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CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 629 CLiN. CHEM. 25/4, 629-638(1979) Agarose Gel Electrophoresis Thxi Submit- J.-O. Jeppsson, C.-B. Laurell, and B. Franz#{233}n ters: Department of Clinical Chemistry, University of Lund, Malm#{244} General Hospital, S-214 01 Malm#{228}, Sweden Eva lua- Chester A. Alper, Center for Blood Research, tors: Boston, MA 02115 Donald T. Forman, Evanston Hospital, Evanston, IL 60201 Ernest S. Tucker and Linda Kruse, Scripps Immunology Reference Laboratory, La Jolla, CA 92038 A. Milford Ward, Hallamshire Hospital - Medical School, Sheffield, England * Present North Carolina Memorial Hospital, Chapel address: Hill, NC 27514 Introduction A system for plasma protein fractionation and for nomen- clature according to charge has been inherited from Tiselius’ technique of moving boundary electrophoresis. The nomen- clature was maintained on transition to zone electrophoresis with filter paper as supporting medium. The clinical use of electrophoresis in plasma-protein analysis is still based on the simple electrophoretic separation of plasma proteins, ac- cording to their relative mobility, into albumin, a1-, a2-, 11-, and ‘y-globulins in spite of the knowledge that each of the classical electrophoretic zones contains two or more major plasma proteins that often are subject to independent meta- bolic regulation. Specific analysis of individual proteins is increasingly used as a supplement to electrophoretic anal- ysis. Thirteen plasma proteins together comprise more than 95% of the serum protein mass. Their distribution on agarose electrophoresis and their average relative concentrations are given in Figure 1. Each classical electrophoretic zone contains between two and six of these 13 components. Some proteins are spread between more than two of the electrophoretic zones. Therefore, scanning the zone pattern gives good in- formation about only two of the predominant proteins, al- bumin and IgG, which represent about 90% of the protein in the albumin and ‘y zones, respectively. With the naked eye it is easier to detect minor mobility and concentration changes (two-dimensional scanning) from comparative electrophoresis on agarose gel with a parallel series of plasma samples than from the conventional one-dimensional scanning diagrams used to calculate proteins in the various zones. With increased complexity in the composition of an electrophoretic fraction there is an increasing risk that the abnormality of a separate component will go undetected by scanning the fraction. Clinical use of electrophoretic results depends on the resolution achieved for the proteins and on the experience of the interpreter. Resolution of the proteins was improved when cellulose acetate, which had lower protein absorption, was substituted for filterpaper as the supporting medium. Techniques such as electrophoresis on starch gel and poly- acrylamide, which add amolecular sievingeffect,have been of great importance in biochemistry. Nevertheless, identifi- cation of components is often complicated if the analyst does not have access to information of clinical diagnostic value. Wieme (1) introduced cooled agarasa support for clinical electrophoresis. The negativelycharged polysaccharide units cause disturbing electroosmotic water flow,and they interact with some proteins. Agar is extracted from certain red seaweeds. It can be fractionated into two fractions, one with a high content of sulfate and carboxyl groups (called agaropectin), the otheran almost neutral fraction (agarose). The medium-endosmosis type of agarose is suitable for protein separation from most biological fluids. The low-endosmosis type may be valuable forseparatingcertainproteinssuch as lactoferrin, lysozyme, and cationic proteins from leukocytes. Electrophoresis on agarose gel with one of two buffer sys- tems, barbituric acid-sodium barbiturate (2) or Tris-barbi- turate (3), is a valuable screening method for detection of abnormal distribution of proteinswithinand between various zones. The agarose electrophoresiscan easilybe combined with immunofixation (4) for identification of M-components where the separation system has higher resolution than is the case for ordinary immunoelectrophoresis. Grossly abnormal concentrations of proteins such as prealbumin, albumin, ai-antitrypsin, a2-macroglobulin, a- and fl-lipoproteins, transferrin, C3-complement, and IgG are detected by visual inspection. Principle The galactopyranose network of a 10 g/L agarose gel allows relatively free electrophoretic mobility, according to charge, of proteins with sizes up to more than 106 daltons. For larger, fibrillar molecules, an increasing resistance to mobility is observed up to five to ten million daltons, where migration is completely inhibited. Thus the gel allows almost free migra- tion of all plasma proteins except chylomicrons, very-low- density lipoproteins, immune complexes, and fibrin(ogen) polymers. The plasma proteins are separated by zone elec- trophoresis at pH 8.6, so that all major proteins (except some IgG) migrate toward the anode if the gel is free from endos- motic water flow. The plasma protein separation achieved at a given volts per centimeter value depends mainly on buffer composition and gel temperature. Increasing the concentration of the buffer from 0.04 to 0.10 mol/L decreases the resolution in the l-’y

Transcript of Agarose Gel Electrophoresis - pdfs.semanticscholar.org · electrophoresis inplasma-protein...

CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 629

CLiN. CHEM. 25/4, 629-638(1979)

Agarose Gel Electrophoresis

Thxi

Submit- J.-O. Jeppsson, C.-B. Laurell, and B. Franz#{233}nters: Department of Clinical Chemistry,

University of Lund, Malm#{244}GeneralHospital, S-214 01 Malm#{228},Sweden

Eva lua- Chester A. Alper, Center for Blood Research,tors: Boston, MA 02115

Donald T. Forman, Evanston Hospital,Evanston, IL 60201

Ernest S. Tucker and Linda Kruse, ScrippsImmunology Reference Laboratory, LaJolla, CA 92038

A. Milford Ward, Hallamshire Hospital- Medical School, Sheffield, England

* Present North Carolina Memorial Hospital, Chapeladdress: Hill, NC 27514

IntroductionA system for plasma protein fractionation and for nomen-

clature accordingto charge has been inherited from Tiselius’technique of moving boundary electrophoresis. The nomen-clature was maintained on transition to zone electrophoresiswith filter paper as supporting medium. The clinical use ofelectrophoresis in plasma-protein analysis is still based on thesimple electrophoretic separation of plasma proteins, ac-cording to their relative mobility, into albumin, a1-, a2-, 11-,and ‘y-globulins in spite of the knowledge that each of theclassical electrophoretic zones contains two or more majorplasma proteins that often are subject to independent meta-bolic regulation. Specific analysis of individual proteins isincreasingly used as a supplement to electrophoretic anal-ysis.

Thirteen plasma proteins together comprise more than 95%of the serum protein mass. Their distribution on agaroseelectrophoresis and their average relative concentrations aregiven in Figure 1. Each classical electrophoretic zone containsbetween two and six of these 13 components. Some proteinsare spread between more than two of the electrophoreticzones. Therefore, scanning the zone pattern gives good in-formation about only two of the predominant proteins, al-bumin and IgG, which represent about 90% of the protein inthe albumin and ‘y zones, respectively. With the naked eye itis easier to detect minor mobility and concentration changes(two-dimensional scanning) from comparative electrophoresison agarose gel with a parallel series of plasma samples thanfrom the conventional one-dimensional scanning diagramsused to calculate proteins in the various zones. With increasedcomplexity in the composition of an electrophoretic fractionthere is an increasing risk that the abnormality of a separatecomponent will go undetected by scanning the fraction.

