Fatty acid distribution in systems modeling the
normal and diabetic human circulation. A 13C
nuclear magnetic resonance study.
D P Cistola, D M Small
J Clin Invest.
1991;
87(4)
:1431-1441.
https://doi.org/10.1172/JCI115149
.
A nonperturbing 13C nuclear magnetic resonance (NMR) method was used to monitor the
equilibrium distribution of carboxyl 13C-enriched fatty acids (FA) between distinct binding
sites on human serum albumin, native human lipoproteins, and/or phospholipid model
membranes, under conditions that mimic the normal and diabetic human circulation. Two
variables pertinent to the diabetic circulation were examined: FA/albumin mole ratio (as
elevated in insulin deficiency and/or nephrosis) and pH (as decreased in acidosis). 13C
NMR spectra for samples containing carboxyl 13C-enriched palmitate, human serum
albumin, and phospholipid vesicles or native lipoproteins (all samples at pH 7.4, 37 degrees
C) exhibited up to six carboxyl NMR resonances corresponding to FA bound to distinct
binding sites on albumin and nonalbumin components. When the sample FA/albumin mole
ratio was 1, three FA carboxyl resonances were observed (182.2, 181.8, and 181.6 ppm;
designated peaks beta, gamma, and beta', respectively). These resonances corresponded
to FA bound to three distinct high-affinity binding sites on human serum albumin. When the
sample mole ratio value exceeded 1, additional carboxyl resonances corresponding to FA
bound to phospholipid vesicles (179.0 ppm, peak phi), lipoproteins (180.7 ppm, peak
sigma), and lower affinity sites on albumin (183.8 ppm, peak alpha; 181.9 ppm, peak
gamma'), were observed. The intensity of peaks phi and sigma increased with increasing
mole ratio or decreasing pH. Using Lorentzian lineshape […]
Research Article
Fatty Acid Distribution in Systems Modeling the Normal
and
Diabetic Human Circulation
A 13CNuclear Magnetic Resonance Study
DavidP.CistolaandDonald M.Small
Departments ofBiophysics, Biochemistry, and Medicine, Housman Medical Research Center, Boston UniversitySchool ofMedicine, Boston, Massachusetts 02118
Abstract
A nonperturbing
'3C
nuclear magnetic resonance (NMR) method was used tomonitortheequilibrium distribution of car-boxyl"C-enriched
fatty acids (FA) between distinct binding siteson human serum albumin, native humanlipoproteins, and/or phospholipid model membranes, under conditions that mimic the normal and diabetic human circulation. Two vari-ablespertinenttothediabeticcirculationwereexamined:
FA/
albumin mole ratio (as elevated in insulin deficiency and/or
nephrosis) and pH (as decreased in acidosis).
'3C
NMR spectraforsamplescontaining carboxyl
"C-enriched
palmitate,human serumalbumin, and phospholipid vesicles or native lipopro-teins(allsamplesatpH 7.4,370C)
exhibiteduptosixcarboxyl NMR resonancescorrespondingto FAboundtodistinct bind-ing sites on albumin and nonalbumin components. When the sampleFA/albuminmole ratio was 1, three FA carboxyl reso-nances wereobserved (182.2,181.8, and 181.6 ppm; designated peaksft,
y,
andif',
respectively). These resonances corre-sponded to FA bound to three distinct high-affinity binding sites on human serumalbumin. When the sample mole ratio valueexceeded 1, additional carboxyl resonances correspond-ing to FA bound to phospholipid vesicles (179.0 ppm, peakl),
lipoproteins (180.7 ppm, peaka),
and loweraffinitysites on albumin (183.8 ppm, peakca,
181.9 ppm, peakY),
were ob-served. Theintensity ofpeaks 4 andaincreasedwithincreasingmoleratio or decreasing pH. Using Lorentzian lineshape
analy-sis, the relative mole quantities ofFA bound to albumin and nonalbumin bindingsitesweredetermined. Plots of the fraction of FAassociatedwithnonalbumin components as a function of FA/albumin mole ratio were linear and extrapolated to the
ab-scissaat amole ratio valueof1.This pattern of FA distribution was observed regardless of the type of nonalbumin acceptor used(phospholipidvesicles, human high- or low-density
lipo-proteins) or the typeofFA used(palmitate, oleate, or stearate), andprovidedevidence for negative cooperativity for human
serumalbumin uponbinding of1molof FA per mole albumin.
Theseinvitro NMR results suggest that the threshold
FA/al-buminmoleratio value for alterations in FA distributions in the
Address reprint requests to Dr. Cistola. Present address: Department of Biochemistry and Molecular Biophysics, Box 8231, Washington Uni-versity School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110.
Receivedfor publication31August1989and inrevisedform2 Oc-tober 1990.
humancirculationmaybe 1, rather than3,aspreviouslyheld. Thepathophysiologicalimplicationsofthesefindings are dis-cussed.(J. Clin.Invest.1991.87:1431-1441.) Keywords:
dia-betes mellitus*fattyacids-ipoproteins* model membrane. nuclear magnetic resonance * serum albumin
Introduction
Inhumans, - 200goffatty acidsaremobilized from
adipose
tissue eachdayandtransportedin the circulationat
concentra-tions -100-1,000-fold higherthantheirmonomer
solubility
limit.'
Solubilization and transport is mediatedprimarily
byserumalbumin,athree-domainproteinwithatleast six high-and
medium-affinity binding
sites for long-chain fatty acids(1-4). Albuminprevents theself-association of fatty acids into liquid-crystalline orcrystallineaggregates at neutral pH(5, 6)
and provides tissues with a readily available source offatty acids forenergyproduction and
lipid
synthesis (7).Under normal conditions in
humans,
it isthoughtthat>99%ofthecirculatingfattyacidsareboundtoserum
albu-min(7-9). However, plasma
lipoproteins
and cellularmem-branesalso havea
high affinity
forfatty
acids(8, 10)
and may,under certain normal orabnormal
conditions,
competewith albumin for fatty acid binding. Such conditions could include elevatedcirculating fatty
acid levels(secondary
touncontrolled diabetesmellitus,
myocardial infarction, hyperthyroidism,
orsepsis),
decreased albumin levels(secondary
tonephrotic
syn-drome, liver diseases,
genetic defects),
and/or altered albuminbinding properties (secondary
toacidosis, drug administration,
nonenzymatic glycosylation,orhyperbilirubinemia). Themost severeabnormalities infatty
acidtransportareexpected
in dia-betic ketoacidosiscomplicated bynephrosis,
since largeeleva-tions in
circulating fatty
acidsaresuperimposed
with decreased albuminlevels andacidosis.In vitro, fatty acids in blood cells, endothelial cells, and
lipoproteins havebeen showntoinduceavariety of
detrimen-talfunctional effects
(reviewed
inreference 7)that maycontrib-ute to the
pathogenesis
of vascularcomplications
indiabeticsubjects.
However, the in vivosignificance
of these effectsre-mainsunknown, largely because the distribution of
fatty
acidsin the humancirculation isnoteasily determinedorpredicted.
Anumber of variables, suchasthose mentioned above, may
affectthedistribution offatty acidsbetweenalbumin and
non-albumincomponents, but the
quantitative
contribution of each variable isnotknown.Inaddition,themolecularbasis for1.Theterm"fatty acid,"rather than"nonesterifiedfattyacid"or"free
fatty acid,"is usedthroughout thismanuscript.Useoftheterm"fatty
acid" isnot meant toimplyany informationregardingtheionization
stateof thecarboxylgroup. J.Clin.Invest.
