The role of stable isotopes in the investigation of plasma lipoprotein metabolism

5 The role of stable isotopes in the investigation of plasma lipoprotein metabolism C H R I S T O P H E R J. P A C K A R D

Each day grams of lipid, mainly cholesterol and triglyceride, are carried by the plasma lipoprotein transport system from sites of absorption (gut) and synthesis (gut, liver) to peripheral tissues that require it for cell growth, for hormone production, for energy or to lay down as depot fat. The system is in constant, sometimes apparently futile, flux. For example, adipose tissue releases triglyceride as fatty acids which are taken up by the liver, re-esterified and secreted in the form of very-lowdensity lipoprotein (VLDL) triglyceride. VLDL is subsequently lipolysed by lipoprotein lipase on the endothelial cells of adipose tissue and the fatty acids returned to storage. Cycling such as this permits subtle regulation of lipid transport by catecholamines, insulin and other hormones and enables fuel and nutrients to be directed where they are required by the body. Measurement of concentrations of plasma lipid and lipoproteins, therefore, provides only limited information--a snapshot of the various processes. Assessment of the dynamics of the situation by investigation of lipid and apolipoprotein synthesis and catabolic rates reveals the complexities of the system and the co-ordinate control of the various elements. The present concept of lipoprotein metabolism has been derived principally by examining the behaviour of radio-iodinated lipoproteins in normal subjects and those with aberrations such as receptor and enzyme deficiencies (Soutar et al, 1977; Stalenhoef et al, 1984; Demant et al, 1988). Arising from these and other studies are questions regarding the nature and control of lipoprotein synthesis. Some have been addressed in in vitro experiments with cultured hepatocytes and other cell lines, but others can be answered only in man. The most appropriate methods to do this employ labelled precursors to measure the synthesis of lipids and apolipoproteins directly. It is timely, therefore, that recent improvements in mass spectrometers (particularly gas chromatography/mass spectrometry (GC/MS)) in terms of their ease of use and computer-enhanced sensitivity coupled with a widespread availability of inexpensive, labelled compounds (amino acids and lipid precursors) have ushered in a new era in the application of stable isotopes to human physiological research. The techniques are still labour-intensive and more complex than those which employ radio-active Baillidre's Clinical Endocrinology and Metabolism755 Vol. 9, No. 4, October 1995 Copyright © 1995, by Bailli~re Tindall ISBN 0-7020-1982-8 All rights of reproduction in any form reserved

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tracers, but it is now clear that they can be used successfully to determine the kinetics of lipoprotein production. The following discussion reviews the current status of methods based on stable isotope-labelled compounds as they have been applied, so far, to the investigation of lipid and apolipoprotein metabolism.

A P P R O A C H E S TO THE STUDY OF L I P O P R O T E I N KINETICS Detailed studies of lipoprotein metabolism are possible because a substantial number of processes affecting these particles, such as interconversion and lipid exchange, occur in the circulation. Furthermore, the system can be studied either in steady state (i.e. when the concentration of the substance of interest is constant) with radio-active or stable-isotope tracers or in acute perturbation where a stimulus is applied and the return to steadystate monitored. Examples of the latter are the response to alimentary lipaemia, to the extracorporeal removal of lipoproteins or to the introduction of a 'flooding' dose of an amino acid tracer to measure protein synthesis. The analysis of non-steady-state kinetics is complex (Toffolo et al, 1993), however, and the focus of the present discussion will be on the use of tracers which are introduced into a steady-state system to follow the fate of specific components. The division of tracer labels into radio-active and non-radio-active is important because the amounts given and the means of detection differ significantly and this has an impact on the subsequent mathematical manipulation of the kinetic data (Cobelli et al, 1992). Radioactive tracers can be further sub-divided into those introduced into the lipoprotein of interest ex vivo, e.g. low-density lipoprotein (LDL) radiolabelled with 125I(Caslake et al, 1992) and those which are precursors and incorporated into particles endogenously e.g. 3H-leucine (Fisher et al, 1991) and 3H-glycerol (Zech et al, 1979); stable-isotope-labelled compounds have, to date, fallen into the latter category. Comparisons of the usefulness of endogenous versus exogenous, and radio-active versus stable isotope, tracers as applied to the study of the lipoprotein system are presented in Table 1. Each has advantages, and the approach employed depends on the question posed. For example, if the conversion of one LDL subfraction into another is to be followed then a radio-active tracer (125I) should be used to label specifically the particle of interest. On the other hand, measurement of the relative rates of production of apoB in different VLDL fractions is best achieved with an endogenous, stable-isotope-labelled amino acid. It is comforting to note, that in studies performed to date, kinetic variables measured by both radio-active and stable-isotope-based methods have been in good agreement (Cryer et al, 1986; Ikewake et al, 1993). In experiments from our own laboratory, we noted that the decay curves generated by both techniques were very similar in appearance (Figure 1) and yielded comparable production interconversion and catabolic rates (unpublished observations).

