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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

(2)

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 spectra

forsamplescontaining 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 peaks

ft,

y,

and

if',

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, peak

l),

lipoproteins (180.7 ppm, peak

a),

and loweraffinitysites on albumin (183.8 ppm, peak

ca,

181.9 ppm, peak

Y),

were ob-served. Theintensity ofpeaks 4 andaincreasedwithincreasing

moleratio 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 mediated

primarily

by

serumalbumin,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 cellular

mem-branesalso havea

high affinity

for

fatty

acids

(8, 10)

and may,

under certain normal orabnormal

conditions,

competewith albumin for fatty acid binding. Such conditions could include elevated

circulating fatty

acid levels

(secondary

touncontrolled diabetes

mellitus,

myocardial infarction, hyperthyroidism,

or

sepsis),

decreased albumin levels

(secondary

to

nephrotic

syn-drome, liver diseases,

genetic defects),

and/or altered albumin

binding properties (secondary

to

acidosis, drug administration,

nonenzymatic glycosylation,orhyperbilirubinemia). Themost severeabnormalities in

fatty

acidtransportare

expected

in dia-betic ketoacidosiscomplicated by

nephrosis,

since large

eleva-tions in

circulating fatty

acidsare

superimposed

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 may

contrib-ute to the

pathogenesis

of vascular

complications

indiabetic

subjects.

However, the in vivo

significance

of these effects

re-mainsunknown, largely because the distribution of

fatty

acids

in 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 for

1.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

(3)

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. Thesample

pHwasadjustedto7.4, 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

(4)

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 mole

ratio on the equilibrium distribution of

fatty

acids between binding sites on human serum albumin and model

mem-branes, '3CNMR spectra wereobtainedfor samples containing albumin, phospholipid vesicles,and

increasing

amountsof

car-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

resonancesfor

fatty

acidsbound tophospholipid

vesi-cles are narrow,unlike those forfatty acids boundto redblood

cells.

Finally,

carboxylresonancesforfatty acidsboundto vesi-cles,which occurat 179 ppm atpH 7.4, arewell resolved

from 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

vesiclesasacceptorstothoseobtained

using

native

accep-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).

Resonances

fl,

y,andf3are

analogous

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. The

chemi-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

(5)

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 for

publi-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 thesepeaks

wasidentical (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 ppmatpH7.4to177.3

ppm 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%atpH

7.4, 7.2, 7.0, and 6.8, respectively. In contrast, the chemical

shifts of peaks

Al,

y,and 'didnotchangebetweenpH7.4 and

6.8.Therefore, essentially 100% of thepalmitateboundto

hu-man serumalbuminatequilibriumwasanionicatallfourpH

(6)

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

178 174 170

Chemical

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'

0.2

IL

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.

(7)

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, except

forthefollowing. 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 forpeaks

f3/,y/f'

and

gIoT

were 1.1 and 1.0 s,respectively,for the

sampleshown 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(<< 10

s-')

were slow on the NMR chemical shift time scale. In addition, changesin

fatty acid/albumin

moleratioandsample pH

re-sulted in nodetectable changesin the upperlimits forthese rates.

Plotsofthefraction oftotalfatty acidsbound to lipopro-teins (HDLorLDL)as afunctionoftotalsample

fatty

acid/al-buminmole

ratio,

are shown in

Fig.

7.Plotsfor

samples

con-taining

HDLat24

mg/ml (-)

and 9

mg/ml

(U)

werelinear

(r

= 0.98and0.99,respectively)andextrapolatedtothex-axisat

amoleratiovalueof1.1. Theseplotswereverysimilartothose

obtained for samples

containing phospholipid vesicles,

rather thanHDL,asnonalbumin

fatty

acidacceptors

(cf. Fig.

4).For asample

containing

LDL,thefraction of

palmitate

associated

withLDLwas0.19atpH7.4

(0)

and0.28atpH 6.8

(E),

allat a

6:1 moleratio.