©TheAmericanSocietyfor Clinical Investigation,Inc.
0021-9738/91/04/1431/11 $2.00
fatty acid interactions with binding sitesonhumanserum
albu-min, cell membranes, and lipoproteins is only partially under-stood. Moreover, nonperturbing techniques for quantitating fatty acid distributions between albumin and nonalbumin bind-ing sites under physiological conditions have not been
avail-able.
Inthe present study, a new13Cnuclearmagnetic resonance (NMR)2 modelsystemapproachwasused tomonitorthe
equi-librium distribution of carboxyl13C-enrichedfatty acids in re-constituted and whole human plasma and blood under condi-tions that mimic steady-state condicondi-tions in the normal and diabetic human circulation. This NMR approach, based on
earlier work(1 1), is nonperturbing and provides physicochemi-cal information regarding the distribution of fatty acids be-tweenindividual albumin andnonalbumin binding sites and the mechanisms that govern thisdistribution. The effects of twovariablespertinent to the diabetic circulation were exam-ined: fatty acid/albumin mole ratio (increased during insulin deficiency and/or nephrotic syndrome), and pH (decreased during ketoacidosis). The influence of these variables on fatty acid distributions have previously been examined using bio-chemical approaches (1, 7, 9). In the present study, the 13C
NMR approach has been applied to reexamine the quantitative contributions of these variables and toinvestigate the molecu-larmechanisms governing fatty acid transport in the normal and abnormal human circulation.
Methods
Materials. Palmitic acid [1-_3C,90% enriched] waspurchased from KOR Stable Isotopes,Cambridge,MA(LotDM-I-89),andwas >99% purefatty acid bythin-layerchromatography and>95% purepalmitic
acid bygas-liquidchromatography. Egg yolkphosphatidylcholinewas
purchased fromLipidProducts, Nutley,England,andwas>99% pure bythin-layer chromatography.
Crystallized, lyophilized fattyacid-free humanserumalbuminwas
purchased fromSigmaChemical Co. St.Louis,MO(A-3782,Lot 76F-9335), and analyzedas follows. Sodium dodecyl
sulfate-polyacryl-amide gelelectrophoreticanalysisof humanalbuminusing3-24% gra-dientgels (25 ug humanserumalbumin perlane)demonstrateda
ma-jorbandat66 kD andseveral minor bands(totaling - 5%)with
molecularmassescorrespondingtoalbuminoligomers.Previous stud-ies indicated that the presence ofcovalently linked albuminoligomers
didnotaffect 13C NMR spectra offattyacid/albumin complexes (3).In
addition, 13CNMRcarboxylresonancesforfattyacids boundto
hu-man serumalbumin fromlyophilizedpreparationswereidentical to
those observed in spectra of whole humanplasmafreshlyisolated from individual healthy donors. Noimpuritybandswereobservedat a
mo-lecularmasscorrespondingtoapoproteinA-I. "Fatty acid-free" albu-minwasanalyzed for residual fattyacidcontentusing gas-liquid
chromatography. Fatty acids wereextracted twiceusingabenzene/ chloroform/methanolprocedure( 12)andtransesterifiedusing
boron-triflouride-methanol. Gas-liquid chromatographic analysisof the methyl esterderivatives, using heptadecanoic acidas aquantitative
internal standard, indicated that the albuminpreparationcontained
<0.01 molfattyacid per mole albumin.
Native human HDL (d= 1.081-1.21) and LDL (d=1.025-1.050)
fractionswerepurified by ultracentrifugation from one unit of plasma
2.Abbreviations used in this paper:NMR,nuclearmagneticresonance; NOE, nuclear Overhauserenhancement;
T,,
spin-latticerelaxation time.aftera 12-hfast. Samples of heparinizedwhole humanplasma used for
NMRanalysisweredrawnfrom healthy donors aftera12-hfast, and levels ofalbumin, total protein, total cholesterol, HDLcholesterol, totalbilirubin, triglycerides,andnonesterified fattyacidswere
mea-sured in eachplasma sample.
Thebuffer solution used throughout this study contained 135mM
NaCl,4 mMKCl,0.8mMMgSO4, 2mMCaCl2, 0.1mMNaN3,0.1
mMascorbicacid,and40 mM
N-tris[hydroxymethyl]methyl-2-ami-noethanesulfonic acidbuffer,atpH7.4.
PreparationofNMRsamplescontainingfattyacid, human albu-min, and model membranes. The general approach for equilibrating
potassiumsalts offattyacids with albumin(3)and the
physical-chemi-calbasisfor that approach (5, 6) have been discussed in detail else-where.In short, stocksolutions of'3C-enrichedfatty acids in chloro-formwereprepared and their concentrationsweredetermined by
mea-suring dry weights on a microbalance (Cahn Instruments, Inc.,
Cerritos, CA).Anappropriate aliquot(typically400Mug)waspipetted directly intoa10-mmNMRtube and the solventwasevaporated underastreamofnitrogen.A I00-Mlaliquotof H20orD20(forNMR
locksignal) and 1.2 equivalents of 1NKOHwereaddedtotheNMR
tube, and the samplewasmixed andequilibratedat35-450Cuntil all
crystalline-oroil-phasefattyacidwascompletelyconvertedtothe po-tassium salt and dissolved in the aqueous phase. Theresulting sample consisted ofanopticallyclearmicellarsolution (- 15mM).For potas-siumpalmitate,thesamplesunderwentareversiblegelto micellar phase transitionat - 30°C and werewarmed ina37°Cwaterbath
beforemixingwithalbumin, vesicle,lipoprotein,orplasma samples.
Unilamellarphospholipidvesicles(100 mg/mlinbuffer)were pre-paredbysonication(50min, 25 watt, 35%dutycycle)underN2inan
ice-water bathusingaSonifier (Branson SonicPowerCo., Inc.,
Dan-bury,CT)equippedwithamicroprobe tip.Thephospholipid prepara-tions containednodetectablefattyacidsorlysolecithin by thin-layer chromatography (100Mgofphospholipid spotted),eitherbeforeorafter sonication.Aliquotsofphospholipidvesicles(0.8ml) and albumin(0.8
ml, 100mg/ml)werecombined ina 10-mm NMRtube, andasmall
quantityofD20(100
M1,
forNMRlocksignal)wasadded. ThesamplepHwasadjustedto7.4, anda'3CNMRspectrumwasacquired.Then thesamplewastransferredto asecondNMRtubecontaining carboxyl 13C-enriched potassium palmitatein 100 Ml ofH20, equilibratedfor fiveminutes,and thepHwasadjustedto7.4. Thissample containeda
fattyacid/albuminmole ratio of1:1,analbumin/phospholipid weight
ratio of 1:1, andan albuminconcentration of47 mg/ml.An NMR
spectrumwasacquired, andadditional '3C-enrichedpalmitatewas
addedusingtheprocedurenoted abovetoyieldtotal
palmitate/albu-min moleratio valuesof 2:1 and 3:1.In asimilar manner, a separate samplewasprepared withstartingmoleratioof 4:1, and palmitate was addedtoyieldsamplescontaining5:1and 6:1fattyacid/albumin mole ratio values.NMRspectrawereacquiredateach moleratioincrement.
