Lipoprotein profiling by high performance gel chromatography

b Clinica Chimica Acta 228

@

( 1994)171- 179

Lipoprotein profiling by high performance gel chromatography G.A. Tallis*, M.D.S. Shephard, M.J. Whiting Department

of Biochemistry and Chemical Pathology. Australia

Flinders Medical

Centre. Bedford Park. South

5042, Auwalia

(Received I2 January 1994; revision received I I April 1994: accepted I3 April 1994)

High performance gel chromatography (HPGC) was used to separate lipoproteins on the basis of their size and to generate lipoprotein profiles for plasma collected from patients with different lipoprotein phenotypes. These profiks provided a direct measurement of low density lipoprotein (LDL)-choksterol which was more precise than LDLcholesterol values calculated by the Friedewald equation. IO addition, LDLcholesterol conantrations were obtained in patients with combined hyperlipidemia in whom LDLcholesterol could not be accurately calculated by the Friedmald equation. The response of LDL-cholesterol to the drug gemfibrozil was reliably monitored and io addition changes in LDL particle size could be assessed from the LDL apolipoproteio B/cholaterol ratio. HPGC also assisted in the diagnosis of type III hyperlipidemia by revealing a characteristic lipoprotein profile. HPGCderived lipoprotein profiks provided additional useful clinical information for combined hyperlipidemia (Fredrickson lipoprotein phenotypes Ilb. III). Ke~~ordr: High hyperlipidemia

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1. IntrodWtioo The separation of lipoprotein particles may be based upon differences in hydrated density, electrical charge or size. Although ultracentrifugation is a widely used technique for isolating serum lipoproteins according to their density [I], two recent l

Corresponding author.

0009-8989LWSO7.00 Elscvicr Science B.V. SSDI 0009-8981(94)05873-Q

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reports have described the use of high performance gel chromatography (HPGC) to separate lipoprotein particles according to their size [2,3]. When used with on-line measurement of cholesterol, a lipoprotein profile can be produced with the advantage that it enables a direct measurement of low density lipoprotein (LDL)cholesterol to be made rather than estimating LDL-cholesterol using the Friedewald formula [2,3]. Another advantage of HPGC is that it is an analytically robust technique with excellent precision, recovery and accuracy. In this report lipoprotein profiles have been produced which represent the most common Fredrickson lipoprotein phenotypes seen in a general hospital Lipid Clinic. The complex effect of the drug gem~bro~l on the lipoprotein profile of a patient with severe combined h~rlipide~a (Fredrickson lipoprotein phenotype IIb) is also demonstrated. These results highlight the potential benetits of HPGC lipoprotein profiles in the evaluation and monitoring of patients with combined hyperlipidemia; the technique may have a role in the setting of a specialist Lipid Service. 2. Methods 2.1. Separation of lipoproteins Lipoproteins were separated using a Pharmacia HPGC system equipped with a Superose 6 Column (containing cross-linked agarose beads (13 pm in diameter) and a bed size of 300 x 10 mm, Pharmacia Australia Pty. Ltd., North Ryde, New South Wales). Filtered plasma (300 ~1)was loaded onto the column. With a constant flow rate of 0.5 ml/min, lipoproteins were sequentially eluted from the column with 0.15 moi/l NaCI (pH 7.0). The run time for complete elution of plasma proteins was 55 min. The timing of fraction collection and order of elution was established by eluting purified very low density lipoprotein (VLDL), LDL and high density lipoprotein (HDL) (prepared by ultracentrifugation). VLDL particles were eluted in fractions 14-19, with the peak maximum being observed in fraction 16, LDL eluted in fractions 20-29 (peak 25), while HDL eluted in fractions 30-37 (peak 32). The first 7 ml of eluent were discarded and then 24 x 0.5 ml fractions (corresponding to fraction numbers 14-37) were collected and transferred for subsequent Iipid analysis. Following elution, pools of each lipoprotein class were prepared for subsequent quantitative analysis by mixing 100 ~1 of each representative fraction prior to lipid and apohpoprotein measurements. The column has a long life-span and is capable of analysing around 300 samples, providing the manufacturers recommended maintenance procedures are followed. 2.2. Lipid and apolipoproteinanalysis The cholesterol and triglyceride concentrations in each of the fractions collected (and in subsequent lipoprotein pools) were measured on a Cobas Bio centrifugal analyser (Roche Diagnostic Systems, Nutley, NJ, USA) with use of Boehringer Mannheim (Mannheim, Germany) enzymatic kit methods (Monotest Cholesterol CHOD-PAP, Catalogue No. 236691, and Boehringer Mannheim Triglycerides GPO-PAP, Catalogue No. 701910, respectively). The concentrations of apolipoproteins (apo) A,, B and (a) in the individual frac-