Clinical use of electrophoretic results depends on theresolution achieved for the proteins and on the experience of

the interpreter. Resolution of the proteins was improved whencellulose acetate, which had lower protein absorption, wassubstituted for filterpaper as the supporting medium.Techniques such as electrophoresis on starch gel and poly-acrylamide, which add a molecular sievingeffect,have beenof great importance in biochemistry. Nevertheless, identifi-cation of components is often complicated if the analyst doesnot have access to information of clinical diagnostic value.

Wieme (1) introduced cooled agar as a support for clinicalelectrophoresis.The negativelycharged polysaccharide units

cause disturbing electroosmotic water flow,and they interactwith some proteins.

Agar is extracted from certain red seaweeds. It can befractionated into two fractions, one with a high content ofsulfate and carboxyl groups (called agaropectin), the otheranalmost neutral fraction (agarose). The medium-endosmosistype of agarose is suitable for protein separation from mostbiological fluids. The low-endosmosis type may be valuableforseparatingcertainproteinssuch as lactoferrin,lysozyme,and cationic proteins from leukocytes.

Electrophoresis on agarose gel with one of two buffer sys-tems, barbituric acid-sodium barbiturate (2) or Tris-barbi-turate (3), is a valuable screening method for detection ofabnormal distribution of proteinswithinand between variouszones. The agarose electrophoresiscan easilybe combinedwith immunofixation (4) for identification of M-componentswhere the separation system has higher resolution than is thecase for ordinary immunoelectrophoresis. Grossly abnormalconcentrations of proteins such as prealbumin, albumin,ai-antitrypsin, a2-macroglobulin, a- and fl-lipoproteins,transferrin, C3-complement, and IgG are detected by visualinspection.

Principle

The galactopyranose network of a 10 g/L agarose gel allowsrelatively free electrophoretic mobility, according to charge,of proteins with sizes up to more than 106 daltons. For larger,fibrillar molecules, an increasing resistance to mobility isobserved up to five to ten million daltons, where migration iscompletely inhibited. Thus the gel allows almost free migra-tion of all plasma proteins except chylomicrons, very-low-density lipoproteins, immune complexes, and fibrin(ogen)polymers. The plasma proteins are separated by zone elec-trophoresis at pH 8.6, so that all major proteins (except someIgG) migrate toward the anode if the gel is free from endos-motic water flow.

The plasma protein separation achieved at a given volts percentimeter value depends mainly on buffer composition andgel temperature. Increasing the concentration of the bufferfrom 0.04 to 0.10 mol/L decreases the resolution in the l-’y

I ALBJ ci J a2

1gA CRP

I IAIb IgG

630 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

GC CIG fiLp 1gM

I .!.\I6 .IcL2M l’c C3

IHp2-1 Tf Fg

Fig. 1. Electrophoretic distribution and relative concentrationof the major plasma proteins compared with the classicalelectrophoretic fractionsAbbreviations: Aib, Albumin; Lp, Lipoproteins; Om, Orosomucoid;a,-At, a1-antitrypsin; a2-M, a2-macroglobulin;GC, group-specific components; !,haptoglobin; GIG, cold-insoluble globulin; Hz, hemopexin, Tf, transferrin; C3,C3 complement; Fg, fibrinogen; Ig, immunoglobuIin; and CAP, C-reactive pro-tein

zone and increasesit in the albumin-a2 zone. If Ca2+ ions areadded to the buffer, resolution of the major a-globulins(transferrin, 1i-lipoproteins, C3, and IgA) is increased. Acooling system aids in balancing the heat production in thegel during the run. After electrophoresis is finished the pro-teins are denatured and made visible by staining. Series ofsamples are run in parallel to facilitate a comparative evalu-ation (Figure 2).

Materials and Methods

Reagents

Agarose SeakemlM (ME), manufactured by FMC Corp.,

Marine Colloids Division, Rockland, ME 04841. Severalbatches with electroendosmosis (-me) 0.13-0.19 were suit-able.’

Electrophoresis buffer: Barbiturate-sodium barbiturate

buffer, pH 8.6, 75 mmol/L, with calcium lactate 2 mmolfL.Prepare the buffer with diethylbarbiturate, 10.35 g in ap-proximately 1 L of distilled water heated to boiling; add 2.88g of calcium lactate and 65.7 g of sodium diethylbarbituratewhen the barbital has dissolved, and dilute to 5 L.

Fixing solution: Add 15 g of picricacid (Merck) to 1 L ofwater. After storage overnight at room temperature, filter thesolution and add 200 mL of concentrated acetic acid.

Destaining solution: Mix 200 mL ofglacialaceticacid,900mL of methanol, and 900 mL of distilled water. This solution

‘Agarose HSA, Litex, Glostrup, DK-2600, Denmark, or Agarosetype A, Pharmacia Fine Chemicals, Piscataway, NJ, are equiva-lent.

J can be reused repeatedly after passage through a column with

activated charcoal to remove the dye.Staining solution: Dissolve 5 g ofAmido Black lOB (Merck)in 2 L of deataining solution by heating to 60 #{176}C.Filter thesolution after cooling to room temperature and before use.

Indicator solution: Dissolve2 g ofbromphenol blue in 100mL of ethanol/water (95/5 by vol).

Apparatus

Electrophoresis chamber with a water-thermostated cell

(5). Available from Behring Diagnostics; Bio-Rad Laborato-ries; LKB, Rockville, MD; or MRA Corp., Clearwater, FL.

Power supply, minimum 300 V and capacity of 150 mA perelectrophoresis cell.

Circulating cooler with flow rate of 1-2 L/min and adjust-able between 0 and 20 #{176}C.

Flexible polyester film, Gelbond, 7 mm thick, availablein rolls from FMC Corp., Marine Colloids Division, Rockland,ME.

Sample application foil, available from LKB, Rockville,MD, or Pharmacia Fine Chemicals, Piscataway, NJ. A similarapplication foil is also included in the precast and prebufferedagarose gel system PanagePTM, from Worthington Diagnostics,Freehold, NJ 07728.

Whatman No 1 filter paper, 1 cm width, in rolls.U-frame, for casting gels, 205 X 110 X 1 mm, available from

Bio-Rad Laboratories or Behring Diagnostics. Glass plate is205 X 1IOX 1.5 mm from Bio-Rad Laboratories or BehringDiagnostics.

Sample-application syringe, 1-5 iL, with stoppers, ob-tainable from Hamilton or SMI.

Electrode wicks, seven layers of Whatman no. 1 papers,“Wetex” cloths, or “Ultra wicks,” from Bio-Rad Laboratories;alternatively, vertical agarose wicks can be used (5).

Collection and Handling of Specimens

Venous blood is drawn into Vacutainer Tubes containingdisodium ethylenediaminetetraacetate (Becton-Dickinson).After centrifugation, the supernatant fluid is stored at 5 #{176}Cif analysisis to be performed within the next two to three days.Storage at -20 to -26 #{176}Cwill disturb some of the patterns,but at -70 #{176}Call the native electrophoretic pattern is pre-served. Serum may be used if analyzed on the day of bloodsampling.

