Phenotyping of type II and IV hyperlipoproteinemias by a simple, quantitative agarose gel lipoprotein electrophoresis

CLINICA CHIMICA ACTA

PHENOTYPING A SIMPLE,

30.5

OF TYPE

QUANTITATIVE

II AND

IV HYPERLIPOPROTEINEMIAS

.AGAROSE

GEL

LIPOPROTEIN

BY

ELECTRO-

PHORESIS

CESARE SIRTORI*, BAD1 M. BOULOS Clinical Pharmacology

Biometry,

liniversity

(Received

Septrmber

KHATAB

M. HASSANEIN,

RUTH

HASSANEIN

AND

and Toxicology Center. Defartments of Medicine, Pharmacology and of Kansas Medical Center, Kansas City, Kansas 66103 (U.S.A .) 15,

1970)

SUMMARY

From the statistical analysis of the densitometric percentage counts derived from 97 lipoprotein electrophoreses from patients with Type II or Type IV hyperlipoproteinemia, we developed a simple sequential screening approach. The method is highly accurate in assigning patients to their proper diagnostic classification. Also, the method is nonparametric, i.e., distribution-free, and considers one variable at a time for discrimination. This method, apart from sampling errors, does not give misclassified patients, but may give some undecided cases.

Since the original introduction, by Fredrickson and Lees’, of a method for phenotyping hyperlipoproteinemias, analyses of blood lipids and lipoprotein (LP) electrophoresis have become of almost routine use. However, in some instances, it still remains difficult for the managing physician to reach the correct interpretation of a phenotype. This difficulty is, in part, attributable to the electrophoresis of lipoproteins on paper. This method is time consuming and especially unsatisfactory because of the poor separation of /?- and pre$-LP, often resulting in purely subjective interpretations of the pattern. Recently, Havel* has pointed out that only a combination of techniques, giving priority to the blood lipid pattern and the clinical picture, can provide a reliable answer to the problem. The LP electrophoresis would in this way become only an additional technique. Newly developed techniques for LP electrophoresis on medias such as cellulose acetate3 and agarose* allow considerably better separation of the LP and an easier quantitation of the bands by densitometry. Postma and Stroe9 and Winkelman et aL6 have reported some suggestions for the use of the quantitative data obtained by densitometry. More recently Winkelman et al.‘, using cellulose acetate medium, reported l

Merck International

Fellow in Clinical Pharmacology. Clin. Chim. Acta,

31 (1971) 305-310

SIRTORI

306

tt al.

quantitative measurements of the LP bands and their range of distribution by calculating the percentages of the scan counts given by an integrator for each band. These data are not completely satisfactory, primarily because the electrophoresis on cellulose acetate medium does not allow a satisfactory definition of the pattern of the Type III abnormality. It was our intention to find a simple statistical method for the evaluation of the LP electrophoresis by using agarose gel medium, which best separates the bands (Dyerberg and Hjrne* and Zollner et akQ). Since Types I, III, and V are rare, and can be distinguished by clinical features and by other characteristics (milky plasma and heavy chylomicron bands in Type I and V, milky layer floating on top of the plasma and one single p-pre-/I band in Type III), we restricted our attention to the distinction between Type II and Type IV patterns. In our experience, these types constitute more than 90% of LP abnormalities seen clinically. MATERIALS

AND METHODS

Ninety-seven patients, of both sexes, aged from 4 to 75, were randomly selected from those being followed in the Lipid Clinic of the University of Kansas Medical Center. Clinical features and blood lipid values (obtained on at least three successive visits) allowed them to be classified as Type II and Type IV according to the criteria proposed by Fredrickson and Lees’. Laboratory