G.A. Tallis ef al. / Clin. Chim. Acta 228 (1994) 171-179

173

tions and pools were measured by i~unonephelomet~ on a Behring Nephelometer (Behring Diagnostics Australia Pty. Ltd., Kingsgrove, New South Wales). All assays were performed using Behring antisera and calibration materials. Manufacturerrecommended instrument settings for each assay were modified to enable measurements within the concentration range of 0.05- 1.5 mmol/l for lipids and 5-250 mg/l for apolipoproteins to be made in individual and pooled fractions. Quantitative data on the % recovery, amount, % dist~bution and concentration of lipid and apolipoprotein in each lipoprotein class were calculated after the analysis of pooled fractions. 2.3. Patient samples Fasting plasma samples were obtained from patients attending the Flinders Medical Centre Lipid Clinic. Patients were selected so that the common lipoprotein phenotypes were represented. 3. Results 3.1. Performance churacter~tics of HPGC Between-run precision studies for cholesterol measurement in different lipoprotein fractions were performed by analysing a plasma pool five times over a 3 week period. Coefficients of variation (C.V.%) for the VLDL, LDL and HDL cholesterol concentration in these pools were 5.2%, 1.2% and 5.9%, respectively. The mean % recoveries (and standard deviations) of lipids and apolipoproteins using this technique were cholesterol 108% ( f 13%), triglyceride 103% ( f 17%), apo B 103% (i 7%) and apo Ai 83% (a 7%). The correlation coefficients (r) between VLDL-cholesterol, LDL-cholesterol and HDLcholesterol measured directly by HPGC, and calculated using the Friedewald formula and by polyethyleneglycol precipitation, respectively, were 0.96, 0.99 and 0.96 (n = 29). 3.2. Lipoprotein cholesterol and triglyceride profiles of t& different lipoprotein phenotypes

Fig. 1A demonstrates the HPGC lipoprotein cholesterol and triglyceride profiles of a normal subject, followed by Fredrickson classification lipoprotein phenotypes Ifa, IIb, III and IV (Figs. 1B-1E). Fig. 1F shows the lipoprotein profile of a patient with previously diagnosed Tangier Disease. As expected, there was virtually no HDL peak and a small increase in remnants between the VLDL and LDL peaks. 3.3. Apolipoprotein profile in a patient with type IIb hyperlipidemia

Fig. 2A demonstrates the apo A,, B and (a) profiles in a patient with type IIb hy~rli~proteinemia. There was no overlap of apo At and B~ontaining lipoproteins; apo(a)-containing particles (Lp(a)) eluted between the VLDL and LDL peaks. 3.4. LDL-cholesterol measured by HPGC and calculated by the Friedewald equation in a patient with type IIb hyperlipidemia

Table 1 demonstrates that in a patient with type IIb hy~rlipidemia

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G.A. Tallis et al. / Clin. Chim. Acta 228 (1994)

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TaBis et al. I C&t. Chitn. Acta 2.28 (1994) 171-179

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triglyceride of 4.5 mmolil (pre-gemtibrozil) there was a significant discrepancy of 1.2 mmol/l between LDLcholesterol measured by HPGC and that calculated by the Friedewald equation. However, this discrepancy did not occur in the post-gemfibrozil sample which had a plasma triglyceride of 1.9 mmol/l. 3.5. Effect of gemfibrozil on the lipoprotein cholesterol profile of a patient with type Iib hyperlipidemia

Fig. 2B and Table 1 demonstrate the effect on the lipoprotein profile of treatment with gemfibrozil600 mg b.d, for 3 months in a patient with type IIb h~rlipidemia. ~~brozil therapy resulted in a 58% decrease in plasma t~~yce~de but was accompanied by a 23% increase in LDL-cholesterol as measured by HPGC. There was a 19% decrease in the ratio LDL apo B/cholesterol suggesting that LDL particles had become significantly larger. This presumed increase in LDL size resulted in only a marginal left shift in the elution of the LDL peak (Fig. 2B).

