4 SALT MICRO-ANALTTICAL METHODS [Vol. 7s

Micro-Analytical Methods for Proteins in Blood Plasma A Critical Review

BY HAllO1,T) R. SALT

SUMMAR\- 01; cos'ri<s.rs Introduction. Estimation of total protein in plasma or serum.

By physical methods. By nitrogen content. By colour reactions for molecular groiipings.

Electrophoretic separation. Differential precipitation by alcohol. Fractionation by salting-out.

Fibrinogen by coagulation. Cryoglobulins by cold precipitation. Globulins by viscosity measurements. Metal-combining globulin by absorptiometric titration. Albumin by combination with haemin. Acid-soluble mucoproteins. Specific protein fractions by immunoche~nical precipitation.

The differential determination of plasma protcins.

The separation and determination of certain proteins by their special properties.

Normal values.

1IEcm;T advances in our knowledge of the proteins of human blood plasma have shown the value of analysing plasma samples in detail and thereby delineating the various protein patterns that occur in health or in disease. Normally the plasma contains large amounts of albumins, globulins and fibrinogen, smaller amounts of lipoproteins and mucoproteins, and many antibodies, hormones and enzymes. Disease states are often accompanied by quantitative changes in the amounts or proportions of these plasma proteins, and some diseases are characterised by the appearance of abnormal proteins.

Our more intimate knowledge has come about largely through the application of elaborate new techniques for the separation, identification and estimation of the plasma protein com- ponents. Ultracentrifuga1,l electrophoretic2 y3 and physico-chemical preparative procedures4 p5 have each helped to elucidate the problems, whilst other techniques, appropriately related to physiological or immunological mechanisms,6 have been applied to the study of those proteins that possess specific biological properties. The elaboration of these varied techniques continues to be the subject of important fundamental research.

In the clinical laboratory, where the problems of human disease most urgently confront the chemist, detailed analyses for plasma proteins demand simpler and less costly methods than those proper to research. The complex nature of the proteins themselves and the necessary limitations of technical practice lead inevitably to an acceptance of analytical methods that are in greater or less degree empirical, and this has fostered, in turn, the develop- ment of many different procedures. Descriptions of these and discussions of their merits are widely distributed ; they form an extensive and perplexing literature.

Most of the methods published up to 1047 were reviewed by Kirk7 in a discussion on the chemical determination of proteins. Other analytical considerations have been included by Edsall,8 reviewing the fractionation of plasma protein, and by Gutman9 in a survey of plasma proteins in disease.

In this review only the simpler procedures acceptable to the clinical chemist will be discussed. Special attention will be directed to those model-n refinements of technique that best demonstrate the whole pattern of the plasma proteins and reveal any such particular abnormality as may be expected in any particular disease state. First, however, the problem of estimating the plasma total protein (or any one protein) must receive adequate consideration.

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Jan., 19631 FOR P R O T E I N S IS BLOOD PLASMA. A CRITICAL REVIEW 5 ESTIMATION OF TOTAL PROTEIN I N PLASMA OR SERUM

B Y PHYSICAL METHODS-

The effects of proteins on certain physical properties of their solutions provide simple methods for their estimation which avoid the need for first isolating them. Such methods are applicable to blood plasma, in which variations of physical properties are correlated closely with quantitative changes in protein content and but little affected by fluctuations in other constituents. The methods are especially useful when large numbers of observations are to be made, but they demand careful standardisation and the maintenance of exact techniques.

Procedures based on specific gravity measurements have been described by Kagan,lo who timed the fall of a drop of serum through a fixed distance in oil, and by Lowry and Hunter,ll who noted the flotation of a drop of serum in an organic liquid mixture having its specific gravity graded throughout its height. A more recent technique, which has been widely used and studied, is that of Van Slyke, Hiller, Phillips, Hamilton, Dole, Archibald and Il:der,12 who compute the plasma total protein from the specific gravity determined by flotation of drops of plasma in solutions of copper sulphate. Sunderman13 has recommended that total protein be estimated by measurement of the refyactiue index of the plasma. The method is less sensitive than those based on specific gravity and may be inaccurate when plasma lipids are high.

Other physical methods have been applied to the protein after its precipitation from solution. In the method of Looney and Walsh,14 the protein was precipitated from diluted serum by means of salicylsulphonic acid in the presence of ghatti gum, and the optical turbidity of the suspension was measured in a photelometer. This technique has been refined by Salt15 and described along with a similar method for serum globulins. Light dispersion methods of this kind are both accurate and sensitive, provided that the particle-size of the dispersed proteins are standardised by exact control of reaction conditions, of which the most important is the controlling effect of a protective colloid.

Separation of proteins from solution, with subsequent purification, drying and weighing, was put forward as a pav ime t r i c method by Robinson and Hogden.16 Unlike what has happened for most analytical methods, the gravimetric procedure has not been generally accepted as the most reliable standard reference method, owing to the difficulties of purifying and drying the protein precipitates. If these difficulties can be surmounted, the method has merit for reference purposes. I t is free from errors due to variations in protein nitrogen content (which also affect the accuracy of the Kjeldahl procedure, more often used as a reference method) but i t requires large quantities of material.