To assesstheirspectral resolution and appearance in the

carboxyl/carbonyl

region,

samples ofwhole human plasma

andwhole bloodwithadded

13C-enriched

palmitate

were

pre-paredandexamined by 13CNMR

(Fig. 8, A-C). Carboxyl

reso-nances

corresponding

to

fatty

acidsbound tohuman serum

albumin (peaks

3,

oy,

f',

a) and

lipoproteins (peak a)

were ob-served and thedegree of

spectral

resolutionwassimilartothat observed for the reconstituted systems shown above. As

previouslyobservedfor samples

containing

fractionatedHDL orLDL, resolution of

peaks

aand gwas

improved by lowering

thepH from7.4to6.8.Aspectrumfora

sample

ofwhole blood

(Fig.

8 D)wassimilartothatof

plasma,

exceptforthe greater

intensity of natural abundance carbonyl resonances

resulting

from

hemoglobin

in redblood cells.

Carboxyl

resonancesfor

(8)

fatty 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 and

endo-(E)

6/1

pH

6.

0/I

186

182

178 174

170

170

166

16

10

0.5-0.

0

c,, 0.4

:5 3:

-a0.3-.o

,0.

.-0.

° 0.2

'<

0.1

C

Of

tv 0.0

-U- 2 3 4 5 6 7 8

FA/Albumin Mole Ratio

8

Figure

7. Plotsof the fraction of

fatty

acids(FA)associated with humanplasma lipoproteinsas afunction of the total samplefatty acid/albuminmoleratio. The fractions offattyacids associated with

lipoproteinswerecalculatedfrom 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 this

sample (not shown)weresimilar to thoseshown inFigs. 8,A-C andexhibited carboxylresonancescorrespondingtopeaks

#,

y,

ff,

a, a, and

0.

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

NMRapproachtodeterminedthe

equilibrium

distribution of

"C-enriched

fattyacids in reconsti-tuted and whole human plasmaand bloodunder conditions

Chemical 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 albumin

concentration,4.2 g/dl; erythrocytesurface area, 163jUm2(reference 15, p.69);meanwhite blood celldiameter, 12

Mm;

meanplatelet diame-ter, 3

Mm;

totalendothelial surface area, 1.1x

10'

m2 (see reference52).

(9)

(D)

blood

6/1,

pH

7.4

y

(C)

plasma

(B)

plasma

6/1,

pH

7.4

(A)

plasma

2/1,

pH

7.4

186 182 178 174 170 166

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,

the

highest affinity class containstwo or three binding

sites,

the

mediumaffinity class, three to five, and the lowest

affinity

class,>20(reviewedin reference7).Each class is describedby

anaverageaffinityconstant.The Scatchard model is

oversim-plifiedin thatitdoes not accountforpossible

cooperativity

or

configurational adaptability,and the

validity

of

grouping

indi-vidualbinding sites intoclasseshas beenquestioned (7, 9).A

secondmodel,the

stepwise

association

model,

ismoregeneral

and makesno

assumptions

about the

groupings

of sites into

classes (7, 9). Association constants foreach mole of bound

ligandare

obtained,

ratherthan average constants foraclass of

sites.For

palmitate

binding

tohumanserum

albumin,

the

step-wise association constants decrease, but show no

abrupt

changes,with

increasing fatty acid/albumin

mole ratio

(7).

(10)

The NMR results supportcertainfeaturesofboth models whileprovidingnewevidence for negative

cooperativity

in the

interactions of fatty acids with human serum albumin. At a

mole ratio value of 1,palmitate wasdistributed amongthree

structurally distinct

binding

sites in the

population

ofalbumin

molecules(peaks

#,

'y,

and , inFigs.1 B,5B,and6B). Hence,

the NMR resultsprovided

independent physicochemical

ratio-nale for thegroupingofsites into ahigh

affinity

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 the

affin-ity of humanserumalbumin forthe second moleof

fatty

acid

per 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 albumin

forfatty acid

following

the

binding

of1 mol

fatty

acidper mole HSA. These findings provide evidence for negative

coopera-tivityin theinteractions of long-chain

fatty

acidswith human

serumalbumin.

Two possible explanationshave been proposedforthe

cur-vilinear Scatchard plots obtained for fatty acid bindingdata(7):

(a) fatty acid binding sitesareheterogeneous, and/or

(b)

fatty

acid binding is associated with negative

cooperativity.