Negative-stainelectronmicrographsof the 6:1 mole ratio sampleafter
NMRanalysiswereessentiallyidentical to thosefor the vesicle prepara-tion withnoaddedalbuminorpalmitateandrevealedarelatively ho-mogeneouspopulation of unilamellar vesicles witha meandiameter of
30nm.
In asimilar manner, otherwise identical samples containing less
phospholipid (final concentrations,24 and 11 mg/ml)wereprepared. PreparationofNMRsamplescontainingfattyacid, albumin, and
nativehumanlipoproteins.HDLand LDLfractionswerepressure
dia-lyzedagainstthree volumesofsamplebufferusinga 10-ml Amicon ultrafiltration apparatus(Amicon Corp.,Danvers,MA)equippedwith
aXM300filter. HDL- andLDL-protein concentrationswere deter-mined byamodified Lowry method(13).Human serumalbuminand HDL(0.8mleach)werecombined ina10-mmNMRtubesothattheir finalproteinconcentrationswere 47and24mg/ml,respectively.A'3C
NMRspectrum wasacquiredatpH7.4before the addition offatty acid. The samplewassubsequentlytransferred toasecond 10-mm
NMRtubecontaining0.1 mlof aqueous'3C-enriched potassium pal-mitatetoyieldatotalfatty acid/albuminmoleratio of 2:1. Samples with mole ratio values of 4:1 and 6:1 were prepared in a similar
ner. Inaddition, separatesamplescontainingadifferent concentration ofHDL(final HDLproteinconcentration, 9mg/ml)orcontaining
LDL(final LDLproteinconcentration,11mg/ml)wereprepared using identical procedures.
Gas-liquidchromatographic analyses of HDL and LDL fractions before addition offattyacidoralbumin contained 0.39 and 0.32 mol of naturalabundance (no '3C enrichment) fattyacid, as expressed per moleof albumin in the finalNMRsamples. Thereportedfatty
acid/al-bumin mole ratio values include the endogenous unenriched plus the added"3C-enrichedfattyacids.Negative-stainelectronmicrographsof thesamplescontaining thehighest fatty acid/albuminmoleratiowere
essentially identicaltothosefor isolatedHDL or LDLwithnoadded fatty acid oralbumin and showedarelatively homogeneouspopulation
ofround particles witha meandiameters of 1 1 and 22 nm,respectively. '3CNMRspectroscopy. '3C NMR spectrawererecordedon amodel WP-200 NMR spectrometer(Bruker Instruments, Inc.,Billerica, MA; 50.3MHzfor `3C)asdescribed elsewhere (14) andahome-built 360
MHz NMRspectrometer(90.58MHzfor`3C)attheMolecular Bio-physics Laboratory,Francis BitterNational Magnet Laboratory, Mas-sachusetts Institute ofTechnology.NMRinternalsampletemperatures werecontrolled to 37±1 'C.Thechemical shiftofthenarrow resonance
from albumin e-Lys/fi-Leucarbons(3)wasusedas aninternal
refer-enceaftercalibratingthisresonance(39.52ppm)againstexternal
tetra-methylsilane. Theestimated uncertainties in chemical shift valueswere
±0.1 ppm.Insome cases,relative peak intensitiesweremeasuredusing
the integration routinesprovided within the BrukerDISNMRand
M.I.T.RNMR software packages.Inordertodeconvoluteoverlapping
resonancesandmeasureindividual peak intensities, Lorentzian spec-tralsimulations weregenerated usingthe Bruker GLINFIT program. Ninety-degree pulseswereusedthroughout andspectral pulse intervals (4.82 s;>4-5 xspin-lattice relaxation time [T.])weresufficientlylong
toobtain full relaxation and equilibrium NMR intensities for palmitate and oleate carboxyl resonances.Spin-lattice relaxation and nuclear Overhauser enhancement(NOE)valuesweremeasuredaspreviously
described (3). The minimum time betweensample mixingand the
beginning ofNMRdata collectionwas 30 min. NMRresultswere
independent ofequilibration time for times 2 30 min.
pHmeasurements. Allsample pH measurements were made di-rectly in the NMR tubeusingathin glass combination electrode (Micro-electrodes,Inc., Londonderry,NH).Measurementsmade at room tem-perature(21-24°C)werecorrectedtovaluesat37°Cusing the follow-ing conversion factor.ApH/AT= -0.0 146 (15). All reported pH values inthis manuscript represent values corrected to 37°C.
Results
Toexamine the effect of increasing
fatty
acid/albumin moleratio on the equilibrium distribution of
fatty
acids between binding sites on human serum albumin and modelmem-branes, '3CNMR spectra wereobtainedfor samples containing albumin, phospholipid vesicles,and
increasing
amountsofcar-boxyl'3C-enrichedfattyacid,all at pH 7.4 and37°C. Sonicated phospholipid vesicleswere chosen as model systems to mimic thefatty acid binding properties ofcell membranes and
lipo-protein surfaces forthefollowingreasons.First, phospholipid modelmembranesystems haveaffinities for fattyacidssimilar
tothose ofredbloodcellsandplatelets (10). Secondly,the
13C
carboxyl
resonancesforfatty
acidsbound tophospholipidvesi-cles are narrow,unlike those forfatty acids boundto redblood
cells.
Finally,
carboxylresonancesforfatty acidsboundto vesi-cles,which occurat 179 ppm atpH 7.4, arewell resolvedfrom those for fatty acids bound to human serum albumin
(181.6-183.8 ppm). Thebiological relevance of vesicles as a
modelfatty acidacceptorwas assessed by comparing the results
using
vesiclesasacceptorstothoseobtainedusing
nativeaccep-tors(isolatedhuman lipoprotein fractions and whole human
plasma).
The carboxyl/carbonyl regionof'3CNMRspectrafor
sam-plescontaininghumanserumalbumin, phospholipid vesicles,
andincreasingamountsofcarboxyl '3C-enriched palmitateare
shown in Fig. 1. Withnoadded palmitate (Fig. 1 A), several
resonances were observed in addition tothe broadcarbonyl
fringe centeredat175ppm.The latterrepresentsnatural abun-danceresonancesfrom the carbonyl and carboxyl carbons of the protein polypeptide backbone and aspartate side-chain resi-dues (16, 17). Theresonance at181.0ppm,designatedg, repre-sents glutamate side-chain carboxyl carbons of albumin (3),
and thenarrowdoubletat173.6 and 173.3ppm,thecarbonyl
carbons ofphosphatidyl-choline molecules ontheouterand inner monolayer leaflets, respectively, of phospholipid
vesi-cles (18).
Whenthefattyacid/albumin mole ratio in the samplewas
1:1 (Fig. 1 B), a partially resolved triplet was observed at
182.14, 181.82, and 181.58 ppm, in additionto the peaks noted above. These three resonances (designated peaks
fl,
y,and#,' respectively) werealsoseenin samples containing
pal-mitate and human albumin but nophospholipid. Theywere
assigned to the carboxyl carbons of palmitate bound to the three highest-affinity fatty acid binding sitesonhumanserum
albumin based onindependent results presented elsewhere (D. P. Cistola and J. A. Hamilton, manuscript submitted for
publication).