BY NITKOGEN CONTEST--

Determination of total nitrogen in purified protein fractions (or in cruder material with a suitable correction for non-protein nitrogen) has been adopted generally as the standard method to which others can be referred. Brand, Kassell and Saidell7 determined the percentages of nitrogen in separated fractions of normal human plasma by the micro-Dumas combustion method with the following results: albumin 15.95, P-globulin 14-84, y-globulin 16-03, fibrinogen 16.90.

In the Kjeldahl technique as applied to plasma total protein, a constant protein-nitrogen content of 16.0 per cent. (factor 6.25) is usually assumed. The acceptability of this assumption has been confirmed by Hiller, Plazin and Van Slyke,l* who recently reviewed the experimental conditions variously recommended for Kjeldahl determinations of protein nitrogen. These authors concluded that mercury, which was first introduced as a catalyst only two years after Kjeldahl's original publication in 1883, is the only satisfactory accelerator of the digestion process. Their micro-Kj eldahl procedure makes use of sulphuric acid, potassium sulphate arid mercuric sulphate to effect complete digestion within 30 minutes of the clarification of tlie solution. Before distillation of the ammonia, zinc dust is added to reduce mercuric oxide to mercury, as otherwise a considerable fraction of the ammonia is bound by the mercuric oxide (precipitated by the added sodium hydroxide) and cannot be liberated by boiling. A convenient new form of all-glass apparatus for steam distillation has been described by Markham. l9

Despite criticisms1* of selenium as a catalyst, the colorimetric method of Campbell and Haniia,2" comprising micro-digestion and direct nesslerisation, has been found convenient and satisfactory in clinical work.

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6 SALT: MICRO-ANALYTICAL METIIO1)S [Vol. TS

Several colour reactions, specific for characteristic groups occurring in all proteins, have led to colorimetric micro-methods in which preliminary destruction of the proteins is unnecessary. The most successful methods have been based on the constant occurrence of tyrosine or arginine, or of the “twin” peptide linkages found in the amino-acid chains of the protein molecule.

The tyrosine reactio:t--In the method of Greenberg,’l the phenol reagent of Folin and Ciocalteu was used to give a blue colour with the tyrosine moiety of the proteins, and the colour was compared with that from a pure tyrosine standard. As tryptophan also gives some colour, the method is not specific, and an empirical relationship between the amounts of each protein and the colour intensities produced has to be established. )Yokes and Still22 investigated the method and found that it gave results lower than those given by Kjeldahl nitrogen determinations and that the values increased with time of storage of the sera before analysis. Several improvements to Greenberg’s technique were subsequently described and various procedures were developed including a preliminary heating of the protein in sodium hydroxide solution before proceeding with the colour reaction. This simple modifica- tion overcame the difficulties mentioned, although the method still remained empirical. I have found the technique of Minot and Keller32 to be both the simplest and the most reliable.

An interesting further development of the reaction between proteins and the phenol reagent has been reviewed by Lowry, Rosebrough, Farr and Kanda11,24 and applied by them to a method for estimating as little as 0.2 pg of protein. By a preliminary reaction for 1 0 minutes a t room temperature, the protein is held in an alkaline copper solution. The phenol reagent is then added and a blue colour appears in 30 minutes and, with serum proteins, has an intensity seven times that produced when the preliminary reaction is omitted. Although the procedure suffers from the disadvantage that the intensity of the colour varies with different proteins and is not strictly proportional to the concentration of protein, the greater sensitivity achieved makes i t especially useful for the determination of small amounts of protein of fairly standard composition.

The arginine reaction-The Sakaguchi reaction for arginine has been developed quantita- tively by Albanese, Saur and I r b ~ . ~ ~ In this micro-method, the plasma sample is diluted with 10 per cent. sodium hydroxide solution; water and ethanolic cc-naphthol are then added, and finally 0.06 N sodium hypochlorite and 20 per cent. urea solutions. Keyser26 found it necessary to increase the hypochlorite concentration to 0.15 2c’ to achieve satisfactory estimations of total protein, and he experienced other difficulties when attempting to estimate albumin and globulin separately. I have not found any special advantage in this nietliod; moreover, it cannot be said that the arginine content of the various plasma proteins is niore constant than the tyrosine content.

The ninhydrin reactioir-The colour reaction2’ between cc-amino-acid groups and ninhydrin has been applied by Kunkel and Ward28 to the determination of protein, especially the sinall quantities obtain able fractionally from serum after immunochemical precipitation. A1 t hougl1 non-specific, inasmuch as peptides, amino-acids, amines and ammonia also interact , and although variable, in that different proteins give different intensities of colour, this simple method is highly sensitive and especially useful for minute quantities of particular proteins, for as little as 3 pg of albumin can be determined accurately by it.

