1

HORMONE MEASUREMENT GUIDELINES 1

Tracing lipid metabolism: the value of stable isotopes 2

A Margot Umpleby 3

Diabetes and Metabolic Medicine, Faculty of Health and Metabolic Sciences, University of Surrey, 4

Guildford, UK 5

6

Key words: stable isotopes, tracers, fatty acids, cholesterol, lipoproteins 7

8

Address for correspondence 9

Professor Margot Umpleby 10

Diabetes and Metabolic Medicine, 11

Leggett Building, University of Surrey 12

Daphne Jackson Rd, Manor Park, Guildford GU2 7WG, UK 13

14

Phone: 01483 688579 Fax: 01483 688501 15

16

E mail:m.umpleby@surrey.ac.uk 17

18

Word count: 4,859 19

20

21

Page 1 of 24 Accepted Preprint first posted on 5 June 2015 as Manuscript JOE-14-0610

Copyright © 2015 by the Society for Endocrinology.

2

Abstract 22

Labelling molecules with stable isotopes to create tracers has become a gold standard method to 23

study the metabolism of lipids and lipoproteins in humans. There are a range of techniques which 24

use stable isotopes to measure fatty acid flux and oxidation, hepatic fatty synthesis, cholesterol 25

absorption and synthesis and lipoprotein metabolism in humans. Stable isotope tracers are safe to 26

use, enabling repeated studies to be undertaken and allowing studies to be undertaken in children 27

and pregnant women. This review provides details of the most appropriate tracers to use, the 28

techniques which have been developed and validated for measuring different aspects of lipid 29

metabolism and some of the limitations of the methodology. 30

31

32

33

Page 2 of 24

3

Introduction 34

The global increase in obesity has led to an increased focus on understanding the regulation of lipid 35

metabolism. Obesity is associated with insulin resistance, hyperlipidaemia, non-alcoholic fatty liver 36

disease (NAFLD), type 2 diabetes mellitus and atherosclerotic cardiovascular disease (1,2). Fatty 37

acids are one of the mediators of insulin resistance (3) and an imbalance in fatty acid metabolism 38

results in the accumulation of ectopic fat which can lead to NAFLD, fatty infiltration of the pancreas 39

and epicardial fat (4). Hyperlipidaemia, ie increased levels of triacylglycerol (TAG) and cholesterol, is 40

a major risk factor for atherosclerosis, the major cause of cardiovascular disease (5). This review will 41

provide an overview of the techniques that can be used to measure lipid metabolism using stable 42

isotopes in humans. 43

The concentration of lipids in a blood sample can provide information about whether levels are 44

abnormal but cannot provide information about the underlying mechanisms for any abnormality. 45

The concentration of any lipid in a blood sample is a product of its local synthesis and influx into the 46

circulation, absorption from dietary sources and efflux (and local degradation) over time. Therefore, 47

a high concentration of plasma cholesterol, for example, could be a result of its increased synthesis 48

and/or absorption or decreased removal from the circulation. To gain an understanding of lipid 49

metabolism and how it is regulated we need to be able to measure these fluxes. To do this, the lipid 50

of interest needs to be labelled so that it is indistinguishable from the naturally occurring lipid in the 51

body but is distinct in some way that enables it to be detected and measured accurately. Since the 52

labelled lipid needs to behave metabolically like the unlabelled lipid, the labelling must not change 53

its metabolic characteristics. One way to achieve this is to use stable or radioactive isotopes. Stable 54

isotopes are not a source of ionising radiation, unlike radioactive isotopes. Labelling with stable 55

isotopes has become a gold standard method to study the metabolism of lipids and lipoproteins in 56

humans. The quantities of stable isotopes that are administered are non-toxic and studies can be 57

safely undertaken in humans, including pregnant women and children. 58

Theory 59

Some elements are composed of atoms that are chemically identical but have a different mass due 60

to different numbers of neutrons. These are termed isotopes. In the biological environment, the 61

most abundant isotopes of the major elements, ie hydrogen (H), carbon (C), nitrogen (N) and oxygen 62

(O) are 1H,

12C,

14N and

16O. These are stable but there are also other stable isotopes of these 63

elements which are much less common (ranging from 0.02-1.1%). These are 2H (deuterium),

13C,

15N, 64

17O and

18O. In this review the term “stable isotope” will refer to these less common isotopes. When 65

one of these less abundant isotopes is substituted for the common form in a molecule, this is known 66

as “labelling” and creates a “tracer”. The original molecule is known as the tracee. The tracer and 67

tracee have the same chemical properties. The amount of tracer relative to the amount of tracee is 68

expressed as the tracer:tracee ratio (TTR). TTR corrected for the background TTR is referred to as 69

enrichment. An alternative way of expressing enrichment is as mole percent excess (MPE) or atom 70

percent excess (APE). This is the amount of tracer (in moles or atoms) relative to the sum of tracer + 71

tracee multiplied by 100. The different mass of stable isotopes enables them to be detected and 72

accurately measured by mass spectrometry. 73

One of the first studies where stable isotopes were used to study lipid metabolism was published by 74

Shoenheimer and Rittenberg in 1937 (6). In this study, deuterium labelled linseed oil was fed to mice 75

Page 3 of 24

4

and it was shown that 33% of the labelled fatty acids could be detected in adipose tissue (6). 76

However stable isotope labelled tracers were not easily obtainable and the mass spectrometers 77

needed for their measurement were costly so this methodology was of limited use for most 78

researchers. However there was a plentiful supply of radioisotopes, which could be used for 79

creating radioactive lipid tracers. These could be measured by scintillation counters which were 80

readily available. The health risk associated with radioactive tracers in humans and the development 81

of affordable mass spectrometers led to the greater use of stable isotope tracers in the late 1960s 82

and early 1970s. Stable isotopes are now widely used for lipid tracer studies although many of the 83

techniques that are used were developed using radioactive tracers. 84

Measurement 85

Isotopic enrichment of biological samples can be measured by nuclear magnetic resonance (NMR) 86

or mass spectrometry (MS); the latter is often interfaced with an instrument which first separates 87

the molecule of interest eg gas chromatography (GC), liquid chromatography (LC) etc. In GCMS and 88

