Methods in cell biology 80, mitochondria, 2nd ed l pon, e schon (AP, 2007)

The protocol used to measure ATP synthesis is similar to that describedabove for permeabilized cells, with the omission of the digitonization step, andusing 30 mg of liver or 100 mg of b

12 Rinse the slides with dH2O several times.

13 Counterstain the slides briefly with hematoxylin

14 Rinse the slides with dH2O, dehydrate through ascending ethanol series,and clear in xylene

15 Mount the slides with synthetic resin (Permount)

IV In Situ Hybridization to mtDNA

ISH is a technique that permits the precise cellular localization and tion of cells that express a particular nucleic acid sequence The essence of thismethod is the hybridization of a nucleic acid probe with a specific nucleic acidsequence found in a tissue section ISH has been used extensively in humanpathologic conditions to correlate mitochondrial abnormalities with the presence

identifica-of mutated mtDNAs, an analysis that provides strong support for a pathogenicrole of a specific mitochondrial genotype As the method relies on sequencehomology, it has been applied mainly to diVerentiate mtDNAs with large-scaledeletions from wild-type sequences in samples from patients with KSS or isolated

OM In these studies, two probes have been used to determine the distribution

of wild-type and D-mtDNAs: one probe, in the undeleted region, hybridizes to

both wild-type and D-mtDNAs (the ‘‘common’’ probe); the other, correspond

to mtDNA sequences located within the deletion, hybridizes only to wild-typemtDNAs (the ‘‘wild-type’’ probe) Typical results on serial muscle sections showedabundant hybridization signal (focal accumulations of mtDNAs) with the com-mon probe, but not with the wild-type probe, in COX-deficient RRF of thepatients (Bonilla et al., 1992; Moraes et al., 1992) These observations indicatedthat the predominant species of mtDNAs in COX-deficient RRF of KSS and OMpatients areD-mtDNAs Furthermore, the concentration of D-mtDNAs in these

RRF reached the required threshold level to impair the translation of the chondrial genome because they were characterized immunohistochemically by alack or a marked reduction of the mtDNA-encoded COX II polypeptide (Bonilla

mito-et al., 1992; Moraes mito-et al., 1992)

An important extension of ISH to mtDNA is regional ISH, which has beenapplied to determine the spatial distribution of multiple D-mtDNAs in samples

from patients with Mendelian-inherited OM These are disorders characterized

by progressive external ophthalmoplegia (PEO) and mitochondrial myopathy

In Mendelian OM, hundreds of diVerent deletions coexist within the same muscle

in aVected family members Thus, as opposed to the single deletions found in

sporadic KSS and OM, Mendelian OM is associated with multiple D-mtDNAs

that are apparently generated over the life span of the individual The geneticdefects in these disorders cause mutations in nuclear gene products that regulatethe mitochondrial nucleotide pool (DiMauro and Bonilla, 2004) ISH of serialmuscle sections from these patients, in which a diVerent mtDNA regional probe

was used on each section, showed specific, and diVerent, RRFs losing hybridization

signal with each specific probe, while the remaining RRFs hybridized intensely.These observations have provided the strongest evidence to date that each RRF inMendelian-inherited OM contains a clonal expansion of a single species of

D-mtDNA (Moslemi et al., 1996; Vu et al., 2000).

Although ISH utilizing RNA probes is more widely used in typical cell andmolecular biology applications, most of our recent experience derives from theuse of digoxigenin (DIG)-labeled DNA probes to visualize mtDNA (Manfredi

et al., 1997; Vu et al., 2000) We describe here a method that we employ for theidentification of RRF (Fig 4) and for the detection of depletion of mtDNA onmuscle-frozen sections Although tissue samples can be frozen or fixed andparaYn-embedded, we described a procedure optimized for frozen sections that

has been tested thoroughly on muscle sections from patients with mitochondrialmyopathies Because of the focal pattern of distribution of mutant and wild-typemtDNAs in muscle fibers, one must make sure that serial sections above andbelow the ones used for ISH are characterized histochemically by staining forCOX and SDH activities (Fig 4)

The size of the labeled probe is very important and should be optimized to promotespecificity and tissue penetration Sizes between 300 and 400 nucleotides are opti-mum, even though we have obtained excellent results with 500-nucleotide probes.DNA probes can be prepared in diVerent ways, but we routinely use PCR-generatedDIG-labeled mtDNA probes Kits and polymerases for DNA labeling are avail-able from most molecular biology companies It is important that prior toperforming ISH experiments, the concentration of the probe be determined bydot blot and that the specificity of the probe be tested by Southern blot

1 Method

1 Collect 8-mm-thick frozen sections on poly(L-lysine)-coated (0.1%) slides

2 Fix the sections with 4% paraformaldehyde for 30 min at RT

3 Rinse the slides with PBS containing 5-mM MgCl2(PBS–MgCl2, pH 7.4)three times for 5 min at RT

4 Incubate the sections with 5-mg/ml proteinase K for 1 h at 37
C

5 Place the slides in PBS–MgCl2at 4
C for 5 min to remove the proteinase K

6 Acetylate the sections by incubating in 0.1-M triethanolamine containing0.25% acetic anhydride for 10 min at RT

7 Treat the slides with 5-mg/ml RNase (DNase-free) in RNase buVer (50-mM

NaCl and 10-mM Tris–HCl, pH 8) for 30 min at 37
C

8 Rinse the slides briefly with PBS–MgCl2

9 Dehydrate the sections through ascending series of ethanol (optional)

10 Incubate with hybridization buVer without probe (prehybridization) for1–2 h at 37
C (In our experience, this step can be eliminated.) Hybridiza-tion solution: 50% deionized formamide, 20-mM Tris–HCl, 0.5-mM NaCl,10-mM EDTA, 0.02% Ficoll, 0.02% polyvinylpyrollidone, 0.12% BSA,

Fig 4 Cellular localization of mtDNA by ISH Serial muscle sections from a patient with MERRF were stained for SDH (A) and COX (B) activity, and for mtDNA localized by ISH using digoxigenin- labeled probes (C) The ISH signal is seen as the red material A control section subjected to ISH without the denaturing step (D) shows no hybridization signal Note the strong mtDNA signal in

an RRF (asterisk) characterized by increased SDH activity and lack of COX activity Bar ¼ 50 m.

0.05% salmon sperm DNA, 0.05% total yeast RNA, 0.01% yeast tRNA,and 10% dextran sulfate.

11 Denature the probe/hybridization solution (20 ng/ml) at 92
C for 10 minand immediately place the solution on ice until it is applied to the sections

12 Blot oV excess hybridization solution and apply probe/hybridization solution

to the sections

13 Denature the sections covered with the hybridization solution at 92
C for10–15 min

14 Hybridize overnight at 42
C

15 Rinse the slides briefly with 2 SSC (3-M NaCl, 0.3-M sodium citrate,

pH 7.0) at RT followed by rinsing twice with 1 SSC for 15 min at 45
C,

and once with 0.2 SSC for 30 min at 45
C.

16 Rinse the slides with PBS for 5 min at RT

17 Incubate the slides with 1% BSA for 1 h at RT

18 Incubate the slides with alkaline phosphatase-conjugated anti-DIG antibody(1:1000–1:5000) for 1 h at RT

19 Rinse the slides with PBS three times for 5 min each, at RT

20 Incubate the slides with color-substrate solution (SIGMA FASTTM FastRed TR/Naphthol AS-MX Alkaline phosphatase substrate tablets) untilthe desired strength of the signal is obtained

21 Mount the sections with glycerol-PBS, 1:1

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I Introduction

II ATP Synthesis AssaysIII Methodological Considerations

A Cell Permeabilization (Detergent Titration in Cultured Cells)

B ATP Detection by Luciferase–Luciferin

C Specificity of the Assay

IV Experimental Procedures

A Measurement of ATP Synthesis in Cultured Cells

B ATP Synthesis in Mitochondria Isolated from Animal Tissues

V Measurement of High-Energy Phosphates in AnimalTissue and Cultured Cells by HPLC

A Apparatus and Reagents

B Preparation of Biological Samples

Copyright 2007, Elsevier Inc All rights reserved. 155 DOI: 10.1016/S0091-679X(06)80007-5

glycolysis in the cytoplasm Therefore, the measurement of mitochondrial ATPsynthesis can be considered a pivotal tool in understanding many importantcharacteristics of cellular energy metabolism, both in the normal physiologicalstate and in pathological conditions such as mitochondrial disorders In the firstsection of this chapter, we will discuss approaches to measure ATP synthesis frommammalian cells and tissues In the second section, we will discuss procedures toestimate the steady-state content of ATP and other high-energy phosphates byhigh-performance liquid chromatography (HPLC).

