Determination of fasting and postprandial lipoprotein cholesterol concentrations in pigs: A comparison of methods

Nutrmon

Research, Vol. 20, No. 11, pp. 1621-1631. 2000 Copyright 0 2000 Elsewer Science Inc. Printed m the USA. All nghts reserved 027 I-53 I7/00/$-we front matter

PII: SO271-5317(00)00247-5

ELSEVIER

DETERMINATION OF FASTING CONCENTRATIONS

AND POSTPRANDIAL LIPOPROTEIN CHOLESTEROL IN PIGS: A COMPARISON OF METHODS

Frazer J Allan* B.V.Sc., M.V.Sc., Roger N Johnson1 B.Sc. Ph.D, Prudence V. McNuttl, Kerry A.C. James2 BSc. Ph.D., Keith G. Thompson B.V.Sc. Ph.D., B. William Manktelow B.V.Sc. Ph.D. Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand, 1National Women’s Hospital, Department of Clinical Biochemistry, Auckland, New Zealand, 2New Zealand Institute for Crop & Food Research Limited, Palmerston North, New Zealand

ABSTRACT We examined serum specimens collected in the fasting and postprandial states from five g-week-old Large White pigs to determine whether cholesterol fractions can be Se uential ultracentrifugation was used estimated without use of the ultracentrifuge. to determine cholesterol in VLDL (VLDL Puge), LDL (LDLfuge) and HDL (HDLfuge) fractions. VLDLfuge was compared with VLDL cholesterol concentration estimated as serum triglyceride concentration divided by four (VLDLtrlg). HDLfuge was compared with cholesterol remaining in the supematant after precipitation of apolipoprotein B-containing lipoproteins with Mn2+ and heparin (HDLPPt). LDLfuge was compared with the concentration determined from the Friedewald formula (total cholesterol less HDLppt less VLDLtrig). After correcting the centrifuged fractions for recovery of total cholesterol, the mean difference between the LDLfriede and LDLfuge of fastin samples was ~5% and the mean difference between fasting HDLPPt and HDL ? uge was ~8%. Fasting VLDLtrlg was more than twice VLDLf”ge, after correction for cholesterol recovery, possibly because of very low recoveries in the ultracentrifuge or because of an incorrect divisor of total serum triglyceride. We conclude that whereas HDL and LDL cholesterol can be reliably estimated in these specimens by simple methods, VLDL cholesterol estimation requires further investigation. A secondary finding was that feeding significantly reduced the concentrations of total cholesterol and LDL cholesterol but raised HDL cholesterol. 0 zoo0Elsevler sc,ence1°C KEY WORDS:

Swine, Lipoproteins,

Cholesterol

Determination,

Postprandial

INTRODUCTION In 1972, Friedewald lipoprotein (LDL) Friedewald formula

et al (1) proposed a formula for estimating the fasting serum low density cholesterol concentration in human beings. Laboratories adopted the because it avoided the laborious, time-consuming ultracentrifugation steps

*Corresponding author: Ph: +646 350 5799 ext. 7898, Fax: +646 350 5616, e-mail: F.J.Allan@,massey.ac.nz 1623

F.J. ALLAN et al.

1624

required to isolate LDL cholesterol. The formula requires the measurement of three parameters: the fasting total serum cholesterol concentration, the serum triglyceride concentration and the high-density lipoprotein (HDL) cholesterol concentration (Fig. 1). Fasting total cholesterol and triglyceride concentrations are determined by enzymatic methods. In human beings, HDL cholesterol concentrations are measured in the supematant following precipitation of apolipoprotein-B containing lipoproteins (predominantly LDL and very low-density lipoprotein (VLDL)), or by direct assay (2).

FIG. 1, The Friedewald formula. The triglyceride concentration, [Triglycerides], multiplied by 0.46 provides an estimate of the very low-density lipoprotein cholesterol concentration of the sample. [LDL-C], low-density lipoprotein cholesterol concentration; [HDL-C], high-density lipoprotein cholesterol concentration. All concentrations are in mmol/L.