This study evaluated the possible role of HPGC-derived lipoprotein profiles in the Lipid Clinic of a general hospital, and in particular in the evaluation and management of patients with combined hyperlipidemia. Although the procedure is time consuming and labor intensive and in our laboratory a rn~irn~ of six li~protein profiles were obtained per day, the technique does offer significant advantages over other methods for separating lipoproteins. Our study confirms earlier reports that HPGC-derived lipoprotein profiles demonstrate acceptable imprecision, recovery and accuracy [2,3]. A major advantage of HPGC is that it permits direct measurement of plasma LDL-cholesterol concentrations. This contrasts with the usual clinical laboratory approach of measuring total cholesterol, triglyceride and HDL-cholesterol and calculating LDL-cholesterol by the Friedewald formula [4]. The combining of three measurements necessarily results in an increase in analytical imprecision. Analytical imprecision for measurement of LDL-cholesterol may range from C.V. 2.0% to 8.0% [S]. In this study the between-run analytical C.V. for LDL-cholesterol measurement was 1.2%. Although we did not fully assess the accuracy of HPGC LDL~holesterol measurements by

Fig. 1. HPGC lipoprotein cholesterol (-•-) and triglyceride (-0-) protiles from: (A) normal subject (choIesterolS.4 mmolll, triglyceride 1.1 mmoM and HDL~holesterol 1.1 mmolil);(8) patient with Fredrickson type IIa lipoprotein phenotype (cholesterol 8.1 mmol/I, triglyceride 1.0 mmoM and HDLcholesterol 1.5 mmov1); (C) patient with Fredrickson type IIb phenotype (cholesterol 7.0 mmoy1, triglyceride 3.5 mmobl, HDLcholesterolO.8 mrnol/I); (D) patient with Fredrickson type III phenotype (cholesterol 9.2 mmol/l, triglyceride 6.0 mmoliI, HDL-cholesterol 1.0 mmoM); (E) patient with Fredrickson type IV phenotype (cholesterol 5.6 mmol/l. triglyceride 8.4 rnmol/l, HDL-cholesterolO.5 mmol/l); (F) patient with Tangier Disease (cholesterol 3.8 mmoyf, triglyceride 2.8 mmolfi and HDL-cholesterol < 0.1 mmoM). Note that the scales for lipid ~~ntmtion are the same in all patients, except (B). All elution profiles have been corrected for recovery of cholesterol and triglyceride.

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GA. Tallis ef al. I Clin. C&m. Aria 228 (1994) 171-179

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Fig. 2. (A) WCC apolipoprotein profile in a patient with Fredrickson type Ilb lipoprotein phenotype (apoA1 1.5g/l,apoBl.8g4andapo(a)320mg/lsee Fig. 1C for corresponding lipoprotein lipid profile). The ape(a) elution profIle is shown for fractions 18-23 only, because the remaining fractions had ape(a) concentrations less than the lower limit of detection of the method (21 mgil). (B) Effect of gemfibrozil treatment on the lipoprotein cholesterol profde of a different patient with type IIb hy~riipi&~a (see Table 1 for lipid values). The eiution proftle has been corrected for recovery of cholesterol.

G. A. Tallis et al. / Clin. Chim. Acta 228 (I 994)

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Table 1 Effect of gemtlbrozil on the lipid profile of a patient with type Ilb hyperlipidemia

Total choksterol (mmol/I) Triglyceride (mmol/l) HDLcholesterol (mmoy1) LDL-choksterol (mmov1) LDLcholesterol (mmol/l) LDL-apo Et/cholesterol