II ATP Synthesis Assays

Two methodological issues need to be addressed when measuring drial ATP synthesis The first one is how to deliver reaction substrates to themitochondria Because of the low permeability of the plasma membrane to somehydrophilic substrates, such as adenosine diphosphate (ADP), some investigatorsprefer to analyze isolated mitochondria (Tatuch and Robinson, 1993; Tuena deGomez-Puyou et al., 1984; Vazquez-Memije et al., 1996) However, for ADPphosphorylation to take place, mitochondria need to be maintained intact and

mitochon-in a coupled state Therefore, ATP synthesis can only be measured on freshlyisolated mitochondria Moreover, the isolation of highly coupled mitochondriafrom cultured cells involves delicate and time-consuming procedures, and theresults are sometimes inconsistent, leading to a potential lack of reproducibility.Measurement of ATP synthesis on whole cells requires a permeabilization step toallow for the penetration of hydrophilic substrates through biological membranes.Cell membranes can be permeabilized with detergents such as saponin (Kunz

et al., 1993) or digitonin (Houstek et al., 1995; Wanders et al., 1993, 1994, 1996)

In addition, the use of permeabilized cells rather than isolated mitochondriareduces substantially the number of cells required for each assay

The second issue is how to detect and quantify ATP Fluorimetry is a commonlyused method to detect ATP produced by isolated mitochondria (Houstek et al.,1995; Tatuch and Robinson, 1993; Wanders et al., 1993, 1994, 1996) Anothermethod employed on isolated mitochondria is the incorporation of [32]Pi intoADP and its subsequent transfer to glucose-6-phosphate by hexokinase, followed

by extraction of unincorporated [32]Pi and measurement of radioactivity in ascintillation counter (Tuena de Gomez-Puyou et al., 1984)

Typically, for steady-state ATP measurements based on fluorescence or assays, isolated mitochondria or permeabilized cells are incubated with appropri-ate substrates followed by a lysis extraction in strong acid conditions to inactivatecellular ATPases With this approach, in order to obtain kinetic measurements ofATP synthesis, replicate tests have to be run at various time intervals Alterna-tively, it would be convenient to perform measurements of ATP synthesis, which

radio-do not require repeated sampling and which allow for kinetic measurements to beperformed on single samples In this chapter, we describe a rapid kinetic approach

to monitor continuous ATP synthesis in mammalian cells that takes advantage ofthe luciferase–luciferin system.

Firefly luciferase is widely used as a reporter for gene expression to studypromoter regulation in mammalian cells, but its bioluminescence properties havealso been used to measure ATP content in isolated mitochondria (Lemasters andHackenbrock, 1973; Strehler and Totter, 1952; Wibom et al., 1990, 1991) and inpermeabilized cells (James et al., 1999; Maechler et al., 1998; Ouhabi et al., 1998).The reaction catalyzed by luciferase is:

Luciferaseþ Luciferin þ ATP ! Luciferase - Luciferyl - AMP þ PPiLuciferase - Luciferyl - AMPþ O2! Luciferase þ Oxyluciferin þ AMP þ CO2þ hvThe reaction produces a flash of yellow-green light, with a peak emission at

560 nm, whose intensity is proportional to the amount of substrates in the reactionmixture (Deluca, 1976)

III Methodological Considerations

A Cell Permeabilization (Detergent Titration in Cultured Cells)

Although more convenient than isolating coupled mitochondria, tion procedures also require standardization InsuYcient permeabilization could

permeabiliza-result in an underestimation of ATP synthesis due to lack of available substrates.Conversely, excessive permeabilization leads to mitochondrial membrane damageand uncoupling For these reasons, it is important to establish the optimal amount

of detergent needed per unit of cell protein We use digitonin as the detergent ofchoice because it results in plasma membrane permeabilization at concentrationsthat do not aVect mitochondrial membranes significantly Alternatively, saponinmay be used as a detergent (Kunz et al., 1993) particularly in muscle cells Due tovariation in membrane cholesterol content, the parameters for digitonin treatmentneed to be optimized for each cell type In our experience, the digitonin concen-tration at which HeLa and COS-7 cells (1-mg/ml cell proteins) become permeable

to substrates and achieve the maximal ATP synthesis rate is 50mg/ml, while N2Amouse neuroblastoma cells require 25 mg/ml and HEK 293T (HEK, humanembryonic kidney cells) cells require 75mg/ml (Fig 1)

B ATP Detection by Luciferase–Luciferin

A number of variables need to be taken into consideration in setting up aluminescence assay First, depending on the ATP concentrations, firefly luciferaseshows two diVerent rates of light production, possibly due to the binding of sub-

strates at two diVerent catalytic sites At high concentrations of ATP, a short flash

of light is produced followed by enzyme inactivation At low concentrations ofATP, the flash is less intense but more prolonged (DeLuca and McElroy, 1974).

We observed that in the range of ATP concentrations normally present in culturedcells, the initial flash and the ensuing inactivation of luciferase could be prevented

by preincubating luciferase with luciferin for 10 min on ice, prior to the assay (see

buVer B in Table I) Second, the kinetics of luciferase is unstable at low

concen-trations of ATP and high concenconcen-trations of luciferin (Lembert and Idahl, 1995),and its activity decreases over time Therefore, the concentration of luciferin in thereaction mixture has to be appropriate for the levels of ATP present in cells.Furthermore, buVer B (containing the luciferase–luciferin mix) has to be protectedfrom ambient light and kept on ice until the beginning of the measurement

In order to test for the stability of the luciferin–luciferase complex in buVer B, measurethe luminescence derived from a fixed ATP standard in between measurements ontest samples

C Specificity of the Assay

ATP is formed not only through mitochondrial oxidative phosphorylation, butalso through other metabolic pathways such as glycolysis and adenylate kinase.Therefore, to obtain a specific measurement of mitochondrial ATP synthesis, theseother sources of ATP must be either accounted for or, preferably, eliminated

Fig 1 Titration of digitonin HeLa, COS-7, HEK 293T, and N2A cells were incubated with ing concentrations of digitonin All measurements were performed after 1 min of incubation with digitonin, followed by a wash in buVer A ATP synthesis was performed using malate/pyruvate as

increas-substrates The maximal ATP synthesis in HeLa and COS-7 cells is obtained with 50- mg/ml digitonin.

In contrast, N2A cells show maximum ATP production after being permeabilized with 25- mg/ml digitonin, while HEK 293T require 75 mg/ml.

Adenylate kinase catalyzes the following reversible reaction:

2ADP!ATPþ AMPWhen ADP is added to the cells, the luminescence response shows an initial rapidphase due to adenylate kinase activity and a second slow phase derived fromoxidative phosphorylation For these reasons, P1,P5-di(adenosine) pentapho-sphate, an inhibitor of adenylate kinase (Kurebayashi et al., 1980), is added to thereaction mixture to eliminate the initial rapid phase of light emission Moreover,prior to the measurement, the cells are rinsed and maintained in glucose-free buVer

to deplete most of the residual intracellular glucose By assaying luminescence with

Store at
20 C; at room temperaturefor the assay

Digitonin 5 ml (see Fig 1 for

the appropriate concentration)

10 mg/ml in DMSO Store at
20 C; at room temperature

for the assay

Di(adenosine

pentaphosphate)

thaw and dilute

thaw and dilute

thaw and dilute

Oligomycin b 2 ml (2 mg/ml) 0.2 mg/ml in ethanol Store at
20 C; thaw and keep on ice

Tris–acetate, pH 7.75

Store at
20 C; thaw and keep on ice

BuVer B 10 ml 0.5-M Tris–acetate, pH

7.75, 0.8-mM luciferin, 20- mg/ml luciferase

Prepare the same day; keep on ice

and without inhibitors of mitochondrial ATP synthesis, such as atractyloside(an inhibitor of the adenine nucleotide translocator) or oligomycin (an inhibitor

of the ATP synthase), it is possible to determine how much luminescence derivesfrom the ATP that is not produced by oxidative phosphorylation The inhibitor-insensitive ATP synthesis is subtracted from the rate of synthesis of total ATPmeasured

IV Experimental Procedures

This section describes the assay of ATP synthesis in five cell types: HeLa, COS-7,N2A, HEK 293T, and 143B-derived cytoplasmic hybrids (cybrids; King andAttardi, 1989) harboring either wild-type mitochondrial DNA (mtDNA) or theT8993G mtDNA mutation in the ATPase 6 gene, which is responsible for a mito-chondrial disorder characterized by neuropathy, ataxia, and retinitis pigmentosa(NARP; Holt et al., 1990)