757

PLASMA LIPOPROTEIN METABOLISM Table 1. Characteristics of tracers. Advantages and disadvantages of endogenous versus exogenous tracers

Exogenous Labels specific lipoprotein apoproteins or lipoprotein subfractions Labelled according to mass distribution Measures synthesis indirectly by inference from steady state calculation Measures interconversions and catabolic events well Tracer does not recycle Urine data useful for assessing catabolism

Endogenous Labels all lipoprotein apoproteins Labels according to production rate Measures synthesis directly by tracer incorporation Measures interconversions and catabolic events poorly Tracer recycles Excretion data of little value

Radio-active versus stable-isotope-labelled endogenous tracers

Stable isotope Safe Applicable to all, including women and children Can be repeated many times Difficult to measure with sufficient precision Capital cost high

Radio-active Radiation hazard Limited applicability Limited repeatability Easy to measure Inexpensive apparatus and tracers

Tracer - D3-1eucine

T r a c e r - 125 VLDL2 10

10

,

..~. --4 ....

~., ~r - * -

ta "

"~

._

~

"-

"6 .1-

.-,_ -

LDLapoB

:

LDLapoB

~

'

, - .__,

E

Z

,' ~ ~,t.. IDL a p o B k r

LL

,,.

I.

.01

0

~ ~ IDL apoB ~ .,, V L D L 2 a p o B

25

510

a~lk___

~ ~ A

7'5

VLDL2 apo

100 .01 0

- i-

. . . .

7'5

•

lo0

Time (hours)

Figure 1. Comparison of radio-active and stable-isotope tracers in apoB metabolism. The left-hand panel shows apolipoprotein B decay curves in VLDL2 (Sf 20-60), IDL and LDL following injection of a tracer of '25I-VLDL2into a normal subject as described in Gaw et al (1993). The fight-hand panel depicts the appearance and disappearance of tri-deuterated leucine in VLDL2-, IDL- and LDL- apoB (prepared by the same method) following intravenous injection of approximately 500 mg of the amino acid into another normolipaemic subject.

The principal benefits and drawbacks of the use of stable-isotopelabelled compounds in comparison to traditional radio-iodinated lipoprotein tracers are considered to be: 1.

The exact nature of the tracer is known---deuterated leucine will mix with the body leucine pool and be indistinguishable from it. Its appearance in an apolipoprotein can only be due to synthetic events. In

758

.

.

.

.

c.J. PACKARD contrast, radio-iodination of a lipoprotein may introduce label into protein, lipid and possibly carbohydrate. Even within the protein, while most iodine atoms will be covalently attached to tyrosine residues others will be introduced into other amino acids such as histidine. If the protein is modified naturally during its lifetime in the plasma, it is conceivable that label could be lost in an unexpected way. However, it should also be borne in mind that some stable-isotope-enriched compounds are labile and may lose label without the necessity of protein degradation. Production rates are measured directly from the incorporation of amino acid into apolipoproteins. Furthermore, the relative production rates of all lipoproteins of interest can be studied simultaneously. With radioiodinated tracers only two useful labels are available, 1~I and 1311, and if these were administered in the form of, for example, VLDL and intermediate density lipoprotein (IDL), then the LDL production rate would have to be inferred from the LDL apoB mass not accounted for by IDL to LDL conversion (Gaw et al, 1993). Lipoprotein lipid and protein production rates can be measured simultaneously in a wide variety of situations by the co-administration of tracers such as acetate and leucine without additional risk to the subject. This yields valuable insight into the way in which lipoproteins are assembled and secreted in vivo. A potential problem with stable-isotope-labelled compounds is that they must be given in large doses. The term 'tracer' is something of a misnomer when it is considered that administration of a usual 6.0 mg/kg body weight of d3-1eucine expands the leucine plasma pool five to ten times. The impact of this tracer mass complicates the calculation of kinetic variables from enrichment data obtained from the mass spectrometer (Cobelli et al, 1992). Perhaps the greatest problem with stable-isotope-labelled precursor tracers is that of recycling. Amino acids are used to synthesize body protein and when, after 20-40 hours, the initial input of tracer has passed through the lipoprotein system, tracer returns from large body pools at a slow rate that becomes the dominant feature of the decay seen in apoB and other apolipoproteins (Figure 2). The disappearance curves in VLDL and other lipoprotein fractions eventually parallel the decline in plasma leucine. If experiments are kept short (< 10 hours) then this phenomenon has little impact. However, when slowly turning over apolipoproteins (apoB in LDL, apoAI in high-density lipoprotein (HDL)) are studied then it must be accounted for otherwise erroneous values are obtained for fractional synthetic rates (FSRs) and other parameters.