These two explanations could not be

distinguished

using previous equilibrium binding data. The present NMR results indicate that,atleastfor humanserum

albumin,

bothexplanationsare

valid.

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 pH

7.2, 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

acidtransportand

uptake

invivo.It

has often been assumed that >99% of the

circulating

fatty

acidsarebound toalbumin under normalconditions in

hu-mans

(7,

8).

The presentNMRresults supportthis

assumption

as

long

as

fatty acid/albumin

mole ratio values remain < 1. It has also been

suggested,

based onthe concept ofthree

high

affinity

fatty

acid

binding

sites,

that

fatty

acid/albumin

mole

ratio valuesmustexceed threeorfour before

lipoproteins

and cellmembranescan

effectively

compete for

fatty

acid

binding

(1,

7,

9). However,

the present NMR resultsindicatethat

lipo-proteins

and membranescan compete for

fatty

acid

binding

when the

fatty acid/albumin

mole ratiovalueexceeds 1, before the three

"high-affinity" binding

sites are

completely

filled.

Therefore,

thethreshold moleratiovaluefor alterationsin

fatty

acid distributions in the human circulation appearsto be

1,

rather than 3.

This finding has important implications forboth normal andabnormaltransportof

fatty

acidsin thecirculation.Under

most

physiological conditions,

steady-state ratios of

plasma

fatty

acidstoalbuminare < 1

(7,

35,

36). Although

the distri-butionof

fatty

acids (governed by

thermodynamic

processes)is

heavily weighted

towardalbumin

binding,

efficientremoval of

fatty

acidsfromthecirculationoccursbecauseof

rapid

kinetic

processes, that

is, rapid

dissociation from albuminand

clear-ance from the circulation

(7,

9). However,

the ratios of

fatty

acidstoalbuminin

plasma

can

transiently

exceed 1 in

healthy

individuals, particularly following

prolonged exercise

and/or

fasting (19,

37,

38).

In this case, there may be an additional

thermodynamic

contributionto

fatty

acidclearance.This

con-tribution,

mediated

by

the

negative cooperativity

property of humanserumalbumin,wouldtransientlyshiftthedistribution of

fatty

acids from albumintowardcellmembranesand

facili-tate their clearance from the circulation under conditions

where

fatty

acids are

being

rapidly metabolized by tissues. Therefore, the negative cooperativitypropertyof albumin

pro-videsafine

tuning

capability that allows adjustment of its affin-ities for

fatty

acidstothepresentmetabolic needs. Sucha

nega-tive

cooperativity

and fine

tuning

capability is analogous to

that observedforoxygeninteractions with

hemoglobin.

In severalabnormal conditions, steady-statefatty

acid/al-buminratiosinplasmabecomeacutelyorchronically elevated

above

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

(11)

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 C

facility

for

performing gas-liquid

andthin-layer

chro-matographic

analyses,

and Mr.Michael

Gigliotti

forisolation of

lipo-proteins

from

plasma.

We also thankMr.DonaldGantzfor electron

micrographs

of

lipoprotein

and vesicle

preparations

and Dr.JamesA.

Hamiltonfor

helpful

discussions.

This researchwas

supported by

U.S.Public HealthService grants HL-26335andHL-07224.A

portion

of this workwas

performed

using

NMRinstrumentationattheFrancis Bitter NationalMagnet

Labora-tory, Massachusetts Institute ofTechnology,

Cambridge,

MA,

sup-ported

by

National Institutes of Health Grant RR-00995.Dr.Cistola

wasthe

recipient

of the Andrew Costello Research Fellowshipof the

Juvenile Diabetes Foundation International.

References

1.Spector,A. A. 1975.Fattyacidbindingtoplasmaalbumin.J.LipidRes.

16:165-179.

2.Brown,J.R.,andP.Shockley.1982.Serumalbumin:structureand charac-terization of itsligand bindingsites.InLipid-ProteinInteractions,Volume 1.

P.C. Jost and0.H.Griffith,editors. JohnWiley&Sons, Inc.,NewYork. 26-68. 3.Cistola,D.P.,D. M.Small,andJ. A.Hamilton. 1987. 13CNMRstudies of saturatedfattyacids boundtobovineserumalbumin.I.Thefillingof individual

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References

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