[LDL-C] = [Total cholesterol] - [HDL-C] - ([Triglycerides]

x 0.46)

In human beings, this formula reliably estimates serum LDL cholesterol concentrations provided it is applied to fasting blood samples, the triglyceride concentration is less than 4.5 mmol/L and the patient is not suffering from dysbetalipoproteinaemia (1,3). Following a meal, a large proportion of circulating triglycerides is found within chylomicrons. In this circumstance, the Friedewald formula will tend to overestimate the VLDL cholesterol concentration and underestimate the LDL cholesterol concentration. Individuals with dysbetalipoproteinaemia have VLDL that is abnormally enriched with cholesterol relative to triglyceride and consequently, the Friedewald formula will tend to underestimate VLDL cholesterol concentrations and overestimate LDL cholesterol concentrations (3). Pigs are often used as a model for studying the effects of dietary lipids on lipoprotein metabolism in human beings. The principal objective of this study was to evaluate whether cholesterol fractions can be estimated in pig serum samples without the use of the ultracentrifuge. A second objective was to determine the effect of feeding on porcine serum lipoprotein cholesterol concentrations.

MATERIAL AND METHODS Experimental

procedure:

Five, S-week-old Large White pigs were used in this study. Blood samples were taken from the pigs following an overnight fast and 2 hours after they had been given ad-libitum access to commercial grower ration. The blood samples were collected from the right brachiocephalic or jugular vein, allowed to clot and spun at 2250 xg for 10 min (11105 rotor, Centra -3C centrifuge, International Equipment Co, Needham Heights, MA, USA). Serum was harvested and refrigerated until analysis, which was initiated within 24 hours of blood collection.

CHOLESTEROL

Analvtical

MEASUREMENT

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IN PIGS

Methods

Determination of IiDoprotein cholesterol concentrations bv sequential ultracentrlfugation: The lipoprotein fractions were prepared using an ultracentrifuge (Centrikon T-2070, Kontron Instruments, Zurich, Switzerland, fitted with either a TFT 80.4 or TFT 45.6 rotor). Ultracentrimgation was performed as described by Mills et al (4) Initially, serum was overlaid with NaCl solution (0 196 molal, density 1.006 kg/L at 200C). The sample was spun for 15 hours at 117 000 xg at 2OOC and the supernatant was harvested by a tube-slicing technique. The infranatant was then adjusted to a density of 1 063 kg/L using NaBr solution (4 778 molal, 1 3 199 kg/L at 200C) and overlaid with NaBr solution (0 844 molal, 1.063 kg/L at 200C). The sample was spun for 20 hours at 191 500 xg and the supernatant was harvested. A second supernatant layer was also harvested by tube slicing. The remaining infranatant was then adjusted to a density of 1 21 kg/L using NaBr solution (7.593 molal, 1.4795 kg/L at 2OoC), overlaid with NaBr solution (2.973 molal, 1.21 kg/L at 200C) and spun for a further 21.5 hours at 205 000 xg. The supernatant was harvested. All solutions contained EDTA (1 mmol/L) and azide (2 mmol/L) Each fraction was weighed and the concentration of cholesterol determined by the enzymatic method of Roche (formerly Boehringer Mannheim) using cholesterol esterase, cholesterol oxidase and peroxidase The lipoprotein content of each fraction was determined qualitatively by electrophoresis on agarose gel using a barbitone buffer (76 mmol/L, pH 8.6) and staining with Fat Red Calcrllation of liyovrotem cholesterol concentrations by .smlyle method. HDL cholesterol lHDLPPt) ApoB-containing lipoproteins (LDL and VLDL) were precipitated from 1 ml of serum at 4OC by the addition of 50 PL of sodium heparin at a concentration of 4000 USP/ml and 100 uL of 1 mol/l MnC12 (5). Precipitation methods for determining the HDL content of pig sera have been previously validated (6,7) Afler mixing, the sample was allowed to stand for 30 min at 4oC and then centrifuged at 15000 xg for 5 min in a microcentrimge The supernatant was removed and its cholesterol concentration was determined by the automated enzymatic method described above using a cholesterol reagent modified by the further addition of EDTA to a final concentration of 8 mmol/L VLDL cholesterol (‘VLDLtW] The ratio of the mass of triglyceride to that of cholesterol in porcine VLDL is approximately 9 1 (8) corresponding to a molar ratio of 4 1 Consequently, in this study, we have estimated the molar concentration of VLDL cholesterol by multiplying the serum triglyceride concentration by 0.25 The serum triglyceride concentration was determined by the enzymatic method of Roche (formerly Boehringer Mannheim) using lipase, glycerol kinase, glycerol phosphate oxidase and peroxidase LDL cholesterol (LDLklede) LDL cholesterol

was determined

by applying

a modification

of the Friedewald

formula

(Fig

2)

1626

F.J. ALLAN et al.

