J. clin. Path. (1957), 10, 360.

THE PROTEINS OF NORMAL URINE*BY

GREGOR H. GRANTWiTH THE TECHNIcAL ASSISTANCE OF PHILIP H. EVERALLFrom the Department of Pathology, Royal Salop Infirmary, Shrewsbury

(RECEIVED FOR PUBLICATION JULY 8, 1957)

It has been known since the end of the lastcentury that normal urine contains traces of pro-

teins from both blood and urinary tract (Morner,1895); yet many of the textbooks have ignoredthis, as well as the fact that pathological protein-uria must merge imperceptibly with the normal.One protein apparently arising from the urinarytract has been clearly defined, namely the muco-

protein intensively studied by Tamm and Horsfall(1952).

Electrophoresis was first used to study thenature of the proteins of normal urine by Rigasand Heller (1951). They used the moving boun-dary method after first concentrating 72-hourspecimens of urine by ultrafiltration. The nor-mal daily excretion of protein was found to beabout 40 mg., and to consist of components withthe same range of mobilities as those found inblood. Unlike blood, however, the globulin com-

ponents were less well defined, exceeded thealbumin in quantity, and were mainly a globulinsrather than and y globulins. Their findings,which were strikingly uniform and similar in bothsexes, have since been confirmed by two othergroups of American workers, whose measure-ments of daily output vary from about 25 to90 mg. (Boyce, Garvey, and Norfleet, 1954;McGarry, Sehon, and Rose, 1955).These authors were unable to distinguish clearly

between the proteins arising from the blood andthose coming from the urinary tract. Here an

attempt has been made to do this as well asto identify the individual proteins more clearlyafter their electrophoretic separation, by using animmunochemical technique in which rabbit anti-sera prepared against the serum proteins andagainst the urinary tract proteins respectively pre-

cipitate proteins from the two sources separately.The use of this method to study primarily the pro-teins arising from the blood is described below.On testing unconcentrated normal urines

immunologically by the agar gel diffusion tech-*Based on a paper read at the meeting of the Association of

Clinical Pathologists at Torquay on April 12, 1957.

nique (Ouchterlony, 1949), it has always been pos-sible to demonstrate the presence of albumin, butthe other proteins are usually present in too smallamounts to be detected unless the urine is firstconcentrated.

Previously described methods of concentrationhave not proved entirely satisfactory (McGarryet al., 1955). The essentials of any such methodare that it should not denature the proteins, thatit should be simple to perform, capable of the con-tinuous concentration of large volumes of urine,and fast enough to concentrate a 24-hour specimenin, say, 48 hours. A method is described here,based on the use of a large filtering area, whichappears to satisfy these criteria.Having concentrated the urine proteins it is

possible to analyse them by the immuno-electro-phoretic method of Grabar and Williams (1953,1955). This technique was first introduced to usby Dr. Gell, who was kind enough to show usthe details of his modifications (1955). Briefly,the proteins of a mixture are first separated inagar by the physical process of electrophoresisand then identified biochemically by precipitatingthem with their homologous antibodies.

Methods and MaterialsUrines Examined.-Pooled urine from normal male

adults was collected, and also individual samples fromfour normal men, four normal women, and fournormal boys aged between 3 and 9. All the adultswere members of the laboratory staff. The sampleswere not taken by catheter, but simple precautionswere taken to avoid contamination. Urines from themales, apart from the pooled sample, were mid-streamspecimens. All samples were preserved with sodiumazide (1 g. per litre) until their colloids were concen-trated by ultrafiltration.

Concentration of Urine Proteins.-Small quantitiesof protein solutions can be concentrated satisfaGtorilyby dialysis against concentrated dextran or polyvidonesolutions, and this might be the most convenientmethod for the investigation of cases of grossproteinuria.

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THE PROTEINS OF NORMAL URINE

It was, however, necessary to concentrate normal FROMurine about 1,000 times, and for this purpose the RESERVOIRfollowing technique was evolved.