Procedures

Casting the gel. Agarose solution is prepared as a 8 g/Lsuspension of agarose in barbital buffer. Heat to boiling todissolve, using an electric heater equipped with a magneticstirrer. Cool the clear solution to about 50 #{176}Cand pipet it intoa mold consisting of two thin glass plates enclosing a Gel-bond1Mpolyester sheet. Spray 0.5 mL of ethanol/water (95/5by vol) on one glass plate and place the polyester sheet ofsimilar size with its hydrophobic side down. (This is tested byseeing the behavior of a drop of water on both sides; the waterwill “spread” on the hydrophilic surface, whereas it will “bead”on the hydrophobic one.) Make sure that no air bubbles re-main between the plastic sheet and the glass plate. Place theU-frame and then the second glass plate, and clamp themtogether with strong paper clamps. Pour 20 mL of warmagarose solution between the plates. Allow the agarose to so-lidify, cover the open upper edge with tape, and store in therefrigerator until use. Plates can be stored for at least a week,but resolution is better if the agarose plates are prepared onlyone day before use.

Sample application. Apply a slip of Whatman No 1 filterpaper about 2.5 cm from one long side of the gel where thesamples will be applied. Remove the strip immediately after

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

CLINICAL CHEMISTRY, Vol. 25,No.4,1979 631

Fig. 2. Examples of patterns

1. Slightly increased a-Iipoprotein, Hp 2-2, and increased CAP.2. Decreased a-lipoprotein andstrongly increased 3-llpoproteln.3. Increased a-lipoprotein.4. Increased acute-phasereactants and fast albumin(penicillin).5. Hypoalbuminemia,decreased a-lipoproteln and acute phase reactants,

hypotransferrinemia.and increased polyclonal IgG(liver cirrhosis).6. Increased acute-phasereactants with strongly increased polyclonal lgG.7. Normalpattern.8. Hypoalbuminemia,increased proteins of high relative molecular mass

(glomerular protein losses).9. Lipolysis, increasedacute-phasereactants.

10. GenetIc variant, double C3.11. Decreased Hp and C3. Increaseda2M and strongly increased polyclonal

lgG(systemic lupus erythematosis).12. Normal pattern.

justtouching the geland drying the surface (Figure 3). Thesample-application foil is placed on top of the gel with the slits

in the middle of the wiped area.Apply the applicationfoilwiththe side downwards where the edges of the slits protrude. Add20 iL of bromphenol blue solution to 0.5 mL of plasma, anduse this sample as a marker. This color will make the albuminfraction visible during the run. With the sample-applicatorsyringe, apply 2-4 iL of plasma over each slit, withouttouching the gel surface (Figure 4). The sample solution willbe absorbed by the gel within 4 to 5 mm. Peel off the foil whenthe last sample has beencompletely absorbed.

Elect rophoresis. Immediately transfer the gel to the coolingplate in the electrophoresis cell. Spread 5-6 mL of nonionicdetergent solution along one side of cooling surface and placethe plate with one long sideincontactwith thewater and thenslowly lower it until it rests completely on the cooled surface.A thin film of water, free of air-bubbles, should remain be-tween the cooling plate and the polyester sheet.

Contact wicks forming bridges from buffer vessels to the

gel should extend 10 mm onto the gel.

Fig. 3. Fast drying of the agarose gel surface by use of aWhatman no. 1 filter paper strip

13. Normalpattern.14. Increasedpolyclonal IgA and strongly increased cathodal 190.15. DecreasedHp. strongly increased polyclonal anodal lgG (lgG3).16. M-component. IgA.17. Increaseda-lipoprotein and IgA (alcohol abuse).18. Increased a2-macroglobulin (child).19. Normal pattern.20. Serum samplewithhypoalbtsninemia, decreaseda-lipoproteinsandacute

phasereactantsandstronglyincreasedpolyclonal lgG(livercirrhosis).21. Strongly increased acute phase reactants with increased polyclonal lgG.22. M-component, IgG,cathodal to fibrinogen.23. a1-Antitrypsin variant (P1-typeFM) increased IgA.24. Normal pattern.

Cool water should be circulated through the cell and ad-justed to 10-14 #{176}C,to keep the geltemperature during runningnear room temperature. Electrophoresis is done at 20 V/cmin the gelfor45 (40-60) mm, when the blue-stained albuminband has migrated 5.5 cm.

Fixing, drying, and staining. Immediately transfer theplate to the picric acid-acetic acid solution for 15 mm. Coverthe gel with wet Whatman no. 1 filterpaper, taking care toavoid air bubbles between paper and gel. The drying time canbe shortened by applying a 1-cm-thick layer of paper towels,upon which is placed a glass plate and a weight of 1-2 kg forabout 10 mm. Remove the softpapers and dry the gel in astream of hot air.

Then place the dried gel in the staining solution for 10 mm.Rinse the gel plate in tap water and soak it in two baths ofdestaining solution during 10 mm until the background iscolorless. Finally, dry the plate again in a stream of hot air.

Technical Discussion

Electrophoresis of plasma proteins does not require a highlypurified agarose. Most important is reproducible quality frombatch to batch. We therefore recommend that a one- to two-year supply be stocked. Most agarose qualities are not as re-producible as that of Seakem ME. If other batches of agaroseare used itisadvisable to perform test runs with agaroseconcentrations ranging from about 7 to 12 g/L, to ascertainoptimum separation conditions and efficient tensile strengthforeasy handling of the gel.A good indicatorof resolutionisthe distinctness of the group-specific globulin bands in frontof the a2-macroglobulin fraction(Figure1). The mobilityoflipoproteinsand C-reactiveprotein is related to the agarosequality. Use of the Gelbond polyester film forgelsupport fa-cilitates handling and storing the finished product.

The sample-application system we recommend is thatsuggested by Cawley (6) and simplifies the procedure ascompared to the well-forming system (5). The application foilis rinsed in isotonic saline and can be reused 100-200 times.

Effective cooling by good contact between the geland thecooled surface is important. A potential gradient in the gel of15-20 V/cm may be used in most apparatuses. This corre-sponds toa totalfield strength of 250-300 V and a current of

Prealbumin

a1-Antitrypsin

GC-globulins

<1-1 -5

0.40-1.05

0.8-1.90.2-0.55

0.2-0.7

0.22-0.461.2-2.4

0.40-2.9

0.1-0.3

1.7-2.7

2.2-7.4

2.2-4.40.6-2.26.5-15.5

<0.00 1<0.02

632 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

Fig. 4. Application of 2 to 4 zL of plasma in the slits of theoverlayered application foil

Table 1. Range of Normal VarIation, for Adults, ofPlasma Proteins InfluencIng the Electrophoretic

Plasma Protein PatternProtein Normal rang, g/L

0.15-0.36Albumin 39-51a-Lipoproteins (3-7)

(a_Fetoproteins)a(Orosomucoid)

(Inter-a 1-trypsin inhibitors)

(Ceruloplasmin)

aMacrogIobuIinsHaptoglobins (1-1, 2-2, 2-1)Cold-insoluble globulinTransferrin

fl-LipoproteinsC3, third component of complement 0.6-1.4

100-140 mA, depending on the conductivity of the electrodewicks.

Resolution can be slightly improved if gel thickness is de-creased to 0.5 mm and the potential gradient in the gel in-creased to 30-35 V/cm. However, most commercially availableequipment has too low a heat conductivityto give good resultsat voltages higher than those recommended here.

