540-550 mp)“. They showed that absorbance was stable with time at 540 m,u; but at 625 rnp it varied with time, though absorption values were greater than at 540 m,u (Fig. 2). Using o.r-ml serum samples as recommended, absorbance is low (Fig. I). Only a good instrument can be used for measuring absorbance in the 0.030.10 range, and even so is being used in an inherently inaccurate range. Statistical analysis (F test; Table I) showed that the ferric chloride method3 gave a significantly greater precision than the new method, and that the difference between the means was significant (t test), so that some explanation for the higher values obtained with the latter technique has to be sought. The authors assume, on the basis of Carr and Drekter’s works, that both free cholesterol and its esters have equal chromogenicity, thereby avoiding the need for saponification. Webster4 found this to be incorrect, his Liebermann-Burchard reagent overestimating total cholesterol by 16% in unsaponified sera. A further source of error is that cholesterol intermediates, such as cholestanol and 7-dehydrocholesterol, have collectively greater chromogenicity with Liebermann-Burchard reagent than with FeCl 3 reagent 6. I wish to thank Dr. Vincent Marks for many helpful suggestions about this paper. The Area Laboratory at West Park Hospital, Epsom, Surrey (Great Britain) I 2 3 4
A. A. N. D.
P. J. N.
HOWORTH
T. NESS, J.V.
PASTEWKA AND A. C. PEACOCK, Clin.Chim. Acta, IO (1964) 229. ZLATKIS, B. ZAK AND A. J. BOYLE, J. Lab.CZin. Med., 41 (1953) 486. CHIAMORI AND R. J. HENRY, Am. J. C&z. Pathol., 31 (1959) 305. WEBSTER, Cl&. Chim. Ada, 8 (1963) 731. 5 J. J. CARR AND I. J. DREKTER, Clin.Chem., 2 (1956) 353. 6 R. J. HENRY, ClinicalChemistry, Harper, New York, 1964, p. 860.
Received October rzth, 1965 Clin. Chim.
Acta,
13 (1966) 669-670
Les ho-enzymes
de I’amylase
Plusieurs travaux ont ete consacres a l’isolement et a la caracterisation de l’amylase par electrophorese 1- 4. Apres avoir decrit deux iso-enzymes de l’amylase, l’une au niveau des albumines, l’autre en front des y-globulines, tous les auteurs ont conclu a l’existence dune seule zone a activitt! amylasique au niveau des y-globulines, l’autre activite s’etant rev&e un artefact. Une activite amylasique se retrouve au niveau des y-globulines quel que soit le milieu biologique CtudiC (salive, sue gastrique, secretion exocrine du pancreas, serum et urines). Un seul auteur, Ctudiant un extrait pancreatique du rat, a rapport4 Clin. Chim. Ada,
13 (1966) 670-674
COMMUNICATIONS
6%
Comparison of precision of the Ness method for serum total cholesterol with a ferric chloride method A new quick method for serum total cholesterol has recently been presented’, but few data were given. Moreover, whilst the authors recommended that o.I-ml samples be used their data were determined using o.z-ml samples, a procedure which they advised against in their paper. Hence a small trial was carried out to estimate the precision of the method. Thirty routine laboratory sera were analysed in duplicate by the new method, whilst the ferric chloride method of Zlatkis et aL2 as modified by Chiamori 3, was used as a comparison method of similar complexity. The same cholesterol standards in glacial acetic acid were used for both methods. RESULTS
Typical standard curves for the two methods are shown in Fig. I, readings being on a Hilger Uvispec spectrophotometer. Fig. z shows absorbance wavelength curves for the doe-mg cholesterol standard. Table I gives statistical data derived from the results of serum analyses on thirty sera.
0
V
Fig. I. Standard
curves for the Ness method
Fig. 2. Absorbance-wavelength
TABLE
L
0.1 0.2 a3 0.4 Cholesterol, g per 100 ml
460
500 550 600 Wavelength in rnp
650
and the ferric chloride method.
curves for the 4oo-mg cholesterol
standard solution.
I
RESULTS OFDUPLICATE ANALYSES ON THIRTY RANDOM SERAFOR THE NESS et al.’ METHOD AND A FERRIC CHLORIDE METHODS
Cholesterol mgjroo Mean (5) Standard deviation Coefficient of variation
SERUM
TOTAL
CHOLESTEROL
BY
ml
Ness method
FeCl, method
258 r7.r 6.6%
240 9.70 4.0%
F test: F = 3.10, P < 0.01 f test: t = 6.28, P < 0.001
CZin. Chim. Acta. 13 (1966) 669-670
SHORT
670
COMMUNICATIONS
DISCUSSION
Ness et al. read absorbance of their reaction mixture at 540 m,u, this being “the best compromise for the isosbestic point of pure cholesterol (535 mp) and of sera 540-550 mp)“. They showed that absorbance was stable with time at 540 m,u; but at 625 rnp it varied with time, though absorption values were greater than at 540 m,u (Fig. 2). Using o.r-ml serum samples as recommended, absorbance is low (Fig. I). Only a good instrument can be used for measuring absorbance in the 0.030.10 range, and even so is being used in an inherently inaccurate range. Statistical analysis (F test; Table I) showed that the ferric chloride method3 gave a significantly greater precision than the new method, and that the difference between the means was significant (t test), so that some explanation for the higher values obtained with the latter technique has to be sought. The authors assume, on the basis of Carr and Drekter’s works, that both free cholesterol and its esters have equal chromogenicity, thereby avoiding the need for saponification. Webster4 found this to be incorrect, his Liebermann-Burchard reagent overestimating total cholesterol by 16% in unsaponified sera. A further source of error is that cholesterol intermediates, such as cholestanol and 7-dehydrocholesterol, have collectively greater chromogenicity with Liebermann-Burchard reagent than with FeCl 3 reagent 6. I wish to thank Dr. Vincent Marks for many helpful suggestions about this paper. The Area Laboratory at West Park Hospital, Epsom, Surrey (Great Britain) I 2 3 4
A. A. N. D.
P. J. N.
HOWORTH
T. NESS, J.V.
PASTEWKA AND A. C. PEACOCK, Clin.Chim. Acta, IO (1964) 229. ZLATKIS, B. ZAK AND A. J. BOYLE, J. Lab.CZin. Med., 41 (1953) 486. CHIAMORI AND R. J. HENRY, Am. J. C&z. Pathol., 31 (1959) 305. WEBSTER, Cl&. Chim. Ada, 8 (1963) 731. 5 J. J. CARR AND I. J. DREKTER, Clin.Chem., 2 (1956) 353. 6 R. J. HENRY, ClinicalChemistry, Harper, New York, 1964, p. 860.
Received October rzth, 1965 Clin. Chim.
Acta,
13 (1966) 669-670
Les ho-enzymes
de I’amylase
Plusieurs travaux ont ete consacres a l’isolement et a la caracterisation de l’amylase par electrophorese 1- 4. Apres avoir decrit deux iso-enzymes de l’amylase, l’une au niveau des albumines, l’autre en front des y-globulines, tous les auteurs ont conclu a l’existence dune seule zone a activitt! amylasique au niveau des y-globulines, l’autre activite s’etant rev&e un artefact. Une activite amylasique se retrouve au niveau des y-globulines quel que soit le milieu biologique CtudiC (salive, sue gastrique, secretion exocrine du pancreas, serum et urines). Un seul auteur, Ctudiant un extrait pancreatique du rat, a rapport4 Clin. Chim. Ada,
13 (1966) 670-674