Resonancesfl,
y,andf3areanalogous
topeaks b,d, and b' that represent fatty acids boundtothe threehighest
affinitybinding sitesonbovineserumalbumin (3, 4). Withan
increase in sample fatty acid/albumin mole ratioto2/1(Fig. 1 C), peaks ,B, y,and, increasedinintensity,and anadditional resonance wasmarginally detectedat 179.1 ppm (designated peak4). At higher mole ratio values (Fig. 1, D-F), peak4 was
unequivocally observed and increased inintensitywith in-creasing mole ratio. The chemical shift ofpeak b was identical
to apeak observedinsamples containing palmitate and phos-pholipid vesicles, but no albumin, and was assigned to the car-boxyl carbons of palmitate associated with the phospholipid bilayer (see Fig. 2 and reference 1 1).
Todetermine whether peak4 wasreproducible at 2:1 mole ratio and to improve its signal-to-noise ratio, an NMR
spec-trum wasacquired for a sample prepared in an identical
man-ner to that shown in Fig. 1 C, except that four times more NMR transients were acquired. A small, but clearly defined reso-nance at 179.0±0.1 ppm was observed (spectrum not shown). In addition, an NMR spectrum for a third, identically prepared sample also exhibited a resonance corresponding to peak4 at 179.1±0.1 ppm.
To determine the ionization state of fatty acids correspond-ing to peak 4, an NMR titration curve was obtained for palmi-tateassociated with phospholipid vesicles under conditions
identical to those used in Fig. 1, except that no albumin was present. This titration curve and a selected NMR spectrum are shown inFig. 2. The carboxyl chemical shift of palmitic acid/ palmitate bound to phospholipid vesicles increased from 175.9 ppm at pH 3.3 to 181.2 at pH 10.5andexhibited a sigmoidal
titrationcurvewithan apparent
pK.
value of- 7.3. Thechemi-cal shift at pH 7.4 was 179.0 ppm, essentially identichemi-cal to that forpeakq inFig. 1.Thisresultindicated that, in samples
con-taining both albumin and vesicles, - 58% of the fatty acid
albu-186 182 178 174 170
Chemical Shift(ppm)
Figure 1.Carboxyl/carbonylregion of 50.3-MHz'3CNMRspectra for samplescontaininghumanserumalbumin,sonicated
phospho-lipidvesicles,andincreasing quantities of carboxyl'3C-enriched
pal-mitate,allatpH 7.4 and370C.Thenumericalratios above the right edge of each spectrum indicate the total mole ratio ofpalmitateto
humanserumalbumin in each sample. The letter g denotes natural abundanceglutamate carboxylresonancefrom humanserum albu-min (see Results). The lettersfl, y, and , denote carboxylresonances
of'3C-enriched palmitateboundtothe threehigh-affinity binding
siteson human serumalbumin.Athighermoleratio values, peak oy may also contain acontribution fromanoverlappingresonance
representing fattyacid boundtomedium-affinity bindingsites (peak
y';
D. P.Cistola and J.A.Hamilton,manuscriptsubmitted forpubli-cation).Theresonancelabelled 4 representspalmitateassociated with
phospholipidvesicles. The doubletat173.6/173.3 ppm corresponds
tophospholipidcarbonyl carbons in theouterandinner monolayer
leaflets,respectively, ofphospholipidvesicles.NMRspectrawere
recorded after2,000 accumulationsusing900pulses andapulse in-terval of 4.82s.The T. values forpeaks
#/'y/j'
and Xwere1.1±0.1 and0.9±0.1 s,respectively,andthe NOE valuesfor all of thesepeakswasidentical (1.4±0.1).Alinebroadeningfactor of 3 Hzwasused in spectral processing. Spectra shown inDandE wererecorded after
4,000accumulations. Theconcentrations of humanserumalbumin andphosphatidylcholinewere 47mg/ml, each.
181.0-
180.0-t2L 184 160 176 172 166
,179.0- CHEMICALSHFT (PPM)
IJ tpK,-7.3
178.0-I
177.0-
176.0-2.0 3.0 4.0 5.0 6.0 7.0 8..0 id.o 11.0 pH
Figure 2. NMRtitration curve for sonicated phospholipid vesicles containing 5 mol% '3C-enriched palmitate at 370C, and a selected
"3CNMRspectrum at pH 7.3 (inset).Corresponding spectra were re-cordedafter 400-1,600 accumulations, and the spectrum shown in the inset,after 1,200 accumulations. Reported sample pH values were correctedtovaluesat370Casdescribed in Methods. Each data point wasderived from a single NMR spectrum.
min binding sites in the same sample, which, at equilibrium, werefully ionized at pH 7.4 (D. P. Cistola and J. A. Hamilton, manuscript submitted for publication).
Toestimate the exchange rates of palmitate between hu-man serum albumin and phospholipid vesicles and between individual binding sites on human albumin, NMR spectra in Fig. 1 were compared with spectra for samples containing
pal-mitateand human serum albumin (but no vesicles) and palmi-tateand vesicles(butnoalbumin). The correspondingcarboxyl resonances had essentially identical chemical shifts and line-widths in all cases. Therefore, palmitate appeared to be in slow exchange, on theNMRchemical shifttime scale, between
bind-ingsites on humanalbuminandphospholipidvesicles(<< 140
exchangespersecond)andbetweenindividual siteson human albumin (4 10exchanges per second). No detectable changes wereobservedinthese exchange rateupper limits with
increas-ingfatty acid/albumin moleratio.
Toexamine theeffectof small decreases in pH on the distri-bution offatty acids,13C NMR spectra were obtained for sam-plescontaininghuman serumalbumin,phospholipid vesicles, andpalmitateatpHvalues of7.4, 7.2,7.0,and 6.8(Fig. 3).The fattyacid/albuminmole ratiointhesamplewas 4:1.This mole ratio value was chosen because the ratio of plasma free fatty acidstoalbumininpatients with diabetic ketoacidosis averages
4(19-21).WithdecreasingpH, the relativeintensityofpeak X increased, indicative of anincreasingfraction ofpalmitate
associated withphospholipidvesicles. Inaddition,the
chemi-cal shift of
peaks
decreased from 179.1 ppmatpH7.4to177.3ppm atpH6.8, consistent withachangein theionizationstate
ofpalmitate bound to phospholipidvesicles(referto Fig. 2).
The percentage ofpalmitate bound tophospholipid vesicles
thatwasionized
(anionic)
was58%,46%, 37%,
and 27%atpH7.4, 7.2, 7.0, and 6.8, respectively. In contrast, the chemical
shifts of peaks
Al,
y,and 'didnotchangebetweenpH7.4 and6.8.Therefore, essentially 100% of thepalmitateboundto
hu-man serumalbuminatequilibriumwasanionicatallfourpH
values, consistent with ourprevious studies with bovineand human albumins (reference 4 and D. P. Cistola and J. A. Hamilton, manuscript submitted for publication).