T?ze biziret reactiout-In my experience, the biuret reaction, in its modern form, has proved to be the most reliable absorptiometric method for direct determinations of protein in clinical work. All plasma proteins give the colour response at approximately the same intensity and the reaction is unaffected by small amounts of ammonium ions, which may remain after ammonium sulphate has been used for fractionation. Various biuret reagents and practical procedures have been devised by different analysts during the past four decades. The reaction itself has been re-examined recently by Mehl, Pacovska and Winzler,29 who show it to be similar to, but not identical with, that used by Lowry et ~ ~ 1 . 2 ~ in their preliminary treatment of protein, before colour reaction with the phenol reagent, by the method noted above.

A biuret technique has been adapted for visual colorimetry by making use of the Lovibontl comparator. This simple application gives an approximate determination of protein, but the method is not without its difficulties. and sekeral improvements have been introduced by Love and I i m i ~ d e n ~ ~ in a recent revision of the technique.

BY COLOUR REACTIONS FOR MOLECULAR GROUPINGS-

Some of these were studied by Wokes and

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Jan., 19531 FOR PROTEINS IS BLOOD PLASMA. A CRITICAL REVIEW 7

For accurate protein determination, refined absorptiometry of the biuret colour is essential. 'This is achieved by use of the Ilford No. 605 colour filter in the photo-electric instrument described by Salt,15 as slightly modified to accommodate optical cells of 13 mm optical length. Extensive use of the method of Kingsley31 has in the past provided excellent results, but this has now been relinquished in favour of a newer procedure based on the studies of Weich~elbaum.~~

In recent years many analysts have sought to improve the biuret technique by establishing optimal reaction conditions and to simplify i t by devising a reagent containing the copper and alkali together in one solution. A later technique of KingsleyS was criticised by Gornall, Bardawill and Davida and was further modified by who chose to apply the biuret reaction in highly alkaline medium and to use a solution of potassium permanganate as an artificial colour standard. Levin arid B r a ~ e r ~ ~ have also devised a 11 ighly alkaline biuret reagent containing both sodiuni and ammonium hydroxides. They found that ammoniiim hydroxide by itself failed to bring about the reaction.

In contrast, Weichselbaum32 has put forward a biuret reagent h ~ i n g a high copper content, stabilised with sodium potassium tartrate and potassium iodide, and containing a small amount of sodium hydroxide. The method was devised for visual colorimetry, but was also described in a footnote32 in a suitably modified form for photo-electric ahsorptiometry. Experience with these several reagents has led me to adopt the weak117 alkaline type of reaction, despite the claima that optimal conditions are only established by the use of a higher concentration of sodium hydroxide. Weichselbaum's absorptiometric reagent has been effectively applied by Wolfson, Cohn, Calvary and Ichiba3' to the determination of serum protein and protein fractions in an analytical scheme that is described below (p. 9). The reagent is invariable, keeps indefinitely and rarely shows any turbidity even with highly lipaemic sera. If a turbidity does appear, the coloured solution can be clarified by shaking with ether and centrifugation. This procedure is more effective than the method of correcting for turbidity described by Keyser and \Taughn3s in connection with a biuret reaction of ;t

different type.30 939

T H E DIFFEREXTIAL DETERMIXAl3OX OF PLASMA I'lIOTEISS

In many types of disease the blood plasma is found to contain normal proteins in dis- proportionate amounts, and refined methods oi separation are therefore needed before exact analytical assessment of the several fractions can be made. Many methods of separation have been devised and comparative studies have been undertaken to (letermine in detail the protein patterns characteristic of health and of disease.

E:LECTIIOPIIORETIC SEPARATION-

In recent years, the moving-boundary method of electrophoretic :~nnlysis, based on tiit. principles laid down by Ti~elius,~O has become a standard method by which to evaluate other procedures for plasma protein fractionation. In the electrophoretic technique, the proteins are separated on the basis of their differential mobility in an electric field, and the determinations are made refractometrically. Other modes of differential deterniinatioii have been compared with the electrophoretic method by Edsall,8 Gutman!) and by Marracli and H o ~ h , ~ l with conclusions that generally testify to the superiority of electrophoresis. The moving-boundary electrophoretic procedure is, however, too elaborate and costly for routine use in clinical analysis, so that i t will not be considered further here.

elegant new technique for micro-electrophoresis of proteins on filter-paper, wl iicli has recently become available, is attractively simple and deserves detailed consideration. In principle, a strip of filter-paper is soaked in barbitone buffer a t a pH of 8.6, a small volumc (as little as 0-01 ml) of serum is applied at a point on the paper, and a direct current (at, say, 100 volts) is passed for a sufficient time (about I8 hours) through the paper. After the period of electrophoresis, the paper is dried and the proteins are stained with a suitable dye, where- upon it is seen that the albumin fraction has migrated furthest along the paper strip towards the anode, and the ul-, u2-, 18- and y-globulins have migrated through progressively shorter distances, in that order.