LCMS the labelled molecule eg [1-13

C]palmitate, (the tracer) is measured relative to the unlabelled 89

palmitate (ie the tracee). A major advantage of stable isotopes over radioactive isotopes is that both 90

tracer and tracee are measured simultaneously with very high precision. An isotope ratio mass 91

spectrometer (IRMS), measures the isotopic enrichment of a gas. This technique is very sensitive so 92

can be used when enrichment is low. Samples are converted to gases, through combustion or 93

pyrolysis which are then separated. Samples containing 13

C labelled molecules can be converted to 94

CO2 by combustion while deuterium containing molecules can be converted to hydrogen by 95

pyrolysis. An IRMS is also used for measuring expired 13

CO2 generated from the oxidation of a 13

C 96

labelled molecule and for measuring the enrichment of labelled plasma water (2H2O) which is used in 97

the measurement of fatty acid synthesis. For more details on measurement techniques see 98

Schierbeek H et al (7). 99

Lipid metabolism can be measured by the administration of a tracer either orally or intravenously. 100

There is a natural abundance of stable isotopes so any isotopic tracer that is administered will add to 101

the existing background. It is therefore essential that the background levels are measured before 102

administration of the tracer. Because stable isotope tracers have mass the quantities used should be 103

low so that the pool size of the tracee is not affected as this would perturb the kinetics of the system 104

being studied. This requirement is usually fulfilled since measurement techniques are very sensitive. 105

Tracer supply and choice 106

Stable isotope tracers can be purchased from a number of suppliers. For human studies they are 107

relatively expensive because of the quantity required. The cost varies depending on the number of 108

labels and the site of labelling in the molecule. If they are to be administered intravenously in 109

humans, they must be either purchased sterilised and prepared for human use or prepared in this 110

way in a hospital Pharmacy. This adds another layer of cost. 111

The standard notation for a stable isotope tracer indicates the mass number as a superscript to the 112

left of the atom and the number of the atoms substituted with the isotope as a subscript to the 113

right; for example [2H4] palmitic acid has four atoms of

2H. Uniformly labelled, represented by “U”, 114

means all the atoms are substituted with the isotope. Thus [U-13

C] palmitic acid indicates that every 115

carbon has been replaced by 13

C. The choice of tracer is determined by the level of sensitivity 116

Page 4 of 24

5

required, whether oxidation rate is to be measured, whether other tracers are to be administered in 117

the same study, whether tracer recycling is likely to occur and other factors such as cost. More detail 118

on the most appropriate tracers to use, are given when describing each method below. 119

120

Stable isotope techniques for measuring lipid kinetics 121

There are four techniques which are used for measuring lipid kinetics a) dynamic isotope dilution b) 122

the product precursor method c) tracer conversion and d) the dual isotope method. The choice of 123

technique is dependent on the metabolic pathway to be measured. Some of the tracers used to 124

study lipid metabolism, and the points where they are utilised are shown in Figure 1. 125

Adipose tissue lipolysis 126

The technique of dynamic isotope dilution can be used to measure whole body production rates of 127

metabolites which are secreted into the circulation. Since the whole body production rate of non-128

esterified fatty acids (NEFA) into the blood from tissues is mainly from peripheral adipose tissue in 129

the fasting state, this is used as an index of whole body adipose tissue lipolysis of stored TAG. 130

Although intravascular lipolysis of plasma lipids can contribute, in a fasting state the former is 131

considered to make only a minor contribution (8). In a postprandial state this becomes more 132

significant (9). In a fasting state the rate of production of fatty acids is equal to the rate of whole 133

body uptake which can also be of interest. 134

A palmitate tracer is most commonly used for measuring non-esterified fatty acid (NEFA) production 135

rate as it is the cheapest tracer. It is assumed that palmitate is representative of all fatty acids. The 136

tracer can be administered as an intravenous bolus or as a constant intravenous infusion. The latter 137

is the most frequently used since a bolus requires very rapid blood sampling as the clearance of 138

NEFA from the circulation is very fast (10). With the constant infusion method, a known amount of 139

the fatty acid tracer eg [1-13

C] palmitate is infused intravenously at a constant rate. Samples are 140

collected ideally from arterialised venous blood or from an artery. In a metabolic steady state eg 141

during fasting, the TTR increases with time until a plateau is reached indicating an isotopic steady 142

state is achieved (Figure 2). At this point the rate of removal of the tracer from the system is equal to 143

the rate that it appears ie the infusion rate. In the plasma pool the tracer is mixed with tracee 144

entering the system from adipose tissue and measurement of its dilution provides a measure of the 145

rate at which palmitate is appearing in the body pool. This technique requires the unlabelled 146

palmitate to be constant during the study ie in a steady state. It is assumed that the tracer is diluted 147

in a single body pool eg plasma. The dilution of the tracer by the tracee at isotopic steady state is a 148

measure of the rate of appearance (Ra) of palmitate into the pool which in a steady state is equal to 149

the rate of loss from the pool (also known as the flux) (Figure 3). 150

Ra (mg/min) =Tracer infusion rate (mg/min)/TTRSS-TTR (t=0) 151

where SS=isotopic steady state 152

To convert palmitate Ra to NEFA Ra, the concentration of palmitate relative to total NEFA needs to 153

be measured. Other fatty acids can be used as tracers; palmitate, oleate and linoleate have all been 154

shown to provide good estimates of NEFA Ra whereas myristate and stearate do not (10). Since long 155