Recently, it has become apparent that the application of certain unique features of stable-isotope methodology may yield kinetic information that cannot be acquired using radio-active tracers. When the radioactive content of a substance is measured it is not possible to discern on which molecules within the tracee the label exists. For example, if 14C-acetate

759

PLASMA LIPOPROTEIN METABOLISM Tracer- D3-1eucine 100. 10' 1. E o

I--

.01

VLDL1apoB

.001 0

25 '

5o

75 '

100

Time (hours) Figure 2. The effect of recycling on VLDL~ apoB decay. A subject was given an injection of about 500 mg d3-1eucine. Disappearance of the tracer from plasma was initially rapid and then, after 25 hours, a long, slow decay was observed. This was due to the return of d3-1eucine released during the turnover of body protein pools. The recycled tracer provided continuous low-level input into VLDL apoB (in this case the curve for the VLDL~ Sf 60-400 fraction is shown) and hence a slow decline in VLDL apoB tracer content became apparent. This feature must be taken into account in long-term stable-isotope experiments, usually by performing multicompartmental analysis (Parhofer et al, 1991).

is given and its incorporation into palmitate followed over time, the amount of total label in the fatty acid can be determined but not its abundance within individual molecules. In contrast, if 13C-acetate is given and the enrichment in palmitate measured by GC/MS it is possible to determine the relative proportions of the fatty acid that contain one, two, three or more 1~C units. These data--obtained from a mass isotopomer distribution analysis (MIDA)---can be converted into kinetic information which reveals the FSR of the product without having to measure precursor enrichment (Hellerstein et al, 1991a,b; Lee et al, 1991). It can be applied to the determination of cholesterol and fatty acid synthesis rates and, theoretically, to any other biopolymer.

M E T H O D S F O R THE GENERATION AND INTERPRETATION OF STABLE-ISOTOPED-BASED KINETIC DATA A number of issues need to be addressed when designing and executing a stable isotope-based turnover: first, the nature of the system under study; second, the choice of tracer and its administration; third, the analytical approach for determining enrichments with sufficient precision over the appropriate time period, and fourth, the mathematical technique to be used for deriving physiologically meaningful kinetic parameters. Careful consideration of these matters can considerably enhance the value of the information acquired.

760

C.J. PACKARD

The system under study Stable-isotope-labelled tracers are usually administered intravenously and equilibrate rapidly with the body's own pool of the substance, e.g. an amino acid or acetate. The site of synthesis of the protein or lipid of interest takes up the tracer and the product appears, perhaps after a brief delay, in the circulation where it can be sampled. Alternatively, tissue biopsies may be taken to provide information on the turnover of body protein (muscle) or lipid (adipose). In many ways, apolipoprotein B-100 in VLDL is an ideal candidate for a stable-isotope-based kinetic study. It is made at a single site using an amino acid precursor pool that equilibrates rapidly with that in plasma. The protein has a rapid turnover and so a substantial amount of enrichment occurs, even during short-term experiments. The product is secreted rapidly into the circulation and is restricted to this physical compartment, making it easy to quantify without making extra measurements of, or assumptions about, the volume of distribution of the protein. Furthermore, VLDL undergoes metabolism in the circulation to IDL and LDL (Figure 1) and thus further, useful information can be gained about apoB kinetics from a single introduction of tracer. Other lipoprotein components present theoretical as well as practical problems if they are to be studied. Apolipoprotein AI is known to be synthezied at two sites, gut and liver. Thus, tracer amino acids will be incorporated into the plasma protein from pools of potentially differing enrichment since the gut receives amino acids from the diet as well as from endogenous protein turnover. This unmeasurable heterogeneity in the precursor pools seriously affects the confidence with which kinetic constants can be defined. In the conduct of experiments using stable-isotope-labelled (as well as radio-active) tracers, known physiology should be incorporated into both design and interpretation. In particular, careful attention should be paid to establishing that the assumptions which underpin mathematical models for interpreting kinetic data are upheld. As pointed out recently (Foster et al, 1993), following infusion of amino acid it is possible to measure the enrichment within LDL apoB and, using linear regression, calculate an FSR. However, the answer is likely to be erroneous because application of this mathematical approach demands that the protein be produced directly by the liver whereas LDL is known to be made in the circulation, albeit from VLDL apoB which was synthesized by the liver. A further, significant problem depends on the metabolic characteristics of the product, especially where absolute synthesis rates (in mg/day) are to be estimated. They require accurate measurement of tracee mass, which is not always possible. Apolipoprotein concentrations in the plasma are readily determined and, in short-term experiments, little penetration of extravascular space occurs so absolute rates (the product of the FSR and the tracee mass) can be calculated with precision. In contrast, while the plasma cholesterol FSR can be determined using a number of stable-isotope techniques, determination of the tracee mass is difficult since cholesterol in plasma equilibrates rapidly with that in liver and red cells, so that absolute cholesterol synthesis rates in short-term studies have to be based on possibly inaccurate pool sizes

PLASMA LIPOPROTEIN METABOLISM

761

(Faix et al, 1993). Another approach would need to be adopted if subtle changes in cholesterol absolute synthesis rates were sought. Choice of tracer and its administration