Approximately 6 ft. of dialysis* tubing (A. Gallen-kamp and Co. Ltd. " visking ") are encased in " tube-gauz" (Scholl). This is done by first sliding the"tubegauz " on to the handles of a long Spencer Wellsforceps. One end of the dialysis tubing is gripped bythe forceps and the " tubegauz" slid on. A shorttapered piece of glass tubing is then pushed into oneend; this, with the tubing and gauze wrapped roundit, is inserted into the rubber bung, tapered end first. -i(The inner end of the glass tube is covered with ashort piece of rubber tubing to prevent damage to TUBES SEALEDthe dialysis tubing while doing this.) The remainder BETWEE

*Tubing of i in. flat width was used. For smaller quantitics NECKI in. tubing can be used and does not require support bytubegauz."

TUBEGAUZ - i

DIALYSISTUBING

FIG. 2.-Enlarged drawing of portion A-B of ultrafiltration apparatusin Fig. 1.

of the gauze is wetted and pulled out over the dialysistubing until it is fully extended. The tubing andgauze are then coiled up and placed in the aspirator,

A the free end being clipped between the neck and thebung (Figs. 1 and 2).The reservoir containing the urine is connected to

the glass tube and suction applied to the jar so thatthe tubing gradually fills with urine. Hot moltenvaseline" is meanwhile poured over the bung to

form an airtight seal. The suction is stopped when-. ~~a negative pressure of approximately 60 cm. of mer-

cury has been reached. The apparatus is then left onthe bench to filter; at intervals the filtrate is drawnoff and the reservoir refilled with urine until the wholesample has been processed. In this way very largequantities of urine can be filtered if the vacuum isoccasionally renewed.When only a little urine is left in the tubing the

free end is withdrawn leaving a single U of tubingcontaining all the concentrate, which is then reduced

to the desired final volume. During this stage bar-i0 T biturate buffer pH 8.6 is poured into the bottom of

Pum the aspirator so that the concentrate is dialysedagainst this buffer during its final concentration.The brown viscous concentrate is removed by

cutting the tube on both sides of the filuid, removingthe gauze, placing the tube in a centrifuge tube with

Fio. l Ultrafiltration apparatus one end turned back over the rim, and centrifuging.

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GREGOR H. GRANT'

These concentrates were preserved by adding strep-tomycin and penicillin each to a concentration of100 units per ml.Preparation of Antisera.-Adjuvants were used to

stimulate antibody production (Kidd, 1955), rabbitsbeing injected intramuscularly weekly for six to 10weeks with 2 or 3 ml. of a mixture of

I part protein mixture (10-20 mg. per ml.).1 part " arlacel A " C' mannide mono-oleate Honeywill and

Stein Ltd.)3 parts " wyrol CX " (an " esso " light paraffin oil) to which

is added 5 mg. per ml. killed dried Mycobacteriumtuberculosis.

They were bled 10 days after completion of thiscourse.When further supplies of antiserum were required,

the rabbits were re-stimulated by intraperitonealfollowed by intravenous injections, as recommendedby Wootton (1950). The antisera were preserved withstreptomycin and penicillin each at a concentrationof 100 units per ml.; sodium azide was found not todiffuse fast enough to prevent the growth of somestrains of B. subtilis in the agar plates.

In this way three types of antisera were prepared(I) Anti-human-serum-proteins.-This antiserum

was prepared by injection of normal human serum(about 25 samples pooled), and is obviously anexcellent tool for investigating those urine proteinswhich are related to plasma proteins, since it willignore all other urine proteins. The particular anti-serum used here is capable of revealing at least 17protein fractions in normal human serum, namely,pre-albumin (p), albumin, X, three a1 globulins, sixa2 globulins, five f globulins, and y globulin (cf.Williams and Grabar, 1955). This nomenclature issomewhat arbitrary, as some of the fractions occupyintermediate positions between the main groups. Atthe optimum ratio 13 can usually be distinguished:two a globulins are faint, X is obscured by the albu-min, and the pre-albumin was only visible when excessantiserum was used.

(2) A nti-human-urine-proteins.-This antiserum,prepared by injection of normal urine colloids con-centrated as above, may contain antibodies to highmolecular weight polysaccharides as well as toproteins. When it is absorbed with normal humanserum to remove antibodies to serum proteins, anantiserum specifically against urinary tract colloids isobtained.