Page 5 of 24

6

chain fatty acids are insoluble in aqueous solution, the stable isotope tracer of a fatty acid salt eg 156

potassium palmitate is complexed with human albumin to aid solubility. Deuterated tracers are 157

cheaper but if [U-13

C] palmitate is used the infusion rate can be reduced if GC-combustion IRMS is 158

used to measure enrichment. This reduces the amount of albumin which needs to be infused. If [U-159 13

C] palmitate is used palmitate oxidation rate can also be measured. 160

NEFA production rate can underestimate adipose tissue lipolysis because some NEFA released from 161

lipolysis is re-esterified within adipose tissue. The production rate of glycerol measured with a 162

glycerol tracer provides a more accurate measure of lipolysis since glycerol kinase levels are very low 163

in adipose tissue so there is very little recycling (11). As with using NEFA production rate as a 164

measure of lipolysis, intravascular lipolysis of plasma lipids can also contribute some glycerol to the 165

plasma pool although in a fasting state this would be a minor contribution (12). The technique used 166

is identical to the one described above for palmitate. There are several options in the choice of 167

tracer- [2H5] glycerol, 2-

13C1 and U-

13C glycerol have all been shown to give similar measurements of 168

whole body glycerol production rate (13). 169

The isotope dilution technique can also be used in the non-steady state. With the tracee in a steady 170

state eg during fasting, an isotopic steady state is achieved first with the tracer infusion. The steady 171

state can then be disturbed by, for example, the subject exercising or with the infusion of a 172

hormone. The changes in TTR and concentration with time are used to calculate the change in the 173

flux of the tracee using non steady state equations or mathematical modelling (14). Using this 174

technique the insulin sensitivity of lipolysis can be measured by combining a constant iv infusion of 175

labelled glycerol or labelled palmitate with a low dose insulin infusion (15) (Figure 4). 176

NEFA Ra can also be measured during the postprandial period, using non-steady state equations. 177

However, in the postprandial period, some fatty acids hydrolysed from dietary fat (chylomicrons) 178

can directly enter the plasma pool, a process known as “spillover”, so NEFA Ra is a measure of both 179

lipolysis and spillover (16). 180

The isotope dilution technique can be combined with the measurement of arterio-venous balance 181

measurements in specific tissues eg subcutaneous adipose tissue. This can provide measurements of 182

tissue specific rates of production and uptake (16). This method requires the measurement of blood 183

flow through the tissue. 184

Fatty acid oxidation 185

Whole body NEFA oxidation can be measured using the technique of tracer conversion. If 13

C 186

labelled palmitate is administered the appearance of 13

C in CO2 can provide a measure of palmitate 187

oxidation rate. Small bags or tubes are used to collect expired air. The 13

CO2 content can then be 188

measured by IRMS. To calculate the 13

CO2 expiry rate, CO2 production rate (VCO2) must also be 189

measured. This can be obtained using a gas analyser when collecting the breath samples. A 190

quantitative measure of palmitate oxidation rate can be determined if a steady state of 13

CO2 is 191

achieved. An isotopic steady state of 13

CO2 can take up to 8 hours due to a slow rate of exchange of 192 13

CO2 with the bicarbonate pool. This can be shortened by priming the bicarbonate pool by 193

administering an iv bolus of 13

C sodium bicarbonate when the tracer infusion is initiated. The other 194

consideration with this method is correction for any fixation of 13

CO2 in the body bicarbonate pool 195

and loss of 13

C during the TCA cycle. A method for correcting for this has been developed using 13

C 196

Page 6 of 24

7

labelled acetate (17). In this method 13

C labelled acetate is infused in a separate study, which mimics 197

the conditions employed for the determination of palmitate oxidation (17). Acetate enters the TCA 198

cycle as acetyl CoA and the difference between the amount of 13

C acetate infused and the amount 199

recovered in13

CO2 is a measure of the amount of fixation which needs to be corrected for (Figure 5). 200

The need for an additional study adds an extra burden to this measurement. 201

Lipoprotein metabolism 202

Lipoprotein particle kinetics can be measured by labelling one of the apolipoprotein components 203

with an isotopically labelled amino acid eg 13

C leucine. Lipoprotein TAG kinetics can be measured by 204

labelling TAGin the TAG rich lipoproteins ie VLDL and/or chylomicrons. To measure the synthesis 205

rates of macromolecules such as proteins and triglycerides the precursor-product method is used. A 206

stable isotope labelled precursor is administered either orally or intravenously and the incorporation 207

of the precursor into the macromolecule is measured over a period of time. If the TTR of the 208

precursor is known or can be measured the fractional rate of synthesis (FSR) of the product can be 209

determined. In a steady state ie when the protein mass or TG pool is constant, the FSR is equal to 210

the fractional catabolic rate (FCR), a measure of metabolic clearance. FSR and FCR have units of time-

211 1. An absolute secretion rate (eg mmol/hour) is calculated by multiplying the FSR by the product pool 212

size. 213

Apolipoprotein kinetics 214

ApoB100 is a large structural protein in VLDL, IDL and LDL. There is one apoB100 215

molecule/lipoprotein particle. VLDL, IDL and LDL apolipoprotein (apo) B100 FCR and absolute 216

secretion rate (ASR) can be measured with either an intravenous bolus or constant infusion of a 217

labelled amino acid (18,19). Most researchers use [1-13

C] leucine or [2H3] leucine, although labelled 218

valine and lysine have also been used. 219

With the infusion method the rate of increase in apoB enrichment measured relative to the 220

precursor pool is a measure of apoB fractional secretion rate (FSR). With the bolus method, the 221

decrease in apoB enrichment over time is measured. With both methods the change in enrichment 222

with time is not linear so fitting the data with linear regression is inappropriate. A monoexponential 223

function can be fitted to the enrichment data (20) to calculate FSR but a mathematical model can 224