A wide variety of tracers have been applied to the study of apolipoprotein and lipid metabolism. The earliest apoprotein turnovers were performed with 15N-glycine whereas, lately, the use of d3-1eucine has been favoured. Ideally, the tracer amino acid employed to assess production rates should be relatively abundant in the protein of interest, should not undergo excessive metabolism itself and should be affordable. A further issue is whether the amino acid is singly or multiply-enriched with the heavy isotope; compounds of the latter variety, though usually more expensive, permit a higher sensitivity of detection (see below). Direct comparisons of the various amino acids reveal that, for VLDL apoB, virtually all give similar results. Lichtenstein et al (1990) administered d3-1eucine, d3-valine and d2-13C2lysine at the same time to normal subjects and reported that VLDL apoB100 production rates determined by the three tracers differed less than 3.5%. If the initial rate of rise in product enrichment is to be used as the mathematical method to determine FSR then a further consideration in choosing a tracer is the precision with which the hepatic precursor pool can be monitored. It may be necessary to follow the enrichment in an easily prepared metabolite as well as the original tracer. For example, hippurate has been used to determine the enrichment in the precursor glycine pool (Cryer et al, 1986) and similarly, ketoisocaproate is a marker of intracellular leucine (Arends and Bier, 1991). However, with careful choice of amino acid tracer, the plasma level of enrichment may be as good a guide as any to intracellular precursor kinetics (Ikewaki et al, 1993). D3-1eucine is emerging as the tracer of choice for the study of apoB kinetics. It is one of the most abundant amino acids in the protein and can be detected with good precision and sensitivity in the mass spectrometer. It is an essential amino acid and, therefore, is not produced in the body. Further, since it is metabolized to ketoisocaproate in muscle, the extent of recycling to the hepatic precursor pool in long-term experiments is minimized. These features commend its use, and from the data of Arends and Bier (1991) and our own unpublished studies, it appears that plasma enrichment is an acceptable reflection of the hepatic precursor pool. Cholesterol synthesis rates in man have been determined using two stable-isotope tracers, D20 and 13C-acetate; each has its advantages and drawbacks. With D20 the rapid equilibration with total body water obviates the need to measure the tissue precursor pool. However, enrichment is low and requires the more demanding technique of isotope ratio mass spectrometry to obtain accurate results (Wong et al, 1991; Jones et al, 1992). ~3C-acetate can be given orally, its incorporation into cholesterol followed by standard GC/MS and an FSR determined by MIDA (Faix et al, 1993), a method which, again, does not require direct assessment of the tissue precursor pool. Furthermore, the same 13C-acetate tracer can be used to determine lipogenesis rates in vivo.

762

c.J. PACKARD

In a number of situations, the nature of the system under study or the choice of tracer dictates the method of administration. For measurement of protein synthesis rates, however, alternative methods of introducing the tracer exist, i.e. as a bolus, as a primed constant infusion or as a flooding dose. The last has been recently employed for body proteins and seeks to overcome the need to determine intracellular precursor enrichment by introducing such a large amount of labelled compound into the plasma that it dominates intracellular amino acid pools (Toffolo et al, 1993). So far, the technique has not been employed in apolipoprotein studies. Bolus and primed constant infusion methods have been compared in a recent investigation of apolipoprotein B metabolism by Parhofer et al (1991). When the data were analysed by multicompartmental analysis, essentially the same result was obtained for the VLDL apoB FSR, a finding we have been able to confirm recently for VLDL subfractions (unpublished). It has been suggested (Schaefer et al, 1992) that while bolus injection necessitates compartmental analysis, infusion methods can be interpreted by noncompartmental means and this is an advantage of the latter. Foster et al (1993) point out that this is a misconception and that the two methods, in theory, are amenable to both forms of mathematical treatment.

Analysis of mass isotope enrichments Formal description of mass spectrometry techniques is beyond the remit of the present discussion. A few practical issues, however, are worth addressing in that they impact upon the design of experiments and the nature of the results obtained. The detection of enrichment in an amino acid residue of a protein occurs against the background of na~ral abundance. For example, when the mass distribution of the TBDMS (tert-butyldimethylsilyl) derivative of leucine is analysed, the base ion m0 (formed by the loss of a 75 mass unit fragment from the parent ion) has an abundance of 73% while the m + 1, m + 2 and m + 3 ions have abundances of 18.4, 7.4 and 1.2% respectively, In an experiment where 13C~-leucine is used as tracer, its presence in a product is monitored against the high background of the m + 1 peak limiting the level of enrichment that can be detected with precision. If, however, d3-1eucine is administered much lower amounts can be detected with adequate precision above the m + 3 peak (Lichtenstein et al, 1990; Walsh et al, 1991). Hence, the latter tracer can be used to follow the synthesis of proteins which turn over more slowly, such as LDL apoB, where the enrichment levels will be low. It is possible to enhance further the sensitivity of GC/MS by selectively monitoring the m + 3/m + 2 ratio rather than the m + 3/mo ratio. In this modification (Demant et al, 1994), the amount of material presented to the GC/MS is increased and the detector optimized for the measurement of the m + 3 and m + 2 ions. This permits enrichments of as little as 0.01% to be determined with confidence (Demant et al, 1994) and if more highly substituted amino acids such as ds-phenylalanine are employed even higher sensitivity is possible (Calder et al, 1992). An alternative way of improving the sensitivity of detection is to use an