(3) Anti-Tamm-Horsfall-mucoprotein. This anti-serum was prepared by injecting this protein whichhad been salted out from normal urine (Tamm andHorsfall, 1952). It was found to be contaminatedwith some anti-albumin, so this was removed beforeuse by absorbing it with a little normal serum.Immuno-electrophoretic Analysis. In this method

a drop of the protein mixture to be analysed is placedin a hole cut in a rectangular plate of agar about3 mm. thick, and a potential difference applied acrossthe plate for several hours, so that the differentproteins separate as in paper electrophoresis. Then,

instead of staining them, antiserum is placed in atrough cut in the agar parallel to the direction of thecurrent and about + cm. to one side of the row ofproteins already separated electrophoretically. Theplate is incubated for about 48 hours during whichtime the separated proteins and the antiserum diffusetowards one another and form arcs of precipitatewherever a protein meets its homologous antibody.The detailed technique used was substantially that

of Grabar and Williams (1955) and Gell (1955) apartfrom the following small modifications:A 0.8% solution of New Zealand agar (Davis Gela-

tine Company) was used, clarified by filtering whilehot through a bed of "hyflo super cell" (cf. Fein-berg, 1956); barbiturate buffer in the solid state wasthen added to it to give a pH of 8.6 and an ionicstrength of 0.05 (10.3 g. of sodium diethylbarbiturateand 1.84 g. of diethylbarbituric acid per litre). Toprevent the growth of bacteria and moulds, sodiumazide was added to a concentration of 1 in 1,000 andthe agar finally sterilized at 5 lb. pressure for 15minutes.The apparatus was made entirely of perspex, the

platinum wire electrodes being contained in separatecompartments connected to the inner buffer com-partments by glass U-tubes containing barbiturateagar. A direct current supply at 200 to 250 voltswas used and the current adjusted to about 30 milli-amps for an agar plate measuring 61 x 43 in., byvarying the number of barbiturate agar bridges.The results can be recorded photographically by

direct contact printing, or the agar sheet with its linesof precipitate may be preserved by drying and stain-ing with azocarmine as described by Uriel and Grabar(1956).

It was found more convenient to dry the agar sheeton stretched cellophane than on glass, and, afterstaining, to differentiate by washing in tap water ratherthan dilute acetic acid-glycerol solution.The dried agar sheet on its cellophane can be cut

out and filed or it can be mounted as a lantern slide.If projected on a screen details are particularly clearlyseen.When using this immuno-electrophoretic technique

certain limitations of the method must always beborne in mind:

(1) The relative densities of the lines are no indica-tion of the concentrations of the different proteins inthe mixture. They depend on the response of theinoculated rabbit, and each rabbit behaves differently.This means that each antiserum must be regarded asa different reagent. It does not, however, prevent onecomparing two protein mixtures, provided that eachis examined with the same antiserum.

(2) The number of lines formed represents theminimum number of proteins in the mixture, as therabbit may not have made antibodies to all of them.

(3) It is often impossible to arrange the quantitiesof protein mixture and antiserum used, so that allcomponents of the mixture have antigen / antibody

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THE PROTEINS OF NORMAL URINE

ratios suitable for the formation of clear lines ofprecipitate. This means that several proportions ofprotein mixture and antiserum should always be used.

ResultsUltrafiltration Technique.-In order to make

certain that the ultrafiltration did not in itself alterthe proteins, some normal human serum wasdiluted 2,000 times with Ringer solution, and afterreconcentration to its original volume was com-pared with an untreated sample of the same serum.