provide a better curve fit and more accurate measurements of FSR (21). Because VLDL is 225

metabolised to IDL and then LDL in the circulation and during this process apoB100 is retained within 226

the lipoprotein, if apoB100 enrichment is measured in each fraction it is possible to describe this 227

whole metabolic pathway using a multicompartmental mathematical model (Figure 6). A number of 228

different model structures have been proposed (18,22). If VLDL apoB100 enrichment achieves an 229

isotopic steady state during a constant infusion study, this plateau of enrichment is a measure of the 230

enrichment of the precursor pool. If this is not the case, an alternative measure of the precursor 231

pool is used. Plasma leucine does not provide an appropriate measure of the precursor pool since 232

hepatic intracellular leucine (the true precursor pool) is diluted by intracellular proteolysis. Within 233

hepatic cells, leucine is deaminated to α ketoisocaproate (α KIC) and the enrichment of α KIC, which 234

can be found in the circulation, has been shown to provide a good estimate of hepatic intracellular 235

leucine enrichment (23). 236

Page 7 of 24

8

VLDL and IDL apoB100 enrichment can be measured by GCMS, but there is considerable dilution of 237

the tracer in the large pool of LDL apoB100. This necessitates the measurement of LDL apoB100 238

enrichment by GC-combustion IRMS which has a much higher sensitivity than GCMS. In several 239

studies VLDL has been subfractionated and the enrichment and kinetics measured of two forms of 240

VLDL, VLDL1 a large triglyceride rich lipoprotein and VLDL2 a less triglyceride rich lipoprotein (24). 241

VLDL2 can be formed by direct secretion from the liver or from catabolism of VLDL1 and both these 242

pathways can be determined by mathematical modelling. Unlike VLDL, LDL has a very slow turnover 243

so a constant infusion of tracer over a single day, provides a limited description of the LDL apoB 244

enrichment curve. An iv bolus of tracer provides an alternative approach which enables LDL apoB 245

enrichment to be measured over several days if volunteers are willing to return to the clinical 246

research centre for a single blood sample over the following 12-14 days. This provides a more 247

accurate measurement of LDL kinetics (19). 248

In studies where constant meal feeding is used to achieve a postprandial steady state, labelling of 249

apoB48, the structural protein of chylomicrons, with a labelled amino acid such as [1-13

C] leucine can 250

provide a measure of chylomicron particle kinetics. The methodology is identical to that described 251

for VLDL apoB100. Both apoB48 and apoB100 are isolated by SDS polyacrylamide gel electrophoresis 252

(25). 253

By labelling the main protein component, apoA1, HDL particle kinetics can be measured (26). This 254

has been measured with a constant intravenous infusion of a labelled amino acid over a single day. 255

However HDL has a half-life of 5 days, which means a very limited description of the apoA1 256

enrichment curve is gained in one day. As with measuring LDL kinetics, the iv bolus technique with 257

measurement of enrichment over several days combined with mathematical modelling should 258

provide a more robust measurement of HDL apoA1 kinetics. There are however no published 259

studies which have investigated this. 260

VLDL TAG kinetics 261

VLDL TAG kinetics can be measured with an iv bolus or constant infusion of labelled glycerol (27) or a 262

fatty acid, usually palmitate (28). Glycerol is the preferred tracer since recycling of a fatty acid tracer 263

can occur which can result in an underestimation of VLDL TAG synthesis rate (28). Administration of 264

an iv bolus of 2H5 glycerol with measurement of the rise and decline in glycerol enrichment in 265

triglyceride over 8-12 hours is the most widely used method (27). The FSR (and FCR) can be 266

determined by mathematical modelling as described above for apoB100 kinetics (21,24). Depending 267

on the ultracentrifugation methods used to isolate VLDL, either total VLDL TAG kinetics or VLDL1 and 268

VLDL2 TAG kinetics can be measured (Figure 7). 269

An iv bolus of 2H5 glycerol has been shown to be incorporated into chylomicron triglyceride enabling 270

chylomicron triglyceride synthesis to be measured in a constant feeding study (Figure 8) (29). This is 271

a laborious method since chylomicron particles must be separated from VLDL particles. This cannot 272

be achieved by ultracentrifugation. In the above study an immunoaffinity technique using 3 273

antibodies to apoB100 was used to isolate VLDL from chylomicrons (29). 274

Hepatic fatty acid synthesis 275

Page 8 of 24

9

The precursor-product method is also used for measuring hepatic fatty acid synthesis. Since fatty 276

acids are synthesised from multiple units of acetyl CoA an intravenous infusion of 13

C acetate (the 277

precursor) should enable the determination of hepatic fatty acid synthesis (frequently referred to as 278

de novo lipogenesis; DNL) if the TTR of the precursor (acetate) is known and the TTR of the product, 279

eg palmitate the major product of hepatic synthesis, is measured in VLDL TAG (which is produced by 280

the liver). However, because there are different pools of acetyl CoA in the liver used for different 281

metabolic pathways, the plasma acetate TTR cannot be assumed to be the true precursor. One way 282

to calculate the enrichment of the true precursor uses mass isotopomer distribution anlaysis (MIDA). 283

This method determines the precursor enrichment from the pattern of labelling in the product (30). 284

A constant iv infusion of the acetate tracer is administered for 6-8 hours with measurement of the 285

enrichment of palmitate in VLDL TAG at the end of the infusion period. The frequency of double 286

labelled relative to single labelled palmitate is measured and the TTR of the precursor is calculated 287

using an algorithm. This method has been used in both fasting and feeding studies. In feeding 288

studies repeated mixed meal feeding is used to achieve a postprandial steady state in VLDL TAG (31). 289

With an infusion period of 8 hours the fractional synthesis of palmitate is determined as 290