The somewhat elaborate apparatus of Cremer and T i ~ e l i u s , ~ ~ and the simpler forms described by Grassmann, Hannig and Knede1,43 Goa44 and Kunkel and T i s e l i ~ s , ~ ~ all contain the paper strip in a horizontal position. L a t r ~ e r ~ ~ has described an apparatus tl7iCt holds the paper sloping downwards at an angle of 24", while Durrum47 and Flynn and de Mayoj*'

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8 SALT : MICRO-ANALYTICAL METHODS [Vol. 78

have achieved protein separation on folded paper strips held in a steeply inclined position. Staining of the proteins has been accomplished by means of azocarmine,4Q J % ~ ~ bromo-

phenol blue,42s44@s47s48 or Amidoschwarz 10B (Naphthalene Black 12B, 200).83s48 Ultimate differential determination of the proteins was completed by elution of the dye from cut portions of the paper when bromophenol blue was used, or by an elegant method of direct photometry of the coloured portions of the paper when the black dyea or azocarmine50 was used. An ingenious process of “retentiometry” was devised by Wieland and Wirth51 in connection with their azocarmine method.

I have obtained good results, especially when using Whatman No. 100 filter-paper in the horizontal or slightly sloping positions, in apparatus similar to that of Grassman et U Z . , ~ and without the complication of any cooling device. The determinations have been completed successfully by using the acid-aqueous bromophenol blue staining method and subsequent washing with 0.5 per cent. aqueous acetic acid,& and also by staining with naphthalene black48 and estimating the proteins by direct photometry.&

Micro-electrophoresis on paper, besides being simple, provides a means for the absolute separation of the several protein components from minute amounts of serum, and also an opportunity for analysing the fractions for components other than the proteins. For a general account of the possibilities of this technique the comprehensive paper of Kunkel and Tiselius& is especially recommended.

DIFFERENTIAL PRECIPITATION BY ALCOHOL-

The large-scale procedures, devised by E. J. Cohn and his colleagues (see review by Edsalls) for the separation of protein and other components of plasma, by means of various concentrations of ethanol at low temperatures with controlled pH and ionic strength, have now been applied to smaller volumes of plasma by Cohn et d5 Their new scheme, “Method 10,;’ has been further adapted by Lever, Gurd, Uroma, Brown, Barnes, Schmid and S c h ~ l t z ~ ~ to provide detailed analyses of 5.0-ml samples of plasma for their contents of various proteins, protein-bound lipids and carbohydrates. Some special equipment is required, but the scheme has the advantage that the biochemical properties of the separated proteins are unaltered. An application of this scheme of analysis to series of pathological plasma samples would be a valuable extension of this wwk and likely to produce important new knowledge.

In a similar, but simpler, manner Pillemer and Hutchinson= effected separation of serum albumin and globulin at 0” C by means of 42.5 per cent. methanol at a pH of 6.8. Whereas these authors completed the determinations by Kjeldahl digestion, Christensen” demonstrated that the biuret reaction could be applied to the methanolic filtrate containing albumin.

Salt15 has described a dispersimetric method for the direct determination of globulin in diluted serum, wherein the globulins are precipitated by 45 per cent. methanol at a pH of 6.6 in the presence of ghatti gum in 1 hour at 37°C. Under these conditions a uniform colloidal suspension of denatured globulin is formed, having considerable light-dispersing power especially for the violet waveband, so that a high degree of sensitivity is achieved.

FRACTIONATIOK BY SALTING-OUT-

In clinical chemistry, salting-out techniques have been both widely used and vigorously criticised. Often they have been modified or elaborated inorder to yield protein fractions closely in agreement with those obtained by electrophoretic procedures. Most interest has been centred around the globulin components of the serum, some of which are now known to have special biochemical functions in vizo.

Ammonium sulphate-The classical method of separating plasma proteins by salting-out with ammonium sulphate is now regarded as unreliable except for the precipitation, by 0.34 saturation, of the y-globulin fraction in a fairly pure ~ t a t e . ~ The difficulties of nitrogen estimation, as a result of using ammonium sulphate precipitation of the protein, have been avoided in determinations of serum y-globulin by the use of the biuret r e a ~ t i o n , ~ ~ , ~ ~ or by a turbidimetric t e c h n i q ~ e . ~ 6 ~ ~ ~ These simple methods are of practical value, despite failure to obtain exact correlation between amounts of precipitated protein determined t urbidime t ric ally and y -globulin isolated electrophoretic ally , either quali t a t ively58 or q~antitatively.~~ 969

Sodium suZphate-The classical method of Howeso for separating serum proteins into albumin and three globulin fractions by means of sodium sulphate at final concentrations of 21-5, 17.4 and 13.5 per cent. has been widely used until recent times, when it became evident

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Jan., 19533 FOR PROTEINS I N BLOOD PLASMA. A CRITICAL REVIEW !I

that there were discrepancies between the fractions so obtained and those given by electro- phoresis. For example, Peterman, Young and Hogness61 were able to show that albumin values obtained by the Howe technique were in close agreement with the sum of the albumin and a-globulin concentrations determined electrophoretically. Later investi- g a t o r ~ ~ ~ , ~ ~ , ~ ~ , 6 ~ , ~ 6 , 6 7 have obtained results which, although riot altogether in agreement, have indicated that a better separation of serum proteins can be achieved by salting-out witl; sodium sulphate a t the higher concentrations of 26-0, 1 9 4 and 15.0 per cent., the resulting iractions being then similar to the albumin and the three main globulins as separated 11) electrophoresis.