Nitrogen estimations of the reconcentrated anduntreated sera by the micro-Kjeldahl methodshowed that there had been no appreciable loss onor through the membrane, and comparison of thesamples by immuno-electrophoresis showed thatthe protein composition was almost unchangedqualitatively. The results obtained are reproducedin Fig. 3, which is a direct print from a driedplate stained with azocarmine. When a similarplate was stained for fat with Sudan black, it wasfound that the fl-lipoprotein, although present inthe reconcentrated serum, formed a line of pre-cipitate which was more hazy than that formed bythe untreated serum. Possibly the dilution hadcaused some degree of denaturation. (Both speci-mens were stored under similar conditions in therefrigerator at 2 to 4° C. and not frozen.) Itseems, therefore, that the ultrafiltration process

FIG. 3.-Comparison between normal human serum (cup 2) and thesame serum diluted 2,000-fold and reconcentrated by ultra-filtration (cup 1). The centre trough contained antiserumagainst normal human serum proteins. (Direct print from driedagar plate stained with azocarmine.)

FIG. 4.-Analyses with antiserum against normal human serum

proteins: Left: Pooled normal serum (S). Centre: Normalurine colloids (U). Right: A mixture of pooled normal serum

and normal urine colloids (S+U). (Direct print after stainingwith azocarmine.)

causes no important qualitative change, althoughthe loss of some minor component on or throughthe membrane cannot be excluded.Naked-eye Appearance of Concentrated Nor-

mal Urine Colloids.-The depth of the browncolour and the viscosity varied from one subjectto another. Some samples formed precipitates on

standing; the supernatant was then used foranalysis. Others were too viscous for a precipitateto settle. Microscopically such precipitates con-sisted of mucoid material with a few crystals,including calcium oxalate and a variable numberof epithelial cells, polymorphs, and erythrocytes(see Rofe, 1955).

Quantitative Analyses of Total Protein inNormal Urine.-Nitrogen estimations carried outon the colloid concentrates from the 24-hourspecimens of two of the adult male subjects gavefigures of 51 and 61 mg. protein respectively, onthe assumption that 1 mg. nitrogen is equivalentto 6.25 mg. protein. As these results are withinthe range found by previous workers both by thisand by the surface tension method (Tarnoky,1951), no further estimations were made.Immuno-electrophoretic Analyses.-These were

done using the following antisera, whose prepara-tion is described above:

(1) Antiserum Prepared Against Normnal HumaniSerum Proteins.-All the urine samples gave

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GREGOR H. GRANT

essentially similar results when analysed with thisantiserum. There were differences in deta;il, butthese were no more marked between samples frommales and females than between samples fromsubjects of the same sex. Fig. 4 shows an exampleof such an analysis, in which a sample of concen-trated adult male urine is compared with poolednormal human serum. It will be seen that eightout of the 13 protein fractions demonstrable inserum are also demonstrable in this urine, namely,albumin, four a globulins, two /3 globulins, andy globulin. Not only do the urine concentrate andserum produce a similar pattern of lines whenthey are analysed separately but when mixedtogether the lines of precipitate from the twosources are seen to coincide. This confirms thatthe precipitin lines obtained with the urine repre-sent substances identical electrophoretically andimmunologically with the proteins of normalserum. Further, when the antiserum trough wasarranged to form a rectangle around theseparated urine and serum proteins, the albumin

FIG. 5.-The immunological identity of the albumin, al globulin andV globulin fractions from urine (U) and serum (S). The anti-serum was contained in a rectangular trough enclosing the areashown in the photograph. (Direct print after staining withazocarmine.)

and y globulin lines from urine and serum respec-tively were found to join, showing, as on anordinary agar diffusion plate, that ttey are iden-tical antigenically (Fig. 5). By suitable arrange-ments other fractions can be made to behavesimilarly.One traction of the / globulins, the ,B lipo-

protein, has not been demonstrated in urine.Staining the dried agar plate with Sudan black forlipid shows a conspicuous /8 lipoprotein fractionin the serum but no corresponding band in theurine. Most of the a globulins of serum can bedemonstrated in urine, but their concentrationscompared with those of the other proteins do notappear to be any greater in urine than in serum.This suggests that the " a globulins," which Rigasand Heller found to predominate on movingboundary electrophoresis of normal urine protein,do not arise from the blood.Two common but inconstant anomalies of the

urine immuno-electrophoretic pattern are tailingof the albumin line and duplication of the yglobulin line. The cause of the former is notclear: the latter is presumably due to partialbreakdown of the y globulin fraction in the urine.Neither phenomenon was seen in the serumreconcentrated after 2,000-fold dilution, so thatthey cannot be due to dilution alone, but arepresumably due to interaction with other sub-stances sometimes present in the urine. It isinteresting that so few of the proteins in urineappear to be altered by such substances.There is almost always some pigmented material

left unmoved in the hole into which the urineconcentrate was placed before electrophoresis,especially if the sample is very viscous. If theurine becomes infected, all discrete lines may dis-appear; and, even if stored sterile, the lines tendto become hazy after a week in the refrigerator.