PLASMA LIPOPROTEIN METABOLISM

763

isotope ratio mass spectrometer (IRMS) rather than GC/MS. These instruments have very high precision at low enrichments but require analytes to be presented as a gas (N2, CO2) and thus organic compounds must be fully combusted (Bennett et al, 1990). This is a tedious initial preparative step limiting the through-put of the method, although the recent availability of gas chromatography--combustion--IRMS instruments considerably enhances productivity and convenience. The other principal drawbacks to the use of IRMS are the substantial cost of the equipment and the fact that sample sizes are considerably higher than those for GC/MS, e.g. in the assay of cholesterol enrichment following D20 administration (Jones et al, 1992), milligram amounts of the pure sterol were required to be prepared at each time interval.

Mathematical approaches to the interpretation of stable-isotope kinetic data Several methods have been employed to derive the primary kinetic parameter, the fractional synthetic rate, from tracer incorporation curves during a primed constant infusion. These include (a) simple regression fit of the initial rate of rise of enrichment in VLDL apoB, (b) application of an appropriate mono-exponential equation, and (c) multicompartmental analyses (Table 2). Foster et al (1993) reviewed the pitfalls of the first two methods and provided examples to demonstrate that metabolic heterogeneity in lipoproteins undermines assumptions that are basic to the application of a simplistic non-compartmental interpretation. They also demonstrated that linear regression analysis using enrichment data for a product protein whose direct precursor is not the hepatic amino acid pool (e.g, LDL apoB) is likely to give erroneous results. The most rigorous method for calculating apolipoprotein kinetics from stable-isotope data is that of multicompartmental analysis with a computer program such as CONSAM (Parhofer et al, 1991; Foster et al, 1993). Enrichment data are converted into tracer/tracee ratios using the equation of Cobelli et al (1992); the latter and not the 'percent enrichment' is the stable-isotope equivalent of specific activity in radio-activity experiments since it takes into account the fact that a non-negligible mass of tracer is introduced into the system under study. It is the basic datum used in multicompartmental analysis. Tracer/tracee ratios may then be multiplied by the amount of tracee in a compartment to yield the tracer mass A further benefit of applying this mathematical approach is that it permits evaluation not only of the increase in tracer content in a component but also its disappearance on stopping the infusion or following a bolus (Figures 1 and 2). This extra information gives valuable insight into lipoprotein metabolic heterogeneity (Foster and Barrett, 1990). Where the synthesis of the lipid components of a lipoprotein are to be determined both linear regression and compartmental analysis have been used. An alternative mathematical approach is available if the lipid has a polymeric structure, e.g. a fatty acid or cholesterol which is synthesized

N/A 11.4 + 5.8 6.4 + 1.7 9.9 + 1.7 14.6 + 6.5 9.3 + 4.1 11.6, 9.5 28.4 + 9.39 12.7 + 7.5 4.7 + 1.7

9.2 + 2.4

3.2 + 0.5

5.1 + 0.6

4.6 + 0.4

8.6 + 2.1

13.0 + 6.7

9.1, 9.7

5.4 + 2.0

11.2 + 6.6

4.2 + 0.6

VLDL apoB Absolute production rate (mg/kg/day)

Linear regression

Mono-exponential function

Mono-exponential function

Compartmental analysis

Mono-exponential function

Compartmental analysis

Linear regression

Linear regression

Linear regression

Linear regression

Calculation method

Venkatesan et al (1993)

Cortner et al (1992)

Lichtenstein et al (1992)

Krul etal (1992)

Cortner et al (1991)

Parhofer et al (1991)

Cohn et al (1990)

Cohn et al (1990)

Lichtenstein et al (1990)

Cryer etal (1986)

Reference

* Typical values for VLDL apoB fractional catabolic rates = (FSRs) in pools/day determined by radio-iodinated tracers are 5.7 + 0.4 (Howard etal, 1987), 4.3 + 1.4 (Berman et al, 1978) and 12.5 + 5.1 (Packard et al, 1980). N/A = not available.