*4s a precipitant, sodium sulphate has not been entirely satisfactory in my laboratory, because long and difficult filtrations in the warm are necessary and some inaccuracy can aristi from adsorption of proteins on the filter-paper.6s The separation of globulin from albumiii by means of sodium sulphate can be done, however, without adsorption error by the technique of Kingsley,G9 in which ether is also gently shaken into the mixture and separation of the. globulin effected in the centrifuge.

Sodium sulfihite-Other neutral salts have been used for fractionation of serum proteins, notably sodium sulphite a t reagent concentrations of 21.0, 18.0 and 15-0 per cent., as described by Campbell and Hanna.'O Although the 21-0 per cent. reagent is now regarded as insufficiently concentrated to precipitate all of the globulins from serum, the scheme of Campbell and Hanna gave fractions comparable with those prepared by the Howe technique, and without the need of working in the warm. The clinical usefulness of sulphite fractionation has been demonstrated by Salt7* for the serum proteins of patients with chronic rheumatic disorders.

Covzbinatioizs o j saltiizg-ozd @vocedures-In 1948, Cohn and W0lfson7~ introduced the use of sodium sulphite at the higher concentration of 28.0 per cent. for precipitation of ali globulins from serum. This method was further studied73 and was finally elaborated to a form37 that provided rapid estimations of serum albumin and of the globulins in threc fractions. For the differentiation of the globulins, use was made of sodium sulphate at ;L

reagent concentration of 234 per cent. and of an ammonium sulphate - sodium chloridc solution. This last reagent was devised as 19.3 per cent. ammonium sulphate in 4.0 per cent. sodium chloride, but was later modified to include only 3.0 per cent. of sodium chloride, as noted by de la Huerga and Popper.56

I have made extensive trials of the procedure of Wolfson et aL37 with satisfactory i-esult,c. I t should be noted, however, that the biuret reagent described by the authors of the method is stated to contain 90 g of Rochelle salt, whereas I prefer only 30 g in 2 litres of reagent. The lower quantity appears to be more satisfactory, and it gives results that agrec with the original biuret procedure of Wei~hse lbaum.~~ The separations of precipitated proteins are all quickly made in the centrifuge, ether being used as an aid in the sodium sulphate fractionation, and in the sodium sulphite fractionation ether containing 1.0 per cent. of Span 20. Several other non-ionic surface-active agents are equally effective, Empilan AQlOO being the one I normally use.

Although this simple and useful scheme is recommended, it must be recognised that the separation of serum proteins into characteristic fractions, each in a state of purity, is inipossiblc by any kind of salting-out process. 'The comparative studies of Jager, Schwartz, Smith, Xickerson and Hr0wn7~ revealed that the protein obtained in filtrates from four procedures for the salting-out of total globulin in no instance consisted solely of albumin, as shown elec trophoretically. Despite this, evidence for the reliability of the methods of Wolfson et aZ.37 for determining the albumin and the albumin plus a-globulin fractions was recorded, and similar support was provided by I-evin, Oberholzer and Whitehead75 in respect of these €ractioiis and of y-globulin.

Studies were also made by Popper, de la Huerga, Franklin, Bean, Faul, Routh and Schaffner7'j in order to evaluate the chemical methods of Wolfson et aZ.,37 the electrophoretic process being used for reference. The results led to a recommendation of the chemical methods for albumin, total globulins and y-globulin, but to only a cautious acceptance oE the determinations of K- and ,8globulins. These two fractioiis are both obtained by thc method of difference, and this, together with the fact that the globulins have lipid and carbohydrate moieties combined with them, may account for the larger experimental errors. I t may be recalled that 0 t h e r s ~ 6 , ~ ~ who used sodium sulphate for fractionation, also recorded poor results for the determinations of a- and ,&globulins.

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10 SALT : MICRO-ANALI’TICAL METHODS [Vol. ‘78

When the lipid content of serum is greatly increased, special difficulties are encountered in both separating and estimating the protein fractions. Popj Ak and McCarthy77 have shown that complete saturation of lipaemic or of normal sera with magnesium sulphate successfully separates the globulins from albumin. after a few trials, questioned this claim, but Jager et aZ.7* established its validity. The main disadvantages of the magnesium sulphate procedure are that 24 hours are required for salting-out and that the biuret reaction is inapplicable, as the alkaline reagent gives a precipitate of magnesium hydroxide in the reaction mixture.