methods

Glyceride glycerol (GG) was analyzed according to the method of Azarnoffio were performed according to and cholesterol by Autoanalyzer ll. LP electrophoreses the method of Noblea, modified as follows: Plasma was immediately separated from blood samples collected in Vacutainer tubes containing EDTA, and processed within 36 h; 300 mg of agarose (Seakem) were boiled in 50 ml of 0.05 M barbital buffer (pH 8.6) without addition of human albumin. Approximately 4-5 ml of the mixture were spread on 3 x 15 cm strips of Cronar-C 41 Polyester film (Photo Products Department, Du Pont, Wilmington, Delaware), and a r-cm long piece of a 21-g needle was immediately pushed into the gel layer in the middle of each strip. After the gel had solidified, the piece of needle was removed with a magnet and each resulting -4 drop of an albumin solution trough filled with approximately IO ~1 of plasma. stained with ioscine was placed alongside each trough as an indicator of the distance of LP migration. The electrophoreses were run in either a Gelman electrophoretic chamber or in a Beckman Model R Cell, using barbital buffer, 0.05 M (pH 8.6). Good separation was obtained by applying a voltage around 200, with IO PA per strip, for 30-40 min. The albumin was allowed to migrate 4 cm. The strips were fixed in an ethanol-methanol-isopropanol-water solution (67 : 1.5 : 1.5 : 30) for I h, dried on a warm plate and stained with Sudan Black B (I g per 1 of the fixative solution) for 30 min. Destaining was accomplished with 2-3 washings in plain fixative solution. The strips were scanned with a Spinco Model RB Analytrol densitometer, with a disc integrator. The scans for each lipoprotein fraction were demarcated as recommended by Noble ct a1.l2, and each value recorded as percent of the total scan.

Clin. Chim. Acta,

31 (1971) 305-310

TYPE

II AND IV HYPERLIPOPROTEINEMIAS

307

We were given a sample of 68 patients from the population of Type II hyperlipoproteinemias and a sample of 29 from the population of Type IV disorders. and “percent ,!?’ lipoValues of the three variables, “percent cc,” “percent pre$,” protein were determined for each patient. The problem was to find a method of assigning a new patient to the correct population on the basis of the values of these three variables, Usually such a problem is referred to as a problem of discrin~ination. Our approach was sequential screening, using one variable at R time. Since the sum of the values of the three variables is IOO for any patient, we decided to use two variables only, percent p and percent pre-fl, for assigning a patient to either group. Decision rule was always made according to the variable which gave the greatest number of true positives.

I.5 X ff

THE 02NSITOYET6lC

COUNTS

f

7.5

IS.5

IS.5

2w

315

37s

OF THE DENSITOMETRIC COUNTS

26

c20 z Y L *

10

5 wp 15 ~285

363

485

66.8.) 66.5

X OF THE MHSlTONETRlC

Fig. I. Distribution of the densitometric of abnormalities studied.

perqntages

76.5

6G

COUNTS

of a, pre-/I, and )? bands in the two types C&n. Chim. A&,

31 (1~~71)305-310

308

SIRTORI

et id.

From inspection of the data (Fig. I), it can be seen that the two groups of patients differ more in pre-/l than @ or a values. We constructed a frequency distribution for pre-/I values (Table I). It was easy to see that the distributions of the two TABLE

I

FREQUENCY

DISTRIBUTION

<.s

OF TYPE

IT AND

TYPE

IV FOR

“/o PRE-,!-LIPOPROTEIN

2

5--

7

TO-

‘3

15 -

‘3 ‘7 ‘3 3

2O25-30.35 -,o -‘-

-1550 55.60 65 -7” -

0

3 68

Total

29

groups for pre-/!Ioverlapped in the range 30 to 35. Outside of this range were 65 (total 68) patients of Type II and 27 (total 29) patients of Type IV. Our first rule for discrimination : allot to Type II allot to Type IV refer to second variable (percent 8)

pre-/3 < 30 pre-B > 35 30 < pre-B < 35

There were five patients for which pm-G lay in the common range 30 to 35. For these 5 patients, we constructed a distribution for percent j3 (Table II). Proceeding as before, we added the second rule for discrimination: percent /l < 40 percent /I > 40

allot to group IV allot to group II

These two rules leave no case undecided, and we have 95% of the patients TABLE

IT

FREQUENCY

DISTRIBUTION

% ir’

Type II

OF

5 PATIENTS Type IV

25.-

3035 -40-45S556065 .-

I I I I

1

7Q-Total

Clin. Chdm. Acta,

3

2

31 (1971) 305-310

NOT DISCRIMINATED

BY

:&

PRE-B

TYPE II AND IV HYPERLIPOPROTEINEMIAS

309

phenotyped by the percentage of pre-b LP alone, only the remaining the percentage of fl-LP in addition for discrimination.