Normal (fasted) (n = 5) Normal (fed) (n = 3) Normal (fasted) (n = 6) Normal (fed) (n = 6) Normal (fasted) (n = 4) Normal (fasted) (n = 4) Hypobetalipoproteinaemic (n = 2) Normal (fed) (n = 8) Normal (fasted) (n = 13) Normal (fasted) (n = 7)

Patient type

VLDL apoB FSR (pools/day)

Table 2. Very-low-density lipoprotein apolipoprotein B-100 synthesis rates in normals as determined by stable-isotope-labelled endogenous tracers.*

7,

.-,1

PLASMA LIPOPROTEIN METABOLISM

765

from acetate units. Mass isotopomer distribution analysis (Hellerstein et al, 1991b) is a procedure which determines the enrichment of the precursor monomer (acetate) pool based on the abundance of labelled monomers in the product of interest. MIDA allows direct estimation of the FSR after correction for the background levels of naturally occurring heavy isotopes. APPLICATION OF STABLE ISOTOPE TECHNIQUES TO PLASMA APOLIPOPROTEIN METABOLISM

Apolipoprotein B metabolism Early reports of the use of stable-isotope-labelled amino acid tracers in the determination of apolipoprotein turnover focused on demonstrating the potential of the technique and revealed the general comparability of the results with those obtained using radio-iodinated tracers. Cryer et al (1986) were the first to show the incorporation of ~SN-glycine into VLDL apoB, and Patterson et al (1991) later presented methods for following the enrichment of d4-1eucine in apoB-100, apoE, apoAI, apoAII, apoCI, apoCII and apoCIII over prolonged periods. The FSRs and absolute synthesis rates for VLDL apoB-100 from studies published to date are given in Table 2. Values for the former parameter range in normal subjects from 3.2 (Lichtenstein et al, 1990) to 13.0 (Cortner et al, 1991) and appear to be influenced by the method of analysis with investigators who used linear regression, in general, reporting lower values than those who employed other methods of calculation. Patient-to-patient variation undoubtedly accounted for some of these differences in FSR since numbers are small, but it is predictable that the regression method would be prone to underestimate the initial slope (Foster et al, 1993). Examination of VLDL apoB-100 kinetics in groups of normolipidaemic and hypertriglyceridaemic subjects has revealed that both the FSR (equal to the fractional catabolic rate (FCR) at steady state) and the absolute synthesis rate are strongly related to plasma triglyceride and VLDL apoB pool size. In the 13 normal subjects studied by Cortner et al (1992), the correlation between VLDL apoB pool and FSR was r = -0.58 (P = 0.037) and between VLDL apoB pool and absolute synthesis rate it was r = 0.90 (P = 0.0001). These workers (Cortner et al, 1991) and, more recently, Venkatesan et al (1993) also measured VLDL apoB production in subjects with familial combined hyperlipidaemia. Both groups observed a substantial increase in VLDL apoB synthesis in these subjects compared to normals--26.3mg/kg/day vs 9.4mg/kg/day (Cortner et al, 1991) 12.8 mg/kg/day vs 4.7 mg/kg/day (Venkatesan et al, 1993)--indicating that the basis of the dyslipidaemia may be VLDL overproduction in the liver. However, it should be noted also that the VLDL apoB FSR (FCR) was subnormal, and defective clearance may have played a role in generating the elevated levels of the lipoprotein. Walsh et al (1991) were the first workers to document the metabolic behaviour of VLDL subfractions using a stableisotope tracer (d3-1eucine). They studied post-menopausal women before

766

c.J. PACKARD

and then during oestrogen replacement therapy and observed that the hormone increased the production rate of large VLDL (Sf 60-400) but not that of small VLDL (Sf 20-60). A number of investigators have monitored the appearance of tracer in LDL during infusion of amino acid and determined LDL apoB FSRs from increase in enrichment relative to the VLDL-100 plateau. The values, again, compare favourably with those obtained using radiolabelled tracer lipoprotein, e.g. 0.30 _+0.08 pools/day in Lichtenstein et al (1990), 0.24 + 0.06 pools/day in Walsh et al (1991), and 0.48 _+0.03 pools/day in Cohn et al (1990); however, they must be viewed with caution for the reason cited above (see 'Mathematical approaches to the interpretation of stable-isotope kinetic data', above). To date, only two studies have used multicompartmental analyses to derive LDL apoB FSRs. These were reported as 0.65 + 0.38 pools/day by Parhofer etal (1991) and 0.42 _+0.50 pools/day by Krul et al (1992). In an investigation that demonstrated the utility of endogenous labelling with stable isotopes, Cohn et al (1990) studied VLDL and LDL apoB-100 metabolism in the fasted and continuously fed states. They reported increased VLDL apoB-100 production when subjects were fed (9.9 _+1.7 mg/kg/day) compared with fasted (6.4 _+1.7 mg/kg/day). A further advantage of stable-isotope-based experiments is that they permit simultaneous evaluation of the synthesis of multiple proteins. Thus, direct comparison can be made of the metabolism of apolipoprotein isomers, such as B-48 and B-100, or of mutant and native forms of a protein in subjects heterozygous for an inherited defect. Lichtenstein et al (1992) measured, in fed subjects, the individual production rates of apoB-48 (from intestine) and apoB-100 (from liver). They found that the plateau enrichment for B-100 was 72.5% of the plasma d3-1eucine level whereas B-48 rose to only 39.7%. This relative difference was confirmed in a later study by Ikewaki et al (1993) which employed '3C6-phenylalanine, and both groups of investigators concluded that it was due to dilution of the amino acid precursor pool in the intestine due to the influx of dietary amino acids. It was also a consistent observation that the FSR of apoB-48 in the VLDL density range was less than that of apoB-100 for reasons that are not yet clear. In an elegant study by Krul et al (1992), the metabolic characteristics of both normal apoB-100 and mutant apoB (apoB-75) were determined simultaneously in subjects heterozygous for hypobetalipoproteinaemia. Affected individuals have about 30% of normal levels of apoB and the normal and mutant proteins are found throughout the VLDL-LDL density spectrum. In vitro, B-75-containing LDL bound more effectively than did B-100 LDL to fibroblast receptors. This translated, in vivo, into enhanced removal rates for both VLDL and LDL apoB; FCRs for apoB-75 in VLDL and LDL were 2.0 and 1.3-fold higher than those for apoB-100 in the same fraction. A low production rate for apoB-75 was also noted compared to apoB-100 in the same subject.