I t is preferable to remove the lipids from lipaemic sera before analysing for proteins. The removal of lipids is done most successfully by shaking the serum with ether, freezing solid, then thawing ,and centrifuging; the whole process is repeated five times, each with fresh ether, as described by Popjiik and M~Carthy.~7 The cleared serum is then analysed without difficulty, either by the chemical method37 or by micro-electrophoresis on filter-paper.&

Martin and

THE SEFARATIOS AXD DETERMISATION OF CERTAIN PROTEINS BY THEIR SPECIAL PROPERTIES

FIRRISOGEN BY COAGULATIOK-

The classical method78 for the separation of fibrinogen from plasma, in which the addition of an excess of calcium ions activates the enzymic precipitation of fibrin, is an early example of a procedure that makes use of a special property of a particular protein. Subsequently, salting-out with 12.5 per cent. sodium sulphite7O or buffered sodium ~ulphate7~ was devised in order to include determinations of fibrinogen along with other plasma protein fractions in cwmprehensive schemes for differential analysis.

Several of these methods were critically reviewed by Morrison,80 who then proceeded to a careful study of the clotting process brought about by the addition of a thrombin preparation derived from blood plasma. He defined the optimal conditions for coagulation of fibrinogen, such that complete separation of fibrin was effected, with but minimal occlusion of other proteins. Morrison’s method for fibrinogen has been usefully adapted by Ratnoff and Menziesl for the analysis of small clinical samples of plasma.

Another methods2 that has been used successfully for the determination of plasma ‘tibrinogen depends upon the unique precipitability of fibrinogen when a solution of protamine and subsequently one of calcium acetate are added to dilute plasma.

CRYOGLOBULINS BY COLD PRECIPITATIOK-

Morrison’s studies80 on the coagulation of fibrinogen led also to the recognition, in normal plasma, of minute amounts of a protein that forms a reversible gel when a solution of it is cooled to 0” C. Similar proteins possessing the property of separating from solution when plasma is cooled had previously been discovered occasionally in the blood of persons with certain diseases. The earlier literature has been reviewed by Lerner and Watson,83 who described in detail a cold-precipitable globulin. The term “cryoglobulin” was proposed for this type of protein and further researches were recorded by Lerner and Greenberg.84 By a suitably devised technique, Lerner, Barnum and Wats0n8~ showed the presence of small amounts of serum cryoglobulin in many different disease states and its absence from the serum of normal controls.

Subsequent contributions to our knowledge of the cryoglobulins have been reviewed and new data recorded in several recent publications.86~87~88 Evidently the cryoglobulins comprise a group of proteins with variable properties, but having one common property, zliz., that of spontaneous precipitation from solution at temperatures below 37” C, with the formation of a gel, an amorphous deposit or a crystalline precipitate. In all, the cryo- globulin can be separated from the cooled plasma and estimated, directly if the amount is large or by difference if small. When gel formation occurs in the plasma, the cryoglobulin may be absent from the serum, owing to occlusion with fibrin during the blood-clotting process. Hence, both serum and plasma samples should be examined for the presence of cryoglobulin by the methods indicated.85 787

GLOBULINS BY VISCOSITY MEASUREMEXTS--

high viscosity. Closely related to the occurrence of cryoglobulins is the existence of globulins of unusually

Descriptions of this rare abnormality have been published,sg sgo and the

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J;tn., 19531 FOR TJHOTI<INS I N BLOOD PLASMA. A CRITICAL R E V I E W 11

teclmique of investigation , by measurement of serum viscosity at various temperatures, has been d e ~ c r i b e d . ~ ~ ~ ~ ~ By the method of Woodmansey and 1&’ilsong2 for the measurenaent of viscosity, I have confirmed the value of that technique in three instances, but as the enhanced viscosity in these rare cases is related to the nature of the globulins rather than to their absolute quantity, the method is chiefly of qualitative value.

Small increases in the viscosity of serum or plasma may be found in a wide variety of disease states, owing mainly to increased concentrations of the normal globulins or of fibrinogen. The usefulness of single viscosity measurements is illustrated by results of the recent publications of Salt71 and of Houston, Whittington, Cowan and H a r k n e ~ s . ~ ~ Obviously, it is impossible to establish strict quantitative relationships, as abnormal proteins may also be present and others, especially albumin, may be depleted by disease.

L a w r e n ~ e ~ * , ~ ~ has used viscometry for the assessment of the whole plasma and of tlie plasma proteins obtained in solution in four fractions after separation by chemical means, but does not advise conversion of the measured values to viscosity units or to protein concentrations.

The interesting technique of Foster and Biguriag6 depends on the production of an increase in the viscosity of serum by the addition of formaldehyde. This increase is due to interaction with the globulin moiety only, and the measured increase in viscosity may thus be related to the globulin content of the serum.

~IT<TAI.-COMBTXING GLOl3ITLIX BY ABSOKPTIOMETKIC TITRATION-

A &-globulin constantly present in the plasma and possessing unique biochemical properties has been described by Surgenor, Koechlin and Strongg7 This protein, although present normally in only small amounts, is responsible for the transport of inorganic iron in the body and can be estimated readily by means of its iron-binding capacity. In the technique described by Rath and Finch,gs the existing serum iron is determined absorptio- metrically by means of the dipyridyl reactiong9 and the residual iron-binding capacity of the serum by absorptiometric titration with an acid solution of ferrous ammonium sulphate. The sum of the two values gives the total iron-binding capacity of the serum and, as this property is due entirely to the metal-combining /3,-globulin, its specific estimation is achieved.