FSR (%/hour) =(TTRpalmitate (t8)- TTRpalmitate (t0))x 100/ TTRPrecursor x (t8-t0) 291

Where TTRpalmitate is the enrichment of palmitate in VLDL TAG at time 0 (before the tracer infusion) 292

and 8 hours and TTRPrecursor is the enrichment of the precursor pool (acetate) at 8 hours. 293

An alternative method for measuring hepatic fatty acid synthesis uses labelled water. During 294

palmitate synthesis, H from NADPH and H2O is added and this can be utilised to measure palmitate 295

synthesis rate (32). When 2H2O is given orally it rapidly equilibrates with total body water and

2H is 296

incorporated into palmitate as it is synthesised. A plateau of deuterium enrichment is reached within 297

12 hours. In the most widely used method an oral dose (3g/kg body weight) of 2H2O which aims to 298

produce 0.45% enrichment of body water is given in the evening, half with the evening meal and half 299

at 10pm. Subjects drink only water (0.45% enriched), to prevent dilution of the labelled body water, 300

with ingested water, until after a blood sample is taken the following morning, for the measurement 301

of VLDL TAG-palmitate enrichment and plasma water enrichment. There are 31 hydrogens in 302

palmitate which could potentially be labelled. However not all hydrogens are equivalent. Seven H 303

are directly from H2O, 14 are from NADPH and 10 are from acetyl CoA. Complete equilibration with 304

the labelled body water pool does not occur with the latter 2 pools. It has been shown, using MIDA 305

that the maximum number of labelled H that can be incorporated into palmitate is 21 (33). 306

If all VLDL TAG-palmitate are derived from DNL, the enrichment in TAG-palmitate will be equal to the 307

enrichment of the plasma water multiplied by the maximum number of labelled H that can be 308

incorporated. 309

Maximum theoretical palmitate TTR= TTR2H2O x 21 310

The maximum theoretical proportion of VLDL TAG-palmitate which can be derived from DNL is 311

100%. The actual proportion is calculated from the ratio of the observed VLDL TAG-palmitate 312

enrichment (TTR) to the maximum theoretical palmitate enrichment (the theoretical enrichment if 313

all VLDL TAG-palmitate is derived from DNL). 314

%DNL= VLDL TAG-palmitate TTR/ theoretical maximum palmitate TTR x100 315

Page 9 of 24

10

The absolute rate of VLDL TAG synthesis from DNL can be calculated by multiplying %DNL by total 316

VLDL TAG synthesis rate (eg measured using a glycerol or pamitate tracer as described in the section 317

on VLDL TAG kinetics). 318

319

De novo cholesterol synthesis and absorption from the diet 320

A high cholesterol level is a strong independent risk factor for cardiovascular disease. Understanding 321

the regulation of cholesterol metabolism is therefore of major importance. Plasma cholesterol is 322

derived from endogenous synthesis and exogenous sources (diet and bile acids). Stable isotope 323

tracers can be used to measure cholesterol absorption from the diet and from de novo synthesis. 324

Cholesterol absorption 325

The dual isotope method is used for measuring oral absorption of cholesterol (34). It is based on a 326

method previously developed by Zilversmit using radioisotopes (35). An oral dose of 2H5 cholesterol 327

(or 2H6 or

2H7 cholesterol ) in a meal and an iv injection of

13C5 cholesterol (or

13C6 or

13C7 cholesterol) 328

is administered on day 0, and on day 3 the plasma enrichment of the two tracers is measured. At this 329

point it has been shown that the ratio of enrichment of the oral/iv tracer in the plasma is stable (34). 330

A limitation of the method is that the labelled cholesterol absorption is not necessarily the same as 331

cholesterol absorption from a food. Percentage cholesterol absorption is calculated from the ratio 332

of plasma 2H5 cholesterol TTR /

13C5 cholesterol TTR x 100 on day 3. This method has been shown to 333

have good precision and repeatability (34) An alternative method has used 2H5 cholesterol as the 334

tracer and 2H4 sitostanol as a non-absorbable marker given orally for 7 days. Cholesterol absorption 335

was calculated from faecal samples collected over days 4-7 and measured by GCMS (36). 336

Cholesterol synthesis 337

The precursor-product method is used for measuring free cholesterol synthesis. There are 2 principal 338

methods which are very similar to methods used for measuring fatty acid synthesis as described 339

above. An intravenous infusion of 13

C acetate combined with the MIDA method to determine the 340

true precursor pool has been used to measure the FSR of cholesterol (33). To measure the absolute 341

synthesis rate blood samples can be collected over the next few days to measure the decay of the 342

labelled cholesterol with time. The rate constant for this removal rate provides an estimate of the 343

cholesterol pool size. The absolute synthesis rate of cholesterol can then be calculated as 344

FSR (%/day) x cholesterol pool size. 345

The pool size is assumed to be free cholesterol in liver, plasma and red cells, as these free 346

cholesterol pools are in rapid equilibrium in humans. What is measured by this method is the 347

synthesis of cholesterol (which is predominantly in the intestine and liver) which is secreted into the 348

plasma cholesterol pool. Total body cholesterol synthesis is not measured by this method, because 349

tissue cholesterol synthesis that does not leave the cell or equilibrate with the plasma pool is not 350

measured. While some researchers have used an 8h iv infusion of 13

C acetate, a 24h infusion is 351

recommended. 352

Page 10 of 24

11

An alternative method to measure the fractional synthesis rate (FSR) of cholesterol uses the oral 353

administration of 2H2O and measurement of the incorporation of the deuterium tracer into plasma 354

cholesterol or erythrocyte cholesterol (38). The deuterated water equilibrates across the body water 355

pool and intracellular NADPH pools which are precursors for 22 of the 46 hydrogens in cholesterol. 356

Measurements are usually made over 24h to account for the diurnal rhythm in cholesterol synthesis. 357