HPGC Friedewald

Pre-gemfibrozil

Post-gemfibrozil

o/uChange

8.5 4.5 1.0 4.3 5.5 0.85

7.2 1.9 1.1 5.3 5.2 0.69

-15% -58% +IO% +23X -5% -19%

comparisons with the LDL-cholesterol from a combined ultracentrifugation and precipitation method, the results of such comparisons have been reported to be excellent [2,3]. In patients with severe combined hyperlipidemia the LDL-cholesterol is often not available since the Friedewald formula is invalid when the triglyceride is > 4.5 mmol/l. In addition, for triglyceride concentrations in the range of 2.2-4.5 mmol/l only 72% of estimates of LDL-cholesterol using the Friedewald equation are within f 10% of LDL-cholesterol values obtained by direct measurement using a combined ultracentrifugation and precipitation method. As the triglyceride increases through the range 2.2-4.5 mmol/l, accurate estimation of LDL-cholesterol by the Friedewald equation decreases from 76 to 61% [a]. This is illustrated by the pretreatment data shown in Table 1 which show a significant discrepancy between LDL-cholesterol calculated by the Friedewald equation (5.5 mmol/l) and LDLcholesterol measured by HPGC (4.3 mmol/l). HPGC permits direct monitoring of the change in LDL-cholesterol in response to drug therapy in patients with combined hyperlipidemia. As an example, the effect of gemtibrozil therapy in a patient with type IIb hyperlipidemia is demonstrated in Fig. 2B and Table 1. The increase in LDL-cholesterol concentration and in LDL particle size following treatment of hypertriglyceridemic patients with gemfibrozil is well recognised [7]. However, the extent of increase in LDL-cholesterol following treatment with tibrates is often not known in routine clinical practice because the baseline plasma LDL-cholesterol concentration of patients with severe combined hyperlipidemia either cannot be calculated by the Friedewald formula or is inaccurately measured. HPGC lipoprotein profiles may therefore be useful for direct measurement of plasma LDL-cholesterol in patients with combined hyperlipidemia who have been referred for evaluation and management by a specialist Lipid Service. The patient described in Fig. 2B and Table 1 is likely to have had small dense LDL (pattern B of Austin et al. [8]) which reverted to normal size (pattern A) following treatment with gemfibrozil [9]. These particle size changes are most reliably diagnosed by LDL gradient gel electrophoresis [8]. Inspection of Fig. 2B shows that the LDL peak moved marginally to the left (larger particles) following treatment with gemfibrozil. While it is interesting that the shift observed in this patient is consistent with that expected, we believe that the technique of HPGC is not sufficiently sensi-

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tive to reliably diagnose differences in LDL particle size. An increase in LDL particle size is more reliably inferred by examining the LDL apo B/cholesterol ratio which decreased by 19% following gemfibrozil treatment. If it is assumed that LDL is perfectly spherical, this equates to an approximate 6% increase in LDL particle diameter which is very close to the 5.6% increase in diameter of pattern A LDL compared with pattern B LDL [8]. The diagnosis of type III hyperlipidemia in patients with combined hyperlipidemia is possible with the use of the HPGC technique. Firstly, as shown in Fig. 1D there is a marked increase in remnant lipoprotein particles intermediate in size between LDL and VLDL particles which provides a characteristic appearance to the lipoprotein profile. Secondly, if a characteristic lipoprotein profile is observed, the VLDL cholesterol/triglyceride ratio may be measured. For VLDL separated by ultracentrifugation, this ratio has been reported to be >0.8 in cases of type III hyperlipidemia [lo]. In the case illustrated in Fig. 1D the HPGC VLDL cholesterol/triglyceride was 1.34. We did not consider that the HPGC technique provided additional useful information for Fredrickson phenotype IIa where the Friedewald equation accurately estimates LDLcholesterol. Similarly diagnosis of type IV/V hyperlipidemia may be inferred from the baseline lipid data and stored plasma test. However, in patients with type IV/V hyperlipidemia the HPGC technique should permit the LDL response to drug therapy to be accurately assessed. It was of considerable interest to examine the lipoprotein profile of a patient with Tangier Disease and to note the slight excess of remnant particles, as well as the absent HDL peak (Fig. 1F). The remnant particles in Tangier Disease are also qualitatively abnormal [l l] and this technique, whereby fractions are recovered, permits further characterisation of the lipoprotein particles. In conclusion, the HPGC-derived lipoprotein profile was able to provide additional clinically useful information in cases of combined hyperlipidemia. Firstly, it permitted a direct measurement of LDLcholesterol which enabled the LDL response to treatment to be monitored. Secondly, by calculating the LDL apo B/cholesterol ratio it enabled comment to be made regarding LDL particle size. Thirdly, it was useful in suggesting and confirming the diagnosis of type III hyperlipidemia. Further studies are required in patients with combined hyperlipidemia to determine whether the actual shape of the lipoprotein profile (for example, the distance above the baseline of the nadir between VLDL and LDL - representing remnant particles) is predictive of response to the various lipid-lowering drugs. Acknowledgement The authors are grateful to Diana Tanevski for her assistance in the preparation of the manuscript. References [l] Have1 RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-1353.

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[6]

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[IO]

(I I]

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