A Measurement of ATP Synthesis in Cultured Cells

1 Cell Culture

Cells are grown in 100-mm culture dishes in Dulbecco’s Modified Eagle’s Medium(DMEM) containing high glucose (4.5 mg/ml), 2-mM L-glutamine, 110-mg/litersodium pyruvate, and supplemented with 5% fetal bovine serum (FBS) at 37Cand 5% CO2 Cells are harvested by trypsinization when they reach80% confluence.Cells are then sedimented by centrifugation at 800 g at RT, and the cell pellet iswashed with glucose-free, serum-free DMEM Cells are counted in a hemocytometer

or an automated cell counter, and resuspended in glucose-free, serum-free DMEM

at a concentration of 1.5 106

cells/ml

2 Measurement of ATP Synthesis

Media and reagents used for these assays are shown in Table I For HeLa, COS-7,and 143B-derived cybrids, 1.5 106

cells are used in each assay Cells are trated by centrifugation at 800 g at RT, and the cell pellet is resuspended in 160 ml

concen-of buVer A (Table I) at RT In the case concen-of HEK 293T and N2A cells, 2  106

cells areused in each assay BuVer A-containing cells are incubated with the appropriateamount of digitonin (Fig 1) for 1 min at RT, with gentle agitation Digitonin isremoved by washing cells with 1 ml of buVer A Cells are concentrated by centrifu-

gation at 800 g at RT, and the cell pellet is resuspended in 160 ml of buVer A

and P1,P5-di(adenosine) pentaphosphate (to 0.15 mM) Add 10 ml of buVer B

(containing luciferin and luciferase), ADP (to 0.1 mM), and either malate pluspyruvate (both to 1 mM) or succinate (to 5 mM) plus 2-mg/ml rotenone to the cellsuspension to obtain the baseline luminescence corresponding to nonmitochondrialATP production, one replicate tube for each sample is prepared containing the

components described above plus either 1-mg/ml oligomycin or 1-mM atractyloside.These inhibitors have no eVect on luciferase activity.

Cells are transferred to a luminometer cuvette and, after a gentle mixing with avortexer for 2 sec, they are placed in a counting luminometer and light emission isrecorded We use an Optocomp I luminometer (MGM Instruments, Inc., Hamden,CT), which allows for multiple recordings in the kinetic mode The integration timefor each reading is set at 1 sec and the interval between readings at 15 sec, for a totalrecording time of 3.5 min (15 readings) Other luminometers can be used, but thosethat allow for kinetic protocols are preferable It is also useful to interface theinstrument with a computer to facilitate data collection and analysis Total cellularprotein content is measured on the digitonized cells using the Bio-Rad DC proteinassay kit (Bio-Rad Laboratories, Hercules, CA) Serial dilutions of bovine serumalbumin (BSA) are used as standards

Figure 2 shows a representative assay on HeLa cells The luminescence curvesdescribe the kinetics of ATP synthesis in cells energized with malate plus pyruvate

in the presence and absence of oligomycin Luminescence increases linearly for

3.5 min It reaches a peak at 4–5 min, followed by a progressive decrease (notshown) The decrease in luminescence is probably due to the chemical properties ofluciferin, which is converted progressively into the inactive derivate deoxyluciferin(Lembert and Idahl, 1995) The linear portion of the curve is used to extrapolate thevariation in luminescence per unit of time (change in relative light units,DRLU).

TheDRLU measured in the presence of the inhibitor is subtracted from the total DRLU in order to obtain the proportion of DRLU derived from mitochondrial

ATP synthesis The change in luminescence is then converted to ATP concentration

200 400 600 800

1000 1200 1400 1600

0 1800

50 100 150

Time (sec) Oligomycin

based on an ATP standard curve, since the luminescence is directly proportional tothe ATP concentration in the physiological concentration range (Strehler, 1968).

A standard ATP–luminescence curve is constructed by measuring flash cence derived from ATP solutions containing 0-, 0.05-, 0.1-, 0.5-, 1-, 5-, and 10-mMATP in buVer A plus 10 ml of buVer B, using a single point reading (Fig 3) ATPsynthesis rates with malate plus pyruvate in HeLa, COS-7, N2A, HEK 293T areshown in Fig 4A, and ATP synthesis in 143B-derived cybrid cells in Fig 4B

lumines-B ATP Synthesis in Mitochondria Isolated from Animal Tissues

We use the luciferin–luciferase method to measure ATP synthesis in mitochondriafreshly isolated from mouse liver and brain

1 Isolation of Mitochondria

Reagents

BuVer H

0.22-MD-mannitol0.007-M sucrose20-mM HEPES1-mM EGTA1% BSA

pH 7.2

Fig 3 ATP standard assay Linear regression analysis for the correlation between ATP concentration and luciferin/luciferase-dependent luminescence (RLU) Each point indicates the value corresponding

to the mean luminescence  SD (n ¼ 5); R 2 ¼ 0.9991.

fuged at 10,000 g for 10 min at 4C The mitochondria-rich pellet is resuspended in

a small volume (250 ml) of buVer H and kept on ice The final protein concentration

Fig 4 (A) ATP synthesis rates in HeLa, COS-7, HEK 293T, and N2A cells using malate/pyruvate as substrates Bars indicate the values corresponding to the mean activity  SD (n ¼ 3) (B) ATP synthesis

in wild-type and T8993G mutant cytoplasmic hybrids (cybrids) resulting from fusion of mtDNA-less 143B osteosarcoma cells ( r 0 cells) with platelets from individuals aVected by heteroplasmic T8993G

mtDNA mutations associated with the mitochondrial disease NARP (neuropathy, ataxia, and retinitis pigmentosa) ATP synthesis was measured using either malate/pyruvate as substrates Bars indicate the values corresponding to the mean activity  SD (n ¼ 3).

is 40–60 mg/ml Higher dilutions are not recommended because they tend to causeuncoupling of mitochondrial respiration.

Mitochondria from whole mouse brain are isolated by homogenization in 2 ml

of ice-cold buVer H The homogenate is centrifuged at 1500  g for 5 min at 4C.

The supernatant is kept on ice, while the resulting pellet is resuspended in 1 ml ofbuVer H and subjected to a second centrifugation of 1500  g at 4C The two

supernatants are combined and centrifuged at 13,500 g for 10 min at 4C The

mitochondria-rich pellet is resuspended in 50–100ml of buVer H and kept on ice.

The final protein concentration is 20–40 mg/ml

2 Measurement of ATP Synthesis

Reagents

BuVer R

0.25-M sucrose50-mM HEPES2-mM MgCl2

1-mM EGTA10-mM KH2PO4

pH 7.430-mM glutamate30-mM malate300-mM ADP1-mg/ml oligomycinProtocol

Before starting to measure the ATP synthesis, we test the coupling state ofmitochondria by polarography Approximately 600mg of liver and 400 mg of brainmitochondrial proteins are resuspended in 0.3 ml of buVer R Oxygen consump-tion is measured in a Clark-type electrode oxygraph (Hansatech, UK) using either20-mM succinate or 30-mM glutamate plus 30-mM malate in the absence ofexogenous ADP (state 2 respiration) and after addition of 300-mM ADP (state 3respiration) The addition of 1-mg/ml oligomycin inhibits the mitochondrial ATPsynthase and reduces oxygen consumption to a rate similar to that preceding theaddition of ADP Respiratory control ratios (RCRs, the ratio between state 2 andstate 3 respirations) should be above 3 (with succinate) and 7 (with glutamate/malate) in liver, and 2.5 (with succinate) and 4 (with glutamate/malate) in brain.Lower RCRs suggest mitochondrial uncoupling

The protocol used to measure ATP synthesis is similar to that describedabove for permeabilized cells, with the omission of the digitonization step, andusing 30 mg of liver or 100 mg of brain mitochondrial proteins resuspended in

buVer A (Table I).