Apolipoprotein A metabolism Apolipoprotein AI and apoAH, the major proteins in HDL, are more

767

PLASMA LIPOPROTEIN METABOLISM

difficult to study with stable-isotope-labelled amino acid precursors for both practical and theoretical reasons. First, they have a lower FSR--0.20 to 0.25 pools/days--than VLDL or LDL apoB and so, during a short-term infusion, will incorporate less tracer with consequent difficulties for making precise measurement of enrichment. It is necessary to employ techniques with multiply-labelled amino acids to achieve the necessary sensitivity. Second, the proteins are made at two major sites, gut and liver, and this, potentially, invalidates basic assumptions in applying linear regression or mono-exponential analysis to derive FSRs from the data (Foster et al, 1993). Either both tissues should be known to have identical precursor pool enrichments or each should be assessed separately and then applied to the proportion of protein judged to come from that site. To this end, Ikewaki et al (1993) used apoB-100 and apoB-48 plateau values during a constant infusion as estimates of liver and gut precursor enrichments respectively. Assessment of apoAI production (Table 3) using stable-isotope-labelled amino acids was first reported by Cohn et al (1990). As for apoB, these workers (Lichtenstein et al, 1990), observed that the FSR for HDL apoAI was the same whether leucine, valine or lysine were used as tracers. The FSR (FCRs) obtained were similar to those seen with radio-iodinated apoAI, i.e. approximately 0.2 pools/day. In a later publication from this laboratory (Cohn et al, 1990), it was reported that the apoAI FSR was not significantly altered if subjects were studied in the fed or fasted state although, as the investigators emphasized, the values obtained must be interpreted with caution because of the theoretical problems noted above. Ikewaki et al (1993) undertook an evaluation of endogenous versus exogenous tracer methods for measuring apoAI kinetics. They administered '25I-apoAI and 13C6-phenylalanine and analysed the first by classical means and the second by fitting a mono-exponential function which used the VLDL apoB-100 plateau value as a measure of precursor pool enrichment. This, of course, assumed that the bulk of apoAI synthesis occurs in the liver. The mean residence time calculated by the two methods was highly correlated (r = 0.87) with the endogenous technique giving slightly higher Table 3. High-density lipoprotein apolipoprotein AI synthesis rate in normals as determined by stableisotope-labelled endogenous tracers.

Patient type Normal (fasted) (n = 6) Normal (fed) (n = 3) Normal (fed) (n = 2) Normal (fed) (n = 4) N/A = not available.

ApoAI FSR (pools/day)

ApoAI production rate (mg&g/day)

0.22 + 0.02

Calculation method

Reference

11.7 - 1.1

Linear regression

Cohn et al (1990)

0.19 + 0.04

9.7 + 0.2

Linear regression

Lichtenstein et al (1990)

0.23, 0.29

9.7, 10.7

Linear regression

Schaefer et al (1992)

0.19

N/A

Mono-exponential function

Ikewaki et ai (1993)

