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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:[email protected] 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
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12
References 383
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491
492
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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