The temperature of the cooling water must be adjusted toexclude overgeneration of heat, which is detected by thecondensation of water on the underside of the glass plate thatcovers the electrophoresis chamber. Overcooling, however,gives rise to fluid drops on the gel surface, resulting in ablurred band pattern. The buffer can be reused several times,but the polarity of the current has to be changedevery run toavoid pH shifts in the buffer vessels. Albumin focuses in arelatively narrow band, and the high local protein concen-tration makes it important to have an effective precipitatingagent such as picric acid, to avoid blurring of the pattern.Amido Black lOB is used because it gives distinct zones andfacilitates the visualization of a- and -lipoprotein. CoomassieBrilliant Blue is two to three times more sensitive and is ofgreat value when one is analyzing a more dilute protein solu-tion.

Interpretation of the Agarose GelElectropherogram In Terms of Variation ofIndividual Proteins

The ability of the agarose gel electrophoresis to reveal dif-ferent proteins varies with protein concentration, dye-bindingcapacity, and degree of electrophoretic homogeneity. Thedye-binding capacity generally decreases with increasing ol-igosaccharide and lipid content. Figure 1 gives a schematicpresentation of the relative concentration and the electro-phoretic heterogeneity of the major plasma proteins in ahealthy subject having the most frequent phenotypes.

The individual concentration-mobility profiles are drawnto indicate the degree of electrophoretic heterogeneity and toshow the discrepany between the demarcation of electro-phoretic fractions and the range of individual proteins.

Table 1 lists the individual proteins that may be present insuch a concentration that they influence the electrophoreticpattern. Those given in parentheses normally are present insuch low concentration that they do not influence the patternsignificantly. Examples of abnormal electrophoretic proteinpatterns are given in Figure 2.

IgA 1.0-3.0Fibrinogen

(1gM)

lgG(Light immunoglobulin chains)

(C-reactive protein)

a componentsin parenthesesmay influence the pattern visibly when theirconcentration increasesin disease.

The Prealbumin Zone

The proteins in this zone are barely discernible, diffuselydemarcated, and vary slightly in mobility. It is difficult todetect any proteinsif the pattern is not inspected against awhite background under reflected light. Normally the thy-roxine- and a2-microglobulin-binding prealbumin is the onlyprotein discerned in this zone. Prealbumin shows a variationof migration and band spread, depending on the degree ofcomplex formation and probably on genetic polymorphism.So far, only the total relative concentration of prealbumin isof clinical interest. In disorders of intermediary triglyceridemetabolism or in secondary lipolysis, a-lipoproteins maymigrate in the prealbumin zone. Albumin molecules to whichacid substances (e.g., drug metabolites) are firmly linked mayalso appear here.

Pathology. An increased prealbumin fraction may be ob-served in alcoholics. A remarkable concentration decreasewithin one day is a regular finding as part of the acute in-flammatory response (cause unknown). The concentrationremains low in active chronic inflammation. Decreasedprealbummn is a characteristic finding early in liver cirrhosis.The principle clinical use of prealbumin estimation is in theearly detection of decrease in functioning liver-cell mass.

The Albumin Zone

This fraction is broad because the concentration of albuminis higher than that of other proteins, and also because of itselectrophoretic heterogeneity, both acquired and of geneticorigin. In healthy subjects the albumin fraction has the samewidth except in the relatively uncommon heterozygotes(prevalence <1:100) for fast or slow albumins. The mostcommon genetic variant is characterized by a cathodalbroadening of the albumin fraction. A series of albumin var-iants with anodal or cathodal widening of the fraction has beenobserved. Heterozygotes with clear separation of the albumin

CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 633

into two bands are said to have “bisalbuminemia” (prevalence<1:10 000).

Pathology. Decreased albumin concentration is a commonfinding caused by changes in intra- to extravascular distri-bution, water retention, or decreased synthesis. Examples areconditions of acute or chronic inflammatory response, cardiacor renal insufficiency, abnormal intestinal or other externallosses, advanced cirrhosis, and advanced stages of malignantdisease.

Hypoalbuminemia may also be a sequel to the infusion ofdextran or of such high production of immunoglobulins thatthey contribute to the colloidal osmotic pressure. Hyperal-buminemia is a sign of dehydration.

The electrophoretic mobility and spread of albumin maybe influenced by bound exogenous and endogenous chargedorganic substances. Bromphenol blue, which is often used asa marker during electrophoresis, is recognized in the albuminfront. The most common endogenous causes of increased al-bumin mobility are hyperbilirubinemia, the presence ofnonesterified fatty acids, or high intake of aspirin. Pen icillicacids are covalently bound to albumin, and a fast albuminfraction with a normal biological half-life is often recognizedweeks after parenteral treatment with high doses of peni-cillin.

Decreased mobility of part of the albumin fraction is usuallyhereditary, but contamination with heavy metals such ascopper may give a similar pattern.

The Albumin-a1 Interzone

This area is usuallyevenly stained,owing to the presence

of the heterogeneous a-lipoproteins.In exceptional cases,a-fetoproteinmay appear asa faintband inthe middle ofthezone. Rapid-migrating aj-antitrypsinvariantsmay appearfrom the middle of the cathodal limit of the zone. In the latter

the color may be abnormally intensebecause of atypicala-lipoproteinsor very high concentrationsoforosomucoid.

a1-Lipoproteins comprise one of the most heterogeneousplasma proteingroups electrophoreticallyand chemically.The

usual positionisfrom the albumin zone intothe a1 band. Themean mobility increaseswith increasingconcentration ofnon-esterifiedfattyacids.Normally, the colorintensityof thezone between albumin and the a1 band mainly reflectsthea-lipoproteinconcentration.This intensityishigh inwomen

during puberty, after the intake of estrogens,and during

pregnancy.Pathology. a-Lipoproteins on the anodal sideofalbumin

indicateabnormal lipolysis.A high a-lipoproteinfractionwith

normal or slow mobilityisa common findinginchronicalco-

holics. Moderate decrease in concentration of the a-lipopro-tein fraction is part of the inflammatory response.

A marked decrease of the a-lipoproteins with decreasedmean mobility and altered immunoreactivity is a regularfinding in acute hepatitis and precedes hyperbilirubinemiaduring a relapse. Similarly, the a-lipoprotein zone is “faded”in patterns from cases of liver cirrhosis, particularly duringa relapse. The a-lipoprotein zone also is less intense duringbiliary occlusion, in which the concentration of the fl-lipo-protein band is increased and the presence of lipoprotein-Xmay be demonstrated.

Decreased mobility of the anodal a-lipoprotein fractionresults in a blurred aj band. This may be seen in severe mal-nutrition and in alcoholics, particularly after drinking bouts.A high a-lipoprotein fraction with normal or slow mobility isa common finding in chronic alcoholics. Moderate decreaseof the a-lipoprotein fraction belongs to the inflammatory re-sponse.

Decrease of the a-lipoproteins accompanies several inher-ited lipoprotein disorders, such as lecithin:cholesterol acyl-

transferase (EC 2.3.1.43) deficiency and Tangier disease.a-Feto protein is a normal trace component migrating in

the zone between albumin and the a1 band.Pathology. a-Fetoprotein may cause a faint band when

present in 100-fold excess of normal, in sera from cases ofprimary cancer of the liver or embryonic tumors.