Cholesterol enrichment can be measured by GCMS and plasma water by IRMS or by GC-MS, via 358

acetone exchange (39). FSR is calculated as 359

FSR (%/d) = cholesterol TTR (t24-t0)/ (plasma water TTR x 0.478) 360

The factor 0.478 is the fraction of hydrogen atoms/ cholesterol molecule that can become enriched 361

with deuterium during cholesterol synthesis. This can be converted to an absolute synthesis rate if 362

assumptions are made about the size of the free cholesterol pool. 363

The FSR measured by an iv infusion of 13

C acetate and the MIDA method agrees well with FSR 364

measured with oral 2H2O, showing a significant correlation (r = 0.84, p = 0.0007) in 12 subjects (40). 365

Conclusion 366

Measuring lipid concentrations in blood samples provides only a static measurement of lipid 367

metabolism. This is provides inadequate information for understanding lipid metabolism in health 368

and disease in humans. Using stable isotope tracers the fluxes of molecules through lipid metabolic 369

pathways can be measured in vivo. There are many different methods which can be used and the 370

choice of method and tracer depends on the lipid metabolite to be studied. The techniques and 371

tracers can be used individually or several techniques and tracers can be combined in a single study 372

enabling multiple metabolic pathways to be measured simultaneously. 373

374

375

Declaration of interest: 376

The author declares that there is no conflict of interest that could be perceived as prejudicing the 377

impartiality of the research reported. 378

379

Funding: 380

This research did not receive any specific grant from any funding agency in the public, commercial or 381

not-for-profit sector 382

Page 11 of 24

12

References 383

1. Hubert HB, Feinleib M, McNamara PM and Castelli WP 1983 Obesity as an independent risk factor 384

for cardiovascular disease: a 26- year follow-up of participants in the Framingham Heart Study. 385

Circulation 67:968-977 386

2. de Marco R, Locatelli F, Zoppini G, Verlato G, Bonora E, Muggeo M. Cause-specific mortality in 387

type 2 diabetes. The Verona Diabetes Study. Diabetes Care 1999; 22(5):756-61. 388

3. Boden G. Free fatty acids (FFA), a link between obesity and insulin resistance. Front Biosci. 389

1998;3:169-75 390

4. Morelli M, Gaggini M, Daniele G, Marraccini P, Sicari R, Gastaldelli A. Ectopic fat: the true culprit 391

linking obesity and cardiovascular disease? Thromb Haemost. 2013;110(4):651-60. 392

5. Duffield RG, Lewis B, Miller NE, Jamieson CW, Brunt JN, Colchester AC. Treatment of 393

hyperlipidaemia retards progression of symptomatic femoral atherosclerosis. A randomised 394

controlled trial. Lancet. 1983;2(8351):639-42. 395

6. Schoenheimer R. The Investigation of Intermediary Metabolism with the Aid of Heavy Hydrogen. 396

Bull N Y Acad Med. 1937;13:272-95 397

7. Schierbeek H, van den Akker CH, Fay LB, van Goudoever JB. High-precision mass spectrometric 398

analysis using stable isotopes in studies of children. Mass Spectrom Rev. 2012 Mar-Apr;31(2):312-30 399

8. Wolfe RR, Shaw JHF, Durkot MJ: Effects of sepsis on VLDL kinetics: responses in basal state and 400

during glucose infusion. Am J Physiol248 :E732 –E740,1985 401

9. Evans K, Clark ML, Frayn KN. Effects of an oral and intravenous fat load on adipose tissue and 402

forearm lipid metabolism. Am J Physiol. 1999;276(2 Pt 1):E241-8. 403

10. Mittendorfer B, Liem O, Patterson BW, Miles JM, Klein S. What does the measurement of whole-404

body fatty acid rate of appearance in plasma by using a fatty acid tracer really mean? Diabetes. 405

2003;52(7):1641-8. 406

11. Miles JM, Park YS, Walewicz D, Russell-Lopez C, Windsor S, Isley WL, Coppack SW, Harris WS. 407

Systemic and forearm triglyceride metabolism: fate of lipoprotein lipase-generated glycerol and free 408

fatty acids. Diabetes. 2004;53(3):521-7. 409

12. Robinson J, Newsholme EA. Glycerol kinase activities in rat heart and adipose tissue. Biochem J 410

1967; 104: 2c-4c 411

13. Wolfe Robert R. Radioactive and stable isotope tracers in biomedicine. Principles and Practice of 412

Kinetic Analysis. Wiley Liss Inc. 1992 413

14. Steele R, Bishop JS, Dunn A, Altszuler N, Rathbeb I, Debodo RC (1965) Inhibition by insulin of 414

hepatic glucose production in the normal dog. Am.J.Physiol 208: 301-306 415

Page 12 of 24

13

15. Shojaee-Moradie F, Baynes KCR, Pentecost C, Bell JD, Thomas EL, Jackson NC, Stolinski M, Whyte 416

M, Lovell D, Bowes SB, Gibney J, Jones RH, Umpleby AM Exercise training reduces fatty acid 417

availability and improves insulin sensitivity of glucose metabolism. Diabetologia 2007;50:404-413. 418

16. Bickerton, A. S., R. Roberts, B. A. Fielding, L. Hodson, E. E. Blaak, A. J. Wagenmakers, M. Gilbert, 419

F. Karpe and K. N. Frayn Preferential uptake of dietary Fatty acids in adipose tissue and muscle in the 420

postprandial period. Diabetes 2007; 56(1): 168-176. 421

17. Schrauwen P, van Aggel-Leijssen DP, van Marken Lichtenbelt WD, van Baak MA, Gijsen AP, 422

Wagenmakers AJ. Validation of the [1,2-13C]acetate recovery factor for correction of [U-423