Toquantitate the percentage offattyacids associatedwith non-albumin components, theintensities(areas)ofindividual
carboxylresonances were obtainedbyLorentzianspectral
sim-ulation and deconvolution (see Methods). Since NMRpulse intervals were chosen to be > 4 x T1 forpalmitate carboxyl resonances(longest T. = 1.1 s), and since nuclear Overhauser enhancement values for the observed fatty acid carboxyl
reso-nances wereequal (NOE = 1.4),the relative intensities of pal-mitate carboxyl resonances could be directly converted into relative mole quantities ofpalmitate bound to albumin binding
I I I I I I I I I 1
186
182
178 174 170Chemical
Shift
(ppm)
Figure3. Carboxyl/carbonyl region of50.3-MHz'3C NMRspectra
forsamples containinghumanserumalbumin, sonicated
phospho-lipidvesicles,and'3C-enriched palmitateatfourdifferentpH values.
Allspectrawereaccumulatedat37°C forasinglesamplewitha
palmitate/albuminmoleratioof4/1. Spectrawererecordedafter 2,000accumulationsand processed with 3-Hz linebroadening.
Chemical shift values inppmarenoteddirectlyabove peak0in each spectrum. All othernomenclatureasinFig. 1legend.
co 0.5
10
co 0
' 0.4m
I 0.3
0 0 co
r'
0.2IL
0-0
C.0
IL .U0.0
pH 6.8A 16:0 18:1 pH 7.0A
pH 72
pH 7.4
D 1 2 3 4 5 6 7 8 FA/Albumin MoleRatio
Figure4.Plotsof the fraction offattyacid(FA)moleculesassociated withphospholipidvesiclesas afunctionof the totalsamplefatty acid/albuminmole ratio andpH.Thefractions offattyacid molecules associated withphospholipidvesicleswerecalculated from the relative intensities of'3CNMRcarboxylresonancescorrespondingtofatty
acids boundtobindingsitesonvesicles and albumin. Peak intensities werequantitatedusing Lorentizianlineshape analysesof spectra shown inFigs. I and3 and other spectranotshown. Each data point wasderived fromasingleNMR spectrum. The data forpalmitateat
2/1 mole ratiowas notobtained from the spectrum shown inFig. 1 C, but from asimilar spectrumcontaining 8,000transients anda
twofold greatersignal-to-noiseratio. NoT.orNOEcorrectionswere
requiredto convertrelativepeakintensities into relative mole quan-tities offattyacids. Allsamplescontained humanserumalbumin(47
mg/ml),phospholipidvesicles (47mg/ml),anddifferent types and amounts of '3C-enrichedfattyacids.All NMRspectrawereobtained
at37°C and pH 7.4, except where noted otherwise.(-) Palmitate, (-)
oleate,(0) stearate, (A)palmitateatfour differentpHvalues. (o)
Samplecontainingpalmitateandalowerconcentration of
phospho-lipid vesicles(24 mg/ml). The linedesignated 16:0 representsalinear least squares fitof the solidcircles,withaslopeof0.068,acorrelation coefficient of0.99,andanx-interceptof 0.97. The linedesignated
18:1 representsalinear least squares fit of the solid squares, witha
slope of0.063,ancorrelationcoefficient of0.99,andanx-intercept
of1. 1.Theestimated uncertainties inxand y valuesare±0.1 and ±0.02,respectively.
sites andphospholipid vesicles.3Aplot of the fraction of total
fatty acidsassociated withphospholipid vesicles as a function of total samplefattyacid/albumin mole ratio and pH is shown in Fig. 4.The fraction of palmitate associated with
phospho-lipid vesiclesincreased from 0.07 at2:1 mole ratio to 0.34 at 6:1 moleratio,all at pH 7.4 (Fig. 4, 0).A least squares fit of these
points yieldeda straight line with aslope of0.068, a correlation
coefficientof 0.99,and anx-intercept of 1.0. Data for similar systemscontaining oleate (Fig. 4, *) or stearate (Fig. 4, 0) yieldedresults qualitatively andquantitatively similar to those ofpalmitate (Fig.4, 0). In addition,the percentage ofpalmitate
associated with phospholipid vesicles increased from 0.22 at
3.Inthe present study, no attempt was made to quantitate the intensi-ties ofindividual carboxyl resonances corresponding to fatty acids boundtodistinctbindingsitesonhuman serum albumin, since
signifi-cantuncertantieswereassociated with individual measurements under theseconditions. Rather, Lorentzian lineshape analysis was performed
toquantitatethe total carboxylintensity associated with albumin vs. nonalbumin binding sites. The occupation of individual binding sites
onhumanserumalbuminwasassessedqualitatively by inspection of
NMRspectra.
pH 7.4 to 0.39 at pH 6.8, all at 4/1 mole ratio (Fig. 4, *). For a similar sample containing one-half the concentration of phos-pholipid (24 mg/ml) and a palmitate/albumin mole ratio of 6.0, thefraction of palmitateassociated with the vesicles was 0.28 at pH 7.4 (Fig. 4, 0).
To determine whether the distribution of fatty acids was
affectedby the typeof non-albumin fattyacid acceptor used,
"3C
NMR spectra were accumulated for samples containing human serumalbuminandnativehuman HDL with increas-ing amountsofcarboxyl 13C-enrichedpalmitate, all at pH 7.4 and 370C. The carboxyl/carbonyl region ofthese spectra, showninFig.5, weresimilarto those shown in Fig. 1, exceptforthefollowing. First,the narrow resonances between 171.2 and 173.9 ppm represent carbonyl carbons of triglycerides,
cholesteryl esters, andphospholipids in HDL (22). Secondly,
186 182 178 174 170
Chemical Shift (ppm)
Figure 5.Carboxyl/carbonylregion of 50.3MHz"3CNMRspectra for samplescontaininghumanserumalbumin (47mg/ml),native human
HDL(24 mgprotein/ml), andincreasing quantitiesof
'3C-enriched
palmitateatpH7.4(A-D) and pH 7.0(E). Peakarepresents
palmi-tateassociated withlipoproteins. Where peaks g andaoverlapped,
thecompositepeak is notedasg/a.Unlikebilayeredvesicles(Figs.
1-3),phospholipids in HDL exhibited onlyonecarbonyl carbon res-onancebecause HDL hasamonomersurface. All other nomenclature
asinFig. 1legend.NMRspectrawererecordedafter2,000
accumu-lationsusing
900
pulses andapulse interval of 4.82s.TheT. values forpeaksf3/,y/f'
andgIoT
were 1.1 and 1.0 s,respectively,for thesampleshown inD.
peak g representsglutamate carboxyl carbons fromboth hu-manalbumin and the apoproteins ofHDL. Finally, peak o,
which overlaps with peak g at pH 7.4, represents palmitate
bound to the monolayer surface ofHDL. This assignment is
based on spectra not shown for otherwise identical samples
containing palmitate andHDLbut noalbumin; thesespectra exhibit a major fatty acid carboxyl resonance at 180.8 ppm at pH 7.4.
For samples containing added '3C-enriched palmitate,
peaks y,a,and, were observed(Fig. 5). Asdiscussedabove, peaks yS,a, and , representpalmitate bound to the three high
affinity sitesonhuman serumalbumin.Theintensity of peak g/uo increased with increasing fatty acid/albumin mole ratio, as showninFig.5, B-E,indicating an increase in the amount of
palmitate associated withHDL. Adecrease in sample pH from 7.4 (Fig. 5 D) to 7.0 (Fig. 5 E) resulted in a decrease in the chemical shiftofpeakorfrom180.8 to 180.1 ppm. Since peak g
remainedunchanged at 180.9 ppm, peaks g and awere better
resolved at pH 7.0 than at 7.4.