Orosomucoid is slightly heterogeneous and spreads on theanodal side of the ai-antitrypsin band. Because of its lowdye-binding capacity (high sialic acid content) it is usually notrecognized.

Pathology. The at band may appear broadened and slightlyfuzzy on the anodal side if the orosomucoid concentrationexceeds 2 g/L. It is impossible to judge by appearance whetheran anodally fuzzy a1 band is a result of increased a1-lipopro-teins or of increased orosomucoid. An increase of the oroso-mucoid fraction is a regular finding within one to two days ofonset of an inflammation. Increased values are also seenduring glomerular filtration insufficiency and often duringtreatment with prednisolone and other corticosteroid analogs.One of its catabolic routes is disappearance through glomer-ular filtration because of its low relative molecular mass(45 000). Anodally fuzzy a1 bands are common in uremia.

The a1 Zone

a1-Antitrypsin is responsible for the normal a1 band, whichhas a minor adjacent cathodal satellite band. This is an ex-pression of the microheterogeneity of a1-antitrypsin. Inheritedand acquired mobility variants are common and may causebroadening, slight displacement, or doubling of the a1band.

About 15% of Caucasians have genetic a1-antitrypsin(protease inhibitor, Pi) variants detectable on agarose gelelectrophoresis. Most of these individuals are heterozygotes:half their a1 -antitrypsin appears in the normal Pi M band, halfin a second band on its cathodal or anodal side. These variantsare of no clinical significance. The most frequent cathodalvariants are S with slightly and Z with markedly decreasedconcentration (60 and 15% of the average, respectively).Homozygotes for S and Z have no band in the normal a1-antitrypsin position. Subjects with the classic a1-antitrypsindeficiency have a scarcely discernible band with slightly de-creased mobility. aj-Antitrypsin has a reactive SH-group, andfor this reason thiol compounds may be bound and sometimesalter the mobility.

Pathology. a1-Antitrypsin belongs to the acute-phasereactants, increasing one to two days after tissue injuries.Selective or dominating a1-antitrypsin increase is a remark-ably valid sign of liver injury and a sensitive sign of increasedestrogen, and it doubles, e.g., during pregnancy. A decreaseda1 band is an indication for specific measurement of a1-anti-trypsin and genetic typing (Pi-typing) (8).

The a1-a2 lnterzone

On the anodal side of the usually well-demaracted a2 band,one to three adjacent faint bands are recognized. They rep-resent primarily the group-specific component, inter-a-trypsin inhibitor, and pregnancy zone protein in pregnantwomen. When present, haptoglobin of type 1:1 overlaps theseminor bands on the anodal side of a2-macroglobulins. In suchcases the cathodal part of the a2 zone appears empty.

Pathology. During acute inflammatory response a new bandmay appear on the cathodal side of a1-antitrypsin. The proteinresponsible has not been identified.

The bands inthiszone are more darkly stained during anacute inflammatory response and during pregnancy. Inspec-tion is not sufficient to ascertain which of the proteins hasincreased. Complexes between K-chains and a1-antitrypsin

634 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

or albumin may be found in myeloma patients with K-chainBence Jones proteinuria. They are linkedthough an SS-bridgeand are cleaved on mild reduction. Free light chains of A-typemay also appear in this zone.

The Broad a2 Band

The complex protein composition of the broad a2 band zonecannot be settled by inspection, although two proteins dom-inate, a2-macroglobulins and haptoglobins. Both show suchan individual variation and varying zone spread that a quan-titative analysis of one of them is necessary for an interpre-tation of the composition of the fraction. Haptoglobins alsoshow a wider normal range than most other plasma proteins.A distinct front-sharpening of the a2 band, however, indicatesa major a2-macroglobulin fraction. This front demarcationis usually quite apparent in children and often in the aged. Thea1-macroglobulin concentration is also greater during child-hood.

a2-Macroglobulin is so dominant in the a2 zone during earlyinfancy that variation of other a2-globulins is not recogniz-able.

Pathology. Increase of the a2-macroglobulin fraction hasa low diagnostic significance. It occurs regularly in cases ofselective glomerular protein loss, but not in intestinal proteinlosses.

A slight increase is common in liver cirrhosis, in diabetes,in malignancy, and in older people. Low a2-macroglobulinvalues also have low diagnostic significance in spite of the keyposition of the protein as the major acceptor of proteases re-leased into the intercellular space and the short biologicalhalf-life of these complexes. No apparent decline of the con-centration in plasma is observed in acute pancreatitis, in spiteof the increased intra-abdominal a2-macroglobulin catabo-lism. Low values are common during the final days of life.a2-Antiplasmin decreases earlier and more markedly thandoes a2-macroglobulin in pancreatitis.

The great increase of plasma haptoglobin during inflam-matory response and the decrease during increased intra-vascular hemoglobin release (e.g., slight hemolysis, B12andfolic acid deficiency, cancer metastases in liver and bonemarrow, sepsis, hepatitis, liver cirrhosis, splenomegaly) makethe haptoglobin contribution to the a2 zone most variable anda quantitative analysis advisable. Treatment with 17-hy-droxylated androgens augments the inflammatory increaseof plasma haptoglobin. Malignant plasma cell proliferationmay cause an M-component of the IgA class to migrate in thea2 zone.

Several other a2-globulins show a disease-related variation,but their concentration is too low to influence the appearanceof the a zone appreciably.

The intensity of the a2 band may be increased and extendedcathodally when hemoglobin-haptoglobin complexes havebeen formed. In this case the sample is usually red.

The a2-f31 Interzone

The backgroundstain of this zone has low intensity. Thecold-insoluble globulin (fibrinolignin)of fibroblastorigincauses a weak, narrow band in the middle of the zone. Itsconcentration declines when plasma clots, because of itslinkage to the fibrin. It also constitutes the bulk of thecryoprecipitate in heparin-treated plasma at 4 #{176}C.

Fuzzy bands and abnormally high mobility may appearwhen concentrations of pre-/3- and -lipoproteins are low,especially after storage of samples.

On electrophoresis of sera with apparent hemoglobin con-tamination (>1 g/L), the free hemoglobin migrates in thecathodal a2-1 interzone, overlapping the anodal part of the!- band.

Pathology. The concentration of cold-insoluble globulinis slightly influenced by disease, but tends to decrease duringthe inflammatory response. Increased concentration is regu-larly found in cholestasis of pregnancy. M-components (nar-row and broad) of all Ig classes and of light-chain type mayappear in this zone.

The fl Zone

The color intensity of this band varies roughly propor-tionally to the transferrin concentration. Splitting of thefraction into one slower or one faster band is the expressionof heterozygosity for a transferrin variant. In such cases thetwo bands have similar and half-normal intensity. Thesevariants are of no clinical significance.

After cleavage of complement component C3, the C3cfragment migrates overlapping or adjacent to the anodal sideof the i band. This is usually a sampling and storage artifact,which is avoided by collecting blood directly into a tube withethylenediaminetetraacetate. C3 cleavage regularly occurs onstorage of serum. The cleavage rate is faster in sera fromsubjects with inflammatory response.