FIG. 2 The modified Friedewald formula used in this study. The concentration, [Triglycerides], multiplied by 0.25 provided an estimate low density lipoprotein cholesterol concentration. [LDL-C], lipoprotein cholesterol concentration; [HDL-C], high-density cholesterol concentration. All concentrations are in mmol/L. [LDL-C] = [Total cholesterol] - [HDL-C] - ([Triglycerides]

triglyceride of the very low-density lipoprotein

x 0.25)

Expression of Results The total cholesterol concentration was determined directly from the serum samples by the enzymatic method outlined above (chol total) and from the data obtained from sequential ultracentrifugation (cholsum ). The latter method involved weighing the VLDL, LDL and HDL fractions, determining the volume and number of moles of cholesterol in each and expressing the result as a concentration relative to the volume of the original sample. The proportion of cholesterol recovered from the fractions was then calculated by dividing cholsum by choltotal. The raw VLDL, LDL and HDL cholesterol concentrations obtained from sequential ultracentrifugation were corrected for cholesterol recovery by dividing the raw concentrations by the proportion recovered. The difference between the serum LDL cholesterol concentration, obtained by applying the Friedewald formula (LDLfrtede) and the serum LDL cholesterol concentration, determined by sequential ultracentrifugation (LDLmge), was calculated and expressed as percentage of the LDLfuge (Fig. 3) This was performed on both the raw data and the recovery-adjusted data. Similar differences were calculated for the HDL and VLDL cholesterol concentration data. Student’s two-tailed t-test for paired samples was used to evaluate the effect of feeding on the total cholesterol, LDL cholesterol, VLDL cholesterol and HDL cholesterol concentrations PC0 05 was considered significant FIG. 3 Formula used to calculate the percent difference between the LDL cholesterol concentration as determined by sequential ultracentrifugation and derived from the Friedewald formula LDL% difference = (LDLfrledr - LDL”‘g”)/LDL”‘gr x 100

RESULTS

After removal of the supernatant fraction floating at a density of 1 006 kg/L, electrophoresis of the floating layer obtained at a density of 1 063 kg/L showed only a prominent B-migrating band. The layer immediately below contained significant quantities of cholesterol and triglyceride and had an identical electrophoretic pattern It was concluded that these layers were respectively LDL and a For analytical purposes they were combined dense subfraction of LDL, possibly LDL2. Electrophoresis of the floating layer obtained following ultracentritugation at a density of 1.21 kg/L showed only an a-migrating band, consistent with HDL and free of contaminating species

CHOLESTEROL

MEASUREMENT

TABLE

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1

(Chol”““‘) Determined Enzymatically and the Mean Cholesterol Concentration Determined by Summation of the Cholesterol Content of the VLDL, LDL and HDL Fractions Following Sequential Ultracentrifugation (Chol”““‘)

Mean

Total

Cholesterol

Concentration

Cho,‘“‘“l

C hols’“”

Recovery

(mmol/L) Fasting

2.21 (0 17)

1.90 (0.18)

0.86 (0.03)

Fed

1.92 (0.16)

1 64 (0.13)

0 85 (0 01)

0 03

0 03

p-value

Recovery refers to the amount of cholesterol recovered following sequential ultracentrifugation expressed proportion of the Chol’“‘“‘. Standard deviations are shown m brackets. significance for the comparison between the fasting and fed data

The p-value

as a is the level of statistical

Approximately 86% of the total serum cholesterol was recovered during sequential ultracentrifugation from each sample (Table 1) Duplicate Cholsum fractionations gave a coeffkient of variation of 1 4%. Coeffkients of variation for the individual VLDLfUge, LDLdfuge and HDLfuge fractions were 6 8%, 2.0% and 2 5% respectively.

TABLE 2 Mean Serum LDL Cholesterol Concentrations Prepared From Sequential Ultracentrifugation (LDL”‘“) and the Mean Serum LDL Cholesterol Concentrations Derived From the Modified Friedewald Formula (LDLfiled”).

~~

Fed p-value

090(0 0.002

11)

0 76 (0 08)

0.89 (0.09)

0 002

0.001

18.1 (4 0)

2 9 (1.7)

Ultracentrifugedvalues, which have been correctedfor recovery, are also shown Difference (raw and corrected) 1s the mean absolute difference between the LDL”led’ and LDLh8’, esprcssed as a percentage of the LDL? Standard deviations are shown in brackets. The p-value IS the level of statistlcal srgnificance for the comparison between the fasting and fed data.