,ALRUMIN B Y COMBINATION W I T H HAEMIN-

Iron in organic combination is not normally present in the plasma, but if haemin (ferri- protoporphyrin IX chloride) is added to plasma or serum, a coloured compound is formed specifically with the albumin fraction. The compound methaemalbumin has been studied by Rosenfeld and Surgenorloo and a method for the determination of albumin, based on its haemin-binding property, has been proposed. The method involves titration with haemin at a pH of 7.3, the titration being followed absorptiometrically at 403 mp. More complete details have now appeared,lol and the method has been used by Lever et for the determination of albumin in serum or, preferablv, in a serum fraction containing all the albumin together with only about 20 per cent. of the other proteins.

ACID-SOLUBLE MUCOPROTEIKS-

Protein-bound carbohydrate is present in the serum, being distributed generally on tlie globulins, and more especially throughout the a-globulins. These compounds, which have been variously designated as seromucoid, glycoprotein, mucoprotein and so on, are still only vaguely defined, although abnormalities in their concentration occur in certain diseases and these are of considerable importance.

A distinct advance was made by Winder, Devor, Mehl and Smyth,l02 who used percliloric acid solution to precipitate the main plasma proteins and give an acid filtrate containing a mucoprotein fraction. The mucoprotein was then precipitated by means of phosphotungstic acid, the protein being estimated by the biuret reaction or from its tyrosine or nitrogen content. Alternatively, the characteristic carbohydrate fraction of the mucoprotein was determined by the colour reaction it gives with orcinol in sulphuric acid solution. This reaction has been studied by Friedmannlm and compared with similar reactions in which carbazole or skatole were used. Her results confirm the view that the hexose moiety of serum mucoprotein consists of mannose and galactose.

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12 SALT : MICRO-ANALYTICAL METHODS [I‘ol. 7s

Greenspan, Lehman, Graff and Schoenbachlo4 have published careful details of their scheme for the assessment of serum “polysaccharide” (in arbitrary units), and for the cleter- mination of the carbohydrate of ethanol-precipitated serum protein and of separated miico- protein by condensation with tryptophan in the presence of sulphuric acid. In certain disease states, distinct abnormalities were revealed, including that of the ratio of hexose to protein (biuret value), which was frequently disproportionate, so indicating qualitative as well as quantitative changes in the mucoprotein fraction.

Graff, Greenspan, Lehman and Holechek105 have adopted the anthrone reaction for the estimation of hexoses in protein and in the serum mucoprotein fraction. By this method a green colour is developed without the need for elaborately controlled reaction conditions, and all interference by non-specific reaction products is eliminated when measurements are made in the region of 620mp. I favour the use of the anthrone reaction, especially as it gives results agreeing closely with those by the tryptophan reagent.

Besides mannose and galactose, glucosamine is also present in serum mucoprotein, although it is not estimated by any of the reactions above mentioned. West, Clarke and KennedylOG devised a method for hydrolysing diluted serum by boiling it with AT hydrochloric acid for 5 hours. The glucosamine in the neutralised filtrate is then determined absorptiometrically at 525 mp, after reaction with acetylacetone and condensation with 9-dimethylaminobenzaldehyde in hydrochloric acid.

SPECIFIC I’KOTEIS FRACTIONS BY IMMUSOCHEMIC‘AL PRECIPITATIOS----

Since it became possible to prepare albumin and certain globulins of high purity, they have been available for the preparation of specific precipitating anti-sera from animals after suitable immunisation procedures. These anti-sera possess the property of precipitating specifically the homologous antigen from the plasma being analysed ; the amount of protein so obtained can then be determined by any suitably sensitive technique.

In the method of Chow,107 plasma albumin is precipitated by this means and determined either turbidimetrically or by micro-Kjeldahl estimation of the nitrogen in the precipitate. Later, Chow and his colleaguesloS confirmed the value of the turbidimetric method, which avoids the necessity for separating the immunochemically precipitated protein and washing it before analysis. Moreover, the procedure was found to give results closely in agreement with those of electrophoretic analysis and superior to those obtained by salt-fractionation. Kunkel and Ward28 have also described a determination of serum albumin by precipitation with a specific anti-serum and estimation of the precipitated protein absorptiometrically after applying the ninhydrin reaction. In a similar way, Bendich and KabatlOQ used an anti-serum to precipitate y-globulin from plasma or serum samples and completed the deter- mination by nitrogen analysis of the precipitated protein.

Provided that cross-reactions can be minimised between anti-sera and antigens closely related to the specific protein to be determined, immunochemical methods of serum protein fractionation are of great value. This is especially true when the amounts of serum available are minute, or when the particular protein is present in amounts too small for determination by any other technique. An immunochemical method for demonstrating the presence of Bence- Jones protein in serum, and for determining its concentration, has been described by Moore, Kabat and Gutman.llo In this procedure, the preparation of a potent anti- serum is difficult, but the method appears to be the only successful one for estimating this particular protein in serum.