Pathology. Intensely or weakly stained th bands indicatehigh and low transferrin concentrations, respectively. Thetransferrin increase during pregnancy, after estrogens, andin iron deficiency is apparent to the naked eye. The decreaseduring inflammatory response usually follows the hypoal-buminemia but the transferrin decrease is a slightly moresensitive marker of malnutrition than hypoalbuminemia,especially in alcoholics.

Broad M-components, predominantly of the IgA class,appear in this zone. Their molecular size heterogeneity andcomplex formation are the cause of their extension in theanodal and cathodal direction. The other M-componentsusually have a sharp demarcation.

The /l-fl2 lnterzone

A slightly wavy 3-lipoprotein band with a sharper cathodalthan anodal demarcation dominates this zone, where thebackground stain is mainly caused by IgA. The mobility of thefl-lipoproteins increases with the amount of nonesterified fattyacids bound and decreases with increasing concentration ofthe fraction. This concentration-dependence of the mobilityis caused by interaction with an agarose contaminant andtherefore varies from batch to batch. The mobility of the f3-lipoproteins decreases with increasing concentration of Ca2+in the buffer.

Pathology. Comparative electrophoresis of fresh sera ren-ders it possible to judge whether fl-lipoprotein concentra-tions are high or low. A cathodally displaced band of high in-tensity indicates an abnormally high -lipoprotein concen-tration; a low amount results in faster mobility.

High color intensity over this zone, extending into the an-odal y-zone, is a characteristic finding with increased IgA.

M-components of all classes may be found in this zone.

The fl2 Zone

This zone is mainly caused by the third complement factor(C3). A series of genetic variants occurs with slightly higheror lower mobility. In the heterozygotes the amount of C3 isequally divided between the two bands. Deficiency variantsexist but are extremely rare. Normally the C3 band has a smallbump on the anodal side. C3 is cleaved during storage of serumand the predominant conversion product is C3c, which has afast fl mobility. During the early conversion a weak bandappears on the cathodal side of C3. This product is very la-bile.

Pathology. C3 is one of the acute-phase reactants, in-creasing in concentration with a lag phase of some five to seven

CLINICALCHEMISTRY, Vol. 25, No. 4, 1979 635

days. Italsoincreasesduring biliaryobstruction.Normal or

low C3 values after the first week of inflammatory responsesuggest an abnormal C3 activation. Conversion products ofcomplement activation in vivo are rarely found in bloodsamples because of the short biological half-life of the cleavageproducts. A weak C3 band in combination with the occurrenceof conversion products suggests in vitro activation. Low valuesowing to increased complement activation in vivo may be seenearly in acute nephritis, membranoproliferative glomerulo-nephritis, systemic lupus erythematosis, and similar condi-tions with intravascular immune complexes. The concomitantsigns of inflammatory response may be slight. Complementactivation following the classical pathway in antigen-antibodyreactions-e.g., in hemolytic anemia-is often followed by norecognizabledecrease in C3 in spite of a marked decrease inC4.

M-components are common in the C3 zone.

The y Zone

On the cathodal side of the C3 band follows the extended-y zone. On analysis of serum, the intensity and distributionof color mainly depend on the concentration of the varioussubclasses of IgG. Gross abnormalities are observed with thenaked eye. Not even faint bands are accepted as a normalfinding in the -y zone. High 1gM concentration has some in-fluence on the color intensity in the anodal part of the -yzonewhere fibrinogen migrates if plasma is being analyzed.

Fibrinogen causes a sharp band with an adjacent fraction

of 10 to 20% on its anodal front. The intensity of the bandreflects adequately the fibrinogen concentration. The largesize and slow mobility of the molecules probably precludedetection of genetic variants with single amino acid substi-tutions. Fuzzy demarcation of the fibrinogen band withouttrailing is sometimes found in subacute pancreatitis.

Pathology. Faint single bands may appear in the y zonesecondary to the proliferation of selected Ig-producing cellclones or as a result of the appearance of certain other pro-teins.

A labile conversion product of C3 in the anodal -yzone hasbeen mentioned earlier. C-reactive protein migrates on thecathodalside of the bulk of IgG and often reaches a concen-trationsuch that a visibleband appears incasesof acute andchronic inflammatory response. Interaction of C-reactiveprotein with agarose gel contaminants causes variations inband intensity from batch to batch of agarose.

Another cause of a single band, or more often two or threefaint bands (<2 g/L), in the -y zone is oligoclonal proliferationof some Igcell clones. This is often seen after viral infectionsand in malignancy.

Dark, narrow bands (M-components) are seen in myeloma,macroglobulinemia, and in other conditions with more or lessmalignant monoclonal immunocyte proliferation. Immuno-logic classification of the M-component is necessary to de-termine its class.Monoclonal Ig increase is usually but notregularly characterized by a sharply demarcated band. A seriesof narrow bands with stepwise increasing or decreasing IgGconcentration may be found in the middle or the cathodal yzones in monoclonal Ig cell proliferation. It is seldom difficultto decide if poly- or monoclonal plasma cell increase hascaused an atypical protein partition in the -y zone, becauselimited polyclonal origin always causes a much fuzzier de-marcation than does monoclonal origin. If any doubt remains,the problem is solved by light-chain typing, which will giveonly one type if the origin is monoclonal cell proliferation.

The concentration of the M-component roughly indicates

the size of the proliferating immunocyte clone. Immuno-globulins of the classes A, M, D, and E have half or shortersurvival times in plasma than do the predominant immuno-

globulins of IgG subclasses 1,2, and 4. This must be consideredwhen one is estimating the cell mass from the concentrationof the M-component. Increasing M-component concentrationindicates increasing cell mass. It is important to recognize aconcomitant decrease in concentration of immunoglobulinsderiving from the other immunocyte populations, as a gen-erally depressed Ig value suggests malignant growth of theclone producing the M-component. The concentration ofBence Jones protein in urine is correlated to malignancy ofthe cell clone.

M-components in a concentration at or below 10 g/L in oldpeople are usually a sign of comparatively innocent immu-nocyte proliferations. M-components of class 1gM in this lowconcentration interval arouse suspicion of malignant lym-phoma or reticulosis if there are concomitant signs of in-flammatory response with no apparent cause.

Polyclonal Ig cell stimulation, apparent as increasedstaining of part or all of the y zone, regularly indicates in-creasedIgG synthesis. Grossly increased concentrations (>25g/L) primarily suggest hyperimmune states. The most com-mon causes are systemic lupus erythematosus, chronic activehepatitis, abscess formation of microorganism origin, ormalignancy with bone marrow involvement. It may be a rareaccidental finding in subjectively healthy individuals.

Moderate hypergammaglobulinemia develops in responseto various antigenic stimuli, but never reaches abnormalconcentrations that are recognizablein electrophoreticpat-terns earlier than three weeks.

Hypogammaglobulinemia is easily recognized on compar-ative electrophoresisand usually indicates serious diseases(e.g., diabetes, myelosclerosis, malignant plasma- or reticu-lar-cell proliferation, widespread metastases) if losses (in-testinal or renal) and disturbances of the glucosteroid me-tabolism can be excluded. Hypogammaglobulinemia in adultswithout apparent cause is an indication to search for BenceJones proteinuria.