V Measurement of High-Energy Phosphates in Animal Tissue and Cultured Cells by HPLC

Among the diVerent methods used to assay high-energy phosphates in biologicalsamples, HPLC has the advantage of high sensitivity and eYciency because it allowsfor the simultaneous analyses of all species of phosphorylated nucleotides in oneanalysis

DiVerent HPLC techniques are commonly used to separate nucleotides (Zakaria

and Brown, 1981) Nucleotide phosphates are charged molecules that can beseparated well by ion-exchange HPLC However, the separation time is relativelylong, and the columns are less stable than are reversed-phase ones Reversed-phaseHPLC separates compounds based on hydrophobic interactions and is less suitablefor charged nucleotide phosphates However, ion-pair reagents, such as tetrabutyl-ammonium hydrogen sulfate and tetrabutylammonium dihydrogen phosphate,when added to the phosphate-buVered mobile phase can complex the chargedmolecules and enable reversed-phase HPLC to be used in nucleotide separation,thereby allowing for shorter separation times and a better reproducibility thanion-exchange HPLC (Meyer et al., 1999) After adding an ion-pair reagent, theelution order of nucleotides is reversed compared with reversed-phase HPLCwithout ion-pair reagent (Uesugi et al., 1997)

Because of the high rate of conversion of ATP to ADP and adenosine phosphate (AMP) in fresh samples by cellular ATPases, protocols for the quickinactivation of phosphatases are essential for ATP measurement Rapid-heating

mono-or flash-freezing procedures have been used to preserve high-energy phosphates inpreparing biological samples The method of focused microwave irradiation,which can rapidly kill small rodents and irreversibly inactivate enzymes, hasbeen used in sample preparations for investigating rapidly modulated neuro-chemicals such as neurotransmitters and high-energy phosphorylated nucleotides(McCandless et al., 1984; Schneider et al., 1982) The microwave method can also

be used for the extraction of cellular ATP (Tsai, 1986) This method is also usefulfor preserving the in vivo protein phosphorylation state of many phosphoproteins(O’Callaghan and Sriram, 2004) Due to the resistance of phosphorylated com-pounds to heat, denaturing tissue culture samples in boiling distilled water canproduce high yield and good reproducibility for assays of phosphorylated nucleo-tides (Ozer et al., 2000) Alternatively, flash-freezing of tissues or small animals inliquid nitrogen is, in our experience, a simple but eVective way to instantly preventthe inactivation of ATPases However, because the inactivation of enzymes isreversible, maintaining subfreezing conditions during dissection and homogeniza-tion of the samples is critical A cold chamber with a cryoplate can be used todissect target tissues from the frozen animal body For enzyme inactivation in cellculture samples, following quick removal of the culture medium, concentratedperchloric acid or alkaline KOH can be added directly to cells A proteinaseK-based extraction technique has been reported to generate consistently higher

adenylate yields from a broad range of cellular samples compared to perchloricacid or the boiling method (Napolitano and Shain, 2005).

We have tested diVerent sample preparation procedures on rat and mousetissues as well as on cultured cells, and have developed an ion-pair reversed-phase HPLC system, using a phosphate-buVered acetonitrile gradient mobilephase, to simultaneously determine the levels of creatine, its high-energy phos-phate, and all of the three adenylates (AMP, ADP, and ATP) With this system,high-energy phosphates separated into well-resolved, tight, reproducible peaks,allowing for a reliable quantification

A Apparatus and Reagents

A Perkin–Elmer (Norwalk, CT) M-250 binary LC pump, a Waters (Milford,MA) 717 plus autosampler, a Waters 490 programmable multiwavelength UVdetector, and an ESA (Chelmsford, MA) 501 chromatography data processsystem are used in the HPLC assay A mechanically refrigerated thermal platform(Sigma, San Diego, CA) is used for frozen tissue dissection All chemicals are fromSigma (St Louis, MO)

B Preparation of Biological Samples

Rats are anesthetized with pentobarbital (64.8 mg/ml, 50 mg/kg) and decapitated(according to institutional protocols), and the heads are immediately snap-frozen inliquid nitrogen Mice are anesthetized with isoflurane delivered by a vaporizer(VetEquip, Inc., CA) and the animal is immediately frozen in liquid nitrogen.Striatum, cortex, and cerebellum are quickly dissected on a cold plate at
20C.

Frozen tissues are transferred to a 1.5-ml microfuge tube and 10 ml of ice-cold0.4-M perchloric acid are added per milligram wet weight The tissue is immediatelyhomogenized with a pellet pestle The acidic homogenate is kept on ice for 30 min andthen centrifuged at 18,000 g in a microfuge at 4C for 10 min An aliquot of the

pellets is set aside for protein measurements The supernatant (100ml) is neutralizedwith 10ml of 4-M K2CO3, kept on ice for 10 min, and then at
80C for 1–2 h, to

promote precipitation of the perchlorate Finally, the mixture is centrifuged, asdescribed above Supernatants are stored at
80C until HPLC assay.

Cultured cells are grown in six-well dishes, as described above Culture medium

is removed by aspiration, followed by immediate addition of ice-cold 0.4-Mperchloric acid (500ml per 1  106

cells) The culture dish is sealed tightly withParafilm and cooled to
80C Cell lysates are thawed on ice, scraped oV thor-

oughly from the wells, and transferred to 1.5-ml microfuge tubes Samples arecentrifuged at 18,000 g at 4C for 10 min and supernatants are neutralized with

K2CO3, as described above

External standards are prepared in 0.4-M perchloric acid, neutralized, and treated

in exactly the same way as the tissue and cells samples Protein measurements areperformed using a Bio-Rad protein analysis protocol (Bio-Rad Laboratories)

C Chromatography

ReagentsBuVer A25-mM NaH2PO4

100-mg/liter tetrabutylammonium, pH 5

BuVer B

10% (v/v) acetonitrile in 200-mM NaH2PO4

100-mg/liter tetrabutylammonium, pH 4.0

BuVers are filtered through a Rainin 0.2-mm, Nylon-66 filter (Woburn, MA)

and degassed in a flask linked to a vacuum pipe

ProcedureThe gradient elution is performed on a 4.6-mm i.d. 250-mm, 3-mm-particle-sizeYMC C18 HPLC column (Waters, Milford, MA) with buVer A and B, at a rate of

1 ml/min The gradient is:

100% buVer A from 0 to 5 min100% buVer A to 100% buVer B from 5 to 30 min100% buVer B to 100% buVer A from 30 to 31 min100% buVer A from 31 to 45 min for column reequilibration, which is

suYcient to achieve stable baseline conditions.

Fifty microliters of prepared sample or standard mixture are autoinjected andmonitored by UV at 210 nm from 0 to 15 min (for detection of creatine andphosphocreatine) and at 260 nm from 15 to 45 min (for phosphorylated nucleo-tides) Peaks are identified by their retention times and by using co-chromatographywith standards

D Standard Curves

Each standard of interest is first subjected individually to chromatography todetermine its retention time and later identify each compound in a standardmixture A standard curve for each compound is constructed by plotting peakheights (mV) versus concentration (10–1000 mM for creatine and phosphorylatedcreatine; 5–500mM for phosphorylated nucleotides) Linear curves are obtained(R2 values are 0.98) from which the detection limits of the HPLC method areestimated to be fivefold above baseline noise The quantification of creatine, phos-phocreatine, and phosphorylated nucleotides in the sample is carried out using theexternal standard calibration (i.e., by co-chromatography of the mixed standardsolution and samples), integrating sample peak heights against correspondingstandard curves

E Measurement of Creatine, Phosphocreatine, and Phosphorylated Nucleotides

Figure 5 shows the separation of creatine, phosphorylated creatine, AMP,ADP, and ATP from a rat brain tissue sample (Fig 5A) and a standard mixture(Fig 5B) One single run takes 45 min; an average of 30 min is suYcient for theseparation of adenosine nucleotides Retention times are: creatine 3.5 min, phos-phocreatine 11 min, AMP 22.5 min, ADP 24.5 min, and ATP 26 min All thecompounds of interest in the tissue samples are resolved clearly and have verysharp peaks In cultured cell samples, there is no detectable creatine and phospho-creatine peak, since the cells do not synthesize creatine (not shown) Figure 6shows an example of a standard curve for ATP Detection limits for the threephosphorylated nucleotides are200 pmol and are 500 pmol for creatine andphosphocreatine Calculations are performed by peak height calibrations andvalues are expressed asmmol/g of wet tissue or nmol/mg of protein The followingvalues are obtained from rat brain (mmol/g wet wt.): creatine 9.07  0.52, phos-phocreatine 2.2 0.23, AMP 0.91  0.08, ADP 0.71  0.03, and ATP 1.23  0.18;

Time (min) 5

100 mM (5).

from mouse brain (nmol/mg protein): creatine 68.0 4.9, phosphocreatine 18.2 1.5, AMP 14.4  2.1, ADP 7.3  1.1, and ATP 16.0  2.8; from cell culturesamples (nmol/mg protein): AMP 14.8 1.2, ADP 9.1  0.5, and ATP 21.5  1.3.All these values are in good agreement with those reported in the literature (Carterand Muller, 1990; Horn et al., 1998; Mitani et al., 1994; Pissarek et al., 1999).

high-Deluca, M (1976) Firefly luciferase Adv Enzymol Relat Areas Mol Biol 44, 37–68.