NORMAL VALUES

A critical approach to the many techniques for separating and determining plasma or serum proteins and an appraisal of their several merits and difficulties clearly show that it is impossible to define exact limits for normality. Some analytical empiricism is inevitable and every result has to be related to the method by which it has been derived.

Many of the publications already mentioned include valuable data about normal con- centrations of plasma protein components and tabulated values are presented in some of t h e ~ e . ~ , ~ , ~ ~ $2 The appropriate sections of a reviewlll of the chemical composition of blood plasma and serum also include values for electrophoretic and ethanol-separated protein fractions in normal plasma ; a recent publication112 on paper electrophoresis provides a useful comparison of the results produced by that technique with those by the original electrophoretic method of Tiselius.

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Jan., 19531 FOR PROTEINS I N BLOOD PLASMA. ‘4 CRITICAL REVIEW 13

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Pillemer, L., and Hutchinson, M. C., J . Biol. Chem., 1945, 158, 299. Christensen, H. N., J . Lab. Clin. Med., 1946, 31, 916. Jager, B. V., and Nickerson. M., J . Biol. Chem., 1948, 173, 683. de la Huerga, J., and Popper, H., J . Lab. Clin. Med., 1950, 35, 459. Levin, B., Oberholzer, V. G., and Whitehead, T. P., J . Clin. Path., 1950. 3, 284. de la Huerga, J., Popper, H., Franklin, M., and Routh, J. I., J . Lab. Clin. Med., 1950, 35, 466. Ricketts, W. E., Sterling, K., and Levine, R. S., Ibid., 1951, 38, 153. Howe, P. E., J . Bid. Chem., 1921, 49, 93, 109 and 115. Peterman, M. L., Young, N. F., and Hogness, K. R., Ibid., 1947, 169, 379. Majoor, C. L. H., Ibid., 1947, 169, 583. Milne, J., Ibid., 1947, 169, 595. Kibrick, A. C., and Blonstein, M., Ibid., 1948, 176, 983. Martin, N. H., and Morris, R., J . Clin. Path., 1949, 2, 64. Martin, N. H., Morris, R., and Smith, M., Ibid., 1950, 3, 266. Baker, R. W. R., and Merrivale, W. H. H., Scand. J . Clin. Lab. Invest., 1951, 3, 273.

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14

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Popjhk, G., and McCarthy, E. F., Biochem. J . , 1946, 40, 789. Cullen, G. E., and Van Slyke, D. I)., J . Biol. Cheulz., 1920, 41, 587. Hill, R. M., and Trevorrow, V., J . Lab. Clin. Med., 1941, 26, 1838. Morrison, P. R., J . Awer . Chem. Soc., 1947, 69, 2723. Katnoff, 0. D., and Menzie, C., J . Lab. Clin. Med., 1951, 37, 316. Mylon, E., Winternitz, M. C., and de Siito-Naigy, G. J., .J. Bid. C h e w , 1942, 143, 21. Lerner, A. B., and Watson, C. J., Atnev. J . Med. Sci., 1947, 214, 410. Lerner, A. B., and Greenberg, G. R., J . Biol. Chem., 1946, 162, 429. Ixrner, A. B., Barnum, C. P., and Watson, C. J., Anier. J . Med. Sci., 1947, 214, 116. Hill, R. M., lhinlop, S. G., and Mulligan, R. M., .I. Lab. Clin. Med., 1949, 34, 1057. Harr, D. P., Header, G. G., and Wheeler, C. H., Ann. Intern. Med., 1950, 32, 6. Rorvik, K., Acta Med. Scand., 1950, 137, 390. Shapiro, S., lioss, V., and Moore, D. H., J . Clin. Invest., 1043, 22, 137. Lucey, H. C., Leigh, E., Hoch, H., Marrack, J. R., and Johns, 13. G. S., Brit . J . Ex?. Pccth., 1930,

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, , Ibid. , 1952, 199, 911. Winzler, R. J., Devor, A. W., Mehl, J. W., and Smyth, I. XI., J . Clin. Invest., 1948, 27, 609. Friedmann, R., Biochem. J . , 1949, 44, 117. Greenspan, E. M., Lehman, I., Graff, M. M., and Schoenbach, E. B., Cancer, 1951, 4, 972. Graff, M. M., Greenspan, E. M., Lehman, I. R., and Holechek, J . J., J . Lab. Clin. Med., 1951,

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Bendich, A., and Kabat, E. A., Ibid., 1949, 34, 1066. Moore, D. H., Kabat, E. A., and Gutman, A. B., J . Clin. Invest., 1943, 22, 67. Krebs, H. A., Ann. Rev. Biochem., 1950, 19, 409. Koiw, E., Wallenius, G., and Gronwall, A., Scand. J . Clin. Lab. Invest., 1952, 4, 47.

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31, 380.

--

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1052.

THE BIOCHEMICAL LABORATORY THE ROYAL INFIRMARY

WORCESTER August 28th, 19.52

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