(2) The Same Antiseruim After Absorptionwith Urine Colloids.-This absorbed antiserum,although now producing no precipitin lines whentested against urine concentrates, was found toshow six lines with normal human serum, namely,X, two a globulins, and three /3 globulins. Inother words these six protein fractions, which arepresent in normal serum, are absent or nearlyabsent from normal urine. By staining withSudan black it was confirmed that one of thesesix protein fractions was 8 lipoprotein (Fig. 6).

(3) Antisera Prepared Against Human UrineColloids.-Two rabbits were used for the prepara-tion of antisera against normal human urine col-loids and both produced potent antisera as judgedby the content of anti-albumin etc.; yet on testing

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THE PROTEINS OF NORMAL URINE 365

mobility by this technique is much less than whenpurified and measured by moving boundaryelectrophoresis (Perlmann, Tamm, and Horsfall,1952). The precipitin line extends over the wholea globulin range. There seems little doubt that it

FIG. 6.-Inability of the kidney to excrete c lipoprotein. In trough:antiserum (against normal human serum proteins) absorbed withnormal urine colloids. In cup (S): Normal human serum.(Direct print after staining with Sudan black to show the j-lipo-protein fraction. The five other lines present before stainingare no longer clear.)

them against normal human serum, some of theplasma protein antibodies were found to be miss-ing, including that against the f8 lipoprotein frac-tion. This is further evidence for its absencefrom normal urine.

(4) Antisera Specific for Urinary Tract Colloids.-Analyses have also been carried out with theantisera prepared against urine colloids andagainst the Tamm-Horsfall mucoprotein afterserum protein antibodies had been removed fromthem by absorption with normal human serum.They reveal the presence of several proteins in theurine of normal men and women which do notoccur in the blood, the principal one being theTamm-Horsfall mucoprotein. Fig. 7 shows theprecipitin line obtained with the antiserumagainst this mucoprotein. It will be seen that its

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FIG. 7.-Mobility of Tamm-Horsfall mucoprotein. Left trough:Antiserum against human serum proteins. Middle trough:Empty. Right trough: Antiserum against Tamm-Horsfallmucoprotein. Serum in left cup (S). Urine colloids in rightcup (U). (Direct print of unstained plate.)

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GREGOR H. GRANT

must be the cause of the blurred pattern and theapparent increase in a globulins when normalurine proteins are examined by moving boundaryor paper electrophoresis. The elongated appear-ance of the line may be due to absorption of themucoprotein on to the agar or to the mucoproteinbeing a mixture of molecular species of varyingmobility but similar antigenic properties, cf., they globulin fraction. It is hoped to settle this pointand to report further details of the urinary tractproteins at a later date.

DiscussionNormal urine evidently contains small quan-

tities of many different proteins. The majorityare electrophoretically and immunochemicallyidentical with the plasma protein fractions, and sopresumably arise from the blood. Others, amongwhich the mucoprotein described by Tamm andHorsfall is probably the most abundant, have notbeen found in plasma and so presumably arisefrom the urinary tract. Several previous authorshave suspected that this mucoprotein must comefrom the urinary tract; the present findings pro-vide clear evidence in favour of this view.The presence of plasma protein fractions in