13C]palmitate oxidation rates in humans. J Physiol. 1998;513:215-23. 424

18. Millar JS, Lichtenstein AH, Cuchel M, Dolnikowski GG, Hachey DL, Cohn JS, Schaefer EJ. Impact of 425

age on the metabolism of VLDL, IDL, and LDL apolipoprotein B-100 in men. J Lipid Res. 1995 426

Jun;36(6):1155-67. 427

19. Demant T, Packard CJ, Demmelmair H, Stewart P, Bedynek A, Bedford D, Seidel D, Shepherd J. 428

Sensitive methods to study human apolipoprotein B metabolism using stable isotope-labeled amino 429

acids. Am J Physiol. 1996 Jun;270(6 Pt 1):E1022-36. 430

20. Cummings MH, Watts GF, Umpleby M, Hennessy TR, Quiney JR, Sönksen PH. Increased hepatic 431

secretion of very-low-density-lipoprotein apolipoprotein B-100 in heterozygous familial 432

hypercholesterolaemia: a stable isotope study. Atherosclerosis. 1995 Feb;113(1):79-89 433

21. Parhofer KG, Hugh P, Barrett R, Bier DM, Schonfeld G. Determination of kinetic parameters of 434

apolipoprotein B metabolism using amino acids labeled with stable isotopes. J Lipid Res. 1991 435

Aug;32(8):1311-23. 436

22. Duvillard L, Pont F, Florentin C, Galland-Jos P, Gambert P, Verges B. 2000 Metabolic 437

abnormalities of apolipoprotein B containing lipoproteins in non-insulin dependent diabetes: a 438

stable isotope kinetic study. Eur J Clin Invest 30:685-694. 439

23. Barazzoni R, Meek SE, Ekberg K, Wahren J, Nair KS. Arterial KIC as marker of liver and muscle 440

intracellular leucine pools in healthy and type 1 diabetic humans. Am J Physiol. 1999 Aug;277(2 Pt 441

1):E238-44. 442

24. Adiels M, Packard C, Caslake MJ, Stewart P, Soro A, Westerbacka J, Wennberg B, Olofsson SO, 443

Taskinen MR, Boren J 2005 A new combined multicompartmental model for apolipoprotein B-100 444

and triglyceride metabolism in VLDL subfractions. J Lipid Res 46:58-67. 445

25. Lichtenstein AH, Hachey DL, Millar JS, Jenner JL, Booth L, Ordovas J, Schaefer EJ. Measurement 446

of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-447

2H3]leucine. J Lipid Res. 1992 Jun;33(6):907-14. 448

26. Cohn JS, Wagner DA, Cohn SD, Millar JS, Schaefer EJ. Measurement of very low density and low 449

density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in 450

human subjects using deuterated leucine. Effect of fasting and feeding. J Clin Invest. 1990;85(3):804-451

11. 452

Page 13 of 24

14

27. Sarac I, Backhouse K, Shojaee-Moradie F, Stolinski M, Robertson MD, Bell JD, Thomas EL, 453

Hovorka R, Wright J, Umpleby AM. Gender differences in VLDL1 and VLDL2 triglyceride kinetics and 454

fatty acid kinetics in obese postmenopausal women and obese men. J Clin Endocrinol Metab. 455

2012;97(7):2475-81. 456

28. Patterson BW, Mittendorfer B, Elias N, Satyanarayana R, Klein S. Use of stable isotopically labeled 457

tracers to measure very low density lipoprotein-triglyceride turnover. J Lipid Res. 2002 458

Feb;43(2):223-33. 459

29. Shojaee-Moradie F, Ma Y, Lou S, Hovorka R, Umpleby AM. Prandial hypertriglyceridemia in 460

metabolic syndrome is due to an overproduction of both chylomicron and VLDL triacylglycerol 461

Diabetes. 2013;62(12):4063-9 462

30. Hellerstein MK, Neese RA. Mass isotopomer distribution analysis at eight years: theoretical, 463

analytic, and experimental considerations. Am J Physiol. 1999;276(6 Pt 1):E1146-70. 464

31. Stanhope KL, Schwarz JM, Keim NL, Griffen SC, Bremer AA, Graham JL, Hatcher B, Cox CL, 465

Dyachenko A, Zhang W, McGahan JP, Seibert A, Krauss RM, Chiu S, Schaefer EJ, Ai M, Otokozawa S, 466

Nakajima K, Nakano T, Beysen C, Hellerstein MK, Berglund L, Havel PJ. Consuming fructose-467

sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases 468

insulin sensitivity in overweight/obese humans. J Clin Invest. 2009 May;119(5):1322-34 469

32. Leitch CA, Jones PJ. Measurement of human lipogenesis using deuterium incorporation. J Lipid 470

Res. 1993;34(1):157-63. 471

33. Diraison F, Pachiaudi C, Beylot M. In vivo measurement of plasma cholesterol and fatty acid 472

synthesis with deuterated water: determination of the average number of deuterium atoms 473

incorporated. Metabolism. 1996 Jul;45(7):817-21. 474

34. Bosner MS, Ostlund RE, Osofisan O, et al. Assessment of percent cholesterol absorption in 475

humans with stable isotopes. J Lipid Res 1993;34:1047–53. 476

35. Zilversmit DB, Hughes LB. Validation of a dual-isotope plasma ratio method for measurement of 477

cholesterol absorption in rats. J Lipid Res 1974;15:465–73. 478

36. Lutjohann D, Meese CO, Crouse JR, et al. Evaluation of deuterated cholesterol and deuterated 479

sitostanol for measurement of cholesterol absorption in humans. J Lipid Res 1993;34:1039–46. 480

37. Neese RA, Faix D, Kletke C, Wu K, Wang AC, Shackleton CH, Hellerstein MK. Measurement of 481

endogenous synthesis of plasma cholesterol in rats and humans using MIDA. Am J Physiol. 482