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mean values (5.14 days) than the exogenous (4.80 days). Despite the fact that turnovers were conducted in the fed state the authors concluded, on the basis of modelling the potential contributions of gut and liver (based on B-48 vs B-100 plateau enrichment values), that the best estimate for the contribution of the latter was 90% of overall apoAI synthesis. Further investigations of apoAI metabolism using stable-isotope tracers have revealed that FH homozygotes have an increased FCR for HDL apoAI (0.38 pools/day compared to 0.26 pools/day in normals) coupled with a reduced synthesis rate (Schaefer et al, 1992). Subjects with partial or complete LCAT deficiency (Fish-eye disease) have high clearance rates of apoAI and apoAII but normal production rates of these proteins (Rader et al, 1994). APPLICATION OF STABLE-ISOTOPE-BASED TECHNIQUES TO THE STUDY OF PLASMA LIPID METABOLISM Cholesterol in the body is derived from two sources, intestinal absorption and de novo synthesis which occurs in most tissues but mainly gut and liver. Two recent publications have explored alternate ways of using stableisotope-labelled compounds to estimate the percentage cholesterol absorption in man. Lutjohann et al (1993) modified the continuous isotope feeding method of Crouse and Grundy (1978) by giving multiply deuterated cholesterol and sitostanol in a known ratio orally for one week. The plant sterol was not absorbed and, therefore, the altered ratio of the tracers in faecal samples collected on days 5 to 7 after starting ingestion provided a direct measure of cholesterol absorption. It was demonstrated that samples could be mailed and that the reproducibility of the technique was excellent. This approach, therefore, offers a convenient method for remote testing. Bosner et al (1993) adapted the technique of Zilversmit (1972) to the use of stable isotopes. They examined the ratio of orally versus intravenously administered deuterated cholesterol in the plasma 3 days after giving the tracers. GC/MS analysis provided the necessary sensitivity and precision and the method was, again, relatively easy to perform and highly reproducible with a standard deviation of 2.8% between replicate tests. In both experimental approaches, the use of multiply labelled sterols permitted analysis with a standard GC/MS where previous investigations with single-labelled compounds required the precision of an IRMS instrument. Measurement of whole-body cholesterol synthesis using a standard experimental design still requires the use of an IRMS. Typically, a large dose of D20 is given and its enrichment of body water and incorporation into blood cholesterol estimated at frequent time intervals thereafter (Jones et al, 1992). The method permits evaluation of FSRs within 4 hours and compares successfully with plasma mevalonate assay for its ability to track diurnal variation in sterol production in humans (Jones et al, 1992). An altemative and perhaps more promising technique is that used by Faix et al (1993) to estimate menstrual and diurnal periodicities in cholesterol synthesis. MIDA was employed with oral or intravenous administration of

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769

13C-acetate to demonstrate that serum cholesterol production was 1.5-3.0 times higher at night than during the day but did not vary with menstrual cycle. The method again allowed rapid evaluation of FSR but overcame many of the theoretical uncertainties that apply to repeated consecutive testing of FSR with the classical D20 technique (Foster et al, 1993). Hellerstein and co-workers (Hellerstein et al, 1991a; Faix et al, 1993) have also applied MIDA to an evaluation of lipogenesis. In the first report of the rates of de novo VLDL-palmitate production in man, they established that lipogenesis is a minor quantitative pathway accounting for less than 2% of VLDL palmitate secretion in fasted and fed states (Hellerstein et al, 1991a). These workers proceeded to show, using other stable-isotopebased techniques, a lack of effect of cigarette smoking on hepatic lipogenesis but a substantial impact of the habit on fatty acid mobilization from adipose tissue (Hellerstein et al, 1994). Smoking increased acutely the flux of free fatty acid by 77% with an attendant rise in plasma levels of 73%. Hepatic re-esterification of this fatty acid was also elevated and this would be predicted to have important consequences for VLDL synthesis. A final example of the potential breadth of application of stable isotopebased methods is the work of Hachey et al (1987) in which multiply deuterated triglycerides were fed to lactating women. Over a period of 3 days the transit of triglyceride was followed from the diet through plasma triglyceride-rich lipoproteins to breast milk. The authors observed that dietary fat contributed 10-20% of fatty acids in human milk. Clearly, the study would have been impossible with radioactive tracers, and other techniques such as arterio-venous differences across the breast would be traumatic. SUMMARY

The last 5 years have seen promising beginnings of the application of stable-isotope-based methods to the study of lipoprotein metabolism. Many aspects of plasma lipid transport make it an attractive system for investigating by this means. While early efforts borrowed from standard techniques of generating and interpreting kinetic parameters (FSRs), the shortcomings of these procedures as applied to lipoproteins are now appreciated. Lipoprotein heterogeneity requires that multicompartmental analysis and relatively long-term studies be employed if the information obtained is going to be a useful adjunct to that produced by radio-iodinated lipoprotein tracers. Both approaches--stable-isotope-labelled endogenous tracer and ex vivo radio-iodinated lipoprotein experiments--must be considered complementary. The first provides direct information on lipoprotein synthesis pathways while the latter is superior at following interconversions and catabolic events. Excellent agreement has been demonstrated where the methods have been used simultaneously to estimate the same kinetic parameter. Many of the questions that have arisen from decades of radioactive tracer studies relate to the nature and rate of lipoprotein synthesis, e.g. what kinds of particles are produced by the liver when different diets are taken, and

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how are the synthetic pathways altered in dyslipidaemic states? Endogenous tracers can address these issues, and methods are presently available which provide the means for measuring the production of all apolipoproteins and for estimating cholesterol and fatty acid biosynthesis. When techniques are developed to examine triglyceride, cholesteryl ester and phospholipid production then a much clearer picture of how lipoproteins are assembled in vivo will emerge. This kind of information will be essential to an understanding of regulation of plasma lipid transport and the subtle changes that occur in those at risk for coronary heart disease. Acknowledgements The excellent secretarial help of Mrs Nancy Thomson is gratefully acknowledged. The author's own cited research was supported by a grant from the British Heart Foundation (190/1242) and performed in collaboration with J. Shepherd, P. Stewart, D. Bedford and Th Demant.

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