Qualitatively and quantitatively similar results were
ob-tained for samples containinghuman LDL,rather than HDL, as anonalbuminfatty acidacceptor(Fig.6). Theintensity of
thecarboxyl resonancecorresponding topalmitatebound to LDL(peak v) increasedwith increasing mole ratio and its
chemical shiftdecreased from 180.6 ppm at pH 7.4 to 179.5 ppm at pH 6.8. Peak awas assignedbased on independent
spectrafor samples containing fatty acid andLDL,butno
al-bumin (J.A.Hamilton, personalcommunication).
As observedforsamples containing human serum albumin andphospholipid vesicles,the exchange ratesof palmitate be-tweenalbumin andnativehuman lipoproteins
(<35
s-')
and between individual binding sitesonalbumin(<< 10s-')
were slow on the NMR chemical shift time scale. In addition, changesinfatty acid/albumin
moleratioandsample pHre-sulted in nodetectable changesin the upperlimits forthese rates.
Plotsofthefraction oftotalfatty acidsbound to lipopro-teins (HDLorLDL)as afunctionoftotalsample
fatty
acid/al-buminmoleratio,
are shown inFig.
7.Plotsforsamples
con-taining
HDLat24mg/ml (-)
and 9mg/ml
(U)
werelinear(r
= 0.98and0.99,respectively)andextrapolatedtothex-axisat
amoleratiovalueof1.1. Theseplotswereverysimilartothose
obtained for samples
containing phospholipid vesicles,
rather thanHDL,asnonalbuminfatty
acidacceptors(cf. Fig.
4).For asamplecontaining
LDL,thefraction ofpalmitate
associatedwithLDLwas0.19atpH7.4
(0)
and0.28atpH 6.8(E),
allat a6:1 moleratio.
To assesstheirspectral resolution and appearance in the
carboxyl/carbonyl
region,
samples ofwhole human plasmaandwhole bloodwithadded
13C-enriched
palmitate
werepre-paredandexamined by 13CNMR
(Fig. 8, A-C). Carboxyl
reso-nances
corresponding
tofatty
acidsbound tohuman serumalbumin (peaks
3,
oy,f',
a) andlipoproteins (peak a)
were ob-served and thedegree ofspectral
resolutionwassimilartothat observed for the reconstituted systems shown above. Aspreviouslyobservedfor samples
containing
fractionatedHDL orLDL, resolution ofpeaks
aand gwasimproved by lowering
thepH from7.4to6.8.Aspectrumforasample
ofwhole blood(Fig.
8 D)wassimilartothatofplasma,
exceptforthe greaterintensity of natural abundance carbonyl resonances
resulting
fromhemoglobin
in redblood cells.Carboxyl
resonancesforfatty acids bound to red blood cell membranesare extremely broad andnotobservable underthese conditions.
To quantitate the distribution ofadded
13C-enriched
palmi-tateunder conditions that mimic thefattyacidbinding proper-ties of the intact human circulation, a sample was prepared containing whole human plasma, phospholipid vesicles, and '3C-enrichedpalmitate. This samplewaspreparedsothatthe ratio ofphospholipid vesicle surfaceareapermole ofhuman serum albumin wascomparableto the ratio ofblood andendo-(E)
6/1
pH
6.
0/I
186
182
178 174170
170166
1610
0.5-0.
0
c,, 0.4
:5 3:
-a0.3-.o
,0.
.-0.
° 0.2
'<
0.1C
Of
tv 0.0
-U- 2 3 4 5 6 7 8
FA/Albumin Mole Ratio
8
Figure
7. Plotsof the fraction offatty
acids(FA)associated with humanplasma lipoproteinsas afunction of the total samplefatty acid/albuminmoleratio. The fractions offattyacids associated withlipoproteinswerecalculatedfrom the relative intensities of'3CNMR
carboxylresonances asdescribed in theFig.4legend and in thetext.
Each datapointwasderived fromasingleNMRspectrum.(.) Sam-ples containingHDLataconcentration of 24 mg protein/ml and correspondtovalues obtained from spectra shown inFig. 5,B-D.(i) Samples containingHDL at aconcentration of 9 mgprotein/mland correspondto NMRspectranotshown. (oo)Asamplecontaining
LDLat aconcentration of 12 mgprotein/ml,and spectra shown in Fig. 6, DandE, respectively. (A) Data fromanNMRspectrum of whole plasma from a healthy,nondiabetic human donor containing added'3C-enriched palmitateandaccumulatedatpH 7.4 and370C.
This spectrum was similar to that shown in Fig. 8B.(v)Datafrom
an NMRspectrum of thesameplasma sample with added
'3C-enrichedpalmitate,except that itwasaccumulatedatpH 6.8. This spectrumwassimilartothat shown inFig. 8 C. The plasma fatty
acid/albuminmoleratiobefore the addition of"C-enrichedfatty acid was0.5:1. Other laboratory values were as follows: total cholesterol, 275mg/dl; HDL cholesterol, 49 mg/dl; triglycerides, 156 mg/dl;
al-bumin,5.2 g/dl.
thelial cell membrane surface area per mole of albumin found in the normal human circulation.4
`C
NMR spectra for thissample (not shown)weresimilar to thoseshown inFigs. 8,A-C andexhibited carboxylresonancescorrespondingtopeaks
#,
y,ff,
a, a, and0.
At apH valueof7.4and a moleratiovalueof 5.9,the totalfraction ofpalmitatebound tononalbumin com-ponents(lipoproteinsandmembranes)was0.31 (Fig. 7, V).In the same plasma sample containing no added phospholipid vesicles,thetotalfraction of palmitateassociatedwith non-al-bumin components(lipoproteins only)was0. 13(Fig. 7, A).Discussion
Wehave used anovel
"C
NMRapproachtodeterminedtheequilibrium
distribution of"C-enriched
fattyacids in reconsti-tuted and whole human plasmaand bloodunder conditionsChemical Shift (ppm)
Figure 6. Carboxyl/carbonyl region of 50.3 MHz13CNMR spectra for samples containing human serum albumin (42 mg/ml), native human
LDL(12mg protein/ml) and increasing amounts of
"3C-enriched
palmitate,atpH 7.4 (A-D) and 6.8(E). All peak nomenclature, sam-ple, and spectral conditions as in Fig. 1 and 5 legends. Spectra for
samplesobtainedat90.6 MHz (not shown) were qualitatively and
quantitativelysimilar.