1993;264:E136-47. 483

38. Jones, P. J. H., A. M. Scanu, and D. A. Schoeller. Plasma cholesterol synthesis using deuterated 484

water in humans: effect of short-term food restrictions. J Lab Clin Med 1988; 111: 627-633. 485

39. Shah V, Herath K, Previs SF, Hubbard BK, Roddy TP. Headspace analyses of acetone: a rapid 486

method for measuring the 2H-labeling of water. Anal Biochem. 2010 Sep 15;404(2):235-7 487

Page 14 of 24

15

40. Di Buono M, Jones PJ, Beaumier L, Wykes LJ. Comparison of deuterium incorporation and mass 488

isotopomer distribution analysis for measurement of human cholesterol biosynthesis. J Lipid Res 489

2000;41:1516–23. 490

491

492

Page 15 of 24

16

Figures 493

Figure 1: Sites of utilisation of palmitate, glycerol, water and acetate tracers used for the 494

measurement of lipid metabolism. 495

Figure 2: Schematic example of a constant infusion of tracer to achieve an isotopic steady state. 496

Figure 3: Calculating rate of appearance (Ra) at isotopic steady state. Rd, rate of disappearance; 497

black circles represent the tracer; white circles represent the tracee (adapted from Wolfe R. 498

Radioactive and stable isotope tracers in biomedicine. Principles and Practice of Kinetic Analysis. 499

Wiley Liss Inc. 1992. Page 120) 500

Figure 4: Glycerol production rate in a healthy subject measured with a constant infusion of [2H5] 501

glycerol during fasting and following an infusion of insulin. The decrease in glycerol production rate 502

is a measure of the insulin sensitivity of lipolysis. 503

Figure 5: Plasma NEFA oxidation calculated using the acetate correction factor when infusing [U-504 13

C]palmitate. The correction factor, is the fractional

13C label recovery in breath CO2, observed after

505

an infusion of [1,2-13

C]acetate. (from Figure 4, Schrauwen P, van Aggel-Leijssen DP, van Marken 506

Lichtenbelt WD, van Baak MA, Gijsen AP, Wagenmakers AJ. Validation of the [1,2-13C]acetate 507

recovery factor for correction of [U-13C]palmitate oxidation rates in humans. J Physiol. 508

1998;513:215-23) 509

Figure 6: Typical curve fit for leucine tracer:tracee ratio in VLDL, IDL and LDL apoB100 using 510

mathematical modeling following an infusion of [1-13

C] leucine. VLDL shown as large squares. IDL 511

shown as triangles. LDL shown as circles. The solid lines indicate the curve fit. The modelling 512

provides a measure of VLDL, IDL and LDL apoB100 fractional catabolic rate and production rate. 513

Figure 7: Typical curve fit of VLDL1 and VLDL2 glycerol tracer:tracee ratio in TAG, following an iv bolus 514

of [2H5] glycerol, using mathematical modeling. VLDL1 shown as large squares. VLDL2 shown as small 515

circles. The solid lines indicate the curve fit. The modelling provides a measure of VLDL1 and VLDL2 516

TAG fractional catabolic rate and production rate. (from Sarac I, Backhouse K, Shojaee-Moradie F, 517

Stolinski M, Robertson MD, Bell JD, Thomas EL, Hovorka R, Wright J, Umpleby AM. Gender 518

differences in VLDL1 and VLDL2 triglyceride kinetics and fatty acid kinetics in obese postmenopausal 519

women and obese men. J Clin Endocrinol Metab. 2012;97(7):2475-81. ) 520

Figure 8: Glycerol tracer:tracee ratio in chylomicron TAG in healthy subjects (○) and subjects with 521

MetS (●) following an iv bolus of [2H5]glycerol. (mean±SEM) Mathematical modelling of this data 522

provides a measure of chylomicron TAG fractional catabolic rate and production rate. (Figure 3 from 523

Shojaee-Moradie F, Ma Y, Lou S, Hovorka R, Umpleby AM. Prandial hypertriglyceridemia in 524

metabolic syndrome is due to an overproduction of both chylomicron and VLDL triacylglycerol 525

Diabetes. 2013;62(12):4063-9) 526

527

528

Page 16 of 24

13C acetate

U-13C palmitate

2H2O

2H5glycerol

VLDL1TAGFFAs

AcetylCoA

VLDL2

SystemicFFAs

Page 17 of 24

6

5

4

3

Plas

ma

enric

hmen

t (AP

E)

2

1

00 50 100

Infusion time (min)150 200 250

Page 18 of 24

Rd Tracer

infusionrate

Ra

Ra (mg/min) =Tracer infusion rate (mg/min)/TTR

Page 19 of 24

3.0

2.5

2.0

1.5

Gly

cero

l pro

duct

ion

rate

mg/

min

/kg

1.0

0.5

00 80 100

Time (min)120 140 240160 180 200 220

Constant infusion of [2H5] glycerolInsulin infusion 0.3 ml/min/kg

Page 20 of 24

5.0

4.0

3.0

2.0

Plas

ma

FFA

oxid

atio

n (µ

mol

kg–1

min

–1)

1.0

0.5

0.00 30 60

Time (min)90 120

4.5Acetate correctionNo correction

3.5

2.5

1.5

Page 21 of 24

0.004

0.003

0.002

Trac

er: t

race

e ra

tio

0.001

00 50 100

Time (min)150 200 450250 300 350 400

Page 22 of 24

0.10

0.07

0.04

Trac

er: t

race

e ra

tio

0.02

0.000 100

Time (min)200 600

0.08

0.09

0.05

0.06

0.01

0.03

300 400 500

Page 23 of 24

0.0030

0.0020

Trac

er: t

race

e ra

tio

0.0005

0.00000 100

Time (min)200 500

0.0025

0.0015

0.0010

300 400

Page 24 of 24