M-components of 1gM or IgG classes may appear on thecathodal side of the normal -y zone in rare cases. Lysozymenormally migrates here, but seldom increases to a concen-tration causinga visible band except in myeloproliferativedisease.

Some examples of common electrophoretic patterns aregiven in Figure 2. A more detailed collection is given in ref.7.

A summarized scheme for analysis of electrophoretic pat-terns is given in Table 2.

Comparison of the Electrophoretic Pattern ofProteins in Plasma and Cerebrospinal Fluid

Normally, some 80% of the proteins of cerebrospinal fluid(CSF) originate from plasma, and the rest have been syn-thesized locally. The biological half-life for the plasma pro-teins entering the subarachnoid space is about one week, andtherefore the composition of CSF proteins follows that ofplasma, but with some delay. The filtration resistance forplasma proteins rapidly increases with molecular size andtherefore the ratio of large proteins (1gM, -lipoproteins, anda2-macroglobulins) to albumin normally is much lower in CSFthan in plasma. This molecular size discrimination diminisheswith increasing CSF protein concentration.

The locally synthesized share of the proteins normally di-minishes with increasing concentration of CSF proteins. TheCSF has to be concentrated at least 150-fold before electro-phoresis.

The electrophoretic pattern of concentrated CSF is char-acterized by a strong prealbumin fraction, which migratesslightly faster than in plasma. The albumin and ai zone aresimilar to those in plasma, but the share of a-lipoproteins is

Table 2. Analysis of Electrophoretic Patterns Table 2. ContInuedEl.ctrophoretlc Electrophoretlc

zones Look for Possible causes zones Look for Possible causes

Prealbumin zone Decrease Low liver cell-mass, 132 ZOflO

inflammatory IgA Selective Malignancies,response, increase mucosalmalnutrition Infection, rheumatoid

Albumin zone Decrease Inflammatory arthritis, alcoholresponse, abusemalnutrition, losses, C3 Polymorphism Genetic variantsmalignancy, Position In vivo/invitro con-increased version of C3 to C3c

extracellular volume. Increase Chronic Inflammatory

Fast anodal front Drugs (penicillin), response, biliaryjaundice obstruction

Albumin-a1 Decrease Complementinterzone activationa1-Llpopro- Decreased Liver cell lesions Y zoneteins intensity Fibrinogen Increase Inflammatory

Increased Malnutrition, alcohol response

intensity abuse, Relative Fibrinolysis,postmenopausal, decrease inadequatehigh estrogen level sampling procedure

a1 zone Immuno- Sharp bands M-componenta1-Antitrypsin Polymorphism Genetic variants globulins

Increase Inflammatory Diffuse increase Polyclonal IncreaseBanded Oligoclonal increase.

response,liver cell injury appearance viral infection,

Relative Genetic deficiency malignancydecrease Losses

a2 zonea2-Macro- Increase Age dependent in decreasedand the al band is more diffuse. The a2 zone ap-

pears faint and fuzzy (owing to its very low relative cr2Mglobulin children content), with more proteins in the a2-f31 zone. The $ bandand elderly (>70), is similar to that for plasma, while the /32 band is doubled

selective proteinuria becauseof C3 and of a locally produced carbohydrate-defi-

I-Iaptoglobin Increase Inflammatory cient transferrin. A tendency to band sharpening is oftenresponse recognizedin the middle of the -yzone.It is IgG in nature, but

Relative In vivo hemolysis, no corresponding concentration gradient is observed in

decrease hepatosplenomegaly, plasma.At the cathodal end of the -y zone a faint band is located,Increased ineffective

which is caused by the small (10 000 daltons), labile “-y-trace”erythropoiesis fraction.

/31 zone Clinical interest in electrophoretic analysis of CSF is limitedTransferrin Polymorphism Genetic variants, to the immunoglobulin pattern, in search of signs of local

(Bence immunoglobulin production in or around the central nervous

Jones protein, system. Mono- and oligoclonal pattern in the -y zone of CSFM-component, when the plasma pattern is polyclonal is a reliable indicator

of local immunocyte response to microorganisms, viruses, orhemoglobin) antigen-antibody complexes.This typical, usually oligoclonal,

Increase Iron deficiency, band pattern is blurred when concomitant inflammatory re-estrogens action results in increasedprotein leakageof plasma proteins

Decrease Malnutrition, losses, into CSF, i.e., in an increased protein concentration ininflammatory CSF.response Electrophoretic comparative CSF and plasma protein

/3-Lipoprotein Increase Hypercholesterolemia analysis is of no interest when the CSF protein concentrationPosition Decreased mobility exceeds2 g/L, becauseeven high local Ig production can nolonger be distinguished from the electrophoretic pattern due

with to presenceof plasma proteins.an increased Intensitysuggests biliary Comparison of the Electrophoretic Pattern ofobstruction Proteins In Urine and Plasma

continued Primarily determinant for the urinary protein pattern are

the composition and amount of proteins permeating the gb-

636 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 637

merular filter in relation to the tubular reabsorptive capacity.Normally, thisislimited to some 200 mg/day.

On urinary loss of larger amounts of protein, the compar-ative electrophoretic analysis distinguishes the three majorpossibilities: nonselective gbomerular proteinuria, selectiveglomerular proteinuria, and Bence Jones proteinuria.

In nonselective gbomerular proteinuria, the lesions of theglomerular membranes are so severe that there is no dis-crimination in filtration rate between molecules in the sizerange of albumin and IgG (70 000 to 150 000 daltons).

The amount of protein (albumin) in the urine tells us moreabout the lesion than doesa urinary pattern that mimics theserum pattern.

Albumin normally passesthe gbomerular filter five to 10times more readily than does IgG. The degreeof gbomerularselectivity is better reflected in the albumin/IgG ratio thanby a comparative electrophoretic analysis.

In selective gbomerular proteinuria the electrophoreticurinary protein patterns diverge more from the plasma pat-tern.

In severe selective gbomerular proteinuria the plasma isdepleted of the proteins of low relative molecular mass,whichare enriched in the urine. The urine pattern is dominated byalbumin, the a1 band (a1-antitrypsin), and the /3 band(transferrin), while the cathodal part of the pattern is empty.Simultaneously, the plasma pattern shows the pattern typicalof gbomerular protein loss with decreased albumin and in-creased concentration of all large proteins (a2-macroglobulins,

cold-insoluble globulin, 9-lipoproteins, 1gM, and fibrin-ogen).

Mild, selective glomerular proteinuria (<1 g/L) is a commonfinding in inflammatory conditions and in circulatory insuf-ficiency and is therefore of marginal clinical interest. Theinflammatory response gives increased orosomucoid excretion,causing the a1 band to blur in the anodal direction. Theinter-a zone is partly occupied by Zn-a2-glycoprotein andother small proteins.The small loss of proteins in urine hasno feedback effect on the plasma protein pattern. Reasons forrunning electrophoretic analysis on urine in casesshowingmild proteinuria are limited to the suspicion of Bence Jonesproteinuria or of proximal tubular proteinuria.

