**2. Principles of tracer technology**

Exogenous and endogenous labelling techniques have been used to study the in vivo metabolism of an endogenous molecule, the tracee (see review [4]). In the exogenous method, the same molecule, in form of a usually radioactively labelled tracer, is introduced into the bloodstream [9]. In lipoprotein studies, this methodology first requires purification of the target molecule or particle and ex-vivo radiolabelling followed by reinfusion into the circulation. The physological integrity of the target molecule might, however, suffer from such procedure. Furthermore, in case of multiprotein complexes (which most lipoproteins are), the kinetics of individual protein components cannot be investigated by this approach. As an example, the investigation of in vivo kinetics of both protein components of Lp(a), as described in this article, to study its assembly mechanism would not be possible with the exogenous labelling approach.

In contrast, in endogenous labelling, a labelled precursor of the molecule of interest, in case of proteins usually a labelled amino acid, is used to label the target molecule by infusion into the circulation of a suitable proband. Ideally, the tracer can easily be detected and quantified, has the same kinetic behaviour as the tracee, and does not perturb the system. Usually, kinetic studies are performed in steady state, where the rates of input and output for a given unlabelled tracee substance are equal and time invariant. Thus, the information provided by the tracer reflects the behaviour of the tracee [10, 11]. At various times, the target protein or particle has to be purified from the blood of human probands and the amount of tracer is quantified to provide a kinetic curve. A mathematical model is then constructed to extract all the information contained in the kinetic curve. By fitting a model to the data, it is possible to calculate the parameters of the model that characterize the flux of molecules between kinetically homogeneous pools. For example, it is thus possible to investigate the whole pathway including production, conversion or catabolism of lipoprotein particles, information that cannot be obtained by static measurements alone.

The term stable isotope refers to a non-radioactive isotope of a given atom that is less abundant in a molecule within a biological system than the lightest naturally occurring isotope. The most common stable isotope used as metabolic tracer for apolipoprotein kinetic studies is [2H3]-leucine. Stable isotope tracers are much safer than radioactive tracers for both the study subject and the investigator. Furthermore, the duration of stable isotope experiments is normally less than 24 hours which is much shorter compared to radiotracer techniques which may need up to 14 days of examination [9].

#### **2.1. Tracer administration**

22 Lipoproteins – Role in Health and Diseases

laboratory animals such as mice and rats which – unless genetically modified or induced by special diet - do not develop atherosclerosis (see review [6]). The same argument is valid for investigations using cellular model systems. Since the liver is the central organ responsible for lipoprotein metabolism and primary human hepatocytes are of only limited use in research, most cellular studies in lipoprotein metabolism have been conducted in human hepatoma cells lines. These lines express, secrete and assemble a lipoprotein pattern which

For all these reasons, the in vivo investigation of metabolic pathways in human subjects is the ultimate approach to elucidate physiological or pathological functions of metabolites in the human body. Historically, such human kinetic studies were performed using radioactive tracers; this methodology is, however, nowadays of only restricted use. Therefore, stableisotope tracer kinetic studies in human subjects with clear advantages regarding safety and technical issues have replaced the radiotracer methods to become an important research tool for achieving a quantitative understanding of the dynamics of metabolic processes in vivo. The aim of this review is to shortly describe the methodology and illustrate how the approach has expanded our understanding of physiological mechanisms as well as the pathogenesis of disorders of human lipoprotein metabolism. We will then specifically address the assembly mechanism of the atherogenic Lp(a) complex and focus on the kinetics of apoB-containing lipoproteins in patients with chronic kidney disease. This patient group is well-known for its high risk for atherosclerotic complications and a 10- to 20-fold

Exogenous and endogenous labelling techniques have been used to study the in vivo metabolism of an endogenous molecule, the tracee (see review [4]). In the exogenous method, the same molecule, in form of a usually radioactively labelled tracer, is introduced into the bloodstream [9]. In lipoprotein studies, this methodology first requires purification of the target molecule or particle and ex-vivo radiolabelling followed by reinfusion into the circulation. The physological integrity of the target molecule might, however, suffer from such procedure. Furthermore, in case of multiprotein complexes (which most lipoproteins are), the kinetics of individual protein components cannot be investigated by this approach. As an example, the investigation of in vivo kinetics of both protein components of Lp(a), as described in this article, to study its assembly mechanism would not be possible with the

In contrast, in endogenous labelling, a labelled precursor of the molecule of interest, in case of proteins usually a labelled amino acid, is used to label the target molecule by infusion into the circulation of a suitable proband. Ideally, the tracer can easily be detected and quantified, has the same kinetic behaviour as the tracee, and does not perturb the system. Usually, kinetic studies are performed in steady state, where the rates of input and output for a given unlabelled tracee substance are equal and time invariant. Thus, the information provided by the tracer reflects the behaviour of the tracee [10, 11]. At various times, the target protein or particle has to be purified from the blood of human probands and the

is substantially different from the respective human counterpart [7].

increased cardiovascular mortality compared to the general population [8].

**2. Principles of tracer technology** 

exogenous labelling approach.

A tracer can be administered intravenously as either a single bolus injection, a primed constant infusion (i.e., a constant infusion given immediately after a priming bolus), or as a combination of both. The tracer bolus administration offers superior dynamics compared with the primed constant infusion, because the enrichment curves (the tracer tracee ratios) after a bolus injection correspond to the impulse response of the system. It is therefore suitable to study components of lipoprotein metabolism with a slow rate of turnover. Another advantage of bolus administration is that it facilitates the determination of newly synthesized particles, as the intracellular precursor enrichment is greater at the start of the study. This argument therefore counts particularly when investigating kinetics of particle assembly, as described in 3.1.1. Practically, the bolus infusion is also most convenient for both subjects and investigators.

#### **2.2. Multicompartment models for data analysis**

Multicompartment modelling is a superior method to dissect the complexities of lipoprotein metabolism, and has been widely applied to systems in which material is transferred over time between compartments connected in a specific structure to permit the movement of material amongst the compartments [12].

Each compartment is assumed to be a homogenous entity within which the entities being modelled are equivalent. For instance, the compartments may represent different types of lipoprotein particles that are kinetically homogeneous and distinct from other material in the system. Very often, the data can be described by more than one model. To ensure that the best model is selected, it is necessary to carefully examine the fitting of the kinetic curve, to determine the precision of the parameter estimates, and to perform statistical tests to compare results obtained with different models. However, the complexity of a multicompartment model is usually a compromise for what is practically possible. A very simple model may not adequately describe the kinetic heterogeneity present within the system. A model that is too complex, on the other hand, will not be supported by

experimental data and, hence, will have little predictive value. Furthermore, even if the development of models is based on experimental data, several assumptions are required in order to derive the model that is to be used. Thus, mathematical models do not determine the kinetics of lipids directly; rather, they derive an indirect approximation.

The software SAAM (Epsilon Group, Charlottesville, VA, USA) has become the first choice for modelling lipoprotein kinetic studies. The SAAM II program was recently developed by SAAM Inst., Inc., Seattle, WA, USA, and is frequently used to analyse lipoprotein tracer data using compartmental models [13, 14]. The primary kinetic parameter resulting after modelling with SAAM II is the fractional synthesis rate (FSR) which, under steady state conditions, is identical to the fractional catabolic rate (FCR) and has the dimension of pools/day. The reciprocal value of FSR/FCR is called retention time (RT, given in days) and indicates the residence time of the investigated tracee (the target apolipoprotein in our cases) in the circulation. The product of FSR multiplied by the concentration of tracee is called production rate (PR) and is usually expressed as mg/kg body weight/day.