5% requiring

DISCUSSION

Noble zt al.12 have shown a very good correlation between LP fractions separated by agarose gel electrophoresis and those obtained by analytical ultracentrifugation. From our data, we found a very simple quantitative method for distinguishing by agarose gel electrophoresis the two most common LP abnormalities. This method allows one to confirm the “classical” pictures of LP abnormalities, as well as to diagnose cases where the LP pattern is less typical (Figs. z-5).

-_.

.‘..,,.-

Fig. Z. Typical Type II LP pattern. Patient V.P.: cholesterol 281 mg/roo Densitometric percentages: fi 68, pre-,!I 4, cc 28. Fig. 3. Less typical Type II LP pattern. Patient G.K.: cholesterol IOO ml. Densitometric percentages: B 60, pre-/3 35, tc 5.

Fig. 4. Typical Type IV LP pattern. Patient D.R.: cholesterol ml. Densitometric percentages: /I 34, pre-,!? 47, a 19.

ml, GG 7.7 mg/roo

450 mg/roo

250 mg/roo

ml.

ml, GG 20.5 mg/

ml, GG 54.6 mg/roo

Fig. 5. Less typical Type IV LP pattern. Patient D.M. : cholesterol 208 mg/roo IOO ml. Densitometric percentages: ,J 39, pre-j3 31, ct 29.

ml, GG 23.8 mg/

We purposely avoided testing our method on LP electrophoreses of people with normal lipid values, since the majority of authors agree that these people should not be examined by this technique. We are currently using the method in the routine analysis of our LP electrophoreses with very satisfactory results. ACKNOWLEDGMENTS

This study

was supported,

in part, by grant

GM 15956 from USPHS. C&L Chim.

Acta,

31 (1971)

305-310

SIRTORI et al.

310

Also, thanks are given to Dr. D. L. Azarnoff for his careful reading of this manuscript and valuable suggestions. REFERENCES I D. S. FREDRICKSON AND R. S. LEES, Circulation, 31 (1965) 321. z R. J. HAVEL, Atherosclerosis, II (1970) 3. 3 H. P. CHIN AND D. H. BLANKENHORN, Clin. Chim. Acta, 20 (1968) 305. 4 R. P. NOBLE, J. Lipid Res., g (1968) 693. 5 T. POSTMA AND J. A. STROES, Cl&. Chim. Ada, 22 (1968) 569. 6 J. WINKELMAN, F. A. IBBOTT, C. SOBLE AND D. R. WYBENGA, Cl&. Chim. Acta, 26 (Ig6g) 33. 7 J. WINKELMAN, D. R. WYBENGA AND F. IBBOTT, Clin. Chim. Acta, 27 (1970) 181. 8 J. DYERBERG AND N. HJRNE, Clin. Chim. Acta, 28 (1970) 203. g N. ZOLLNER, W. GROBNER, C. BERGER AND G. WOLFRAM. Z. Klilz. Chem. Klin. Biochem., 7

(1969) 525. IO D. L. AZARNOFF, J. Lab. Clin. Med., 60 (1962) 331. II Symposium on Atromid, J. Atheroscler. Res., 3 (1963) 347. 12 R. P. NOBLE, F. T. HATCH, J. A. MAZRIMAS, F. T. LINDGREN, L. C. JENSEN AND G. L. AUAMSON, .&%dS, 4 (1969) 55. Clin. Chim. Ada,

31 (1971) 305-310