normal urine, especially the globulins, would bedifficult to explain on the older view that thenormal glomerulus is impervious to albumin orlarger molecules (unless they are foreign). Duringthe last few years, however, it has seemed increas-ingly probable that the glomerular capillaries aresimilar to other capillaries and produce a filtratecontaining about 15 to 20 mg. of protein per 100ml. similar to C.S.F. and other biological filtrates(Rather, 1952). Almost all this protein, amount-ing to about 30 g. daily, must be reabsorbed by thetubule cells; it is then probably broken down toamino-acids by an enzyme system of their mito-chondria (Oliver, MacDowell, and Lee, 1954;Sellers, 1956). On this theory the plasma proteinsin normal urine are easily explained as proteinsfrom the glomerular filtrate which have not beencompletely reabsorbed by the tubules.The proportions of these proteins excreted will

depend on the characteristics of tubular reabsorp-tion as well as on the degree of glomerular per-meability. Sellers (1956) assumed that the reversedA/G ratio of normal urine found by Rigas andHeller meant that the tubules must reabsorbalbumin more completely than the globulins;the present findings, however, suggest that thisassumption is unnecessary. the low A /G ratiobeing due to the presence of urinary tract protein.In the nephrotic syndrome, in which the inter-fering effect of urinary tract protein is dwarfed by

large quantities of excreted plasma proteins,Hardwicke and Squire (1955) found that, whentheir patients were given infusions of humanserum albumin, there were increases in theclearances of the four electrophoretic globulinfractions as well as the expected increase in thealbumin clearance, all five in definite proportionsto one another, suggesting that the tubulesreabsorb all proteins without selection up to theirmaximum capacity (threshold). As they point out,if in fact the tubules do reabsorb the differenttypes of protein without selection, the proportionsof plasma proteins excreted in the urine willmirror those in the glomerular filtrate, and thestudy of urine and serum proteins will provide asimple way of assessing the permeability of bothnormal and diseased glomerular capillaries.

For example, it should be possible to deducesome information about normal glomerular per-meability from the present immuno-electro-phoretic analyses by comparing the proteinsexcreted by the kidney with those not excreted,and seeing if the results are consistent with infor-mation available about capillary permeability ingeneral. The glomerular capillaries differ fromothers in being covered by the visceral layer ofBowman's capsule, but this layer probably onlyacts as a support, for by electron microscopy boththis epithelial layer and the endothelium appearto have pores large enough to allow any plasmaprotein molecule through, the real filtering mediumapparently being the continuous basement mem-brane between them (Pease, 1955). In any case,whether or not the capillaries are covered by anepithelial layer as in the glomerulus or choroidplexus, all the evidence suggests that the ease withwhich protein molecules pass through capillarymembranes depends primarily on the relative sizeof the molecules (see Yoffey and Courtice, 1956).The explanation of this on the " pore " theory ofcapillary permeability does not necessarily implythat the pores vary in size. Pappenheimer (1953-)and Pappenheimer, Renkin, and Borrero (1951)pointed out that even if the pores are all the samesize there will not be a clear-cut size of moleculewhich will just pass completely through the poresor not pass through at all. The more nearly thediameter of a molecule approaches that of thepore, the more will its passage tend to be restricted,until it is too large to pass at all.The molecular dimensions of a few of the better

known plasma proteins have been calculated(Hughes, 1954). Albumin, y globulin, fibrinogen,a lipoprotein, and transferrin are all elongatedwith short diameters of the order of 35 to 50 A,whereas 13 lipoprotein is spherical with a diameter

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of 185 A. Pappenheimer, who studied diffusionrates through the muscle capillary wall in cats,found that his results could be explained byuniform cylindrical pores 60 to 90 A in diameter.Such pores would stop the ,P lipoprotein moleculecompletely, but would allow the other moleculesabove to pass through in varying amounts accord-ing to their size. Our finding that /3 lipoproteinis not detectable in normal urine is in agreementwith this and with the findings of Baudouin,Lewin, and Hillion (1953) and Gavrilesco,Courcon, Hillion, Uriel, Lewin, and Grabar (1955),who investigated the proteins of C.S.F. by paperelectrophoresis and immuno-electrophoresis re-spectively. Morris and Courtice (1955), on theother hand, have found this lipoprotein by paperelectrophoresis in the lymph of some animals,especially cholesterol-fed rabbits, so that it canevidently pass through the capillary walls of theseanimals. Its absence from human urine in thenephrotic syndrome, a disease in which particu-larly large amounts of this lipoprotein occur inthe serum (Hardwicke, 1954), is further evidencethat it probably cannot pass through the normalglomerular capillary wall in man, and that theglomerular pores are therefore smaller than 185A. The alternative possibilities that it is com-pletely destroyed as it passes down the urinarytract, or is completely reabsorbed by the tubulecells, seem unlikely.Hardwicke and Squire (1955) have pointed out