DeLuca, M., and McElroy, W D (1974) Kinetics of the firefly luciferase catalyzed reactions Biochemistry

1271, 349–357.

Fig 6 ATP standard curve obtained by HPLC ATP concentrations range from 5 to 500 mM.

James, A M., Sheard, P W., Wei, Y H., and Murphy, M P (1999) Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations Eur J Biochem 259, 462–469.

King, M P., and Attardi, G (1989) Human cells lacking mtDNA: Repopulation with exogenous mitochondria by complementation Science 246, 500–503.

Kunz, W S., Kuznetsov, A V., Schulze, W., Eichhorn, K., Schild, L., Striggow, F., Bohnensack, R., Neuhof, S., GrasshoV, H., Neumann, H W., and Gelleric, F N (1993) Functional characterization

of mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers Biochim Biophys Acta 1144, 46–53.

Kurebayashi, N., Kodama, T., and Ogawa, Y (1980) P1, P5-Di (adenosine-50) pentaphosphate (Ap5A)

as an inhibitor of adenylate kinase in studies of fragmented sarcoplasmic reticulum from bullfrog skeletal muscle J Biochem (Tokyo) 88, 871–876.

Lemasters, J J., and Hackenbrock, C E (1973) Adenosine triphosphate: Continuous measurement in mitochondrial suspension by firefly luciferase luminescence Biochem Biophys Res Commun 55, 1262–1270.

Lembert, N., and Idahl, L A (1995) Regulatory eVects of ATP and luciferin on firefly luciferase

Meyer, S., Noisommit-Rizzi, N., Reuss, M., and Neubauer, P (1999) Optimized analysis of cellular adenosine and guanosine phosphates in Escherichia coli Anal Biochem 271, 43–52 Mitani, A., Takeyasu, S., Yanase, H., Nakamura, Y., and Kataoka, K (1994) Changes in intracellular

intra-Ca2þand energy levels during in vitro ischemia in the gerbil hippocampal slice J Neurochem 62, 626–634.

Napolitano, M J., and Shain, D H (2005) Quantitating adenylate nucleotides in diverse organisms.

J Biochem Biophys Methods 63, 69–77.

O’Callaghan, J P., and Sriram, K (2004) Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: Comparison with other methods of sacrifice and analysis of multiple phosphoproteins J Neurosci Methods 135, 159–168.

Ouhabi, R., Boue-Grabot, M., and Mazat, J P (1998) Mitochondrial ATP synthesis in permeabilized cells: Assessment of the ATP/O values in situ Anal Biochem 263, 169–175.

Ozer, N., Aksoy, Y., and Ogus, I H (2000) New sample preparation method for the capillary electrophoretic determination of adenylate energy charge in human erythrocytes J Biochem Biophys Methods 45, 141–146.

Pissarek, M., Reinhardt, R., Reichelt, C., Manaenko, A., Krauss, G., and Illes, P (1999) Rapid assay for one-run determination of purine and pyrimidine nucleotide contents in neocortical slices and cell cultures Brain Res Brain Res Protoc 4, 314–321.

Schneider, D R., Felt, B T., Rappaport, M S., and Goldman, H (1982) Development and use of a nonrestraining waveguide chamber for rapid microwave radiation killing of the mouse and neonate rat J Pharmacol Methods 8, 265–274.

Strehler, B L (1968) Bioluminescence assay: Principles and practice Methods Biochem Anal 16, 99–181.

Strehler, B L., and Totter, J R (1952) Firefly luminescence in the study of energy transfer mechanism.

I Substrates and enzyme determinations Arch Biochem Byophys 40, 28–41.

Tatuch, Y., and Robinson, B H (1993) The mitochondrial DNA mutation at 8993 associated with NARP slows the rate of ATP synthesis in isolated lymphoblast mitochondria Biochem Biophys Res Commun 192, 124–128.

Tsai, T L (1986) A microwave method for the extraction of cellular ATP J Biochem Biophys Methods 13, 343–345.

Tuena de Gomez-Puyou, M., Ayala, G., Darszon, A., and Gomez-Puyou, A (1984) Oxidative phorylation and the Pi-ATP exchange reaction of submitochondrial particles under the influence of organic solvents J Biol Chem 259, 9472–9478.

phos-Uesugi, T., Sano, K., Uesawa, Y., Ikegami, Y., and Mohri, K (1997) Ion-pair reversed-phase performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion J Chromatogr B Biomed Sci Appl 703, 63–74.

high-Vazquez-Memije, M E., Shanske, S., Santorelli, F M., Kranz-Eble, P., Davidson, E., DeVivo, D C., and DiMauro, S (1996) Comparative biochemical studies in fibroblasts from patients with diVerent

forms of Leigh syndrome J Inherit Metab Dis 19, 43–50.

Wanders, R J., Ruiter, J P., and Wijburg, F A (1993) Studies on mitochondrial oxidative ylation in permeabilized human skin fibroblasts: Application to mitochondrial encephalomyopathies Biochim Biophys Acta 1181, 219–222.

phosphor-Wanders, R J., Ruiter, J P., and Wijburg, F A (1994) Mitochondrial oxidative phosphorylation in digitonin-permeabilized chorionic villus fibroblasts: A new method with potential for prenatal diagnosis J Inherit Metab Dis 17, 304–306.

Wanders, R J., Ruiter, J P., Wijburg, F A., Zeman, J., Klement, P., and Houstek, J (1996) Prenatal diagnosis of systemic disorders of the respiratory chain in cultured chorionic villus fibroblasts by study of ATP-synthesis in digitonin-permeabilized cells J Inherit Metab Dis 19, 133–136 Wibom, R., Lundin, A., and Hultman, E (1990) A sensitive method for measuring ATP-formation in rat muscle mitochondria Scand J Clin Lab Invest 50, 143–152.

Wibom, R., Soderlund, K., Lundin, A., and Hultman, E (1991) A luminometric method for the determination of ATP and phosphocreatine in single human skeletal muscle fibres J Biolumin Chemilumin 6, 123–129.

Zakaria, M., and Brown, P R (1981) High-performance liquid chromatography of nucleotides, nucleosides and bases J Chromatogr 226, 267–290.

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CHAPTER 8

Measurement of the Ratio of Lactate to Pyruvate in Skin Fibroblast Cultures

Nevi Mackay and Brian H Robinson

Department of Pediatric Laboratory Medicine and the Research Institute Hospital for Sick Children

University of Toronto Toronto, Ontario, Canada

Department of Biochemistry University of Toronto Toronto, Ontario, Canada

I Introduction

II PrincipleIII Procedure

A Sample Preparation

B Determination of Lactate

C Determination of Pyruvate

IV ResultsReferences

I Introduction

A group of inborn errors of metabolism exists that result in the condition ofchronic lactic academia of childhood Nearly all of the defects that can be identifiedoccur in mitochondrial proteins, and many can be demonstrated in skin fibroblastcultures established from the patients concerned One approach to diagnose thesedefects is to measure production of lactate and pyruvate from fibroblast culturesafter incubation in glucose-containing medium The total amount of lactate andpyruvate and the ratio between them is diVerent in cells from patients with defects in

the pyruvate dehydrogenase complex (PDH) or the respiratory chain compared tocells from unaVected individuals.