LDL cholesterol concentrations estimated by the modified Friedewald formula were consistently greater than the raw concentrations derived from sequential ultracentrifugation (Table 2). Correcting the LDLfuge data for cholesterol recovery greatly improved the agreement between the methods Serum HDL cholesterol concentrations derived from the precipitation method agreed closely with the concentrations determined following sequential ultracentrifugation (both raw and corrected) (Table 3) The HDLppt was marginally higher than the raw HDLfuge but marginally lower than the corrected HDLfuge

F.J. ALLAN et al.

1628

TABLE 3 Mean Serum HDL Cholesterol Concentrations Prepared From Sequential Ultracentrimgation (HDLfug”) and the Mean Serum HDL Cholesterol Concentrations Derived Following the Precipitation of Apolipoprotein B-Containing Lipoproteins (HDLpp’). HDLPP’

HDL”“’ raw

HDLf”kY corrected

Difference raw

corrected

(mmol/L)

W)

Fasting

0.78 (0.06)

0.74 (0.05)

0.84 (0.07)

5.4 (3.8)

7.8 (4.5)

Fed

0.91 (0 07)

0 83 (0.06)

0.97 (0.08)

10.1 (1 7)

5.8 (1.6)

0 03

0 05

0.06

p-value

Ultracentrifugedvalues, wlrichhave been corrected for recovery, are also shown. Difference (raw and corrected) is the meCan absolute difference between the HDLpP’ and HDLhpe, expressed as a percentage of the HDLrup” Standard deviations are shown m brackets. The p-value IS the level of statistical significance for the comparison between the fasting and fed data

There was significant disparity between the methods used to determine the VLDL cholesterol concentration (Table 4) The VLDL cholesterol concentration, calculated from the serum triglyceride concentration, was 22 times greater than the raw VLDL cholesterol concentrations The agreement improved when the raw data was derived from sequential ultracentrimgation There was closer agreement between the methods in the corrected for cholesterol recovery. postprandial samples than in the fasting samples.

TABLE 4 Mean Serum VLDL Cholesterol Concentrations Prepared From Sequential Ultracentrifugation (VLDLfuge) and the Mean Serum VLDL Cholesterol Concentrations Calculated by Dividing the Serum Triglyceride Concentration By Four (VLDLtrlg). VLDL’“g

V,JDL”‘a” raw

VLDLfi’g”

Difference

corrected

raw

(mmol/L)

corrected W)

Fasting

0.10 (0.02)

0 03 (0.01)

0 04 (0 01)

210 (74)

170 (73)

Fed*

0.11 (0 04)

0 06 (0.02)

0.07 (0 02)

98 (29)

67 (27)

0.5

0 009

0.009

p-value

Ultracentrifuged values, which have been corrected for recovery, are also sl~own Difference (raw and corrected) IS the mean absolute difference between the VLDLD’R and VLDLhgc, expressed as a percentage of the VLDL”? Standard deviations are sl~own in brackets The p-value is the level of statistical slguificance for the comparison between the fasting and fed data *Values followmg fccdmg represent the cholesterol concentration of the combined VLDL/chylomicron fraction

Regardless of the method of determination, the total cholesterol concentration and the LDL cholesterol concentration were significantly lower in the postprandial samples than in the fasted samples Conversely, the HDL cholesterol was significantly higher in the fasted samples than in