how, on the hypothesis of non-selective tubularreabsorption, comparison of the excretion of largeand small molecule proteins in the urine could beused to study changes of glomerular permeabilityin disease, so that the relative importance ofglomerular leakage and tubular malabsorption ina given case of proteinuria could be deduced fromquantitative measurements of the serum and urineproteins. Studying cases of gross proteinuria inwhich the proportion of urinary tract proteinwould be too small to affect the results, they foundby paper electrophoresis that the clearances of thehigh molecular weight globulin fractions y anda2 were much less than that of the albumin frac-tion in nephrosis but became relatively higher inacute nephritis, a disease in which gross glomer-ular damage is known to occur.

It should be possible to apply this principlesatisfactorily to less severe cases of proteinuria,if the urine protein fractions are estimated bysome method which excludes the interfering effectof urinary tract protein. Uncorrected urine A/Gclearance ratios measured by paper electrophoresisare difficult to interpret (Wolvius and Verschure,

1957). It is hoped that measurement of theclearance ratio of albumin to y globulin by thistechnique may be more satisfactory, since neitherof these fractions overlaps with the urinary tractprotein; or the clearance ratio of albumin tosome convenient protein fraction of larger mole-cular weight could be estimated immunologicallyby such a technique as that recently described byGell (1957).

SummaryThe proteins of normal urine have been con-

centrated by ultrafiltration under negative pres-sure and then analysed by the immuno-electro-phoretic technique of Grabar and Williams.By using rabbit antisera raised both against

normal human serum and against normal humanurine colloids respectively, it has been shown notonly that normal urine contains small quantitiesof many different protein fractions, but also thatthese proteins are divisible into two groups:

(1) A group of protein fractions which, on thegrounds of their apparent identity with the corre-sponding plasma protein fractions, have probablyfiltered from the blood through the glomeruli.

Although these protein fractions of urine includemost of the protein components of plasma,3 lipoprotein and several other fractions presentin plasma were not detected in urine. It seems,therefore, that, although the glomerular poresmust be much larger than used to be supposed,they are not wide enough to let through everyplasma protein.

(2) A smaller group of proteins not demon-strable in blood and presumably arising from theurinary tract; the mucoprotein described byTamm and Horsfall was found to be a member ofthis group of urinary tract proteins.The high proportion of " a globulin " found in

normal urine protein by moving boundary orpaper electrophoresis is probably not due todifferential tubular reabsorption of the plasmaproteins but to the presence of this urinary tractprotein.We should like to thank Mr. G. Wright and Miss

M. Carpenter for technical assistance and Mr. R. Rossfor Figs. 1 and 2.

REFERENCES

Baudouin, A., Lewin, J., and Hillion, P. (1953). C.R. Soc. Biol.(Paris), 147, 1036.

Boyce, W. H., Garvey, F. K., and Norfleet, C. M. (1954). J. clin.Invest., 33, 1287.

Feinberg, J. G. (1956). Nature (Lond.), 178, 1406.Gavrilesco, K., Courcon, J., Hillion, P., Uriel, J., Lewin, J., and

Grabar, P. (1955). Bull. Soc. Chim. biol. (Paris), 37, 803.

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Gell, P. G. H. (1955). J. clin. Path., 8, 269.-(1957). Ibid., 10, 67.Grabar, P., and Williams, C. A. (1953). Biochim. biophvs. Acta, 10,

193.(1955). Ibid., 17, 67.

Hardwicke, J. (1954). Proc. roy. Soc. Med., 47, 832.-- and Squire, J. R. (1955). Clin. Sci., 14, 509.Hughes, W. L. (1954). In The Proteins, Vol. 2, Pt. B, Ch. 21.

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