Copyright 2007, Elsevier Inc All rights reserved. 173 DOI: 10.1016/S0091-679X(06)80008-7

II Principle

When glucose is metabolized by skin fibroblasts, the end product of glycolysis ispyruvic acid This molecule has two major fates in most oxidative tissues It can beoxidized to acetyl coenzyme A (CoA) through the PDH, or it can be reduced toform lactic acid by NADH The extent to which this latter reaction takes place isgoverned by two elements: the rate of flux of pyruvate through PDH into the citricacid cycle, and the extent of reduction of the NADH/NAD couple in the cytosol.The cytosolic redox couple is again in equilibrium with the mitochondrial NADH/NAD couple through the glutamate/aspartate shuttle system (Fig 1)

Glucose

Glucose

Pyruvate

ASP 2-OG

GLU

NADH

Respiratory chain complexes

Pyruvate

Mitochondria

ATP ADP

NAD OAA

NAD/NADH = 70

ASP 2-OG AcCoA

GLU

Pyruvate dehydrogenase complex

Krebs cycle

OAA

II III IV

The lactate dehydrogenase (LDH) equilibrium in the cytosolic compartment isdescribed by:

Hþþ Pyruvate þ NADH$Lactateþ NADþThis emphasizes the fact that the equilibrium of the LDH lies to the right, evenmore so in acidic conditions The redox ratio of the [NADH]c/[NAD]ccouple inthe cytosol can be calculated from:

½Lactate

½Pyruvate
 KLDH¼½NADH
c

½NAD
cwhere KLDHis the equilibrium constant for the LDH reaction, having a value of1.11 104(Krebs, 1973) The lactate/pyruvate (L/P) ratio both in the intracellularcompartment and in the surrounding body fluids or tissue culture medium is usuallybetween 10:1 and 25:1 in favor of lactate By calculation, the NADH/NAD ratio inthe cytosol is about 1:300 in most cell types In contrast, the NADH/NAD ratio

in mitochondria of most cell types is 1:10 (Williamson et al., 1973)

The measurement of lactate and pyruvate accumulated after incubating cellswith glucose can give an indication of the absolute rate of production of lactate

It is also a measure whether the flux into the respiratory chain is compromised by

a defect in pyruvate dehydrogenase (normal L/P ratio) or by a defect in therespiratory chain (high L/P ratio) (Robinson, 1996; Robinson et al., 1985, 1986).This latter application has been documented for defects in complex I, complex IV,and multiple defects of the respiratory chain

III Procedure

Fibroblast cells are grown on 9-cm Petri dish tissue culture plates or 25-ml tissueculture flasks (T25) Use one plate or flask per sample The cells should besubcultured 1:4 from a confluent culture and their growth should be monitored

so that the cells are used once confluence is reached The test should be done nolater than 2 weeks after the cell have been subcultured Cells in the growth phaseare not suitable for this assay, as they have somewhat lower L/P ratios, depending

on the growth rate

3 Add 1-ml PBS (sterile) to each plate, and incubate for 1 h at 37C after, remove medium by aspiration, as above.

There-4 Add 1-ml PBS and 1-mM glucose to each plate and incubate for 1 h

5 Add 50 ml of 1.6-M perchloric acid (PCA), to stop metabolism

6 Pipette extract from the plate into one Eppendorf tube

7 Add 1 ml of biuret reagent to each plate and determine the protein content ofthe sample, using 1 and 2 mg/ml protein standards

B Determination of Lactate

1 For 20 samples, combine:

20-ml hydrazine buVer (0.1-M Tris, 0.4-M hydrazine, 0.4-mM EDTA,10-mM MgSO4, pH 8.5)

200-ml NAD, 80 mg/ml200-ml LDH, 50 mg/10 ml in distilled water

2 Pipette 1 ml of the above mixture into a set of tubes (Eppendorf 1 ml)

3 Add 100ml of cell extract (Section III.A, step 6), vortex, and incubate at RTfor 1 h For controls, which should be carried out in duplicate, use 100-ml PBSinstead of cell extract For standards, addL-Lactate to final concentrations of

25 and 50 nmol, instead of cell extract

4 Measure the amount of lactate produced in each sample using a photometer at a wavelength of 340 nm The blanks can be subtracted auto-matically if a split-beam spectrophotometer is used If only a single-beamspectrophotometer is available, measure the blank samples against water andsubtract from experimental samples

spectro-C Determination of Pyruvate

1 Adjust a fluorimeter to the following settings:

excitation wavelength: 340 nmemission wavelength:>400 nmsensitivity (for 2- to 4-nmol NADH): full scale, using either computerizedrecordings or a chart recorder

2 Transfer 1-ml 0.1-M potassium phosphate buVer pH 7.0 to a fluorimeter

cuvette

3 Add 100ml of acidified cell extract (Section III.A, step 6) to the cuvette

4 Add 4-nmol NADH, oVset the fluorescence background, and read baseline

Calculate the amount of pyruvate, which is almost stoichiometric underthese conditions.

IV Results

In the set of measurements shown in Fig 2, the amount of lactate produced bycontrol cells is 400–500 nmol/min/mg protein This rate is increased in PDHdeficiency, COX deficiency, and complex I deficiency Since the pyruvate produc-tion rate in control cells is 20–25 nmol/min/mg protein, the L/P ratio for controlcells is typically 20:1 In PDH deficiency, pyruvate production is increased and L/Pratios are normal or low In COX deficiency and complex I deficiency, pyruvateproduction is markedly decreased, and L/P ratios are elevated

The change in redox state, as measured by L/P ratios, is usually proportional tothe severity of the defect This can be seen in Fig 3, where the measured L/P ratiosfor a series of cell lines with COX deficiency are plotted against residual cytochromeoxidase activity It would seem that little change in redox state occurs until theCOX activity has fallen to40% of control values This implies that cytochromeoxidase activity is present in a roughly 2.5-fold excess over the rate limiting step, sothat overall oxidation is little aVected by residual values higher than this

Since redox state and demands for ATP production are quite diVerent inquiescent and actively growing cells (Erecinska and Wilson, 1982), care should

be taken to ensure that cells are confluent when these measurements are made.Cells that have not reached confluency tend to have an abnormally low L/P ratio

In contrast, cells that are over confluence have an abnormally high L/P ratio

COX 100

Fig 2 The Lactate and pyruvate produced in confluent fibroblast cultures after 1 h of incubation

in glucose-containing medium The lactate and pyruvate production rates are documented for control ( n), PDH-deficient (y), cytochrome oxidase-deficient (l), and NADH-CoQ reductase- deficient ( m) cell lines Values are given as the mean  SEM for at least four determinations.

Robinson, B H., McKay, N., Goodyer, P., and Lancaster, G (1985) Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia Am J Hum Genet 37(5), 938–946.

Robinson, B H., Ward, J., Goodyer, P., and Baudet, A (1986) Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia J Clin Invest 77(5), 1422–1427.

Williamson, J R., Safer, B., LaNoue, K F., Smith, C M., and Walajtys, E (1973) cytosolic interactions in cardiac tissue: Role of the malate-aspartate cycle in the removal of glycolytic NADH from the cytosol Symp Soc Exp Biol 27, 241–281.

I Introduction

II Metabolite Measurements

A Acylcarnitine Profiles and Total and Free Carnitine Levels

B Measurement of Urine Organic Acids and AcylglycineIII Enzyme and Transporter Assays

I Introduction

Mitochondrial fatty acid
-oxidation represents an essential pathway of energymetabolism for tissues such as liver, muscle, and kidney during periods ofincreased demand due to fasting, febrile illness, reduced caloric intake due togastrointestinal illness, and when cold In heart muscle, fatty acid oxidation may

be as important as carbohydrate metabolism for normal cardiac function The endproducts of fatty acid oxidation in the liver are ketone bodies, which are trans-ported in the circulation to tissues such as brain, which has minimal
-oxidationcapacity In nonketogenic tissues, such as muscle and kidney, the end product of

-oxidation is acetyl-coenzyme A (CoA), which is used directly as an energy

Copyright 2007, Elsevier Inc All rights reserved. 179 DOI: 10.1016/S0091-679X(06)80009-9

source within the TCA cycle (Rinaldo et al., 2002; Stanley et al., 2006) In recentyears, it has also been recognized that gastrointestinal, fetal, and placental metab-olism also uses the fatty acid
-oxidation pathway as a source of fuel (Oey et al.,2005; Shekhawat et al., 2003) However, the extent and necessity of use of thispathway in these tissues remains to be established.

The fatty acid oxidation pathway is divided into a number of cellular nents (Fig 1) Typically, long-chain fatty acids of chain lengths C16–C18 arereleased at the plasma membrane following the action of lipoprotein lipase activity

compo-on circulating triglycerides, or are derived from albumin-bound ncompo-onesterified fattyacids The fatty acids are then transported across the plasma membrane andactivated to their acyl CoA derivatives at the outer mitochondrial membrane.Evidence suggests that CD36, the fatty acid transporter (also known as FAT) may

Carnitine

Carnitine

Carnitine Carnitine

− C − CH2−O

II

I

Fig 1 Interactions of mitochondrial fatty acid oxidation and the respiratory chain OMM, outer mitochondrial membrane; IMM inner mitochondrial membrane [Reprinted with permission from

Rinaldo et al (2002) # 2002 by Annual Reviews www.annualreviews.org]

play an important role in this process, and provide a direct connection with theouter mitochondrial membrane, where the fatty acids are activated to becomeacyl CoAs (Campbell et al., 2004) The plasma membrane carnitine transporter,which provides carnitine, an essential cofactor, is also necessary for normal fattyacid oxidation.