4.Theratioofphospholipid vesicle surface area per mole albumin in these samples ranged between 0.23 and 0.45x 107m2/mol.Theratio of bloodandendothelial cellsurface areapermolalbumin in healthy humansubjectsis - 0.3 x 107m2/mol. Thesecalculations assumed
thefollowing:meanvesiclediameter,
250A;
normalplasma albuminconcentration,4.2 g/dl; erythrocytesurface area, 163jUm2(reference 15, p.69);meanwhite blood celldiameter, 12
Mm;
meanplatelet diame-ter, 3Mm;
totalendothelial surface area, 1.1x10'
m2 (see reference52).(D)
blood
6/1,
pH
7.4y
(C)
plasma
(B)
plasma
6/1,
pH
7.4(A)
plasma
2/1,
pH
7.4186 182 178 174 170 166
Chemical Shift (ppm)
Figure 8. Carboxyl/carbonyl region of 90.6-MHz'3CNMRspectra of whole human plasma (A-C) and whole human blood (D) with added '3C-enriched palmitate, all at370C. The total fatty acid/albumin mole ratioand sample pH are shown above theright edge of each spectrum. Theresonance labeled c represents carboxyl carbons of citrate, the anticoagulant used in this sample. The plasma and blood samples wereobtained from a healthy, nondiabetic human donor after a 14-h fast.The plasmafattyacid/albumin moleratiopriortotheaddition of '3C-enrichedpalmitatewas 1.0. Otherlaboratory valueswere as
follows: albumin, 4.7 g/dl; total cholesterol, 164 mg/dl;HDL choles-terol,39 mg/dl;triglycerides,65mg/dl; totalbilirubin,0.5mg/dl; total protein 6.7g/dl.
thatmimic steady-state conditions in the normal and diabetic humancirculation. Theeffectoftwovariablesrelevant to the diabetic circulation were examined: fatty acid/albumin mole ratio (asincreased in insulin deficiency and/ornephrosis)and pH(asdecreased in acidosis).
13CNMR has several advantages over other methods for
monitoringfatty aciddistributions(11).First, distributionscan bequantitated undernonperturbing conditions, withoutneed forseparationofcomponentsbycentrifugationor
chromatog-raphy.Earlier studiesutilizingultracentrifugation (23-26) pro-ducedartifactualresults, inpart, becauseof the high salt
con-centrations utilized to separate lipoproteins. Even using "milder" conditions, it was notcertain that the separationof
components did not alter theequilibrium distribution of fatty acids (8). Secondly, physiological conditions oftemperature,
saltcomposition, salt concentration, and albumin
concentra-tioncanbeutilized, includingthe useofwhole humanplasma.
Some studies have beenperformedatnon-physiological
tem-peratures (8, 27), or in the absence of calcium (24-29). De-creasesin calcium concentration below physiological levels alter theconformationofalbumin nearphysiological pH (30)
and may affect its binding properties. Thirdly, structural and kinetic information can be obtained from NMR spectra, such asthe relative occupation of individual binding sites on albu-min,lipoproteins,andmodelmembranes, theionizationstates offattyacidsboundtoeachsite,andestimatesof the exchange ratesof fatty acids between albumin and nonalbumin binding sites(3, 4, 11).
Alimitation ofthis
13C
NMR approach is that fatty acids associated with red blood cells cannot be quantitated. Because of their largediameter, redblood cells tumble slowly in solu-tion, resulting in broad unobservablecarboxyl NMR reso-nances for boundfatty acids. To circumventthisproblem, small unilamellarphospholipid vesicles (diameter - 30 nm)wereused as a model system to simulate the fatty acid binding properties of cell membranes (see Results).
Molecular mechanisms governing fatty acid distribution.
The equilibrium distribution of fatty acids between human serum albumin, model phospholipid membranes,and/or na-tivehuman lipoproteins as a function of total fatty acid/albu-min mole ratio exhibited the followinggeneral pattern. At a moleratiovalueof1,essentiallyallofthefattyacidwas asso-ciated with albumin and distributed among its three highest affinity binding sites (Figs. 1 B, 5 B, and 6 B). At mole ratio values > 1, fatty acidsassociatedwithboth albumin and nonal-bumin components, and the fractionassociatedwith nonalbu-min componentsincreased linearly with increasingmole ratio (Figs. 4 and 7). At the highest mole ratio examined, a large fraction of the totalfattyacid wasassociated withnonalbumin components. The samegeneralpattern offattyacid distribu-tion was foundregardlessofthe type offattyacid acceptor used
(phospholipid vesicles, native human HDL, or LDL) or the type offatty acid used (palmitate, oleate,orstearate).
There-fore,thisdistribution patternappearedtobegoverned primar-ilyby thebinding properties ofhuman serumalbumin,rather thanthe propertiesof aparticular nonalbuminacceptor or
fattyacid.
Twomathematicalmodels have beenoftenused todescribe
the fatty acid binding properties of albumin andto analyse bindingdata. The Scatchard model assumes thatfattyacid
binding sites fall into distinct classes with respecttobinding
affinities. Forpalmitatebindingtohumanserum
albumin,
thehighest affinity class containstwo or three binding
sites,
themediumaffinity class, three to five, and the lowest
affinity
class,>20(reviewedin reference7).Each class is describedbyanaverageaffinityconstant.The Scatchard model is
oversim-plifiedin thatitdoes not accountforpossible
cooperativity
orconfigurational adaptability,and the
validity
ofgrouping
indi-vidualbinding sites intoclasseshas beenquestioned (7, 9).A
secondmodel,the
stepwise
associationmodel,
ismoregeneraland makesno
assumptions
about thegroupings
of sites intoclasses (7, 9). Association constants foreach mole of bound
ligandare
obtained,
ratherthan average constants foraclass ofsites.For
palmitate
binding
tohumanserumalbumin,
thestep-wise association constants decrease, but show no
abrupt
changes,withincreasing fatty acid/albumin
mole ratio(7).
The NMR results supportcertainfeaturesofboth models whileprovidingnewevidence for negative
cooperativity
in theinteractions of fatty acids with human serum albumin. At a
mole ratio value of 1,palmitate wasdistributed amongthree
structurally distinct
binding
sites in thepopulation
ofalbuminmolecules(peaks
#,
'y,
and , inFigs.1 B,5B,and6B). Hence,the NMR resultsprovided
independent physicochemical
ratio-nale for thegroupingofsites into ahighaffinity
class in accor-dancewiththeScatchardanalysis,and alsoindicated thatthere arethree,rather than two,high-affinity
sites forhuman serum albumin (7, 31, 32).However,at amoleratiovalueof 2,before thehighaffinity sites weresaturated,fatty
acidwasalso asso-ciated with nonalbumincomponents,indicating
that theaffin-ity of humanserumalbumin forthe second moleof
fatty
acidper mol albumin was lower than thatofthefirst. Moreover,
plotsof fatty acid distributionas afunction ofmoleratio (Figs.
4and7)intersectedthex-axisat 1,rather than at 0 or3,and
indicated adiscontinuous decrease in the
affinity
of albuminforfatty acid
following
thebinding
of1 molfatty
acidper mole HSA. These findings provide evidence for negativecoopera-tivityin theinteractions of long-chain
fatty
acidswith humanserumalbumin.
Two possible explanationshave been proposedforthe
cur-vilinear Scatchard plots obtained for fatty acid bindingdata(7):
(a) fatty acid binding sitesareheterogeneous, and/or
(b)
fattyacid binding is associated with negative
cooperativity.
These two explanations could not bedistinguished
using previous equilibrium binding data. The present NMR results indicate that,atleastfor humanserumalbumin,
bothexplanationsarevalid.
The uptakeoffatty acidsas afunction of increasing fatty acid/albuminmoleratiohas beenmonitoredinprevious
stud-ies using bovineserumalbuminandErlich ascitestumor cells (9, 33,34).Theobservedpatterns weresimilar insome respects to the distributionpatternsin the present study, exceptthat negative cooperativityat 1:1 mole ratio wasnotreadily
appar-ent.However, areexamination of this earlierwork inlight of
the present results seems toindicate the possibility ofabiphasic pattern, withmostofthedatapoints extrapolatingin alinear fashionto 1:1 moleratio. (See Fig.4of reference 33).