CHOLESTEROL

the postprandial samples. doubling of the VLDLfuge

Feeding

MEASUREMENT

did not significantly

IN PIGS

change

VLDLtrig

1629

but it resulted

in a

DISCUSSION

This study has shown that porcine serum HDL and LDL cholesterol concentrations, as determined by the precipitation method and the modified Friedewald formula respectively, agree well with concentrations determined by sequential ultracentrifugation. With the exception of the fasting HDL cholesterol concentration, the agreement was better when the ultracentrifuged concentrations were corrected for cholesterol recovery. Conversely, for both the fasting and postprandial samples, there was poor agreement between the VLDLtrrg and the VLDLfuge cholesterol concentrations. This occurred despite the fact that the Friedewald formula was modified to reflect the higher ratio of triglyceride:cholesterol in the VLDL of pigs compared to human beings. While only small amounts of VLDL (30-80 mgidl) are to be expected in pig serum (6), any loss of VLDL during tube-slicing would lower the concentration of VLDLfuge even further Repeating fractionation with larger samples, which should allow for better recovery, might reveal the significance of such losses. However, since the quantity of cholesterol contained within the VLDL fraction is <5% of the total cholesterol concentration, any error inherent in either of the methods used to determine the VLDL cholesterol concentration had little bearing on the agreement between LDLfriede and LDLfuge, Since neither of the methods for VLDL cholesterol determination used in this study takes into account postprandial chylomicronaemia, the VLDL cholesterol concentration after feeding is effectively the cholesterol concentration in both the VLDL and chylomicron fractions We found that following feeding, the mean VLDLfuge doubled but the VLDLt’ig remained unchanged. Luhman et nl found that the cholesterol concentration of a VLDL/chylomicron fraction in pigs tended to be greater four hours after feeding than after a 12-hour fast (9) which is consistent with our observation of a significant increase in the VLDLfUge following feeding Conversely, Luhman et nl found that compared to fastin g samples, the plasma triglyceride concentration was significantly lower four hours after feeding but there was no difference in the plasma triglyceride concentration one hour after feeding (9). Given that in our study, the VLDLt”g is determined from the serum triglyceride concentration, the results of Luhman ef czl may explain why the VLDLtrig was not affected by feeding Consequently, using the serum triglyceride concentration to determine VLDL/chylomicron cholesterol concentrations should be avoided on postprandial samples. We have chosen to present both the raw and corrected sequential ultracentrifugation data because both sets of values are inherently subject to error. The quantity of cholesterol recovered from the various lipoprotein fractions following sequential ultracentrifugation was approximately 15% less than the quantity of cholesterol determined by assay of the original sample This ‘loss’ of cholesterol is recognised as a shortcoming of lipoprotein preparation following extensive ultracentrifugation (lo,1 1) and consequently, the raw data may underestimate the actual cholesterol concentration in each of the lipoprotein fractions. By correcting for recovery however, we have assumed that the cholesterol loss following sequential ultracentrifugation is the same in each lipoprotein fraction This may not be the case and for some classes of lipoprotein, correction may have led to overestimation of the actual cholesterol concentration The density HDL (10).

range of porcine LDL2 has been shown to overlap This has led some workers to employ differential

the lower density limit of porcine flotation to separate LDL2 from

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F.J. ALLAN et al.

HDL (10). In our study, contamination of the LDL layer with HDL (and vice versa) was not detected by electrophoresis. We proposed that the second layer that was harvested following ultracentrifugation at a density of 1 063 kg/l might have contained the LDL2 subfraction thereby preventing HDL contamination. Feeding significantly reduced the serum total cholesterol and LDL cholesterol concentrations but increased the I-IDL cholesterol concentration This is in contrast to the findings of Luhman et nl (9) who found that feeding had no effect on these parameters in pigs. Luhman et al, however, fed high fat diets (20 or 40% calories from fat) compared to the standard commercial grower ration used in the current study, which may explain the difference between the results of these studies. In human beings, the postprandial HDL cholesteryl ester concentration appears to be inversely related to the postprandial rise in plasma triglyceride concentrations (12) Despite an increase in 1ecithin:cholesterol acyl transferase (LCAT) activity within HDL postprandially, the presence of increased levels of substrate for cholesteryl ester transfer protein (CETP) results in a net transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins (13) In contrast to human beings, pigs lack CETP activity (14,15), which may explain why we observed an increase in the postprandial HDL cholesterol concentration Cohn et nl found that LDL cholesterol concentrations in human beings were significantly lower 3 and 6 hours after eating a fat-rich meal (11). This was associated with a decrease in the plasma concentration of apolipoprotein B and the authors hypothesised that the postprandial rate of catabolism of LDL exceeded its synthesis. Although we were unable to measure apolipoprotein concentrations in this study, a similar mechanism may explain our findings in pigs. In summary, we have shown that in porcine serum samples, a precipitation method for determining the HDL cholesterol concentration and a modification of the Friedewald formula for estimating the LDL cholesterol concentration closely approximate values obtained from sequential ultracentrifugation. Conversely, the VLDL cholesterol concentration, determined by dividing the triglyceride concentration by 4, did not agree with concentrations determined by sequential ultracentrifugation. More work is required to address this issue We have also documented significant postprandial changes in lipoprotein cholesterol concentrations when pigs are fed lowfat commercial pig food

ACKNOWLEDGEMENTS

The authors would like to thank the Foundation Zealand for funding the study.

of Research,

Science and Technology,

New

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CHOLESTEROL

MEASUREMENT

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