Long-chain acyl CoA species cannot be transported directly across the chondrial membrane Substrate transfer is dependent on the carnitine cycle(McGarry and Brown, 1997) In this cycle, long-chain acyl CoAs are first converted

mito-to their respective acylcarnitine species by the action of carnitine palmimito-toyltrans-ferase 1 (CPT 1) at the outer mitochondrial membrane The acylcarnitine species istransported through the inner mitochondrial membrane by the bidirectional carni-tine:acylcarnitine translocase (CACT), a carrier that also transports free carnitineand acylcarnitine out of the mitochondria Finally, the acylcarnitine is reconverted

palmitoyltrans-to the acyl CoA species by the action of carnitine palmipalmitoyltrans-toyltransferase 2 (CPT2)

at the inner mitochondrial membrane Free carnitine is recycled through CACT

to provide substrate for CPT1 Medium-chain length fatty acids (C6–C10) appear toenter into the mitochondrial matrix by a carnitine-independent process that is notyet characterized Medium-chain fatty acids are frequently provided as a nutri-tional supplement in the form of medium-chain triglycerides, and are presumablymetabolized entirely within the mitochondrial matrix

The process of acyl CoA
-oxidation involves four enzymatic steps First, adouble bond is inserted into the 2,3 position by an acyl CoA dehydrogenase(ACD), to generate a 2,3-enoyl-CoA species This is an energy-generating stepand electrons are transferred by electron transfer flavoprotein (ETF), an FAD-dependent pathway, to ETF: coenzyme Q (CoQ) oxidoreductase in the respiratorychain The second step involves hydration across this double bond by a hydratase,which generates a stereospecificL-3-hydroxyacyl CoA species The third enzymaticreaction involves further reduction in the 3-position by an NAD-requiring L-3-hydroxyacyl CoA dehydrogenase Electrons from this step are transferred to therespiratory chain through Complex I, which recycles NAD for further metabolic use.The product of this third reaction, a 3-ketoacyl CoA species, is then thiolyticallycleaved by a 3-ketoacyl CoA thiolase into acetyl-CoA and a two-carbon chain-shortened acyl CoA, which continues to reenter the spiral until all of the originalfatty acid has been converted to acetyl-CoA

The process of complete oxidation of a long-chain fatty acid requires theparticipation of a series of genetically similar enzymes, each with diVerent chain-length specificities Long-chain acyl CoAs are metabolized near the inner mito-chondrial membrane by a family of long-chain-specific membrane-bound enzymes,while medium- and short-chain intermediates are metabolized in the mitochondrialmatrix by a group of soluble enzymes (Table I) Three of the enzymes involved

in membrane-associated metabolism of long-chain intermediates are part of amultifunctional enzyme complex known as the mitochondrial trifunctional protein(TFP) This complex is a hetero-octamer consisting of four-subunits and four

-subunits The -subunit has both long-chain 2,3 dienoyl-CoA hydratase

(LHYD) and long-chain-L-3-hydroxyacyl CoA dehydrogenase (LCHAD) ities, while the
-subunit has long-chain-3-ketoacyl CoA thiolase (LKAT) activity.Little is known about the organization of the enzymes within the mitochondrialmatrix, but there is increasing evidence that the enzymes are organized as a uniquelystructured and closely associated metabolome, which is in a required order for thenecessary eYcient substrate flow.

activ-Genetic defects that impair flux through the pathway have been identified formost of the well-characterized steps in fatty acid oxidation The clinical pheno-types of the genetic disorders have been described in a number of recent reviews(Rinaldo et al., 2002; Roe and Ding, 2001; Stanley et al., 2006) In this chapter, wedescribe methods to measure unique metabolites, specific enzymes, and genes infatty acid oxidation, and to monitor metabolic flux through the entire pathway

We also describe methods for the diagnosis of diseases of fatty acid oxidation.Please refer to reviews that have been cited for full clinical descriptions of thediseases

Table ICharacterized Components of Mitochondrial Fatty Acid Oxidationa

Carnitine uptake and cycle

Inner mitochondrial membrane enzymes (long-chain fatty acids)

and four
47-kDa subunits

Mitochondrial matrix enzymes (medium- and short-chain fatty acids)

a CPT1A, carnitine palmitoyltransferase 1A (hepatic); CACT, carnitine: acylcarnitine translocase; CPT2, carnitine palmitoyltransferase 2; VLCAD, very-long-chain acyl CoA dehydrogenase; LHYD, long-chain enoyl-CoA hydratase; LCHAD, long-chain L -3-hydroxyacyl CoA dehydrogenase; LKAT, long-chain 3-ketoacyl CoA thiolase; MCAD, medium-chain acyl CoA dehydrogenase; M/SCHAD, medi-

um and short-chain l-3-hydroxyacyl CoA dehydrogenase; SCAD, short-chain acyl CoA dehydrogenase; SKAT, short-chain 3-keto acyl CoA thiolase (also known as
-ketothiolase) LHYD and LCHAD are both components of the -subunit of the mitochondrial trifunctional protein; LKAT is a component of the

-subunit.

II Metabolite Measurements

A Acylcarnitine Profiles and Total and Free Carnitine Levels

Transport of long-chain fatty acids into the mitochondrion for fatty acidoxidation depends on the essential cofactor carnitine The carnitine transporterthat is present in muscle and kidney is a member of the organic cation transporterfamily, OCTN2, which is a high-aYnity sodium cotransporter that can concen-

trate carnitine within the cell to levels that are 50 times higher than those observed

in the circulation OCTN2 is competitively inhibited by certain acylcarnitinespecies, which results in renal tubular loss of carnitine, and eventual depletion oftissue carnitine levels (Stanley et al., 1993) In genetic OCTN2 deficiency, there ismassive renal loss and marked deficiency in tissues, leading to symptoms ofcardiac and skeletal muscle dysfunction similar to those seen in patients withprimary defects which, if untreated, are fatal Measurement of total and free car-nitine levels is important in the evaluation of patients who may have a fatty acidoxidation defect or an OCTN2 deficiency Moreover, evaluation of acylcarnitineprofiles and identification of specific acylcarnitine species may provide clues to thesite of a fatty acid oxidation defect For example, elevations of the level ofmedium-chain C8, C10, and C10:1acylcarnitine species is diagnostic for medium-chain acyl CoA dehydrogenase (MCAD) deficiency The majority of newborns inthe United States and other western countries are screened for this disorder andother disorders of fatty acid oxidation at birth In this screen, acylcarnitine levelsare measured in blood samples using tandem mass spectrometry The sensitivity ofthis technique is suYcient for acylcarnitine analysis in small blood samples or

in blood that has been collected as spots onto filter paper The method is used inwhole population screening (Chace et al., 2003; Frazier et al., 2006)

For the measurement of total and free carnitine, a small blood sample isrequired The sample is divided analyzed in two aliquots The level of freecarnitine is quantified in one aliquot by the same technology as for the acylcarni-tine analysis The second aliquot is subjected to alkaline hydrolysis to liberate anyacylated carnitine, and the level of total carnitine is measured The diVerencebetween the total and free carnitine represents the acylcarnitine fraction Indivi-duals with a carnitine transporter defect have markedly reduced plasma totalcarnitine levels (to less than 10% of normal), and carriers for the defect have levelsthat are intermediate Most patients with disorders of fatty acid oxidation havereduced total carnitine levels

Protocol for measurement of total and free carnitine and acylcarnitine in plasma

1 Separate plasma into 20- to 50-ml aliquots

2 Add a series of stable-isotope-labeled acylcarnitine internal standards(including free carnitine) to each sample (Deuterated standards are availablefrom Cambridge Isotope Laboratories Inc., Product No NSKB.)

3 Dilute each sample with 450ml of ethanol and vortex mixed for 30 sec

4 Centrifugation at 13,000
g for 10 min at RT to separate the deproteinizedplasma.

5 Transfer 300ml of the supernatant from each sample into wells in 96-wellplates, and evaporate to dryness under a steady stream of nitrogen

6 Prepare butyl-derivatives by the addition of 100ml of butanolic hydrochloricacid, heating at 65C for 15 min (Not all laboratories choose to makebutylated acylcarnitine and carnitine derivatives, claiming that nonderivati-zation is a simpler process We find that butylation is preferable, particularlyfor measurement of dicarboxylic acid carnitine conjugates, such as glutaryl-carnitine.)