Bence Jones Proteinuria

Urinary excretion of K or A immunoglobulin light chains ofmonoclonal origin (Bence Jones proteinuria) results in theappearance of a more or less dark single (or usually double)band somewhere in the ai to the slow y zone. Its nature maybe demonstrated by immunoelectrophoresis but preferablyis shown by immunofixation of an agarose electrophoresis withanti-K and anti-A antisera. The result of conventional immu-noelectrophoretic analysis is more difficult to interpret thanthe immunofixation result if the concentration of the mono-clonal light chain is low, because polycbonal light chains alwaysoccur in the urine.

The excretion of increased amounts of light chains throughthe glomeruli causes competition with other proteins of lowrelative molecular mass for the limited reabsorptive capacityin the proximal tubular section of the nephron. Therefore, theprotein pattern in Bence Jones proteinuria often shows bandssuggesting insufficient tubular reabsorption.

Pure tubular proteinuria is a mild proteinuria of less than0.5 g/day, characterized by excretion of the smaller (<50 000daltons) proteins that normally permeate the gbomerular filterand that are normally reabsorbed by the proximal tubularcells.

The predominant proteins of low molecular mass giving riseto electrophoretic bands in concentrated urine are a2-micro-

globulin (retinol binding protein), /3-microglobulin (constantpieceof the HLA-antigen), and “-y-trace.” Equally abundant,but electrophoretically heterogeneous, are protein HC (9) andpolycbonal light chains.

The proportions of these proteins vary in disease. The puretubular lesions are rare, but a protein pattern similar to“tubular proteinuria” is common in advanced glomerularfiltration insufficiency. This is explained by the increasingconcentration in plasma, when filtration is insufficient, of allmolecules that normally are eliminated by filtration. The loadof small proteins increases in the few remaining functioningnephrons and thus surpasses the reabsorptive capacity of theproximal tubular cells.Primary proximal tubular insufficiencyis combined with normal glomerular filtration (normal plasmacreatinine) in contrast to secondarytubular insufficiency withprimary glomerular filtration insufficiency (uremia). In casesof normal plasma creatinine values and slight proteinuriaelectrophoretic analysis of concentrated urine may be ofsupplementary interest if other laboratory signs of proximaltubular insufficiency exist.

In conclusion, urine electrophoresis is of great clinical valuewhere Bence Jones proteinuria is suspected, but otherwise isof marginal interest.

Contributed Comments (from Evaluators E.S.T.and L.K.)

We evaluated the described technique with use of an LKBelectrophoresis chamber 2117 Multiphor. The power supplywas a Searle Model 3-1155, current and voltage regulated.Temperature was maintained at 10-14 #{176}Cby use of a recir-culating pump connected to an ice bath reservoir.

We have the following comments regarding specific aspectsof the method.

Reagents: Picric acid is difficult to obtain because it is nolonger manufactured by many companies. It is available fromMCB Chemicals(cat. no. PX1165-01).

Apparatus: The cooling device for the electrophoreticchamber does not have to be a cryostat temperature-regulateddevice. An acceptable substitute is an ice bath with a recir-culating pump such as the type used for an aquarium or dec-orative fountain. One can be obtained at a local hardware store

for about $30.00 to $40.00. This performs well when connectedto a recirculating water bath ina largeStyrofoam container.Crushedice can be added as needed to maintain the temper-ature in the range of 10-14 #{176}C.

Fixing, drying, and staining: Problems developed in re-moving the wet Whatman no. 1 filter paper after the dryingand removal of picric acid. The gel had a tendency to pull awayfrom the polyester sheet. A helpful hint is to spray the filterpaper with distilled water before attempting to remove it.

Accuracy: Because the method is largely qualitative andsubjectively semiquantitative, accuracy was estimated bycomparing the electrophoretic pattern with immunochemicalquantitation of specific proteins. In general, we observed thatabnormalities appearing in the electrophoretic pattern werereflected by abnormalities in concentrations of the majorserum proteins.

Precision: Subjective evaluation of precision as determinedby different observers appeared to be excellent. The repro-ducibility of patterns within a given run, as well as betweenruns, appeared quite good.

Reliability: The method is reliable when the materials anddirections specified are followed.It is important that a powersupply of good quality and an electrophoretic chamber of goodconstruction be used.

Normal ranges and abnormal ranges in disease: Themethod relies on visual inspection of the stained electropho-

638 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

retic pattern. As such, it is a qualitative procedure; assessmentof. normal vs. abnormal patternsissubjective. In evaluatingthis aspect of the method, electrophoretic patterns we judgedto be normal were compared with quantitative values formajor serum proteins in the patients’ samples under study.Similar comparison was made with quantitative protein valueswhen there were abnormal electrophoretic patterns. In mostinstances, it was possible to determine an abnormality inpattern by visual comparison with a selectednormal. Theabnormalities appeared either as an increase or decrease instaining intensity of individual bands in the electrophoreticstrip. Based on the relative intensity of staining of a band, aprediction was then made as to the underlying protein ab-normality. This was then correlated with the quantitative dataand in most instances good correlation was noted, similar tothe correlations discussed by the Submitters.

Conclusion: The method exhibits good characteristics ofworkability, accuracy, precision, and reliability. It can berecommended as a qualitative screening procedure for de-tecting abnormalities of the major proteins and as an impor-tant adjunct to specific quantitation of serum proteins.

References

1. Wieme,R. J., Agar Gel Elect rophoresis. Elsevier, Amsterdam, NewYork, 1965.

2. Laurell, C-B., Laurell, S., and Skoog, N., Buffer composition inpaper electrophoresis. Clin. Chem. 2,99-111 (1956).3. Axelsen, N. H., KrIl, J., and Weeke, B., In Quantitative Immu-noelectrophoresis, Universitetsforlaget, Oslo, 1973, p 25.4. Ritchie, R. F., and Smith, R., Immunofixation. General principlesand application to agarose gel electrophoresis. Clin. Chem. 22,497 -499 (1976).5. Johansson, B., Agarose gel electrophoresis. Scand. J. Clin. Lab.Invest. 29, Suppl. 124, 7-20 (1972).6. Cawley, L. P., Elect rophoresis and Immunoelectrophoresis, Little,Brown and Co., Boston, MA, 1969, p 230.7. Laurell, C.-B., Composition and variation of the gel electrophoreticfractions of plasma, cerebrospinal fluid and urine. Scand. J. Clin. Lab.Invest. 29, Suppl. 124, 71-82 (1972).

8. Pierce, J., Jeppsson, J.-O., and Laurell, C.-B., ai-Antitrypsinphenotypes determined by isoelectric focusing of the cysteineanti-trypsin mixed disulfide in serum. Anal. Biochem. 74, 227-241(1976).

9. Tejler, L., and Grubb, A. 0., A complex-forming glycoproteinheterogeneous in charge and present in human plasma, urine andcerebrospinal fluid. Biochim. Biophys. Acta 439,82-96 (1976).

Editor’s note: The reader is reminded that Selected Methods donot bear the official imprimatur of the Association. They aremethods that seem durable and generally useful, and that have beenchecked by several evaluators. As detailed elsewhere [Clin. Chem.19, 1207 (1973)], these methods are offered here for criticism by theworld community of users, and will be revised appropriately beforebeing collected into a bound volume, Selected Methods of ClinicalChemistry. The last such volume was published by the Associationin 1977.

No reprints of these papers will be available, because they are notregarded as necessarily being final versions.