Small decreasesinpHanalogoustothoseseenin moderate
to severeacidosis resulted inanadditional shift in the distribu-tion of fatty acids from albumintononalbumin components.
Forexample,at afatty acid/albuminmoleratio of4, approxi-mately23%, 31%,and39% ofthe totalfatty acidwasassociated with phospholipid vesiclesatpH7.2, 7.0,and6.8, respectively (Figs. 3 and 4). Concurrently, thepercentage of fatty acids
boundto
phospholipid
vesiclesthat wereionized (anionic) de-creasedfrom - 50% atpH 7.4 to46%, 37%,and 25% at pH7.2, 7.0,and6.8, respectively.Incontrast,thefatty acids bound
toalbuminbinding sitesatequilibriumwereessentially 100%
ionizedat all pH values between pH 7.4 and 6.8.Basedon these
distributionandionizationstatevalues, amechanismthat
in-volves a muchhigher affinity ofprotonated fatty acidsfor
phos-pholipid vesicles relative to human serum albumin seems
likely. Analysisoftheseand otherrelated resultsusing mathe-matical models will help to further evaluate thisproposed mechanism. As has beensuggested, small decreasesin pH in the extracellularfluids in certain tissues may promotethe up-take of fatty acidsby cells byalteringthedistributionof fatty
acids between albuminand cellmembranes
(18,
34).Implications
forfatty
acidtransportanduptake
invivo.Ithas often been assumed that >99% of the
circulating
fatty
acidsarebound toalbumin under normalconditions in
hu-mans
(7,
8).
The presentNMRresults supportthisassumption
as
long
asfatty acid/albumin
mole ratio values remain < 1. It has also beensuggested,
based onthe concept ofthreehigh
affinity
fatty
acidbinding
sites,
thatfatty
acid/albumin
moleratio valuesmustexceed threeorfour before
lipoproteins
and cellmembranescaneffectively
compete forfatty
acidbinding
(1,
7,
9). However,
the present NMR resultsindicatethatlipo-proteins
and membranescan compete forfatty
acidbinding
when thefatty acid/albumin
mole ratiovalueexceeds 1, before the three"high-affinity" binding
sites arecompletely
filled.Therefore,
thethreshold moleratiovaluefor alterationsinfatty
acid distributions in the human circulation appearsto be
1,
rather than 3.This finding has important implications forboth normal andabnormaltransportof
fatty
acidsin thecirculation.Undermost
physiological conditions,
steady-state ratios ofplasma
fatty
acidstoalbuminare < 1(7,
35,
36). Although
the distri-butionoffatty
acids (governed bythermodynamic
processes)isheavily weighted
towardalbuminbinding,
efficientremoval offatty
acidsfromthecirculationoccursbecauseofrapid
kineticprocesses, that
is, rapid
dissociation from albuminandclear-ance from the circulation
(7,
9). However,
the ratios offatty
acidstoalbuminin
plasma
cantransiently
exceed 1 inhealthy
individuals, particularly following
prolonged exerciseand/or
fasting (19,
37,
38).
In this case, there may be an additionalthermodynamic
contributiontofatty
acidclearance.Thiscon-tribution,
mediatedby
thenegative cooperativity
property of humanserumalbumin,wouldtransientlyshiftthedistribution offatty
acids from albumintowardcellmembranesandfacili-tate their clearance from the circulation under conditions
where
fatty
acids arebeing
rapidly metabolized by tissues. Therefore, the negative cooperativitypropertyof albuminpro-videsafine
tuning
capability that allows adjustment of its affin-ities forfatty
acidstothepresentmetabolic needs. Suchanega-tive
cooperativity
and finetuning
capability is analogous tothat observedforoxygeninteractions with
hemoglobin.
In severalabnormal conditions, steady-statefatty
acid/al-buminratiosinplasmabecomeacutelyorchronically elevatedabove
L.'
Chronic elevationstovaluestypically between 1 and 2 occurin obesity (21, 35, 36, 39), hypertriglyceridemia (40),andinsulin-treated diabetes mellitus (21, 36).Acuteelevations
occur in uncontrolleddiabetes mellitus (19-21, 36),
gram-negative infection and sepsis (41), nephrotic syndrome (24), hyperthyroidism (35), myocardial infarction (42, 43), and acute emotional orpsychological stress (44-46). The highest moleratio valuesareobservedin uncontrolleddiabetes, where
valuesoften exceed 2 and occasionally reach 6 (19-21, 36). In
patients with diabetic ketoacidosis complicated bynephrosis,
mole ratiovalues may exceed 6, and pH values below 7.2 are not uncommon.
5. Theplasma fatty acid/albuminmole ratio values reported here are based on an assumednormal plasmaalbumin of 4.2 g/dl. Sinceplasma fattyacidmeasurements neglectthe potential fatty acid fraction asso-ciated with blood and endothelial cell membranes, it is likely that
plasma fattyacid/albumin mole ratio values underestimate the total
Extrapolating from thepresentin vitro NMR results, we
predict that a substantial fraction (up to 50%) ofthe total circu-latingfatty acid pool may associate with plasma lipoproteins and the membranes of blood cells and vascular endothelial cellsduring uncontrolled diabetes in humans. The degree of fatty acid association with nonalbumincomponentsdepends
onthe value of thefatty acid/albumin mole ratio and the
pres-enceand severity of acidosis, in accordance with previous
re-sults(7). However,wealsopredict that significant alterations in fatty acid distributions may occurinlessextremeconditions such asobesity, nephrotic syndrome, and diabetes controlled withconventional insulin therapy.
Possible consequences ofabnormalfatty acid distributions.
Fatty acidsare known to exert avariety of effects on cellsin
vitro. For example, fatty acids inhibit neutrophil chemotaxis (47), enhance platelet aggregation (48, 49), and increase the transfer of macromoleculesacrossendothelialcellsinculture (50). In addition, fatty acids have been recently shownto
in-hibit the bindingof LDL to human fibroblastLDLreceptors (51). Interestingly,the inhibitionwas observed atfatty acid-al-bumin mole ratios atand above 2, which corresponds to the values where abnormal distributionswere observed in the pres-entstudy.Such afatty acid-mediatedinhibitionof LDLuptake could haveimportant pathological consequences in abnormal states suchasdiabetesmellitus.
Acknowledaments
The authors thank Mr. Ronald P.Coreyand Ms.CherylOliva of the
Biophysics
Core Cfacility
forperforming gas-liquid
andthin-layerchro-matographic
analyses,
and Mr.MichaelGigliotti
forisolation oflipo-proteins
fromplasma.
We also thankMr.DonaldGantzfor electronmicrographs
oflipoprotein
and vesiclepreparations
and Dr.JamesA.Hamiltonfor
helpful
discussions.This researchwas
supported by
U.S.Public HealthService grants HL-26335andHL-07224.Aportion
of this workwasperformed
usingNMRinstrumentationattheFrancis Bitter NationalMagnet
Labora-tory, Massachusetts Institute ofTechnology,
Cambridge,
MA,sup-ported
by
National Institutes of Health Grant RR-00995.Dr.Cistolawasthe
recipient
of the Andrew Costello Research Fellowshipof theJuvenile Diabetes Foundation International.
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