7 Thereafter, evaporate to dryness under a steady stream of nitrogen at RT

8 Resuspend the material in each well with 100-ml acetonitrile:water (50:50)

9 Ten microliters of this material is injected directly into the tandem massspectrometer via a column-free HPLC system

Analysis of the mass spectroscopy dataAnalyze the parent compounds for a fragment of m/z 85 These masses areusually characteristic of carnitine and carnitine-conjugated compounds that can

be associated with disease Occasionally, two acylcarnitine compounds have thesame mass but are associated with diVerent conditions These isobaric compoundscannot be diVerentiated using tandem mass spectrometry In this case, alternatemethods for analysis of the metabolite profile, including urine organic acid analy-sis, are required to diVerentiate the species and identify the disease.

Determine the level of these carnitine fragments using known concentrations ofthe stable isotope-labeled internal standards that are added at the outset Isotope-labeled internal standards are not commercially available for all acylcarnitinespecies When an immediate internal standard is not available for a particularcompound, it is acceptable, but not ideal, to quantitate using the nearest massequivalent internal standard

B Measurement of Urine Organic Acids and Acylglycine

Methods for the analysis of urine organic acids and acylglycines have been wellestablished as a clinical tool, and usually involve acidification of urine, addition ofsuitable internal standards, extraction into an organic solvent, and derivatizationand analysis by gas chromatography/electron impact mass spectrometry, withdata collection in the mass range 50–600 amu Please refer to an online documentfor procedures for this assay: http://www.aacc.org/AACC/members/nacb/LMPG/OnlineGuide/PublishedGuidelines/Maternal_Fetal_Risk/

1 Organic Acids

The analysis of urine organic acids has become a standard tool for investigation

of suspected metabolic disease patients showing a wide variety of clinical signs(Kumps et al., 2002) In mitochondrial fatty acid oxidation defects, a number of

specific and nonspecific metabolites can be detected in urine, using gas tography/mass spectrometry In the event that flux through the fatty acid oxida-tion pathway is impaired, long-chain fatty acids can be partially oxidized withinperoxisomes to chain lengths C6–C10 Further metabolism of these medium-chainspecies takes place in the microsomes, where o and o-1 oxidation products are

chroma-produced Thus, theo-oxidation products adipic, suberic, and sebacic acids, which

are also known as medium-chain dicarboxylic acids, are prominent but nonspecificmarkers of impaired mitochondrial
-oxidation, as are the o-1 products of

5-hydroxyhexanoic, 7-hydroxyoctanoic, and 9-hydroxydecanoic acids In addition,

a number of 3-hydroxydicarboxylic acids up to chain length C14may be detected

by this process Interpretation of 3-hydroxydicarboxylic aciduria may includedefects at the level of 3-hydroxyacyl CoA dehydrogenase and 3-ketoacyl CoAthiolase Longer chain-length dicarboxylic acids are not found in urine They areprobably circulated as protein-bound metabolites, which are not filtered by therenal glomeruli

Protocol

1 Add 50ml of an internal standard of hendecanedioic acid (1 mg/ml) and 50 ml

of methoxylamine hydrochloride (25 g/liter) to a volume of urine that tains 2-mg creatinine (The methoxylamine protects ketoacids from beingconverted to their respective hydroxy acids.)

con-2 Bring volume up to 3 ml with distilled deionized water, alkalinized with —three to four drops of 2-M sodium hydroxide, and heat at 70C for 30 min tocomplete the oximation

3 Cool the samples and then acidify them with 5-M HCl to pH 1–2

4 Extract sample three times with ethylacetate Pool the organic phases anddry them at under a steady stream of nitrogen at RT

5 Trimethylsilyl-derivatives are made by adding 150-ml bis trifluoroacetamide:trimethylchlorosilane (BSTMS:TMCS, 99:1 obtainedfrom Pearce Chemical Company) and heating for 30 min at 70C

(trimethylsilyl)-6 Inject the derivatized extract into the gas chromatograph-mass spectrometerand generate a total ion chromatogram (mass range 50–600 amu) for theorganic acid profile

2 Acylglycines

The extraction procedure that isolates and measures organic acids in urine alsoisolates acylglycines These compounds are the products of mitochondrial glycineconjugation of acyl CoA intermediates, including hexanoylglycine in MCADdeficiency and butyrylglycine in SCAD deficiency These markers are probablypathognomic for the particular disorders, but are present at lower levels, necessi-tating the approach of stable isotope dilution and selected ion monitoring massspectrometry for accurate quantitation The acylglycine internal standards for this

procedure are [13C,15N]-labeled acylglycine species, which generate a mass that is

2 amu greater than that of the naturally occurring species (Rinaldo et al., 1988)

III Enzyme and Transporter Assays

A Carnitine Uptake

Carnitine uptake can only be measured in living intact cells The muscle andkidney carnitine transporter, which presently represents the only genetic carnitinetransporter defect, is present in cultured skin fibroblasts, and this represents themajor tissue in which carnitine transport is evaluated There is a second carnitinetransporter that is expressed primarily in liver, and in the case of a suspected trans-porter defect for this isoform, the assay would need to be performed in culturedhepatocytes As with all assays that require the use of cultured skin fibroblasts,testing should be performed at the lowest possible cell passage number, since fibro-blasts can transform over time and certain properties regarding metabolism maychange The assay monitors intracellular uptake of radiolabeled [14C]-carnitineand is capable of diVerentiating homozygous deficient, normal, and heterozygous

carriers (Treem et al., 1988)

to deplete the intracellular stores of carnitine

3 Replace the medium with the same medium also containing 0.5-mM [3

carnitine (0.5mCi/ml)

H]-4 At 0, 1, 2, and 4 h time points, terminate the reaction by repeated washing

4 times with ice-cold 0.1-M MgCl2

5 Extract intracellular carnitine from each well with 0.5-ml ethanol, and mine the amount of radiolabeled carnitine in the extract using a scintillationcounter The rate of carnitine uptake is determined by kinetic analysis of thelabeled intracellular carnitine at the various time points

deter-B Carnitine Cycle

The individual elements of the carnitine cycle include carnitine transferase 1 (CPT1), carnitine: acylcarnitine transferase (CACT), and carnitinepalmitoyltransferase 2 (CPT2) CPT1 and CPT2 activities can be measured

simultaneously using the method of McGarry and Brown (1997) In this assay,the conversion of palmitoyl-CoA to palmitoyl carnitine is monitored usingradiolabeled carnitine.

Palmitoyl-CoAþ ½14C-carnitine!Palmitoyl-½14C-carnitine þ CoA

Protocol for measurement of CPT1 and CPT2 activityThe assay is performed in two parts In the first part, total CPT activity (CPT1plus CPT2) is measured, in the forward direction for CPT1 and the reversedirection for CPT2 In the second part of the assay, CPT1 activity is inhibited

by the addition of malonyl-CoA at a final concentration of 50 mmol/liter Thisassay determines the contribution toward total CPT activity by CPT2, which isnot inhibited by malonyl-CoA at this concentration

The enzymes are stable when tissues are stored at80C, and the assay can be

applied to all tissue types, with the understanding that the kinetics of CPT1 bition by malonyl-CoA diVers between the muscle and liver CPT1 isoforms, and

inhi-that appropriate inhibiting malonyl-CoA concentrations are required To date,the only genetic cause of CPT1 deficiency that has been described involves CPT1A,the hepatic isoform (Bennett and Narayan, 2005) This is the isoform that is ex-pressed in cultured skin fibroblasts, making this a useful tissue for clinical diag-nostics CPT2 is a systemic gene product and can be measured in any available andsuitable tissue Because one of the clinical phenotypes of CPT2 deficiency includesexercise-induced rhabdomyolysis in young adults, the enzyme is frequentlymeasured in biopsied muscle tissue

Assay cocktailBSA (200 mg)Glutathione (1.5 mg)KCN (2.6 mg)ATP (480ml of a 100-mg/ml stock solution)14-ml Trizma pH 7.4, 0.15 M

34.2-ml magnesium chloride (2.45-M stock solution)20-ml rotenone (400 mg/10 ml acetone stock solution)This cocktail is made fresh daily

Substrate mixture320-mlL-carnitine (100 mM)20-mCi [14

C]-carnitine400-ml palmitoyl-CoA (20 mM)Bring up to 15 ml with distilled water

This is stable for 6 months and can be aliquoted and frozen